Abscisic Acid Analogues That Act as Universal or Selective

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Abscisic acid analogs that act as universal or selective antagonists of phytohormone receptors Nandhakishore Rajagopalan, Ken M Nelson, Amy F Douglas, Vishal Jheengut, Idralyn Q Alarcon, Sean A. McKenna, Marci Surpin, Michele C. Loewen, and Suzanne R Abrams Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00605 • Publication Date (Web): 15 Aug 2016 Downloaded from http://pubs.acs.org on August 17, 2016

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Abscisic acid analogs that act as universal or selective antagonists of phytohormone receptors Nandhakishore Rajagopalana, Ken M. Nelsona,b, Amy F. Douglasa, Vishal Jheenguta, Idralyn Q. Alarconb, Sean A. McKennac, Marci Surpind, Michele C. Loewene,f,* and Suzanne R. Abramsb a

National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, Canada S7N

0W9 b

Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK,

Canada S7N 5C9 c

Department of Chemistry, University of Manitoba,144 Dysart Road, Winnipeg, MB, Canada

R3T 2N2 d

Valent BioSciences Corporation, 870 Technology Way, Libertyville, Illinois 60048 USA

e

National Research Council of Canada, 100 Sussex Dr, Ottawa, ON, Canada K1N 5A2

f

Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon SK,

Canada S7N 5E5 * To whom correspondence should be addressed (email: [email protected]). National Research Council of Canada, 100 Sussex Drive, Ottawa, ON, K1K 3G6

Funding Source Statement. This work was funded by the National Research Council of Canada (core-funding) to MCL and SRA, Natural Sciences and Engineering Research Council of Canada

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DG and RTI grants (#s RGPIN-2015-06142 and 406378-2011repsectively) to SAM and DG grant # 261683-2012 to MCL, and by Valent Biosciences Corporation to MS, MCL and SRA.

Abbreviations ABA, abscisic acid; RCAR, Regulatory Component of ABA Receptor; PYR1/PYL, Pyrabactin Resistant 1 / Pyrabactin Resistant 1-Like; CCD, carotenoid cleavage dioxygenase; GA, gibberellic acid; IPTG, isopropyl-beta-D-thiogalactopyranoside; DTT, dithiothreitol; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; BN-PAGE, blue native – polyacrylamide gel electrophoresis; ITC, isothermal titration calorimetry.


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Abstract The plant hormone abscisic acid (ABA) plays many important roles in controlling plant development and physiology, from flowering to senescence. ABA is now known to exert its effects through a family of soluble ABA receptors, that in Arabidopsis thaliana has thirteen members divided into three clades. Homologs of these receptors are present in other plants, also in relatively high numbers. Investigation of the roles of each homolog in mediating the diverse physiological roles of ABA is hampered by this genetic redundancy. We report herein the in vitro screening of a targeted ABA-like analog library and identification of novel antagonist hits including the analog PBI686 that had been developed previously as a probe for identifying ABAbinding proteins. Further in vitro characterization of PBI686 and development of second generation leads, yielded both receptor-selective and universal antagonist hits. In planta assays in different species have demonstrated that these antagonist leads can overcome various ABA induced physiological changes. While the general antagonists open up a hitherto unexplored avenue to control plant growth through inhibition of ABA-regulated physiological processes, the receptor-selective antagonist can be developed into chemical probes to explore the physiological roles of individual receptors.


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The plant hormone ABA is a monocyclic sesquiterpenoid carboxylic acid that is found in all plants. ABA regulates a wide range of physiological and developmental processes over the plant’s life cycle, from seed development and germination, to fruit ripening, and to amelioration of abiotic and biotic stress

1,2

. It has taken over forty years since the discovery of ABA to

identify its receptors in plants, due in large part to the genetic redundancy of the genes encoding ABA receptors. The first ABA receptors were identified in Arabidopsis thaliana and named RCARs (Regulatory Component of ABA Receptors) or PYR1/PYLs (Pyrabactin Resistant 1 / Pyrabactin Resistant 1-Like) in 2009

3,4

. They were originally identified as a group of fourteen

soluble cytosolic proteins in A. thaliana. Interestingly, RCAR7 (PYL13) was initially shown to be inactive when assayed with HAB1 phosphatase, but more recently was found to mediate function through other downstream phosphatases 5. The receptors fall into three general evolutionary clades 3. While the receptors belonging to clade I and II exist as monomers in solution, the members of clade III form dimers 6. Homologs of these proteins have subsequently been identified from other species like rice, grape, strawberry, citrus, cucumber and soybean and shown to be functional ABA receptors

7–11

. The mode of action of a number of these receptors

has been elucidated and the structure and function of individual receptors investigated 12–17. Modulation of ABA levels and its responses in plants can be a beneficial approach to control various physiological processes in plants. Genetic strategies have been successful in altering ABA levels and responses in plants, including manipulation of carotenoid cleavage dioxygenases (CCDs) that regulate ABA biosynthesis and the hydroxylases that regulate ABA degradation

18,19

. Similarly, modulation of a farnesyl transferase (ERA1) has been shown to

amplify the ABA response and increase stress tolerance

20

.

Modulation of the genetic

components of ABA transport and signal transduction, have also been reported to alter ABA Page 4 of 36 ACS Paragon Plus Environment

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content and responses 15,21–23. Genetic means for countering ABA effects in plants have also been achieved in some circumstances by enhancing other hormonal systems that function to antagonize the effects of ABA, the classical example being the competition between gibberellic acid (GA) and ABA in barley seed alpha-amylase degradation

24–26

. Chemical manipulation of

ABA levels in plants has been achieved by inhibiting the CCDs using specifically designed inhibitors

27,28

and by treatment with ABA itself. Currently, ABA is in commercial use for

promotion of ripening of red table grapes 29. ABA-based analogs have been developed that have greater potency and resistance to plant degrading enzymes

30–36

. ABA analogs have also been

shown to be effective for elucidation of aspects of the mechanism of action of ABA 37,38. Recent in vitro receptor screening of commercial chemical libraries has led to the discovery of non-ABA based small molecules that substitute for ABA in subsets of ABA receptors in A. thaliana

31,32

.

Recently, there have been several reports of direct chemical approaches to antagonize ABA perception in plants. Pyrabactin is a selective antagonist of RCAR14 (PYL2) but a potent agonist of other RCARs

12

. The 3’-thioalkyl ASn series of ABA analogs are partial agonists of ABA

receptors that can antagonize ABA mediated processes in plants

39

. The recently reported

bicyclic ABA analog PAO4 is another antagonist of ABA receptors showing a preference for the monomeric clade receptors like RCAR8 (PYL5) over the dimeric clade receptor like RCAR11 (PYR1) 40. We report the discovery of ABA analogs that inhibit ABA receptor activity. These were identified through in vitro screening of a library of ABA-related analogs against select ABA receptors from A. thaliana. Individual analogs show a range of antagonistic potencies and receptor-selectivity, including molecules that inhibit all clades of receptors and act as universal antagonists of ABA receptors. In planta testing of a potent analog for effects on germination and Page 5 of 36 ACS Paragon Plus Environment

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stomatal opening confirmed the physiological relevance of the antagonists. A potential mode of action of the ABA antagonists and possible applications are discussed.

Materials and Methods ABA analog chemical synthesis All chemicals were purchased from Sigma–Aldrich unless otherwise stated. The synthesis of PBI664, PBI690 and PBI691 are reported in the supplementary section, from intermediates described in 33,41. The analog PAO4 was prepared by the method reported by 40. Plasmids and protein expression RCAR3, RCAR8, RCAR10 and HAB1 were cloned into pDEST 17 vector (Life Technologies) and were introduced into E. coli BL21 AI cells (Life Technologies). RCAR11 was cloned into pET 100 vector (Life Technologies) and introduced into E. coli BL21 DE3 pLysS (Life Technologies). Expression of the desired protein was induced by adding 0.2 % arabinose for RCAR3, RCAR8, RCAR10 and HAB1. Expression of RCAR11 was induced by adding 1 mM IPTG. The cultures were incubated overnight at 16 °C post-induction. The cell pellets were frozen in liquid nitrogen and stored at -80 °C. Protein purification Frozen cell pellets were thawed and the final volume was made up to 20 mL using the lysis buffer (100 mM Tris pH 7.9, 100 mM NaCl, 0.3 mM MnCl2 and 4 mM DTT) containing 10 mM imidazole. The cells were lysed using the French press at 1000 psi. The cell lysate was clarified by centrifuging at 17,000 rpm for 25 min at 4 °C. The supernatant was quickly removed Page 6 of 36 ACS Paragon Plus Environment

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and used for further purification. 4 mL of Ni-NTA (50 % suspension) (QIAGEN) was taken in a plastic column and washed thrice with 5 mL of lysis buffer containing 10 mM imidazole. The washed Ni-NTA resin was added to the cell lysate supernatant and incubated at 4 °C for 30 min. Elution of the bound protein was performed by adding 4 mL fractions of lysis buffer containing 150 mM imidazole till all proteins were eluted from the column. The eluted protein was further purified using size exclusion chromatography. A 26/600 Superdex 200 column (GE Healthcare life sciences) was equilibrated with GF buffer (100 mM Tris pH 7.9, 100 mM NaCl, 0.3 mM MnCl2 and 0.25 mM TCEP). 10 to 20 mg of protein in 1.5 to 2 mL sample volume was loaded on the column and proteins separated using the GF buffer at a flow rate of 2.5 mL/min. Phosphatase assay The phosphatase assay was based on published protocols in 3. Briefly, the receptor, phosphatase and ABA / analogs were mixed together in a 50 µL volume mixture in a buffer containing 100 mM Tris pH 7.9, 100 mM NaCl, 0.3 mM MnCl2 and 4 mM DTT. This mixture was incubated for 15 min at 30 °C. 50 µL of substrate (1 mM 4-Methylumbelliferyl phosphate) was added to the enzyme receptor mix to start the reaction and the assay mix was incubated for 15 min at 30 °C. The final amounts of the phosphatase and receptor were 0.4 µg and 2.4 µg, respectively. The fluorescence intensity of the product formed was measured at 15 min after initiation of the assay. The excitation wavelength was set at 355 nm and the emission wavelength was 460 nm and a measurement time of 0.1 s was used. For agonist assays, 0.1 µM of ABA or its analogs were added to the assay mix and antagonist assays contained a fixed concentration of ABA (0.1 µM) and 100 µM of analogs. Blue Native – Polyacrylamide Gel Electrophoresis (BN-PAGE)


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The method was adapted from published protocol in 42. Briefly, a 12 % PAGE resolving gel was cast without adding detergent. The sample loading dye contained Coomassie blue G-250 and was detergent-free. The gel running buffer also contained Coomassie blue G-250. After incubation, the samples were mixed with the loading dye and immediately loaded onto the gel and electrophoresis was carried out. Isothermal Titration Calorimetry (ITC) RCAR8 and HAB1 were purified by His-tag affinity purification followed by size exclusion chromatography (Superdex 200 pg HiLoad 26/600, GE Healthcare) and dialyzed against ITC buffer for 12 hours (100 mM Tris pH 7.9, 100 mM NaCl, 0.3 mM MnCl2 and 0.25 mM TCEP). A Microcal iTC200 instrument (GE Healthcare) with a cell volume of 200 µL 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. The cell contained 200 µL of 13.5 µM RCAR8. A 40 µL injector was used to deliver 15 to 30 injections (1 to 2 µL each) of 0.25 mM (+)-ABA or analogs into the sample cell. The first injection (0.4 µL) was excluded from data processing. The reaction was continuously stirred at 500 rpm. The data were processed using the Origin for ITC software. Molecular Modeling RCAR11, HAB1 and (+)-ABA structure coordinates were obtained from PDB ID # 3K90 and PDB ID# 3QN1. Coordinates for PBI413 and PBI690 were obtained from ChemDraw (Perkin Elmer, USA). Analog structures were overlaid on (+)-ABA manually, matching the


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positions of the ABA ring and side chain, and distances measured using Coot

43

. Images were

rendered using VMD software 44. Seed germination assays Arabidopsis thaliana CVI (Cape Verde Island) seeds were a gift from Dr. Allan Feurtado, NRC Saskatoon. Lettuce (Grand Rapids) seeds were purchased from Early’s garden store (Saskatoon SK, Canada). In a standard petri plate two whatman I filter paper (70 mm) were placed and soaked with 2 ml water or different concentrations of ABA or analogs. ABA and analog stocks were prepared by adding equimolar NaOH solution and diluted using sterile water 45

. For Arabidopsis seed germination assays, 40 seeds were placed in each plate and the lettuce

seed germination study had 25 seeds per plate. The lids were sealed with film. The Arabidopsis plates were incubated for 16 h in light at 25ºC and 8 h in dark at 20ºC. Lettuce plates were covered with aluminum foil and incubated at 33ºC. Three and four replicates per treatment was used for the Arabidopsis and lettuce studies, respectively. The number of seeds that germinated in each plate was recorded every day. Arabidopsis thaliana Col-0 (Columbia) seeds were purchased from Lehle Seeds, Inc. (Round Rock, TX). Seeds were sterilized in a 10% bleach solution and rinsed in sterile distilled and deionized water, and stratified for three days at 4°C. The sterilized seeds were sown on Petri dishes containing ½ x Murashige and Skoog basal salts media, pH 5.8, supplemented with Gamoborg’s vitamins, and sealed with surgical tape. The plates were incubated for 12 h in the light at 24°C and 12 h in the dark at 20°C. Three replicates of 50 seeds each were plated. Starting at two days after sowing, the number of seeds germinated was recorded every day.

Stomatal aperture assay


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Arabidopsis thaliana Col plants (4 weeks old) were incubated in dark early in the day. Lower epidermal peels were prepared and incubated with lower surface facing down on phosphate buffer saline (control), or 100 µM (+)-ABA or 100 µM PBI664 in phosphate buffer saline. These epidermal peels with various treatments were incubated in dark for a further 30 min. The peels were placed on a glass cover slide and photographs were obtained using a light microscope. The length and width of each stoma were measured using the ImageJ software.

Results ABA analog chemical library A targeted ABA analog library was developed that comprised 240 compounds, all structural variants of the base molecule S-(+)-ABA. The molecules had been synthesized for specific structure/activity studies to determine the requirements of a number of features of the ABA molecule: double bond geometry in the side chain, oxidation levels at C-1 and C-4’ of ABA, stereochemistry at C-1’, additions and deletions to the carbon framework, and molecules that were intermediates in the syntheses of the above. The set also included molecules designed as photoaffinity probes to identify ABA binding proteins, and molecules in the ABA biosynthetic and catabolic pathways (Figure 1).


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Figure 1. Structures of ABA and tetralone analogs from the primary chemical library and second generation library. 1, S-(+)-ABA; 2, (+)-PBI413; 3, (-)-PBI414; 4, (+)-PBI686; 5, (±)PBI664; 6, (+)-PBI690; 7, (-)-PBI691; 8, (±)-PBI911 (reported as PAO4 by 40); AS6 as reported in reference 39. Page 11 of 36 ACS Paragon Plus Environment

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Identification of antagonist hits by in vitro screening First generation antagonist hit The synthetic ABA analog library was screened for agonist and antagonist activities against 16 Arabidopsis ABA receptor-phosphatase combinations using phosphatase enzyme activity assays. ABA receptors RCAR3 (PYL8), RCAR8 (PYL5), RCAR10 (PYL4) and RCAR11 (PYR1) were selected as representatives from all different ABA receptor clades. RCAR3 belongs to clade I, RCAR8 and RCAR10 represent clade II and RCAR11 belongs to clade III 3. Four of the best characterized clade A phosphatases, known to mediate ABA receptor activity were selected for this study, ABI1, ABI2, HAB1 and AHG3. The agonist and antagonist activities of each compound were assayed separately. S-(+)-ABA and several other agonist analog compounds facilitated the inhibition of downstream phosphatases by ABA receptors (Supplementary figure S1). Interestingly, one compound among the 240 ABA analogs screened showed a recovery of ABA-inhibited phosphatase activity in the antagonist assays with representative members from all ABA receptor clades (Figure 2A). This analog PBI686 had been designed as a probe for identifying ABA-binding proteins 41,46,47. Structurally, PBI686 was based upon PBI413, which is a potent agonist hit (Supplementary figure S1)

38

. PBI686 incorporates

all the essential features of ABA needed for recognition by the receptor, the ketone at C-4’, the stereochemistry at the stereocentre, and the intact ABA sidechain, with the addition of an aromatic ring replacing the 2’,3’-double bond and 7’-methyl group and with the addition of a long tether ending in a biotin moiety to immobilize this compound on a streptavidin column


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(Figure 1). However, unlike PBI413, PBI686 had no agonist activity against any of the receptors tested (Supplementary figure S1). Second generation antagonist hits A second generation of compounds was developed based upon the hit structure PBI686. Molecules based on the tetralone scaffold were synthesized with intermediate chain length between the parent PBI413 and the primary antagonist hit PBI686 (Figure 1), starting with a racemic intermediate in the synthesis of the latter 33. The racemic carboxylic acids PBI664 (OH terminus) and PBI911 (methyl terminus; this compound is identical to the recently reported antagonist PAO4

40

) (Figure 1) showed antagonist activity similar to PBI686 (Figure 2B),

indicating that a shortened tether retained the antagonist activity. Interestingly, while PBI664 showed universal antagonist activity against representatives of all receptor clades tested here, PBI911 (PAO4

40

) with its methyl terminus showed receptor-selectivity and had no antagonist

activity against RCAR11 at the concentration tested in the biochemical assay (Figure 2B). Further assessment of the pure enantiomers of racemic PBI664, demonstrated that only the (R)(+) enantiomer, PBI690, is active in the mixture as an antagonist, with (S)-(-) enantiomer, PBI691, being inactive (Figure 2B).


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Figure 2. Antagonist activity of ABA analogs against RCARs and HAB1. (A) Antagonist activity of 100 µM first generation analog PBI686 in the presence of 0.1 µM (+)-ABA. (B) Antagonist activities of 100 µM second generation analogs in the presence of 0.1 µM (+)-ABA. The phosphatase activity of RCARs + HAB1 shown by the black bars was considered as the Page 14 of 36 ACS Paragon Plus Environment

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maximum activity and was set as 100 %, the inhibition of phosphatase activity in the presence of ABA and receptor is shown by light grey bars and the phosphatase activity of 100 µM of various analogs in the presence of 0.1 µM (+)-ABA is shown by dark grey bars. A thin dashed line placed at the level of the average value of ABA inhibition is provided to indicate the antagonist activity of the analogs. Any analog that shows a recovery of inhibited phosphatase activity represented by a bar higher than this reference line can be considered an antagonist. Each bar shows the average % activity of three replicates and the standard deviation is shown on top.

Antagonists prevent complex formation between RCARs and PP2Cs Toward investigating the mechanism of antagonism, we employed blue native – polyacrylamide gel electrophoresis (BN-PAGE) to analyze receptor-phosphatase complex formation in the presence of some of these analogs. The phosphatase HAB1 and the receptor RCAR8 ran as two distinct bands on the gel (Figure 3A, lanes 1 and 2, respectively). They did not form a complex when mixed together at equimolar ratio (Figure 3A, lane 3). A distinct upper band indicating the complex appeared above the HAB1 band only when the two proteins were incubated with (+)-ABA or the strong agonist PBI413 (Figure 3A, lanes 4 and 5). Addition of antagonists to the mix led to a partial reduction of the intensity of the upper complex band in the case of PBI686 (Figure 3B, lane 5) and a total disappearance of the complex when PBI664 was added (Figure 3B, lane 6). These results indicate that both PBI686 and PBI664 act as antagonists and dissociate the RCAR8 – ABA – HAB1 complex.


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Figure 3. BN-PAGE analysis of receptor-phosphatase complex formation. (A) Promotion of RCAR8 – HAB1 complex by (+)-ABA and the strong agonist PBI413. (B) Dissociation of RCAR8 – ABA – HAB1 complex by PBI686 and PBI664. The proteins and analogs were mixed and incubated at room temperature for 30 min. Native sample loading dye was added to the samples and immediately loaded on a 12 % native gel.

Antagonists bind to RCAR directly and prevent its association with the phosphatase Isothermal titration calorimetry (ITC) experiments were performed to further probe the mechanism of binding of the agonist and antagonist hits, in particular assessing the ability of the antagonists to bind directly to the receptor. From the ITC results (+)-ABA was determined to bind to RCAR8 with a kD of 1.2 µM and to RCAR8 + HAB1 with a kD of 41.3 nM (Supplementary figures S2 A and B). This is in good agreement with previously published ITC data involving the same molecules

48

. The potent agonist hit, PBI413, showed similar binding

characteristics like ABA. It bound to RCAR8 alone with a kD of 0.7 µM and bound to RCAR8 + HAB1 with a kD of 63.7 nM (Supplementary figures S2 C and D), explaining its relative


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potency. On the other hand its (-) enantiomer, PBI414 didn’t show any measurable binding to RCAR8 alone and had reduced binding affinity to RCAR8 + HAB1 (kD = 2.2 µM; Supplementary figure S3), consistent with its weak agonist activity (Supplementary figure S1) and highlighting the correlation between binding affinity and biochemical activity. ITC analysis of the first generation antagonist hit, PBI686, showed a similar binding affinity to RCAR8 alone like its parent agonist analog PBI413 (kD = 0.6 µM) (Figure 4A). However, unlike PBI413 which showed an increased affinity when titrated against RCAR8 + HAB1, PBI686 showed no remarkable increase in binding affinity when both phosphatase and receptor were present in the reaction cell (kD = 0.2 µM) (Figure 4B). Interestingly, the second generation antagonist hit PBI664 showed a kD of 1.2 µM for RCAR8 alone and a kD of 2.1 µM for RCAR8 + HAB1 (Figures 4 C and D). This latter result highlights a unique relationship between the antagonistbound receptor and the phosphatase that is distinct from the relationship between agonists-bound receptors and phosphatases. Together this data suggests that the antagonistic activity of PBI664 is not related to its interaction with the receptor, which is similar to the native agonist, but rather to modulation of the interaction of the antagonist-bound receptor with HAB1.


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Figure 4. Isothermal Titration Calorimetry of ABA receptor antagonists. Raw ITC data of sequential injections of ABA analogs. (A) PBI686 titrated against RCAR8 alone, (B) PBI686 titrated against (RCAR8 + HAB1), (C) PBI664 titrated against RCAR8 alone and (D) PBI664 titrated against (RCAR8 + HAB1). The processed data is shown below each binding isotherm. The binding parameters are shown in the inset. Sequential injections of the ligands were performed till saturation was reached. The data were fitted using the ‘one set of sites’ in the Origin software and the best fit is represented by the black line.

None of the above compounds had any measurable binding affinity when titrated against the phosphatase HAB1 alone, showing that they interact directly with the receptor (Supplementary figure S4).

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We tested if the universal antagonist PBI664 could promote germination of naturally dormant seeds of Arabidopsis thaliana CVI ecotype which is caused by elevated ABA levels 49. While only a low percentage of control seeds imbibed in water germinated at the end of 8 days, seeds imbibed in 100 µM PBI664 showed rapid seed germination starting at day 5 and by day 8 almost 95 % of the seeds germinated (Figure 5A). We also tested the effect of PBI664 on induced seed dormancy of A. thaliana Col-0 ecotype. These seeds do not display natural dormancy and it can be induced by addition of exogenous ABA (Figure S6). Co-application of PBI664 with ABA relieved this induced dormancy and promoted seed germination (Figure S6). We further tested if PBI664 can break induced thermodormancy of lettuce seeds caused by a spike in ABA levels when the seeds are imbibed at higher temperatures 50. We observed a dosedependent promotion of seed germination in the presence of PBI664 (Figure 5B). Lettuce seeds treated with water almost completely failed to germinate at the same temperature.


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Figure 5. Physiological effects of ABA receptor antagonists. (A) Effect of ABA and antagonist analogs on germination of naturally dormant seeds of A. thaliana CVI ecotype. Each point represents the average and standard deviation of 3 replicates. (B) Effect of ABA and PBI664 on thermodormancy of lettuce seeds at 33ºC. Each point represents the average and standard deviation of four replicates. (C) Stomata of A. thaliana Col. epidermal peels treated with buffer (control), (+)-ABA and PBI 664. The stomata are highlighted by red arrows. (D) Width/length ratios of A. thaliana Col. epidermal peels treated with buffer (blue), (+)-ABA (red) and PBI 664 (green). At least 40 stomata were measured for each data.

Opening of stomatal aperture caused by ABA antagonist treatment Increased levels of ABA caused by drought stress lead to a closure of stomata 2. We hypothesized that an ABA receptor antagonist will lead to an open stomata state in plants. We measured the stomatal aperture size in abaxial epidermal peels from A. thaliana Col. Ecotype plants incubated in dark earlier in the day when the stomata will generally remain closed. The control leaves as well as the ABA treated leaves had mostly closed stomata represented by the oval shape of the guard cells and a lower width to length ratio (Figures 5 C and D). However, in leaves that were treated with PBI664, almost all stomata were open represented by almost circular guard cells and the stomatal width to length ratio approaching 1 (Figures 5 C and D).

Discussion Tetralone analogs are highly effective ABA receptor ligands Alteration of the tether attached to the aromatic ring of a potent ABA receptor agonist, the tetralone PBI413, completely reversed the biological activity and resulted in the identification of an antagonist PBI686, active against all three clades of RCAR receptors. The analogs PBI686 and PBI664 have no agonist activity of their own, but are effective antagonists Page 20 of 36 ACS Paragon Plus Environment

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of ABA receptors and are able to block phosphatase binding to the receptor. The key to understanding how the tetralone analogs act as effective agonists or antagonists lie in deciphering their nature of interaction with the receptor. The change in enthalpy (∆H) during protein-ligand complex formation is considered a good indicator of the nature of binding, where lower ∆H values mean a more favorable interaction, usually associated with an net increase in non-covalent interactions between the binding partners 51. While the ∆H of (+)-ABA to RCAR8 is – 8.9 kcal/mol, it is lower for the PBI413-RCAR8 interaction (- 11.6 kcal/mol) (Supplementary figure S2) as well as for PBI686-RCAR8 interaction (∆H = -10 kcal/mol) (Figure 4). Interestingly, if we look at the binding properties of ABA and PBI686 to RCAR3, these differences become striking. ABA shows very weak binding to RCAR3 (kD = 7.5 µM, ∆H = ~ - 5 to 6 kcal/mol), whereas, PBI686 displays a high affinity interaction with RCAR3 (kD = 0.7 µM, ∆H = - 8.7 kcal/mol) (Supplementary figure S5). However, the binding parameters of the second generation tetralone analog PBI664 does not follow this trend (kD = 1.2 µM, ∆H = 4.3 kcal/mol). PBI664 is a mixture of (+) and (-) enantiomers while the above-mentioned tetralone analogs PBI413 and PBI686 were pure (+) enantiomers, which are generally the bioactive forms. PBI690 which is the (+) enantiomer of PBI664 is the active antagonist (Figure 2B). We can expect the pure and active (+) enantiomer to show a better binding affinity and a lower enthalpy than PBI664. Thus, it appears that the extra bulk from the aromatic ring structure in the tetralones contribute to increased molecular interactions with the ABA binding pocket in the receptors to enhance binding parameters of these analogs. This increased interaction with the ABA binding pocket of the tetralone antagonists is clearly reflected in their physiological activities. While the tetralone antagonist molecules PBI664 and PBI911 (or PAO4 40) were able


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to induce seed germination in naturally dormant A. thaliana CVI seeds, the non-tetralone 3’modified ABA analog AS6 failed to show similar activity (Figure 5A). In solution, the parent tetralone analog, PBI413, adopts a similar conformation as ABA, with the side chain axial

33

. The aromatic ring fused to the ABA ring maintains the same

geometrical relationship as the vinyl methyl of the ABA molecule and the additional atoms are in the same plane as the vinyl methyl group. The cyclohexenone ring in ABA has greater conformational mobility than the bicyclic skeleton of PBI413, as observed by variable temperature NMR studies

33

.

It appears that all the Arabidopsis ABA receptors can

accommodate the extra carbon atoms as the in vitro and in planta activities of ABA and PBI413 are similar 38. Consideration of an overlay of the crystal structure of ABA bound RCAR11, with PBI413 in the approximate position occupied by ABA in the binding pocket, suggests that with the core ABA ring and side chain making the expected contacts, the tetralone ring is situated such that it extends toward the mouth of the cavity making strong interactions with residues of the closed conformation gate and latch loop regions. In particular, PBI413 could make strong van der Waals interactions with conserved side chains Val 83 (4.6 Å) and Leu 87 (2.1 Å) located in the gate region, as well as a weaker interaction with the side chains of Phe 61 (2.5 Å) and also Val 163 (4.3 Å) in the terminal helix, without affecting the gate-latch mechanism (Figure 6A). Together all these interactions likely contribute to a stronger overall interaction between PBI413 and the RCARs (compared to ABA), which is clearly reflected in the ITC data (as discussed above).


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Figure 6. Overlay of agonist and antagonist ABA-like analog hits on S-(+)-ABA in the binding pocket of the crystal structure of RCAR11. (A) PBI413 (yellow) overlaid on the S(+)-ABA (grey) molecule of RCAR11 from PDB ID# 3K90, showing potential additional contacts with the RCAR gate and terminal helix regions that the tetralone ring may afford this agonist, yielding a stronger overall interaction, as measured by ITC. (B) PBI 690 overlaid on the S-(+)-ABA molecule of RCAR11 from PDB ID# 3QN1, showing the general orientation of the tether extending out of the mouth of the gate-latch region of the RCAR toward the associated HAB1. (C) PBI 690 overlaid on the S-(+)-ABA molecule of RCAR11 from PDB ID# 3K90, showing the potential clashes the tether makes with gate and pre-gate residues, which likely preclude closure of the gate-latch, leading to the analogs antagonist activity. (D) PBI 690 overlaid on the S-(+)-ABA molecule of RCAR11 from PDB ID# 3QN1, showing its distal proximity from the HAB1 Trp 385 that is inserted into the receptor gate-latch region.

Another point of interest with respect to the tetralone antagonist compound PBI686 is that while it may bind effectively and antagonize the activity of ABA against the RCAR receptors, it was originally designed as a mimic of ABA and used for the affinity capture of several other ABA binding proteins from plants

46,47

. Interestingly, our previous report showed

that PBI686 was more potent than ABA in inducing 3-ketoacyl-CoA synthase (FAE) expression


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in microspore-derived embryos of Brassica napus

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. This finding suggests the interesting

possibility that the physiological control of lipid deposition and modification by ABA might be perceived and mediated through an entirely different molecular pathway and may be independent of the RCAR receptor signaling pathway. Further work needs to be done to confirm this possibility. The tether dictates the receptor selectivity of antagonists In this work we show that the choice of the terminus of the tether is important in determining the receptor selectivity of the analog. The tether in PBI664 ends with a polar alcohol group and is active against RCARs 3, 8, 10 and 11. On the other hand, PBI911 which is structurally identical to PAO4 reported independently by Takeuchi and co-workers 40, contains a hydrophobic tether and showed selective antagonism for RCARs 3, 8 and 10 but not 11 (Figure 2B). Within the family of ABA receptors, a subset of these has been shown to be dimeric in the ‘inactive’ state, requiring higher concentrations of ABA for stimulation 52. RCAR11 is unique in the group of four RCARs used in this work, in that it is the only one of the four known to homodimerize 6. Thus in the case of RCAR11 only (and possibly all other homodimerizing RCARs), the polar terminus of the PBI664 tether group may be instrumental in disrupting the homodimer to gain access to the binding site.

Of course this mechanism remains to be

investigated and characterized. By using quinabactin, a selective agonist of dimeric ABA receptors like RCAR11, it was demonstrated that activation of dimeric ABA receptors was sufficient for substituting the physiological effects of ABA in plants with reduced ABA biosynthesis

31

. As a corollary to this observation, we hypothesized that, for an exogenous

antagonist molecule to be more effective in planta, it should act effectively against all the functionally relevant receptors perceiving that molecule and not against only a subset of the Page 24 of 36 ACS Paragon Plus Environment

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receptors. Hence, we wanted to test if the universal receptor antagonist PBI664 will be more effective in planta compared to the monomeric receptor-specific antagonist PBI911 (or PAO4 40

). When the efficacy of these analogs was compared in A. thaliana CVI seed germination

studies, PBI664 showed greater efficacy than PBI911 (or PAO4 40) at the same dose, proving our hypothesis (Figure 5A). Mechanism of ABA receptor antagonism The ABA binding pocket is flanked by two loop regions that are highly conserved in all RCARs. These two loops form the ‘gate’ and ‘latch’ structures 12. In the ABA-free receptor these loops are extended apart and this represents the open state. Upon ABA binding, the gate loop undergoes a conformational change and closes over the ABA binding pocket to interact with the latch, forming the closed state of the receptor. The closed receptors can then dock onto the active site region of the phosphatase and inhibit its activity. As a final ‘lock’ mechanism, a conserved tryptophan residue from the interacting phosphatase inserts into the ABA binding pocket and seals the closed state of the receptor 12–14,53,54. We anticipate that these antagonists function by a similar antagonism mechanism to that described for pyrabactin, which binds to the ABA binding pocket of RCAR14 and keeps the receptor in an open inactive state. This prevents the receptor from docking with the PP2C and hence there is no inhibition of phosphatase activity

55

. Like

pyrabactin, PBI686 and PBI664 have been shown here to bind directly to the ABA binding pocket of RCARs with similar affinity as (+)-ABA. Thus they may also be acting by keeping the gate – latch loops apart through steric hindrance caused by the tethers hanging out the entrance to the binding cavity. This is supported by consideration of an overlay of PBI690 with ABA in the active site of closed conformation RCAR11, which shows that the tether extends directly off the tetralone ring further out toward the closed gate and latch (Figure 6 B and C). In particular the Page 25 of 36 ACS Paragon Plus Environment

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ester group itself and third carbon following the ester along the tether come into direct steric clash, squeezed between the side chains of gate residue Leu 87 (1.2 Å away in the model), and ring of Phe 61 (2.5 Å away in the model) respectively. Due to the high affinity binding and rigidity of the tetralone analog in contrast to the flexibility of the RCAR loops, these clashes are more likely to force the gate-latch loops out and away, rather than preclude entry of the analog into the binding site. Thus PBI690 should bind effectively to the ABA-binding site and disrupt the surface of the RCAR presented to interact with the phosphatase, inhibiting the RCARphosphatase interaction. Finally, none of the tetralone based antagonists showed any agonist activity against the receptors in the in vitro phosphatase assays. Taken together, these results suggest that the tetralone antagonists might act by stabilizing the open conformation of the receptor leading to disruption of its interaction with the downstream phosphatase. This mechanism is unlike the one described for the ASn molecules

39

. Although the ASn molecules

displayed antagonistic effects in planta, mechanistically they are partial agonists that bound and stabilized the closed ABA-like state of the receptors

39

. However, structural studies on RCARs

bound to the tetralone antagonists will shed additional light on their exact mechanism of action.

Conclusion In conclusion, through a screen of 240 ABA analogs, we identified and developed compounds that showed universal ABA receptor antagonism as well as receptor/clade-selective antagonism. Classically, ABA is known for its positive effects in stress responses and plant development. Indeed, increasing ABA biosynthesis and signal transduction to elicit stronger stress resistance (both biotic and abiotic) are major internationally recognized objectives.


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However ABA is being linked to a growing number of physiological effects where down regulation of ABA signaling could be desirable. We have shown that the chemical nature of the tether of the tetralone analogs affects their receptor-selectivity. Further, such receptor-selective ABA antagonists could be used as research tools for better understanding of the physiological roles of individual ABA receptors in plants or even create ABA antagonists selective for certain species of plants. These molecules are key leads for further structure/activity studies that could lead to the development of a new class of plant growth regulators for agriculture and horticulture applications.

Acknowledgements This manuscript represents NRC Communication # 56219.

Supporting Information Available Supplementary Figures Figure S1. Agonist activity assay of (+)-ABA and some ABA-like analogs from the library. Figure S2. Isothermal Titration Calorimetry of ABA receptor agonists. Figure S3. Binding isotherms of PBI414. Figure S4. Binding isotherms of ABA and analogs with HAB1. Figure S5. ITC experiments with RCAR3.


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Figure S6. Effect of (+)-ABA and antagonist analog PBI664 on germination of A. thaliana Col-0 ecotype. Supplemental General Chemistry

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(42) Wittig, I., Braun, H.-P., and Schägger, H. (2006) Blue native PAGE. Nat. Protoc. 1, 418– 428. (43) Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 486–501. (44) Humphrey, W., Dalke, A., and Schulten, K. (1996) \uppercase{VMD}: visual molecular dynamics. J. Mol. Graph. Model. 14, 27–28,33–38. (45) Balsevich, J. J., Cutler, a. J., Lamb, N., Friesen, L. J., Kurz, E. U., Perras, M. R., and Abrams, S. R. (1994) Response of Cultured Maize Cells to (+)-Abscisic Acid, (-)-Abscisic Acid, and Their Metabolites. Plant Physiol. 106, 135–142. (46) Kharenko, O. a, Boyd, J., Nelson, K. M., Abrams, S. R., and Loewen, M. C. (2011) Identification and characterization of interactions between abscisic acid and mitochondrial adenine nucleotide translocators. Biochem. J. 437, 117–123. (47) Kharenko, O. a, Polichuk, D., Nelson, K. M., Abrams, S. R., and Loewen, M. C. (2013) Identification and characterization of interactions between abscisic acid and human heat shock protein 70 family members. J. Biochem. 154, 383–391. (48) Santiago, J., Rodrigues, A., Saez, A., Rubio, S., Antoni, R., Dupeux, F., Park, S.-Y., Márquez, J. A., Cutler, S. R., and Rodriguez, P. L. (2009) Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs. Plant J. 60, 575–588. (49) Ali-Rachedi, S., Bouinot, D., Wagner, M.-H., Bonnet, M., Sotta, B., Grappin, P., and Jullien, M. (2004) Changes in endogenous abscisic acid levels during dormancy release and maintenance of mature seeds: studies with the Cape Verde Islands ecotype, the dormant model of Arabidopsis thaliana. Planta 219, 479–488.


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Biochemistry

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Biochemistry

For Table of Contents Use Only Abscisic acid analogs that act as universal or selective antagonists of phytohormone receptors Nandhakishore Rajagopalana, Ken M. Nelsona,b, Amy F. Douglasa, Vishal Jheenguta, Idralyn Q. Alarconb, Sean A. McKennac, Marci Surpind, Michele C. Loewene,f,* and Suzanne R. Abramsb

100

(±)-PBI664

Control (water)

Germination (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABA (100 uM)

80

AS6 (100 uM) 60

PBI911 (100 uM) PBI664 (100 uM)

40 20 0 0

1

2

3

4

5

6

7

8

Days after sowing


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Abscisic Acid Analogues That Act as Universal or Selective

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