Abscisic acid analogs as chemical probes for dissection of abscisic acid responses in Arabidopsis thaliana.

Phytochemistry xxx (2014) xxx–xxx

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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Abscisic acid analogs as chemical probes for dissection of abscisic acid responses in Arabidopsis thaliana Chantel L. Benson a,1, Michal Kepka b,1, Christian Wunschel b, Nandhakishore Rajagopalan a, Ken M. Nelson a, Alexander Christmann b, Suzanne R. Abrams c,⇑, Erwin Grill b, Michele C. Loewen a,d a

National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada Lehrstuhl für Botanik, Technische Universität München, D-85354 Freising, Germany Department of Chemistry, University of Saskatchewan, Saskatoon, SK, Canada d Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, Canada b c

a r t i c l e

i n f o

Article history: Available online xxxx In honor of the 60th birthday of professor Vincent de Luca Keywords: Arabidopsis thaliana Cruciferae Abscisic acid analogs RCAR/PYR/PYL receptor PP2C phosphatases Structure–activity–function relationships

a b s t r a c t Abscisic acid (ABA) is a phytohormone known to mediate numerous plant developmental processes and responses to environmental stress. In Arabidopsis thaliana, ABA acts, through a genetically redundant family of ABA receptors entitled Regulatory Component of ABA Receptor (RCAR)/Pyrabactin Resistant 1 (PYR1)/Pyrabactin Resistant-Like (PYL) receptors comprised of thirteen homologues acting in concert with a seven-member set of phosphatases. The individual contributions of A. thaliana RCARs and their binding partners with respect to specific physiological functions are as yet poorly understood. Towards developing efficacious plant growth regulators selective for specific ABA functions and tools for elucidating ABA perception, a panel of ABA analogs altered specifically on positions around the ABA ring was assembled. These analogs have been used to probe thirteen RCARs and four type 2C protein phosphatases (PP2Cs) and were also screened against representative physiological assays in the model plant Arabidopsis. The 10 -O methyl ether of (S)-ABA was identified as selective in that, at physiologically relevant levels, it regulates stomatal aperture and improves drought tolerance, but does not inhibit germination or root growth. Analogs with the 70 - and 80 -methyl groups of the ABA ring replaced with bulkier groups generally retained the activity and stereoselectivity of (S)- and (R)-ABA, while alteration of the 90 -methyl group afforded an analog that substituted for ABA in inhibiting germination but neither root growth nor stomatal closure. Further in vitro testing indicated differences in binding of analogs to individual RCARs, as well as differences in the enzyme activity resulting from specific PP2Cs bound to RCAR-analog complexes. Ultimately, these findings highlight the potential of a broader chemical genetics approach for dissection of the complex network mediating ABA-perception, signaling and functionality within a given species and modifications in the future design of ABA agonists. Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.

Introduction The plant hormone (S)-abscisic acid (1, (S)-ABA, (+)-ABA; Fig. 1) is a key signaling molecule employed by all plants for both amelioration of responses to abiotic stress and modulation of general Abbreviations: ABA, abscisic acid; ABI, ABA insensitive; A. thaliana, Arabidopsis thaliana; HAB, homology to ABA insensitive; ITC, isothermal titration calorimetry; RCAR, Regulatory Component of ABA Receptor; PP2C, type 2C protein phosphatases; PYL, Pyrabactin Resistant-Like; PYR1, Pyrabactin Resistant 1. ⇑ Corresponding author. Address: Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK S7N 5C9, Canada. Tel.: +1 306 966 1719; fax: +1 306 966 1702. E-mail address: [email protected] (S.R. Abrams). 1 These authors contributed equally to this work.

plant growth and development (Wasilewska et al., 2008). Although the complete mechanism of ABA signal transduction mediating this breadth of physiological functions remains unclear, recent advances in the understanding of ABA perception have helped clarify some of the earlier steps (Cutler et al., 2010; Raghavendra et al., 2010). In particular, two proteins, RCAR1 (Regulatory Component of ABA Receptor 1)/PYL9 (Pyrabactin Resistant 1-Like 9) and RCAR11/PYR1 (Pyarabactin Resistant 1) were identified independently, using protein interaction analyses and chemical genetics approaches respectively, to be members of a family of fourteen homologues in Arabidopsis thaliana(A. thaliana), forming the RCAR/PYR1/PYL family of ABA receptors (Ma et al., 2009; Park et al., 2009).

http://dx.doi.org/10.1016/j.phytochem.2014.03.017 0031-9422/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Benson, C.L., et al. Abscisic acid analogs as chemical probes for dissection of abscisic acid responses in Arabidopsis thaliana. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.03.017

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C.L. Benson et al. / Phytochemistry xxx (2014) xxx–xxx

Fig. 1. Signal transduction by ABA (1) and associated assays. ABA (1) binding to the RCAR receptor, leads to closure of the gate and latch lid over the active site. The affinity of the receptor–ABA or receptor–analog interaction is measured by isothermal titration calorimetery herein. The complex presents a surface that includes the gate and latch regions, that has high affinity for the PP2C, sequestering it away from SnRK2 and inactivating it. The degree of sequestration of the PP2c can be assessed by measuring its phosphatase activity in the presence of the ligandstimulated RCAR. Once released from the inhibitory effect of the PP2C, SnRK2 stimulates downstream signalling, leading to well document physiological effects. Some these include, inhibition of seed germination, stomatal closure and inhibition of root elongation, all which can be measured.

The genetic and functional redundancy of the members of this family of receptors, which made initial identification by classical genetics impossible, was further highlighted later by the need to knock out at least three of the family members at once to elicit a change in phenotype (Park et al., 2009). This redundancy has made further functional characterization of the individual family members difficult. Indeed to date, only a few targeted studies have linked particular members of this family to specific physiological effects. For example, RCAR1 was recently shown to modulate downstream phosphorylation of the guard cell linked anion channel SLAH3, but this was only demonstrated in vitro to date (Geiger et al., 2011). Other reports link RCAR10 (PYL4) over-expression to regulation of jasmonic acid signaling (Lackman et al., 2011) and RCARs 8 and 10 (PYLs 4 and 5) over-expression to increased drought resistance (Santiago et al., 2009b; Pizzio et al., 2013)). At the same time RCAR 8 has been linked to modulation of root growth (Antoni et al., 2013). Additionally, RCAR7 (PYL13) was shown to modulate classic ABA-sensitive physiological effects, through interactions with PP2Cs, but independently of any interaction with ABA itself (Zhao et al., 2012). However, another report documents the characterization of triple, quadruple, quintuple and even sextuple RCAR mutants, targeting RCARs 3, 8, 10, 11, 12 and 14, and concluded that the family members contribute additively to roles in regulation of seed germination, plant growth and reproduction, stomatal aperture, and transcriptional response (Gonzalez-Guzman et al., 2012). Finally orthologs of the A. thaliana

receptors have been reported in a variety of other plant species including rice (Kim et al., 2012), strawberry (Chai et al., 2012; Jia et al., 2011; Li et al., 2011), grape (Boneh et al., 2012; Li et al., 2012), citrus (Romero et al., 2012), cucumber (Wang et al., 2012) and soy bean (Bai et al., 2013), with roles for these receptors broadly correlated to ABA sensitivity, ripening and stress perception processes. In general, the functional roles of individual ABA receptor family members remain to be deciphered. The regulation of ABA-mediated RCAR signaling downstream of perception appears to be very complex. On the one hand, as many as seven different members of the clade A PP2C family in A. thaliana have been implicated in ABA responses, each with independent and overlapping functions (Merlot et al., 2001; Kuhn et al., 2006; Robert et al., 2006; Saez et al., 2006; Yoshida et al., 2006; Nishimura et al., 2007; Antoni et al., 2012). While some of these PP2Cs have been shown to interact with multiple RCAR receptors, they, like the receptors themselves, are also differentially expressed throughout plant tissues during different developmental stages (Nishimura et al., 2010; Szostkiewicz et al., 2010). On the other hand, recent reports suggest that the same receptor surface that binds to PP2Cs also mediates homodimerization of a subset of the RCAR receptor family (Dupeux et al., 2011; Hao et al., 2011). While such receptor dimerization has been linked to inhibition of basal receptor activity against the PP2Cs as well as a decreased sensitivity to ABA (1) in general, a more recent report questions the biological relevance of this interaction (Antoni et al., 2012). Additionally, in contrast to inactive ABA metabolites, specific hydroxylated catabolites of ABA have been shown to interact with the receptors and inhibit the activity of associated PP2Cs, introducing the possibility of a role for ABA catabolites in regulation of signaling (Kepka et al., 2011). Together these findings suggest a complex network of interplay mediating ABA-perception and signaling that relies on spatially and temporally regulated gene expression of the genetically redundant receptors and PP2Cs, as well as regulation of signaling through protein–ligand and protein–protein interactions. Mechanistically, structural analyses of RCAR receptors have demonstrated conformational differences in ABA-bound and ABA-free receptor forms, highlighting open access of the ligand to an internal binding cavity in the unbound form (Melcher et al., 2009; Melcher et al., 2010; Miyazono et al., 2009; Nishimura et al., 2009; Santiago et al., 2009a; Yin et al., 2009; Shibata et al., 2010; Soon et al., 2012; Miyakawa et al., 2012). However, once ABA (1) has entered, and docked with its side-chain carboxyl group deepest into the cavity, two loops that are located at the entrance of the protein’s ABA binding pocket (termed the gate and latch), close over the 2,6,6-trimethylcyclohexenone ABA ring to form a ‘lid’ on the cavity, thus encapsulating ABA (1) within the receptor. The resulting hydrophobic area on the receptor surface formed by the ‘lid’ binds to a specific group of type 2C protein phosphatases (PP2Cs) including a direct interaction between a PP2C tryptophan residue and ABA (1). This tight interaction causes inactivation of the PP2C co-receptor, effectively removing the brake on ABA signal transduction and leading to well documented ABA responses (Raghavendra et al., 2010; Miyakawa et al., 2012; Fig. 1). Studies using small molecule ligands are shedding light on the structural requirements of the binding site in the cavities of the RCAR ABA receptors. Screening of large chemical libraries has led to the identification of several synthetic aromatic sulfonamides, non-ABA-like, small molecules selective for groups of receptors and physiological effects (Okamoto et al., 2013; Cao et al., 2013). One of these non-ABA related chemicals, pyrabactin, activates two of the RCAR receptors, while another, quinabactin, activates an additional three RCARs. Pyrabactin affects seed ABA processes while quinabactin has effects on stomatal closure in a number of plant species.



An alternate targeted approach to probe the structural requirements of the receptors builds on ABA as the lead molecule and alteration of the ligand’s structure to learn about the binding site of RCAR receptors. From the first studies and discovery of the RCAR/PYR/PYL receptors the stereoisomers and geometrical isomers of ABA have been employed as probes and controls in experimental design. The unnatural (R)-ABA (2) enantiomer, differing from natural ABA (1) only in the stereochemistry of C-10 , elicits responses in some, but not all, processes known to be ABA-regulated. This phenomenon has been used in a genetic screen for ABA mutations (Nambara et al., 2002). These studies have shown that the binding pockets of the individual RCAR proteins have different capacities to bind the (S)- and (R)-stereoisomers of ABA (Ma et al., 2009; Park et al., 2009; Szostkiewicz et al., 2010; Zhang et al., 2013). Most recently, an in-depth investigation focused on biochemical characterization of (R)-ABA (2) binding in nine RCARs with HAB1, as well as a crystal structure of (R)-ABA (2) bound in RCAR8, have been reported (Zhang et al., 2013). Differences in the receptor binding pockets were observed between RCARs and in the conformation adopted by the ABA ligands within the receptor. The authors suggest that alteration of the substituents on the ABA ring could lead to selective ABA agonists useful for agrochemical development. Consistent with this approach, a panel of structural analogs was developed to exploit and amplify differences in binding of ligand to receptors and in complexes with phosphatases, to tease apart the initial ABA signaling process. The panel was chosen to incorporate both (S)- and (R)-enantiomers of ABA analogs, each with single changes to the ABA ring, which would be expected to make significant differences in the fit within the binding pocket (Zaharia et al., 2005; Fig. 2). One analog pair, PBI 413 (3) and its enantiomer PBI 414 (4), differs from ABA (1) in that the vinyl methyl (C-20 , 30 and 70 ) of ABA (1) is replaced with an aromatic ring which is in the same plane as the vinyl methyl of ABA (1) and has greater steric bulk. This analog had been developed to trap and assess the biological activity of an intermediate in ABA catabolism. The bicyclic analog PBI 413 (3) and its 80 -hydroxylated analog of PBI 413 were found to have ABA activities. A second analog pair, PBI 352 (5) and its enantiomer PBI 354 (6), differ from ABA (1) in that the proton of the hydroxyl group of the ABA molecule is replaced with a methyl group. PBI 352 (5) had exhibited ABA activity in growth inhibition and stomatal aperture in wheat seedlings. A third analog pair, PBI 425 (7) and PBI 426 (8), differ from ABA (1) in that the 80 -methyl group is replaced with an acetylene group. In the conformation ABA (1) has been shown to adopt within the receptor, PBI 425 (7), in a similar conformation, would extend the carbon chain below the plane of the ABA ring, towards the lid of the binding cavity. This molecule had been shown to have significantly longer biological activity than ABA (1) itself due, at least in part, to its resistance to oxidation by plant P450 enzymes that degrade ABA (1). In Arabidopsis, PBI 425 (7) has been shown to induce drought tolerance genes and physiological responses (Huang et al., 2007). An additional analog pair (PBI 694 (9) and its enantiomer PBI 695 (10)) differed from ABA (1) by replacing the 80 -methyl group of ABA (1) with a cyclopropyl group, increasing the bulkiness around the 80 -carbon atom. The final analog pair (PBI 514 (11) and 515 (12)) which had been found to act as an irreversible inhibitor of ABA 80 -hydroxylase, differed from ABA (1) in that the 90 -methyl group was replaced with a bulkier propargyl group, that could affect the closing of the lid of the receptor. In this paper, both of the early stages of ABA perception were probed by comparative analysis of ABA (1) or analogs binding to RCAR receptors, of enzyme activity with purified ABA (1) or analog-bound A. thaliana RCAR–PP2C pairs in vitro, including all thirteen relevant RCARs, and activity in physiological assays for germination inhibition, root growth inhibition and stomatal

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Fig. 2. Structures of (S)-ABA (1), (R)-ABA (2) and (S)-ABA like analogs PBI 413 (3), 352 (5), 425 (7), 514 (11) and 694 (9). The structures are drawn in the approximate conformation adopted by (S)-ABA (1) bound to PYL1 (Miyazono et al., 2009). The dashed line represents the added steric bulk of the analogs relative to (S)-ABA (1). The corresponding (R)-ABA-like analogs PBI 414 (4), PBI 354 (6), PBI 426 (8), PBI 515 (12) and PBI 695 (10) are not shown.

aperture, all in A. thaliana. The study was, however, confined to the model system A. thaliana and its RCAR phosphatase complexes to eliminate confounding factors including species differences. Results Comparison of the effects of ABA (1) and ABA analogs on key physiological processes The responses of ABA (1) and the analogs were tested in three different physiological assays so that differences in RCAR responses in ABA-analog-induced phosphatase activity profiles could be correlated to specific physiological plant responses. First, the effect of ABA (1) and analogs on germination inhibition of A. thaliana – Ler seeds was measured using analog concentrations of 3 lM (Fig. 3A). After 3 days, (S)-ABA (1) reduced germination by almost 100% relative to the control. For most of the pairs of analogs studied, the (S)-ABA-like enantiomers gave results comparable to, or better than (S)-ABA (1), while the (R)-ABA-like enantiomers were relatively ineffective. This was the case for (S)-ABA (1) and (R)-ABA (2), as well as analog pairs substituted at the 80 position (PBI 425 (7)/PBI 426 (8) and PBI 694 (9)/PBI 695 (10)). However, the bicyclic compound PBI 414 (4) was moderately active and (R)-ABA-like 90 -propargyl PBI 515 (12) was comparable to the (S)-analogs. The 10 -methyl ether enantiomers PBI 352 (5) and PBI


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Fig. 3. Physiological response of A. thaliana seedlings to (S)-ABA (1) and ABAanalogs. (A) Germination of A. thaliana – Ler seeds in presence of 3 lM (S)-ABA (1) and the enantiomeric pairs of ABA-analogs within 3 days (n > 100). (B) Root growth of 5-day old seedlings in the presence of 3 lM ligand within 3 days. (C) Stomatal aperture of epidermal peels exposed to (S)-ABA (1) and diverse ABA-analogs (3 lM).

354 (6) did not exhibit any activity, illustrating that either a proton donor at the C-1 position is critical for this ABA response or that the bulky methyl group has an effect on binding in the active site. The effects of ABA (1) and analogs on root growth inhibition of 5 day old A. thaliana seedlings were measured and compared (Fig. 3B). After 3 days, the roots of the control seedlings grew to an average length of 13 mm. (S)-ABA (1) treated samples (3 lM) exhibited extremely reduced growth with roots reaching only 2 mm in length. Once again, (S)-ABA like analogs PBI 413 (3), PBI 425 (7), PBI 514 (11), and PBI 694 (9) had similar activity to ABA (1), although none of them outperformed the natural compound in this assay. The (R)-ABA like enantiomers PBI 414 (4), PBI 426 (8), PBI 515 (12) and PBI 695 (10), as well as the enantiomeric pair PBI 352 (5)/PBI 354 (6), were at least as ineffective as (R)-ABA (2). Finally, the direct effects that ABA (1) and analogs (3 lM) had on stomatal aperture in leaf epidermal peels were measured (Fig. 3C). The stomatal aperture (ratio of pore width to pore length) in untreated samples was found to be 0.63, while in (S)-ABA (1) treated tissue it was 0.28. Treatment with (R)-ABA (2) had relatively little effect. The (S)-ABA like analogs PBI 413 (3), PBI 425 (7), and PBI 514 (11) all displayed very similar effects as (S)-ABA (1), while (R)-ABA like analogs were comparable to (R)-ABA (2). Interestingly, PBI-352 (5), the analog with the methyl group replacing the proton of the hydroxyl group of ABA (1), had as strong or

Fig. 4. ABA (1) and synthetic analogs differentially regulate PP2Cs via RCAR1. (A) ABI2 activity regulated by RCAR1 through various enantiomeric ABA analog pairs in vitro. The protein phosphatase activity was analyzed in the presence of 10 lM ligand at a constant molar ratio of RCAR1 and ABI2 of 2 to 1. (B) Inhibition of ABI2 phosphatase activity by increasing concentrations of (S)-ABA (1) and various enantiomeric ABA-like analog pairs (n = 2). (C) Physiological activity of ABA-like analogs in regulating ABA-responsive reporter expression. The ABA-induced upregulation of gene expression was monitored using the ABA-responsive reporter constructs pRD29B::LUC in A. thaliana protoplasts and was measured as relative light units (RLU/RFU). Each data point represents the mean value of three independent transfections at a ligand concentration of 3 lM. (D) ABI1, PP2CA and HAB1 activity regulated by RCAR1 against various enantiomeric ABA analog pairs. The protein phosphatase activity was analyzed in the presence of 10 lM ligand at a constant molar ratio of RCAR1 to phosphatase of 2:1 in vitro.

stronger effects on stomatal aperture as the natural hormone. The O-methyl ether analog had little effect on germination and root growth inhibition, and no apparent toxicity to the seed or seedling.



Comparison of the effects on ABI2 activity mediated by ABA (1) or analogs and RCAR1 In order to compare the effects of the selected analogs in vitro, measurements were made of the extent of inhibition as a result of analog treatment in the presence of RCAR1 (Fig. 4A). (S)-ABA (1) strongly reduced ABI2 activity (95% residual activity) relative to the ‘no-analog’ control (100% ABI2 activity). This indicates enantioselectivity of the RCAR1 receptor for (S)-ABA (1) in accordance with previous results (Szostkiewicz et al., 2010). The (S)-ABA like analogs, PBI 413 (3), PBI 425 (7) and PBI 514 (11) were all found to be good receptor agonists, inhibiting ABI2 through RCAR1, with enzyme activity dropping to 5% or less. PBI 694 (9) was slightly less potent, yielding 20% residual activity. The corresponding (R)-ABA like enantiomers, PBI 414 (4), PBI 426 (8), PBI 515 (12) and PBI 695 (10) were all about equally ineffective in reducing ABI2 activity through RCAR1, showing 40–60% residual activity. Interestingly, the substitution of the hydroxyl proton with a methyl group has the greatest effect in reducing activity. The 10 -methyl ether analog PBI 352 (5) was relatively weak yielding only 40% ABI2 residual activity in the presence of RCAR1, while its enantiomer, PBI 354 (6), was virtually inactive (90% residual activity) in the ABI2 inhibition assay. Evaluation of the concentration dependence of some of the more effective ABA analogs for the inhibition of ABI2 in the presence of RCAR1 yielded similar IC50 values for (S)-ABA (1), PBI 413 (3), PBI 425 (7) and PBI 514 (11) at 58, 56, 44 and 51 nM, respectively (Fig. 4B). PBI 694 (9) was slightly less potent with half maximal inhibition at 116 nM and PBI 352 (5) was the least potent with an IC50 measuring 1.4 lM. These activities generally reflect the relative ability of each of the analogs to induce the ABA-responsive reporter construct pRD29B::LUC (Fig. 4C) (Uno et al., 2000). While the tetralone analog PBI 413 (3) gave a stronger response than (S)ABA (1), this value falls within the statistical error of the values obtained for PBI 425 (7) and PBI 514 (11), at approximately equal to (S)-ABA (1). On the other hand, PBI 694 (9) showed a slight gene induction relative to controls. Comparison of the effects of different phosphatases on RCAR1/ABA and analog activity profiles In order to gain insight into the relationship between ABA-analog activities and the different PP2Cs, the same series of analogs was screened for their ability to inhibit the activity of three additional PP2Cs, ABI1, PP2CA (AHG3) and HAB1, in the presence of the same

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RCAR1 (Fig. 4D). All three phosphatases were similarly affected by (S)-ABA (1), and its analogs through RCAR1, compared to ABI2. This trend held for most analogs tested, with a few minor exceptions. Amongst the (R)-like analogs (e.g. (R)-ABA (2), PBI 414 (4)) PP2CA activity was mildly more sensitive in the presence of RCAR1. This finding correlates with a previous report showing PP2CA as a strong negative regulator of ABA signal transduction (Kuhn et al., 2006). Interestingly, HAB1 shows selective insensitivity to PBI 352 (5), while ABI1 shows selective insensitivity to PBI 515 (12), similar to ABI2. Comparison of effects on PP2C enzyme activity of different RCARs with respect to ABA (1) and analogs In order to probe the effects of ABA-analogs and different RCAR receptors, the same set of ABA-analogs was tested with ABI2 in the presence of each A. thaliana RCAR (Fig. 5), excluding RCAR7 (Xhao et al., 2013; Fujii et al., 2009). It is important to note that 100-fold lower concentrations of ligands were applied in this experiment to afford the most detailed possible assessment of relative ligand activities. (S)-ABA (1) showed relatively strong and, in some cases, even near complete inhibition of ABI2 across all RCARs tested. On the other hand, (R)-ABA (2) did not inhibit ABI2 to any great extent through any RCAR, under the conditions tested here. Similar to the ‘all on’ or ‘all off’ activity of the ABA enantiomers, the (S)-ABA-like analogs PBI 413 (3), PBI 425 (7) and PBI 514 (11) were found to be good inhibitors of ABI2 activity for most of the RCARs tested, showing residual ABI2 activities ranging from 5% to 45%. However, (S)-ABA-like analogs PBI 352 (5) and PBI 694 (9) showed a greater range of activity against the receptors (15–90% residual activity), both being relatively potent against RCAR 8, but PBI 694 (9) showing additional potency against RCAR 9. This is representative of an observed trend in which clade II members (RCARs 5–10) are generally more sensitive to (S)-ABA (1) and synthetic compounds, with clade I (RCARs 1–4) and clade III (RCARs 11–14) members being generally less sensitive. Interestingly, (R)-analogs PBI 354 (6), PBI 414 (4), PBI 426 (8), PBI 515 (12), and PBI 695 (10) were significantly less active against the RCARs than (S)-ABA-like analogs across the board. In particular PBI 426 (8), like PBI 694 (9), was found to be selectively more potent against RCARs 8 and 9. The (R)-ABA like 80 -cyclopropyl substituted PBI 695 (10) was for the most part not very effective, but did show slightly more potency (down to 70% residual ABI2 activity) against four of the receptors, compared to (R)-ABA (2). Finally, PBI 354 (6) and PBI 414 (4) both showed only weak activity against the phosphatases, but did break with the clade II potency trend, showing marginally stronger activity against select clade I and III receptors.

Fig. 5. ABA analog activity profiles are dependent on the identity of the RCAR pair. ABI2 activity regulated by A. thaliana RCARs against various enantiomeric ABA analog pairs. The protein phosphatase activity was analyzed at a constant molar ratio of RCAR to ABI2 of 2:1 in vitro at a constant analog concentration of 100 nM (n = 3).


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comparison of phosphatase inhibition induced by ABA (1) with four different phosphatases and either clade II RCAR8 or clade I RCAR1. Differences are observed (Fig. 6A) with the RCAR1–ABA complex having slightly lower potency against HAB1 (IC50 0.10 ± 0.01 lM) compared to either of the ABI1/2 (IC50 of 0.07 ± 0.01 and 0.06 ± 0.01 lM) or PP2CA phosphatases (IC50 0.08 ± 0.01 lM). In contrast the RCAR8–ABA (Fig. 6B) complex had the lowest potency against the PP2CA phosphatase (IC50 0.05 ± 0.01 lM compared to 0.02 ± 0.01 lM for the others). This trend is amplified in the comparable experiments in which PBI 352 (5) is tested in the place of ABA (1), where an IC50 of 3.5 ± 0.50 lM was detected for RCAR1– PBI 352–HAB1 compared to 1.2 ± 0.3 lM for all the others three PP2Cs tested against RCAR1–PBI 352 (Fig. 6C); and where an IC50 of 1.0 ± 0.10 lM was detected for RCAR8–PBI 352-PP2CA compared to 0.07 ± 0.01 lM for all the others three PP2Cs tested against RCAR8–PBI 352 (Fig. 6D).

Correlation of ABA (1) and analogs binding affinity with RCAR8

Fig. 6. Dose responses of RCARs 1 and 8 to ABA (1) and PBI 352 (5) are dependent on the identity of PP2Cs. The dose response of RCAR 1 (A) and RCAR8 (B) to ABA (1) was tested against phosphatases ABI1, ABI2, HAB1 and PP2CA. The dose responses of RCAR1 (panel C) and RCAR 8 (panel D) to PBI 352 (5) was also tested. The protein phosphatase activity was analyzed at a constant molar ratio of RCAR to PP2C of 2:1 in vitro at the indicated concentrations of ligand (n = 3).

A more detailed evaluation of the dose responses of the phosphatase–receptor–analog interactions is revealed in the

The in vitro binding affinities of RCAR8 to (S)-ABA (1) and selected ABA analogs were determined by isothermal titration calorimetry (ITC; Fig. 7). Titration of (S)-ABA (1) into a solution of purified RCAR 8 yielded a saturating binding isotherm showing large heat release and a dissociation constant (Kd) of 1.2 lM with an enthalpy (DH) of 9.0 kcal mol1 and an entropy value (DS) of 3.1 cal mol1 deg1 (Fig. 7A). The stoichiometry (N) of binding was approximately 0.5, which suggests a 1:1 binding of RCAR8 to (S)-ABA (1), assuming some degree of receptor proteolysis or aggregation during the measurement. These results correlate very well with the observations of Rodriguez and coworkers, who

Fig. 7. Isothermal titration calorimetric analysis of the binding ABA (1) and select ABA-analogs to RCAR8. Raw data of sequential injections of (A) ABA (1), (B) PBI 413 (3), (C) PBI 414 (4), (D) PBI 425 (7), (E) PBI 426 (8), (F) PBI 352 (5) and (G) PBI 354 (6) into RCAR8 (13.5 lM). The processed results are shown below each isotherm. 1 ll injections of the ligands were performed until the binding saturation was reached. The data were fitted using the ‘one set of sites’ option in the Origin software and the best fit is represented by the black line.



reported a Kd of 1.1 lM for the same receptor–ligand combination (Dupeux et al., 2011). The (S)-enantiomer of the tetralone derivative PBI 413 (3) yielded a Kd of 0.7 lM (DH = 11.6 kcal mol1 and DS = 10.8 cal mol1 deg1; Fig. 7B). This represents a slightly higher affinity compared to the natural ligand. The enthalpy gain in the interaction of PBI 413 (3) with the receptor (11.6 kcal mol1; N = 0.6) compared to that observed in the case of ABA (1) (9.0 kcal mol1) suggests that there might be additional noncovalent interactions involved in the PBI 413 (3) interaction. The (R)-enantiomer PBI 414 (4) did not show any measurable binding (Fig. 7C), correlating with its relatively weak activity in the in vitro receptor assay (Fig. 5). It is important to note that this ITC result does not mean that PBI 414 (4) does not bind to RCAR8. It likely does still bind (as demonstrated by the in vitro activity data), but with a Kd > than 100 lM. The (S)-ABA like 80 -acetylene analog PBI 425 (7) showed slightly weaker binding, with a Kd of 6.3 lM (Fig. 7D) while the (R)-ABA like 80 -acetylene analog PBI 426 (8) showed significantly weaker binding with a Kd of approximately 67 lM (Fig. 6E). This correlates with the observed potency of PBI 426 (8) against RCAR8 (Fig. 5) compared to PBI 414 (4). Finally a comparison of PBI 352 (5) (Fig. 7F) and PBI 354 (6) (Fig. 7G) binding characteristics yielded Kd’s of 34 and 31 lM, respectively. While this PBI 352 (5) value correlates with its intermediate in vitro activity against RCAR8 in vitro (Fig. 5) that of PBI 354 (6) does not. This suggests that PBI 354’s (6) lack of potency against RCAR8 in vitro is not related to reduced ligand binding affinity, but possibly to some other factor, such as ineffective modulation of the interaction with the PP2C.

Discussion Now with the screening of ABA receptors and phosphatase pairs in hand, plant growth regulator development can be accelerated and deeper investigations into specific processes in the ABA signal transduction pathway can be elucidated. The synthetic analogs of (S)-ABA (1) used in this study have previously been reported to retain (S)-ABA-like activity in physiological assays measuring effects on plant stress tolerance, growth, germination, and induction of genes associated with ABA response (Zaharia et al., 2005). A selected series of enantiomeric pairs of ring carbon substituted ABA analogs were tested directly on a series of A. thaliana ABA receptor complexes in vitro, as well as in planta to gain a clearer perspective of ABA structure–activity and ABA receptor–physiological relationships. In the instance of physiological assays conducted over several days, differential effects related to uptake or rate of metabolism of (S)-ABA (1) versus analogs cannot be ruled out. However the stomatal assay is rapid, minimizing the effects of metabolism. The observation that (S)-ABA (1) and (S)-ABA like analogs have similar activities in inhibition of root growth and germination, suggests uptake is not an issue. As well, previous work showed uptake of (R)-ABA (2) and select ABA analogs comparable to (S)-ABA (1) (Perras et al., 1997; Cutler et al., 2000; Huang et al., 2007). From a structure–activity perspective, the alterations of (S)-ABA (1) at C-80 , C-90 and with the C-30 –C-70 aromatic ring as in analogs PBI 425 (7), PBI 514 (11) and PBI 413 (3) respectively, with addition of bulk to the parent ABA molecule at the 80 - and 90 -carbon atoms or at the 20 -, 30 -carbon atoms, did not lead to any significant reduction in activity compared to (S)-ABA (1) in either the in vitro receptor assay or in the physiological assays. PBI 694 (9), modified with a larger cyclopropyl group at C-80 , maintained strong in vitro activation capabilities, particularly for the clade II members RCAR8 and 9. This observation correlates with a selective reduction in regulation of root growth responses only, with this compound showing (S)-ABA-like regulation of germination and stomatal aperture.

7

These latter effects correlate to observed reduced potency of the PBI 694 (9) in quantitative dose response experiments (Fig. 4B), suggesting limitations in the bulk that can be accommodated at C-80 . In contrast, substituting the hydrogen of the OH on the C-10 with a methyl group, in PBI 352 (5), affords an analog selective in inducing stomatal closure but not affecting germination or root growth of Arabidopsis seedlings. This selectivity has potential utility for practical applications wherein temporary drought protection could be provided to a plant without reducing concomitant root growth. This analog PBI 352 (5) reduced receptor activity more significantly compared to (S)-ABA (1) for all of the RCARs tested. Thus, the extra bulk on the oxygen of PBI 352 (5) seems to be somewhat more deleterious to ligand binding processes in which germination inhibition and root growth inhibition are the resulting physiological effects, likely related to space in the receptor being limiting in the region of the hydroxyl group and possibly disrupting the hydrogen bond network in the active site. From a structure–function perspective, attempts at computationally docking of the ABA analogs into RCAR binding sites using available 3D structures of RCARs (RCBS Protein Data Bank; http://www.rcsb.org/) and the AutoDock software (Morris et al., 1998), yielded an uninformative variety of equally favorable binding conformations for each analog (data not shown); highlighting that one cannot assume that the (S)- and (R)-analogs are binding in the same orientation as (S)- and (R)-ABA respectively. Thus reliable structural insight about the analogs’ binding awaits ongoing co-crystallization of RCARs with the ABA analogs, and we refrain from speculative discussion at this time. However, differential effects and binding affinities for different analogs are noted against different receptors in vitro and these may at least be explained in part by evaluation of conservation, localization and dynamics of amino acids comprising the A. thaliana RCAR (S)-ABA (1) binding site (Mosquna et al., 2011), and recent comparison of these to the (R)-ABA (2) binding site crystal structure (Zhang et al., 2013). Together these suggest the possibility of a selectivity filter modulating interactions of the receptor with the ABA-analogs. Such a selectivity filter would include variable residues located in the ligand binding site, across all thirteen RCARs. Some obvious variable sites in the binding pocket include I90 and V111 in the gate region, F136 and V138 in the latch region and V185, I188 and V189 in the final helix (RCAR8 numbering; Fig. 8A). These residues are situated across the ABA binding site from each other and the variations in amino acid identities here could be expected to yield differential constraints on the size and shape of the binding site in terms of accommodating extra bulk and the (R)-ABA like stereoisomers (Fig. 8B). Comparisons of representative members from the three evolutionary clades of this family suggest extra space in RCAR1 around the ABA side-chain, compared to either RCARs 8 or 11, related mainly to an F136I variation. More relevant to the discussion here on ring-modified analogs, extra space in RCAR8 around the ABA ring region is predicted compared to RCAR1 due to a V138I variation. While RCAR11 also maintains the larger I138 side chain variation like RCAR1, this bulk may be partially offset with the variation to A in place of V across the pocket at position 185. Thus we predict from this in silico analysis that RCAR8 and possibly RCAR11 might better accommodate ring modifications and the (R)-ABA like stereoisomers than RCAR1. Indeed this concept was recently demonstrated, where variations at residue equivalents to RCAR8 positions 138 and 188 in PYL9 (RCAR 1) and PYL3 (RCAR13) were shown to dictate the receptor’s ability to bind (R)-ABA (2) (Zhang et al., 2013). Smaller V side chains at these positions allowed the reoriented 80 and 90 ring methyl groups of the (R)-ABA (2) to be accommodated, while larger I’s and L’s at these sites inhibited the (R)-ABA interaction.


8


Fig. 8. Variations in the ABA binding-sites of RCARs 1, 8 and 11. (A) Tabulation of the only variations that occur among the ABA-binding site/ABA-interacting residues in A. thaliana RCARs. Sequences were aligned using ClustalW (Larkin et al., 2007). Residue numbering is according the RCAR8. (B) Structural comparison of the five variable ABA-active site residues in RCAR1 (pink), RCAR8 (green) and RCAR11 (orange). Numbering is according to RCAR8. Distances measured within given structures are indicated with dashed lines in the associated color. The hydrogen bond with conserved residue Lys87 is shown in black. Coordinates were obtained from the PDB (RCBS Protein Data Bank; http://www.rcsb.org/) including PDB ID’s: 3OQU (RCAR1), 3QRZ (RCAR8) and 3K90 (RCAR11). Structures were superposed and distances measured using Coot (Emsley et al., 2010). High resolution images were produced using VMD (Humphrey et al., 1996). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Analysis of the experimental results obtained here for in vitro screening further supports this model. At both high and low (R)-ABA (2) levels (Fig. 4D and Fig. 5) only very weak inhibition of ABI1, ABI2 and HAB1 are observed for the constrained RCAR1. This correlates with the report by Zhang et al. (2013), which in particular demonstrated that RCAR’s 1, 4, 5 and 6 (all of which maintain a larger I residue at RCAR8 138 equivalent positions) were insensitive to (R)-ABA (2), while RCAR’s 8, 9, 10 and 13 (all with a small V residue at RCAR8 138 equivalent positions) were potently modulated by (R)-ABA (2). However these were only tested against HAB1. Interestingly the combination of RCAR1 with (R)-ABA (2) was more potent against PP2CA, than either ABI1 or HAB1 (Fig. 3D), highlighting that a receptor ‘selectivity filter’ cannot account for the entire regulatory mechanism. Indeed this latter observation supports a case for (R)-ABA (2), and potentially other analogs, in modulating the RCAR–PP2C interface more directly and interacting with residues of the phosphatase directly, much as (S)-ABA (1) does, but yielding different effective/selective interactions. Such selective interactions by (R)-ABA (2) may be responsible for the very weak and selective effect observed for (R)-ABA (2) against germination (Fig. 3) and as reported by others (Milborrow, 1974; Nambara et al., 2002; Cutler et al., 2010). This general idea of analog-selective physiological effects being mediated by selective RCAR–PP2C–analog interactions is further evidenced by the effect of the O-methyl substituted ring hydroxyl (S)-analog, PBI 352 (5), which generally is not a very potent agonist against ABI2 regardless of the RCAR, with the exception of RCARs 8

or 9 (Fig. 5). Further examination of the case of PBI 352 (5) and RCAR8 demonstrated that this combination is moderately potent against HAB1, as well as ABI 1 and 2, compared to (S)-ABA (1) (Fig. 4 and Fig. 6D), but is only a weak inhibitor of PP2CA. Thus one could conclude that the observed physiological selectivity of PBI 352 (5) for regulation of stomatal function, having no impact on germination or root growth, is the result of the selective interaction of PBI 352 (5) for RCAR8 with HAB1, ABI1 or ABI2. However when tested against knockdowns of RCARs 8 and 9 (data not shown), the effect of PBI 352 (5) was identical to its effect against WT Arabidopsis, suggesting that the situation is more complicated. Indeed the observed potency of PBI 426 (8) against the RCAR8– ABI2 complex (Fig 5), but its lack of any effect on stomatal function, further emphasizes this. In addition, while not evident in the broad all-RCARs versus ABI2 screen, some selective potency was observed in the case of RCAR1, where PBI 352 (5) was moderately potent against ABI1 and PP2CA, but relatively ineffective against HAB1 and ABI2 (Figs. 4D, 5 and 6), compared to (S)-ABA (1). Thus, together these results highlight that in the RCAR8 knockdown experiment, a potential interaction of PBI 352 (5) with RCAR1 in complex with ABI1 or PP2CA might be contributing to the observed selective stomatal regulation. Testing of a double RCAR8, RCAR1 knockdown might shed additional light on this hypothesis; however in this instance it might be more efficient to screen all the RCAR–PP2C complexes against PBI 352 (5) and then initiate targeted in planta work to validate the screening results. Similarily, other discrepancies observed in our results, for example between PBI 426 (8) and PBI 515 (12), which show similar potencies in vitro against ABI2 (Fig. 5) and similar effects on root growth and stomatal aperture, but differential effects on inhibition of germination, may be explained by the opposing effect of these analogs on PP2C selectivity when assayed against RCAR1 and diverse PP2Cs (Fig. 4D). Again a broader screening of all RCAR–PP2C complexes against these two analogs could provide the mechanism of this physiological difference. Overall these results emphasize the complexity of the redundant network of protein and protein–ligand– protein interactions mediating the ABA response, but also highlight the potential RCAR/PP2C selectivity that can be achieved in an ABA-analog, and the potential in subsequently applying such an analog to deciphering mechanistic aspects (Lin et al., 2005). In this context of using ABA analogs as chemical probes in resolving ABA-related redundancies and mechanisms of action, the following are noted: The effects of ABA analogs are likely species-specific. For example as above, PBI 352 (5) selectively elicits (S)-ABA-like stomatal aperture responses in A. thaliana. This is in contrast to the effect reported for PBI 352 (5) in wheat, where it had weak effects on both germination and transpiration (i.e. stomatal regulation) (Rose et al., 1996b). Similarly, PBI 354 (5) was found to be a very potent inhibitor of germination in wheat (Rose et al., 1996b), but had no effect in the work reported here on germination in A. thaliana. Thus analog profiles obtained related to receptor–physiological relationships should not be extrapolated to other species. However, the identification of trait specific probes for a given species, such as PBI 352 (5) in A. thaliana, can serve as powerful analytical tools. Two other ABA analogs, PBI 414 (4) and PBI 515 (12) are moderately potent in the A. thaliana germination assays and relatively ineffective in the root growth and stomatal aperture assays. While the potency of these are both weak at 100 nM ligand concentrations, much higher potency is observed at 10 lM concentrations, including some distinct PP2C selectivity (Fig. 4D). While a full in vitro screen of the fourteen receptors against all known related PP2Cs against these analogs would again likely reveal the select RCAR–PP2C pair(s) relevant to the selective activity of PBI 414 (4) and PBI 515 (12), as also suggested for PBI 352 (5) above, such an expanded screen goes beyond the scope of this work which was to conduct a survey of the analogs’



potential application. Indeed an expanded screen might best be conducted with respect to a specific study focusing on one trait, analog, receptor or phosphatase, and should take the wealth of available transcriptomic data into consideration. Together in A. thaliana, PBIs 352 (5), 414 (4) and 515 (12) could be used as probes to study aperture-specific versus germination-specific ABA mechanisms of action. It should also be noted that correlating broader transcript profiling of plants treated, respectively with these compounds could lead to the identification of novel gene targets involved uniquely in one or the other physiological effect. Indeed such experiments were recently reported wherein application of chemically more stable and potent ABA-mimetics, PBI 425 and and its racemic analog PBI 429, led to the identification of various novel genes in ABA signaling and metabolism, including a putative hydroxysteroid dehydrogenase involved in inhibition of germination (Li et al., 2005, 2007; Huang et al., 2007). Conclusions Overall, genetic redundancy within the ABA receptors and associated signaling components has made it difficult to unravel individual functions of components as a basis for understanding the intricacies of ABA signaling across the phytohormone’s diverse functionalities. The findings reported herein highlight how a series of synthetic ABA analogs can be used as chemical probes to dissect functional redundancy within the ABA receptor family. In particular these findings demonstrate how these analogs can lead to novel hypotheses related to the roles of specific RCAR–PP2C complexes and how they can serve as physiological trait-specific probes for A. thaliana. It can be speculated that with expanded screening, it may be possible to use these distinctive analog activity patterns to map the functional activities of additional players in the complex network of ABA signaling and transport in A. thaliana (Kang et al., 2010; Kuromori et al., 2010; Kharenko et al., 2011). Earlier reported variations in analog interactions across species further suggest merit in applying such an expanded screen to tease out species-specific mechanisms and related functionalities in the longer term.

9

Sequences accession numbers Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: RCAR1 – At1g01360; RCAR2 – At4g01026; RCAR3 – At5g53160; RCAR4 – At4g27920; RCAR5 – At5g46860; RCAR6 – At5g45870; RCAR7 – At4g18620; RCAR8 – At5g05440; RCAR9 – At2g40330; RCAR10 – At2g38310; RCAR11 – At4g17870; RCAR12 – At5g46790; RCAR13 – At1g73000; RCAR14 – At2g26040; PP2CA (AHG3) – At3g11410; ABI2 – At5g57050; ABI1 – At4g26080; HAB1 – At1g72770. Plasmid constructs The pRD29B::LUC reporter plasmid used in this work has been described previously (Ma et al., 2009; Moes et al., 2008). All RCARs and ABI1/2 and PP2CA constructs used in this study were generated as described by Ma et al. (2009) and Szostkiewicz et al. (2010) (Kepka et al., 2011). For ITC analyses, the cDNA of RCAR8 was amplified with the primer pair 50 -CACCATGAGGTCACCG GTGCAACTCCAAC-30 and 50 -TTATTATTGCCGGTTGGTACTTCGA GCCAGAG-30 . The PCR fragment was cloned into the pENTR.D.Topo vector (Invitrogen) and subsequently recombined into the pDEST17 vector (Invitrogen) according to the manufacturer’s specifications, yielding pDEST17-RCAR8. Expression and purification of RCARs and PP2Cs

Chemicals were obtained from Sigma–Aldrich (http://www.sigmaaldrich.com), Fluka (part of Sigma–Aldrich), Roth (http://www.carlroth.com), AppliChem (http://www.applichem.com), and J.T. Baker (http://www.mallbaker.com). (S)-ABA (1) was purchased from Lomon Bio Technology (http:// www.lomonbio.com). The ABA analogs were obtained by methods previously described; (R)-ABA (2) (Dunstan et al., 1992), PBI 352/ 354 (5/6) (Rose et al., 1996a), PBI 425/426 (7/8) (Rose et al., 1997), PBI 514/515 (11/12) (Cutler et al., 2000b) and PBI 413/414 (3/4) (Nyangulu et al., 2006). The 80 -cyclopropyl ABA analogs PBI 694 (9) and 695 (10) were synthesized and resolved similar to the method previously described for PBI 425/426 (7/8) by substituting ethynylmagnesium bromide with cyclopropylmagnesium bromide.

His-tagged RCARs and ABI1/2 as well as PP2CA proteins were expressed in Escherichia coli strain M15, BL21De3Star or BL21-AI as deemed appropriate essentially as described previously (Ma et al., 2009; Kepka et al., 2011). Briefly, cells were grown overnight in Luria Bertani medium with appropriate antibiotic selection and used for inoculations of 1 L of culture. The cells were grown at 37 °C with shaking until an optical density at 600 nm of 0.5–0.6 was reached. Protein expression was induced by administration of isopropyl-b-D-thiogalactopyranoside (0.5 mM final concentration) or in the case of BL21-AI cells L-arabinose (0.2% final concentration). The cells were harvested at 4 °C and 4000g for 30 min at 2 h (PP2Cs), 4 h (RCARs for activity analyses) and 16 h (RCAR8 for ITC; induced at 16 °C) after induction. The cell pellet was used directly for purification. The pellet was dissolved in lysis buffer (10 mL, 50 mM NaH2PO4, 300 mM NaCl, and 5 mM imidazole, pH 8.0) supplemented with lysozyme (1 mg mL1 final concentration) for 30 min. Cells were subsequently disrupted by sonication on ice (six times for 10 s). The protein lysate was obtained after centrifugation at 4 °C and 25,000g for 30 min and loaded onto a nickel–Tris(carboxymethyl)ethylene diamine column (Macherey– Nagel; http://www.macherey-nagel.com). Washing buffer (8 mL, 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole, pH 8.0) were applied to the column to remove non-speecifically bound proteins. Proteins of interest were eluted with of elution buffer (4 mL, 50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole, pH 8.0). Fractions of eluate (1 mL) were collected and dialyzed twice against dialysis buffer (100 mM Tris–HCl, 100 mM NaCl, and 2 mM dithiothreitol, pH 7.9). Fraction 2 was used in the phosphatase assays.

Plant material

Phosphatase assays

A. thaliana lines used in this work were ecotype Columbia and Landsberg erecta (Ler). Plants used for protoplast isolation were grown for 4 weeks in a perlite–soil mixture in a controlled growth chamber at 23 °C under long-day conditions with 16 h of light (250 lE m2 s1; (Moes et al., 2008)).

Phosphatase activity was measured using 4-methyl-umbelliferyl-phosphate as a substrate (Meinhard and Grill, 2001; Ma et al., 2009). Values are means ± SD of three–four replicates. Regulation of PP2C activity is expressed relative to the RCAR-dependent inhibition at 1 mM (S)-ABA. Control experiments of ABI2 activity in the

Experimental procedures Chemicals


10


presence of ABA analogs showed no changes (less than 3%) in activity in the absence of RCARs.

measured on digitized images using ImageJ software ((Moes et al., 2008); http://rsbweb.nih.gov/ij/).

Protoplast analysis

Acknowledgements

Preparation and analysis of A. thaliana protoplasts was performed as described (Moes et al., 2008). A. thaliana protoplasts were transfected with DNA (10 lg) of the reporter construct (pRD29B::LUC) and 2 lg of p35S::GUS plasmid as a control for internal normalization of the expression. Protoplast suspensions were incubated in the presence or absence of ABA and ABA analogs after transfection.

We thank A. Cutler (National Research Council (NRC) of Canada) and M. Surpin (Valent Biosciences Corp.) for critical comments on the manuscript. We are grateful to S. McKenna, University of Manitoba, for discussions and the use of his iTC200 instrument. We thank J. Boyd (NRC), for cloning of RCAR8 and in silico docking of ABA-analogs. We thank C. Gordon and F. Ball (NRC) for discussions regarding ABA- and phosphatase-binding sites. This work was supported by the Deutsch Forschungsgemeinschaft (grant Nos. GR938/6 to E.G. and CH182/5 to A.C.), by the Bayerisches Staatsministerium für Wissenschaft, Forschung und Kunst (FORPLANTA; to E.G.), the National Research Council (NRC) of Canada Plants for Health and Wellness program (to S.R.A., and C.B.) and the NRC Genomics and Health Initiative with Valent BioSciences Corp. (to S.R.A., M.C.L., and N.R). This article is National Research Council of Canada paper #54662.

Isothermal titration calorimetry RCAR8 to be used for isothermal titration calorimetry (ITC) was purified by His-tag affinity purification followed by size exclusion chromatography (Superdex 200 pg HiLoad 26/600, GE Healthcare) and dialyzed against ITC buffer (100 mM Tris pH 7.9, 100 mM NaCl, 0.3 mM MnCl2 and 0.25 mM TCEP) for 12 h. A Microcal iTC200 instrument (GE Healthcare) with a cell volume of 200 ll was employed for this study. Stock solutions (100 mM) of ABA and analogs were diluted to the required concentration using the ITC buffer. The ITC experiments were performed at 25 °C. The protein, ligands and buffer were equilibrated to room temperature and de-gassed before performing the experiment. The cell contained 200 ll of 13.5 lM RCAR8. A 40 ll injector was used to deliver 20–39 injections (1 ll each) of 0.25 mM ABA or analogs into the sample cell. The first injection (0.5 ll) was excluded from data processing. The reaction was continuously stirred at 500 rpm. The data were processed using the Origin for ITC software. Bioassays of stomatal closure in epidermal strips Strips of abaxial epidermis were prepared from A. thaliana leaves by mounting 5 mm 5 mm leaf samples on glass coverslips with the help of a medical adhesive, Telesis V (Premiere Products), and transferring the coverslips to 3 cm diameter petri dishes containing incubation medium (3 mL, 10 mM MES–KOH, pH 6.15, and 50 mM KCl) and removing the mesophyll layer using a scalpel. The strips were then exposed to white light (150 lmol m2 s1) in fresh incubation medium for 2 h, with the light filtered through a water jacket. Photon flux was measured with a Li-Cor quantum sensor (Li-Cor Instruments). The temperature was maintained at 25 ± 1 °C. Test compounds were added to the medium, and the strips were kept under the same conditions for another 2 h before measuring the stomatal aperture. The width of the stomatal aperture was measured with a research microscope (Nikon Eclipse TE 200) fitted with a camera and connected to an image-analysis system. Seed germination and root elongation assays Under sterile conditions, 100–150 seeds were plated on Murashige and Skoog agar medium containing tested compounds and incubated at 4 °C for 2 d in the dark to break dormancy. The plates were then transferred to a culture room with continuous light (60 lE m2 s1) at 22 °C. After 3 d, seeds were examined with a stereo microscope. Seeds were counted, and germination rate was calculated as percentage of the total number of seeds. For root elongation assays, 5-d-old seedlings were transferred in a row to petri dishes with solidified Murashige and Skoog medium supplemented with 5 g sucrose/L and ABA or ABA analog as specified, and kept in a vertical position at 22 °C in continuous light for 3 d. Root tip position was marked every 24 h, and root lengths were

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Abscisic acid regulates root elongation through the activities of auxin and ethylene in Arabidopsis thaliana.

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Knockout of AtDjB1, a J-domain protein from Arabidopsis thaliana, alters plant responses to osmotic stress and abscisic acid.

Abscisic acid inhibits root growth in Arabidopsis through ethylene biosynthesis.

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Abscisic acid deficiency increases defence responses against Myzus persicae in Arabidopsis.

The receptor kinase IMPAIRED OOMYCETE SUSCEPTIBILITY1 attenuates abscisic acid responses in Arabidopsis.

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Designed abscisic acid analogs as antagonists of PYL-PP2C receptor interactions.

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Accumulation of eicosapolyenoic acids enhances sensitivity to abscisic acid and mitigates the effects of drought in transgenic Arabidopsis thaliana.

Major latex protein-like protein 43 (MLP43) functions as a positive regulator during abscisic acid responses and confers drought tolerance in Arabidopsis thaliana.

Abscisic acid, abscisic acid-esters and phaseic acid in vegetative terminal buds of Acer pseudoplatanus during emergence from winter dormancy.