A thermodynamic switch modulates abscisic acid receptor sensitivity

Abscisic acid (ABA) is a key hormone regulating plant growth, development and the response to biotic and abiotic stress. ABA binding to pyrabactin resistance (PYR)/PYR1-like (PYL)/Regulatory Component of Abscisic acid Receptor (RCAR) intracellular receptors promotes the formation of stable complexes with certain protein phosphatases type 2C (PP2Cs), leading to the activation of ABA signalling. The PYR/PYL/RCAR family contains 14 genes in Arabidopsis and is currently the largest plant hormone receptor family known; however, it is unclear what functional differentiation exists among receptors. Here, we identify two distinct classes of receptors, dimeric and monomeric, with different intrinsic affinities for ABA and whose differential properties are determined by the oligomeric state of their apo forms. Moreover, we find a residue in PYR1, H60, that is variable between family members and plays a key role in determining oligomeric state. In silico modelling of the ABA activation pathway reveals that monomeric receptors have a competitive advantage for binding to ABA and PP2Cs. This work illustrates how receptor oligomerization can modulate hormonal responses and more generally, the sensitivity of a ligand-dependent signalling system.     

Identification of an important site for function of the type 2C protein phosphatase ABI2 in abscisic acid signalling in Arabidopsis

It is known that the clade A protein phosphatase 2Cs (PP2Cs), including ABI1 and ABI2 and other PP2C members, are key players that function directly downstream of the PYR/PYL/RCAR abscisic acid (ABA) receptors. Here, identification of a crucial site for function of ABI2 protein phosphatase in ABA signalling is reported. It was observed that a calcium-dependent protein kinase (CDPK) phosphorylation site-like motif (CPL) in the ABI2 molecule is required for the interactions of ABI2 with the two members of the ABA receptors PYL5 and PYL9 and with a downstream protein kinase SnRK2.6, and for the catalytic activity of ABI2 in vitro, as well as for the response of ABI2 to the ABA receptors PYL5/PYL9 in relation to the ABA receptor-induced inhibition of the ABI2 phosphatase activity. Further, genetic evidence was provided to demonstrate that this CPL is required for the function of ABI2 to mediate ABA signalling. These data reveal that this CPL is an important site necessary for both the phosphatase activity of ABI2 and the functional interaction between ABI2 and PYL5/9 ABA receptors, providing new information to understand primary events of ABA signal transduction.     

PYR/PYL/RCAR family members are major in‐vivo ABI1 protein phosphatase 2C‐interacting proteins in Arabidopsis

Abscisic acid (ABA) mediates resistance to abiotic stress and controls developmental processes in plants. The group‐A PP2Cs, of which ABI1 is the prototypical member, are protein phosphatases that play critical roles as negative regulators very early in ABA signal transduction. Because redundancy is thought to limit the genetic dissection of early ABA signalling, to identify redundant and early ABA signalling proteins, we pursued a proteomics approach. We generated YFP‐tagged ABI1 Arabidopsis expression lines and identified in vivo ABI1‐interacting proteins by mass‐spectrometric analyses of ABI1 complexes. Known ABA signalling components were isolated including SnRK2 protein kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA‐signalling kinases OST1, SnRK2.2 and SnRK2.3 in plants. Interestingly, the most robust in planta ABI1‐interacting proteins in all LC‐MS/MS experiments were nine of the 14 PYR/PYL/RCAR proteins, which were recently reported as ABA‐binding signal transduction proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1 in Arabidopsis. ABI1–PYR1 interaction was stimulated within 5 min of ABA treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 co‐immunoprecipitated equally well in the presence and absence of ABA. To investigate the biological relevance of the PYR/PYLs, we analysed pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in ABA‐induced stomatal closure and ABA‐inhibition of stomatal opening. These findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR family members in Arabidopsis, that PYR1–ABI1 interaction is rapidly stimulated by ABA in Arabidopsis and indicate new SnRK2 kinase‐PYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCAR‐mediated ABA signalling.     

ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA

Hormone‐ and stress‐induced shuttling of signaling or regulatory proteins is an important cellular mechanism to modulate hormone signaling and cope with abiotic stress. Hormone‐induced ubiquitination plays a crucial role to determine the half‐life of key negative regulators of hormone signaling. For ABA signaling, the degradation of clade‐A PP 2Cs, such as PP 2 CA or ABI 1, is a complementary mechanism to PYR / PYL / RCAR ‐mediated inhibition of PP 2C activity. ABA promotes the degradation of PP 2 CA through the RGLG 1 E3 ligase, although it is not known how ABA enhances the interaction of RGLG 1 with PP 2 CA given that they are predominantly found in the plasma membrane and the nucleus, respectively. We demonstrate that ABA modifies the subcellular localization of RGLG 1 and promotes nuclear interaction with PP 2 CA . We found RGLG 1 is myristoylated in vivo , which facilitates its attachment to the plasma membrane. ABA inhibits the myristoylation of RGLG 1 through the downregulation of N‐myristoyltransferase 1 ( NMT 1 ) and promotes nuclear translocation of RGLG 1 in a cycloheximide‐insensitive manner. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP 2 CA protein levels and the formation of RGLG 1–receptor–phosphatase complexes. We show that RGLG 1 ^Gly2Ala mutated at the N‐terminal myristoylation site shows constitutive nuclear localization and causes an enhanced response to ABA and salt or osmotic stress. RGLG 1/5 can interact with certain monomeric ABA receptors, which facilitates the formation of nuclear complexes such as RGLG 1– PP 2 CA – PYL 8. In summary, we provide evidence that an E3 ligase can dynamically relocalize in response to both ABA and increased levels of its target, which reveals a mechanism to explain how ABA enhances RGLG 1– PP 2 CA interaction and hence PP 2 CA degradation.     

Thirsty plants and beyond: structural mechanisms of abscisic acid perception and signaling

Abscisic acid (ABA) is a plant hormone with important functions in stress protection and physiology. Recently, the PYR/PYL/RCAR family of intracellular ABA receptors was identified. These receptors directly link ABA perception to a canonical ABA signaling pathway, in which ABA-bound receptors bind and inhibit type 2C phosphatases. High resolution crystal structures of members of this family have been solved in all relevant states: as apo receptors, bound to ABA, and as receptor-ABA-phosphatase complexes. Together, these structures provide a detailed gate-latch-lock mechanism of ABA recognition, receptor-PP2C interaction, and inhibition of the PP2C phosphatase activity and provide a basis for the design of synthetic ABA agonists for stress protection of crop plants.     

Catalytic mechanism and kinase interactions of ABA-signaling PP2C phosphatases

Abscisic acid (ABA) is an essential hormone that controls plant growth, development and responses to abiotic stresses. ABA signaling is mediated by type 2C protein phosphatases (PP2Cs), including HAB1 and ABI2, which inhibit stress-activated SnRK2 kinases and whose activity is regulated by ABA and ABA receptors. Based on biochemical data and our previously determined crystal structures of ABI2 and the SnRK2.6–HAB1 complex, we present the catalytic mechanism of PP2C and provide new insight into PP2C–SnRK2 interactions and possible roles of other SnRK2 kinases in ABA signaling.     

Protein Phosphatases 2C Regulate the Activation of the Snf1-Related Kinase OST1 by Abscisic Acid in Arabidopsis

The plant hormone abscisic acid (ABA) orchestrates plant adaptive responses to a variety of stresses, including drought. This signaling pathway is regulated by reversible protein phosphorylation, and genetic evidence demonstrated that several related protein phosphatases 2C (PP2Cs) are negative regulators of this pathway in Arabidopsis thaliana. Here, we developed a protein phosphatase profiling strategy to define the substrate preferences of the HAB1 PP2C implicated in ABA signaling and used these data to screen for putative substrates. Interestingly, this analysis designated the activation loop of the ABA activated kinase OST1, related to Snf1 and AMPK kinases, as a putative HAB1 substrate. We experimentally demonstrated that HAB1 dephosphorylates and deactivates OST1 in vitro. Furthermore, HAB1 and the related PP2Cs ABI1 and ABI2 interact with OST1 in vivo, and mutations in the corresponding genes strongly affect OST1 activation by ABA. Our results provide evidence that PP2Cs are directly implicated in the ABA-dependent activation of OST1 and further suggest that the activation mechanism of AMPK/Snf1-related kinases through the inhibition of regulating PP2Cs is conserved from plants to human.     

Enhancement of abscisic acid sensitivity and reduction of water consumption in Arabidopsis by combined inactivation of the protein phosphatases type 2C ABI1 and HAB1

Abscisic acid (ABA) plays a key role in plant responses to abiotic stress, particularly drought stress. A wide number of ABA-hypersensitive mutants is known, however, only a few of them resist/avoid drought stress. In this work we have generated ABA-hypersensitive drought-avoidant mutants by simultaneous inactivation of two negative regulators of ABA signaling, i.e. the protein phosphatases type 2C (PP2Cs) ABA-INSENSITIVE1 (ABI1) and HYPERSENSITIVE TO ABA1 (HAB1). Two new recessive loss-of-function alleles of ABI1, abi1-2 and abi1-3, were identified in an Arabidopsis (Arabidopsis thaliana) T-DNA collection. These mutants showed enhanced responses to ABA both in seed and vegetative tissues, but only a limited effect on plant drought avoidance. In contrast, generation of double hab1-1 abi1-2 and hab1-1 abi1-3 mutants strongly increased plant responsiveness to ABA. Thus, both hab1-1 abi1-2 and hab1-1 abi1-3 were particularly sensitive to ABA-mediated inhibition of seed germination. Additionally, vegetative responses to ABA were reinforced in the double mutants, which showed a strong hypersensitivity to ABA in growth assays, stomatal closure, and induction of ABA-responsive genes. Transpirational water loss under drought conditions was noticeably reduced in the double mutants as compared to single parental mutants, which resulted in reduced water consumption of whole plants. Taken together, these results reveal cooperative negative regulation of ABA signaling by ABI1 and HAB1 and suggest that fine tuning of ABA signaling can be attained through combined action of PP2Cs. Finally, these results suggest that combined inactivation of specific PP2Cs involved in ABA signaling could provide an approach for improving crop performance under drought stress conditions.     

Insulin/insulin-like growth factor (IGF) stimulation abrogates an association between a deubiquitinating enzyme USP7 and insulin receptor substrates (IRSs) followed by proteasomal degradation of IRSs

Abscisic acid (ABA) is one of the most essential phytohormones, and plays an important role in growth and development regulation, as well as in stress responses. The PYR/PYL/RCAR family (PYL for short)—comprised of 14 proteins in Arabidopsis—was recently identified as soluble ABA receptors that function in the perception and transduction of ABA signaling. In this work, the crystal structures of PYL10 were determined in the apo- and ABA-bound states, with respective resolutions of 3.0 and 2.7 Å. Surprisingly, a closed CL2 conformation was observed in the apo–PYL10 structure, which was different from a previously reported open CL2 conformation. A putative two-conformation dynamical equilibrium model was proposed to explain PYL10’s constitutive binding to PP2Cs in the apo-state and its increased PP2C binding ability in the ABA-bound state.     

Selective inhibition of clade A phosphatases type 2C by PYR/PYL/RCAR abscisic acid receptors

Clade A protein phosphatases type 2C (PP2Cs) are negative regulators of abscisic acid (ABA) signaling that are inhibited in an ABA-dependent manner by PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) intracellular receptors. We provide genetic evidence that a previously uncharacterized member of this PP2C family in Arabidopsis (Arabidopsis thaliana), At5g59220, is a negative regulator of osmotic stress and ABA signaling and that this function was only apparent when double loss-of-function mutants with pp2ca-1/ahg3 were generated. At5g59220-green fluorescent protein and its close relative PP2CA-green fluorescent protein showed a predominant nuclear localization; however, hemagglutinin-tagged versions were also localized to cytosol and microsomal pellets. At5g59220 was selectively inhibited by some PYR/PYL ABA receptors, and close relatives of this PP2C, such as PP2CA/ABA-HYPERSENSITIVE GERMINATION3 (AHG3) and AHG1, showed a contrasting sensitivity to PYR/PYL inhibition. Interestingly, AHG1 was resistant to inhibition by the PYR/PYL receptors tested, which suggests that this seed-specific phosphatase is still able to regulate ABA signaling in the presence of ABA and PYR/PYL receptors and therefore to control the highly active ABA signaling pathway that operates during seed development. Moreover, the differential sensitivity of the phosphatases At5g59220 and PP2CA to inhibition by ABA receptors reveals a functional specialization of PYR/PYL ABA receptors to preferentially inhibit certain PP2Cs.     

ABA signal transduction from ABA receptors to ion channels

The plant hormone abscisic acid (ABA) is involved in regulating a number of major processes such as seed dormancy, seedling development, and biotic and abiotic stress responses. The function and effect of ABA on pathogens are still unclear, but the roles of ABA in seed germination and abiotic stress responses have been well characterized. Abiotic stresses elevate ABA levels and activate ABA signaling; thus, inducing a variety of responses, including the expression of stress-related genes and stomatal closure. The past decade has witnessed many significant advances in our understanding of ABA signal transduction due to application of a combination of approaches including genetics, biochemistry, electrophysiology, and chemical genetics. A number of proteins associated with the ABA signal transduction pathway such as PYR/PYL/RCAR family of START proteins, have been identified. These ABA receptors bind to ABA and positively regulate ABA signaling via inactivation of PP2C phosphatase activity, which inhibits SnRK2-type kinases by direct interaction and dephosphorylation. Additionally, SnRK2-type kinases and PP2Cs interact with one another and with other components of ABA signaling and function as positive and negative ABA regulators, respectively. In this review, we focus on ABA function to abiotic stresses and highlight each component in relation to ABA and its interactions.     

Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling pathway to cause disease

We have found that a major target for effectors secreted by Pseudomonas syringae is the abscisic acid (ABA) signalling pathway. Microarray data identified a prominent group of effector-induced genes that were associated with ABA biosynthesis and also responses to this plant hormone. Genes upregulated by effector delivery share a 42% overlap with ABA-responsive genes and are also components of networks induced by osmotic stress and drought. Strongly induced were NCED3, encoding a key enzyme of ABA biosynthesis, and the abscisic acid insensitive 1 (ABI1) clade of genes encoding protein phosphatases type 2C (PP2Cs) involved in the regulation of ABA signalling. Modification of PP2C expression resulting in ABA insensitivity or hypersensitivity led to restriction or enhanced multiplication of bacteria, respectively. Levels of ABA increased rapidly during bacterial colonisation. Exogenous ABA application enhanced susceptibility, whereas colonisation was reduced in an ABA biosynthetic mutant. Expression of the bacterial effector AvrPtoB in planta modified host ABA signalling. Our data suggest that a major virulence strategy is effector-mediated manipulation of plant hormone homeostasis, which leads to the suppression of defence responses.     

Abscisic acid receptors: past, present and future

Jin-Gui Chen (Corresponding author) Abscisic acid (ABA) is the key plant stress hormone. Consistent with the earlier studies in support of the presence of both membrane- and cytoplasm-localized ABA receptors, recent studies have identified multiple ABA receptors located in various subcellular locations. These include a chloroplast envelope-localized receptor (the H subunit of Chloroplast Mg(2+) -chelatase/ABA Receptor), two plasma membrane-localized receptors (G-protein Coupled Receptor 2 and GPCR-type G proteins), and one cytosol/nucleus-localized Pyrabactin Resistant (PYR)/PYR-Like (PYL)/Regulatory Component of ABA Receptor 1 (RCAR). Although the downstream molecular events for most of the identified ABA receptors are currently unknown, one of them, PYR/PYL/RCAR was found to directly bind and regulate the activity of a long-known central regulator of ABA signaling, the A-group protein phosphatase 2C (PP2C). Together with the Sucrose Non-fermentation Kinase Subfamily 2 (SnRK2s) protein kinases, a central signaling complex (ABA-PYR-PP2Cs-SnRK2s) that is responsible for ABA signal perception and transduction is supported by abundant genetic, physiological, biochemical and structural evidence. The identification of multiple ABA receptors has advanced our understanding of ABA signal perception and transduction while adding an extra layer of complexity.     

Action of natural abscisic acid precursors and catabolites on abscisic acid receptor complexes

The phytohormone abscisic acid (ABA) regulates stress responses and controls numerous aspects of plant growth and development. Biosynthetic precursors and catabolites of ABA have been shown to trigger ABA responses in physiological assays, but it is not clear whether these are intrinsically active or whether they are converted into ABA in planta. In this study, we analyzed the effect of ABA precursors, conjugates, and catabolites on hormone signaling in Arabidopsis (Arabidopsis thaliana). The compounds were also tested in vitro for their ability to regulate the phosphatase moiety of ABA receptor complexes consisting of the protein phosphatase 2C ABI2 and the coreceptors RCAR1/PYL9, RCAR3/PYL8, and RCAR11/PYR1. Using mutants defective in ABA biosynthesis, we show that the physiological activity associated with ABA precursors derives predominantly from their bioconversion to ABA. The ABA glucose ester conjugate, which is the most widespread storage form of ABA, showed weak ABA-like activity in germination assays and in triggering ABA signaling in protoplasts. The ABA conjugate and precursors showed negligible activity as a regulatory ligand of the ABI2/RCAR receptor complexes. The majority of ABA catabolites were inactive in our assays. To analyze the chemically unstable 8'- and 9'-hydroxylated ABA catabolites, we used stable tetralone derivatives of these compounds, which did trigger selective ABA responses. ABA synthetic analogs exhibited differential activity as regulatory ligands of different ABA receptor complexes in vitro. The data show that ABA precursors, catabolites, and conjugates have limited intrinsic bioactivity and that both natural and synthetic ABA-related compounds can be used to probe the structural requirements of ABA ligand-receptor interactions.     

ROP11 GTPase Negatively Regulates ABA Signaling by Protecting ABI1 Phosphatase Activity from Inhibition by the ABA Receptor RCAR1/PYL9 in Arabidopsis

The phytohormone abscisic acid (ABA) regulates many key processes in plants, such as seed germina- tion, seedling growth, and abiotic stress tolerance. In recent years, a minimal set of core components of a major ABA signaling pathway has been discovered. These components include a RCAR/PYR/PYL family of ABA receptors, a group of PP2C phosphatases, and three SnRK2 kinases. However, how the interactions between the receptors and their targets are regulated by other proteins remains largely unknown. In a companion paper published in this issue, we showed that ROP11, a member of the plant- specific Rho-like small GTPase family, negatively regulates multiple ABA responses in Arabidopsis. The current work demonstrated that the constitutively active ROP11 (CA-ROP11) can modulate the RCAR1/PYL9-mediated ABA signaling pathway based on reconstitution assays in Arabidopsis thaliana protoplasts. Furthermore, using luciferase complementation imaging, yeast two-hybrid assays, co- immunoprecipitation assays in Nicotiana benthamiana and bimolecular fluorescence complementation assays, we demonstrated that CA-ROP11 directly interacts with ABI1, a signaling component downstream of RCAR1/PYL9. Finally, we provided biochemical evidence that CA-ROP11 protects ABI1 phosphatase activity from inhibition by RCAR1/PYL9 and thus negatively regulates ABA signaling in plant cells. A model of how ROP11 acts to negatively regulate ABA signaling is presented.     

Protein phosphatase 2C (PP2C) function in higher plants

In the past few years, molecular cloning studies have revealed the primary structure of plant protein serine/threonine phosphatases. Two structurally distinct families, the PP1/PP2A family and the PP2C family, are present in plants as well as in animals. This review will focus on the plant PP2C family of protein phosphatases. Biochemical and molecular genetic studies in Arabidopsis have identified PP2C enzymes as key players in plant signal transduction processes. For instance, the ABI1/ABI2 PP2Cs are central components in abscisic acid (ABA) signal transduction. Arabidopsis mutants containing a single amino acid exchange in ABI1 or ABI2 show a reduced response to ABA. Another member of the PP2C family, kinase-associated protein phosphatase (KAPP), appears to be an important element in some receptor-like kinase (RLK) signalling pathways. Finally, an alfalfa PP2C acts as a negative regulator of a plant mitogen-activated protein kinase (MAPK) pathway. Thus, the plant PP2Cs function as regulators of various signal transduction pathways.     

Modulation of abscisic acid signaling in vivo by an engineered receptor-insensitive protein phosphatase type 2C allele

The plant hormone abscisic acid (ABA) plays a crucial role in the control of the stress response and the regulation of plant growth and development. ABA binding to PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS intracellular receptors leads to inhibition of key negative regulators of ABA signaling, i.e. clade A protein phosphatases type 2C (PP2Cs) such as ABA-INSENSITIVE1 and HYPERSENSITIVE TO ABA1 (HAB1), causing the activation of the ABA signaling pathway. To gain further understanding on the mechanism of hormone perception, PP2C inhibition, and its implications for ABA signaling, we have performed a structural and functional analysis of the PYR1-ABA-HAB1 complex. Based on structural data, we generated a gain-of-function mutation in a critical residue of the phosphatase, hab1(W385A), which abolished ABA-dependent receptor-mediated PP2C inhibition without impairing basal PP2C activity. As a result, hab1(W385A) caused constitutive inactivation of the protein kinase OST1 even in the presence of ABA and PYR/PYL proteins, in contrast to the receptor-sensitive HAB1, and therefore hab1(W385A) qualifies as a hypermorphic mutation. Expression of hab1(W385A) in Arabidopsis (Arabidopsis thaliana) plants leads to a strong, dominant ABA insensitivity, which demonstrates that this conserved tryptophan residue can be targeted for the generation of dominant clade A PP2C alleles. Moreover, our data highlight the critical role of molecular interactions mediated by tryptophan-385 equivalent residues for clade A PP2C function in vivo and the mechanism of ABA perception and signaling.     

Interactions between soybean ABA receptors and type 2C protein phosphatases

The plant hormone abscisic acid (ABA) plays important roles in regulating plant growth, development, and responses to environmental stresses. Proteins in the PYR/PYL/RCAR family (hereafter referred to as PYLs) are known as ABA receptors. Since most studies thus far have focused on Arabidopsis PYLs, little is known about PYL homologs in crop plants. We report here the characterization of 21 PYL homologs (GmPYLs) in soybean. Twenty-three putative GmPYLs can be found from soybean genome sequence and categorized into three subgroups. GmPYLs interact with AtABI1 and two GmPP2Cs in diverse manners. A lot of the subgroup I GmPYLs interact with PP2Cs in an ABA-dependent manner, whereas most of the subgroup II and III GmPYLs bind to PP2Cs in an ABA-independent manner. The subgroup III GmPYL23, which cannot interact with any of the tested PP2Cs, differs from other GmPYLs. The CL2/gate domain is crucial for GmPYLs-PP2Cs interaction, and a mutation in the conserved proline (P109S) abolishes the interaction between GmPYL1 and AtABI1. Furthermore, the ABA dependence of GmPYLs-PP2Cs interactions are partially correlated with two amino acid residues preceding the CL2/gate domain of GmPYLs. We also show that GmPYL1 interacts with AtABI1 in an ABA-dependent manner in plant cells. Three GmPYLs differentially inhibit AtABI1 and GmPP2C1 in an ABA-dependent or -enhanced manner in vitro. In addition, ectopically expressing GmPYL1 partially restores ABA sensitivity of the Arabidopsis triple mutant pyr1/pyl1/pyl4. Taken together, our results suggest that soybean GmPYLs are ABA receptors that function by interacting and inhibiting PP2Cs.