Harnessing the Potential of Plant Transcription Factors in Developing Climate-Smart Crops: Future Prospects, Challenges, and Opportunities

Crop plants should be resilient to climatic factors in order to feed ever-increasing populations. Plants have developed stress-responsive mechanisms by changing their metabolic pathways and switching the stress-responsive genes. The discovery of plant transcriptional factors (TFs) as key regulators of different biotic and abiotic stresses have opened up new horizons for plant scientists. TFs perceive the signal and switch certain stress-responsive genes on and off by binding to different cis-regulatory elements. The above 50 species of plant TFs have been reported in nature. DREB, bZIP, MYB, NAC, Zinc-finger, HSF, Dof, WRKY, and NF-Y are important with respect to biotic and abiotic stresses whereas the role of many TFs is yet to explore. In this review, we summarize the role of different stress-responsive TFs with respect to biotic and abiotic stresses. Further, challenges and future opportunities linked with TFs for developing climate-resilient crops are also elaborated.


Introduction
Agricultural crops are important as they represent the in terms of largest source of calories (70%-80%) and protein (60-70%) intake for mankind. However, the changing climate is adversely affecting plant health and causing food insecurity due to activation of multiple biotic and abiotic factors [1]. Plants have adopted different resistance mechanisms for survival under changing environmental conditions. For example, in response to drought stress, plants start developing a strong root system and promote lateral roots to increase the water catchment area. Similarly, in response to terminal heat stress, plants shift their growth patterns from vegetative to reproductive growth to limit the effect of terminal heat stress on reproduction; many other similar examples exist [2]. Plants respond to When plant cells perceive a stress signal, receptors or sensors in the cell wall or membrane detect the stress stimulus, followed by a rapid response that transduces the external signal to intracellular signals. Signal cascades involving intracellular molecules or ions are activated along with kinase cascades, which are generally cytoplasmic. Major cascades are associated with reactive oxygen species (ROS) and calcium ions (Ca 2+ ). Phytohormones, including abscisic acid, jasmonic acid, salicylic acid, and ethylene, are powerful second messengers that coordinate signal transduction pathways during stress responses. These signals activate several parallel transduction pathways, which often involve phosphatases and protein kinases. Following the Figure 2. A detail description of Domain structure, domain composition, and cis-regulatory development of plants, and can reduce up to 50% yields of wheat, rice, maize, and cotton [19]. Plants respond to stress by certain physiological adjustments, i.e., increasing ion fluxes, production of ROS, accumulation of amino acids and soluble sugars, maintaining homeostasis and osmotic potential, and change in phytohormone concentrations ( Figure 1B). The stress-related receptors receive environmental stimuli and activate the stress responsive genes [34]. The role of different genes/TFs in response to different stresses is discussed below (Table 1).

Drought stress
Drought is a devastating abiotic stress, which occurs due to shortage of ground water, high temperature, and/or low rainfall. Drought reduces seedling emergence, germination rate, vegetative growth, root & shoot dry matter, and hypocotyl length [35]. It decreases turgor pressure and limits cell elongation, cell growth, and leaf expansion. Acute shortage of water damages the thylakoid membranes and photosynthetic pigments and slowing the rate of photosynthesis. During drought stress, plants close stomata, thereby reducing intracellular CO2 concentration thus reducing photosynthesis and also inducing oxidative damage [36]. Plants respond to stress by certain physiological adjustments, i.e., increasing ion fluxes, production of ROS, accumulation of amino acids, and soluble sugars and changes in phytohormone levels. TFs play an important role in orchestrating these processes by activating genes that execute stress responses [34].
WRKY TFs play an important role in improving stress tolerance, particularly drought and heat tolerance in various crop plants. TaWRKY1 and TaWRKY33 in Arabidopsis thaliana increases drought tolerance due to overexpression of downstream stress related genes. AtWRKY1 locates in the nucleus and binds to the W-box domains of AtDREB1A, AtMYB2, and AtAB15 to control their transcription and regulate stomatal conductance [28]; in transgenic Arabidopsis ZmWRKY40, activated stress related genes and generation of ROS. Overexpression of TaWRKY2 in wheat increased drought tolerance and grain yield. Arabidopsis AtWRKY30 overexpression in transgenic wheat increased biomass, plant growth, proline concentration, soluble sugar, protein, relative water content, chlorophyll content, and antioxidant enzymes activities to alleviate drought stress [19]. In Arabidopsis thaliana, the bZIP gene, AtABP9 binds to an ABPR motif and increased photosynthetic activity. It increases production of abscisic acid (ABA) and changes composition of photosynthetic pigments. ZmNF-YB16 overexpresses in young seedling under drought conditions, as a result, antioxidant enzymatic activity increases [34].
Similarly, SlWRKY8 overexpressed under drought conditions activates malondialdehyde (MDA), hydrogen peroxide (H2O2) production, and antioxidant enzymatic activity. These changes trigger SIRD29, SIAREB, and SIDREB2A genes, which decreases stomatal aperture, oxidative pressure, and increases relative water and proline contents to alleviate drought stress. DREB1A overexpressed in Arabidopsis thaliana, resulted in high accumulation of solutes and initiation of late embryogenesis abundant protein [37]. Similarly, soybean GmNAC8 overexpressed under drought stress and binds to GmDi19-3 (drought-induced proteins), which activates proline and superoxide dismutase (SOD) activity. Overexpression of ZmNAC111 increases water use efficiency of drought prone maize seedling by upregulating drought responsive genes [38].

Heat stress
Heat stress adversely affects plant growth and quality by changing their morphological, physiological, and biochemical features. It decreases chlorophyll content and simultaneously induces oxidative stress due to accumulation of hydroxyl (OH -), hydroperoxyl (HO 2-), alkoxy (RO -), and superoxide (O 2-) radicals. Oxidative stress hinders photosynthesis and respiratory activities, disrupts protein structure, and membrane integrity [39]. Heat stress causes burning of leaves and branches, patch formation on leaves, reduction in germination and growth, reduced tillering, and reduction of grain size and grain yield [36]. Elevated temperature uplifts the rate of transpiration, which adversely affects root growth. Sucrose phosphate synthase, adenosine diphosphate-glucose pyro-phosphorylase, and invertase are highly sensitive enzymes that disturb sucrose and starch synthesis pathways during heat stress. On the other hand, high temperature increases catalytic activity of Rubisco, but decreases its ability to bind with CO2 and O2, and slows down the photosynthetic rate [40].
Triticum aestivum, Solanum lycopersicum, Cicer arietinum, Glycine max, and Sorghum bicolor are heat-sensitive crops. Similarly, pollens formation, seed setting, and grain filling are highly heatsensitive plant processes [41]. Plants have several adaptations against heat stress, i.e., degradation of oxyradicals, reduction in the lipid membrane transformation stage, and biological metabolism. Epigenetic modifications, i.e., acetylation, methylation, phosphorylation, and ribosylation also help plant survival during heat stress by modifying histone proteins after translation [42]. Another modification is evolution of heat shock factors (HSFs). HsfA1s are prime activators in response to heat stress, while in non-stress conditions; these are suppressed by heat shock proteins, i.e., HSP70 and HSP90. These regulate expression of some TFs, i.e., dehydration responsive element binding 2A (DREB2A), heat shock factors A2 (HsfA2), heat shock factor B (HsfBs), DREB2C, multiprotein binding factor 1C (MBF1C), and NAC. HSPs are involved in homeostasis at the cellular level and plant defence. At the onset of heat stress, inactive HSFs are activated through oligomerization and shuttle signalling between the cytoplasm and nucleus [43].
HSP70 makes a complex with heat stress RNA1 (HSR1) and translation elongation factor (eEF1A), which activates HSF1. HSF1 then activates the cell heat stress responsive machinery [44]. A complex of TFs network consisting of MYB, bZIP, NAC, and a homeobox linked with Leucine zipper is recognized as effective elements in long-term heat stress conditions. General heat stress responsive elements and stabilizers for protein metabolism are HSP10s, HSP20s, HSP60s, HSP90s, and cochaperones [45]. Different WRKY i.e., AtWRKY18, AtWRKY25, AtWRKY33, AtWRKY40, and AtWRKY46 also have roles to play during heat stress. AtWRKY39 imparts heat stress tolerance in Arabidopsis thaliana by binding to a calmodulin binding TF [46]. The AtDREB2A binds to dehydration responsive elements (DRE) at the promoter site of AtHSFA3, and activates a stress response through an ABA-independent pathway [47].

Salinity stress
Around 30% of arable land is affected by salt stress and the proportions continue to increase at a rapid pace due to urbanization. Salt stress inhibits imbibition, and decreases root elongation and germination percentage [48]. Salinity and osmotic stress are related and plants have to undergo drought stress in media or soil causing salt stress [49]. Osmotic stress closes stomata, decreases photosynthetic rate, and disrupts action of the thylakoid membrane or Calvin cycle enzymes [50]. It alters leaf anatomy, i.e., thickness of epidermis, mesophyll, palisade length, and diameter. Plants start producing ROS (O 2ion, H2O2 and OH -) in chloroplast, cytosol, apoplastic space, and mitochondria. Activation of ROS results in oxidation of carbohydrates, lipids, proteins, nucleic acid, and impacts membrane integrity. OHion causes damage to DNA by disrupting purine and pyrimidine [51]. Plants maintain homeostasis within and outside the cytoplasm for normal growth [52]. Proteins, channel protein anti porters and symporters, maintain ion transport during homeostasis. Moreover, compatible osmolytes, i.e., free amino acid sugars, quaternary ammonium compounds, and proline are produced [53]. These osmolytes help in protection of cell structure and maintain osmotic balance by continuous water flux. Glycine betaine, an organic compound, plays a significant role in lowering salt stress by osmotic adjustment, protecting photosynthetic machinery, and protein stabilization.
Various antioxidant enzymes, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione peroxidase (GPX), and non-enzymatic antioxidants, such as carotenoids and tocopherols, act as scavengers against ROS [51]. All of these events are regulated by a large number of salt responsive genes controlled by different TFs, which percept signal and start defense mechanism [54]. Three types of genes play roles in responses to salt stress, which includes genes of sensing and signaling stress, transport regulators, and salt stress-response-related genes. When stress occurs, Na + ion enters the cell via non-selective cation channels and other membrane transporters. These Na + ions are recognized by unknown sensory elements. In the second step, ROS, Ca 2+ , and other hormones act as secondary messengers against salt stress and induce altered transcriptomic profile [55]. AtNAC2, a nuclear localized gene, is upregulated during salt stress; it promoted lateral root development [56]. Similarly, over-expression of AtWRKY46 increases lateral root development through an ABA signaling pathway. Overexpression of Gossypium hirsutum derived GhWRKY34 in Arabidopsis increased salt tolerance by inducing selective uptake of Na + or K + ions in roots and leaves [57]. Moreover Reaumuria trigyna derived RtWRKY1 is overexpressed under salt stress and resulted in increased root growth, increased antioxidative enzymes, decreased Na + or Na + /K + ratio [58]. Overexpression of GmWRKY54 alleviates salt stress in soybean [59]. AtbZIP24 increases salt tolerance by osmotic balance, ion homeostasis in cytoplasm, and increased growth and development, involving homo-and heterodimerization, or post-transcriptional modification [60]. Overexpression of GmERF3 in tobacco increased free proline and soluble carbohydrates and relieved salt stress.

Cold stress
Cold stress affects cellular metabolism by decreasing the rate of biochemical reaction, concentration of nucleic acid, and protein. Plants respond to cold stress by increasing proline contents, membrane fluidity, and ROS activation [61]. ROS causes oxidative damage in cells, reduced enzymatic activity, cause ionic imbalance, damaged cell membranes, reduced respiration rate, and degraded proteins. Plants also face low germination, delayed cell cycle, low leaf development rate, decreased seedling vigor, and yield [62]. Root development is also affected, resulting in lowered biomass and root length, and reduced root volume. Onset of cold stress at the reproductive stage leads to pollen sterility, abortion of flower or ovules, distortion of pollen tubes, poor fruit setting, deformation of panicles, spikelet degeneration, and reduced productive tillers [63]. The photosynthetic rate decreases due to reduced CO2 conductance in the mesophyll and stomata, restricted transport of metabolites and increased photo-inhibition [62]. Different TFs respond to cold stress by regulating expression of cold responsive genes, dehydrin, abscisic acid responsive genes, and late embryogenesis abundant genes. These TFs are present in the nucleus through as they possess nucleus-localization signals (NLSs). Under stress, membrane bound TFs are activated, enter the nucleus, and regulate gene expression. Nuclear-localized SlNAC1 activate stress responsive genes and enhances cold tolerance. Similarly, ZmSNAC1, OsNAC5, and TaNAC57 genes are overexpressed and enhance cold tolerance . Arabidopsis thaliana and grapevine calli show overexpression of VaWRKY12 and regulated expression of the genes encoding antioxidant enzymes, glutathione S-transferases, and peroxidase [64]. OsWRKY71 was upregulated in rice under cold stress. Overexpression of CsWRKY enhanced cold tolerance by affecting root development, germination rate, seed development, flowering, and dormancy in cucumber [65]. OsbZIP73 upregulates and plays an important role at flowering, seedling, and reproductive stages to counter cold stress. It co-expressed with OsbZIP71 and enhanced seed set by affecting pollen fertility through an increase in concentration of soluble sugar in pollen and a decreased level of ABA in anthers [66]. OsbZIP87 and OsbZIP38 also play an important role in enhancing cold tolerance [66]. OsDREB1G regulates expression of cold induced genes present in protoplasts. Arabidopsis thaliana AtDREB1A/CBF1 and AtCBF4 are overexpressed in response to cold stress (Moon et al., 2019). MYBs also play important roles in combating cold stress by affecting cell cycle, cellular morphogenesis, hormonal signaling, secondary metabolism, and gene expression. AtMYB14 down regulates under cold stress and encodes the proteins that act as the R2R3-MYB activator. As a result, CBF proteins (CBF1, CBF2, and CBF3) are activated, which initiate cold responsive genes. In transgenic Arabidopsis GmMYBJ1 is overexpressed and enhances cold tolerance. GmMYBJ1 regulates expression of many stress responsive genes, such as AtCOR15a, AtRD29B, AtP5CS, and AttCOR78, as a result the plant tolerates cold stress. OsMYB4 in Arabidopsis thaliana is also overexpressed and increases freezing tolerance [67].

Heavy metal stress (HMS)
Heavy metals (HMs) are generally defined as metals with relatively high densities, atomic weights, or atomic numbers, i.e., Mo, Ni, Cd, Cr, and Zn. Heavy metal enriched soil results in reduced growth, altered nutrients and water balance, inhibition of photosynthesis and chlorosis. Further, HMs affect chlorophyll content, photosystem II effectiveness, and Rubisco activity [68]. It leads to drop in reductant pool and less ATP production with less CO2 fixation [69]. Heavy metal enhanced protease activity decreases ammonia glutamate dehydrogenase (GDH), glutamine oxoglutarate aminotransferase (GOGAT), glutamine synthetase (GS), nitrite reductase (NiR), and nitrate reductase (NR) activity [70]. ROS starts accumulating, cause oxidative stress by interacting with proteins, DNA, and lipids, and destabilizing cellular organization [71]. Cd stimulates the myelin basic protein (MBP) kinase gene and OsMAPK2 in Oryza sativa. Due to heavy metal stress, different mitogen activated protein kinases (MAPKs) are activated, such as in Arabidopsis thaliana induction of MPK3 and MPK6, due to CuSO4 and CdCl2. Oryza sativa multiple stress responsive MK2 (OsMSRMK2), wound and JA-uninducible MK1 (OsWJUMK1), and OsMSRMK3 are induced in presence of Cd 2+ and Cu 2+ in roots and leaves. Stress activated MAPK (SAMK), Medicago MAP kinase3 (MMK3), Medicago MAP kinase2 (MMK2), and salt stress induced MAPK (SIMK) are activated in Medicago sativa in response to CdCl2 and CuCl2 [72]. Downstream TFs, i.e., activator protein 2 (AP2), WRKY, bZIP, MYB, DREB, ERF, and ZAT (C2H2 type ZF-) transcription factors are stimulated by activation of MAPKs as their targets. Due to Cd and Zn stress, MYB TFs, i.e., AtMYB28, AtMYB72, AtMYB48, AtMYB124, and AtMYB4 are upregulated in Arabidopsis thaliana. A short duration stress of Cu to roots in Arabidopsis activates AtWRKY22, AtWRKY25, and AtWRKY29, whereas long duration stress of Cd activates AtWRKY25 and AtWRKY29. Similarly, in Glycine max, up-regulation of GmbZIP62 and down-regulation of GmbZIP44 and GmbZIP78 is associated with Cd stress. In Arabidopsis thaliana, Cd stress is responsible for expression of ethylene responsive factors, i.e., AtERF1, AtERF5, and AtZAT6 to regulate plant metabolism [73].

Waterlogging stress
Water logging is a combination of two important stresses: submergence when the whole plant is under water and water logging, when only roots are submerged [74]. Water logging results from prolonged rainfall, poor drainage of soil, and its intensity tends to increase every year [75]. Diffusion of gases in water is low when compared to air, which results in decreased oxygen availability and detrimental effects on plant health [76]. Different physiological and biochemical processes are activated, which shift aerobic respiration to anaerobic fermentation. Moreover, certain toxic compounds, such as alcohol and aldehydes are accumulated in the cytoplasm [77]. There are two stages of water logging, hypoxia (partial depletion of oxygen) and anoxia (complete depletion of oxygen). Different plant processes, i.e., cytoplasmic pH, cellular energy, stem elongation, and adventitious root formation decreases. In addition, fresh and dry mass decrease; and the electrontransport chain and CO2 assimilation are also affected [78]. Furthermore, toxic compounds and ROS accumulated. Certain redox enzymes, such as cupredoxins, are activated to maintain ROS balance [79].
Roots also play an important role in response to water logging stress, as aerenchyma and adventitious roots are established. The function of aerenchyma is to increase internal diffusion of oxygen from aerial parts to waterlogged roots to facilitate an aerobic environment [80]. The known transcription factors that play roles in regulation of water logging stress include bZIP, NAC, WRKY, MYB, and ERF families. But the highest number of transcription factors responding to waterlogging stress belong to the MYB and the AP2/EREBP families [81]. Low oxygen-induced genes are characterized by an anaerobic response element (ARE) present in the promoter. ARE has GC and the GT motifs, which are important for gene activity and signal transduction [82]. In Arabidopsis, five ERF VII genes AtHRE1, AtHRE2, AtRAP2.2, AtRAP2.3, and AtRAP2.12 are key regulators of flooding. Rice SUB1A is considered a master regulator against water logging stress. These ERF VII tandem repeats are responsible for increasing inter-nodal elongation and enable the plants to overcome waterlogged conditions [80]. ZmEREB180 increases waterlogging tolerance in maize seedlings. ZmEREB180 has a conserved N terminal motif and its ectopic expression increases tolerance [83]. Overexpression of AtSHYG (AtNAC047) causes hyponastic growth in Arabidopsis [84]. Moreover, SiWRKY51 and SiWRKY65 also play important roles in roots of waterlogged plants [85]. However, exploring the role of TFs under waterlogging stress needs further attention.

Role of Transcriptional Factors under Biotic Stresses
Biotic stresses such as diseases, insects, and nematodes adversely affect plant growth, development, survival, and crop productivity. Reported losses due to biotic stresses are up to 35% [19]. Yield losses in USA due to Fusarium head blight and wheat rust amounted to US $3 billion and US $5 billion, respectively. In the middle of the 19 th century, during the Irish potato famine, the crop were completely destroyed due to late blight (Phytophthora infestans), leaving millions of farmers empty handed [86]. The available data suggest that a decrease in annual crop productivity by arthropods is 18%-20% worldwide, amounting to US $470 billion. The area's most prone to biotic stresses are African and Asiatic countries [87]. Disease negatively affects morphological characteristics, i.e., plant height, chlorophyll content, and leaf architecture [88]. To deal with these challenges, the plant adopt coordination of different physiological, biochemical, and molecular processes through signal transduction mechanisms [89]. Pathogen attack signals are recognized through pathogen-associated molecular patterns (PAMPs) present on the host surfaces that trigger a basic immune response PAMP-triggered immunity (PTI). Plants have resistance proteins (R) that directly or indirectly identify effectors and activate effector-triggered immunity (ETI), such as hypersensitive response (HR). The detailed infection process of bacterial, fungal, and viral pathogens, insects, and nematodes, is described below, along with a variety of defense responses for each infection.  Oryza sativa

Bacterial Infections
More than 200 pathogenic bacterial species have been identified in plants. The most important bacterial infections belongs to genera Pseudomonas, Ralstonia, Agrobacterium, Xanthomonas, Erwinia, Xylella, Pectobacterium, and Dickeya. Pathogenic bacteria produce cell wall degrading enzymes, which provide passage for infiltration and maceration in plant tissue for feeding. Erwinia amylovora causes fire blight of the Rosaceae family, apple, and pear (Mansfield et al., 2012. Ralstonia solanacearum causes bacterial wilt of tomato, tobacco, banana, and the brown rot of potato. Xanthomonas mainly effects rice, banana, tomato, and citrus fruits, and invades mostly xylem or parenchyma tissues [112]. Xylella fastidiosa is a xylem-limited phytopathogen, which causes diseases in grapes, almond, citrus, peach, coffee, and olive trees. Pseudomonas syringea causes infection in the tomato by forming a necrotic lesion surrounded by yellow chlorotic halo on the tomato (termed as bacterial speck) [113]. After a pathogen attack, TFs activate pathogen related (PR) genes and promote HR. HR is responsible for tissue necrosis by systemic acquired resistance (SAR). Defense-related TFs include bZIP, AP2/ERF, NAC, MYB, DOF, and WRKY, which play an important role in defense response against pathogen attacks. Rice OsWRKY80 and OsWRKY4 genes increase resistance against sheath blight. OsWRKY80 attaches to a W-box in the promoter region of OsWRKY4, which activates defense response against Rhizoctonia solani. Moreover, OsWRKY7, OsWRKY58, OsWRKY64, and OsWRKY76 genes are expressed in response to rice blast disease [19]. Overexpression of VaERF20 increases resistance against Pseudomonas syringae and Botrytis cinerea in transgenic Arabidopsis [114]. SlCabZIP and SlERF11 eliminates pathogenicity of Pseudomonas syringae Pv. tomato DC3000 and provide resistance [115]. AtNAC032 represses activation of MYC on pest attack by blocking a coronatine mediated reopening of stomata to thus stopping the entry of Pseudomonas syringae Pv. tomato DC3000 [116]. GhWRKY39-1 provides resistance to Root rot (R. solani) in Gossypium hirsutum [117]. CaWRKY27, CaWRKY6 mediate bacterial wilt while CaWRKY58 resists bacterial spot (Xanthomonas axonopodis) [5]. SINAC35 counter bacterial wilt (R. solanacearum) and bacterial spot (X. compestris) infections in Capsicum annuum [19], as explained in Table 2.

Fungal diseases
Economically important fungal diseases are yellow rust, leaf rust, stem rust, spot rust, red rot, sheath blight, rice blast, powdery mildew, downy mildew, and stem canker. Fungal infestation prevents closing of the stomata, damages the xylem cells, disrupt the cuticle layer, causes extensive water loss, decreases leaf and shoot water potential, decrease fresh weight, root number, and length, produces large numbers of brown roots, and reduces uptake and availability of nutrients [118]. When plants are subject to fungal attacks, they produce plant hormones, i.e., ethylene, salicylic acid, and jasmonic acid. Plant hormones activates expression of TFs, i.e., AP2/ERF, WRKY, NAC, MYB, and MYC [119]. Overexpression of AtWRKY72 enhances resistance against powdery mildew. Similarly, AtWRKY8 and AtWRKY28 enhance resistance against Botrytis cinerea. TaWRKY49, TaWRKY62, and TaWRKY70 combat strip rust (Puccinia striiformis) by activating ROS, jasmonic acid, salicylic acid, and ethylene production [117].
In rice, overexpression of OsWRKY45 and OsWRKY22 enhance resistance to Pyricularia oryzae. OsWRKY45 overexpresses and enhances resistance against fungal pathogen Magnaporthe grisea. OsWRKY4 and OsWRKY80 increase sheath blight resistance in rice. OsWRKY80 binds to the W-box in the promotor region of OsWRKY4 and works as a positive regulator for Rhizoctonia solani resistance. In Brachpodium distachyon, BdWRKY8, BdWRKY50, BdWRKY34, and BdWRKY70 are upregulated and enhance resistance against Fusarium graminearum. In Hordeum vulgare, HvWRKY1 cooperates with HvMYB6 to counter powdery mildew [6]. Similarly, TaNAC6 overexpresses and enhances powdery mildew resistance and decreases fungal haustoria. OsNAC6 shows overexpression and enhances rice blast resistance. In barley, HvNAC6 overexpresses under powdery mildew infection and increased resistance against Blumeria gramini. VaERF20 increases resistance against Botrytis cinerea in transgenic Arabidopsis. In soybean, GmbZIP1, GmbZIP62, GmbZIP105, and GmbZIP2 genes prevent infestation of Asian soybean rust [19].

Viral diseases
Viruses cause a variety of plant diseases. The main symptoms of all diseases are decreased internodal distance, deficiency of chlorophyll, and reduction in growth. Other related symptoms are reduced germination rate, rolled leaf blade, less nodulation, swelling of stem, tumors on stem, roots and leaves, reduced pollen fertility, less reduced set, wilting, and cell death [120]. Economically important viruses are sorghum mosaic virus (SrMV), sugarcane mosaic virus (SCMV), and sugarcane streak mosaic virus (SCSMV). SrMV and SCMV are effective pathogens for Sorghum bicolor, gramineous plants and Zea mays [121]. Other less virulent viruses are yellow vein mosaic virus in okra, urd bean leaf crinkle virus, strawberry mild yellow edge virus, rice stripe mosaic virus, cotton leaf curl Gezira virus, sugarcane yellow leaf virus, barley yellow dwarf virus, and maize chlorotic mottle virus. Rapid mutations of viral strains is a major factor behind failure of breeding programsm [122]. Plant activate hormonal responses, gene silencing, metabolic regulation, cellular protein degradation by the ubiquitin proteasome pathway (UPS), signaling of immune receptors and PAMP-triggered immunity to stop replication of viruses. Accumulation of ROS and plant hormones, i.e., salicylic acid, jasmonic acid, abscisic acid, brassinosteroids, cytokinin, auxin, ethylene, and gibberellin play role in plant defense against viruses [123]. Different TFs play significant roles into resisting virus-induced damage. Overexpression of OsMYB4 is responsible for resistance against viral diseases. The MtWRKY gene of Medicago truncatula provides resistance against tobacco mosaic virus in Nicotiana tabacum. The Gossypium hirsutum based GhWRKY15 gene, when introduced in Nicotiana tabacum, showed activity against the tobacco mosaic virus [5]. NAC TFs play vital roles in plant immunity by specific signals and virulence action of pathogenic effectors. Viral infection proteins sometime hijack NAC TFs to enable viral replication and decrease host immunity. However, some examples of NAC TFs imparting resistance are as follows: SlNAC20, SlNAC24, SlNAC41, and SlNAC61 play a significant role in imparting resistance against Tomato Yellow Leaf Curl Virus (TYLCV) [124]. Triticum aestivum NAC TFs, i.e., AtGRAB1 (Geminivirus Rep A-Binding) and AtGRAB2, interacts with Wheat Dwarf Geminivirus (WDV) Rep A protein and hinder DNA replication of WDV. Arabidopsis thaliana AtAF2 interact with the Tobacco Mosaic Virus (TMV) helicase domain and its overexpression inhibits virus infection [20]. Six WRKY genes, SolyWRKY41, SolyWRKY42, SolyWRKY53, SolyWRKY54, SolyWRKY80, and SolyWRKY81 obtained from tomato reduce TYLCV infection. Interaction analysis provided evidence of interaction between WRKY group III, isochorismate synthase (ICS), and Mitogen-Activated Protein Kinase5 (MAPK) in response to viruses [125].

Nematodes
Plant parasitic nematodes (PPNs) are economically important agricultural pests. Two classes exits i.e., cyst nematodes (CNs) and root knot nematodes (RKNs), causing together an estimated annual loss of US $80 billion. PPNs affect a wide range of hosts among economically important crops, i.e., Solanum lycopersicum, Solanum tuberosum, Gossypium hirsutum, Glycine max, Oryza sativa, Zea mays, and Triticum aestivum [126]. PPNs rich in ascaroside (Ascr# 18) induce the plant immune systems trigger production of jasmonic acid and salicylic acid, as well as trigger PTI and MAPKs. PPN's induce secondary metabolite production in plants, i.e., chlorogenic acid, ethylene, and flavonoids in roots [127]. These secondary metabolites reduce attraction of nematodes towards plant roots. Genes linked with synthesis of cytokinin, gibberellic acid, salicylic acid, jasmonates, and auxin signal responses are activated. SlWRKY75 is activated in Solanum lycopersicum by infection with Meloidogyne javanica and stimulate the JA pathway for regulation of the JA signaling mechanism. CsWRKY23 is overexpressed in cucumber plants during infection of Meloidogyne incognita for contributing to early resistance [128].
AtWRKY23 is overexpressed due to auxin stimulation at the feeding point of cyst nematode Heterodera schachtii. OsWRKY11, OsWRKY70, and OsWRKY62 upregulate in response to Hirschmanniella oryza attack. Similarly, OsWRKY13, OsWRKY59, and OsWRKY62 are upregulated against RKN infection. Nineteen WRKY genes in Solanum lycopersicum responded to nematode infection, i.e., upregulation of SlWRKY70 by salicylic acid, both SlWRKY35 and SlWRKY3 were activated [129]. AtWRKY33 is stimulated by JA and works as a positive regulator against PPN attack. AtWRKY33 overexpression along with different promoters confers resistance against Heterodera schachtii. SlWRKY45 is implicated in signal transduction pathways during accumulation of nematodes in the root zone [130]. Five Glycine max WRKY genes of Glycine max; GmWRKY5, GmWRKY28, GmWRKY36, GmWRKY62, and GmWRKY154 are highly responsive against SCN reducing by the 70% the population, while GmWRKY136, GmWRKY86, GmWRKY53, and GmWRKY52 show moderate response by reducing 40%-60% the population and GmWRKY71 and GmWRKY8 caused a 10%-30% decline in SCN population [131].

Pest attack
Changing climate is promoting the growth of herbivores, as increased in temperature are shortening life cycles and increasing chances of appearance [132]. Insect possess different feeding mechanisms.
Chewing insects such as beetles and caterpillars, consume plant tissues whereas piercing and sucking insects feed on the vascular system for example aphids which inserts their stylets into the phloem. Meanwhile, thrips combine sucking and rasping methods to feed on its host. Some mining type feeders such as larvae of certain beetles, moths, and flies form serpentine cavities when feeding between epidermal cells in leaf tissues and cause twisting or curling of leaves [133]. Moreover, spider mites, a phytophagous acarian belonging to Tetranychus genus, pierce parenchyma cells and suck the contents [134]. Some plants counter attack or activate emergency responses [135]. The plant defense systems are activated when specific pattern recognition receptors (PRRs) detect phytophagous pests through herbivore-associated molecular patterns (HAMPs), microbe-associated molecular patterns (MAMPs), and damage associated molecular patterns (DAMPs). With recognition of molecular patterns, plants activates short-term downstream responses at the membrane levels, i.e., Ca 2+ influx, potential depolarization, and generation of ROS or reactive nitrogen species (RNS) as a result, secretion of JA, SA, and ethylene starts, which activates TFs. TFs regulate expression of downstream-located genes through a cascade of CDPKs. These events, of recognition to response, take place within minutes to hours after herbivore attack [133]. Few TFs have been shown to play a role I responding to herbivore induced plant damage. OsERF3 is upregulated in response to feeding of striped stem borer (SSB) in Oryza sativa, which activates the transcript level of two MAPKs and two WRKY genes. As a result, the concentration of SA, JA, and trypsin protease inhibitor activity increases. OsWRKY45 protects rice against infestation by Brown Plant hopper (Nilaparvata lugens) [136]. TaMYB19, TaMYB44, and TaMYB29 act as co-regulators in phloem based defense response against English grain aphid in wheat. AtMYB75 increases resistance against Pieris brassicae by modulating flavonoid metabolites. Overexpression of GsMYB15 obtained from wild soybean increases Arabidopsis resistance against Helicoverpa armigera by a JA mediated insect response [137]. CmMYB19 increases resistance of Chrysanthemum against aphids by promoting a lignin biosynthesis pathway [138]. NbERF173 obtained from Nicotiana benthamiana provides resistance against Phytophthora parasitica [139]. TaWRKY53 induces resistance mechanisms against Russian wheat aphid in Triticum aestivum [140]. Similarly, OsWRKY53 provides resistance against Chilo suppressalis SSB [97]. Table 2. Role of different transcriptional factor gene families in biotic stress tolerance in plants.

Stress
Crop Disease Gene Reference

Crop Improvement Techniques and TFs
The implication of TFs from signal perception to signal transduction and expression of stress responsive genes, as summarized in this review. A single TF gene may respond to numerous stresses for example SbNAC58 which is effective against drought, cold, and salinity stress [19]. In view of the crucial role of TFs, they have potential to be exploited using different genome modifying molecular techniques for developing climate resilient crops (Table 3) as explained in Figure 3. Gene silencing through RNAi provides a platform for exploring the role of different TFs in plant development and in response to various stresses. RNAi uses double-stranded RNA to activate ribonucleases to target homologous mRNA and degrade it. The resulting phenotypes are either null or partially affected. Thus RNAi can help to elucidate role of different TFs under biotic and abiotic stresses [148]. This knowledge can be exploited by incorporating favorable alleles in suitable genetic backgrounds, and using different biotechnological tools, for stress tolerance. RNAi silencing could be used to knockdown TFs, which promote disease development. It can also be useful for knocking out undesirable TFs, which promote the development of stresses. AtNAC042/JUB1, CBNAC/NTL9 promotes bacterial leaf spot of Arabidopsis. Similarly, TaNAC1, TaNAC21/22, TaNAC30 promotes yellow rust and downy mildew attack on wheat. Knockdown these TFs in separate experiments slowed disease development [20]. Different genome editing tools, such as ZF-nucleases (ZFNs), homing endonucleases or mega nucleases, or transcription activator-like effector nucleases (TALENs) create targeted double-strand breaks that promote recombination at a specific locus and have potential in exploring the role of different TFs [149]. Similarly, Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) are used to establish knockout lines of TF genes for functional genetics. In the CRISPR/Cas9 system, the genomic target site is cleaved by Cas9, located at the site by the guide RNA (gRNA) with which it complexes. As a result, a double stranded break occurs at the target site, the repair of which causes mutations in the form of insertions or deletions or in some cases frameshifts. These mutants can clarify the role of the TF under consideration [150].  Marker assisted breeding (MAB) has a wide variety of applications in stacking of multiple genes in crop plants for various purposes and had been widely used in studying wheat rust [154]. All we need to find out for marker-assisted breeding is the tight linkage of a molecular marker (with TF as our interest). MAB was used previously for MdMYB1, associated with apple fruit skin color. MdMYB1 imparts red color to the fruit whereas its absence results in green color. A dCAPS marker was developed for selection of fruit color in apples at early plant developmental stages using MAB [155]. Another very successful example of MAB is the introduction of the SUB1 region into rice, which increases submergence tolerance without effecting yield, grain quality or development [156]. Similar marker systems could be developed for WRKY and other TFs responding to under various biotic and abiotic stresses for foreground selection prior to plantation of plants in the field. This will save time and labor, and will facilitate stacking of TF genes for multiple responsiveness in crop plants. Development of transgenic plants using TFs has a wide variety of potential applications in development of stress tolerant crops. An example is the production of drought tolerant tobacco plants through the use of MdDREB76 from apple [157], or the use of wheat TaNAC29 to enhance salt and drought tolerance in Arabidopsis [158], and many more examples exist in the literature (Table 3). During development of transgenic plants, TFs is most frequently put under the control of a constitutive promotor, i.e., CAMV35S that is expressed in each cell at all growth stages of the plants. Thus, great potential exists for development of transgenic plants using multiple stresses responsive TFs and evaluating their expression in various crops through development of transgenic crops. However, in successive generations of transgenic, rigorous selection criteria should be used to select stress tolerant plants with no negative effects [36].

Ductility and Flexibility in TFs to Carry Out Their Function
TFs are the most flexible proteins in nature, and this characteristic is very important to conduct regulatory function. It is predicted that 83%-94% of TFs possess extended regions of ductile/disordered residues in eukaryotic organisms. It is observed that organismic complexity is positively and strongly correlated with total number of TFs, the number of their spliced variants, and their total disordered residue content. Transcriptional factor families that take part in cell cycle, cell size, cell proliferation, and cell differentiation have more disordered residues and are more flexible. The evidence suggests that increasing TFs are an important factor for increasing organismic complexity [159]. This ductility of TFs helps plants combat multiple abiotic stress responses by acting as protein chaperones or protecting other cellular components and structures. TFs have complex and versatile networks to efficiently respond to environmental changes. TF disorder plays an important role in plants, providing them with a fast mechanism to obtain complex, interconnected, and versatile molecular networks [160].

Future Prospects, Challenges, and Opportunities
TFs have a great potential for boosting the yield potential and stress tolerance in field crops. Significant achievements have been made in unraveling the potential role of TFs under various biotic and abiotic stresses. However detailed studies of pathways explaining the role of TFs under stress is yet to be explored. Approximately 50 TFs families have been reported with less than ten, i.e., WRKY, DREB, NAC, HSF, MYB, ZF-s, Dof, bZIP, and NAC being functionally characterized under various biotic and abiotic stresses. The Role of the remaining TF families is currently unexplored, which provides a great platform for young researchers to build their future carriers in this field.
There is a need to establish crosstalk between different TF gene families and how they respond under stress conditions [161]. Focus should shift towards development of climate resilient crops with biotic as well as abiotic stress tolerance. In this context, WRKY TFs have a very positive role to play due to their involvement in biotic and abiotic stress tolerance [162]. Not only WRKY, but other TFs also resist biotic as well as abiotic stresses simultaneously. There is a need to identify TF genes that interplay during different stresses. TF genes expressing under multiple stresses should be privileged for breeding climate smart cultivars through conventional as well as modern plant breeding tools. Transgenic crops promises to be a good source of resistance against biotic and abiotic stresses. However, delivery of TFs to various genetic backgrounds using transgenic technologies still pose a great challenge to researchers due to unexplored metabolic pathways. For this reason, development of transgenic plants using TF candidate genes in crops is not yet fully exploited. Overexpressing some TFs improving stress tolerance can lead to impaired growth responses, i.e., low yield, late flowering, and dwarfing, which is still a challenge to be addressed [163]. Use of tissue specific promotors (SynR1 and SynR2 are root specific) as explained by Ali et al. [164] would help in overcoming the off target affects. Although the majority of TFs play a positive role during different biotic and abiotic stresses. However some genes of NAC TFs promote disease development, i.e., TaNAC1 promotes yellow rust and downy mildew in wheat.
At present most functional studies exploring the role of TFs are conducted in model plants, i.e., tobacco [157] and Arabidopsis [158], which are relatively easy to handle. The focus should be shifted to cultivated crops, i.e., wheat, rice, maize, and other field crops for biotic and abiotic stress tolerance. Although reports are available on transformation of TFs in field crops, however stacking of multiple stresses responsive TFs is just a beginning. Similarly, the role of TFs against heavy metal stress and nematode and insect attacks needs more attention. Another area of improvement is development of functional marker systems, i.e., SSR markers, SNPs, or dCAPS for MAB of different TFs. Only a few examples exist in literature for development of functional marker systems for characterization of TFs [154][155][156]. Functional markers will help identification of TFs in successive generations for marker-assisted crop improvement.

Conclusions
Different biotic stresses, i.e., bacterial, fungal and viral diseases, insects and nematodes; and abiotic stresses, i.e., drought, waterlogging, heat, cold, salinity, and heavy metals are becoming an alarming threat to crop productivity with the changing climate. There is an urgent need for development of crops tolerant to biotic and abiotic stresses by targeting different genes and gene families. TFs have emerged as a key potential contributor towards biotic and abiotic stress tolerance in plants, which play a role through signal transduction and activation of stress responsive genes. Different crop improvement approaches, i.e. RNAi silencing, and genome editing promise to deliver safer food to human beings, and ensure food security. Although numerous reports have been available about the use of TFs for crop improvement, the majority of studies are focused on model plants, i.e., Arabidopsis and tobacco. Further, the transgenic plants are frequently evaluated in laboratories under artificial conditions without exposing them to natural environmental conditions for their whole life span. Little is known about engineering of multiple TFs for response against multiple biotic and abiotic stresses. In particular, WRKY TFs have diversified roles in biotic and abiotic stress events that need to be elucidated using different omics approaches to explore the mechanisms underlying these complex responses.

Conflict of Interest
The 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.

Funding
The publication of present work was supported by the National Key Research and Development Program of China (grant no. 2017YFC0504704), and the National Natural Science Foundation of China (51669034, 41761068, 51809224).

Acknowledgments
The Authors are highly thankful to the colleagues at Maize and Millets Research Institute Yusafwala, Sahiwal, Pakistan, and Ayub Agricultural Research Institute, Faisalabad, Pakistan for time-to-time guidance and moral support. Further advanced apologies to the authors whose work has not been cited due to space issues.