**3. ABA in stress signaling**

In plants and other organisms, such as algae, cyanobacteria, and fungi, ABA levels tend to increase with exposure to stress, suggesting a potential role of ABA in stress signal transduc‐ tion [20]. Exogenous ABA application mimics stress conditions in plants and provides a useful means to study the effect of ABA on stress signaling and tolerance [38]. ABA distributes throughout the plant as an inactive glucose sugar conjugate and is converted to the active form by β-glucosidase [4]. ABA acts as an endogenous messenger and salt and drought stress signal transmission to initiate downstream gene expression occurs mainly via ABA signaling. However, cold stress signal transduction occurs in an ABA-independent manner via the C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT BINDING FACTORS (CBFs/DREBs) signaling pathway [39].

A large number of ABA-responsive genes have a common *cis*-element called the ABRE element in their promoter regions. An ABRE together with a Coupling Element (CE) results in ABA induction of gene expression [19]. The **A**BA **R**ESPONSIVE **E**LEMENT **B**INDING FACTOR (AREB/ABF) family of transcription factors are the major transcription factors that regulate ABA-induced gene expression. AREB/ABFs are bZIP transcription factors and their expression is induced by ABA and other potential stress conditions [40]. Different ABFs are induced by ABA at different rates. For instance, *ABF2*, *ABF3*, and *ABF4* are induced faster than *ABF1*. Moreover, *ABF1* is induced only by cold stress, whereas *ABF2* and *ABF3* are induced by salt stress. *ABF4* levels are induced by salt, drought, and cold stress, suggesting that distinct ABFs have roles in various ABA-dependent stress-responsive pathways [40].

There are nine Group A bZIP transcription factors implicated in ABA signaling and they are subdivided into two groups based on where they are mainly expressed. The *ABI5/AtDPBF* family of genes includes *ABSCISIC ACID INSENSITIVE 5 (ABI5), ENHANCED EM LEVEL (EEL),* and *AREB3* and are expressed in the seed during seed maturation [41]. Other AREB/ABF transcription factors are mainly expressed in vegetative tissues [40].

SnRK2 III is the major subfamily of SnRKs involved in abiotic stress responses. SnRK2 protein kinases phosphorylate AREB/ABFs and regulate their function in ABA-regulated gene expression under stress conditions [10]. SnRK2.6/OST1 is an important regulator of stomatal closure in drought stress. The role of SnRK2.2 and 2.3 is mainly to transmit the ABA signal to inhibit seed germination and seedling growth in response to stress. A decuple *snrk2* mutant in Arabidopsis, which carries mutations for all 10 SnRK2 members, was hypersensitive to osmotic stress and also defective in ABA accumulation and ABA-induced gene expression under osmotic stress, indicating the critical role of SnRK2 kinases in osmotic stress signaling and tolerance. Moreover, the *snrk2.2/3/6* triple mutant had impaired accumulation of proline, which is a compatible osmolite [42].

#### **3.1. ABA and abiotic stress tolerance**

binding activity in direct ABA-binding assays. GTGs have GTPase activity and GDP bound GTGs have enhanced ABA-binding ability, which in turn initiates ABA signaling. GTG1/2 bind with GPA1 (G-PROTEIN α SUBUNIT 1), which abolishes its GTPase activity and represses ABA binding. GTP bound GPA1 represses ABA signalling. However, the downstream

Recent findings that several ABA receptors exist in different parts of the cell provide evidence that ABA is active in a variety of subcellular compartments. ABA synthesis enzymes are present in different compartments, suggesting that ABA synthesis occurs in different parts of the cell and that these ABA levels contribute to overall ABA homeostasis. For example, the ABA biosynthesis enzyme AtABA1 is localized in the chloroplast, whereas AtABA2 is in the cytosol [33,34]. It has also been proposed that ABA produced in cytoplasm, plastids, vacuole, and other subcellular organelles may have different physiological roles initiated by signaling

Takeuchi *et al*. [36] identified a potential ABA analog AS6 that can inhibit the activity of PYLs. X-ray crystallography studies showed the structure of ABA facilitates the binding of ABA to PYR/PYL/RCAR receptors and thereby inhibits interaction with PP2Cs. The AS6 ABA analog was able to block PYL-PP2C interaction, indicating that binding of ABA to PYL receptors

Inhibition of PP2Cs results in autoactivation of SnRK2 kinases and thereby positive regulation of ABA signaling. Recently, the crystal structures of SnRK2.3 and SnRK2.6 were elucidated, providing evidence that kinase activation is a two-step mechanism as well as details of how the ABA signal is transmitted to downstream components [37]. This study also showed that

Lumba *et al*. [20] did a comprehensive transcriptomic data analysis in order to generate a mesoscale ABA signaling network. They showed that there are 3 main kinase hubs, MAP3K∂4, SnRK3.15, and SnRK3.22, that interact with PP2Cs and these kinases act as negative regulators of ABA response, in contrast to the SnRK2s involved in ABA signaling. SnRK3.15 and SnRK3.22 also interact with a large number of transcription factors and may have a role in overall ABA

In plants and other organisms, such as algae, cyanobacteria, and fungi, ABA levels tend to increase with exposure to stress, suggesting a potential role of ABA in stress signal transduc‐ tion [20]. Exogenous ABA application mimics stress conditions in plants and provides a useful means to study the effect of ABA on stress signaling and tolerance [38]. ABA distributes throughout the plant as an inactive glucose sugar conjugate and is converted to the active form by β-glucosidase [4]. ABA acts as an endogenous messenger and salt and drought stress signal

components of this pathway are not characterized yet [31,32].

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

**2.3. Recent studies on ABA perception and signaling mechanisms**

networks via different ABA receptors in each specific compartment [35].

autophosphorylation of SnRK2.6 is more efficient than that of SnRK2.3.

initiates ABA responses by repressing PP2Cs.

responses in the plant [20].

**3. ABA in stress signaling**

In *Arabidopsis thaliana*, about 10% of the genome consists of ABA-regulated genes. Approxi‐ mately half of these genes are ABA-induced genes and the rest are ABA-repressed. ABAinduced genes code for proteins that confer stress tolerance such as dehydrins, detoxifying enzymes of reactive oxygen species, regulatory proteins (transcription factors, protein kinases, phosphatases) and enzymes required for phospholipid signaling. Genes that are repressed by ABA are mostly related to growth [5]. ABA biosynthesis mutants identified in Arabidopsis [43] and other crop plants [44] wilt and die under prolonged salt and drought stress, suggesting ABA plays an important role in osmotic stress tolerance.

Drought and high salinity generate osmotic stress in plant cells. Endogenous ABA levels are elevated in response to osmotic stress, which in turn coordinates the plant's response to reduced water availability. In addition, seed maturation and post-germinative growth creates cellular dehydration stress, which again results in accumulation of ABA in cells [45]. The role of ABA in drought and salt stress is twofold: water balance and cellular dehydration tolerance. Water balance is achieved through guard cell regulation and the latter role by induction of genes that encode dehydration tolerance proteins in nearly all cells. ABA accumulation is induced by osmotic stress and this is as a result of activation of ABA biosynthesis as well as inhibition of ABA degradation [46]. Thus, ABA-mediated adaptive stress responses of plants to environmental stimuli occur via ABA-responsive gene expression and regulation of stomatal pore size. ABA-responsive gene expression involves various transcription factors, ABA receptors, secondary messengers, protein kinase/phosphatase cascades, and chromatin remodeling factors [45].

Both drought stress and salinity stress upregulate osmotic stress responsive genes that are ABA-inducible. Most of the high-salinity-induced genes are also induced by drought, sug‐ gesting there is overlap between salt and drought stress tolerance mechanisms [6]. A large number of transcription factors are induced by multiple stress conditions. AREB1/ABF2, AREB2/ABF4, ABF3, and MYB41 are some of the main transcription factors that are induced by both salt and drought in vegetative tissues [45].

Drought and salt stress results in osmotic imbalance; thus, salt and drought stress tolerance mechanisms aim at restoring cellular homeostasis. These mechanisms are adaptive responses that create either stress tolerance or avoidance of stress conditions. Modifications in metabolic pathways, synthesis of new proteins, changes in ion uptake, and free radical scavenging are some of the stress responses at the cellular level, immediately followed by stress signal transduction [47]. High ABA levels in cells result in synthesis of storage proteins, desiccation tolerance, and dormancy via inhibition of seed germination [22]. In the plant as a whole, key adaptive responses include induction of stomatal closure as well as control of seedling growth and lateral root formation. While the balance between ABA and auxin levels slightly affects primary root growth, ABA represses lateral root formation while auxin promotes it [4,48].

Inhibition of seed germination under abiotic stress is another function of ABA. Seed germi‐ nation occurs when there is a balance between germination-promoting gibberellin and dormancy-promoting ABA. During late stages of maturation, seeds accumulate ABI5 which in turn activates transcription of LATE EMBRYOGENESIS ABUNDANT (LEA) proteins. LEA proteins confer osmotolerance to the embryo. ABA is necessary for activation of ABI5 via SnRK2.2 and SnRK2.3 phosphorylation of ABI5 [49,50]. When seeds are in unfavorable environmental conditions, elevated endogenous ABA levels results in ABI5 accumulation, preventing seeds from germinating.

#### *3.1.1. ABA and salt tolerance*

Salt stress severely impacts plant growth by affecting metabolic processes and photosynthetic efficiency. NaCl initially induces osmotic stress and eventually accumulation of both Na+ and Cl ions generates ionic stress [51]. However, some responses are salt-specific and distinct from responses to osmotic stress (reviewed in [52]). High salinity in the soil is first sensed by the plant roots. Salt and drought stress induce a rapid increase in cytosolic Ca2+ levels in the root cells. Ca2+ acts a second messenger, inducing salt- and drought-responsive genes [53,54]. Hyperosmotic stress is coupled with Ca2+ signaling and Reactive Oxygen Species (ROS) signaling, thereby inducing a cascade of signaling events, which results in downstream gene expression [52].

reduced water availability. In addition, seed maturation and post-germinative growth creates cellular dehydration stress, which again results in accumulation of ABA in cells [45]. The role of ABA in drought and salt stress is twofold: water balance and cellular dehydration tolerance. Water balance is achieved through guard cell regulation and the latter role by induction of genes that encode dehydration tolerance proteins in nearly all cells. ABA accumulation is induced by osmotic stress and this is as a result of activation of ABA biosynthesis as well as inhibition of ABA degradation [46]. Thus, ABA-mediated adaptive stress responses of plants to environmental stimuli occur via ABA-responsive gene expression and regulation of stomatal pore size. ABA-responsive gene expression involves various transcription factors, ABA receptors, secondary messengers, protein kinase/phosphatase cascades, and chromatin

Both drought stress and salinity stress upregulate osmotic stress responsive genes that are ABA-inducible. Most of the high-salinity-induced genes are also induced by drought, sug‐ gesting there is overlap between salt and drought stress tolerance mechanisms [6]. A large number of transcription factors are induced by multiple stress conditions. AREB1/ABF2, AREB2/ABF4, ABF3, and MYB41 are some of the main transcription factors that are induced

Drought and salt stress results in osmotic imbalance; thus, salt and drought stress tolerance mechanisms aim at restoring cellular homeostasis. These mechanisms are adaptive responses that create either stress tolerance or avoidance of stress conditions. Modifications in metabolic pathways, synthesis of new proteins, changes in ion uptake, and free radical scavenging are some of the stress responses at the cellular level, immediately followed by stress signal transduction [47]. High ABA levels in cells result in synthesis of storage proteins, desiccation tolerance, and dormancy via inhibition of seed germination [22]. In the plant as a whole, key adaptive responses include induction of stomatal closure as well as control of seedling growth and lateral root formation. While the balance between ABA and auxin levels slightly affects primary root growth, ABA represses lateral root formation while auxin promotes it [4,48]. Inhibition of seed germination under abiotic stress is another function of ABA. Seed germi‐ nation occurs when there is a balance between germination-promoting gibberellin and dormancy-promoting ABA. During late stages of maturation, seeds accumulate ABI5 which in turn activates transcription of LATE EMBRYOGENESIS ABUNDANT (LEA) proteins. LEA proteins confer osmotolerance to the embryo. ABA is necessary for activation of ABI5 via SnRK2.2 and SnRK2.3 phosphorylation of ABI5 [49,50]. When seeds are in unfavorable environmental conditions, elevated endogenous ABA levels results in ABI5 accumulation,

Salt stress severely impacts plant growth by affecting metabolic processes and photosynthetic efficiency. NaCl initially induces osmotic stress and eventually accumulation of both Na+

 ions generates ionic stress [51]. However, some responses are salt-specific and distinct from responses to osmotic stress (reviewed in [52]). High salinity in the soil is first sensed by the plant roots. Salt and drought stress induce a rapid increase in cytosolic Ca2+ levels in the root

and

remodeling factors [45].

by both salt and drought in vegetative tissues [45].

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

preventing seeds from germinating.

*3.1.1. ABA and salt tolerance*

Cl-

Biochemical and molecular mechanisms of salt tolerance in plants include exclusion of salt ions, production of suitable osmolytes, changing the structure of the membranes to control ion uptake, and induction of enzymes that produce antioxidants and phytohormones. To manage salt or drought stress, cellular ABA levels increase dramatically. The plant cuticle has been shown to mediate stress signaling as well as ABA biosynthesis and signaling. In addition to its primary function, providing mechanical support to the cell wall and plasma membrane, the cuticle has been implicated in osmotic stress regulation. CED1 (9-CIS EPOXYCAROTENOID DIOXYGENASE DEFECTIVE 1) is an essential protein in cuticle biogenesis. *ced1* mutants are sensitive to osmotic stress, as they are unable to induce ABA biosynthesis in response to osmotic stress [55].

ABA regulates root growth and architecture in plants under stress. Duan *et al*. [56] showed that salt has a strong inhibitory effect on lateral root growth, while primary roots are less sensitive to salt stress. They also showed that endogenous ABA signaling affects root system architecture under stress conditions using ABA biosynthesis mutants (*aba1, aba2*) as well as signal transduction mutants such as *abi1*. Salt stress results in elevated levels of ABA exclu‐ sively in lateral root cells and induces a quiescent period in postemergence lateral roots. Lateral roots in a quiescent stage form a thick, well-developed Casparian strip, which acts as a barrier to reduce diffusion of Na+ ions through the endodermis. In the presence of Na+ ions, endoder‐ mal cells activate ABA signaling and arrest growth so that lateral roots do not elongate into high saline environments. Therefore, ABA is an important signaling molecule in suppressing lateral root growth during salt stress [56].

ABA regulates expression of many salt-stress-responsive genes via transcription factors that are elevated in response to salt. For instance, ABF2/AREB1, ABF3, ABF4/AREB2, ABRE BINDING PROTEIN 9 (ABP9), and MYC/MYB, WRKY, and APETALA2/ETHYLENE RE‐ SPONSE FACTOR (AP2/ERF) are some of the salt-stress-responsive transcription factors that enhance stress tolerance. A recent study showed that the PYL8/RCAR3 ABA receptor has a role in ABA-mediated inhibition of primary root growth and also recovery of lateral root growth on exposure to ABA. PYL8/RCAR3 combines the action of ABA and auxin through direct interaction with MYB transcription factors during growth recovery of postemergence lateral roots [48,57].

There are proteins in the cell that are produced in an ABA-dependent manner that have a role in osmotic tolerance. For example, ABI5 activates transcription of LEA proteins. LEA proteins are highly hydrophilic small proteins shown to have an osmoprotectant role against cellular dehydration during late embryogenesis. LEA proteins also have a role in salt stress tolerance [58]. Due to their hydrophilic nature, LEA proteins can sequester ions accumulating in the cell, as well as act as chaperones and retain water molecules to prevent protein aggregation and inactivation of cellular enzymes [59]. In Arabidopsis, 51 LEA proteins have been identified that belong to nine different groups [60]. Jia *et al.* [61] showed overexpression of AtLEA14, which belongs to the LEA group 2 proteins, overactivates salt-stress-inducible genes such as *RD29B*, which encode dehydration protective proteins, and subsequently confers salt tolerance in Arabidopsis.

In addition, ABA has been implicated in histone H3 acetylation and methylation, thereby regulating stress-inducible gene expression at the epigenetic level. Chen *et al.* [62] showed that histone modifications by HISTONE DEACETYLASE 6 (HDA6) are involved in inhibition of seed germination, salt stress responses, and ABA- and salt-mediated gene expression in Arabidopsis.
