abscisic acid analogs as chemical probes for dissection of abscisic acid responses in arabidopsis...

Phytochemistry xxx (2014) xxx–xxx

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Phytochemistry

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

http://dx.doi.org/10.1016/j.phytochem.2014.03.0170031-9422/Crown Copyright � 2014 Published by Elsevier Ltd. All rights reserved.

Abbreviations: ABA, abscisic acid; ABI, ABA insensitive; A. thaliana, Arabidopsisthaliana; 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 306966 1719; fax: +1 306 966 1702.

E-mail address: [email protected] (S.R. Abrams).1 These authors contributed equally to this work.

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 Arabidopliana. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.03.017

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, Canadab Lehrstuhl für Botanik, Technische Universität München, D-85354 Freising, Germanyc Department of Chemistry, University of Saskatchewan, Saskatoon, SK, Canadad Department of Biochemistry, University of Saskatchewan, Saskatoon, SK, Canada

a r t i c l e i n f o

Article history:Available online xxxx

In honor of the 60th birthday of professorVincent de Luca

Keywords:Arabidopsis thalianaCruciferaeAbscisic acid analogsRCAR/PYR/PYL receptorPP2C phosphatasesStructure–activity–function relationships

a b s t r a c t

Abscisic acid (ABA) is a phytohormone known to mediate numerous plant developmental processes andresponses to environmental stress. In Arabidopsis thaliana, ABA acts, through a genetically redundant fam-ily 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 concertwith a seven-member set of phosphatases. The individual contributions of A. thaliana RCARs and theirbinding partners with respect to specific physiological functions are as yet poorly understood. Towardsdeveloping efficacious plant growth regulators selective for specific ABA functions and tools for elucidat-ing ABA perception, a panel of ABA analogs altered specifically on positions around the ABA ring wasassembled. 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, itregulates stomatal aperture and improves drought tolerance, but does not inhibit germination or rootgrowth. Analogs with the 70- and 80-methyl groups of the ABA ring replaced with bulkier groups generallyretained the activity and stereoselectivity of (S)- and (R)-ABA, while alteration of the 90-methyl groupafforded an analog that substituted for ABA in inhibiting germination but neither root growth nor stoma-tal closure. Further in vitro testing indicated differences in binding of analogs to individual RCARs, as wellas 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 dissectionof the complex network mediating ABA-perception, signaling and functionality within a given speciesand modifications in the future design of ABA agonists.

Crown Copyright � 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 amelio-ration of responses to abiotic stress and modulation of general

plant growth and development (Wasilewska et al., 2008). Althoughthe complete mechanism of ABA signal transduction mediatingthis breadth of physiological functions remains unclear, recent ad-vances in the understanding of ABA perception have helped clarifysome of the earlier steps (Cutler et al., 2010; Raghavendra et al.,2010). In particular, two proteins, RCAR1 (Regulatory Componentof ABA Receptor 1)/PYL9 (Pyrabactin Resistant 1-Like 9) andRCAR11/PYR1 (Pyarabactin Resistant 1) were identified indepen-dently, using protein interaction analyses and chemical geneticsapproaches respectively, to be members of a family of fourteenhomologues in Arabidopsis thaliana(A. thaliana), forming theRCAR/PYR1/PYL family of ABA receptors (Ma et al., 2009; Parket al., 2009).

sis tha-

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Fig. 1. Signal transduction by ABA (1) and associated assays. ABA (1) binding to theRCAR receptor, leads to closure of the gate and latch lid over the active site. Theaffinity of the receptor–ABA or receptor–analog interaction is measured byisothermal titration calorimetery herein. The complex presents a surface thatincludes the gate and latch regions, that has high affinity for the PP2C, sequesteringit away from SnRK2 and inactivating it. The degree of sequestration of the PP2c canbe assessed by measuring its phosphatase activity in the presence of the ligand-stimulated RCAR. Once released from the inhibitory effect of the PP2C, SnRK2stimulates downstream signalling, leading to well document physiological effects.Some these include, inhibition of seed germination, stomatal closure and inhibitionof root elongation, all which can be measured.

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

The genetic and functional redundancy of the members of thisfamily of receptors, which made initial identification by classicalgenetics impossible, was further highlighted later by the need toknock out at least three of the family members at once to elicit achange in phenotype (Park et al., 2009). This redundancy has madefurther functional characterization of the individual family mem-bers difficult. Indeed to date, only a few targeted studies havelinked particular members of this family to specific physiologicaleffects. For example, RCAR1 was recently shown to modulatedownstream phosphorylation of the guard cell linked anion chan-nel SLAH3, but this was only demonstrated in vitro to date (Geigeret al., 2011). Other reports link RCAR10 (PYL4) over-expression toregulation of jasmonic acid signaling (Lackman et al., 2011) andRCARs 8 and 10 (PYLs 4 and 5) over-expression to increaseddrought resistance (Santiago et al., 2009b; Pizzio et al., 2013)). Atthe same time RCAR 8 has been linked to modulation of rootgrowth (Antoni et al., 2013). Additionally, RCAR7 (PYL13) wasshown to modulate classic ABA-sensitive physiological effects,through interactions with PP2Cs, but independently of any interac-tion with ABA itself (Zhao et al., 2012). However, another reportdocuments the characterization of triple, quadruple, quintupleand even sextuple RCAR mutants, targeting RCARs 3, 8, 10, 11, 12and 14, and concluded that the family members contribute addi-tively to roles in regulation of seed germination, plant growthand reproduction, stomatal aperture, and transcriptional response(Gonzalez-Guzman et al., 2012). Finally orthologs of the A. thaliana

Please cite this article in press as: Benson, C.L., et al. Abscisic acid analogs as cliana. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.03.0

receptors have been reported in a variety of other plant speciesincluding rice (Kim et al., 2012), strawberry (Chai et al., 2012; Jiaet 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 receptorsbroadly correlated to ABA sensitivity, ripening and stress percep-tion processes. In general, the functional roles of individual ABAreceptor family members remain to be deciphered.

The regulation of ABA-mediated RCAR signaling downstream ofperception appears to be very complex. On the one hand, as manyas seven different members of the clade A PP2C family in A. thalianahave been implicated in ABA responses, each with independentand overlapping functions (Merlot et al., 2001; Kuhn et al., 2006;Robert et al., 2006; Saez et al., 2006; Yoshida et al., 2006; Nishim-ura et al., 2007; Antoni et al., 2012). While some of these PP2Cshave been shown to interact with multiple RCAR receptors, they,like the receptors themselves, are also differentially expressedthroughout plant tissues during different developmental stages(Nishimura et al., 2010; Szostkiewicz et al., 2010). On the otherhand, recent reports suggest that the same receptor surface thatbinds to PP2Cs also mediates homodimerization of a subset ofthe RCAR receptor family (Dupeux et al., 2011; Hao et al., 2011).While such receptor dimerization has been linked to inhibition ofbasal receptor activity against the PP2Cs as well as a decreasedsensitivity to ABA (1) in general, a more recent report questionsthe biological relevance of this interaction (Antoni et al., 2012).Additionally, in contrast to inactive ABA metabolites, specifichydroxylated catabolites of ABA have been shown to interact withthe receptors and inhibit the activity of associated PP2Cs, introduc-ing the possibility of a role for ABA catabolites in regulation of sig-naling (Kepka et al., 2011). Together these findings suggest acomplex network of interplay mediating ABA-perception and sig-naling that relies on spatially and temporally regulated geneexpression of the genetically redundant receptors and PP2Cs, aswell as regulation of signaling through protein–ligand and pro-tein–protein interactions.

Mechanistically, structural analyses of RCAR receptors havedemonstrated conformational differences in ABA-bound andABA-free receptor forms, highlighting open access of the ligandto an internal binding cavity in the unbound form (Melcher et al.,2009; Melcher et al., 2010; Miyazono et al., 2009; Nishimuraet al., 2009; Santiago et al., 2009a; Yin et al., 2009; Shibata et al.,2010; Soon et al., 2012; Miyakawa et al., 2012). However, onceABA (1) has entered, and docked with its side-chain carboxyl groupdeepest into the cavity, two loops that are located at the entranceof 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 bythe ‘lid’ binds to a specific group of type 2C protein phosphatases(PP2Cs) including a direct interaction between a PP2C tryptophanresidue and ABA (1). This tight interaction causes inactivation ofthe PP2C co-receptor, effectively removing the brake on ABA signaltransduction 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 thestructural requirements of the binding site in the cavities of theRCAR ABA receptors. Screening of large chemical libraries has ledto the identification of several synthetic aromatic sulfonamides,non-ABA-like, small molecules selective for groups of receptorsand physiological effects (Okamoto et al., 2013; Cao et al., 2013).One of these non-ABA related chemicals, pyrabactin, activatestwo of the RCAR receptors, while another, quinabactin, activatesan additional three RCARs. Pyrabactin affects seed ABA processeswhile quinabactin has effects on stomatal closure in a number ofplant species.

hemical probes for dissection of abscisic acid responses in Arabidopsis tha-17


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 approximateconformation adopted by (S)-ABA (1) bound to PYL1 (Miyazono et al., 2009). Thedashed 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.

C.L. Benson et al. / Phytochemistry xxx (2014) xxx–xxx 3

An alternate targeted approach to probe the structural require-ments of the receptors builds on ABA as the lead molecule andalteration of the ligand’s structure to learn about the binding siteof RCAR receptors. From the first studies and discovery of theRCAR/PYR/PYL receptors the stereoisomers and geometrical iso-mers of ABA have been employed as probes and controls in exper-imental design. The unnatural (R)-ABA (2) enantiomer, differingfrom natural ABA (1) only in the stereochemistry of C-10, elicits re-sponses in some, but not all, processes known to be ABA-regulated.This phenomenon has been used in a genetic screen for ABA muta-tions (Nambara et al., 2002). These studies have shown that thebinding pockets of the individual RCAR proteins have differentcapacities to bind the (S)- and (R)-stereoisomers of ABA(Ma et al., 2009; Park et al., 2009; Szostkiewicz et al., 2010; Zhanget al., 2013). Most recently, an in-depth investigation focused onbiochemical characterization of (R)-ABA (2) binding in nine RCARswith HAB1, as well as a crystal structure of (R)-ABA (2) bound inRCAR8, have been reported (Zhang et al., 2013). Differences inthe receptor binding pockets were observed between RCARs andin the conformation adopted by the ABA ligands within the recep-tor. The authors suggest that alteration of the substituents on theABA ring could lead to selective ABA agonists useful for agrochem-ical development.

Consistent with this approach, a panel of structural analogs wasdeveloped to exploit and amplify differences in binding of ligand toreceptors and in complexes with phosphatases, to tease apart theinitial ABA signaling process. The panel was chosen to incorporateboth (S)- and (R)-enantiomers of ABA analogs, each with singlechanges to the ABA ring, which would be expected to make signif-icant differences in the fit within the binding pocket (Zaharia et al.,2005; Fig. 2). One analog pair, PBI 413 (3) and its enantiomer PBI414 (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 inthe same plane as the vinyl methyl of ABA (1) and has greater stericbulk. This analog had been developed to trap and assess the biolog-ical activity of an intermediate in ABA catabolism. The bicyclic ana-log PBI 413 (3) and its 80-hydroxylated analog of PBI 413 werefound to have ABA activities. A second analog pair, PBI 352 (5)and its enantiomer PBI 354 (6), differ from ABA (1) in that the pro-ton of the hydroxyl group of the ABA molecule is replaced with amethyl group. PBI 352 (5) had exhibited ABA activity in growthinhibition and stomatal aperture in wheat seedlings. A third analogpair, PBI 425 (7) and PBI 426 (8), differ from ABA (1) in that the80-methyl group is replaced with an acetylene group. In the confor-mation ABA (1) has been shown to adopt within the receptor, PBI425 (7), in a similar conformation, would extend the carbon chainbelow the plane of the ABA ring, towards the lid of the binding cav-ity. This molecule had been shown to have significantly longer bio-logical activity than ABA (1) itself due, at least in part, to itsresistance to oxidation by plant P450 enzymes that degrade ABA(1). In Arabidopsis, PBI 425 (7) has been shown to induce droughttolerance 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 ofABA (1) with a cyclopropyl group, increasing the bulkiness aroundthe 80-carbon atom. The final analog pair (PBI 514 (11) and 515(12)) which had been found to act as an irreversible inhibitor ofABA 80-hydroxylase, differed from ABA (1) in that the 90-methylgroup was replaced with a bulkier propargyl group, that could af-fect the closing of the lid of the receptor.

In this paper, both of the early stages of ABA perception wereprobed by comparative analysis of ABA (1) or analogs binding toRCAR receptors, of enzyme activity with purified ABA (1) or ana-log-bound A. thaliana RCAR–PP2C pairs in vitro, including all thir-teen relevant RCARs, and activity in physiological assays forgermination inhibition, root growth inhibition and stomatal


aperture, all in A. thaliana. The study was, however, confined tothe model system A. thaliana and its RCAR phosphatase complexesto eliminate confounding factors including species differences.

Results

Comparison of the effects of ABA (1) and ABA analogs on keyphysiological processes

The responses of ABA (1) and the analogs were tested in threedifferent physiological assays so that differences in RCAR responsesin ABA-analog-induced phosphatase activity profiles could be cor-related to specific physiological plant responses. First, the effect ofABA (1) and analogs on germination inhibition of A. thaliana – Lerseeds was measured using analog concentrations of 3 lM(Fig. 3A). After 3 days, (S)-ABA (1) reduced germination by almost100% relative to the control. For most of the pairs of analogs stud-ied, the (S)-ABA-like enantiomers gave results comparable to, orbetter than (S)-ABA (1), while the (R)-ABA-like enantiomers wererelatively 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



Fig. 3. Physiological response of A. thaliana seedlings to (S)-ABA (1) and ABA-analogs. (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 growthof 5-day old seedlings in the presence of 3 lM ligand within 3 days. (C) Stomatalaperture of epidermal peels exposed to (S)-ABA (1) and diverse ABA-analogs (3 lM).

Fig. 4. ABA (1) and synthetic analogs differentially regulate PP2Cs via RCAR1. (A)ABI2 activity regulated by RCAR1 through various enantiomeric ABA analog pairsin vitro. The protein phosphatase activity was analyzed in the presence of 10 lMligand at a constant molar ratio of RCAR1 and ABI2 of 2 to 1. (B) Inhibition of ABI2phosphatase activity by increasing concentrations of (S)-ABA (1) and variousenantiomeric ABA-like analog pairs (n = 2). (C) Physiological activity of ABA-likeanalogs in regulating ABA-responsive reporter expression. The ABA-induced up-regulation of gene expression was monitored using the ABA-responsive reporterconstructs pRD29B::LUC in A. thaliana protoplasts and was measured as relativelight units (RLU/RFU). Each data point represents the mean value of threeindependent transfections at a ligand concentration of 3 lM. (D) ABI1, PP2CA andHAB1 activity regulated by RCAR1 against various enantiomeric ABA analog pairs.The protein phosphatase activity was analyzed in the presence of 10 lM ligand at aconstant molar ratio of RCAR1 to phosphatase of 2:1 in vitro.


354 (6) did not exhibit any activity, illustrating that either a protondonor at the C-1 position is critical for this ABA response or thatthe bulky methyl group has an effect on binding in the active site.

The effects of ABA (1) and analogs on root growth inhibition of5 day old A. thaliana seedlings were measured and compared(Fig. 3B). After 3 days, the roots of the control seedlings grew to anaverage 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), PBI425 (7), PBI 514 (11), and PBI 694 (9) had similar activity to ABA(1), although none of them outperformed the natural compoundin 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 pairPBI 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) hadon 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 rela-tively 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 replac-ing the proton of the hydroxyl group of ABA (1), had as strong or


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




Comparison of the effects on ABI2 activity mediated by ABA (1) oranalogs and RCAR1

In order to compare the effects of the selected analogs in vitro,measurements were made of the extent of inhibition as a resultof analog treatment in the presence of RCAR1 (Fig. 4A). (S)-ABA(1) strongly reduced ABI2 activity (<5% residual ABI2 activity),whereas activity levels in the presence of the unnatural (R)-ABA(2) were not significantly reduced (>95% residual activity) relativeto the ‘no-analog’ control (100% ABI2 activity). This indicatesenantioselectivity of the RCAR1 receptor for (S)-ABA (1) in accor-dance with previous results (Szostkiewicz et al., 2010). The(S)-ABA like analogs, PBI 413 (3), PBI 425 (7) and PBI 514 (11) wereall found to be good receptor agonists, inhibiting ABI2 throughRCAR1, with enzyme activity dropping to 5% or less. PBI 694 (9)was slightly less potent, yielding 20% residual activity. The corre-sponding (R)-ABA like enantiomers, PBI 414 (4), PBI 426 (8), PBI515 (12) and PBI 695 (10) were all about equally ineffective inreducing ABI2 activity through RCAR1, showing �40–60% residualactivity. Interestingly, the substitution of the hydroxyl proton witha methyl group has the greatest effect in reducing activity. The10-methyl ether analog PBI 352 (5) was relatively weak yieldingonly �40% ABI2 residual activity in the presence of RCAR1, whileits enantiomer, PBI 354 (6), was virtually inactive (�90% residualactivity) in the ABI2 inhibition assay.

Evaluation of the concentration dependence of some of themore effective ABA analogs for the inhibition of ABI2 in the pres-ence 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, respec-tively (Fig. 4B). PBI 694 (9) was slightly less potent with half max-imal inhibition at 116 nM and PBI 352 (5) was the least potent withan IC50 measuring 1.4 lM. These activities generally reflect therelative ability of each of the analogs to induce the ABA-responsivereporter construct pRD29B::LUC (Fig. 4C) (Uno et al., 2000). Whilethe tetralone analog PBI 413 (3) gave a stronger response than (S)-ABA (1), this value falls within the statistical error of the values ob-tained 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 geneinduction relative to controls.

Comparison of the effects of different phosphatases on RCAR1/ABA andanalog activity profiles

In order to gain insight into the relationship between ABA-analogactivities and the different PP2Cs, the same series of analogs wasscreened for their ability to inhibit the activity of three additionalPP2Cs, ABI1, PP2CA (AHG3) and HAB1, in the presence of the same

Fig. 5. ABA analog activity profiles are dependent on the identity of the RCAR pair. ABI2 aThe protein phosphatase activity was analyzed at a constant molar ratio of RCAR to ABI


RCAR1 (Fig. 4D). All three phosphatases were similarly affected by(S)-ABA (1), and its analogs through RCAR1, compared to ABI2. Thistrend held for most analogs tested, with a few minor exceptions.Amongst the (R)-like analogs (e.g. (R)-ABA (2), PBI 414 (4)) PP2CAactivity was mildly more sensitive in the presence of RCAR1. Thisfinding correlates with a previous report showing PP2CA as astrong negative regulator of ABA signal transduction (Kuhn et al.,2006). Interestingly, HAB1 shows selective insensitivity to PBI352 (5), while ABI1 shows selective insensitivity to PBI 515 (12),similar to ABI2.

Comparison of effects on PP2C enzyme activity of different RCARs withrespect to ABA (1) and analogs

In order to probe the effects of ABA-analogs and different RCARreceptors, the same set of ABA-analogs was tested with ABI2 in thepresence of each A. thaliana RCAR (Fig. 5), excluding RCAR7 (Xhaoet al., 2013; Fujii et al., 2009). It is important to note that 100-foldlower concentrations of ligands were applied in this experiment toafford the most detailed possible assessment of relative ligandactivities. (S)-ABA (1) showed relatively strong and, in some cases,even near complete inhibition of ABI2 across all RCARs tested. Onthe other hand, (R)-ABA (2) did not inhibit ABI2 to any great extentthrough any RCAR, under the conditions tested here. Similar to the‘all on’ or ‘all off’ activity of the ABA enantiomers, the (S)-ABA-likeanalogs PBI 413 (3), PBI 425 (7) and PBI 514 (11) were found to begood inhibitors of ABI2 activity for most of the RCARs tested, show-ing residual ABI2 activities ranging from 5% to 45%. However,(S)-ABA-like analogs PBI 352 (5) and PBI 694 (9) showed a greaterrange of activity against the receptors (15–90% residual activity),both being relatively potent against RCAR 8, but PBI 694 (9) show-ing additional potency against RCAR 9. This is representative of anobserved trend in which clade II members (RCARs 5–10) are gener-ally more sensitive to (S)-ABA (1) and synthetic compounds, withclade I (RCARs 1–4) and clade III (RCARs 11–14) members beinggenerally less sensitive. Interestingly, (R)-analogs PBI 354 (6), PBI414 (4), PBI 426 (8), PBI 515 (12), and PBI 695 (10) were signifi-cantly less active against the RCARs than (S)-ABA-like analogsacross the board. In particular PBI 426 (8), like PBI 694 (9), wasfound to be selectively more potent against RCARs 8 and 9. The(R)-ABA like 80-cyclopropyl substituted PBI 695 (10) was for themost 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) bothshowed only weak activity against the phosphatases, but did breakwith the clade II potency trend, showing marginally stronger activ-ity against select clade I and III receptors.

ctivity regulated by A. thaliana RCARs against various enantiomeric ABA analog pairs.2 of 2:1 in vitro at a constant analog concentration of 100 nM (n = 3).



Fig. 6. Dose responses of RCARs 1 and 8 to ABA (1) and PBI 352 (5) are dependenton 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 responsesof RCAR1 (panel C) and RCAR 8 (panel D) to PBI 352 (5) was also tested. The proteinphosphatase activity was analyzed at a constant molar ratio of RCAR to PP2C of 2:1in vitro at the indicated concentrations of ligand (n = 3).


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

Fig. 7. Isothermal titration calorimetric analysis of the binding ABA (1) and select ABA-anPBI 414 (4), (D) PBI 425 (7), (E) PBI 426 (8), (F) PBI 352 (5) and (G) PBI 354 (6) into RCAR8the ligands were performed until the binding saturation was reached. The data wererepresented by the black line.


comparison of phosphatase inhibition induced by ABA (1) with fourdifferent phosphatases and either clade II RCAR8 or clade I RCAR1.Differences are observed (Fig. 6A) with the RCAR1–ABA complexhaving slightly lower potency against HAB1 (IC50 0.10 ± 0.01 lM)compared to either of the ABI1/2 (IC50 of 0.07 ± 0.01 and0.06 ± 0.01 lM) or PP2CA phosphatases (IC50 0.08 ± 0.01 lM). Incontrast the RCAR8–ABA (Fig. 6B) complex had the lowest potencyagainst 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 com-parable experiments in which PBI 352 (5) is tested in the place ofABA (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 threePP2Cs tested against RCAR1–PBI 352 (Fig. 6C); and where an IC50

of 1.0 ± 0.10 lM was detected for RCAR8–PBI 352-PP2CA comparedto 0.07 ± 0.01 lM for all the others three PP2Cs tested againstRCAR8–PBI 352 (Fig. 6D).

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

The in vitro binding affinities of RCAR8 to (S)-ABA (1) and se-lected ABA analogs were determined by isothermal titration calo-rimetry (ITC; Fig. 7). Titration of (S)-ABA (1) into a solution ofpurified RCAR 8 yielded a saturating binding isotherm showinglarge heat release and a dissociation constant (Kd) of 1.2 lM withan enthalpy (DH) of �9.0 kcal mol�1 and an entropy value (DS)of �3.1 cal mol�1 deg�1 (Fig. 7A). The stoichiometry (N) of bindingwas approximately 0.5, which suggests a 1:1 binding of RCAR8 to(S)-ABA (1), assuming some degree of receptor proteolysis oraggregation during the measurement. These results correlate verywell with the observations of Rodriguez and coworkers, who

alogs to RCAR8. Raw data of sequential injections of (A) ABA (1), (B) PBI 413 (3), (C)(13.5 lM). The processed results are shown below each isotherm. 1 ll injections of

fitted using the ‘one set of sites’ option in the Origin software and the best fit is




reported a Kd of 1.1 lM for the same receptor–ligand combination(Dupeux et al., 2011). The (S)-enantiomer of the tetralone deriva-tive PBI 413 (3) yielded a Kd of 0.7 lM (DH = �11.6 kcal mol�1

and DS = �10.8 cal mol�1 deg�1; Fig. 7B). This represents a slightlyhigher affinity compared to the natural ligand. The enthalpy gain inthe interaction of PBI 413 (3) with the receptor (�11.6 kcal mol�1;N = 0.6) compared to that observed in the case of ABA (1)(�9.0 kcal mol�1) suggests that there might be additional non-covalent 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 thein vitro receptor assay (Fig. 5). It is important to note that thisITC result does not mean that PBI 414 (4) does not bind to RCAR8.It likely does still bind (as demonstrated by the in vitro activitydata), but with a Kd > than 100 lM. The (S)-ABA like 80-acetyleneanalog PBI 425 (7) showed slightly weaker binding, with a Kd of6.3 lM (Fig. 7D) while the (R)-ABA like 80-acetylene analog PBI426 (8) showed significantly weaker binding with a Kd of approxi-mately 67 lM (Fig. 6E). This correlates with the observed potencyof PBI 426 (8) against RCAR8 (Fig. 5) compared to PBI 414 (4). Final-ly 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 intermediatein 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 againstRCAR8 in vitro is not related to reduced ligand binding affinity,but possibly to some other factor, such as ineffective modulationof the interaction with the PP2C.

Discussion

Now with the screening of ABA receptors and phosphatase pairsin hand, plant growth regulator development can be acceleratedand deeper investigations into specific processes in the ABA signaltransduction pathway can be elucidated.

The synthetic analogs of (S)-ABA (1) used in this study have pre-viously been reported to retain (S)-ABA-like activity in physiologi-cal 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 ringcarbon substituted ABA analogs were tested directly on a series ofA. thaliana ABA receptor complexes in vitro, as well as in planta togain a clearer perspective of ABA structure–activity and ABA recep-tor–physiological relationships. In the instance of physiological as-says conducted over several days, differential effects related touptake or rate of metabolism of (S)-ABA (1) versus analogs cannotbe ruled out. However the stomatal assay is rapid, minimizing theeffects of metabolism. The observation that (S)-ABA (1) and(S)-ABA like analogs have similar activities in inhibition of rootgrowth and germination, suggests uptake is not an issue. As well,previous work showed uptake of (R)-ABA (2) and select ABA ana-logs 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 analogsPBI 425 (7), PBI 514 (11) and PBI 413 (3) respectively, with additionof bulk to the parent ABA molecule at the 80- and 90-carbon atomsor at the 20-, 30-carbon atoms, did not lead to any significant reduc-tion in activity compared to (S)-ABA (1) in either the in vitro recep-tor assay or in the physiological assays. PBI 694 (9), modified witha larger cyclopropyl group at C-80, maintained strong in vitro acti-vation capabilities, particularly for the clade II members RCAR8and 9. This observation correlates with a selective reduction in reg-ulation of root growth responses only, with this compound show-ing (S)-ABA-like regulation of germination and stomatal aperture.


These latter effects correlate to observed reduced potency of thePBI 694 (9) in quantitative dose response experiments (Fig. 4B),suggesting limitations in the bulk that can be accommodated atC-80.

In contrast, substituting the hydrogen of the OH on the C-10

with a methyl group, in PBI 352 (5), affords an analog selectivein inducing stomatal closure but not affecting germination or rootgrowth of Arabidopsis seedlings. This selectivity has potentialutility for practical applications wherein temporary drought pro-tection could be provided to a plant without reducing concomi-tant root growth. This analog PBI 352 (5) reduced receptoractivity more significantly compared to (S)-ABA (1) for all of theRCARs tested. Thus, the extra bulk on the oxygen of PBI 352 (5)seems to be somewhat more deleterious to ligand binding pro-cesses in which germination inhibition and root growth inhibitionare the resulting physiological effects, likely related to space inthe receptor being limiting in the region of the hydroxyl groupand possibly disrupting the hydrogen bond network in the activesite.

From a structure–function perspective, attempts at computa-tionally docking of the ABA analogs into RCAR binding sites usingavailable 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 bind-ing conformations for each analog (data not shown); highlightingthat one cannot assume that the (S)- and (R)-analogs are bindingin the same orientation as (S)- and (R)-ABA respectively. Thus reli-able structural insight about the analogs’ binding awaits ongoingco-crystallization of RCARs with the ABA analogs, and we refrainfrom speculative discussion at this time.

However, differential effects and binding affinities for differentanalogs are noted against different receptors in vitro and these mayat least be explained in part by evaluation of conservation, locali-zation and dynamics of amino acids comprising the A. thalianaRCAR (S)-ABA (1) binding site (Mosquna et al., 2011), and recentcomparison of these to the (R)-ABA (2) binding site crystal struc-ture (Zhang et al., 2013). Together these suggest the possibility ofa selectivity filter modulating interactions of the receptor withthe ABA-analogs. Such a selectivity filter would include variableresidues located in the ligand binding site, across all thirteenRCARs. Some obvious variable sites in the binding pocket includeI90 and V111 in the gate region, F136 and V138 in the latch regionand V185, I188 and V189 in the final helix (RCAR8 numbering;Fig. 8A). These residues are situated across the ABA binding sitefrom each other and the variations in amino acid identities herecould be expected to yield differential constraints on the size andshape of the binding site in terms of accommodating extra bulkand the (R)-ABA like stereoisomers (Fig. 8B). Comparisons of repre-sentative members from the three evolutionary clades of this fam-ily suggest extra space in RCAR1 around the ABA side-chain,compared to either RCARs 8 or 11, related mainly to an F136I var-iation. More relevant to the discussion here on ring-modified ana-logs, extra space in RCAR8 around the ABA ring region is predictedcompared to RCAR1 due to a V138I variation. While RCAR11 alsomaintains the larger I138 side chain variation like RCAR1, this bulkmay be partially offset with the variation to A in place of V acrossthe pocket at position 185. Thus we predict from this in silico anal-ysis that RCAR8 and possibly RCAR11 might better accommodatering modifications and the (R)-ABA like stereoisomers than RCAR1.Indeed this concept was recently demonstrated, where variationsat residue equivalents to RCAR8 positions 138 and 188 in PYL9(RCAR 1) and PYL3 (RCAR13) were shown to dictate the receptor’sability to bind (R)-ABA (2) (Zhang et al., 2013). Smaller V sidechains at these positions allowed the reoriented 80 and 90 ringmethyl groups of the (R)-ABA (2) to be accommodated, while largerI’s and L’s at these sites inhibited the (R)-ABA interaction.


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


Analysis of the experimental results obtained here for in vitroscreening further supports this model. At both high and low(R)-ABA (2) levels (Fig. 4D and Fig. 5) only very weak inhibitionof ABI1, ABI2 and HAB1 are observed for the constrained RCAR1.This correlates with the report by Zhang et al. (2013), which in par-ticular demonstrated that RCAR’s 1, 4, 5 and 6 (all of which main-tain a larger I residue at RCAR8 138 equivalent positions) wereinsensitive to (R)-ABA (2), while RCAR’s 8, 9, 10 and 13 (all witha small V residue at RCAR8 138 equivalent positions) were potentlymodulated by (R)-ABA (2). However these were only tested againstHAB1. 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 ac-count for the entire regulatory mechanism. Indeed this latterobservation supports a case for (R)-ABA (2), and potentially otheranalogs, in modulating the RCAR–PP2C interface more directlyand interacting with residues of the phosphatase directly, muchas (S)-ABA (1) does, but yielding different effective/selective inter-actions. Such selective interactions by (R)-ABA (2) may be respon-sible 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 beingmediated by selective RCAR–PP2C–analog interactions is furtherevidenced by the effect of the O-methyl substituted ring hydroxyl(S)-analog, PBI 352 (5), which generally is not a very potent agonistagainst ABI2 regardless of the RCAR, with the exception of RCARs 8


or 9 (Fig. 5). Further examination of the case of PBI 352 (5) andRCAR8 demonstrated that this combination is moderately potentagainst 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. Thusone could conclude that the observed physiological selectivity ofPBI 352 (5) for regulation of stomatal function, having no impacton germination or root growth, is the result of the selective inter-action of PBI 352 (5) for RCAR8 with HAB1, ABI1 or ABI2. Howeverwhen tested against knockdowns of RCARs 8 and 9 (data notshown), the effect of PBI 352 (5) was identical to its effect againstWT 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 broadall-RCARs versus ABI2 screen, some selective potency was ob-served in the case of RCAR1, where PBI 352 (5) was moderately po-tent against ABI1 and PP2CA, but relatively ineffective againstHAB1 and ABI2 (Figs. 4D, 5 and 6), compared to (S)-ABA (1). Thus,together these results highlight that in the RCAR8 knockdownexperiment, a potential interaction of PBI 352 (5) with RCAR1 incomplex with ABI1 or PP2CA might be contributing to the observedselective stomatal regulation. Testing of a double RCAR8, RCAR1knockdown might shed additional light on this hypothesis; how-ever in this instance it might be more efficient to screen all theRCAR–PP2C complexes against PBI 352 (5) and then initiate tar-geted in planta work to validate the screening results. Similarily,other discrepancies observed in our results, for example betweenPBI 426 (8) and PBI 515 (12), which show similar potenciesin vitro against ABI2 (Fig. 5) and similar effects on root growthand stomatal aperture, but differential effects on inhibition of ger-mination, may be explained by the opposing effect of these analogson PP2C selectivity when assayed against RCAR1 and diverse PP2Cs(Fig. 4D). Again a broader screening of all RCAR–PP2C complexesagainst these two analogs could provide the mechanism of thisphysiological difference. Overall these results emphasize the com-plexity of the redundant network of protein and protein–ligand–protein interactions mediating the ABA response, but also highlightthe potential RCAR/PP2C selectivity that can be achieved in anABA-analog, and the potential in subsequently applying such ananalog to deciphering mechanistic aspects (Lin et al., 2005).

In this context of using ABA analogs as chemical probes inresolving ABA-related redundancies and mechanisms of action,the following are noted: The effects of ABA analogs are likely spe-cies-specific. For example as above, PBI 352 (5) selectively elicits(S)-ABA-like stomatal aperture responses in A. thaliana. This is incontrast to the effect reported for PBI 352 (5) in wheat, where ithad weak effects on both germination and transpiration (i.e. sto-matal regulation) (Rose et al., 1996b). Similarly, PBI 354 (5) wasfound to be a very potent inhibitor of germination in wheat (Roseet al., 1996b), but had no effect in the work reported here on ger-mination in A. thaliana. Thus analog profiles obtained related toreceptor–physiological relationships should not be extrapolatedto other species. However, the identification of trait specific probesfor a given species, such as PBI 352 (5) in A. thaliana, can serve aspowerful analytical tools. Two other ABA analogs, PBI 414 (4)and PBI 515 (12) are moderately potent in the A. thaliana germina-tion assays and relatively ineffective in the root growth and stoma-tal aperture assays. While the potency of these are both weak at100 nM ligand concentrations, much higher potency is observedat 10 lM concentrations, including some distinct PP2C selectivity(Fig. 4D). While a full in vitro screen of the fourteen receptorsagainst all known related PP2Cs against these analogs would againlikely reveal the select RCAR–PP2C pair(s) relevant to the selectiveactivity of PBI 414 (4) and PBI 515 (12), as also suggested for PBI352 (5) above, such an expanded screen goes beyond the scopeof this work which was to conduct a survey of the analogs’


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potential application. Indeed an expanded screen might best beconducted with respect to a specific study focusing on one trait,analog, receptor or phosphatase, and should take the wealth ofavailable transcriptomic data into consideration. Together in A.thaliana, PBIs 352 (5), 414 (4) and 515 (12) could be used as probesto study aperture-specific versus germination-specific ABA mecha-nisms of action.

It should also be noted that correlating broader transcript pro-filing of plants treated, respectively with these compounds couldlead to the identification of novel gene targets involved uniquelyin one or the other physiological effect. Indeed such experimentswere recently reported wherein application of chemically morestable and potent ABA-mimetics, PBI 425 and and its racemic ana-log PBI 429, led to the identification of various novel genes in ABAsignaling and metabolism, including a putative hydroxysteroiddehydrogenase involved in inhibition of germination (Li et al.,2005, 2007; Huang et al., 2007).

Conclusions

Overall, genetic redundancy within the ABA receptors and associ-ated signaling components has made it difficult to unravel individualfunctions of components as a basis for understanding the intricaciesof ABA signaling across the phytohormone’s diverse functionalities.The findings reported herein highlight how a series of synthetic ABAanalogs can be used as chemical probes to dissect functional redun-dancy within the ABA receptor family. In particular these findingsdemonstrate how these analogs can lead to novel hypotheses relatedto the roles of specific RCAR–PP2C complexes and how they canserve as physiological trait-specific probes for A. thaliana. It can bespeculated that with expanded screening, it may be possible to usethese distinctive analog activity patterns to map the functionalactivities of additional players in the complex network of ABA signal-ing and transport in A. thaliana (Kang et al., 2010; Kuromori et al.,2010; Kharenko et al., 2011). Earlier reported variations in analoginteractions across species further suggest merit in applying suchan expanded screen to tease out species-specific mechanisms andrelated functionalities in the longer term.

Experimental procedures

Chemicals

Chemicals were obtained from Sigma–Aldrich(http://www.sigmaaldrich.com), Fluka (part of Sigma–Aldrich),Roth (http://www.carlroth.com), AppliChem (http://www.appli-chem.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 methodspreviously 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 PBI694 (9) and 695 (10) were synthesized and resolved similar tothe method previously described for PBI 425/426 (7/8) by substi-tuting ethynylmagnesium bromide with cyclopropylmagnesiumbromide.

Plant material

A. thaliana lines used in this work were ecotype Columbia andLandsberg erecta (Ler). Plants used for protoplast isolation weregrown for 4 weeks in a perlite–soil mixture in a controlled growthchamber at 23 �C under long-day conditions with 16 h of light(250 lE m�2 s�1; (Moes et al., 2008)).


Sequences accession numbers

Sequence data from this article can be found in theGenBank/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 beendescribed previously (Ma et al., 2009; Moes et al., 2008). All RCARsand ABI1/2 and PP2CA constructs used in this study were gener-ated as described by Ma et al. (2009) and Szostkiewicz et al.(2010) (Kepka et al., 2011). For ITC analyses, the cDNA of RCAR8was amplified with the primer pair 50-CACCATGAGGTCACCGGTGCAACTCCAAC-30 and 50-TTATTATTGCCGGTTGGTACTTCGAGCCAGAG-30. The PCR fragment was cloned into the pENTR.D.Topovector (Invitrogen) and subsequently recombined into thepDEST17 vector (Invitrogen) according to the manufacturer’s spec-ifications, yielding pDEST17-RCAR8.

Expression and purification of RCARs and PP2Cs

His-tagged RCARs and ABI1/2 as well as PP2CA proteins wereexpressed in Escherichia coli strain M15, BL21De3Star or BL21-AIas deemed appropriate essentially as described previously(Ma et al., 2009; Kepka et al., 2011). Briefly, cells were grown over-night in Luria Bertani medium with appropriate antibiotic selec-tion and used for inoculations of 1 L of culture. The cells weregrown at 37 �C with shaking until an optical density at 600 nm of0.5–0.6 was reached. Protein expression was induced by adminis-tration of isopropyl-b-D-thiogalactopyranoside (0.5 mM final con-centration) or in the case of BL21-AI cells L-arabinose (0.2% finalconcentration). The cells were harvested at 4 �C and 4000g for30 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 pelletwas used directly for purification. The pellet was dissolved in lysisbuffer (10 mL, 50 mM NaH2PO4, 300 mM NaCl, and 5 mM imidaz-ole, pH 8.0) supplemented with lysozyme (1 mg mL�1 final concen-tration) for 30 min. Cells were subsequently disrupted bysonication on ice (six times for 10 s). The protein lysate was ob-tained after centrifugation at 4 �C and 25,000g for 30 min andloaded onto anickel–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 boundproteins. 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 dialyzedtwice against dialysis buffer (100 mM Tris–HCl, 100 mM NaCl,and 2 mM dithiothreitol, pH 7.9). Fraction 2 was used in the phos-phatase assays.

Phosphatase assays

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


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http://www.applichem.com

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

Protoplast analysis

Preparation and analysis of A. thaliana protoplasts was per-formed as described (Moes et al., 2008). A. thaliana protoplastswere transfected with DNA (10 lg) of the reporter construct(pRD29B::LUC) and 2 lg of p35S::GUS plasmid as a control forinternal normalization of the expression. Protoplast suspensionswere incubated in the presence or absence of ABA and ABA analogsafter transfection.

Isothermal titration calorimetry

RCAR8 to be used for isothermal titration calorimetry (ITC) waspurified by His-tag affinity purification followed by size exclusionchromatography (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 em-ployed for this study. Stock solutions (100 mM) of ABA and analogswere diluted to the required concentration using the ITC buffer.The ITC experiments were performed at 25 �C. The protein, ligandsand buffer were equilibrated to room temperature and de-gassedbefore performing the experiment. The cell contained 200 ll of13.5 lM RCAR8. A 40 ll injector was used to deliver 20–39 injec-tions (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 wereprocessed using the Origin for ITC software.

Bioassays of stomatal closure in epidermal strips

Strips of abaxial epidermis were prepared from A. thalianaleaves by mounting 5 mm � 5 mm leaf samples on glass coverslipswith the help of a medical adhesive, Telesis V (Premiere Products),and transferring the coverslips to 3 cm diameter petri dishes con-taining 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 m�2 s�1) infresh incubation medium for 2 h, with the light filtered through awater jacket. Photon flux was measured with a Li-Cor quantumsensor (Li-Cor Instruments). The temperature was maintained at25 ± 1 �C. Test compounds were added to the medium, and thestrips were kept under the same conditions for another 2 h beforemeasuring the stomatal aperture. The width of the stomatal aper-ture was measured with a research microscope (Nikon Eclipse TE200) fitted with a camera and connected to an image-analysissystem.

Seed germination and root elongation assays

Under sterile conditions, 100–150 seeds were plated onMurashige and Skoog agar medium containing tested compoundsand incubated at 4 �C for 2 d in the dark to break dormancy. Theplates were then transferred to a culture room with continuouslight (60 lE m�2 s�1) at 22 �C. After 3 d, seeds were examined witha stereo microscope. Seeds were counted, and germination ratewas calculated as percentage of the total number of seeds. For rootelongation assays, 5-d-old seedlings were transferred in a row topetri dishes with solidified Murashige and Skoog medium supple-mented with 5 g sucrose/L and ABA or ABA analog as specified, andkept in a vertical position at 22 �C in continuous light for 3 d. Roottip position was marked every 24 h, and root lengths were


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

Acknowledgements

We thank A. Cutler (National Research Council (NRC) of Canada)and M. Surpin (Valent Biosciences Corp.) for critical comments onthe manuscript. We are grateful to S. McKenna, University of Man-itoba, for discussions and the use of his iTC200 instrument. Wethank J. Boyd (NRC), for cloning of RCAR8 and in silico docking ofABA-analogs. We thank C. Gordon and F. Ball (NRC) for discussionsregarding ABA- and phosphatase-binding sites. This work was sup-ported by the Deutsch Forschungsgemeinschaft (grant Nos. GR938/6to E.G. and CH182/5 to A.C.), by the Bayerisches Staatsministeriumfür Wissenschaft, Forschung und Kunst (FORPLANTA; to E.G.), theNational Research Council (NRC) of Canada Plants for Health andWellness program (to S.R.A., and C.B.) and the NRC Genomics andHealth 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.

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abscisic acid analogs as chemical probes for dissection of abscisic acid responses in arabidopsis...

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