**5.1. Transgenic approach**

that phosphorylation of a serine residue in the SnRK2 activation loop (S177/176/175 in SnRK2.2/2.3/2.6, respectively) is critical for full kinase activity [61]. Kinases with this serine mutated to alanine are non-phosphorylatable at this position and showed marked decrease, but not complete abolishment of kinase activity, suggesting that unphosphorylated SnRK2s have basal kinase activity [58]. Crystal structures of unphosphorylated SnRK2s have been solved in their active and inactive states, providing structural insights into the mechanisms of the basal activity [58]. The SnRK2 structures revealed a canonical bilobal kinase fold, with a well-ordered SnRK2 box packing closely in parallel to the αC-helix in the N-terminal lobe (schematically illustrated in Figure 5). The structure of the active SnRK2 adopted a closed conformation, with its N-terminal lobe positioned closer to the C-terminal lobe (Figure 5d), whereas that of the inactive SnRK2 exhibited an open conformation (Figure 5c) resembling the structures of active and inactive Snf1 kinases, respectively. The basal kinase activity is attained by the closing of the catalytic cleft, which is regulated by the SnRK2 box interactions with the αC-helix. The SnRK2 box mediates positioning of the αC-helix to form a network of interaction that favours the binding of Mg2+ and ATP in the catalytic cleft. Such a positioning enables full kinase activity to be achieved upon autophosphorylation of the activation loop, which can

**Figure 5.** Mechanisms of kinase activation. a) In basal state, PP2C inhibit SnRK2 by enzymatic dephosphorylation of the kinase's activation loop serine, as well as steric inhibition by physically binding to the kinase's catalytic cleft. The two PP2C-SnRK2 interaction interfaces are shown. First is through the mutual packing of active sites as indicated by the kinase's activation loop and the phosphatase's ABA-sensing tryptophan "lock". Second is through the SnRK2's Cterminal ABA box region. b) When activated by ABA, PYL compete with SnRK2 for PP2C interaction at the same PP2C catalytic region. c) and d) When relieved of PP2C inhibition, unphosphorylated SnRK2s spontaneously adopt inactive and partially active states by the opening and closing of the catalytic cleft, mediated by contacts between the SnRK2 box and αC-helix. e) The closing of the catalytic cleft produces interactions that facilitates the autophosphorylation of

the activation loop serine residue, which is required to attain full kinase activity.

occur intramolecularly or intermolecularly (Figure 5e).

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

Having understood the roles and mechanisms of the key players in the core ABA signalling, one approach to enhance ABA sensitivity is to generate transgenic plants overexpressing the effectors of ABA signalling. While the dimeric PYL members, PYR1, PYL1, and PYL2 require ABA for their activation, the monomeric receptors, PYL5-10, show ABA-independent constit‐ utive activity which is greatly enhanced in the presence of ABA [46]. Thus, the constitutively active monomeric receptors are thought to be suitable candidates for transgenic overexpres‐ sion to enhance ABA sensitivity. This idea is supported by the observation that transgenic Arabidopsis overexpressing PYL5 showed enhanced ABA sensitivity and increased drought stress tolerance [19]. Similar results have been shown in transgenic Arabidopsis overexpress‐ ing PYL13 [53], which has shown ABA independent inhibition of PP2CA [54]. In rice (*Oryza sativa*), the constitutive expression of OsPYL, the rice orthologue of Arabidopsis PYL, has also shown to improve drought and salt stress tolerance [62].

With the known structural mechanisms, genetic modifications of PYL may be incorporated to further enhance their activity. The constitutive expression of a PYL2 mutant designed to stabilise PYL-PP2C interactions has shown increased ABA signalling in transgenic Arabidop‐ sis seeds [63]. However, the expression of the mutant receptor has not been detected in vegetative tissues, thus precluding further analyses in this study. In another study, overex‐ pression of a mutant PYL4 receptor (PYL4A194T), which showed ABA-independent inhibition of PP2CA in vitro, resulted in enhanced drought tolerance in the transgenic plants [64]. However, as ABA is a negative growth regulator, a drawback of constitutively activating ABA responses is the impairment of growth under normal conditions, thus affecting overall yield. Approaches that allow inducible activation of ABA responses only under stressful conditions may be useful to overcome this problem.

Alternatively, the heterologous expression of SnRK2s of wheat (*Triticum aestivum* L.), TaSnRK2.4, and TaSnRK2.8 in Arabidopsis has been tested and the transgenic plants showed enhanced tolerance to drought, salt, and cold stresses [65, 66]. Similarly, transgenic Arabidop‐ sis overexpressing the maize SnRK2 orthologue, ZmSAPK8, exhibited increased tolerance to salt stress [67]. In these studies, the heterologous expression of members of the SnRK2s did not seem to retard the normal plant growth under unstressed conditions, thus appearing to be a potential approach for the development of stress-tolerant transgenic crops.

#### **5.2. Agonist design**

The direct application of ABA in the fields has been shown to promote drought stress tolerance. However, the widespread use of ABA in agriculture is limited due to its chemical instability and difficulty to synthesise. Therefore, there has been much research focus on the development of ABA analogues that are able to elicit the effects of ABA and exhibit better chemical stability.

Pyrabactin is a synthetic seed germination inhibitor that mediates a selective part of ABA's activities. Being a selective ABA agonist, pyrabactin has been used to overcome the problems of genetic redundancy in the identification of ABA receptors [17]. Although the direct application of pyrabactin is not practical for agriculture use, as its effects are most strongly in seeds rather than in vegetative tissues, the study of ABA receptors bound to pyrabactin has provided insights into the design of ABA receptor agonists. While ABA is a pan-agonist of all PYL members, pyrabactin selectively activates few members, including PYR1 and PYL1. Conversely, pyrabactin acts as an antagonist in PYL2. Comparisons between the structures of ABA-bound PYL, pyrabactin-activated PYL and pyrabactin-antagonised PYL complexes have revealed the ligand-receptor interactions that are important to induce the closed gate confor‐ mation necessary for receptor activation [68-71]. In the pyrabactin-activated receptor (PYR1 or PYL1) structures, pyrabactin interacts with receptor pocket residues in a similar manner as ABA, with its sulphonamide group forming a water-mediated hydrogen bond with the conserved lysine residue and its naphthalene double ring interacting with the receptor gate residues, producing the close-gate conformation (Figure 6b). These features provided a framework for the development of novel ABA agonists. On this basis, Melcher et al. screened virtual chemical libraries for compounds containing the naphthalene-1-sulphonamide group and computationally docked them into the known PYL structures [69]. In vitro screening of top candidates identified four compounds with efficacies comparable to that of pyrabactin.

Examination of the structure of the pyrabactin-antagonised PYL2 receptor revealed a flip in the orientation of pyrabactin relative to that in the pyrabactin-activated receptor structures (Figure 6c). In the antagonist position, the naphthalene double ring and pyridine ring are in reversed order, placing the smaller pyridine ring towards the pocket entrance in a distance too far to interact with the receptor gate. Therefore, pyrabactin antagonises PYL2 by occupying the receptor pocket while being unable to produce the closed gate conformation. With this knowledge, an agonist termed AM1/quinabactin was designed with a similar scaffold as pyrabactin but with the sulphonamide group in reversed orientation [72, 73]. The structures of PYL2 bound to AM1/quinabactin showed that the AM1/quinabactin is oriented with its double ring facing the receptor gate, producing a closed gate conformation (Figure 6d). Furthermore, AM1/quinabactin showed potent in vitro efficacies in PP2C interaction, ability to promote drought tolerance when exogenously applied to plants and higher stability than ABA when exposed to mild UV [72, 73]. Therefore, AM1/quinabactin emerged as a highly promising candidate to be further evaluated for agricultural use.

Abscisic Acid Signalling as a Target for Enhancing Drought Tolerance http://dx.doi.org/10.5772/61317 455

**Figure 6.** Mode of receptor binding by various ligands. Ligand binding pockets of a) PYL2–ABA–HAB1 structure (PDB code: 3KB3), b) PYL1–Pyrabactin–ABI1 (PDB code: 3NMN), c) PYL2–Pyrabactin (PDB code: 3NMH), d) PYL2–AM1– HAB1 (PDB code: 4LG5), and e) PYR1MANDI–Mandipropamid–HAB1 (PDB code: 4WVO). PYL ligand pockets are shown as surface presentation in light grey. In (a) to (d), the conserved lysine and glutamate residues that anchor the ligands to the inner end of the receptor pocket are shown as stick models in blue. In all panels, receptor gate loop resi‐ dues are shown in red, while the conserved PP2C tryptophan "lock" is shown as a stick model in green. In (c), pyra‐ bactin antagonises the PYL2 receptor, leaving the receptor gate in an open conformation. In (d), the four mutant PYR1MANDI residues are shown in blue, while the corresponding wild type residues from the PYR1–ABA–HAB1 (PDB code: 3QN1) structure are shown in orange.

#### **5.3. Orthogonal receptor-ligand approach**

not seem to retard the normal plant growth under unstressed conditions, thus appearing to be

The direct application of ABA in the fields has been shown to promote drought stress tolerance. However, the widespread use of ABA in agriculture is limited due to its chemical instability and difficulty to synthesise. Therefore, there has been much research focus on the development of ABA analogues that are able to elicit the effects of ABA and exhibit better chemical stability.

Pyrabactin is a synthetic seed germination inhibitor that mediates a selective part of ABA's activities. Being a selective ABA agonist, pyrabactin has been used to overcome the problems of genetic redundancy in the identification of ABA receptors [17]. Although the direct application of pyrabactin is not practical for agriculture use, as its effects are most strongly in seeds rather than in vegetative tissues, the study of ABA receptors bound to pyrabactin has provided insights into the design of ABA receptor agonists. While ABA is a pan-agonist of all PYL members, pyrabactin selectively activates few members, including PYR1 and PYL1. Conversely, pyrabactin acts as an antagonist in PYL2. Comparisons between the structures of ABA-bound PYL, pyrabactin-activated PYL and pyrabactin-antagonised PYL complexes have revealed the ligand-receptor interactions that are important to induce the closed gate confor‐ mation necessary for receptor activation [68-71]. In the pyrabactin-activated receptor (PYR1 or PYL1) structures, pyrabactin interacts with receptor pocket residues in a similar manner as ABA, with its sulphonamide group forming a water-mediated hydrogen bond with the conserved lysine residue and its naphthalene double ring interacting with the receptor gate residues, producing the close-gate conformation (Figure 6b). These features provided a framework for the development of novel ABA agonists. On this basis, Melcher et al. screened virtual chemical libraries for compounds containing the naphthalene-1-sulphonamide group and computationally docked them into the known PYL structures [69]. In vitro screening of top candidates identified four compounds with efficacies comparable to that of pyrabactin.

Examination of the structure of the pyrabactin-antagonised PYL2 receptor revealed a flip in the orientation of pyrabactin relative to that in the pyrabactin-activated receptor structures (Figure 6c). In the antagonist position, the naphthalene double ring and pyridine ring are in reversed order, placing the smaller pyridine ring towards the pocket entrance in a distance too far to interact with the receptor gate. Therefore, pyrabactin antagonises PYL2 by occupying the receptor pocket while being unable to produce the closed gate conformation. With this knowledge, an agonist termed AM1/quinabactin was designed with a similar scaffold as pyrabactin but with the sulphonamide group in reversed orientation [72, 73]. The structures of PYL2 bound to AM1/quinabactin showed that the AM1/quinabactin is oriented with its double ring facing the receptor gate, producing a closed gate conformation (Figure 6d). Furthermore, AM1/quinabactin showed potent in vitro efficacies in PP2C interaction, ability to promote drought tolerance when exogenously applied to plants and higher stability than ABA when exposed to mild UV [72, 73]. Therefore, AM1/quinabactin emerged as a highly

promising candidate to be further evaluated for agricultural use.

a potential approach for the development of stress-tolerant transgenic crops.

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

**5.2. Agonist design**

Although the discovery of AM1/quinabactin has shown promising results, pushing a new chemical into the market can be a long and costly process as the potential effects on human health and the environment need to be thoroughly assessed. To bypass such a process, an alternative solution is to make use of currently approved agrochemicals. Park et al. has demonstrated that the ABA receptor can be engineered to be activated by existing agrochem‐ icals [74]. This effort has identified a hextuple mutant PYR1MANDI (PYR1(Y58H/K59R/V81I/ F108A/S122G/F159L)) that showed strong PP2C inhibition with the agrochemical mandipro‐ pamid at nanomolar sensitivity. To understand the mode of the orthogonal receptor-ligand interactions, the crystal structure of a quadruple mutant PYR1(K59R/V81I/F108A/F159L), which contains 4 of the 6 mutations of PYR1MANDI and yielded higher quality crystals than PYR1MANDI, has been solved in complex with mandipropamid and HAB1 (Figure 6e). The crystal structure revealed that the F108A/F159L mutations created more space in the receptor pocket to fit the larger ligand. The arginine of R59 forms a hydrogen bond with the amide carbonyl of mandipropamid, mimicking the interaction between the carboxylate group of ABA with K59 of wild type PYR1. In vivo studies of transgenic Arabidopsis constitutively express‐ ing PYR1MANDI showed enhanced drought survival with mandipropamid treatment, thus demonstrating the feasibility of such an approach.
