**3.1. ABA receptors**

is the largest consumer of global freshwater resources [3], and it is predicted that in the year 2030, the world will be in a 40% water shortage [4]. Therefore, in view of the water and food crises, solutions that aim to increase crop productivity while limiting agricultural water consumption are the most valuable. Such solutions may potentially arise from the fine

The plant hormone abscisic acid (ABA) was first discovered in the 1960s and shortly after, its role as the central and critical regulator of abiotic stress response has become clear. Under environmental stress such as drought, ABA levels rise strongly and rapidly, triggering stress tolerant effects, such as stomatal closure, to enable the plants to conserve water and survive through the harsh condition [5]. Exogenous ABA treatment has been shown to maintain the survival and quality of plants subjected to drought stress [6]. However, the agricultural use of ABA has been limited by its short-lived bioactivity, which is due to its chemical instability and rapid catabolism in plants [7]. Therefore, there has been much interests in the development of synthetic compounds that functionally mimic ABA but exhibit longer periods of bioactivity. While earlier attempts of designing ABA analogues guided by the hormone's chemical structure and catabolic pathway had not led to any desirable candidates [8], recent approaches based on the molecular mechanisms of ABA receptor signalling has shown more promising results. Such developments have made been possible by advances in the structural mecha‐ nisms of ABA signalling components that have emerged in the past few years, as reviewed in

Stress signals, such as drought, salinity, and temperature extremes, trigger the biosynthesis of ABA from carotenoid precursors [9]. ABA is synthesised in vascular tissues and transported to the roots and leaves where the actions occur [10]. In leaves, ABA induces stomata closure to prevent transpirational water loss and promotes the accumulation of osmocompatible solutes to retain water [11]. ABA inhibits root and shoot growth and promotes seed dormancy. Such inhibitory effects help plants to pull through adverse conditions and germinate only when the conditions are favourable for growth. ABA also confers tolerance to freezing through

The effects of ABA has immense agricultural and economic value. Massive amounts of crops are lost to drought every year and the situation may worsen as climate change persists. The ability to manipulate ABA responses offers an innovative solution to alleviate crop loss and sustain agricultural yield in the face of the inevitable climatic change. Moreover, the activation of ABA responses may enhance agricultural water use efficiency, addressing concerns of the global water scarcity issue. A sound understanding of ABA signalling is critical to the

understanding of how plants perceive and respond to abiotic stress signals.

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

the following sections.

**2. ABA is a regulator of abiotic stress tolerance**

the induction of dehydration-tolerance genes [12].

successful development of approaches to manipulate ABA responses.

"There are things known and there are things unknown, and in between are the doors of perception." ― Aldous Huxley

ABA is a messenger that carries the abiotic stress signal. The first step for the plant cells to be aware of the stress is to perceive the stress signal. The perception and transmission of the signal is carried out by ABA receptors, which recognise the ABA molecule and convey the message to downstream effectors.

Although the importance of ABA and many aspects of its signalling has been established following the discovery of the hormone in the 1960s, the identity of ABA receptors has remained elusive for almost half a century. Early efforts to identify ABA receptors employ forward genetics screens of ABA-insensitive mutants, which have instead identified several mediators of ABA signalling downstream of the receptors, such as PP2Cs and transcription factors [13, 14]. The use of alternative approaches by virtue of ABA binding has led to a number of putative candidates (FCA, CHLH, GCR2, GTG1, and GTG2) but none has been further substantiated [15]. In retrospect, these approaches had been futile for two reasons. First, the true identity of ABA receptors had been masked by genetic redundancy, and had thus eluded identification by classical forward genetic screens. Second, the identification of ABA-binding proteins had not addressed the links of the putative candidates to well-established components of ABA signalling.

The discovery of the PYR/PYL/RCAR family of proteins as bona fide ABA receptors had been different from the earlier attempts. Several groups had independently discovered this family of novel candidates, which fit elegantly into a model that connected the core components of the ABA signal transduction pathway. The first landmark studies were two reports published in Science in May 2009 [16, 17]. To overcome genetic redundancy issues, Park et al. [17] used a selective ABA agonist, pyrabactin, in a chemical genetic screen of mutagenised Arabidopsis seeds and identified *PYR1* (for pyrabactin resistance 1). PYR1 belongs to the START domain superfamily of proteins that includes the mammalian STARD proteins. By homology, 13 other Arabidopsis PYR1-like (PYL) members were identified and named PYL1–13 (Table 1). In a different approach, Ma et al. [16] screened for interactors with the type 2C protein phosphatase (PP2C) ABI2, which is a known component of ABA signalling, and identified the same 14 START domain family members, naming them as RCAR1–14 (Regulatory Component of ABA Response) (Table 1). For simplicity, the PYR/PYL/RCAR members are referred to in this chapter as PYL proteins. Other similar studies have also identified PYL members by their interactions with PP2Cs [18, 19] and collectively, these studies showed that PYL proteins are able to bind to ABA and in the presence of ABA, PYL proteins interact with and inhibit the ABA-signalling PP2Cs.


#### Abscisic Acid Signalling as a Target for Enhancing Drought Tolerance http://dx.doi.org/10.5772/61317 445


**Subset of Group A PP2CS**

**Members of the PYR/PYL/RCAR family**

**Protein Name Solved Structures**

K59R, V81I, F108A,

**Complex with**

H60P ABA HAB1 3ZVU

F159L Mandipropamid HAB1 4WVO

V114I Pyrabactin 3NJ1 A93F Pyrabactin 3NMP A93F Pyrabactin HAB1 3NMT A93F Pyrabactin ABI2 3NMV

Ligand PP2C

ABA 3K3K, 3K90 Pyrabactin 3NJO AS6 3WG8 ABA HAB1 3QN1

ABA 3JRS Pyrabactin 3NEF, 3NEG ABA ABI1 3KDJ, 3JRQ Pyrabactin ABI1 3NMN

ABA 3KB0, 3KDI

Pyrabactin 3NJ0, 3NMH,

ABA HAB1 3KB3 ABA ABI2 3UJL Quinabactin (AM1) HAB1 4LG5, 4LA7 AM2 HAB1 4LGA AM3 HAB1 4LGB

ABA 4DSB, 4DSC

Pyrabactin 3OJI (-)-ABA 4JDA

**PDB ID**

3K3K

3KAY

3KDH, 3KL1, 3KAZ

3NR4, 3NS2

3KLX

**PYR/PYL RCAR Mutation(s)**

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

**PYR1 RCAR11**

**PYL1 RCAR12**

**PYL2 RCAR14**

**PYL3 RCAR13**


**Table 1.** List of members belonging to the components of the core ABA signalling pathway and their solved structures.

#### **3.2. Group 2C Protein Phosphatases (PP2Cs)**

PP2Cs are a group of monomeric Mg2+/Mn2+-dependent serine/threonine phosphatases found in virtually all organisms and are known for their roles in the regulation of cell growth and cellular stress signalling. In Arabidopsis, there are 76 known PP2Cs that are genetically clustered into 10 groups (A-J), with the exception of 6 genes that could not be clustered [20]. At least 6 of the 9 members of group A PP2Cs have been shown to be involved in ABA signalling. Among these, ABI1, ABI2, and HAB1 are the most well-studied members and are known to be the negative regulators of ABA signalling.

The *abi1* and *abi2* (ABA insensitive) mutants were isolated from genetic screens of ABA insensitive mutants [21-24]. HAB1 was subsequently identified by homology to ABI1 and ABI2 [25]. The *abi1-1* (ABI1 G180D), *abi2-1* (ABI2 G168D), and *hab1* (HAB1 G246D) mutants dis‐ played dominant ABA insensitive phenotypes, which are later known to be attributed to the loss of regulation by PYL proteins. The isolation of additional recessive loss-of-function mutations that resulted in ABA hypersensitive phenotype provided the early evidence that PP2Cs are negative regulators of ABA signalling [26-28]. This was further supported by the observations that double- or triple-PP2C-knockout mutants displayed enhanced ABA re‐ sponses while constitutive expression of HAB1 led to reduced ABA sensitivity [28-30].

#### **3.3. Snf1-related protein kinases 2 (SnRK2s)**

While members of PP2Cs are known to play a negative regulatory role in ABA signalling, it is not surprising that a group of protein kinases are conversely the positive effectors. The Arabidopsis Snf1-related protein kinase (SnRK) group of kinases share a high degree of homology with the yeast Snf1 and catalytic subunits of mammalian AMPK. There are three classes of SnRKs in Arabidopsis, namely SnRK1, SnRK2, and SnRK3, which comprises of 3, 10, and 25 members, respectively [31]. SnRK2s are further divided into subclasses I, II, and III. While members of subclass I are not responsive to ABA, subclass II members are weakly activated by ABA. All three members of subclass III, namely SnRK2.2/SRK2D, SnRK2.3/SRK2I, and SnRK2.6/SRK2E/OST1, are strongly activated by ABA and are known to be the main positive regulators of ABA signalling. SnRK2.6 is known for its role in stomata regulation in guard cells [32, 33], while SnRK2.2 and SnRK2.3 are important in the inhibition of seed germination and root growth [34]. Consequently, triple mutants lacking SnRK2.2, SnRK2.3, and SnRK2.6 are deficient in almost all ABA responses, indicating that class III SnRK2s play a central role in the core ABA signalling pathway [35-37].

Active SnRK2s are autophosphorylated and are able to directly phosphorylate target proteins such as ion channels and transcription factors to elicit the ABA response. ABA-induced stomata closure is mediated by the effects of SnRK2.6 on ion channels. Under ABA stimulation, SnRK2.6 activates the slow-type anion channel SLAC1 and inhibits the inward-rectifying potassium channel, KAT1, by the phosphorylation of these ion channels, resulting in stomata closure [38-40].

ABA induction of target gene expression is mediated by the SnRK2 phosphorylation of transcription factors known as ABRE-binding (AREB) proteins or ABRE-binding factors (ABFs). The AREB/ABFs are basic domain leucine zipper (bZIP) transcription factors that recognises the ABA-responsive elements (ABREs) located in the promoters of ABA-responsive genes. Of the nine AREB/ABF homologs found in Arabidopsis, the AREB1/ABF2, AREB2/ ABF4, and ABF3 were found to be master transcription factors responsible for regulating the ABRE-dependent expression of stress-responsive genes [41-44]. Such genes include Late Embryogenesis Abundant (LEA)-class genes, transcription factors, and mediators of ABA signalling.
