RCAR ABA receptors

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PYR/PYL/RCAR ABA receptors Pedro L. Rodrigueza, *, Jorge Lozano-Justea and Armando Albertb a

Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas, Universidad Politécnica de Valencia, Valencia, Spain Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas, Madrid, Spain *Corresponding author: E-mail: [email protected] b

Contents 1. Introduction to ABA receptors 2. Biochemical properties and inhibition of phosphatase activity by ABA receptors 2.1 Arabidopsis clade A PP2Cs 2.2 ABA- and receptor-dependent degradation of PP2Cs 2.3 Structural insights into PP2C inhibition by ABA receptors and mutant PP2Cs refractory to inhibition 2.4 ABA receptor subfamilies and biochemical properties 3. Redundant and differential functions of ABA receptors 4. Regulation of receptor turnover and post-translational modifications of ABA receptors 5. Revisiting structural biology of ABA receptors and PP2C co-receptors 5.1 An updated model to account for the enhancement of ABA binding affinity by PP2C co-receptor 5.2 Conformational changes of receptor gate and latch loops and proactive role of PP2Cs References

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Abstract The clade A protein phosphatases type 2C (PP2Cs) ABA INSENSITIVE 1 (ABI1) and ABI2 were the first components of the ABA signaling pathway identified from single-locus dominant mutations but functional redundancy precluded the identification of ABA receptors by classical forward genetic analyses. However, a chemical genetic approach using a synthetic selective ABA agonist, pyrabactin, led to the discovery of PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL1-13) ABA receptors. Independently, the search for interacting partners of clade A PP2Cs in yeast-two hybrid screenings also led to their discovery, naming them as REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR114). From 2009 many studies have shed light on the biochemical properties of PYR/ PYL/RCAR ABA receptors and their capability to inhibit clade A PP2Cs, the key negative regulators of the pathway. Physiological studies have made use of mutants impaired in several PYR/PYL/RCAR genes in order to break functional redundancy, and in the Advances in Botanical Research, Volume 92 ISSN 0065-2296 https://doi.org/10.1016/bs.abr.2019.05.003

© 2019 Elsevier Inc. All rights reserved.

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appropriated biological context, some specific functions of the receptors have also emerged. The regulation of their half-life through interaction with E3 ubiquitin ligases and the discovery of post-translational modifications of ABA receptors have been active fields as well. It is particularly appealing that ABA perception by PYL8 leads to reduced ubiquitination and specific stabilization of this unique receptor. More recently, translational studies in crops are starting to be developed and biotechnological applications are expected in the next years. The structural studies performed with Arabidopsis receptors are being complemented with studies in other plant species, which offer new insights on the mechanism of ABA perception that highlight the role of PP2Cs as coreceptors.

1. Introduction to ABA receptors The PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) are currently recognized as the canonical ABA receptors based on structural, biochemical and genetic evidences obtained independently by several groups (Gonzalez-Guzman et al., 2012; Ma et al., 2009; Melcher et al., 2009; Miyazono et al., 2009; Nishimura et al., 2009, 2010; Park et al., 2009; Santiago et al., 2009a, 2009b; Yin et al., 2009; Zhao et al., 2018). However, their discovery suffered a 25-year lag after the isolation of the first ABA-insensitive mutants of Arabidopsis (Koornneef, Reuling, & Karssen, 1984) and a 15-year lag after the cloning of the ABI1 phosphatase (Leung et al., 1994; Meyer, Leube, & Grill, 1994), whose activity is inhibited by ABA receptors. Moreover, this 14-member Arabidopsis thaliana family of ABA receptors was published ‘belatedly’ after four previous reports of putative ABA receptors. Unfortunately, the first publication that described FCA as a putative ABA receptor (Razem, El Kereamy, Abrams, & Hill, 2006) was soon questioned (Risk, Macknight, & Day, 2008) and finally retracted (Razem, El Kereamy, Abrams, & Hill, 2008). The second ABA receptor reported was CHLH/ABAR/GUN5 (Shen et al., 2006), previously described as the H subunit of Mg-chelatase responsible for introducing Mg2þ into the precursor of the chlorophyll. Shen et al., used biochemical techniques to identify ABA binding proteins through the coupling of ABA’s carboxyl group with a Sepharose resin. Indeed, such approach prevents interactions of ABA’s carboxyl group with the interacting protein and makes it dispensable for ABA binding, which contrasts with the requirement of ABA’s carboxylate for the bioactivity of the hormone and binding to the genuine PYR/PYL/RCAR receptors (Nishimura et al., 2009; Santiago et al.,

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2009b). The role of CHLH in ABA binding was questioned and not reproduced in Arabidopsis and other plant species (Muller & Hansson, 2009; Tsuzuki et al., 2011). GCR2, a transmembrane receptor coupled to G protein, was reported as the third ABA receptor (Liu et al., 2007). This finding remains controversial because of irreproducible gcr2 phenotype in ABA response and GCR2 binding to ABA (Gao et al., 2007; Guo, Zeng, Emami, Ellis, & Chen, 2008; Johnston et al., 2007; Risk, Day, & Macknight, 2009). GTG1 and GTG2 were identified as the fourth ABA receptors from pharmacological evidences that suggested the participation of receptors coupled to G protein in ABA signaling (Pandey, Nelson, & Assmann, 2009). Unfortunately, the gtg1 gtg2 phenotype in ABA response could not be reproduced by Jaffe et al., (2012). The above mentioned problems contrast with the extreme ABA-insensitive phenotype of the sextuple mutant that lacks PYR1, PYL1, PYL2, PYL4, PYL5 and PYL8 receptors (Gonzalez-Guzman et al., 2012), abbreviated as 112458 (note that first 1 always refers to pyr1), further confirmed in the 112458 pyl3 pyl7 pyl9 pyl10 pyl11 pyl12 duodecuple mutant, abbreviated as 112458 3,7,9,10,11,12 (Zhao et al., 2018), as well as the wealth of structural information available for PYR/PYL/ RCAR receptors (apo form, ABA-bound and in ternary complex with clade A PP2Cs) in Arabidopsis and other plant species (Melcher et al., 2009; Miyazono et al., 2009; Moreno-Alvero et al., 2017; Nishimura et al., 2009; Ren et al., 2017; Santiago et al., 2009b; Yin et al., 2009).

2. Biochemical properties and inhibition of phosphatase activity by ABA receptors 2.1 Arabidopsis clade A PP2Cs ABA signaling is initiated by ABA perception through PYR/PYL/ RCAR receptors, which leads to interaction with and inactivation of clade A protein phosphatases type 2C (PP2Cs), such as ABA INSENSITIVE 1 (ABI1) and ABI2, HYPERSENSITIVE TO ABA (HAB1) and HAB2, and PROTEIN PHOSPHATASE 2CA/ABA-HYPERSENSITIVE GERMINATION 3 (PP2CA/AHG3), thereby relieving their inhibition on three ABA-activated SNF1-related protein kinases (SnRK2s) termed subclass III SnRK2s, i.e. SnRK2.2/SnRK2D, 2.3/I and 2.6/E/OST1 (Umezawa et al., 2009; Vlad et al., 2009) (Fig. 1A). Given the major role of ABA receptors is inhibition of clade A PP2Cs and relief of subclass III SnRK2 repression, the discovery of PYR/PYL/RCARs in 2009 nicely

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Fig. 1 ABA receptors efficiently compete for the active site of the PP2C with SnRK2. (A) At low ABA levels, clade A PP2Cs are effective inhibitors of subfamily III SnRK2s. Monomeric (mPYL) and dimeric (dPYL) receptors are activated when ABA levels increase and efficiently compete for the active site of the phosphatase. The relief of PP2C-mediated repression enables that SnRK2s gain intramolecular stabilization and full activity by autophosphorylation of the activation loop, which is more efficient in SnRK2.6 than in SnRK2.2 and SnRK2.3 (Ng et al., 2011). The scheme indicates that a pool of the receptor-ABA-phosphatase ternary complexes is targeted for PP2C ubiquitination and degradation, which is a complementary mechanism to the competitive inhibition of the phosphatase by ABA receptors (Belda-Palazon et al., 2019; Kong et al., 2015; Wu et al., 2016). (B) Structures of the SnRK2.6-HAB1 complex (SnRK2-PP2C, bottom) and the ternary complex formed by Citrus sinensis (sweet orange) CsPYL1-ABAHAB1 (PYL-PP2C, top) (Moreno-Alvero et al., 2017; Soon et al., 2012).

matched previous genetic evidence on the role of clade A PP2Cs and subclass III SnRK2s in ABA signaling (Leung et al., 1994 and 1997; Meyer et al., 1994; Rodriguez, Benning, & Grill, 1998; Mustilli, Merlot, Vavasseur, Fenzi, & Giraudat, 2002; Fujii, Verslues, & Zhu, 2007 and Fujii & Zhu, 2009a). In Arabidopsis, nine protein phosphatases belong to clade A, which also includes AHG1 and Highly ABA-Induced (HAI) 1e3 phosphatases. AHG1 plays an important role to regulate ABA signaling in seeds and is regulated by a subset of ABA receptors and DELAY OF GERMINATION

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(DOG1), which reveals a critical role of AHG1 to regulate seed dormancy and consequently the timing of seed germination (Nishimura et al., 2007, 2018; Nee et al., 2017; Tischer et al., 2017); whereas HAI1-3 have less impact in ABA sensitivity, more to regulate particular drought resistance traits and are regulated by different ABA receptors (Bhaskara, Nguyen, & Verslues, 2012; Tischer et al., 2017). Clade A PP2Cs are central negative regulators of ABA signaling in both seeds and vegetative tissues (Antoni et al., 2012; Gosti et al., 1999; Leonhardt et al., 2004; Merlot, Gosti, Guerrier, Vavasseur, & Giraudat, 2001; Nishimura et al., 2007; Kuhn, Boisson-Dernier, Dizon, Maktabi, & Schroeder, 2006; Rubio et al., 2009; Saez et al., 2004, 2006; Yoshida et al., 2006a) and are able to regulate subclass III SnRK2s by physically blocking the kinase active site and dephosphorylating the conserved Ser residue (Ser175 for SnRK2.6) in the activation loop of the kinase (Belin et al., 2006; Ng et al., 2011; Soon et al., 2012; Umezawa et al., 2009; Vlad et al., 2009; Yoshida et al., 2006b) (Fig. 1B). Clade A PP2Cs are not only key inhibitors of subclass III SnRK2s but also target important effectors of ABA signaling such as SLAC1 and ABRE-binding transcription factors (Antoni et al., 2012; Brandt et al., 2012, 2015; Lee, Lan, Buchanan, & Luan, 2009; Lynch, Erickson, & Finkelstein, 2012; Maierhofer et al., 2014), and considering their vast array of targets, they also act as a regulatory hub for different abiotic stress responses (Cherel et al., 2002; Forster et al., 2019; Geiger et al., 2009; Guo et al., 2002; Himmelbach, Hoffmann, Leube, Hohener, & Grill, 2002; Ohta, Guo, Halfter, & Zhu, 2003; Peirats-Llobet et al., 2016; Rodrigues et al., 2013; Sheen, 1996; Wang et al., 2018; Yang et al., 2006).

2.2 ABA- and receptor-dependent degradation of PP2Cs In addition to inhibiting PP2C activity, degradation of certain PP2Cs is also dependent on ABA receptors. For example, the pioneering work of Kong et al. (2015) demonstrated that degradation of ABI1 mediated by plant U-box (PUB) E3 ligases PUB12/13 is promoted by ABA receptors and ABA, which are strictly required or enhance PUB13-mediated ABI1 ubiquitination. Therefore, it seems that a double mechanism, i.e. biochemical inhibition and facilitation of degradation, is followed by ABA receptors in order to relief the inhibition of ABI1 on ABA signaling. Since PUB12/13 do not interact with other clade A PP2Cs such as ABI2, HAB1 or PP2CA, it is expected the identification of additional E3 ligases that target them. In this direction, it was demonstrated that RGLG1/5 RING-type E3 ligases target PP2CA for degradation, and ABA enhances this interaction

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through a mechanism recently elucidated (Wu et al., 2016; Belda-Palazon et al., 2019). Myristoylated RGLG1 is localized in plasma membrane whereas PP2CA is predominantly localized in nucleus. Recently, it has been reported that ABA inhibits myristoylation of RGLG1 and promotes cycloheximide-insensitive translocation to nucleus, where RGLG1 recognizes receptor-ABA-phosphatase complexes because the E3 ligase can interact with both PP2CA and certain monomeric receptors (Belda-Palazon et al., 2019). Thus, ABA receptors and ABA can facilitate the recognition/ ubiquitination of the phosphatase by PUB12/13 and RGLG1 E3 ligases, suggesting that although the interaction of ABA receptors with clade A PP2Cs in the presence of ABA might be reversible, it is possible that a pool of these complexes is targeted for PP2C ubiquitination and degradation.

2.3 Structural insights into PP2C inhibition by ABA receptors and mutant PP2Cs refractory to inhibition The PYR/PYL/RCAR proteins belong to the superfamily of START/Bet v proteins, whose members are characterized by the presence of a cavity able to accommodate hydrophobic ligands (Radauer, Lackner, & Breiteneder, 2008). This cavity is the ABA-binding pocket and establishes numerous hydrophobic and polar interactions stabilizing the hormone into the pocket (Santiago et al., 2012; Umezawa et al., 2009) (Fig. 2A). For example, taking the structure of AtPYR1 as a model, the importance of the above mentioned ABA’s carboxyl group is highlighted by the indirect interactions (water-mediated hydrogen bonds) with side chains of Glu94, Glu141, Ser122 and Tyr120, as well as a direct contact with the amine group of Lys59 (Fig. 2B). Additionally, the loops defined by the b3-b4 and b5-b6 regions cover ABA and are named as gate and latch, respectively (Fig. 2B and C). The residues of the gate and latch loops are conserved and have been used as a hallmark to define PYR/PYL/RCAR ABA receptors in different plant species (Fig. 3). Although most of the ABA molecule is buried within the ABA-binding pocket and stabilized by the closed conformation of gate and latch loops, structural studies of the ternary receptor-ABA-phosphatase complexes have revealed that PP2Cs are necessary ABA co-receptors able to perceive the occupancy of the hormone-binding pocket to achieve nM affinity for ABA binding (Ma et al., 2009; Melcher et al., 2009; Miyazono et al., 2009; Moreno-Alvero et al., 2017; Santiago et al., 2009a; Yin et al., 2009). A conserved Trp residue of the PP2Cs (Trp385 in HAB1 and Trp300 in ABI1) points into the hormone-binding pocket and establishes

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Fig. 2 ABA receptors contain a deep cavity to accommodate ABA. (A) Structure of Solanum lycopersicum (tomato) SlPYL1 showing the overall dimeric structure of the ABAbound form. (B) Detail of the ABA-binding pocket in the ABA-AtPYR1 structure, indicating the importance of the ABA’s carboxyl group to establish water-mediated hydrogen bonds with side chains of Glu94, Glu141, Ser122 and Tyr120, as well as a direct contact with the amine group of Lys59. (C) Structure of an individual protomer of SlPYL1 in the apo-form, highlighting latch and gate loops in blue and cyan, respectively.

a water-mediated hydrogen-bond to the ketone group of ABA (Figs. 1B and 4). Specifically this water molecule establishes a conserved network of hydrogen bonds that links the backbone amine group of Arg (Arg116 in AtPYR1) at the latch, the backbone carbonyl group of Pro (Pro88 in AtPYR1) at the gate, the ketone group of ABA and the Trp side chain of the phosphatase (Fig. 1B and zoom in 7C) (Dupeux et al., 2011a). Therefore, this hydrogen bond network connects the PP2C with both ABA

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Fig. 3 The b3-b4 and b5-b6 loops of ABA receptors constitute the gate and latch loops, respectively. The figure shows the amino acid sequence alignment of AtPYR1, AtPYL1, Solanum lycopersicon SlPYL1, Citrus sinensis CsPYL1 and Festuca elata (turfgrass) FePYL1, and was generated with ESPript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/) using crystal structure of AtPYR1 to define the localization of the secondary structure elements. Residues that constitute the gate and the latch loops are enclosed by a box, and some residues discussed in the text are marked with a vertical arrow, such as the Pro88 (gate) and Glu114, His115, Arg116 (latch) residues. Additionally, the location of His60Pro, Val83Phe, Met158Ile and Phe159Val mutations that contribute to constitutive activation of AtPYR1 in the absence of ABA are indicated. TT indicates turn motif.

and the gate/latch loops of the receptor and is crucial for the high stability of the ternary complex that leads to PP2C inhibition. Accordingly HAB1W385A and ABI1W300A phosphatases are refractory to inhibition by ABA receptors and transgenic plants harboring the hab1W385A allele are ABA-insensitive (Dupeux et al., 2011a; Melcher et al., 2009). Therefore, mutations in this conserved Trp residue or neighboring residues could be engineered for the generation of dominant receptor-insensitive PP2Cs. Indeed, such approach has been used by nature in order to generate at least one PP2C that is refractory to inhibition by ABA receptors. Thus, in Striga hermonthica, Fujioka et al. (2019) found one clade A PP2C named ShPP2C1

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Fig. 4 Amino acid sequence alignment of the catalytic core of Arabidopsis clade A PP2Cs and Striga ShPP2C1. The figure was generated with ESPript 3.0, using crystal structure of AtHAB1 to define the localization of the secondary structure elements. Residues are labeled to highlight PP2C-PYL interaction (red double dots), metal contacts (blue triangle), the GlyeAsp hypermorphic mutation encoded by abi1-1D allele (purple triangle), conserved Trp that contacts ABA (orange triangle) and the zinc finger (ZF) present in HAI1, HAI2, HAI3 and PP2CA (green boxes).

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that cannot be inhibited by the 8 ShPYL receptors because it harbors mutations in neighboring residues to the conserved Trp (Fig. 4). Striga is a devastating parasitic plant that efficiently absorbs water from its host even under drought conditions because it maintains active transpiration through open stomata that are insensitive to ABA-mediated closure. Through the characterization of ShPP2C1, Fujioka et al. identified the molecular mechanism that explains the ABA-insensitive phenotype developed by Striga to better parasitize the host crops. Another PP2C mutation that provides escape from receptor-mediated inhibition is found in the abi1-1D and abi2-1D dominant gain of function alleles (Koornneef et al., 1984), which were the first ABA-insensitive mutant plants described in Arabidopsis and paved the way for molecular genetics studies to elucidate ABA signaling (Leung et al., 1994 and 1997; Meyer et al., 1994; Rodriguez et al., 1998). The abi1-1D and abi2-1D alleles harbor missense mutations that replace Gly180 or Gly168 by Asp, respectively (Fig. 4). The phenotype of abi1-1D and abi2-1D plants reveals strong ABA insensitivity both in seed and vegetative tissues, which is due to lack of inhibition by ABA receptors of the encoded mutant phosphatases (Fujii et al., 2009b; Ma et al., 2009; Park et al., 2009; Umezawa et al., 2009; Vlad et al., 2009). The introduction of bulky aspartate residues into the active site of these phosphatases as well as in HAB1G246D likely prevents the b3-b4 gate loop of ABA receptors to establish contact and inhibit phosphatase activity (Fujii et al., 2009b; Ma et al., 2009; Park et al., 2009; Santiago et al., 2009a; Umezawa et al., 2009; Vlad et al., 2009). Therefore, ABI1G180D, ABI2G168D and HAB1G246D proteins are refractory to inhibition by PYR/PYL proteins, whereas these mutant PP2Cs can still interact with downstream targets, such as SnRK2s (Umezawa et al., 2009; Vlad et al., 2009). Finally, since the ABA-insensitive phenotype of abi1-1D, abi2-1D and hab1G246D alleles is just the opposite of loss-of-function alleles, they do not represent dominant negative alleles but hypermorphic ones (Robert, Merlot, N’guyen, Boisson-Dernier, & Schroeder, 2006; Rubio et al., 2009; Saez et al., 2006; Santiago et al., 2012).

2.4 ABA receptor subfamilies and biochemical properties ABA receptors are distributed in at least three distinct subfamilies in flowering plants based on amino acid sequence identity of the receptors encoded in the genomes (Fig. 5A). In Arabidopsis, PYR1 and PYL1-PYL3 constitute a subfamily of dimeric receptors (clade III); whereas PYL4-PYL6 (clade II) and PYL7-PYL10 (clade I) subfamilies comprise monomeric receptors

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Fig. 5 Cladogram of the PYR/PYL/RCAR family in Arabidopsis, rice and Brachypodium. (A) Cladogram obtained by amino acid sequence alignment of Arabidopsis thaliana (left), Oryza sativa (center) and Brachypodium dystachion (right) PYR/PYL/RCAR protein sequences. Nomenclature is according to Ma et al., (2009), Park et al., (2009), He et al., (2014) and Pri-Tal et al. (2017). (B) Alignment of Arabidopsis PYR/PYL/RCAR residues that are in contact with ABA (red circle) or are important for dimerization (dark blue circle). Variations in the consensus sequence are highlighted in blue.

(Dupeux et al., 2011b; Hao et al., 2011) (Fig. 5A). PYL11e13 oligomeric state has not been analyzed yet, perhaps because of poor solubility as reported for recombinant PYL13 (Li et al., 2013). Analysis of their gene expression levels reveals that some of ABA receptors, i.e. PYL3, 10, 11, 12 and 13, are hardly expressed or perhaps only in very specific cells (Gonzalez-Guzman et al., 2012). The relative contribution of the different receptors to global ABA signaling can be figured out by genetic analysis, e.g.

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the pyl3 pyl7 pyl9 pyl11 pyl12 pentuple mutant shows wild-type sensitivity to ABA, whereas pyr1 pyl1 pyl2 pyl4 quadruple mutant shows a clear ABA-insensitive phenotype (Park et al., 2009; Zhao et al., 2018). Finally the PYL11e13 subfamily is not present in some plant species, for example Brachypodium (Fig. 5A) or tomato (Gonzalez-Guzman et al., 2014). In rice, OsPYL12 has been considered as putative ortholog of AtPYL13 (Zhao et al., 2018) and might represent the only member of this subfamily (Fig. 5A). Dimeric receptors have lower intrinsic affinity for ABA in the absence of the PP2C co-receptor (Kd over 50 mM) and likely exist as inactive homodimers in cells, unable to bind or inhibit PP2Cs at basal ABA levels. However, in the presence of submicromolar ABA and the PP2C, they achieve nanomolar affinity for ABA binding (Santiago et al., 2009a), and activation of dimeric receptors by the ABA agonist quinabactin leads to efficient stomatal closure and up-regulation of ABA-responsive genes (Cao et al., 2013; Okamoto et al., 2013). Monomeric receptors show higher intrinsic affinity for ABA in the absence of the PP2C (Kd circa 1 mM), basically because dimeric receptors suffer the thermodynamic penalty imposed by dimer dissociation during the receptor activation process (Dupeux et al., 2011b). Therefore, at basal, nonstress ABA levels, monomeric ABA receptors can preferentially bind to ABA and PP2Cs, although with much lower efficiency than in the presence of high ABA levels. This has led to the proposal of ABA-independent inhibition of PP2Cs by certain monomeric receptors (Hao et al., 2011), which is favoured by high ratios of receptor to phosphatase. However, this proposal remains controversial because the putative ABA-independent PP2C inhibition achieved by PYL10 (Hao et al., 2011) and PYL13 (Li et al., 2013; Zhao et al., 2013) was refuted by experiments from Li et al., (2015) and Fuchs, Tischer, Wunschel, Christmann, and Grill (2014), respectively. A comprehensive biochemical analysis using similar recombinant proteins and a variety of phosphatase substrates that mimic physiological phosphopeptides attacked by clade A PP2Cs could shed light on this subject. The capability of monomeric receptors to interact with PP2Cs in the absence of ABA in yeast two-hybrid assays was very useful to identify ABA receptors as interacting partners of ABI1, ABI2 and HAB1 phosphatases (Ma et al., 2009; Santiago et al., 2009a). However, this interaction is dramatically enhanced by ABA as demonstrated by microcalorimetry analysis of binding affinity in phosphatase-ABA-monomeric receptor complexes (Ma et al., 2009; Santiago et al., 2009a). Co-immunoprecipitation experiments with the dimeric receptor PYR1 and either ABI1, ABI2 or HAB1

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phosphatases also revealed strong enhancement of ABI1-PYR1 interaction by exogenous ABA treatment and within 5 min of exposure to ABA (Nishimura et al., 2010; Park et al., 2009). However, overexpression of ABI1 driven by a 35S promoter can lead to the formation of phosphataseereceptor complexes in the absence of exogenous ABA-treatment, which suggests a proactive role of the phosphatase to facilitate such interaction (Moreno-Alvero et al., 2017; Nishimura et al., 2010). To further investigate the capability of monomeric receptors to interact with PP2Cs in vivo, Antoni et al., (2013) used the ABA receptor PYL8 as bait for tandem affinity purification-MS analyses in Arabidopsis suspension cells. It was found a 30-fold increase in the recovery of interacting PP2C peptides by exogenous addition of ABA (Antoni et al., 2013) and similar results have been obtained using PYL4 or PYL5 as baits (Belda-Palazon et al., 2016; unpublished results). These results suggest that monomeric receptors show weaker interactions with clade A PP2Cs at basal ABA levels but this interaction can be affected by receptor-phosphatase stoichiometry. Finally, reconstitution of ABA signaling into plant protoplasts and in vitro analysis of SnRK2.6 phosphorylation revealed ABA-dependent receptor activity to activate ABAresponsive gene expression and inhibit PP2C activity (Fujii et al., 2009b). Comprehensive experiments in protoplasts that examined the 126 possible receptor-phosphatase pairings revealed that all receptors regulate the ABA response in an ABA-dependent or ABA-enhanced manner (Tischer et al., 2017). However, basal ABA levels are sufficient to mediate ABA response of PYL7-PYL10 subfamily and to lesser extent of PYL4-PYL6 subfamily, but not of dimeric receptors (Tischer et al., 2017). An interesting approach to generate activated dimeric receptors in the absence of ABA was devised by Mosquna et al. (2011), who engineered constitutively active dimeric receptors by introducing specific substitutions that stabilize the agonist-bound conformation. Single mutations in 10 different residues located at the PYR1-HAB1 interface led to a constitutive interaction of both proteins in yeast two-hybrid assays. The combination of 3 or 4 mutations was necessary for a full constitutive activation of PYR1 in PP2C inhibition assays (Mosquna et al., 2011). These mutations were located close to the PYR1’s gate loop and the C-terminal helix, including His60Pro, Val83Phe, Met158Ile and Phe159Val (Fig. 3). The His60 residue, which resides on a loop adjacent to the C-terminal helix, is variable among PYR/PYL proteins and the monomeric PYL7, PYL8, PYL9 and PYL10 receptors carry the His60Pro substitution (Fig. 5B). The His60Pro mutation leads to increased affinity for ABA, destabilizes the PYR1 dimer, and therefore identifies a key

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residue in determining the oligomeric state of dimeric receptors (Dupeux et al., 2011b). Activation of ABA signaling can be achieved by constitutively active receptors but a posttranscriptional mechanism limits accumulation of such versions, suggesting the existence of a proteolytic mechanism to reduce protein levels of activated receptors (Mosquna et al., 2011; see also below, regulation of receptor turnover).

3. Redundant and differential functions of ABA receptors PYR/PYL/RCAR ABA receptors belong to a 14-member gene family in Arabidopsis and in crop species are also present as multi-gene families, ranging from 23 putative members in soybean (Bai et al., 2013), 15 in tomato (Gonzalez-Guzman et al., 2014), 12 in rice (He et al., 2014), 11 in maize (Hauser, Waadt, & Schroeder, 2011), 9 in wheat (Mega et al., 2019), 9 in barley (Seiler et al., 2014) and 11 in sweet orange (Arbona et al., 2017), or in emerging model plants for cereals 9 in Brachypodium distachyon (Pri-Tal, Shaar-Moshe, Wiseglass, Peleg, & Mosquna, 2017) and 8 in Setaria viridis (Duarte et al., 2019). This and the lack of dominant mutations explain that the application of forward genetics to identify ABA receptors was not successful, in contrast to genetic screenings for other hormone receptors (Chow & McCourt, 2006; Santner & Estelle, 2009). Although the targeted generation of at least quadruple, pentuple and sextuple mutants is required to obtain robust ABA-insensitive phenotypes (Gonzalez-Guzman et al., 2012; Park et al., 2009), both biochemical analyses of different receptor-phosphatase complexes and receptor gene expression patterns suggest that the function of ABA receptors is not completely redundant (Antoni et al., 2012; Gonzalez-Guzman et al., 2012; Szostkiewicz et al., 2010; Tischer et al., 2017). Moreover, detailed phenotype analyses in different biological contexts can reveal singular roles of ABA receptors. Thus, the single pyl8 mutant has been reported to show a non-redundant role in root sensitivity to ABA and regulation of lateral root growth (Antoni et al., 2013; Zhao et al., 2014) and pyl9 shows reduced ABA-induced leaf senescence under low light (Zhao et al., 2016). The singular role of PYL8 to mediate root ABA perception involves a non-cell-autonomous mechanism like mobile transcription factors that regulate plant development, and compared to other ABA receptors, PYL8 was unique in showing ABAinduced stabilization and predominant nuclear localization (Belda-Palazon et al., 2018). In other root physiological processes governed by ABA, several

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ABA receptors mediate adaptive responses to water deficit such as hydrotropism and repression of lateral root formation exerobranching response(Antoni et al., 2013; Dietrich et al., 2017; Orman-Ligeza et al., 2018). Few genetic analyses of ABA receptors have been performed in crops, but a non-redundant function of the dimeric receptor BdPYL1 has been reported in Brachypodium (Pri-Tal et al., 2017) and strawberry FaPYR1 plays a non-redundant role for fruit ripening (Chai, Jia, Li, Dong, & Shen, 2011). Recently, knock-outs of rice PYLs have been generated using tandem sgRNAs and multiplex CRISPR/Cas9 technology (Miao et al., 2018). Both redundant and differential functions of rice PYLs have been observed, for instance, group I mutants but not group II show seed dormancy and stomatal movement defects. Interestingly, a pyl1 pyl4 pyl6 line exhibited improved growth and productivity, likely because the growth-repressing function of ABA was diminished in the mutant without impairing stress adaptation. These results indicate that under field conditions, some rice PYLs restrain plant growth and in paddy fields without water limitations, reducing ABA response can improve crop performance (Miao et al., 2018). The multiplex CRISPR/Cas9 technology applied to inactivate Arabidopsis receptors allowed the generation of a quattuordecuple mutant but it was severely impaired in growth and precluded further characterization; however, a viable 112458 3,7,9,10,11,12 duodecuple mutant could be analyzed (Zhao et al., 2018). Interestingly, the authors found that in plants severely inhibited in ABA response (the 112458 sextuple or the 112458 3,7,9,10,11,12 duodecuple mutant), the ABA-independent osmotic stress-induced activation of SnRK2s was strongly enhanced (Zhao et al., 2018). It highlights the existence of the ABA-independent osmotic stress response that likely compensates the lack of ABA-dependent responses in severe ABA-deficient or ABA-insensitive mutants (Fujii, Verslues, & Zhu, 2011; Zhao et al., 2018). Water is usually the major limiting factor for plant productivity under field conditions. In Arabidopsis, combined inactivation of clade A PP2Cs and the overexpression of PYL4 and PYL5 ABA receptors proved useful to enhance drought tolerance (Pizzio et al., 2013; Saez et al., 2006; Santiago et al., 2009a). More recently, Yang et al. (2016) performed a comprehensive analysis of Arabidopsis transgenic lines that overexpress individual ABA receptors and found that some of them combined increased water use efficiency (WUE) without penalty in plant growth. Interestingly, overexpression of monomeric receptors was effective to increase WUE, but dimeric receptors failed to improve WUE. This correlates with the

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observation that basal ABA levels (20e40 nM) are sufficient to mediate ABA response of monomeric receptors but not of dimeric ones (Tischer et al., 2017). However, at micromolar ABA or quinabactin concentration, dimeric receptors efficiently promote stomatal closure, ABA-responsive gene expression and enhance drought tolerance (Cao et al., 2013; Okamoto et al., 2013). It remains to be investigated whether certain ABA receptors perform specific roles for regulation of stomatal aperture in response to ABA or different environmental cues. ABA-induced stomatal closure was severely impaired in the quadruple pyr1 pyl1 pyl2 pyl4 mutant as well as ABA inhibition of stomatal opening after light exposure (Nishimura et al., 2010). ABA induces leaf senescence and growth inhibition as a long-term ABA response for plant survival under severe drought stress, and some monomeric receptors such as PYL9 play an important role in this process (Zhao et al., 2016). In addition to senescence of old leaves, induction of seed and bud dormancy by ABA signaling are required to survive under unfavorable conditions and therefore specific roles for certain ABA receptors might emerge (Gonzalez-Grandio et al., 2017). Finally, a catabolite of ABA, phaseic acid (PA), is able to activate a subset of ABA receptors and particularly PYL3, which is only four fold more sensitive to ABA than PA (Weng, Ye, Li, & Noel, 2016). Therefore, functional diversification of ABA receptors has provided the opportunity to recruit an ABA catabolite as receptor agonist of specific members in the PYR/PYL/RCAR family, which leads to sustained activation of the pathway when ABA levels decay (Weng et al., 2016).

4. Regulation of receptor turnover and posttranslational modifications of ABA receptors Regulated protein turnover plays a critical role for hormone signaling (Santner & Estelle, 2009). Both the ubiquitin-proteasome system and non26S proteasome endomembrane trafficking pathways are involved in regulation of ABA signaling (Belda-Palazon et al., 2016; Bueso et al., 2014; Irigoyen et al., 2014; Yu et al., 2016; Yu & Xie, 2017). In addition to lysine ubiquitination, other posttranslational modifications of ABA receptors have been reported, such as tyrosine nitration, serine/threonine phosphorylation and cysteine nitrosylation (Castillo et al., 2015; Chen, Qu, Xu, Zhu, & Xue, 2018; Li et al., 2018; Wang et al., 2018). S-nitrosylation of cysteine residues does not impair receptor-dependent inhibition of PP2Cs, whereas tyrosine nitration and serine phosphorylation inhibit receptor activity and provide a

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fast posttranslational mechanism to inhibit ABA signaling (Castillo et al., 2015; Wang et al., 2018). Ubiquitination of ABA receptors was detected by Bueso et al. (2014) and Irigoyen et al. (2014) using p62-mediated affinity purification of ubiquitinated proteins in extracts prepared from MG132-treated plants expressing HA-tagged versions of PYR1, PYL4 and PYL8. It opened an avenue of research that has resulted in the identification of different Arabidopsis E3 ubiquitin ligases that target ABA receptors, i.e. CULLIN4-RING E3 ubiquitin ligases (CRL4) CRL4DDA1 and CRL4RAE1, RBR-type RING FINGER OF SEED LONGEVITY (RSL1), U-box, and SCFRIFP1 (Bueso et al., 2014; Irigoyen et al., 2014; Li et al., 2016, 2018; Zhao et al., 2017). A detailed evaluation of each E3 contribution to regulation of receptor levels in different tissues and developmental stages will require the analysis of endogenous receptor levels in loss-of-function backgrounds of the E3 ligases. To add further complexity, the rice APC/C (anaphase promoting complex/cyclosome), which contains a distant cullin member called APC2 and a RING RBX1 protein called APC11, targets the OsPYL/ RCAR proteins (Lin et al., 2015). Although most of the substrates targeted by APC/C are related to control of cell cycle progression, other substrates are emerging. Thus, TE (the Cdh1 ortholog in rice) acts as the substrate recognition factor that activates the APC/CTE complex and targets some ABA receptors for degradation. TE recognizes the destruction box (Dbox: RxxLxxxxN/D/E) that is present in OsPYLs and promotes their degradation by the proteasome. However, experiments with te mutant suggest that besides APC/CTE complex, other E3 ligases are involved in degradation of rice ABA receptors (Lin et al., 2015). In Arabidopsis, DET1-, DDB1-ASSOCIATED1 (DDA1) acts as substrate adapter of the CRL4DDA1 complex and interacts with PYL4, PYL8 and PYL9 ABA receptors in the nucleus (Irigoyen et al., 2014). Using HA-tagged PYL8 lines, it was demonstrated that DDA1 overexpression promotes PYL8 degradation through the 26S proteasome; however, in presence of ABA the CRL4DDA1 complex cannot promote degradation of PYL8 (Irigoyen et al., 2014). In addition to the 26S proteasome pathway, recent evidence has revealed a dynamic turnover of ABA receptors from the plasma membrane through the endosomal/vacuolar degradation pathway (Belda-Palazon et al., 2016; Yu et al., 2016). The non-26S proteasome degradation pathway is triggered by ubiquitination of targets at the plasma membrane and requires trafficking through the endosomal sorting complex required for transport (ESCRT) machinery (Yu & Xie, 2017).

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The internalization signal is provided by the ubiquitination of PYR1 and PYL4 by the RBR-type E3 ubiquitin ligase RSL1 (Bueso et al., 2014). Subsequently, ubiquitinated receptors are recognized by FYVE DOMAINCONTAINING PROTEIN 1 (FYVE1)/FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1) and Vps23 components of the ESCRT machinery that address ABA receptors to vacuolar degradation (Belda-Palazon et al., 2016; Yu et al., 2016). Therefore, turnover of ABA receptors is regulated both at the plasma membrane and nucleus through RSL1 and CRL4DDA1, respectively. Other monomeric or putative subunits of multimeric E3 ligases that interact with ABA receptors have been described (Li et al., 2016, 2018; Zhao et al., 2017). Future experiments should investigate whether endogenous levels of the targets are regulated by the F-box RIFP1, the PUB22/23 E3 ligases and the DWD protein RAE1 (putative substrate receptor of CUL4-DDB1 E3 ligase). Mass spectrometry analysis of ABA receptors expressed in plants has identified different posttranslational modifications. Castillo et al. (2015) identified tyrosine nitration and S-nitrosylation at cysteine residues. Tyrosine nitration reduced receptor activity in PP2C inhibition assays, whereas S-nitrosylated receptors fully inhibited PP2C activity. Additionally, in plants expressing HA-tagged versions of PYR1, PYL4 and PYL8, ubiquitinated lysine residues of these receptors were identified. The inactivation of PYR/PYL/RCAR receptors by tyrosine nitration is likely linked to their degradation because proteins with nitrated tyrosine were also polyubiquitinated. Tyrosine nitration is triggered by the combination of NO with superoxide ions, which suggests a rapid mechanism to inhibit ABA signaling by nitric oxide, in agreement with the ABA-hypersensitive phenotype of NO-deficient plants (Lozano-Juste & Leon, 2010). Finally, phosphorylation is an emerging post-translational modification of ABA receptors that is mediated by different types of kinases, i.e. Target of Rapamycin (TOR), Arabidopsis EL1-like (AEL) casein kinases and Cytosolic ABA Receptor Kinase 1 (CARK1) (Chen et al., 2018; Li et al., 2019; Wang et al., 2018). TOR is a conserved kinase that promotes anabolic functions and growth, and based on studies in mammalian cells, it is known that energy depriving conditions lead to inhibition of the TOR pathway by phosphorylation of regulatory-associated protein of TOR (RAPTOR) (Nukarinen et al., 2016). In plants the balance between stress response and growth is crucial to adapt to changing environment and the SnRK1 pathway (activated in response to energy limitation) inhibits TOR by

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phosphorylation of RAPTOR (Nukarinen et al., 2016). Following a largescale phosphoproteomic study, Wang et al. (2018) uncovered phosphorylation of PYLs at a conserved Ser residue (Ser114 in PYL4, Ser119 in PYL1 and Ser94 in PYL9), which abolishes ABA-dependent inhibition of PP2Cs. This Ser residue is specifically phosphorylated by TOR, which negatively regulates ABA signaling in the absence of stress. Additionally, phosphorylation of ABA receptors by AEL casein kinases leads to their destabilization and suppression of ABA response (Chen et al., 2018). Therefore, in conditions that promote growth, ABA signaling is attenuated by phosphorylation of ABA receptors. In contrast, under stress, ABA receptors and ABA-activated SnRK2s are required for phosphorylation of RAPTOR, triggering inhibition of the TOR pathway (Wang et al., 2018).

5. Revisiting structural biology of ABA receptors and PP2C co-receptors The identification of the core components for ABA signaling fostered studies in Arabidopsis to obtain structural insights into the mechanism of ABA perception, phosphatase inhibition and activation of subfamily III SnRK2s. Thus, it was possible to elucidate in atomic detail the different steps from ABA sensing to SnRK2 activation. Structural studies included the resolution of the apo and ABA-bound forms of the PYR/PYL/RCAR family of ABA receptors (Melcher et al., 2009; Miyazono et al., 2009; Nishimura et al., 2009; Santiago et al., 2009b; Yin et al., 2009), their ABA-bound complexes with the PP2Cs (Dupeux et al., 2011a; Melcher et al., 2009; Miyazono et al., 2009; Yin et al., 2009) and the determination of the SnRK2 structure and its complex with PP2C (Ng et al. 2011; Soon et al., 2012; Yunta, MartinezRipoll, Zhu, & Albert, 2011). These studies showed that the binding of ABA to the ABA receptors produces a structural reorganization that enables the receptor to dock into the PP2C active site to inhibit its activity (Fig. 1B). However, previous results in Arabidopsis and recent results in other plant species also suggest a proactive role of the PP2C as co-receptor and part of the locking mechanism in the ternary receptor-ABA-phosphatase complex (Melcher et al., 2009; Miyazono et al., 2009; Moreno-Alvero et al., 2017). The structures of the PYR/PYL/RCAR receptors display the classical fold of the START/Bet v proteins (Radauer et al., 2008). This consists of a seven stranded anti-parallel beta sheets that oppose to a large C terminal alpha helix, which defines a large central hydrophobic pocket to accommodate ABA. In the ABA-free form, the ABA receptors display a wide

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solvent-exposed empty pocket that is flanked by two conserved loops, named gate and latch (Fig. 2C). To accommodate ABA, the latch loop rearranges to conform the hormone binding site and current models indicate that ABA binding generates a conformational change in the gate loop that folds over the ABA molecule shielding it from the solvent (Fig. 2B). The core of the catalytic domain of PP2Cs consists of a central 10-strand antiparallel b-sandwich flanked by two long a-helices at each side. Additionally, there is a small protruding alpha/beta subdomain that contains the conserved Trp residue and contributes to the locking mechanism. The PP2C active site is located at a cleft between two central b sheets, is capped by the small protruding domain and contains 2e3 atoms of either Mn2þ or Mg2þ ions, which are required for catalysis (Dupeux et al., 2011a; Melcher et al., 2009; Miyazono et al., 2009; Yin et al., 2009). The structures of the receptor-ABA-PP2C complexes show that the gate loop inserts into the PP2C active site and blocks access of the substrate (Fig. 1B). Specifically, a conserved Ser residue of the gate loop (in closed conformation) points toward the metal center, mimicking a phosphatase substrate, and establishes hydrogen bonds with conserved Gly residues of the PP2C active site (Gly180 of ABI1 or Gly246 of HAB1). This interaction with the activesite cleft of the PP2C is further stabilized by the water-mediated network of hydrogen bonds generated from the above mentioned Trp residue. Additionally, Melcher et al. (2009) and Miyazono et al. (2009) showed that insertion of the conserved Trp residue into the ABA binding pocket acts as a locking mechanism, which favors a few additional contacts of the gate and latch loops with the ABA molecule. Indeed, the ABA dissociation constant from the receptor-ABA-PP2C ternary complex is 10e25 times lower (affinity higher) than from the binary receptor-ABA complex (Ma et al., 2009; Santiago et al., 2009a). Thus, ABA receptors in the presence of ABA constitute very effective competitive inhibitors of the physiological substrates of the PP2C, for example the phosphopeptide that represents the activation loop of SnRK2.6 (Melcher et al., 2009). PYR1, PYL1, PYL2 and PYL3 are homodimers in the absence of ABA, but form 1:1 monomeric complexes with the PP2Cs after ABA binding. The dimerization interface overlaps with the PP2C-binding interface of the monomeric receptor (Dupeux et al., 2011b). Therefore, the PP2Cinteracting surface is occluded in the homodimers and receptor dissociation is required for the formation of the ternary complex. Consequently, the ABA perception mechanism of dimeric receptors must include an additional dissociation step after ABA binding. Analysis of the crystal structures of both

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ABA-free and ABA-bound states reveals that the binding of ABA in the dimeric receptors induces an intermonomer swiveling that would favor dissociation of homodimers by a reduction of the interface buried area (Dupeux et al., 2011b; Zhang et al. 2012). An extreme case is the transition from apo-PYL3 cis-homodimers to trans-homodimers upon ligand binding, which facilitates dissociation into monomers (Zhang et al., 2012).

5.1 An updated model to account for the enhancement of ABA binding affinity by PP2C co-receptor It is widely accepted that protein function relies on conformational transitions among different states and requires a balance between stability and flexibility of these conformers (Wilson et al., 2015). Hence, to fully understand the structural basis of ABA sensing, it is required the characterization of the dynamic structural changes along the different steps from ABA-mediated activation of the receptors to PP2C binding. The very first structures of Arabidopsis ABA receptors in complex with ABA and those of the receptorABA-PP2C ternary complexes suggested a two-step mechanism for ABA sensing (Yin et al., 2009). Thus, the hormone binds first to the ABA receptor and this event is followed in second place by PP2C binding (Yin et al., 2009). This model was supported by the structure of ABA-bound receptors, which bury totally the hormone into the ABA-binding pocket, and by assuming the structure of ABA-bound receptors is hardly modified by PP2C binding. However, this model does not account for the enhancement of the ABA binding affinity by the PP2C, which suggests a cooperative effect of the phosphatase as necessary co-receptor for high-affinity ABA binding. As a result from structural studies performed by two independent groups with ABA receptors from different plant species, i.e. Citrus sinensis (sweet orange) and Solanum lycopersicum (tomato) (Moreno-Alvero et al., 2017) and Fetusca elata (turfgrass) (Ren et al. 2017), it was identified a novel receptor intermediate where the gate loop remains open after ABA binding (Fig. 6). These structures do not conform to the dogma established in Arabidopsis, which states that ABA binding and gate/latch closure are coupled events leading to the activation of the receptor (Melcher et al., 2009; Weiner, Peterson, Volkman, & Cutler, 2010). Recently molecular dynamics simulations of ABA binding to PYL5 and PYL10 have revealed that the gating loop is still in equilibrium between the open and closed conformations after ABA is bound (Shukla, Zhao, & Shukla, 2019). Therefore, molecular dynamics studies and receptor structures where ligand binding does not induce gate closure suggest that PP2Cs might drive the transition to a

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Fig. 6 ABA binding does not necessarily induce gate closure in the absence of the PP2C. ABA-bound crop receptors display a structural intermediate that shows gateopen conformation, which is unproductive for PP2C binding. (A) Superimposition of the ABA binding site in apo-Citrus sinensis CsPYL1 (green), the binary complex of CsPYL1 with ABA showing the gate loop in the open conformation (orange) and the CsPYL1ABA-HAB1 ternary complex with the gate loop in the closed/productive conformation (red). (B) Superimposition of ABA-bound binary structures of AtPYR1 (grey, gate closed), SlPYL1 (orange, gate open), CsPYL1 (green, gate open), FePYR1 (magenta, gate open) and the CsPYL1-ABA-HAB1 ternary complex (red, gate closed).

productive gate-closed conformation of the receptors (Moreno-Alvero et al., 2017; Shukla et al., 2019). Thus, the observed increase of the ABA binding affinity in the receptor-ABA-phosphatase ternary complex might be a consequence of the PP2C mediated shifting of the equilibrium between the ABA-bound receptor toward the formation of productive complex with the PP2C (Moreno-Alvero et al., 2017) (Fig. 7A). This model supports that the high-affinity system for ABA binding is the receptor-ABA-PP2C complex (Ma et al. 2009; Santiago et al., 2009a).

5.2 Conformational changes of receptor gate and latch loops and proactive role of PP2Cs The precise structural information about the conformational changes that occur in the receptor prior and upon association with ABA is of great interest to support structure-based drug design efforts. The inspection of the socalled latch-open conformation in apo forms of AtPYL1 and AtPYL2 (Melcher et al., 2009; Yin et al., 2009) reveals that residues of the conserved Glu/Asp-His-Arg motif at the latch (Fig. 3) produce steric hindrances that make the ABA binding cavity inaccessible to the hormone, and

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Fig. 7 PP2C has a proactive role in the selection of latch-closed gate-closed intermediates for the formation of ternary complexes. (A) Scheme for ABA perception where the PP2C drives the equilibrium to the formation of receptor-ABA-phosphatase ternary complexes that bind ABA with nM affinity. (B) The latch-open conformation shows steric hindrance for ABA binding. The superimposition of the structures of apo-CsPYL1 (latch open, green) and the binary complex with ABA (latch closed, orange) illustrates the transition from open to closed conformation of the latch. The open conformation hinders ABA binding whereas the closed conformation is appropriate for ABA binding and required to properly arrange the ABA-binding pocket. Arg, His and Glu residues form the latch and are represented in a stick mode. (C) The superimposition of the structures of CsPYL1-ABA binary complex (gate open, orange) and CsPYL1-ABA-HAB1 ternary complex (gate closed, red) illustrates the transition from open to closed conformation of the gate. HAB1 (green cyan) stabilizes the closed conformation of the latch and the gate. The water-mediated hydrogen bond network linking ABA (yellow), latch, gate and PP2C is highlighted. Residues involved in this network and His from the latch are displayed in stick mode. The arrows illustrate the structural rearrangements of latch and gate loops upon ABA and PP2C binding.

consequently, ABA binding requires a structural reorganization of these residues (Fig. 7B). The analysis of the crystal structures available in the literature for ABA-free forms of Arabidopsis ABA receptors shows that some of them, i.e. PYR1, PYL1 and PYL2, display a variable latch-open conformation incompetent for ABA binding, whereas the structures of PYL3, PYL5 and PYL10 adopt a latch-closed conformation competent for ABA binding. This suggests that ABA-free forms of ABA receptors can adopt both

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conformations and, consequently, ABA is not necessary to induce latch closure. Instead, we suggest the hormone will selectively bind to the closed conformation in a classical conformational selection mechanism (Vogt, Pozzi, Chen, & Di Cera, 2014) (Fig. 7A). Next, once ABA occupies the binding pocket, the receptor should undergo a structural rearrangement leading to gate closing for a successful PP2C interaction. The resolution of ABA-bound receptors displaying either gate-open or gate-closed conformations supports a model in which PP2C shifts the gate-open to gate-closed equilibrium of the binary complex toward the formation of the ternary complex (Fig. 7A and C). This information reveals a structural landscape that includes a variety of diverse intermediates, which were not obvious to be predicted, and makes it possible the design of ABA antagonists that stabilize dead-end conformers or ABA agonists that promote the transition from inactive to active receptor conformations. Additionally, potent ABA antagonists have been developed that abolish the interaction of the PP2C with the ABA’s ketone group through C40 -modified ABA analogues, which were designed to prevent the insertion of the conserved Trp residue of the PP2C into the tunnel adjacent to the ketone group in receptor-ABA-PP2C complexes (Takeuchi et al., 2018).

References Antoni, R., Gonzalez-Guzman, M., Rodriguez, L., Rodrigues, A., Pizzio, G. A., & Rodriguez, P. L. (2012). Selective inhibition of clade a phosphatases type 2C by PYR/PYL/RCAR abscisic acid receptors. Plant Physiology, 158, 970e980. Antoni, R., Gonzalez-Guzman, M., Rodriguez, L., Peirats-Llobet, M., Pizzio, G. A., Fernandez, M. A., et al. (2013). PYRABACTIN RESISTANCE1-LIKE8 plays an important role for the regulation of abscisic acid signaling in root. Plant Physiology, 161, 931e941. Arbona, V., Zandalinas, S. I., Manzi, M., Gonzalez-Guzman, M., Rodriguez, P. L., & Gomez-Cadenas, A. (2017). Depletion of abscisic acid levels in roots of flooded Carrizo citrange (Poncirus trifoliata L. Raf. x Citrus sinensis L. Osb.) plants is a stress-specific response associated to the differential expression of PYR/PYL/RCAR receptors. Plant Molecular Biology, 93, 623e640. Bai, G., Yang, D. H., Zhao, Y., Ha, S., Yang, F., Ma, J., et al. (2013). Interactions between soybean ABA receptors and type 2C protein phosphatases. Plant Molecular Biology, 83, 651e664. Belda-Palazon, B., Rodriguez, L., Fernandez, M. A., Castillo, M. C., Anderson, E. A., Gao, C., et al. (2016). FYVE1/FREE1 interacts with the PYL4 ABA receptor and mediates its delivery to the vacuolar degradation pathway. The Plant Cell Online, 28, 2291e 2311. Belda-Palazon, B., Gonzalez-Garcia, M. P., Lozano-Juste, J., Coego, A., Antoni, R., Julian, J., et al. (2018). PYL8 mediates ABA perception in the root through non-cellautonomous and ligand-stabilization-based mechanisms. Proceedings of the National Academy of Sciences of the United States of America, 115, E11857eE11863.

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