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. Author manuscript; available in PMC: 2011 Oct 9.
Published in final edited form as: Curr Opin Plant Biol. 2010 Oct 9;13(5):495–502. doi: 10.1016/j.pbi.2010.09.007

Structural and Functional Insights into Core ABA Signaling

Joshua J Weiner 1, Francis C Peterson 1, Brian F Volkman 1, Sean R Cutler 2,✉
PMCID: PMC2971662  NIHMSID: NIHMS240762  PMID: 20934900

Summary

A series of papers in the last year reported major advances in our understanding of ABA signaling: the identification of soluble ABA receptors, the elucidation of a core ABA signaling pathway and structural insights into the mechanism of ABA perception and signaling. Here we summarize these advances, which have shown in atomic resolution that the ABA receptors PYR1, PYL1 and PYL2 function as allosteric switches that inhibit type 2C protein phosphatases (PP2Cs) in response to ABA. These receptors function at the apex of a core signaling pathway that regulates ABA responses by controlling SnRK2 kinase activity and the phosphorylation of downstream target proteins such as ABFs, which control nuclear responses, and the ion channel SLAC1, which mediates electrophysiological responses to ABA.

Introduction

Plants synthesize a diverse array of diffusible hormonal signals that work in concert to integrate growth, development and cellular physiology to environmental cues [1]. A key abiotic stress signal is the carotenoid derived molecule abscisic acid (ABA). Originally discovered in the 1960s, physiological, biochemical and genetic analyses have uncovered roles for ABA in numerous stress and developmental processes. Several reviews of ABA biosynthesis and signaling have been published recently[2–5]. Since May 2009, an unprecedented number of advances have occurred, including the discovery of a soluble ABA receptor family and the assembly of numerous pieces of the ABA signaling puzzle into a cohesive “core” pathway. We note that the last year also witnessed important advances in identifying ABA transporters [6**,7**], and the demonstration that ABA catabolism is coupled to high-humidity stress [8*], neither of which can be covered due to space limitations.

New ABA Receptors That Regulate PP2C Activity

Several ABA binding proteins have been described and implicated in ABA signaling including the plasma membrane localized GPCR-type G protein (GTGs) and the chloroplast localized Magnesium Cheletase subunit ChlH (For review see [3)]); given the flurry of activity on soluble receptors, we do not cover these proteins here. The PYR/PYL/RCAR family of ABA receptors was identified by 4 separate research groups [9**, 10**, 11**, 12**], and is comprised of a 14-member gene family in Arabidopsis [1–4]; of which at least 13 function in ABA perception [13**]. Three groups independently identified different members of this new receptor family by virtue of their physical interactions with clade A PP2Cs in yeast two hybrid [10**,11**] or immunoprecipitation experiments [12**]. The Arabidopsis genome encodes 76 PP2Cs. One subfamily of 9 “clade A” PP2Cs, which includes ABA INSENSITIVE 1 (ABI1), ABI2 and HOMOLOG OF ABI1 (HAB1) [14], are well characterized negative regulators of ABA signaling (reviewed in [3]). Taking a different approach, we identified pyrabactin, a selective ABA agonist and determined by genetic analysis that PYRABACTIN RESISTANCE 1 (PYR1) is necessary for pyrabactin action in vivo [9**]. A quadruple pyr1/pyl1/pyl2/pyl4 mutant shows defects in several ABA responses, including ABA-induced gene expression, ABA-mediated SnRK2 kinase activation [9**] and ABA-promoted guard cell closure [12**]. Transgenic plants overexpressing REGULATORY COMPONENT OF ABA RECEPTOR 1 (RCAR1/PYL9) are hypersensitive to ABA-promoted guard cell closure [10**] and overexpression of PYR1-LIKE 5 (PYL5) confers drought tolerance on transgenic Arabidopsis plants [11], which validates the new receptor family as a target for manipulating abiotic stress tolerance. PYR/PYL proteins bind ABA directly, and interestingly, their affinity for ABA is stimulated ~10-fold by the presence of PP2Cs [10**,11**,15**], a point we return to later. The single PYR/PYL mutants characterized to date do not possess ABA phenotypes [9**]; this redundancy likely explains why the gene family evaded detection by earlier genetic screens (reviewed in [16]). The selectivity of pyrabactin, a non-natural agonist, for the receptor PYR1 enabled the genetic redundancy observed for ABA to be bypassed, which illustrates the power of synthetic ligands for dissecting plant signaling networks [9**,17,18*].

The physical interactions of PYR1 and its 4 closest relatives (PYL1 – PYL4), with PP2Cs (ABI1, ABI2 and HAB1) are regulated by ABA, as measured using yeast two hybrid assays [9]. The interactions between PYLs 5 –12 and PP2Cs occur in the absence of exogenously added ABA in yeast two hybrid assays [9**, 10**, 11**], a point that requires further investigation. Upon binding ABA, PYR/PYL proteins inhibit the phosphatase activity of multiple clade A PP2Cs, with IC50 values measured in the range of 18 – 390 nM (+)-ABA, depending on the PYR/PYL-PP2C pair examined [9**, 10**, 11**,15*]. Since 9 clade A PP2Cs and 14 PYR/PYL proteins are encoded by the Arabidopsis genome, 126 PYR/PYL-PP2C combinations could potentially form. While the in vivo significance of this remains to be demonstrated, reports of differences in ABA sensitivity between different combinations are suggestive that the combinatorial interactions between receptors and PP2Cs enable a tunable response to stress signaling [10**,11**,15*].

Structural Insights into Receptor Function

The structures of PYR1, PYL1 and PYL2 in apo, ABA bound and ABI1 or HAB1 complexed forms were reported in late 2009 [19**, 20**, 21**, 22**, 23**]; the conclusions from 5 studies are in general agreement and here we present a consensus view of these studies. The PYR/PYL receptors are members of the START-domain superfamily of lipophilic ligand binding proteins [24–26], which exhibit a “helix-grip” structure, in which N-terminal and C-terminal α helices enclose a seven-stranded β sheet to create a ligand-binding pocket [24]. In the published PYR/PYL structures, two highly conserved loops flank the ligand binding pocket: the SGLPA “gate” loop (also called the “cap”, or CL2 loop) between the β3 and β4 strands and the HRL “latch” loop (also called the lock or CL3 loop) between the β5 and β6 strands. The gate and latch loops undergo significant conformational rearrangement upon ABA binding, which is the primary allosteric mechanism that underlies information transfer (Figure 1). Two mutations in PYR1’s gate (P88S) and latch (H115A) abolish ABA-mediated PP2C inhibition without disrupting ABA binding (as measured using NMR methods) [9**,19**], showing that ABA binding and PP2C inhibition can be uncoupled.

Figure 1. Abscisic acid mediated formation of thePYR/PYL-ABA-PP2C ternary complex.

Figure 1

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PYR/PYL proteins contain a central hydrophobic pocket that is flanked by two mobile loops called the “gate” and “latch”, shown in red. ABA binding triggers closure of the gate, this in turn creates an interaction surface for binding to the PP2Cs, which dock onto the closed form of PYR/PYL proteins. The site of docking is adjacent to the magnesium ion containing active site of PP2Cs (shown in magenta). A conserved tryptophan in the PP2Cs, called the “lock” inserts between the gate and latch and makes a water mediated contact to ABA. Docking of the PYR/PYL proteins into the PP2C active site inhibits PP2C activity by occluding access of target proteins. This figure was made using the coordinates for apo-PYL2, ABA-bound PYL2 and the PYL2-ABA-HAB1 ternary complex (3KAZ, 3KBO 3KB3), described in (**19).

The binding of ABA to PYR/PYL proteins is mediated by a combination of hydrogen bonds and hydrophobic interactions, including direct contacts between ABA and residues in the gate and latch, which stabilizes their closure. A conserved lysine (corresponding to K59 in PYR1, K86 in PYL1, K64 in PYL2) forms a charge interaction with the acidic head group of ABA, which explains the critical necessity of a COOH noted in ABA structure activity relationships (for review see [3,27]). Many of the residues that make contacts to ABA are highly conserved between receptor proteins; however subtle sequence variation in ABA contacting residues does exist. Due to space limitations, we cannot cover recently published structural data for PYR/PYL-pyrabactin complexes [28*, 29**, 30**, 31**], but we note that sequence variation in pocket-lining residues contributes to differences in ligand sensitivity between receptor family members [29**, 30**].

Molecular modeling predicted[19**] and ternary structures confirmed [19**, 22**, 23**] that the altered protein surface created by the motion of gate and latch residues in response to ABA facilitates PP2C docking on to PYR/PYL receptors. ABA-bound PYR/PYL receptors are able to bind several clade A PP2Cs and inhibit their phosphatase activity in vitro [9**, 10**, 11**]. The gate loop of ABA-bound receptors is positioned with its centrally located SGLPA serine inserted into the PP2C catalytic site and apparently acts as a high affinity product mimic to inhibit the enzyme, by blocking normal substrate access (Figure 2); direct measurements have shown that PYL2 acts as a competitive inhibitor of HAB1’s phosphatase activity[19**]. Thus, ABA ultimately inhibits PP2C activity by inducing a conformational change in PYR/PYL proteins that converts them into PP2C inhibitors. Interestingly, clade A PP2Cs interact with PYR/PYL proteins via a small recognition loop that contains a conserved tryptophan (W300 in ABI1) that has been called the “lock” [19**] (Figure 2). This tryptophan inserts between the gate and latch loops [19**,22**,23**] and its indole NH makes a water-mediated hydrogen bond to ABA’s ketone group. Mutation of the tryptophan lock residue abolishes ABA-PYR/PYL-mediated PP2C inhibition [19**,23**]. With the exception of AHG1, the tryptophan lock residue is present in all clade A PP2Cs (Figure 3) and missing in other plant PP2Cs, suggesting that PYR/PYL regulation is restricted to clade A PP2Cs.

Figure 2. The SGLPA gate docks into the PP2C active site.

Figure 2

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Serine of the SGLPA gate loop inserts adjacent to the PP2C active site, acting as a high affinity product-mimic.

Figure 3. The tryptophan lock is part of a plant specific recognition loop in clade A PP2Cs.

Figure 3

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ABI1 and HAB1 dock onto PYR/PYL proteins and insert their conserved tryptophan lock residues between the gate and latch. This recognition module is absent from human PP2C structures and therefore a plant specific modulation of PP2C. Eight of the 9 clade A PP2Cs contain the tryptophan lock residue and it is absent from other plant PP2Cs.

The tryptophan lock’s interaction with ABA’s ketone and enhancement of ABA binding affinity by PP2Cs has led to some discussion as to whether PYR/PYL proteins are best described as co-receptors rather than receptors [2,4,19**]. Since over 20 residues in the PYR/PYL proteins make direct or water-mediated contacts with ABA, and ABA is buried within the ligand-binding cavity of PYR/PYL proteins [20**, 21**, 22**, 23**], the structures imply that the primary site of ABA recognition is by the PYR/PYL receptors. Based on current structural data, the primary basis for the enhancements in ABA binding stimulated by PP2Cs occurs by PP2C-mediated stabilization of the closed form of the receptor [11**,19**,22**,23**], which is expected to lower Kd by lowering the Koff for ABA. Thus, the structures provide a rationalization for the ~10 fold increases in ABA affinity provided by PP2Cs and suggest that PYR/PYL proteins participate as the primary sites for ABA and pyrabactin recognition.

PYR1, PYL1, and PYL2 form homodimers in their ligand-free states, but PYL1 and PYL2 bind PP2Cs at 1:1 stoichiometry in the presence of ABA [19**,22**,23**]. Additionally, most of the residues that make contact in the PYL2 homodimer are also involved in binding to the PP2C in the ternary ABA-bound complex [22**], implying that a receptor dimer dissociation step occurs prior to PP2C binding [22**]. Importantly, PYR1 has been shown to form a dimer in vivo [21**], suggesting that dimer breaking in response to ABA is biologically relevant, at least for PYR1. Both Melcher et al. [19**] and Yin et al. [22**] have noted that ABA binding reduces the buried surface area between PYL protomers, which is also supported by small-angle X-ray scattering measurements made in solution [21**]. Furthermore, Yin et al. have suggested dimer breaking may be initiated by tryptophan lock of the PP2C inserting between the gate and latch loops of ABA bound PYR/PYL proteins [22**]. Elucidating the mechanism and functional relevance of dimer breaking will likely be an important line of future investigation.

Since PP2Cs are conserved throughout eukaryotes and have no known direct protein regulators (besides PYR/PYL proteins), it is interesting to ask if the structural and functional insights from plants illuminate mechanisms of PP2C regulation outside of plants? Human PP2CA aligns very closely with ABI1 (Figure 3) and inspection of the aligned structures reveals that the human PP2CA does not contain the recognition loop that ABI1 and HAB1 use to dock onto ABA-bound PYL proteins (Figure 3). Thus, the recognition loop and conserved tryptophan lock appear to be a plant specific innovation exploited in selective regulation of clade A PP2Cs. However, it will be interesting to determine if PP2C inhibition by product-mimics unrelated to PYR/PYL proteins is exploited in the regulation of other PP2Cs.

Elucidation of The Core ABA Response Pathway

The structural studies have revealed how ABA binding to PYR/PYL receptor proteins leads to PP2C inhibition, but how is this event then conveyed to other outputs? A key clue came from the observation that SnRK2 kinases are not properly activated by ABA in the pyr1/pyl1/pyl2/pyl4 quadruple mutant [9**] and this suggested a model for regulation of SnRK2 kinase activity by the PYR/PYL-PP2Cs [9**], which has been validated and greatly extended by two seminal studies described below [13**,31**].

Members of the plant SnRK2 family were originally identified in wheat as an ABA induced kinase transcript [32], and in Vicia faba as a rapid ABA-activated kinase activity [33]. The participation of SnRK2s in ABA signaling has since been established in many species [33–35] and loss-of-function alleles of OPEN STOMATA 1 (OST1; also known as SnRK2.6 or SRK2E) demonstrated the key role of the SnRK2s in vivo [36]. The Arabidopsis genome encodes 10 SnRK2s, of which OST1 and its two closest relatives (SnRK2.2 and 2.3) participate in ABA signaling. Triple mutants lacking these three kinases are deficient in almost all ABA responses, demonstrating the centrality of these kinases to ABA signaling [37*, 38*, 39*]. Active, phosphorylated OST1 immunoprecipitated from plants can be dephoshphorylated by recombinant ABI1, which in turn reduces OST1’s kinase activity [31**]. The mechanism of SnRK2 activation in vivo may be mediated by autophosphorylation [40], but this point is currently unresolved. In vitro, OST1 can autophosphorylate on at least 5 sites [40], but it appears that a single residue, Ser-175, is critical for OST1 kinase activity. Serine 175 is located within the SnRK2 activation loop, proximal to the kinase catalytic site, and the mutation S175A disrupts OST1 autoactivation and cannot complement an ost1-1 mutant in planta [40]. LC-MS studies suggest that the activation loop (and Ser 175 inside this loop) is both phosphorylated in vivo by ABA stimulation and directly dephosphorylated by ABI1 [31]. Coupled to experiments documenting dephosphorylation and concomitant deactivation of OST1 kinase activity by HAB1 in vitro [41*], it is now well established that clade A PP2Cs directly inhibit SnRK2 kinase activity by dephosphorylating them. Thus, ABA pathway activation leads to the accumulation of active and phosphorylated SnRK2s via PP2C inhibition. Once activated, the SnRK2s are poised to directly phosphorylate numerous target proteins involved in ABA responses, including transcription factors that bind to abscisic acid-responsive promoter elements (ABREs) called ABFs (for ABRE-binding factors) [42,43]. These bZIP-class transcription factors are direct substrates of SnRK2 kinases, as documented in several plant species [44–46].

Two groups [13**,31**] have documented the sufficiency of the core PYR/PYL-PP2C-SnRK2 pathway for mediating an ABA response. In one set of experiments, recombinant PYR1, ABI1 and OST1 (immunoprecipitated from ABA stimulated plants) were sufficient for eliciting ABA-mediated OST1 kinase activation, as measured using phosphorylation of a histone substrate [31**], or in independent experiments by ABA-mediated phosphorylation of the ABRE-binding transcription factor ABF2 [13**]. Since the phosphorylation of ABFs by SnRK2s is critical for their ability to activate ABA-mediated gene transcription [45,47], the recent reconstitution experiments demonstrate that the core pathway provides a minimal set of proteins for linking ABA perception to a nuclear output (Figure 4).

Figure 4.

Figure 4

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The core PYR/PYL —| PP2C —| SnRK2 signaling pathway. In the absence of ABA, PP2C activity is high, and the PP2Cs prevent accumulation of phosphorylated SnRK2 kinases by directly dephosphorylating them. In the presence of ABA, PYR/PYL proteins bind to and inhibit PP2Cs, which leads to the accumulation of phosphorylated and active SnRK2s, possibly by auto-phosphorylation, however this is not currently clear. Once activated by phosphorylation, the SnRK2s can then directly phosphorylate downstream targets, such as the ABFs, SLAC, KAT1, and probably other proteins. Phosphorylation of ABFs is necessary for their ability to activate transcription. OST1 stimulates SLAC channel activity in Xenopus experiments and has been hypothesized to inhibit KAT1 channel activity.

What about the diverse non-transcriptional responses triggered by ABA? Key advances in the last year include the demonstration that OST1 phosphorylates and activates the anion channel SLAC1 expressed in Xenopus oocytes [48**, 49**, 50**] and phosphorylates and deactivates the potassium channel KAT1 when expressed in S. cerevisiae [51*]. These observations provide an appealing mechanism for ABA-mediated control of guard cell physiology by the core pathway, which is consistent with observations that the pyr1/pyl1/pyl2/pyl4 quadruple mutant posses defects in ABA promoted guard cell closure [12**]. Moreover, a recent report has demonstrated OST1-mediated phosphorylation of the Arabidopsis RESPIRATORY BURST OXIDASE SUBUNIT HOMOLOG F (RbohF) in in vitro assays [52*], which suggests a mechanism for ABA-mediated ROS production by the core pathway; however, further work will be needed to investigate the in vivo significance of these observations.

While the core pathway has impressive explanatory power, much work remains to be done towards understanding whether and how numerous well-characterized second messengers, such as Ca++ and NO are integrated with ABA signaling. Towards this, an important paper has shown that the calcium dependent kinase CPK23 can phosphorylate the N-terminus of the anion channel SLAC1 in vitro, and that this response is antagonized by ABI1 [53**]. Moreover, in Xenopus oocytes expressing SLAC1, the introduction of CPK23 (and other CPKs) enhances SLAC1 channel activity [53**]. The addition of ABA to in vitro reactions containing recombinant PYL9, ABI1 and CPK23 promotes phosphorylation of SLAC1’s N-terminus [53**]. Furthermore, ABI1 inhibits CPK23 kinase activity in vitro, which suggests that CPK23 kinase activity is regulated directly by the PYR/PYL-PP2C signaling system. These results suggest that the core pathway may bifurcate at the PP2Cs to regulate both SnRKs and CPKs. While exciting these conclusions were drawn largely from in vitro experiments using recombinant proteins and will benefit from further investigation in planta.

Conclusions

A burst of activity has led to enormous advances in our understanding of ABA signaling. Structural data show that ABA binds within the ligand binding pockets of PYR1, PYL1 and PYL2 and in so doing triggers a conformational change in the gate and latch loops of PYR/PYL receptors. The ABA bound receptors interact with clade A PP2Cs and inhibit their activity by docking within the PP2C active site. This in turn allows the accumulation of active, phosphorylated SnRK2s, and possibly CPKs, which can then phosphorylate and modulate the activity of downstream factors including ABFs, SLAC1, KAT1 and Rboh1. The core pathway provides a powerful starting point for developing an integrated picture of ABA action at the mechanistic level.

Footnotes

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