Abstract
Adverse environmental conditions are a threat to agricultural yield and therefore exert a global effect on livelihood, health and the economy. Abscisic acid (ABA) is a vital plant hormone that regulates abiotic stress tolerance, thereby allowing plants to cope with environmental stresses. Previously, attempts to develop a complete understanding of the mechanisms underlying ABA signaling have been hindered by difficulties in the identification of bona fide ABA receptors. The discovery of the PYR/PYL/RCAR family of ABA receptors therefore represented a major milestone in the effort to overcome these roadblocks; since then, many structural and functional studies have provided detailed insights into processes ranging from ABA perception to the activation of ABA-responsive gene transcription. This understanding of the mechanisms of ABA perception and signaling has served as the basis for recent, preliminary developments in the genetic engineering of stress-resistant crops as well as in the design of new synthetic ABA agonists, which hold great promise for the agricultural enhancement of stress tolerance.
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Introduction-Understanding ABA perception
“There are things known and there are things unknown, and in between are the doors of perception.” —Aldous Huxley
We are constantly surrounded by signals, such as traffic signals, notifications, advertisements, feelings of pain or hunger. However, we are only aware of these signals if we perceive them; we would not know of or respond to these signals if we could not perceive them, even if the signals are always there. Thus, in the process of conveying a signal, the perception of the signal is just as important as the existence of the signal itself.
At the cellular level, extracellular signals are often transmitted to cells via molecules such as hormones. In plants, the levels of the hormone abscisic acid (ABA) increase when environmental conditions are harsh and serve as a signal for the plant cells to adapt as necessary. ABA was discovered in the 1960s, and many details were established shortly thereafter regarding the chemistry and physiological importance of ABA1. Over the ensuing decades, more than 100 mediators involved in ABA signaling have been identified using molecular genetic, biochemical and pharmacological studies2. However, for almost half a century, a critical piece of the puzzle remained missing in our knowledge of ABA signal transduction, namely, how it is that ABA signals are perceived.
The identification of receptors capable of sensing ABA and relaying this signal to other mediators in the pathway has proven to be a daunting task rife with controversy and frustration. Since 2006, several reports have claimed to identify the ABA receptors3, but none of these reports could be substantiated after further investigation. The turnaround came in 2009, when at least two separate findings convincingly implicated the same members of the steroidogenic acute regulatory protein (StAR)-related lipid-transfer (START) domain/ major birch pollen allergen (Bet v 1) superfamily of proteins as candidate ABA receptors4,5,6,7. The 14 members of this group of proteins are named Pyrabactin Resistance 1 (PYR1) and PYR1-like 1–13 (PYL1-PYL13)6 or Regulatory Component of ABA Receptor (RCAR1-RCAR14)4 and are referred to in this article as PYL(s) for simplicity. The initial discovery of PYLs soon led to a series of crystallographic studies that collectively unraveled the mechanistic details underlying ABA recognition and signal transduction by the members of this family of ABA receptors. These findings have fuelled remarkable advancements in the field of ABA signaling and hence were recognized as both one of the scientific breakthroughs8 and one of the signaling breakthroughs9 of the year for 2009.
Since then, the molecular interactions comprising the core ABA signaling pathway, a signaling cascade consisting of the PYL ABA receptors, type 2C protein phosphatases (PP2Cs) and Snf1-related protein kinases 2 (SnRK2s), have been further clarified by multiple structural studies. Studies such as these shed light onto the exquisite controls regulating each level of the ABA signal transduction pathway and open up new technological opportunities for the enhancement of abiotic stress tolerance in agriculture. Here, we present a comprehensive review of the current understanding of ABA, its early signaling mechanisms and recent developments in agricultural applications based on the emerging structural findings.
ABA in plants
Physiological role in abiotic stress tolerance
ABA is a vital phytohormone that confers abiotic stress tolerance in plants10. Under stressful environmental conditions such as water shortage, high salinity and temperature extremes, the ABA content in plants rises significantly, stimulating stress-tolerance effects that help plants adapt and survive under these adverse conditions (Figure 1). It has been demonstrated that mutant plants engineered with enhanced ABA sensitivity adapt to drought conditions better than their wild-type counterparts11. Under drought or osmotic stress conditions, ABA promotes stomatal closure, which prevents water loss through transpiration, and the accumulation of osmocompatible solutes to retain water2,12. It has also been shown that ABA is required for tolerance to freezing which occurs through the induction of dehydration-tolerance genes13.
ABA-mediated abiotic stress response. Overview of the molecular events in ABA-mediated stress tolerance. ABA accumulation, induced by stress signals, activates PYL ABA receptors to inhibit group A PP2Cs. PP2C inhibition in turn allows SnRK2 activation through autophosphorylation. Active SnRK2s mediate the ABA response through the phosphorylation of downstream targets. In guard cells, SnRK2s phosphorylate the SLAC1 and KAT1 ion channels, resulting in stomatal closure to prevent transpirational water loss. In seeds, ABI5 phosphorylation by SnRK2s leads to the inhibition of seedling growth. The phosphorylation of the AREB1 (ABF2), AREB2 (ABF4), and ABF3 transcription factors activates the transcription of target genes such as Late Embryogenesis Abundant (LEA)-class genes as well as transcription factors (TF) involved in stress tolerance. Transcriptional increases in the expression of group A PP2C genes may function as a negative feedback loop in the ABA response pathway by inhibiting SnRK2 activity. Positive regulators of the ABA signaling pathway are shown in green, whereas negative regulators are shown in red.
The role of ABA as a negative regulator of plant growth has also been long established14. The activity of ABA in the induction and maintenance of seed dormancy is attributed to its potent effects on the inhibition of seed germination15. ABA also inhibits the growth and development of whole plants or plant parts and counteracts the effects of growth-stimulating hormones such as gibberellins2. The inhibitory effects of ABA on germination and growth help plants withstand these stressful conditions and germinate only when the conditions are favorable for growth.
Biosynthesis and transport
Stress signals induce the expression of enzymes responsible for ABA biosynthesis. Most of the ABA biosynthetic genes have been identified and cloned, including zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED) and abscisic aldehyde oxidase (AAO3)13,16. The synthesis of ABA from C40 carotenoids occurs via several enzymatic steps (Figure 2)17. First, zeaxanthin, which is formed by the hydroxylation of β-carotene, is converted into violaxanthin by ZEP. This is followed by the synthesis and oxidative cleavage of neoxanthin into xanthoxin, the C15 precursor of ABA. The NCED-catalyzed production of xanthoxin is thought to be the key regulatory step of ABA biosynthesis. Xanthoxin is then converted into abscisic aldehyde, which is oxidized into ABA.
ABA biosynthesis and catabolism. The ABA biosynthetic pathway is shown in the left panel. ABA is derived from C40 epoxycarotenoid precursors through oxidative cleavage reactions that occur in the plastid. The C15 intermediate xanthoxin is exported into the cytosol, where it is converted to ABA through a two-step reaction involving an abscisic aldehyde intermediate. The ABA biosynthetic enzymes zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), short-chain alcohol dehydrogenase and abscisic aldehyde oxidase (AAO3) are shown in pink. In the right panel, three catabolic pathways involving C-7′, C-8′, and C-9′ hydroxylation are shown. ABA is fully inactivated after the formation of the biologically inactive compound DPA.
The endogenous concentration of ABA is determined by the balance between ABA biosynthesis and catabolism, as well as by the rate of ABA transport to its sites of action. Although ABA can diffuse passively across biological membranes, recent evidence suggests that transport proteins are involved in actively shuttling ABA in and out of cells. In 2010, two ATP-binding cassette (ABC) transporters were found to be capable of shuttling ABA: AtABCG25 is mainly expressed in vascular tissues, where ABA is predominantly synthesized, and can function as an ABA exporter18, whereas AtABCG40 is expressed at the sites of ABA action, especially in guard cells, and can function as an ABA importer19. Thus, a simple model of ABA translocation was proposed20 in which ABA is synthesized in vascular cells and actively transported into the extracellular apoplastic space by AtABCG25, after which it is taken up by roots and leaves via AtABCG40. However, whereas AtABCG40 is required for efficient ABA signaling as expected, ABA responses are increased and decreased upon AtABCG25 loss-of-function and overexpression, respectively, which cannot be easily explained by this model. Moreover, a second, unrelated ABA importer, ABA-importing transporter 1 (AIT1), has recently been identified21 and unexpectedly appears to be expressed predominantly at the sites of ABA synthesis, indicating that ABA transport may be more complex than initially envisioned.
Catabolism
When stress signals are attenuated, ABA is metabolized into inactive products. ABA catabolism occurs largely through the hydroxylation of one of the three methyl groups (C-7′, C-8′, and C-9′) on the ring structure (Figure 2). Of these, 8′-hydroxylation is thought to serve as the dominant ABA catabolic pathway22,23. Accordingly, the products of the 8′-hydroxylation pathway, phaseic acid (PA) and dihydrophaseic acid (DPA), are the most abundant ABA catabolites. Furthermore, while 8′-hydroxy ABA exhibits substantial biological activity, the spontaneous cyclization of 8′-hydroxy ABA into PA causes a significant reduction in activity24. PA is further catabolized into the biologically inactive compound DPA.
ABA 8′-hydroxylation is catalyzed by ABA 8′-hydroxylase, a cytochrome P450 encoded by the CYP707A gene family25,26. Transcript levels for the four CYP707A family members, CYP707A1−CYP707A4, increase during drought stress and are strongly induced upon rehydration25, with CYP707A3 showing the highest induction27. Recent findings indicated that CYP707A3 functions in vascular tissues to reduce systemic ABA levels, whereas CYP707A1 catabolizes local ABA pools inside guard cells in response to high humidity23.
In addition to hydroxylation, ABA and its hydroxylated catabolites can be conjugated to glucose. The major glucose conjugate of ABA, ABA glucosyl ester (ABA-GE), is biologically inactive and may function as a storage molecule to form a releasable pool of ABA28. ABA-GE is presumably synthesized in the cytosol and stored in the vacuoles29. The import of ABA-GE into vacuoles has been reported to be mediated by proton gradient-driven and ABC-type transporters30. Under abiotic stress conditions, ABA-GE is hydrolyzed by β-glucosidases to release free ABA28,31,32.
Chemical features
The natural and biologically active isomer of ABA is (+)-2-cis-4-trans-ABA, also commonly known as S-ABA, (+)-ABA or S-(+)-ABA. The molecular structure consists of a cyclohexene ring with a monomethyl group, a dimethyl group, a ketone group, a hydroxyl group and a hydrocarbon side chain conjugated to the carboxylic acid group (Figure 3A). The 2-cis-4-trans side chain geometry can be reversibly isomerized by light to form a mixture containing the inactive 2-trans-4-trans isomer2. Studies using ABA analogs lacking the 7′-, 8′-, or 9′- methyl groups showed that the 7′-methyl group is critical for bioactivity33. Flipping the cyclohexene ring around the chiral carbon produces the unnatural (–)-enantiomer, which has weak biological activity (Figure 3B)34.
Chemical structures of ABA stereoisomers, structural analogs and pyrabactin. Chemical structures of (A) the naturally occurring S-(+)-ABA; (B) the unnatural R-stereoisomer, which differs from the S-isomer by a flip around the chiral carbon (C-1′); (C) PBI-51, a structural analog of ABA; (D) Pyrabactin, a seed-specific selective ABA agonist that does not structurally resemble ABA; and (E) AM1/quinabactin, a recently identified ABA agonist that has been shown to exhibit ABA-like effects in vegetative tissues. Functional groups for all molecules are shown in colors, and the nomenclature for the naming of carbon atom positions is shown for the S-(+)-ABA structure.
Structural and functional analogs of ABA have been used to identify ABA-responsive genes, and some of these analogs are potential plant growth regulators35,36. For instance, PBI-51 (Figure 3C), which is structurally similar to ABA, exhibits ABA-antagonizing effects in Brassica napus and Vicia faba37,38, whereas it is a weak ABA agonist in Arabidopsis39. The use of PBI-51 to screen for ABA-related mutants has led to the isolation of novel ABA hypersensitive mutants39. In addition, pyrabactin (Figure 3D), a selective ABA agonist that does not structurally resemble ABA, was employed in the isolation of PYR1, which led to the identification of the PYL family of proteins as ABA receptors6. Recent efforts to identify new ABA agonists based on structural studies of the activation of PYL by ABA and pyrabactin have led to the isolation of AM1/quinabactin as a promising candidate (Figure 3E)40,41. These studies will be reviewed in further detail in the later sections.
Components of the ABA signaling pathway
PYLs-ABA receptors
Forward genetics approaches are most commonly used in the identification of hormone receptors, whereby mutants are identified based on their hormone-insensitive phenotypes. In the case of ABA, the inhibition of seed germination by the application of exogenous ABA is the most widely used assay for mutational screening. These screens have identified several ABA-insensitive mutants42,43, five of which have been extensively characterized. These five mutants are designated abi1–abi5 (for ABA insensitive). The corresponding ABI1 and ABI2 genes encode PP2Cs44,45,46 (see Section PP2Cs-negative regulators below), while ABI3, ABI4 and ABI5 encode transcription factors involved in the seed-specific ABA signaling pathway47,48,49. None of the genes identified through classical forward genetics approaches had ABA-binding or receptor-like properties. Conversely, the use of alternative approaches has identified several putative ABA receptors, including FCA50, CHLH51, GCR252, GTG1 and GTG253, which all reportedly bind ABA with affinities in the nanomolar range (Kd of 19, 32, 21, 35.8, and 41.2 nmol/L for FCA, CHLH, GCR2, GTG1, and GTG2, respectively). These attempts have not, however, led to the unambiguous identification of convincing ABA receptor candidates that fit with the physiological, genetic and biochemical knowledge that has accumulated over the years3.
The discovery of PYLs as candidate ABA receptors was different from that of the earlier putative ABA receptors. Independent findings from several groups converged upon this novel class of ABA binding proteins, which fit elegantly into a model that connected the core components of the ABA signal transduction pathway.
Genetic redundancy has been one of the major issues hindering the identification of ABA receptors using classical forward genetics. To overcome this problem, Park et al used a synthetic seed germination inhibitor named pyrabactin as a selective ABA agonist6. Pyrabactin's effects on global gene transcription were highly correlated with those of ABA in seeds; however, the correlation was weaker in seedlings, indicating that pyrabactin is a highly selective ABA agonist that affects only a subset of ABA's activities. Forward genetic screens for pyrabactin-resistant mutants identified the Pyrabactin Resistance 1 (PYR1) locus, and 13 additional PYR1-like (PYL) genes were identified by sequence analysis and designated PYL1 through PYL13 (Table 1). Using yeast two-hybrid assays, the authors detected interactions between multiple PP2Cs and several PYL members in the presence of ABA. They also demonstrated that PYR1 inhibits the phosphatase activity of the PP2C HAB1 in the presence of ABA, with an IC50 of 125 nmol/L ABA. The binding of ABA to PYR1 has also been detected in heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) experiments, which probe the chemical shifts of protein amide–NH bonds in response to ligands. Collectively, these data suggest that PYLs are ABA receptors that directly interact with and inhibit PP2Cs upon ABA binding.
Meanwhile, in a study to identify the link between ABA perception and PP2Cs, Ma et al performed a yeast two-hybrid screen for Arabidopsis proteins that interact with the PP2C ABI24. Using this approach, this group discovered an ABI2-interacting protein and named it Regulatory Component of ABA Response 1 (RCAR1). Subsequently, 13 homologous proteins were identified and named RCAR2 through RCAR14 (Table 1). The 14 RCAR members discovered in this study turned out to be identical to the 14 PYL members identified by Park et al. Using isothermal titration calorimetry (ITC), the interaction between RCAR1 and (+)-ABA was shown to possess an apparent Kd of ∼0.66 μmol/L. The binding affinity was approximately 10-fold higher in the presence of ABI2, suggesting the formation of a stable receptor-ABA-PP2C complex. RCAR1 was also shown to interact with and inhibit the phosphatase activities of the PP2Cs ABI1 and ABI2 in the presence of ABA, which is consistent with the ABA-binding and PP2C-inhibitory activities demonstrated by Park et al.
Using a similar approach, Santiago et al identified PYL5, PYL6, and PYL8 in a yeast two-hybrid screen for HAB1-interacting proteins7. Consistent with the findings of Park et al and Ma et al, members of the PYL family inhibited the phosphatase activities of ABI1, ABI2 and HAB1 in an ABA-dependent manner. Using ITC, the apparent Kd of (+)-ABA binding to PYL5 was determined to be 1.1 μmol/L and 38 nmol/L in the absence and presence of HAB1, respectively, supporting the formation of a stable ternary complex as suggested by Ma et al. In addition, Santiago et al observed that, whereas certain PYL members such as PYR1 and PYL4 only interacted with PP2Cs in the presence of ABA, PYL5, PYL6, and PYL8 did not require ABA to interact with HAB1 in a yeast two-hybrid assay, although ABA did enhance the affinity of PYL5 for HAB1. This finding suggests the existence of different roles for the PYL members such that a subset of PYL proteins may constitutively inhibit PP2Cs.
In yet another independent study, Nishimura et al screened for ABI1-interacting proteins in planta and identified 9 of the 14 PYL members using co-immunoprecipitation assays followed by mass spectrometry5. Co-immunoprecipitation experiments showed that ABA induces an interaction between PYR1 and ABI1 in vivo within 5 min of ABA treatment.
PP2Cs-negative regulators
Reversible protein phosphorylation mediated by protein kinases and protein phosphatases is a major and universal component of cellular signal transduction. PP2Cs are a group of single subunit Mg2+/Mn2+-dependent Ser/Thr phosphatases, and PP2Cs represent a major phosphatase family in plants. Of the 112 phosphatases encoded in the Arabidopsis genome, 76 are PP2Cs, which genetically cluster into 10 groups (A–J), with the exception of 6 genes that could not be clustered54. At least 6 of the 9 members of the group A PP2Cs have been shown to be involved in ABA signaling, and of these, ABI1, ABI2, and HAB1 are the most well characterized.
The isolation and characterization of ABA-insensitive Arabidopsis mutants have led to the identification of group A PP2Cs as negative regulators of ABA signaling. The dominant mutants abi1-1 (ABI1 G180D) and abi2-1 (ABI2 G168D), which result in reduced ABA responsiveness, were isolated from genetic screens of mutagenized Arabidopsis seeds44,45,46,55. Both of these mutants display reduced seed dormancy, seedling growth, drought tolerance and stomatal regulation. The ABI1 and ABI2 genes encode homologous PP2C proteins and are transcriptionally upregulated by ABA treatment. HAB1 was subsequently identified based on its sequence homology to ABI1 and ABI256.
Consistent with the phenotypic effects observed in the abi1-1 and abi2-1 mutants, introducing the corresponding mutation (G246D) into HAB1 resulted in strong ABA insensitivity57. The abi1-1, abi2-1, and hab1 G246D mutations correspond to the substitution of a conserved glycine residue with an aspartate in the phosphatase catalytic center. These mutants exhibited dramatically reduced phosphatase activity toward phospho-casein, a heterologous substrate, which was difficult to reconcile with the apparent dominant nature of the mutation. Recessive loss-of-function mutations in the catalytic regions of ABI1, ABI2, and HAB1 resulted in ABA-hypersensitive phenotypes (partially constitutive seed dormancy and drought tolerance), indicating that the ABA insensitivity of the abi1-1 and abi2-1 mutations is due not to the observed reduction in catalytic activity but to the gain of a new function, and provided critical evidence that these PP2Cs negatively regulate ABA signaling58,59,60. This notion was further confirmed using double- or triple-PP2C-knockout mutants, which display constitutively enhanced stress responses/ABA hypersensitivity11,61. Consistent with this, the overexpression of HAB1 (35S:HAB1) in Arabidopsis led to reduced ABA sensitivity, supporting the role of HAB as an inhibitor of ABA signaling58.
Given the negative regulatory roles of the group A PP2Cs, the mechanism by which the abi1-1 and abi2-1 mutations, as well as the corresponding G246D mutation in HAB1, induce dominant ABA-insensitive phenotypes remains an enigma. Interestingly, the mutant abi1-1 protein was as effective at inhibiting ABA signal transduction as the wild-type protein, as shown with an ABA-inducible transcription assay in plant protoplasts62. Although earlier studies indicated the hypermorphic nature of this type of mutation57,63, they were unable to fully explain how such a mutation interferes with ABA signal transduction. Instead, the answer lies in the loss of the ABA receptor-mediated regulation of PP2Cs, as explained by more recent structural findings.
SnRK2s-positive mediators
The identification of PP2Cs indicated that protein phosphorylation events are important in ABA signaling. In line with this concept, the SnRK2 family was identified as group of ABA-activated protein kinases64,65. SnRK2s belong to the SnRK group of protein kinases that are closely related to yeast Snf1 and to the catalytic subunits of mammalian AMPK. The Arabidopsis genome contains 38 SnRKs, which are classified into three groups, namely, SnRK1 (1.1–1.3), SnRK2 (2.1–2.10) and SnRK3 (3.1–3.25)66 (Figure 4A). The SnRK1 group shares the highest degree of homology with Snf1 and AMPK. Similar to its yeast and mammalian counterparts, SnRK1 is best known for its role as a key metabolic regulator67. In contrast, SnRK2 and SnRK3 are unique to plants and are thought to be involved in abiotic stress signaling68.
Classification and domain structure of Arabidopsis SnRK2. (A) Diagram showing the classification of Arabidopsis SnRK groups and the SnRK2 subclasses. (B) Domain structure of Arabidopsis SnRK2s.
There are 10 SnRK2 members in Arabidopsis, which are designated as SRK2I–SRK2J65 or SnRK2.1–SnRK2.1066 and divided into three subclasses: I, II and III (Figure 4A). All SnRK2 members, except for SnRK2.9, can be activated by osmotic stress, as shown in an Arabidopsis protoplast system69. Consistent with this observation, an Arabidopsis decuple mutant lacking all 10 SnRK2s grew poorly under osmotic stress conditions70, revealing the importance of SnRK2s in osmotic stress signaling. However, not all SnRK2 members can be activated by ABA, suggesting that osmotic stress signaling consists of ABA-dependent and ABA-independent pathways. Whereas SnRK2 subclass I members are not activated by ABA, subclass II members, represented by SnRK2.7 and SnRK2.8, are weakly activated by ABA. In contrast, the members of subclass III are strongly activated by ABA69.
Subclass III of the Arabidopsis SnRK2 family contains three kinases, namely, SnRK2.2/SRK2D, SnRK2.3/SRK2I, and SnRK2.6/SRK2E/OST1. The members of this subclass of ABA-responsive kinases have been identified as the main positive regulators of ABA signaling. The physiological role of SnRK2.6 has been initially determined in guard cells. Loss-of-function mutations in SnRK2.6 disrupted ABA-induced stomatal closure in Arabidopsis64,65. On the other hand, a snrk2.2 snrk2.3 double mutant showed strong ABA insensitivity with respect to seed germination and root growth inhibition71. Consequently, triple mutants lacking SnRK2.2, SnRK2.3 and SnRK2.6 were impaired in almost all ABA responses, indicating the centrality of these kinases to ABA signaling72,73,74.
SnRK2s contain a well-conserved kinase catalytic domain and a C-terminal regulatory region that encompasses two domains. Domain I, which is also known as the SnRK2 box, is conserved in all SnRK2s and is required for kinase activity. Domain II is a highly acidic region that is important for PP2C interaction and is only conserved among the ABA-responsive members; it is thus also known as the ABA box75,76 (Figure 4B).
Many reports have demonstrated that phosphorylation is important for the activation of SnRK2s. Active recombinant SnRK2.6 is autophosphorylated, and several phosphorylation sites have been identified, with the phosphorylation of S175 within the activation loop shown to be critical for kinase activity75. Active SnRK2s are able to directly phosphorylate target proteins such as ion channels and transcription factors to elicit the ABA response.
ABA-induced stomatal closure appears to be mediated by ion channels on guard cell membranes. One of these is the slow-type anion channel SLAC1, which has been shown to be essential for stomatal closure in response to various factors such as ABA and CO277,78. SLAC1 is phosphorylated and activated by SnRK2.6, a process that can be inhibited by PP2C79,80. In addition, an inward-rectifying potassium channel, KAT1, is also a target of SnRK2.6. ABA-activated SnRK2.6 can phosphorylate T306 in KAT1, thereby reducing KAT1 activity, which suggests that active SnRK2.6 negatively regulates KAT1 via phosphorylation to promote stomatal closure81. Therefore, SnRK2s are important in mediating ABA-induced stomatal closure through the regulation of ion channels.
ABF transcription factors
The accumulation of ABA in plant cells leads to changes in gene expression that generally contribute to drought stress tolerance. Transcriptome studies in rice and Arabidopsis have shown that exposure to ABA and various abiotic stresses results in changes to approximately 5%–10% of the genome, with more than half of these changes being common to drought, salinity and ABA treatments82,83. ABA-induced genes encode proteins involved in stress tolerance and include (i) dehydrins, which are members of the Late Embryogenesis Abundant (LEA) proteins and function as molecular chaperones to stabilize membranes and protect proteins against aggregation; (ii) enzymes that detoxify reactive oxygen species (ROS); and (iii) regulatory proteins such as transcription factors, protein phosphatases and kinases (Figure 1). ABA-repressed genes are enriched for those encoding proteins associated with cell growth.
Many cis-acting DNA elements have been identified by analyzing the promoters of ABA-responsive genes84. Designated as ABA-responsive elements (ABREs), these commonly contain the PyACGTGG/TC consensus sequence belonging to the G-box family (CACGTG), which has been implicated in a wide range of gene expression mechanisms in plants85. ABA-responsive gene expression requires multiple ABREs or the combination of an ABRE with a coupling element86,87.
The ABRE-binding (AREB) proteins, or ABRE-binding factors (ABFs), were isolated by using ABRE sequences as bait in yeast one-hybrid screens88,89. The AREB/ABFs encode basic-domain leucine zipper (bZIP) transcription factors belonging to the group A subfamily, comprising nine homologs in the Arabidopsis genome that share a highly conserved C-terminal bZIP domain and three additional N-terminal conserved regions designated C1, C2, and C390. These nine homologs can be divided into two groups: the ABI5 family (ABI5, EEL, DPBF2/AtbZIP67, DPBF4, and AREB3), the members of which are mainly expressed in seeds and are involved in seed development and maturation49,91,92,93, and the AREB/ABF family (ABF1, AREB1/ABF2, AREB2/ABF4, and ABF3), the members of which are mainly expressed in vegetative tissues under abiotic stress conditions88,89,94,95,96. While ABF1 is strongly induced by cold, but not by osmotic stress97, AREB1/ABF2, AREB2/ABF4, and ABF3 are induced by dehydration, high salinity and ABA treatment during vegetative growth94. The overexpression of these factors enhances drought stress tolerance94,95,96, and triple mutants exhibit impaired stress-responsive gene expression98, indicating that AREB1, AREB2, and ABF3 are master transcription factors responsible for regulating the ABRE-dependent expression of stress-responsive genes.
Several studies suggest that ABA-dependent phosphorylation is required for AREB/ABFs to be full activated. AREB1 requires ABA to achieve full activation, which is regulated by the ABA-dependent multi-site phosphorylation of the conserved domains in AREB199. ABA-activated SnRK2s, including SnRK2.2, SnRK2.3, and SnRK2.6, have been shown to phosphorylate AREB199. Loss of SnRK2.2 and SnRK2.3 function resulted in reduced ABF phosphorylation71, and ABA-induced gene expression was eliminated in a triple mutant lacking SnRK2.2, SnRK2.3, and SnRK2.672. These results suggest that SnRK2s are essential for ABA-induced gene expression through the phosphorylation and activation of ABFs. However, the molecular events leading to the ABA-dependent activation of SnRK2s had remained elusive until recent years, and the identification of ABA receptors was an important step that helped bridge the gap in our understanding of ABA perception and SnRK2 activation.
Model of the core ABA signaling pathway
Based on the aforementioned studies, which independently showed that PYLs bind ABA and inhibit PP2Cs, a model of the core ABA signaling pathway has emerged that links ABA recognition to the transcriptional activation of ABA-responsive genes (Figure 1). In this model, the ABA response is kept silent under basal condition by PP2Cs including ABI1, ABI2 and HAB1, which negatively regulate ABA responses. These phosphatases act by inhibiting SnRK2s including SnRK2.2, SnRK2.3, and SnRK2.6, which are positive regulators of the ABA signaling pathway. High levels of ABA induce PYLs, which are putative ABA receptors, to bind and inhibit PP2Cs. This relieves the PP2C-dependent inhibition of SnRK2s, thereby allowing these kinases to autophosphorylate and activate downstream effectors such as ion channels and ABF transcription factors, which results in the activation of ABA responses.
This model has been elegantly supported by the in vitro reconstitution of the core ABA signaling pathway. Coexpressing a set of the core ABA signaling components, namely, PYLs, PP2Cs, SnRK2s, and ABF2 transcription factors, in plant protoplasts was found to be necessary and sufficient to induce the expression of an ABA-responsive reporter gene upon treatment with ABA100. In another study, in vitro reconstitution analyses performed using PYR1, ABI1, and SnRK2 showed that PYR1 inhibited the ABI1-mediated suppression of SnRK2 activity in an ABA-dependent manner101, which supports the proposed model of the core ABA signaling pathway. As discussed below, this model has provided the framework for further structural and biochemical studies aimed at addressing the precise regulatory mechanisms governing ABA signaling from ABA perception to SnRK2 activation.
Structural elucidation of the early ABA signaling mechanisms
Helix-grip fold receptors
The first reports identifying PYL family members as candidate ABA receptors were quickly followed by a wave of crystallographic studies, which provided structural evidence supporting the role of this family of proteins as bona fide ABA receptors as well as a mechanistic basis for early ABA signaling events. The earliest PYL structural studies were based on PYR1, PYL1, and PYL2102,103,104,105,106. To date, the structures of PYL3, PYL5, PYL9, PYL10, and PYL13 have also been solved, either in their apo or ligand-bound forms or in complexes with PP2C107,108,109,110. The currently available PYL structures represent more than half of the 14-member family of PYL proteins in Arabidopsis, and all of them exhibit the helix-grip fold, a hallmark of START domain/Bet v 1-fold proteins, which is characterized by a long C-terminal α-helix surrounded by a curved anti-parallel β-sheet and several smaller α-helices.
Transmitting the ABA signal to PP2Cs
A distinctive feature of the PYL structure is the occurrence of a large ligand-binding pocket between the C-terminal helix and the central β-sheet. The entrance of this pocket is guarded by two functionally important β-loops, which have been termed the 'gate' and 'latch' loops102 (alternatively named Pro-Cap and Leu-Lock104, CL2, and CL3106, and the β3–β4 and β5–β6 lid loops103), that possess the conserved amino acid sequences SGLPA and HRL. The conformations of the gate and latch loops play critical roles in transmitting the ABA binding signal to downstream effectors102,104,105,106. Notably, the gate loops of the ligand-free PYR1, PYL1, and PYL2 structures exist in an open conformation that allows ligand entry (Figure 5A, 5B). In contrast, the conformations of these loops are observed to close off the entrance to this pocket in the ABA-bound receptor structures (Figure 5C). ABA binding within the receptor pocket is mediated by a network of polar and non-polar interactions, whereby a conserved lysine residue in the receptor (K59, K86, and K64 in PYR1, PYL1, and PYL2, respectively) anchors the carboxylate group of ABA to the inner end of the pocket while the cyclohexene ring of ABA interacts with the receptor gate near the pocket entrance to pull the gate into a closed conformation (Figure 5D–5F). The closure of the gate loop onto the latch loop forms a new gate and latch interface that is necessary for the interaction with PP2C.
ABA-induced gate closure in PYL. (A) Surface representation of the apo PYL2 (PDB code: 3KAZ) structure with an open ligand binding pocket guarded by the gate (blue) and latch (orange) loops. (B) The same PYL2 structure as in (A), showing ABA entering the pocket. (C) Surface representation of ABA-bound PYL2 (PDB code: 3KB0) with a closed pocket. (D−G) Cartoon illustrations of the ligand pocket region of PYL, highlighting the structural mechanisms of the receptor's interaction with ABA and PP2C. The gate and latch loops of the receptor are shown in blue and orange, respectively. (D) The gate loop of the apo receptor is in an open conformation, allowing access to the ligand binding pocket. (E) ABA enters the receptor pocket and binds to a lysine residue via charge interaction. At the outer end of the pocket, ABA interacts with gate residues through hydrophobic interactions. (F) The gate loop closes and forms interactions with the latch residues. (G) In the PYL-ABA-PP2C complex, the receptor gate is partially opened, allowing the insertion of a tryptophan residue from PP2C, which stabilizes the gate and latch interface through a water-mediated interaction network (shown by dashed lines); thus, this residue is termed the “tryptophan lock”.
The ability to induce receptor gate closure is therefore a key feature of ABA agonist activity, which has been supported by various in vitro and in vivo assays showing that mutations in the receptors' gate and latch loops impair their signal transduction abilities102. Consistent with these findings, structural studies using pyrabactin, a selective ABA agonist, showed that selective receptor activation is related to the closure of the receptor gate. In these studies, pyrabactin–PYL structures in the agonist conformation resemble ABA–PYL structures with the receptor gate in the closed conformation111,112,113. Conversely, in pyrabactin-antagonized PYL2 structures, pyrabactin adopts non-productive configurations within the receptor pocket that fail to pull the receptor gate into the active, closed state, thereby explaining its preferentially antagonistic activity112,113,114.
Crystal structures of PYL-ABA-PP2C complexes102,103,106, represented by the PYL2-ABA-HAB1 structure shown in Figure 6B, revealed that the ABA-bound receptor binds to the active site of PP2C, thereby inhibiting the phosphatase activity both competitively, by blocking the PP2C catalytic site (competition with SnRK2 substrate)115, and non-competitively, via hydrogen-bonding of the gate serine hydroxyl group with the catalytic glutamate of PP2C (E203 in HAB1)116. The PYL proteins interact with PP2Cs at the gate-latch interface induced by ABA-binding. In this interface, a conserved tryptophan residue in PP2C (W385 in HAB1 and W300 in ABI1) forms a water-mediated contact with ABA and interacts with the receptor's gate and latch loops, further locking these loops in a closed conformation (Figure 5G). The stabilization of this interface explains the more than 10-fold increase in the ABA-binding affinity of PYL in the presence of PP2C4,7,103,106. These structures also explain the previously characterized ABA-insensitive abi1-1 and abi2-1 mutations, in which a glycine-to-aspartic acid change in the PP2C active site disrupts receptor-PP2C interactions4,6,7,62. In the PYL-ABA-PP2C structures, this glycine is in close proximity to the receptor gate, and the replacement of the glycine hydrogen with the bulky side chain of aspartate sterically interferes with the gate interaction102,103,106.
PP2C inhibition of SnRK2. (A) Structure of the SnRK2.6−HAB1 complex (PDB code: 3UJG). SnRK2.6 is shown in dark green, with its activation loop in dark blue. HAB1 is shown in red. The side chain of the conserved PP2C “tryptophan lock” is shown as a stick representation in pink. Magnesium ions and sulfate ions are shown as dot and stick models, respectively. (B) Structure of the PYL2-ABA-HAB1 complex (PDB code: 3KB3) showing the molecular mimicry between PYL2 and SnRK2.6 (above) in their interaction with PP2C. PYL2 is shown in light green, with its gate loop in blue. (C) Cartoon illustration of the two interaction interfaces between SnRK2.6 and HAB1. (D) Cartoon illustration of the PYL2-ABA-HAB1 complex showing that PYL2, as with SnRK2.6 above, interacts with HAB1 at the PP2C catalytic site.
Receptor diversity and oligomerization states
The 14 members of the PYL family share between 38% and 84% amino acid sequence identity in the full-length proteins102. Reconstitution assays in Arabidopsis protoplasts showed that all PYL members except for PYL13, which is ABA-insensitive110 and was therefore not tested, are able to activate ABA-induced gene expression100, suggesting functional overlap between the PYL members. In line with this notion, multiple mutations, such as pyr1pyl1pyl2pyl4 quadruple mutations6 and pyr1pyl1pyl2pyl4pyl5pyl8 sextuple mutations117, are required to generate robust ABA-insensitive phenotypes. However, other lines of evidence suggest that the functions of different PYL members are not completely redundant. For instance, gene expression studies have revealed both overlapping and differential PYL expression patterns in different tissues117. Recently, it has been shown that a single pyl8 mutant displayed reduced ABA-mediated inhibition of root growth, indicating a non-redundant role of PYL8 in regulating root sensitivity to ABA118.
PYL members have been classified into two groups based on their oligomeric states (Table 1). Using static light scattering and analytical ultracentrifugation, it has been shown that PYL4–10 (except for the untested PYL7) are monomers107. In contrast, PYR1 and PYL1–3 are largely homodimers in solution in the absence of ABA, but dissociate into monomers in the presence of ABA107,108,119. The structures of apo PYR1 and PYL1–3102,103,104,105,106,109 revealed a cis-homodimer arrangement, in which the two PYL monomers associate in a parallel orientation and thereby block access to their ligand-binding pockets. PYL3 has also been shown to form trans-homodimers that can bind ABA and dissociate into monomers more easily109, consistent with a mixed monomer/dimer distribution119.
Because the ligand-binding pocket is blocked in the cis-homodimer conformation, and because the gate loops in the open conformation form part of the dimerization interface, ABA binding requires dimer dissociation, resulting in an almost two-orders-of-magnitude lower ABA affinity compared with the monomeric ABA receptors107,119 (Kd ∼50–100 μmol/L for dimeric and Kd ∼1 μmol/L for monomeric receptors; Table 1) and a reduced-to-undetectable PP2C affinity in the absence of ABA. Conversely, the monomeric receptors PYL5–10 are able, at sufficient concentrations, to inhibit PP2Cs in the absence of ABA. Moreover, PYL10 has larger hydrophobic residues in the ligand-binding pocket that can partially stabilize gate closure in the apo receptor, which allowed the crystallization of apo PYL10 in both the open107 and closed gate conformations120. It is important to note that these interactions are still greatly enhanced by ABA4,7,107. For instance, the affinity of PYL10 for the PP2C ABI1 is 1.2 μmol/L in the absence of ABA and 18 nmol/L in the presence of ABA107. Thus, the monomeric PYLs may function physiologically as highly sensitive ABA receptors rather than as ABA-independent receptors. While the ABA-unresponsive PYL13 does not homodimerize in solution, it is able to heterodimerize with other PYL members and consequently antagonize their activities in the heterodimeric state121. Notably, PYL10 is incapable of ABA-independent PP2C interactions in the context of the PYL10–PYL13 heterodimer complex110. While it is unclear if PYL receptors do make ABA-independent interactions with PP2Cs under physiological conditions, an understanding the in vitro mechanism of constitutive receptor activation can be applied to the generation of transgenic plants with enhanced ABA sensitivities. The existence of 14 apparently similar PYL proteins with varying properties, such as expression pattern, ABA sensitivity and the extent of PP2C inhibition, therefore provides a complex regulatory system for the fine tuning of the ABA response.
PP2C regulation of SnRK2
The crystal structure of a HAB1-SnRK2.6 fusion protein complex, together with biochemical and mutational analyses, revealed a number of interesting features regarding how PP2Cs inhibit SnRK2s in the absence of an ABA signal115. Two interaction interfaces have been identified, one of which appears in the complex structure as a mutual packing of the phosphatase and kinases active sites. In this interface, the activation loop of the kinase docks into the active site of the PP2C while the conserved ABA-sensing tryptophan of the PP2C inserts into the kinase catalytic cleft (Figure 6A), which resembles the PYL-ABA-PP2C interaction (Figure 6B). This observation revealed a surprising amount of molecular mimicry between the PYL and SnRK2 proteins for occupancy of the same PP2C catalytic site in the PYL-PP2C-SnRK2 signaling cascade.
The second PP2C-SnRK2 interaction interface involves charge interactions between the highly acidic C-terminal ABA box of SnRK2 and a positively charged surface region of PP2C (Figure 6C). This interface, although unresolved in the crystal structure, has been strongly hinted at through extensive biochemical and mutational evidence115,122. Collectively, these data suggest that the inhibition of SnRK2s by PP2Cs occurs via a two-step mechanism. In the first step, the kinase activity is drastically reduced, but not completely inhibited, by enzymatic dephosphorylation of a critical serine residue in the kinase activation loop, which can occur at low PP2C:SnRK2 molar ratios115. The second step, involving the full inhibition of kinase activity, is achieved by steric inhibition through the mutual packing of the active sites, as observed in the crystal structure, and requires the formation of stable, stoichiometric PP2C–SnRK2 complexes115.
SnRK2 activation
Understanding how PP2C inhibits SnRK2 has provided partial explanations as to how SnRK2 gains activity upon the relief of this inhibition. Analyses of SnRK2 structures in active and inactive conformations provided further insights into the mechanisms of kinase autoactivation. Three independent groups reported the structures of ABA-responsive SnRK2s in late 2011 and early 2012122,123,124. SnRK2 kinase domains adopt the canonical bilobal kinase fold, with the smaller N-terminal lobe connected to the larger C-terminal lobe by a flexible hinge, and the cleft formed between the two lobes contains the sites for substrate and ATP binding (Figure 7). In all of these structures, the C-terminal regulatory SnRK2 box forms a well-ordered α-helix that packs in parallel against the αC helix in the N-terminal lobe, forming extensive contacts that stabilize the position of the αC helix.
Unphosphorylated SnRK2s can adopt both active and inactive conformations. Structure of SnRK2.3 in the closed conformation (PDB code: 3UC3) in yellow and SnRK2.6 in the open conformation (PDB code: 3UC4) in orange. The SnRK2 box and αC helices are boxed in red and green, respectively.
The position of the αC helix regulates the closing and opening of the catalytic cleft and thus the alignment of the catalytic residues within the cleft. In many kinases, the position of this regulatory αC helix is adjusted by activating proteins that bind to the kinase domain125,126,127,128,129,130,131. By analogy, the extensive interactions formed between the crucial SnRK2 box helix and the αC helix stabilize the αC helix, consistent with the requirement for the SnRK2 box and the SnRK2 box – αC helix-interacting residues for kinase activity.
Although phosphorylation of the activation loop serine is required for full kinase activation, SnRK2s in which this serine is mutated to alanine still retain basal kinase activity123,132. This basal kinase activity can be explained by the SnRK2 box-stabilized αC helix, which allows unphosphorylated SnRK2s to adopt the conformational characteristics of both inactive and active kinases. This has been illustrated by crystal structures of recombinant unphosphorylated SnRK2s, which adopted either a “closed” conformation (SnRK2.3) and an interaction network characteristic of active kinases or an “open” conformation (SnRK2.6) indicative of inactive kinases (Figure 7)123. In the “closed” active conformation, the N-terminal lobe is positioned closer to the C-terminal lobe, producing a network of interactions that stabilize Mg2+ and ATP in the catalytic cleft130,133,134,135. As SnRK2.2, SnRK2.3, and SnRK2.6 show similar levels of phosphorylation-independent basal activity, it is believed that each of these kinases can spontaneously adopt either an “open” or “closed” conformation123, suggesting that SnRK2s in the partially active state can align the ATP and Mg2+ molecules for activity.
These results therefore illustrate a two-step activation mechanism of SnRK2 kinases. The first step is the ABA-mediated partial activation of SnRK2 kinase through the release of PP2C-mediated inhibition (Figure 8A–8D). This converts the kinase from a completely inactive state to a partly active state with basal kinase activity due to its ability to spontaneously adopt both “open” and “closed” conformations (Figure 8E). This partial activity allows the kinase to undergo further conversion into the fully active state via activation loop autophosphorylation (Figure 8F), which likely occurs both intra- and intermolecularly123.
Cartoon summary of the steps leading to SnRK activation. (A) Under unstressed conditions, PP2Cs (represented by HAB1) silence the ABA response by inhibiting SnRK2 kinase activity through a two-step mechanism: first via the enzymatic dephosphorylation of the SnRK2 activation loop serine residue (S175 in SnRK2.6) and second by sterically blocking the kinase catalytic site. HAB1 and SnRK2 are shown in red and dark green, respectively, with their catalytic sites shown in paler shades. The SnRK2.6 activation loop is shown in dark blue. (B) In the absence of ABA, the receptor gate loop is in an open conformation, hindering PP2C binding. (C) The ABA concentration increases under stress conditions, and ABA is sensed by the receptor when hormone binding induces the closure of the receptor gate. (D) ABA-bound PYL2 competes with SnRK2 for PP2C binding. The formation of a stable PYL2-ABA-HAB1 complex inactivates PP2C. (E) SnRK2 is relieved of steric inhibition by PP2C and gains basal activity by spontaneously alternating between active and inactive conformations. (F) Autophosphorylation of the activation loop serine results in full kinase activity.
Concluding notes on the collective structural findings
Altogether, these structural studies highlight the multiple levels of regulation that act upon the key components of the ABA signaling pathway. The inhibition of SnRK2 by PP2C occurs through the enzymatic dephosphorylation of the kinase activation loop serine residue as well as through the physical blockade of the kinase active site, which is accompanied by an allosteric change in the αC helix position. In the relay of the ABA signal, the inhibition of PP2C by ABA-bound PYL involves both the steric blocking of the phosphatase catalytic site and the direct binding of a key catalytic glutamate residue (E203 in HAB1) by the receptor-gate-loop serine residue116. As a result, the activation of SnRK2 is achieved through the inhibition of PP2C catalytic activity as well as through competition for PP2C binding by ABA-bound PYL. Future efforts will likely unravel further levels of complexity in the core signaling pathway as well as in the regulation of the downstream players and furnish a more complete picture of the ABA pathway.
Use of structural information on ABA signaling in agriculture
Genetic engineering approach
The understanding of how PYL proteins transmit ABA signals provides opportunities for the development of new tools to improve stress tolerance in plants. One such approach involves the genetic engineering of ABA receptors to increase their activity. For instance, transgenic plants expressing PYL2 proteins carrying activating mutations that stabilize the receptor-PP2C interactions have shown increased activation of ABA signaling in seeds136. However, the expression of the constitutively active mutant receptor (PYL2CA) was not detected in vegetative tissues in this study, thereby precluding further analysis in adult plants. In another study, transgenic expression of a PYL4 mutant (PYL4A194T), which had been selected for its ABA-independent inhibition of PP2CA in vitro, resulted in enhanced drought tolerance in plants137. Although this study showed promising results, a constitutively active receptor under a strong promoter might affect plant growth under normal conditions, which could lead to decreases in crop yield. Such a drawback could be overcome through the use of inducible promoters to drive receptor expression only under drought conditions. Alternatively, approaches using ABA agonists have also been explored.
Design of new ABA agonists
The pivotal role of ABA in promoting plant survival under stress conditions has fuelled efforts to use this compound in field trials aimed at improving crop survival. However, the agricultural application of the natural ABA hormone has been largely limited by its short-lived bioactivity, which is due to its instability in solution, susceptibility to photodegradation138 and rapid catabolism in plants139. As such, synthetic compounds that functionally mimic ABA but exhibit longer periods of bioactivity are highly sought after. In addition to agricultural uses, ABA agonists may also have commercial value as holding agents to extend the marketing period for plant seedlings and facilitate the transport of ornamental plants140, further highlighting the importance of identifying a suitable analog capable of mimicking the effects of ABA.
Previously, the rational design of ABA analogs was guided by knowledge of the hormone's chemical structure and catabolic pathway. Chemical libraries of ABA mimetics were generated through the substitution of side groups that are known to be targeted by the plant enzymes involved in ABA catabolism. Compounds such as 3′-fluoro-ABA were designed to resist isomerization into phaseic acid141, while 8′-methylene-ABA and 8′-acetylene-ABA irreversibly inhibit ABA 8′-hydroxylase, which catalyzes the predominant step in the breakdown of ABA142. However, some of these compounds, such as 8′-trifluoro-ABA, which resists hydroxylation at the C-8 position, are difficult to synthesize and hence impractical for agricultural use143. Additionally, analogs that target the ABA catabolic pathway run the risk of producing long-lasting effects on the plant that can eventually lead to sustained growth retardation, which is undesirable for agricultural use140.
The ability of pyrabactin to act as a selective agonist not only played a pivotal role in overcoming the problem of genetic redundancy in the identification of ABA receptors but also enabled an understanding of the fine mechanistic details underlying ABA receptor activation. Although the direct application of pyrabactin in fields is not favorable, given that pyrabactin acts strongly in seeds but not in the vegetative tissues where drought stress tolerance is required, structural studies using pyrabactin have nonetheless provided a valuable framework for the design of novel ABA agonists. Structural analyses of PYL receptors bound to both the pan-agonist ABA and the selective agonist pyrabactin have led to the identification of important interactions within the receptors that are needed to trigger the conformational changes associated with receptor activation102,103,104,105,106,112. Pyrabactin binds to PYR1 in a manner similar to ABA, with its naphthalene double ring positioned akin to the cyclohexene ring of ABA and with the bromide group in this ring interacting with the L-P-A residues in the gate loop to help keep it in the closed conformation (Figure 9A, 9B). The sulfonamide group of pyrabactin functionally mimics the carboxylate group of ABA by forming extensive interactions with the receptor, including a water-mediated hydrogen bond with the conserved K86 residue within the ligand-binding pocket. Based on this rationale, Melcher et al conducted a search for ABA receptor activators containing the naphthalene-1-sulfonamide group using virtual chemical libraries and identified 4 compounds that activated PYR1 in vitro with efficacies comparable to that of pyrabactin112.
Mode of receptor binding by ABA, pyrabactin and AM1/quinabactin. Structures of the ligand-bound pockets of (A) the PYL2-ABA-HAB1 complex (PDB code: 3KB3); (B) the PYL1-pyrabactin-ABI1 complex (3NMN); (C) the PYL2-AMI/Quinabactin-HAB1 complex (PDB code: 4LG5) and (D) the PYL2-AMI/Quinabactin-HAB1 complex (PDB code: 4LA7). Residues that form direct or water-mediated hydrogen bonds (dashed lines) with the ligands are shown in the stick representation. Water-mediated interactions between the ligands' carbonyl oxygen and W385 of HAB1 are shown, which are part of the hydrogen bond network (formed together with P92 and R120 of PYL2, this part of the interaction is not shown) that occurs in the ABA- and AMI/Quinabactin-bound complexes.
More recently, another ABA analog that also contains a sulfonamide group has been reported by two independent groups to be a potent agonist of several ABA receptors41,144. This compound has been named AM1 (ABA mimic-1) and quinabactin (for its dihydroquinolinone ring and similar scaffold as pyrabactin) by the two teams (Figure 3). Structural studies carried out by both teams showed that the binding of AM1/quinabactin to PYL2 resembles the binding of ABA more than it does the binding of pyrabactin. The carbonyl group of AM1/quinabactin mimics that of ABA to form a water-mediated hydrogen bond with the W385 tryptophan lock from HAB1 in the complex, whereas such a contact is not observed in the pyrabactin-activated receptor-PP2C complexes (Figure 9C, 9D compared with Figure 9A, 9B). The sulfonamide group of AM1/quinabactin, which is in a reverse orientation compared with that of pyrabactin, is positioned at the hydrophilic inner end of the pocket and forms water-mediated or direct hydrogen bonds with receptor residues that also interact with ABA and pyrabactin. These structural features suggest that AM1/quinabactin elicits ABA responses through a similar mode of receptor binding as ABA. In vitro, AM1/quinabactin was shown to be more potent than both ABA and pyrabactin in activating PYR1 and other PYL proteins and in inducing their interactions with HAB1. Although AM1/quinabactin has been shown to preferentially activate dimeric ABA receptors such as PYR1, PYL1, PYL2, and PYL4, AM1/quinabactin was able to produce a similar transcriptional profile as ABA in Arabidopsis. A study of the transcriptomes of seedlings treated with AM1/quinabactin and ABA showed highly correlated activation patterns, with more than 80% overlap, and the use of a GUS reporter line demonstrated both ABA- and AM1/quinabactin-triggered transcriptional responses in the guard cells and the vascular tissues of the roots and leaves.
In addition, AM1/quinabactin not only inhibited seed germination to a similar extent as ABA but also increased the drought resistance of plants in the vegetative growth stage. When exogenously applied to wild-type plants subjected to drought conditions, AM1/quinabactin induced reduced water loss, delayed wilting, higher survival rates and better recovery after rehydration compared with non-treated control plants. This compound was also able to rescue the water-loss and wilty phenotype of an ABA-deficient mutant, indicating that AM1/quinabactin functionally mimics ABA to produce similar physiological responses that ultimately increase drought stress tolerance. However, unlike ABA, which is susceptible to photodegradation, AM1/quinabactin was shown in a preliminary study to be more stable with a longer half-life when exposed to mild UV (174 min for AM1 versus 24 min for ABA), making it a more suitable and promising candidate for field applications. It will be necessary to characterize the effects of AM1/quinabactin on crop plants to further substantiate its feasibility for agricultural use.
Concluding remarks
In view of the effects of global climate change and the population boom on food demand and freshwater resources, there is an impending need to increase agricultural yields while limiting agricultural water consumption. In our constant search for solutions to increase crop production, the ability to improve stress tolerance towards water scarcity and salinity in plants is extremely valuable. In addition, enhancing abiotic stress tolerance to extend shelf-life during the long-distance transport of commercial plants is highly beneficial. Therefore, understanding abiotic stress tolerance in plants has important and widespread implications that cannot be understated.
Our understanding of the ABA pathway has progressed rapidly in the years following the discovery of bona fide ABA receptors and the elucidation of their structures. The application of such structural information for the rational design of ABA agonists has shown promising signs in the current stages. Although these newly identified compounds require further validation in the field, these studies have demonstrated the practical use of applying structural information to providing solutions for current problems. With more effort being directed toward unraveling downstream ABA signaling events and cross-talk with other pathways, the future of the ABA signaling field in plant biology and crop management remains both exciting and promising.
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Ng, L., Melcher, K., Teh, B. et al. Abscisic acid perception and signaling: structural mechanisms and applications. Acta Pharmacol Sin 35, 567–584 (2014). https://doi.org/10.1038/aps.2014.5
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