**1. Introduction**

Due to their sessile nature, plants cannot avoid environmental stresses, thus they have evolved mechanisms to overcome the detrimental effects of stress. For example, plant endogenous developmental programs are modified such that structural and metabolic changes assist to overcome adverse environmental conditions such as salinity and drought. Failure to adapt to adverse environmental conditions can significantly reduce yield by impacting plant develop‐ ment and productivity. Abiotic stress conditions initiate a number of molecular, biochemical, and physiological changes at both the cellular and whole plant levels [1]. One major biochem‐ ical change in response to stress is elevation of abscisic acid (ABA) levels, which in turn triggers

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expression of a cascade of stress-responsive genes [2]. Cellular ABA levels are induced by environmental stimuli such as light, water, and salinity stress [3].

The plant hormone ABA has been identified as a key regulator of multiple stresses. In general, the adaptive responses of plants to various stress conditions can be either ABA-dependent or ABA-independent. However, there is no clear boundary between these two pathways and there is lot of crosstalk between the pathways and the components involved. This review will focus on recent advancements in ABA-mediated stress signaling and the role of ABA in abiotic stress tolerance in the model plant *Arabidopsis thaliana*.

#### **2. The phytohormone Abscisic Acid (ABA)**

ABA, a sesquiterpenoid (C15H20O4) with a 15-carbon ring (Figure 1), has a variety of biological functions and is found ubiquitously across several kingdoms, including cyanobacteria, sponges, algae, lichens, mosses, and mammals [4-7]. Discovered in the 1960s and initially named dormin or abscissin, ABA is now established as a widely occurring and important plant growth regulator. Although it was initially identified as an abscission-promoting hormone, later scientists discovered that this was partly due to an indirect effect of inducing ethylene biosynthesis [8]. ABA is an important regulator of plant growth, including embryo and seed development, seedling establishment, vegetative and reproductive growth as well as promot‐ ing seed dormancy [9,10]. Seed maturation and promotion of dormancy are important in preventing preharvest sprouting. In addition, ABA has the ability to antagonize the germina‐ tion promoting effects of gibberellin, regulate guard cells, and regulate stress-responsive gene expression under water-deprived conditions. ABA also has a role in plant pathogen responses in a pathosystem-dependent manner [4,5].

**Figure 1.** Structure of phytohormone abscisic acid S-(+)-ABA

The molecular structure of ABA has several important features that facilitate its biological functions. The side chain with the two double bonds (Figure 1) and ABA's stereocenter are two such important features. Exposure to UV light changes the conformation from active to inactive form [5].

#### **2.1. ABA signaling in plants**

expression of a cascade of stress-responsive genes [2]. Cellular ABA levels are induced by

The plant hormone ABA has been identified as a key regulator of multiple stresses. In general, the adaptive responses of plants to various stress conditions can be either ABA-dependent or ABA-independent. However, there is no clear boundary between these two pathways and there is lot of crosstalk between the pathways and the components involved. This review will focus on recent advancements in ABA-mediated stress signaling and the role of ABA in abiotic

ABA, a sesquiterpenoid (C15H20O4) with a 15-carbon ring (Figure 1), has a variety of biological functions and is found ubiquitously across several kingdoms, including cyanobacteria, sponges, algae, lichens, mosses, and mammals [4-7]. Discovered in the 1960s and initially named dormin or abscissin, ABA is now established as a widely occurring and important plant growth regulator. Although it was initially identified as an abscission-promoting hormone, later scientists discovered that this was partly due to an indirect effect of inducing ethylene biosynthesis [8]. ABA is an important regulator of plant growth, including embryo and seed development, seedling establishment, vegetative and reproductive growth as well as promot‐ ing seed dormancy [9,10]. Seed maturation and promotion of dormancy are important in preventing preharvest sprouting. In addition, ABA has the ability to antagonize the germina‐ tion promoting effects of gibberellin, regulate guard cells, and regulate stress-responsive gene expression under water-deprived conditions. ABA also has a role in plant pathogen responses

The molecular structure of ABA has several important features that facilitate its biological functions. The side chain with the two double bonds (Figure 1) and ABA's stereocenter are two such important features. Exposure to UV light changes the conformation from active to

environmental stimuli such as light, water, and salinity stress [3].

stress tolerance in the model plant *Arabidopsis thaliana*.

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

**2. The phytohormone Abscisic Acid (ABA)**

in a pathosystem-dependent manner [4,5].

**Figure 1.** Structure of phytohormone abscisic acid S-(+)-ABA

inactive form [5].

Although ABA has a broad range of functions in plant growth and development, its main function is to regulate plant water balance and osmotic stress tolerance [11]. Thus, under‐ standing ABA signaling is essential to improving plant performance. Genetic screens in *Arabidopsis thaliana* identified many downstream ABA signaling components. Recent findings in the field of ABA signaling reveal a unique hormone perception mechanism (Figure 2) where ABA binds to the ABA receptors Regulatory Components of ABA Receptor/Pyrabactin Resistance Protein1/PYR-like Proteins (RCAR/PYR1/PYLs). RCAR/PYR/PYL proteins belong to the START-domain superfamily and have soluble ligand-binding properties. RCAR/PYR/PYL receptors are found in the cytoplasm as well as in the nucleus. ABA binding to RCAR/PYR/PYLs leads to inactivation of type 2C protein phosphatases (PP2Cs) such as ABSCISIC ACID INSENSITIVE 1 (ABI1) and its close homolog ABI2 [12]. All 14 members of the RCAR family of proteins bind to ABA and interact with PP2Cs. Except for RCAR7/PYL13, all the other RCAR members are positive regulators of ABA signaling. Among the 80 PP2Cs identified in Arabidopsis, six out of nine clade A PP2Cs act as negative regulators of ABA signaling [13]. These phosphatases and RCAR/PYR1/PYLs function as co-receptors and form a high-affinity ABA-binding site. Inactivation of PP2Cs causes suppression of PP2C-mediated dephosphorylation of Sucrose nonfermenting Kinase-1-Related protein kinase 2s (SnRK2s), which are important positive regulators of ABA signaling. As a result, activated SnRK2s target ABA-dependent gene expression and ion channels [5,11]. Table 1 summarizes the major positive and negative regulatory elements in the ABA signaling pathway. Phosphorylated SnRK2s subsequently phosphorylate ABA-responsive element Binding Factors (ABFs), which are basic leucine zipper transcription factors that bind to ABA-Responsive Elements (ABRE) (PyACGTGG/TC), the major *cis*-element in the promoter region of downstream genes that are induced by ABA [19,20].

**Figure 2.** Main components in the core ABA signal transduction pathway


**Table 1.** Major positive and negative regulators of ABA signaling

Therefore, the ABA signaling complex/ABA signalosome is comprised of three major compo‐ nents: (a) RCAR/PYR/PYLs; (b) PP2Cs; and (c) SnRK2s assembled as a double negative regulatory system [7]. In the absence of ABA, PP2Cs dephosphorylate SnRK2s inhibiting kinase activity and thereby preventing downstream gene expression (Figure 2). Several studies showed that these core components are essential for ABA signaling. For instance, Fujita *et al*. [21] showed ABA signaling is completely blocked and *ABF* genes showed reduced expression in the *snrk2.2/2.3/2.6* triple null mutant but not in single or double mutants. In addition, reduced phosphorylation of other bZip transcription factors such as ABSCISIC ACID INSENSITIVE 5 (ABI5), which is a dormancy promoting transcription factor, was also observed [18,22].

In guard cells (Figure 3), ABA binds to the PYR/PYL/RCAR receptor-PP2C complex and blocks its phosphatase activity. Consequently, activated protein kinase SnRK2.6/OPEN STOMATA 1 (OST1) phosphorylates and regulates the key target ion channels, SLOW ANION CHANNEL ASSOCIATED 1 (SLAC1) and K+ CHANNEL IN ARABIDOPSIS THALIANA 1 (KAT1). SnRK2.6/OST1 acts as a positive regulator of stomatal closure where it activates anion channel SLAC1 and inhibits cation channel KAT1 [23-25].

#### **2.2. ABA-binding proteins and alternate ABA receptors**

Identification of putative ABA receptors using forward genetic approaches was not successful for a long time due to genetic redundancy of the genes encoding ABA receptor proteins. However, biochemical approaches leading to purification and analysis of high-affinity ABAbinding proteins have been successful in identification of potential ABA receptor classes [5,26]. Some of these potential ABA receptors are cytosolic while others are on the cell surface,

**Figure 3.** ABA signaling in guard cells

**Signaling component Regulation Expressed References**

developmental stages

involved in stomatal closure

CHANNEL IN ARABIDOPSIS THALIANA 1 (KAT1).

[13-16]

[15]

**regulators**

HYPERSENSITIVE TO ABA 1/2 (HAB1/2) [17]

SRK2D/SnRK2.2 Seeds and vegetative tissues [18] SRK2I/SnRK2.3 Seeds and vegetative tissues [18]

Therefore, the ABA signaling complex/ABA signalosome is comprised of three major compo‐ nents: (a) RCAR/PYR/PYLs; (b) PP2Cs; and (c) SnRK2s assembled as a double negative regulatory system [7]. In the absence of ABA, PP2Cs dephosphorylate SnRK2s inhibiting kinase activity and thereby preventing downstream gene expression (Figure 2). Several studies showed that these core components are essential for ABA signaling. For instance, Fujita *et al*. [21] showed ABA signaling is completely blocked and *ABF* genes showed reduced expression in the *snrk2.2/2.3/2.6* triple null mutant but not in single or double mutants. In addition, reduced phosphorylation of other bZip transcription factors such as ABSCISIC ACID INSENSITIVE 5 (ABI5), which is a dormancy promoting transcription factor, was also observed [18,22].

In guard cells (Figure 3), ABA binds to the PYR/PYL/RCAR receptor-PP2C complex and blocks its phosphatase activity. Consequently, activated protein kinase SnRK2.6/OPEN STOMATA 1 (OST1) phosphorylates and regulates the key target ion channels, SLOW ANION CHANNEL

SnRK2.6/OST1 acts as a positive regulator of stomatal closure where it activates anion channel

Identification of putative ABA receptors using forward genetic approaches was not successful for a long time due to genetic redundancy of the genes encoding ABA receptor proteins. However, biochemical approaches leading to purification and analysis of high-affinity ABAbinding proteins have been successful in identification of potential ABA receptor classes [5,26]. Some of these potential ABA receptors are cytosolic while others are on the cell surface,

SRK2E/OST1/SnRK2.6 Expressed in guard cells and

ABA INSENSITIVE 1/2 (ABI1/2) Various tissues and

**Group A PP2Cs Negative**

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

**SnRK2 subgroup III Positive regulators**

**Table 1.** Major positive and negative regulators of ABA signaling

ABA HYPERSENSITIVE GERMINATION 1

ASSOCIATED 1 (SLAC1) and K+

SLAC1 and inhibits cation channel KAT1 [23-25].

**2.2. ABA-binding proteins and alternate ABA receptors**

(AHG1)

indicating there are extracellular as well as intracellular sites of ABA perception. Studies indicate that there can be multiple ABA receptors at different locations of the cell [27].
