Transcription Factors and Plants Response to Drought Stress: Current Understanding and Future Directions

1The 21st century agriculture is facing a daunting challenge of attaining nearly up to 70% increase in crop productivity by 2050. Also, crop productivity needs to witness up to 40% increase by 2015 in view of the cultivated area being increasingly affected by stresses. Frequent changes in climatic conditions also affect the crop yield (such as flooding after high temperature), which led scientists to undertake research on this aspect in recent time around the world. Among the various abiotic factors challenging crop production globally, drought stress is increasingly playing a crucial role. Drought is a meteorological term and is commonly defined as a combined interplay of reduced rainfall, decreasing ground water table, limiting water availability with rise in temperature. With global climatic vagaries registering escalated frequency, the global drought incidence is likely to swell beyond 20% by the end of this century, especially in Central and South American corn belt, and Central and Western Europic regions. Similarly in South Asia, where more than 75% of the farmers are dependent on rainfed agriculture, a damage of about US$ 84 billion is predicted owing to the global climate change. Most of the crops are susceptible to drought stress, resulting in more than 50% yield losses. In South and Southeast Asia, drought stress causes about 40% annual loss in productivity, which results in 58% of income loss. Similarly, regional climate change models showed Mediterranean regions and Middle East among the ‘Hot-spots’ of severe drought, directly affecting crop failures and livestock death. Brazil, which is the second highest producer (40%) of soybean has lost greater than 20% of their productivity because of the occurrence of drought during 2003–2005. The uneven distribution of rainfall and ground water shortage often create drought stress conditions in the environment, which eventually leads to an enormous decrease in the grain yield potential.

2Upon exposure to drought, plants manifest multiple impairments including cell injury through reactive oxygen species (ROS) generation and increasing cellular temperature, which result in an increase in the viscosity of cellular contents, alterations in the protein–protein interactions, protein aggregation, and denaturation. Cell shrinkage followed by a marked decline in cellular volume becomes evident as an instant symptom caused by dehydration. Besides, greater accumulation of solutes causes toxicity and negatively affects functioning of some enzymes, often leading to reduced photosynthesis and water use efficiency (WUE). Under prolonged dehydration, plants exhibit leaf rolling followed by wilting and bleaching that eventually result in death of the plant. Reproductive stages, i.e., flowering as well as seed development are especially sensitive to drought stress. A reduction in number of grains per spike, grain filling duration, and dry matter accumulation, leading to decreased grain weight in barley kernels has already been reported earlier. Recently, leaf area, leaf weight as well as leaf growth rate were also reported to hold great relevance while breeding crops grown under Mediterranean environmental conditions such as drought stress. Substantial progress has already been made to enhance crop productivity using conventional breeding methods. However, development of drought tolerant cultivars using conventional breeding approach is greatly restricted by low heritability of drought tolerance, narrow genetic variation existing in the crop’s exploitable genetic pool, complex and multigenic nature of this trait and high magnitude of environmental interactions.

3Considering global food security, immediate assessment of drought stress impact and innovative techniques to breed tolerant and productive cultivars under dramatically changing climate are essentially warranted. As a means to provide crop-based answer to burgeoning issues like food insecurity and malnutrition, cereals constitute a vital role as major fraction in the diet, especially in developing nations. Rice, wheat and maize are major cereal crops in the world; therefore less water availability exerts profound consequences. Development of advanced genotypes with improved stress tolerance and wider adaptability is a simple, cost-effective and eco-friendly approach to cope with drought stressed scenarios.

4Drought tolerance is an outcome of a series of molecular, cellular, and physiological processes including induction/repression of various genes that cause accumulation of various osmolytes, improved antioxidant system, reduced transpiration, inhibited shoot growth, and decreased tillering. Besides, the phytohormone abscisic acid (ABA) is reported to be abundant under water-deficit conditions and this in turn causes stomata closure and induces expression of various stress-related genes. It has been shown that drought inducible gene expression is also governed by ABA-independent regulatory system. Plant’s ability to cope with water deficit depends largely on its water status, which changes with environmental conditions. Thus, incorporating drought stress tolerance in crops becomes imperative under changing climatic conditions and it could be a promising approach to meet the global food demand. In this context, unraveling the molecular mechanisms that control the perception and transduction of stress signals to initiate adaptive responses is crucial for engineering drought stress tolerance in plants.

5Notable progress has been made toward this end by utilizing modern genetics and functional genomics approaches such as transcriptomics, proteomics and metabolomics and consequently, various drought stress responsive genes have been identified and characterized in crops. These key genes mainly code for proteins that have either metabolic or regulatory roles, such as those involved in detoxification, osmolyte biosynthesis, proteolysis of cellular substrates, water channel, ion transporter, heat shock protein (HSP), and late embryogenesis abundant (LEA) protein. On the other hand, the regulatory class primarily comprises of TFs (AREB, AP2/ERF, NAC, bZIP, MYC, and MYB), signaling protein kinases [mitogen activated protein kinases (MAPK), calcium-dependent protein kinases (CDPK), receptor protein kinases, ribosomal protein kinases, and transcription regulation protein kinases] and protein phosphatases (phosphoesterases and phospholipase), which synchronize signal transduction and expression of genes during stress responses. Various desiccation-induced genes have been characterized, including those encoding ABA biosynthetic pathway, osmo-protectants such as proline, trehalose, mannitol, ectoine, glycine betaine providing tolerance to cellular dehydration, cellular enzymes, signaling proteins, and TFs. Several of these regulatory genes including TFs were found to play essential roles in multiple abiotic stress responses via regulating downstream stress-responsive genes. As illustrated in Figure 1, TFs regulate gene expression through binding to cis-regulatory elements in the promoter region of different stress-related genes. Thus, genetic modification of the expression of these regulatory genes can greatly influence plant stress tolerance because they further regulate many downstream stress-responsive genes at a given time. Among these regulatory genes, stress-responsive TFs have gained widespread attention on account of their significant role in plant stress tolerance and allelic variations were examined across Indian wild rice population by researchers in genes with respect to abiotic stresses. In this article, we elaborate on the key TFs that contribute toward drought stress tolerance, and their networks, cross-talks and potential utilization in crop improvement programs.

6The response of any crop to drought stress depends primarily on the growth stage and WUE. For example, reproductive stage is considered critically susceptible to drought stress in various crops. As sessile organisms, plants have evolved several defense mechanisms involving various molecular, physiological and biochemical alterations in response to low soil moisture. Visible symptoms describing drought tolerance include leaf rolling, stay green ability, epicuticular wax deposition, closing of stomata, enhanced root length leading to higher WUE, photochemical quenching, photoinhibition resistance, osmotic adjustment, and membrane stabilization at the cellular level. Leaf rolling, a foremost symptom, constitutes an initial survival mechanism against osmotic stress that can minimize canopy temperature and transpiration rate, thereby improving WUE. Thus, species maintaining higher relative water content (RWC) under osmotic stress are reported to be less susceptible to low water potential, and thus retain their growth and productivity. It was also observed that crop growth is primarily supported by the soil moisture surrounding plant root system. Therefore, total root area being directly affected by the decreased soil water content deserves greater attention. It was reported that osmotic stress-induced premature differentiation caused growth inhibition of primary roots and allows outgrowth of lateral roots. A range of biochemical changes have also shown in response to drought stress, including higher accumulation of osmolytes, ROS, antioxidant species, and antioxidative enzymes. These osmolytes are reported to have principal role in maintaining enzyme activity and membrane stability under osmotic stress.

7Drought stress in plants occurs either due to poor quantity of water, i.e., little rainfall or due to insufficient quality of water, i.e., saline habitats. As a means to survive under water stress conditions, plants respond by alteration in expression of several genes and regulate them through complex transcriptional networks. It is therefore imperative to dissect stress regulatory mechanisms and to pinpoint the key regulators that could eventually be harnessed to breed or engineer stress tolerant plants. By analyzing plant genomes and employing modern OMICS tools, including genomics, transcriptomics and proteomics, significant progress has been made in elucidating the stress signaling pathways involved in drought stress response. Few of these genes associated with these transcriptional networks have been characterized in various molecular studies and have been found to be key contributors toward drought stress tolerance in transgenic plants.

8The signaling pathway of any abiotic stress involves certain key steps such as signal perception, transduction, responsiveness, combined with activation of physiological, and metabolic reactions. In this process, plant cells first perceive stress stimulus through sensors or receptors localized mostly at the cell membrane. Then, the receiver signaling molecules activate the intracellular ones through second messengers like calcium ions, inositol phosphate, ROS, cyclic nucleotides (cAMP and cGMP), sugars and nitric oxide. Subsequently, these second messengers initiate the corresponding signaling pathways to transduce the signals. The phosphorylation and dephosphorylation of proteins regulated by protein kinases and phosphatases, respectively, is a significant and effective mechanism in several signal transduction pathways. For example, the MAPKs and CDPKs are well known for their role in drought stress signaling pathways. At the end of the phosphorylation cascade, TFs are activated or suppressed by protein kinases or phosphatases, and directly regulate the expression of an array of downstream genes by interacting with the specific cis-elements in their promoter region. In addition, TFs themselves are regulated at the transcription level by other upstream components and after several modifications at the post-transcription level, such as ubiquitination and sumoylation, they form a complex regulatory network which modulates the expression of stress responsive genes, regulating various physiological and metabolic processes. In the past few decades, considerable research has been carried out toward identification and characterization of different TFs that contribute toward drought stress response.

9The multifarious signaling pathways of water stress in plants consist of several proteins, i.e., TFs, enzymes, functional proteins, molecular chaperones, and metabolites. Genetic engineering approaches to mitigate the challenge of drought stress in plants involve the overexpression of these TFs, enzymes and other metabolites. In recent years, a wide range of TF families holding relevance with drought stress response have been identified. During the signal transduction, TFs directly regulate the expression of the associated genes via serving as molecular switches. These TFs interact specifically with cis-elements located in the promoter region of the genes they regulate. In plants, a large proportion of genes in the genome (up to 10%) potentially encode TFs, which are categorized into different gene families such as AREB, DREB, MYB, WRKY, NAC, and bZIP based on the distinct structure of their DNA-binding domain. In Arabidopsis nearly 6% of the proteome is dedicated to TFs. Among these TF genes, several have been reported to respond under drought stress through pathways dependent/independent of ABA. Promising results have been obtained concerning engineering of some TF genes with an aim to improve tolerance against drought stress. In this section, we describe major TF families known to be responsible for drought stress tolerance and discuss the scope for manipulating them in order to obtain transgenic plants with enhanced drought tolerance.

10Several research groups have elucidated the molecular mechanism related to drought stress transcriptional networks in plants. Under osmotic stress conditions, detailed molecular analyses have found abscisic acid-responsive element binding protein (AREB)/ABFs (ABRE binding factor) as a major transcriptional activator modulating the expression of genes during ABA signaling. ABA-responsive gene expression is controlled by a conserved ABRE (PyACGTGG/TC) cis-element in its promoter region (Figure 1). Studies have revealed the essential role of several ABRE or its combinations with coupling elements (CE) during ABA-responsive genes expression. Structurally, AREB/ABFs consist of four SnRK2 phosphorylation sites containing conserved domains that regulate ABA-dependent gene expression. It was found that vascular tissue specific cells produce ABA and transfer them to target cells. It was also demonstrated that ABA is synthesized within guard cells and vascular tissue specific cells. In Arabidopsis, five genes are known to code for NCED (9-cis-epoxy carotenoid dioxygenase), a major enzyme in ABA biosynthesis and expression. Of these, NCED3 showed higher expression under dehydration and was reported to be under the regulation of an AG-box recognition sequence located 2248 bp upstream to its transcriptional start site. ABA is perceived by an ABA-bound PYL/PYR/RCARs receptor complex combined with PP2Cs, and subsequently SnRK2s is released. The activated SnRKs phosphorylate the downstream proteins like AREB/ABF TFs, which then bind to the ABRE cis-element (PyACGTGG/TC). In the absence of ABA, PP2Cs inhibit ABA signaling pathway by dephosphorylating SnRK2s. Of the nine AREB/ABF family members, AREB1/ABF2 regulates ABA signaling during drought stress at the vegetative stage (Figure 1). ABA is essential for proper functioning of AREB1, and ABA-dependent phosphorylation regulates its activity. Overexpression of AREB1 was shown to improve drought tolerance in Arabidopsis, rice and soybean. Recently it was shown that Arabidopsis plants overexpressing wheat transcription factor TaAREB3 have enhanced ABA sensitivity and drought tolerance (Table 1). In primary species, group A PP2Cs have evolved as essential regulators providing osmotic stress tolerance.

11APETALA2/Ethylene Response Element binding Factors (AP2/ERF) family covers a large group of plant-specific TFs and is characterized by the presence of a much-conserved AP2/ERF DNA-binding domain. This domain binds with the GCC box, which is a DNA sequence having role in the ethylene-responsive transcription. AP2/ERFBP TFs perform diverse roles in plant biological processes, such as cell proliferation, vegetative and reproductive development, plant hormone responses, and abiotic/biotic stress responses. Genome-wide analyses have resulted in the identification of a multitude of AP2/ERFBP members across several plant species such as 145 in Arabidopsis, 170 in rice, 178 in sorghum, 200 in poplar, 291 in Chinese cabbage, 171 in foxtail millet, and 116 in moso bamboo. Based on the number and similarity of AP2/ERF domains, the family is further categorized into four major subfamilies: AP2 (Apetala 2), RAV (related to ABI3/VP1), DREB (dehydration-responsive element-binding protein), and ERF. Apropos of the plant abiotic and biotic stress response, ERF and DREB subfamilies have been extensively studied. Induced, respectively, by cold and dehydration, DREB1/CBF (with 11 genes in rice) and DREB2 (six genes in rice) function in ABA-independent manner. The promoter analysis of drought stress regulated genes having ABA-independent expression in Arabidopsis illustrated a cis-element with A/GCCGAC sequence (known as DRE/CRT: ). Functional similarities were observed in the downstream genes of DREB1A and DREB2A by microarray analysis, however, differential expression was established during carbohydrate metabolism in transgenic plants. Four orthologs of CBF/DREB1A in rice viz. OsDREB1A, OsDREB1B, OsDREB1C, and OsDREB1D have already been characterized. DREB1/CBF TFs specifically interact with the DRE/CRT and regulate the expression of several abiotic stress-responsive genes. Different studies confirmed that DREB1/CBF genes expression is differentially regulated by other genes like ICE1, HOS1, MYB15, SIZ1, PIF7, CAMTA3, and a clock component. Plants overexpressing DREB1/CBF showed enhanced expression of several stress responsive genes providing improved tolerance toward drought including tomato, chrysanthemum, potato, soybean, rice, tobacco, sugarcane, groundnut, peanut, and wheat. In rice, DREB1/CBF-type TFs conferred higher drought tolerance besides expression of cold-responsive genes. experimentally demonstrated improved survival of NERICA1 (an upland rice cultivar) under drought stress through expressing Arabidopsis DREB1C in the transgenic plants. OsDREB1F is reported to be induced by drought stress and ABA application. Overexpression of OsDREB1G is also known to promote drought tolerance. Similarly, constitutive expression of DREB1/CBF3 in transgenic plants led to improved tolerance against salinity, heat and cold. Higher accumulation of osmoprotectants, such as free proline and soluble sugars were recorded in the transgenic rice plants overexpressing DREB1A. More recently, transgenic tobacco with elevated tolerance level against drought and salt was obtained through over-expression of SsCBF4 from succulent halophyte Suaeda salsa (Table 1). An affymetrix microarray based analysis of transgenic rice plants facilitated the identification of 404 genes as induced or suppressed by DREB1BI when compared to their respective wild types.

12A conserved regulatory mechanism has been established after examining DREB2-type proteins across several crop species such as wheat, maize, rice, barley, and sunflower. Rice harbors six DREB2 family genes while Arabidopsis contains eight DREB2 genes of which DREB2A and DREB2B rendered higher induction under drought stress. The constitutive as well as stress inducible expression of OsDREB2A and OsDREB2B in transgenic plants has resulted in enhanced tolerance toward osmotic stress. Transgenic Arabidopsis plants overexpressing OsDREB2B displayed enhanced expression of DREB2A target genes and improved tolerance toward drought stress. Taken together, these reports suggest that OsDREB2 is a major gene encoding for DREB2-type TF that holds great importance in stress-responsive gene expression. However, in addition to imparting increased drought tolerance, over expression of DREB2A was reported to cause significant growth defects. In contrast, no growth defects were seen in case of Arabidopsis and soybean transgenics where stress-inducible expression of DREB2A led to enhanced drought tolerance. Under normal condition, DREB2A is regulated by two mechanisms (i) GRF7 (growth-regulating factor7) inhibits expression of DREB2A binding to its short promoter region and (ii) targeted degradation by 26S proteasome mediated proteolysis assisted by DRIP1 (DREB2A- interacting protein1) and DRIP2 protein. In other words, conditional expression of DREB2A under drought stress offers a way to minimize the inefficient loss of energy.

13Besides DREB1A and DREB2 members, various other AP2/ERF-type and AP2/ERF-like TFs were also characterized in rice with an aim to improve abiotic stress tolerance. Likewise, overexpression of Arabidopsis HARDY gene (a subgroup of A-4 AP2/ERF family) in Trifolium led to enhanced osmotic stress tolerance. OsDERF1 has been established as a negative modulator in osmotic adjustment by interacting directly with GCC box located in the promoter of OsERF3. Furthermore, OsERF3 contains an ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif, which transcriptionally represses the ethylene production along with drought tolerance. Mutant EAR lines showed enhanced ethylene emission and drought tolerance as compared to overexpressing as well as wild type plants. However, reported OsERF4a as a positive regulator of plant growth and drought tolerance in rice. In addition, ectopic expression of tomato JERF3 alleviates soluble sugars and proline with increased drought tolerance in rice. Similarly, overexpression of another AP2/ERF TF showed increased drought, cold, and salinity stress tolerance in transgenic rice (Table 1).

14The NAC gene family is the largest and plant specific TF family. The NAC acronym is derived from three proteins having a specific NAC domain from petunia, i.e., NAM (no apical meristem) and ATAF1/2 and CUC2 (cup-shaped cotyledon) in Arabidopsis. Characteristically, a NAC TF comprises of a highly conserved DNA binding NAC domain in the N-terminal region and a variable C-terminal transcriptional regulatory region. The NAC domain is linked with DNA binding, nucleus-oriented localization and formation of homodimers or heterodimers with similar domains, while the C-terminal functions in transcriptional regulation. The NAC TFs modulate downstream drought inducible EARLY RESPONSE TO DEHYDRATION1 (ERD1) gene transcription by interacting with NAC recognition sequence (NACRS) having CACG core-DNA binding motif in its promoter. In rice, SNAC1 TFs were reported to confer drought tolerance through regulating downstream genes OsPP18, a PP2C in ABA independent pathways. To date, genome wide characterization has offered numerous putative NAC TFs across different plant species like 204 in Chinese cabbage, 152 in soybean, 152 in maize, 151 in rice, 117 in Arabidopsis and 74 in grape. In rice, several NAC genes were reported to be induced during early stages of salt and drought stresses. The role of NAC TFs in drought stress response is further supported by several transcriptome-based analyses undertaken in different crops; examples include 40 NAC genes in rice, 38 NAC genes in soybean. These stress-responsive NAC genes also showed differential expression patterns such as tissue-specific, developmental stage-specific or stress-specific expression, thereby suggesting their active involvement in the complex signaling networks during plant stress responses. In Arabidopsis, drought tolerance was found to be elevated by overexpressing three genes, i.e., ANACO19, ANACO55, ANACO72. Similarly, overexpression of stress responsive NAC1 (SNAC1) in rice provides tolerance against severe drought stress at the reproductive stage in field conditions without a change in their phenotype or yield penalty. In another study it was found that under drought stress SNAC1 was induced in guard cells and its over-expression reduced transpirational losses due to increased stomatal closure. Highlighting the importance of SNAC1 gene, genetic variation of this gene was assessed among Indian wild rice and as a result, discovery of four haplotypes associated with tolerance might be useful for developing drought tolerant crops. Similarly, OsNAC5 expression was reported to be induced under drought, cold, ABA, and methyl jasmonate (MeJA) treatments. Overexpressors of OsNAC5 were reported to serve as transcriptional activators that regulate the stress-responsive gene expression. Function of OsNAC5 in drought tolerance was examined by RNA interference (RNAi) and overexpression study in transgenic rice and Arabidopsis. In addition, OsNAC6/SNAC2 is also known to be induced by cold, drought, salinity and ABA. Furthermore, overexpressing TaNAC2 and TaNAC29 in Arabidopsis resulted in enhanced drought, salt and cold stress tolerance along with higher transcript levels of stress-responsive genes and improved physiological parameters. Contrary to this, OsNAC6 overexpressing rice plants had reduced growth and low yield under normal conditions even though these plants remained tolerant for dehydration and salinity stresses. Based on these studies, OsNAC6 could be established as a potent functional regulator in drought stress response. Interestingly, overexpression of HvSNAC1 in barley not only improved drought tolerance but also provided resistance against fungal infection of Ramularia collo-cygni causing leaf spot.

15Enhanced abiotic stress tolerance in rice caused by overexpressing SNACs was evident when using a root-specific promoter RCc3. Similarly, thicker roots and higher grain yield under drought stress were observed in transgenic rice plants overexpressing OsNAC10, OsNAC5, and OsNAC9/SNAC1 regulated by root specific promoter. Furthermore, microarray analysis of these transgenic plants revealed 62 downstream genes to be differentially expressed, which included P450, Zn-finger, HAK5, 2OG-Fe(II), NCED, NAC, KUP3, calcium-transporting ATPase, germin-like protein, and meristem protein. Of these, only 17 were found to be upregulated. However, as demonstrated by in soybean GmNAC11 was reported to function as a transcriptional activator, whereas GmNAC20 acted as a mild repressor with C-terminal transcriptional activation activity and its overexpression increases lateral root development in transgenic plants. This study established GmNAC20 as a regulator of stress tolerance, which serves by activating DREB/CBF-COR pathway and lateral root formation by modulating auxin signaling genes (Table 1). Thus, precise manipulation of SNACs with suitable promoter offers a promising way to substantially alter drought stress response in plants.

16The basic leucine zipper (bZIP) family contains a conserved bZIP domain, which is composed of a highly basic nuclear localization and DNA binding region at the N-terminus and a leucine-rich motif for dimerization at the C-terminus. Besides regulating plant growth and development, bZIP TFs remain crucial concerning abiotic stress response such as drought. Members of the bZIP TF family have been isolated and characterized in various eukaryotes. Examples include 17 in Saccharomyces, 31 in Caenorhabditis, 55 in grapevine, 75 in Arabidopsis, 89 in rice, 89 in barley, 92 in sorghum, 96 in Brachypodium distachyon, 125 in maize, and 131 in soybean. Though the physiological role of their homologs with putative zinc finger motif remains unclear, studies on abiotic stress responses have confirmed bZIP TFs to be ABA inducible, i.e., these regulate the expression of stress-related genes in ABA-dependent manner after binding with the promoter region of specific ABRE.

17characterized a novel gene ThZF1 from Thellungiella halophila that encodes a functional TF. It contains two conserved Cys-2/His-2 regions with conserved DNA-binding motif similar to the members of Arabidopsis ZFP family. Drought and salinity are reported to induce the transcription of this gene. Ectopic expression of ThZF1 in Arabidopsis mutant azf2 revealed its similarity with Arabidopsis AZF2 in plant development and downstream gene regulation. Likewise, transgenic rice plants overexpressing OsbZIP16 exhibited significantly higher drought tolerance at both seedling and tillering stages. Under drought stress conditions its downstream drought-inducible genes showed significantly higher expression levels in transgenics than the corresponding wild types. Though OsbZIP16 is reported to be ABA-inducible, overexpression of OsbZIP16 renders transgenic plants more sensitive to ABA. A recent study in Arabidopsis revealed that overexpression of TabZIP60 improved plant’s tolerance against stresses like drought, salt and freezing along with increasing sensitivity to ABA.

18Similarly, OsbZIP23, a close relative of Arabidopsis homologs ABF/AREB, was reported to be a principle regulator of ABA-dependent pathways. Rice plants overexpressing OsbZIP23 caused greater ABA sensitivity at both germination and post-germination stages along with increased drought and salt stress tolerance. Microarray analysis showed differential expression of several downstream genes including stress-related TFs, protein kinases, dehydrins and LEA proteins. OsbZIP46 also belong to the subfamily of OsbZIP23 and its constitutively active form OsbZIP46CA1 was developed by mutating OsbZIP46 domain D. Rice plants overexpressing OsbZIP46CA1 showed enhanced drought tolerance. Differentially expressed downstream genes different from those of OsbZIP23 downstream genes were uncovered through a microarray analysis, which in turn suggested independent regulatory mechanisms of OsbZIP46CA1 and OsbZIP23. In contrast, growth of OsbZIP23 or OsbZIP46CA1 overexpressing rice plants is reduced under drought stress. Furthermore, ABF3 belonging to similar subfamily as OsbZIP46 and OsbZIP23 improved drought tolerance in transgenic Arabidopsis and rice. OsbZIP16 and OsbZIP71 are classified into group IV of the rice bZIP subfamily. After ABA treatment transgenic rice plants overexpressing OsbZIP16 showed enhanced drought tolerance and reduced plant growth. However, OsbZIP71 overexpressing rice transgenics with a constitutive promoter or a stress-inducible RD29A promoter showed enhanced drought, salt, and osmotic stress tolerance. Collectively, these reports confirmed the involvement of bZIP TFs in ABA signaling, which could be harnessed for developing better genotypes endowed with drought tolerance.

19Drought stress is an unpredictable event and it varies in severity and duration. This results in both general and specific effects on plant growth and development. Thus, plant response toward drought stress is dynamic, which involves multiple stress perception and signal transduction pathways, which may crosstalk at various steps in the pathways. For this reason, plants have evolved complex regulatory mehanisms, including metabolic adjustment and gene expression toward physiological and morphological adaptation. Moreover, extensive overlap between signal interactions and adaptation mechanisms can be both synergistic and antagonistic, resulting in positive and negative functional outcomes. This type of regulation allows cell to respond rapidly to changes such as drought stress. TFs are of key importance in these signaling cascades and in generating specificity in stress responses. Interactions among different TFs have made significant advances over recent years and were documented recently by. The TFs control transcription of their downstream genes by interacting with other proteins and binding to a consensus sequence in promoters. Identification of these downstream genes becomes imperative to elucidate molecular machineries of gene activation or repression (Figure 1).

20Drought responsive gene promoters contain a DRE/CRT motif where ABA-independent DREB/CBF TF binds and act as a coupling element for ABRE in ABA-dependent gene expression. It was already shown that DREB1A/CBF3, DREB2A, and DREB2C proteins interact with AREB/ABF. Thus, there exists a crosstalk between ABA-dependent and independent signaling and regulatory pathways (Figure 1). Under osmotic stress conditions, AREB/ABF TFs and SnRK2s regulate the transcriptional activation of DREB2A gene, suggesting a complex interaction between DREB and AREB regulatory regions at the transcript and protein level. Similar interactions were also reported in between AREB/ABFs and NACs. In addition, regulation of ABA-dependent gene expression of ABRE regulons by SNAC TFs was confirmed when reported that Arabidopsis SNAC transcription factor ATAF1 directly modulates ABA biosynthetic gene NCED3. By contrast, SNAC gene promoter contains ABRE region. Under dehydration and osmotic stress, ANAC096 was also found to interact with ABF2/AREB1 and ABF4/AREB2. Furthermore, correlation among DREB/CBFs and AP2/ERFs at the transcript levels was also confirmed when Arabidopsis ERF1 was shown to regulate transcript expression of a gene by coupling with two different cis-elements, the GCC box and DRE/CRT during stress response. Recently, it was shown that bZIP TFs VEG2 and FLOWERING LOCUS D (FD) interact with nuclear protein CONSTANS (CO) and positively regulate signaling of flowering activator FLOWERING LOCUS T (FT) proteins. TFs activate several functional genes encoding wide range of proteins involved in osmolyte production, ROS scavenging and detoxification, macromolecule protection and ubiquitination. As envisaged by, such versatility in TFs functioning might have evolved to ensure plants’ longevity, survival and reproductive success under environmental stresses. Our current knowledge about these target genes stems mainly from the studies performed using large-scale transcriptome analyses in plants that overexpress TFs. Deeper analyses can better elucidate their roles under drought response.

21Here, we highlighted the increasingly important roles of TFs as modern genomic tools to improve plant tolerance toward multiple abiotic stresses. Significant progress has been achieved in deciphering the role of TFs toward tolerance to abiotic stress factors such as drought, and an appreciable number of promising candidates – TF genes have already been identified and validated. Selection of key TFs from such large families and realization of their full potential still remain a strenuous task. Also, development of transgenic plants harboring such TF genes that deliver anticipated level of tolerance in the field conditions still poses a great challenge to the research community. Furthermore, functional redundancy between different TF members might potentially hamper the progress to delineate the functions of an individual member.

22The constitutive over-expression of some TF genes may improve the stress tolerance; however, occasional negative effects in transgenic plants such as dwarfing, late flowering and lower yields should also be taken into account while investing research. Furthermore, the complete regulatory mechanism of individual TF encompassing its upstream and downstream co-regulators, as well as their interactions remain largely obscure. A principle concern is also about the selection of plants, as most of the initial experiments are being performed on model crops like Arabidopsis and tobacco. Focus must be shifted to commercial crops like rice, wheat, maize and other cereal crops, which are predominantly affected by drought stress. In comparison to other crops, rice has shown the highest potential under waterlogging conditions. Although reports are available with improved drought tolerance in transgenic rice under field conditions, further research is required in order to unveil the regulatory mechanism of drought tolerance. Future investigations must focus on pinpointing novel genes that enhance drought tolerance as well as yield. In addition, combination of drought-tolerance with submergence-tolerance in rice is exceptionally useful, as this would allow crop to withstand both drought and waterlogged conditions.

23Another attractive approach for discovering novel candidate genes is to intensively examine the mechanisms that describe drought tolerance in extremophiles, i.e., desert plants. It is important to mention here that up to 38% of total protein functions are still unknown even in well-analyzed plants and a renewed focus in this aspect might led to the discovery of robust candidate genes. Modification in root architecture is another important factor for improving drought tolerant crops. Transcriptome analysis has shown that drought-responsive TFs like REB1/CBF, DREB2, AREB/ABF, and NAC TFs function under drought response and tolerance. Utilizing a balanced crosstalk between these TFs under osmotic stress, overexpression of TFs may affect their signaling pathways. Identification of multiple stress-responsive TF genes and their prioritization by means of comparing expression patterns deserves attention of the scientific community and pinpointing the commonly regulated genes which have been proposed to be essential for universal stress responses or represent points of cross-talk between signaling pathways. Genetic manipulation of these multiple stress-responsive TF genes stands to be a powerful approach for improving plant tolerance than addressing each functional gene individually. Furthermore, the field trials are required to critically evaluate the transgenic plants, especially focusing on their growth and tolerance in the whole life period, which unequivocally remains a deciding factor while developing stress-tolerant crops.

24SLS-P and AP conceived the review topic. RJ, BS, and AB drafted the manuscript. SW, ZD, and AL participated in the discussion of topic. All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

25The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.