Development of Inhibitors of Salicylic Acid Signaling - Journal of

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Development of Inhibitors of Salicylic Acid Signaling Kai Jiang,† Tetsuya Kurimoto,† Eun-kyung Seo,† Sho Miyazaki,† Masatoshi Nakajima,† Hidemitsu Nakamura,† and Tadao Asami*,†,‡,§ †

Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan § Department of Biochemistry, King Abdulaziz University, Jeddah 21589, Saudi Arabia ‡

S Supporting Information *

ABSTRACT: Salicylic acid (SA) plays important roles in the induction of systemic acquired resistance (SAR) in plants. Determining the mechanism of SAR will extend our understanding of plant defenses against pathogens. We recently reported that PAMD is an inhibitor of SA signaling, which suppresses the expression of the pathogenesis-related PR genes and is expected to facilitate the understanding of SA signaling. However, PAMD strongly inhibits plant growth. To minimize the side effects of PAMD, we synthesized a number of PAMD derivatives, and identified compound 4 that strongly suppresses the expression of the PR genes with fewer adverse effects on plant growth than PAMD. We further showed that the adverse effects on plant growth were partially caused the stabilization of DELLA, which is also related to the pathogen responses. These results indicate that compound 4 would facilitate our understanding of SA signaling and its cross talk with other plant hormones. KEYWORDS: salicylic acid, systemic acquired resistance, signaling pathway, chemical inhibitor

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itself is an SA receptor.9 In their SA-perception model, SA binding to NPR1 induces a conformational change in NPR1 and relieves the repression of the transcription activation domain of NPR1 by its autoinhibitory N-terminal domain. Several studies have suggested the existence of other SA receptors,6,7 and further studies are required to comprehensively understand the molecular mechanism of SA signaling. In addition to SA, jasmonic acid (JA) and ethylene (ET) play major roles in plant defense responses to various pathogens and abiotic stresses,10 and other hormones, including brassinosteroid, abscisic acid (ABA), and gibberellin (GA), are also involved in defense mechanisms.10,11 These plant hormones regulate the plant defense systems negatively or positively via complex cross talk between the plant hormonal signals. For example, DELLA proteins, which are the main negative regulators of GA signaling, physically interact with JAZ proteins, consequently activating JA signaling.12 The DELLA proteins are also reported to promote a susceptibility to virulent biotrophs and a resistance to necrotrophs by altering the balance between SA signaling and JA signaling.13 Chemical biology, the application of small bioactive chemicals to investigate the cellular networks in organisms, is a useful technology for understanding plant hormone biosynthesis, signaling, and other mechanisms.14 The chemicals that induce SAR signaling have been identified and include probenazole and its derivatives 1,2-benzisothiazol-3(2H)-1,1dioxide,15,16 benzo-(1,2,3)-thiadiazole-7-carbothioic acid Smethyl ester,17−19 and 2,6-dichloroisonicotinic acid,20,21 but

INTRODUCTION Agricultural crops and stock are seriously damaged by various pathogens, causing severe production losses.1 To avoid or attenuate this damage, the plants themselves have evolved defense mechanisms, including physical defenses such as the cuticle, which acts as a barrier,2 and sophisticated immune defenses, such as pathogen-associated molecular pattern (PAMP)-triggered immunity and effector-triggered immunity.3 The invasion of plants by pathogens induces localized effector-triggered immunity, which leads to systemic resistance in distal plant tissues, a phenomenon called systemic acquired resistance (SAR).4 SAR is a long-lasting broad-spectrum immune response that is dependent on the accumulation of salicylic acid (SA). The study of SA-induced SAR will extend our understanding of the plant defense mechanisms against pathogens and improve the technology available for agricultural production and stock maintenance. The pathogenesis-related (PR) genes, a cluster of genes induced by pathogen infection, encode small secreted or vacuole-targeting proteins with antimicrobial properties.3 PR1 gene expression is promoted by SA and can be used as a molecular marker of the onset of SAR.5 SA binds to several receptors in the plant cell, and the reception mechanism is complex.6,7 Nonexpresser of pathogenesis related gene 1 (NPR1) plays an important role in SA signaling. Increased SA concentrations triggered by pathogen infection lead to the disassembly and translocation of NPR1 from the cytoplasm to the nucleus and promote its interaction with TGA transcription factors to control the expression of defense-related genes, such as PR1. Fu et al. demonstrated that NPR3 and NPR4, which are low- and high-affinity SA receptors, respectively, regulate the proteasomal degradation of NPR1 depending on the SA concentration induced by pathogen infection.8 In contrast, Wu et al. showed that NPR1 © 2015 American Chemical Societ

Received: Revised: Accepted: Published: 7124

March 27, 2015 July 30, 2015 August 3, 2015 August 3, 2015 DOI: 10.1021/acs.jafc.5b01521 J. Agric. Food Chem. 2015, 63, 7124−7133

Article

Journal of Agricultural and Food Chemistry

Hypocotyl Growth. To determine hypocotyl growth during the constant dark treatment, the seeds were germinated on 1/2 MS medium with or without the test compound. After stratification at 4 °C for 2 days, the seeds were light-pulsed for 6 h to improve their germination and then kept in the dark for 5 days at 22.5 °C. The 5 day old seedlings were raised in the dark, and the lengths of the hypocotyls were measured with ImageJ software (National Institutes of Health, Bethesda, MD). For hypocotyl growth under constant light, seedlings of Ler and the 5Δdella mutants were grown on 1/2 MS medium under constant light (18.2−46.5 μmol/m2/s) at 22.5 °C, transferred 3 days after germination (3 DAG) to 1/2 MS medium containing GA4 (10 μM), PAMD (25 μM), 4 (25 μM), or combinations of them, and monitored during culture for 9 days under constant light at 22.5 °C. The lengths of the hypocotyls were measured with ImageJ software. Fresh Weight. The seeds were germinated on 1/2 MS medium with or without the test compound under constant light at 22.5 °C. The seedlings were sampled at 10 DAG or 12 DAG and their weights measured. Colletotrichum higginsianum Infection Assay. Seedlings (5 DAG) germinated and grown on 1/2 MS medium at constant light were transferred to treatment medium 1/2 MS (no sucrose) and further cultivated for 8 days. Seedlings (8 day-after-treatment (DAT)) were sprayed with Colletotrichum higginsianum (C. higginsianum) and further cultivated for 4 days. The leaves of 4 day-after-infection (DAI) seedlings were sampled by liquid nitrogen. The RNA samples were extracted and converted to the cDNA as described above. The actin mRNA of C. higginsianum (Ch-Actin) was quantified by qRT-PCR using primers specific for Ch-Actin (5′- CCGCAGACCGCAATCTT3′ and 5′- AATGGAGGCTGAGAGCTGGTT-3′). The transcript levels of Ch-ACT were normalized against those of Arabidopsis Actin7. Quantification of Endogenous SA and ABA. Chemicals. Deuterium-labeled SA (SA-d 4 ) and deuterium-labeled ABA (ABA-13C2) were purchased from Wako Pure Chemical Industries, Ltd. (Japan). Growth Condition. Seedlings (5 DAG) grown on 1/2 MS medium under constant light at 22.5 °C were transferred to 1/2 MS medium with or without the 25 μM compounds (PAMD or 4) for further cultivation for 8 days under constant light at 22.5 °C. The plants were frozen by liquid nitrogen. Extraction and Purification. The frozen plant tissues were homogenized and purified by using solid-phase extraction (SPE) as described by Miyazaki et al.,23 with the exception that SA or ABA was eluted with 0.2 M formic acid (FA) in acetonitrile (AcCN) in this paper. LC-MS/MS Analysis. LC-MS/MS was equipped as described Miyazaki et al.23 The dried samples were reconstituted in 50 μL of 10 mM FA in 10% (v/v) methanol (MeOH), and 10 μL of each sample was then injected onto a reversed-phase UPLC column (Acquity UPLC BEH C18, 2.1 mm × 50 mm, 1.7 μm, Waters) with a guard column (Acquity UPLC BEH C18 VanGuardTM Precolumn, 1.7 μm, 2.1 mm × 5 mm, Waters) coupled to the ESI-MS/MS system. SA and ABA were analyzed in the negative ion mode as [M − H]−. MS/MS analysis conditions for SA were as follows: cone energy, 24 eV; collision energy, 17 V; and MRM, 137.0 > 93.0 for unlabeled SA and 141.0 > 97.0 for labeled SA. The cone energy and collision energy for ABA and ABA-13C2 were 22 and 24 eV, and 14 and 12 eV, respectively, and those of MRM were 263.2 > 153.0 and 165.2 > 153.0, respectively. SA and ABA were analyzed by a linear gradient of 1% (v/ v) FA in water (A) and 1% FA in MeOH (B) at a flow rate of 0.20 mL/min, from 80:20 A/B (v/v) (0 min), 55:45 A/B (v/v) (1 min), 25:75 A/B (v/v) (8 min), and 0:100 A/B (v/v) (9 min), maintaining this composition until 1 min and equilibrated to initial conditions. Chemicals. PAMD (1, ID: KM05261) was purchased from MayBridge (Cornwall, UK), as described previously.22 The PAMD derivatives were synthesized as follows. 2, 2-((3-(Trifluoromethyl)phenylamino)methylene)cyclohexane1,3-dione. 1,3-Cyclohexanedione (10 mmol), orthoacetic acid triethyl ester (13 mmol), and 10 mmol trifluoromethylaniline were combined and stirred for 5 min at 130 °C. Ethanol (3 mL) was added, and the mixture was stirred for 3 min at 130 °C. The mixture was then cooled

there is very limited information available on the inhibitors of SA signaling. Recently, we screened a chemical library for inhibitors of SA signaling and found that 4-phenyl-2-{[3-(trifluoromethyl)aniline]methylidene}cyclohexane-1,3-dione (PAMD, 1) suppresses the expression of SA-related genes in Arabidopsis and increases its susceptibility to pathogens.22 In addition to the suppressive effects of PAMD on the expression of PR genes, PAMD also inhibits the growth of Arabidopsis, including its root growth, seedling establishment, and fresh weight. These side effects limit the utility of PAMD in studies of the SA signaling pathway. Structure−activity relationship studies of PAMD derivatives predicted that a 2-substituted enamine moiety conjugated with 1,3-cyclohexadione is essential for the inhibition of the SA signal. On the basis of this structure− activity relationship analysis, a number of derivatives of PAMD were synthesized in the present study to develop better candidate SA signaling inhibitors. We ultimately selected a compound that strongly suppresses the expression of the PR genes with fewer adverse effects on the growth of Arabidopsis than PAMD. We also found that the retardation effect of these new compounds on plant growth partly depends on the stabilization of the DELLA proteins, negative regulators of GA signaling, which are also involved in the pathogen defense responses.

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MATERIALS AND METHODS

Plant Materials and Growth Conditions. Most of the experiments were carried out with Arabidopsis thaliana Col-0. Landsberg erecta (Ler) was used as the genetic background of 5Δdella, a mutant with loss-of-function mutations in all five DELLA proteins. RGA:GFP-RGA, a line that expresses GFP-RGA driven by the RGA promoter, was used to detect GA-induced DELLA degradation. The seeds were surface-sterilized with 70% ethanol for 20 min, rinsed with 99% ethanol, dried on sterilized filter paper, and then placed onto culture medium containing half-strength Murashige and Skoog (1/2 MS) Plant Salt Mixture (Wako, Osaka, Japan), 1% sucrose, 0.8% agar, vitamin mixture, and 0.01% myo-inositol (Wako). After stratification under dark conditions at 4 °C for 2 days, the seeds were germinated under constant light (18.2−46.5 μmol/m2/s) at 22.5 °C. For the dark treatment in the hypocotyl experiment, the seeds were light-pulsed (18.2−46.5 μmol/m2/s) for 6 h before they were kept under constant dark at 22.5 °C. SA Treatment. Seedlings grown on plates with or without the test compound, as indicated, were foliar sprayed with a 2 mM SA aqueous solution (0.5% DMSO), cultured for a further 3 days under the same conditions, and then frozen in liquid nitrogen. RT-PCR Analyses. Total RNA was extracted from seedlings treated with or without SA, purified with the Total RNA Extraction Mini Kit (RBC Bioscience, Taipei, Taiwan), and then converted to cDNA with the PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan) according to the manufacturers’ protocols. Quantitative PCR was carried out with Thermal Cycler Dice Real Time System TP800 (Takara Bio) and the SYBR Premix Ex Taq (Takara Bio). The transcript levels of PR genes were normalized against those of Actin7 (encoding actin 7), using primers specific for PR1 (5′-CGTCTTTGTAGCTCTTGTAGGTGC-3′ and 5′-TGCCTGGTTGTGAACCCTTAG-3′), PR2 (5′-CGTCTTTGTAGCTCTTGTAGGTGC-3′ and 5′-TGCCTGGTTGTGAACCCTTAG-3′), PR5 (5′-CGTCTTTGTAGCTCTTGTAGGTGC-3′ and 5′-TGCCTGGTTGTGAACCCTTAG3′), and Actin7 (5′-GATATTCAGCCACTTGTCTGTGAC-3′ and 5′-CATGTTCGATTGGATACTTCAGAG-3′). Phenotypic Observations. Germination Rate. After sterilization (as described in the Plant Materials and Growth Conditions section), more than 30 seeds for each treatment were placed on 1/2 MS medium with or without the test compound. Germination was defined as the emergence of the radicle. 7125

DOI: 10.1021/acs.jafc.5b01521 J. Agric. Food Chem. 2015, 63, 7124−7133

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H, s), 2.37 (3 H, s), 2.42 (2 H, s), 2.47 (2 H, s), 7.09−7.11 (1 H, m), 7.15−7.17 (1 H, m), 7.37 (1 H, d, J = 1.7), 8.56 (1H, d, J = 13.2). 8, 2-((4-Hydroxyphenylamino)methylene)-5,5-dimethylcyclohexane-1,3-dione. 5,5-Dimethyl-1,3-cyclohexanedione (10 mmol), orthoacetic acid triethyl ester (13 mmol), and 4-hydroxyaniline (10 mmol) were combined and stirred for 5 min at 130 °C. Ethanol (3 mL) was added, and the mixture was stirred for 3 min at 130 °C. The mixture was then cooled to room temperature, and 10 mL of distilled water was added to quench the reaction. Suction filtration was carried out with distilled water, then twice with 60% ethanol and once with hexane. The compound was purified by recrystallization with ethyl acetate and hexane. The crystal was collected by suction filtration. 1H NMR (500 MHz, CDCl3): δ 1.08 (6 H, s), 2.42 (2 H, s), 2.45 (2 H, s), 6.91 (2 H, d, J = 9.2), 7.12 (1 H, d, J = 8.6), 8.50 (1 H, d, J = 13.7). 9, 5,5-Dimethyl-2-((3-(trifluoromethyl)phenylamino)methylene)cyclohexane-1,3-dione. 5,5-Dimethyl-1,3-cyclohexanedione (10 mmol), orthoacetic acid triethyl ester (13 mmol), and trifluoromethylaniline (10 mmol) were combined and stirred for 5 min at 130 °C. Ethanol (3 mL) was added, and the mixture was stirred for 3 min at 130 °C. The mixture was then cooled to room temperature, and 10 mL of distilled water was added to quench the reaction. Suction filtration was carried out with distilled water, then twice with 60% ethanol and once with hexane. The compound was purified by recrystallization with ethyl acetate and hexane. The crystal was collected by suction filtration. 1H NMR (500 MHz, CDCl3): δ 1.07 (6 H, s), 2.41 (2 H, s), 2.45 (2 H, s), 7.42 (1 H, s), 7.47−7.52 (3 H, m), 8.55−8.58 (1 H, m). 10, 2-((2-Chlorophenylamino)methylene)-5,5-dimethylcyclohexane-1,3-dione. 5,5-Dimethyl-1,3-cyclohexanedione (10 mmol), orthoacetic acid triethyl ester (13 mmol), and 2-chloroaniline (10 mmol) were combined and stirred for 5 min at 130 °C. Ethanol (3 mL) was added, and the mixture was stirred for 3 min at 130 °C. The mixture was then cooled to room temperature, and 10 mL of distilled water was added to quench the reaction. Suction filtration was carried out with distilled water, then twice with 60% ethanol and once with hexane. The compound was purified by recrystallization with ethyl acetate and hexane. The crystal was collected by suction filtration. 1H NMR (500 MHz, CDCl3): δ 1.08 (6 H, s), 2.42 (2 H, s), 2.48 (2 H, s), 7.15 (1 H, t, J = 8.0), 7.33 (1 H, t, J = 8.0), 7.44 (1 H, d, J = 8.0), 7.46 (1 H, t, J = 8.6), 8.60 (1 H, d, J = 13.2). 11, 2-((2,4-Dimethylphenylamino)methylene)-5,5-dimethylcyclohexane-1,3-dione. 5,5-Dimethyl-1,3-cyclohexanedione (10 mmol), orthoacetic acid triethyl ester (13 mmol), and 2,4-dimethylaniline (10 mmol) were combined and stirred for 5 min at 130 °C. Ethanol (3 mL) was added, and the mixture was stirred for 3 min at 130 °C. The mixture was then cooled to room temperature, and 10 mL of distilled water was added to quench the reaction. Suction filtration was carried out with distilled water, then twice with 60% ethanol and once with hexane. The compound was purified by recrystallization with ethyl acetate and hexane. The crystal was collected by suction filtration. 1H NMR (500 MHz, CDCl3): δ 1.08 (6 H, s), 2.30 (3 H, s), 2.35 (3 H, s), 2.39 (2 H, s), 2.45 (2 H, s), 7.03 (1 H, s), 7.06 (1 H, d, J = 8.0), 7.24 (1 H, d, J = 9.0), 8.56 (1H, d, J = 13.5). 12, 5,5-Dimethyl-2-((2-nitrophenylamino)methylene)cyclohexane-1,3-dione. 5,5-Dimethyl-1,3-cyclohexanedione (10 mmol), orthoacetic acid triethyl ester (13 mmol), and 2-nitroaniline (10 mmol) were combined and stirred for 5 min at 130 °C. Ethanol (3 mL) was added, and the mixture was stirred for 3 min at 130 °C. The mixture was the cooled to room temperature, and 10 mL of distilled water was added to quench the reaction. Suction filtration was carried out with distilled water, then twice with 60% ethanol and once with hexane. The compound was purified by recrystallization with ethyl acetate and hexane. The crystal was collected by suction filtration. 1H NMR (500 MHz, CDCl3): δ 1.09 (6 H, s), 2.45 (2 H, s), 2.52 (2 H, s), 7.33 (1 H, t, J = 8.6), 7.65 (1 H, d, J = 8.0), 7.71 (1 H, t, J = 8.6), 8.27 (1 H, d, J = 9.7), 8.61 (1 H, d, J = 13.2). Quantitative Analysis of Anthocyanin. The frozen plant tissues treated with or without compounds were homogenized, pretreated, and then applied to cation-exchange SPE (Oasis MCX) using the same procedure as described by Miyazaki et al.23 The SPE column was

to room temperature, and 10 mL of distilled water was added to quench the reaction. Suction filtration was carried out with distilled water, then twice with 60% ethanol, and once with hexane. The compound was purified by recrystallization with ethyl acetate and hexane. The crystal was collected by suction filtration. 1H NMR (500 MHz, CDCl3): δ 2.03 (2 H, quin, J = 6.9), 2.55 (2 H, t, J = 6.3), 2.60 (2 H, t, J = 6.9), 7.43 (1 H, d, J = 7.4), 7.48−7.50 (2 H, m), 7.53 (1 H, d, J = 8.6), 8.61 (1 H, d, J = 13.2). 3, 2-((Phenylamino)methylene)cyclohexane-1,3-dione. 1,3-Cyclohexanedione (10 mmol), orthoacetic acid triethyl ester (13 mmol), and aniline (10 mmol) were combined and stirred for 5 min at 130 °C. Ethanol (3 mL) was added, and the mixture stirred for 3 min at 130 °C. The mixture was then cooled to room temperature, and 10 mL of distilled water was added to quench the reaction. Suction filtration was carried out with distilled water, then twice with 60% ethanol and once with hexane. The compound was purified by recrystallization with ethyl acetate and hexane. The crystal was collected by suction filtration. 1H NMR (500 MHz, CDCl3): δ 2.02 (2 H, quin, J = 6.9), 2.54 (2 H, t, J = 6.9), 2.58 (2 H, t, J = 6.9), 7.20−7.25 (3 H, m), 7.40 (2 H, t, J = 8.0), 8.60 (1 H, d, J = 13.7). 4, 5,5-Dimethyl-2-((phenylamino)methylene)cyclohexane-1,3dione. 5,5-Dimethyl-1,3-cyclohexanedione (10 mmol), orthoacetic acid triethyl ester (13 mmol), and aniline (10 mmol) were combined and stirred for 5 min at 130 °C. Ethanol (3 mL) was added, and the mixture stirred for 3 min at 130 °C. The mixture was then cooled to room temperature, and 10 mL of distilled water was added to quench the reaction. Suction filtration was carried out with distilled water, then twice with 60% ethanol and once with hexane. The compound was purified by recrystallization with ethyl acetate and hexane. The crystal was collected by suction filtration. 1H NMR (500 MHz, CDCl3): δ 1.08 (6 H, s), 2.41 (2 H, s), 2.45 (2 H, s), 7.22−7.26 (3 H, m), 7.40 (2 H, t, J = 8.6), 8.60 (1 H, d, J = 13.7). 5, 2-((2,4-Dichlorophenylamino)methylene)-5,5-dimethylcyclohexane-1,3-dione. 5,5-Dimethyl-1,3-cyclohexanedione (10 mmol), orthoacetic acid triethyl ester (13 mmol), and 2,4-dichloroaniline (10 mmol) were combined and stirred for 5 min at 130 °C. Ethanol (3 mL) was added, and the mixture was stirred for 3 min at 130 °C. The mixture was then cooled to room temperature, and 10 mL of distilled water was added to quench the reaction. Suction filtration was carried out with distilled water, then twice with 60% ethanol and hexane. The compound was purified by recrystallization with ethyl acetate and once with hexane. The crystal was collected by suction filtration. 1H NMR (500 MHz, CDCl3): δ 1.09 (6 H, s), 2.43 (2 H, s), 2.49 (2 H, s), 7.32 (1 H, d, J = 11.5), 7.40 (1 H, d, J = 8.6), 7.47 (1 H, s), 8.54 (1 H, d, J = 13.2). 6, 2-((3-Chlorophenylamino)methylene)-5,5-dimethylcyclohexane-1,3-dione. 5,5-Dimethyl-1,3-cyclohexanedione (10 mmol), orthoacetic acid triethyl ester (13 mmol), and 3-chloroaniline (10 mmol) were combined and stirred for 5 min at 130 °C. Ethanol (3 mL) was added, and the mixture was stirred for 3 min at 130 °C. The mixture was then cooled to room temperature, and 10 mL of distilled water was added to quench the reaction. Suction filtration was carried out with distilled water, then twice with 60% ethanol and once with hexane. The compound was purified by recrystallization with ethyl acetate and hexane. The crystal was collected by suction filtration. 1H NMR (500 MHz, CDCl3): δ 1.08 (6 H, s), 2.41 (2 H, s), 2.46 (2 H, s), 7.14 (1 H, d, J = 18.9), 7.21−7.27 (2 H, m), 7.31−7.34 (1 H, m), 8.53 (1 H, d, J = 13.2). 7, 2-((3-Chloro-2-methylphenylamino)methylene)-5,5-dimethylcyclohexane-1,3-dione. 5,5-Dimethyl-1,3-cyclohexanedione (10 mmol), orthoacetic acid triethyl ester (13 mmol), and 3-chrolo-2methylaniline (10 mmol) were combined and stirred for 5 min at 130 °C. Ethanol (3 mL) was added, and the mixture was stirred for 3 min at 130 °C. The mixture was then cooled to room temperature, and 10 mL of distilled water was added to quench the reaction. Suction filtration was carried out with distilled water, then twice with 60% ethanol and once with hexane. The compound was purified by recrystallization with ethyl acetate and hexane. The crystal was collected by suction filtration. 1H NMR (500 MHz, CDCl3): δ 1.09 (6 7126


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Figure 1. Effects of PAMD on Arabidopsis growth. (A) At 12 days after germination (12 DAG), Arabidopsis thaliana Col-0 was grown on 1/2 MS medium with or without PAMD (as indicated) under constant light at 22.5 °C. Scale = 5 mm. (B) Seeds were germinated on 1/2 MS medium with or without PAMD and grown under constant light at 22.5 °C. Error bars indicate the means ± SE of three independent replicates, n > 30. (C) Fresh weights of 10 DAG Col-0 plants grown on 1/2 MS medium with or without PAMD under constant light at 22.5 °C. Error bars indicate means ± SE of three independent replicates, n > 10. Student’s t test was used to determine the significance of differences relative to the mock treatment; *, P < 0.05. (D) After stratification for 2 days at 4 °C, Col-0 seeds were light-pulsed for 6 h and were germinated and grown on 1/2 MS medium with or without the test compound under constant dark at 22.5 °C. Hypocotyl length was measured at 5 DAG. Error bars indicate the means ± SE of three independent replicates, n = 10. Student’s t test was used to determine the significance of differences relative to the mock treatment; **, P < 0.01. washed with 5 mM FA and MeOH successively. Anthocyanins were eluted with MeOH (1% NH4OH) and water/MeOH (40:60 in volume ration, 1% NH4OH) successively. The two successive elutions were combined together and quantified by reading the absorbance at 530 nm (A530) and 657 nm (A657) using GeneQuant (GE Healthcare Life Science). The relative level of anthocyanin was calculated as (A530 − 0.25 × A657) g−1 FW. GA-induced DELLA Degradation. The RGA:GFP-RGA line of Arabidopsis was used to observe the GA-induced degradation of DELLA. After stratification for 2 days at 4 °C in the dark, the RGA:GFP-RGA line was germinated on 1/2 MS medium under constant light at 22.5 °C. The primary roots of the seedlings (2 DAG) were placed on a microscope slide with a drop of liquid-phase 1/2 MS containing GA4 (10 μM) or GA4 combined with PAMD (25 μM) or 4 (25 μM). The green fluorescence of green fluorescent protein (GFP) was observed under a confocal-laser scanning microscope (LSM700, Zeiss, Oberkochen, Germany), and the degradation of GFP-RGA was recorded with time-lapse photography.

suppressing effects of PAMD were relevant to its inhibitory effect on SA signaling. Synthesis of PAMD Derivatives. To investigate the relationship between the chemical structure and SA-signalinhibiting activity of PAMD (1) and to reduce its inhibitory effects on plant growth without affecting its SA-signal-inhibiting activity, several PAMD derivatives with deletions or changes to various groups were synthesized (Figure 2). The PAMD derivatives were synthesized with one-step reactions, which are much simpler than the reaction used to synthesize PAMD (Figure 2A). SA-Signal-Inhibiting Activities of PAMD Derivatives. The effects of the derivatives described above on the suppression of PR1 expression were determined with realtime PCR (Figure 3). Compound 2, in which the 2-phenyl group is deleted, and compound 3, in which both the 2-phenyl group and the trifluoromethyl group on the phenyl group attached to the N atom are deleted, did not suppress PR1 expression, suggesting that the phenyl group on the cyclohexadione group contributes to this activity. The substitution of 3 with a dimethyl group at the 5-position of the cyclohexadione group generated compound 4, which recovered its capacity to suppress PR1 expression, although this activity was weaker than that of PAMD. This suggests that in addition to the phenyl group the dimethyl group on the cyclohexane ring also contributes to the suppression of PR1 expression, perhaps as an electron-donating group. On the basis of the structure of 4, various derivatives with several substituents on the N-attached phenyl group of 4 were synthesized, and their activities were determined. Compound 5, with 2,4-dichloro groups on the Nattached phenyl group, showed more potent activity in suppressing PR1 expression than did 4. Compound 6, with a 3-chloro group, compound 7, with 3-chloro and 2-methyl groups, and compound 8, with a 4-hydroxyl group, each

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RESULTS PAMD Inhibits the Growth of A. thaliana. PAMD was isolated from a chemical library of 9600 randomly synthesized compounds using SA-induced β-glucuronidase (GUS)-expressing plants (PR1::GUS line) and was determined to be an effective inhibitor of SA signaling.22 However, the Arabidopsis seedlings treated with PAMD were smaller than the untreated seedlings, indicating that PAMD suppresses plant growth (Figure 1A). Further studies showed that PAMD had no obvious suppressive effect on the seed germination time or germination rate (Figure 1B). We then investigated the dosedependence of the phenotype of the seedlings treated with PAMD and found a positive correlation between the PAMD concentration and Arabidopsis growth suppression, including rosette size, fresh weight, and hypocotyl growth (Figure 1A,C,D). However, it was unclear whether the plant-growth7127


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Figure 2. Synthesis of PAMD derivatives. (A) Scheme of the synthesis of derivatives with one-step methods. (B) Chemical structure of PAMD (1) and its derivatives newly synthesized in this paper.

lost the capacity to suppress PR1 gene expression. Unexpectedly, compounds 3 and 12 increased PR1 gene expression. It is noteworthy that although substituted with the same atom(s) the activities of compounds 5 and 6 differed significantly from that of compound 10 in suppressing PR1 gene expression suggesting the importance of the positions and the combinations of the substituents on the N-attached phenyl group. Various groups on the N-attached phenyl group also played important roles in suppressing PR1 expression and further study of the structure−activity relationships of the substituents on the N-attached phenyl group should reveal important factors that increase or reduce PR1 expression. Effects of Treatment with PAMD Derivatives on Plant Growth. The side effects of the PAMD derivatives on Arabidopsis growth were investigated (Figure 4). At a concentration of 10 μM, 2 reduced the fresh weight of Arabidopsis less than did PAMD, which indicates that the inhibitory effect may be partly induced by the 2-phenyl group. Compound 3 did not differ significantly from PAMD in reducing the fresh weight of Arabidopsis. However, 4 showed clearly less inhibitory effect on growth than did PAMD. These results indicate that the growth-inhibitory effect may result

Figure 3. Determination of SA signal inhibition by PAMD derivatives. Seedlings grown for 5 DAG on 1/2 MS medium under constant light at 22.5 °C were transferred to 1/2 MS medium containing the indicated compounds (25 μM) for further cultivation for 8 days under constant light at 22.5 °C, followed by foliar-sprayed SA treatment. PR1 expression was determined relative to Actin7. Error bars indicate the means ± SD of three replicates.

showed activity almost equal to that of 4. However, some other derivatives, such as compound 9, with a 3-trifluoromethyl group, compound 10, with a 2-chloro group, and compound 11, with 2,4-dimethyl groups on the N-attached phenyl group, 7128


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Figure 4. Estimation of the inhibitory effects of PAMD and its derivatives. Seedlings (5 DAG) grown on 1/2 MS medium under constant light at 22.5 °C were transferred to 1/2 MS medium with or without the test compound and further cultivated for 11 days under constant light at 22.5 °C. Fresh weight was determined as an estimate of growth. Error bars indicate the means ± SE of three independent replicate measurements of the average weights of 10 seedlings. Student’s t test was used to determine the significance of differences relative to the PAMD (1) treatment; *, P < 0.05.

from the basic structure of 3 and that the 5,5-dimethyl groups reduce the inhibitory effect on growth. The result for 9 showed that the trifluoro group has a negative effect on plant growth when it exists in combination with the 3,3-dimethyl groups. Except for 11, which showed similar levels of growth inhibition as 4, all the derivatives based on the structure of 4, including 5− 8, 10, and 12, inhibited growth more strongly than 4, but most of them, including 5, 8, 10, and 12, inhibited growth less strongly than PAMD. However, at a concentration of 25 μM, all the PAMD derivatives suppressed the growth of Arabidopsis (Figure 4). The growth-inhibitory effect of 4 was smallest of all the PAMD derivatives tested, even though it did not differ significantly from that of PAMD (Figure 4). Compound 4 also suppressed the expression of the PR2 and PR5 genes even more strongly than PAMD (Figure 5), indicating that 4 is not a specific inhibitor of PR1 expression. The Collectotrichum higginsianum (C. higginsianum) infection assay indicated that 4 likely increased the susceptibility of Arabidopsis to C. higginsianum (Figure 6). Because some of the PAMD derivatives showed less growth-inhibitory activity than PAMD when applied at 10 μM, we tested whether a lower concentration of 4 suppressed the induction of PR1 gene expression by SA and found that 4 inhibited SA-induced PR1 expression in a concentrationdependent manner (Figure 7A). This result clearly shows that 4 suppresses SA-induced PR1 expression as strongly as PAMD (1) at 10 μM (Figure 7B) at which concentration 4 affects plant growth less than it does at 25 μM (Figure 4). Compound 4 showed less adverse effects on plant growth than PAMD, including on seed germination, fresh weight, seedling establishment, and root growth under constant light (Figure 8A−E). Remarkably, hypocotyl growth in the dark was only slightly inhibited by 4, even when 25 μM of 4 was applied (Figure 8F). On the basis of these results, 4 could be considered a better inhibitor of SA signaling than PAMD because it suppresses PR gene expression to a similar or greater extent than PAMD but with a less adverse effect on growth. Quantification of Endogenous Salicylic Acid and Abscisic Acid in Arabidopsis using LC-MS/MS. Although the inhibitory effects of PAMD and its derivatives on PR1 expression were determined under exogenous SA treatment, it is still an open question whether PAMD and its derivatives impact SA biosynthesis, which may also contribute to the final

Figure 5. Suppressive effect of PAMD (1) and 4 on the expression of PR2 and PR5. Seedlings (5 DAG) grown on 1/2 MS medium under constant light at 22.5 °C were transferred to 1/2 MS medium containing 25 μM of the test compound, as indicated, for further cultivation for 8 days under constant light at 22.5 °C, followed by foliar-sprayed SA treatment. PR1 expression was determined relative to Actin7. Error bars indicate the means ± SD of three replicates. Statistical differences between the groups were calculated with ANOVA analysis (Posthoc: Duncan). Bars with different letters are significantly different, with P < 0.01.

effects of the compound. To answer this question, the endogenous level of SA in the Arabidopsis treated by PAMD or 4 was quantified. The results showed that treatment by PAMD or 4 increased the SA level compared to mock treatment (Figure 9A). Meanwhile, we also quantified the endogenous level of ABA, which is well-known for its accumulation in plants under stresses and its negative roles in regulating plant growth. The plant treated with PAMD or 4 accumulated a higher level of ABA compared to the mock treatment (Figure 9B). In accordance with the increasing level of ABA, the anthocyanin level was also higher in the plants treated by PAMD (Supporting Information Figure 1). The increasing levels of ABA and anthocyanin by PAMD treatment are considered to be the side effects of the compounds, which caused stresses to the plant. Treatment by 4 induced a moderate increase in the ABA level and no change in anthocyanin level compared to that effected by the treatment with PAMD (Figure 9B, Supporting Information Figure 1). These results further indicate that 4 is a better candidate than PAMD with less adverse effects on plants. Inhibitory Effects of PAMD and Its Derivatives Depend Partly on DELLA. Although 4 is a better SA signal inhibitor than PAMD, it still inhibits plant growth to some extent (Figure 8). The positive regulatory roles of GA in plant growth are well-known, and the binding of GA to its receptor 7129


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proteins, we applied GA4 with PAMD or 4 to the 5Δdella mutant line of Arabidopsis, which is insensitive to GA biosynthesis inhibitors or GA antagonists because it carries loss-of-function mutations in all five DELLA proteins.27 The results showed that the hypocotyl growth of the 5Δdella line was less inhibited by PAMD or 4 than was the wild-type line (Ler), although there was still a significant difference between the effects of the mock treatment and test compound treatments in the 5Δdella line (Figure 10A). The restorative effect of GA on the hypocotyl growth suppressed by PAMD or 4 was observed in Ler plants but not in 5Δdella plants (Figure 10A). We then analyzed the effects of treatment with PAMD or 4 on the accumulation of the DELLA proteins using the RGA:GFP-RGA line. Treatment with either of the two compounds delayed the GA-mediated degradation of the GFP-RGA fusion protein (Figure 10B). These results indicate that PAMD and its derivative 4 alleviate the DELLA degradation induced by GA treatment and GA signaling. Therefore, their effects on plant growth partly depend on the function of the DELLA proteins.

Figure 6. Collectotrichum higginsianum (C. higginsianum) infection assay. Seedlings (5 DAG) were transferred to treatment medium containing compounds as indicated or not and further cultivated for 8 days. The 8 day-after-treatment (DAT) seedlings were sprayed by 1 × 107 spores/ml of C. higginsianum and followed by 4 days’ cultivation. Ch-Actin expression was determined relative to Arabidopsis Actin7 (AtActin7). Error bars indicate the means ± SD of three replicates. Statistical differences between the groups were calculated with ANOVA analysis (Posthoc: Duncan). Bars with different letters are significantly different, with P < 0.01. Scale = 5 mm.

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DISCUSSION Not only plant hormone mimics, but also chemical inhibitors such as paclobutrazole, an inhibitor of GA biosynthesis, brassinazole, an inhibitor of brassinosteroids biosynthesis,28 and naphthylphthalamic acid, an inhibitor of auxin transport,29 are useful in studies on plant hormone signaling. Until recently, there has been limited information about inhibitors of SA signaling. In a previous paper, we reported that PAMD (1) inhibits SA signaling in Arabidopsis and increases the susceptibility of the plants to pathogens,22 suggesting that PAMD is an inhibitor of SA signaling and may be a useful tool for determining the SAR mechanism. However, besides its effects on SA signaling, PAMD adversely affected plant growth. In the present study, we prepared several derivatives of PAMD and found that one of them compound 4 suppressed the expression of the SA-response-marker PR genes with a smaller adverse effect on growth than PAMD. Therefore, we selected 4 as a better SA-signaling inhibitor than PAMD. LC-MS/MS analysis confirmed that PAMD and 4 increased the endogenous level of SA, which should have induced the expression of PR gene. This inconsistency between the increasing SA level and the decreasing PR gene expression level in the Arabidopsis treated by PAMD or 4 indicated that these compounds affect the SA signaling pathway and resistance to C. higginsianum by impairing the PR gene expression but not through an impact on the biosynthesis of SA. The induction of SA level could be considered negative feedback resulting from the inhibitory effects of PAMD and 4 on the SA signaling pathway. Our study of the structure−activity relationships of the PAMD derivatives demonstrated that the suppression of PR1 expression by the PAMD derivatives is not directly related to their inhibition of plant growth and that structural modification of the PAMD derivatives dramatically altered their effects on PR1 expression. These results suggest that further chemical modification of the PAMD derivatives may identify more specific inhibitors of SA signaling. The side effects of PAMD on growth have also been investigated here. PAMD severely impacted seedling establishment and root growth and induced an accumulation of a higher level of ABA and anthocyanin compared to mock treatment. These limitations were overcome to some extent by structure− activity relationship modification, and compound 4 was

Figure 7. Suppressive effects of PAMD or 4 on the expression of the PR1 gene. (A) Dose-dependent inhibition of SA-induced PR1 expression by 4. (B) Suppressive effect of PAMD (1) or 4 at the concentration of 10 μM. Seedlings (5 DAG) grown on 1/2 MS medium under constant light at 22.5 °C were transferred to 1/2 MS medium containing a serial concentration of test compound, as indicated, for further cultivation for 8 days under constant light at 22.5 °C, followed by foliar-sprayed SA treatment. After treatment with SA for 3 days, the seedlings were frozen in liquid nitrogen. PR1 expression was determined relative to Actin7. Error bars indicate the means ± SD of three replicates. Statistical differences between the groups were calculated with ANOVA analysis (Posthoc: Duncan). Bars with different letters are significantly different with P < 0.01.

GID1 triggers the degradation of the DELLA proteins, negative regulators of GA signaling, by 26S proteasomes.24 The defective GA biosynthesis in the ga1-3 mutant or the loss of DELLA degradation in the gai-1 and rga-Δ17 mutants, in which the DELLA domain is deleted, cause a severe dwarf phenotype in Arabidopsis.25,26 Therefore, suspecting that PAMD and 4 might inhibit plant growth by compromising GA signaling, we tested whether the growth suppression effects of 1 and 4 were restored by treatment with GA (Figure 10A). Both PAMD and 4 suppressed hypocotyl growth under constant light, and GA treatment partly restored this growth-suppression effect in wildtype plants (Ler). To further investigate whether this restorative effect of GA on hypocotyl growth is related to the DELLA 7130


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Figure 8. Inhibitory effects of PAMD and 4 on Arabidopsis growth. (A) Seedlings (5 DAG) of Arabidopsis grown on 1/2 MS medium containing PAMD or 4 under constant light at 22.5 °C. (B) Seeds were germinated on 1/2 MS medium with or without 4 under constant light at 22.5 °C. Error bars indicate the means ± SE of three independent replicates, n > 30. (C) Fresh weight of seedlings (12 DAG) grown on 1/2 MS medium with or without the test compound under constant light at 22.5 °C. Error bars indicate the means ± SE of three independent replicates, n > 30. Student’s t test was used to determine the significance of difference relative to the mock treatment; **, P < 0.01. (D) Seeds were germinated on 1/2 MS medium with or without the test compound under constant light at 22.5 °C. Seedling establishment was determined as the appearance of the first pair of true leaves at 5 DAG. Error bars indicate the means ± SE of three independent replicates, n = 10. (E) Primary root lengths of seedlings (6 DAG) that were germinated on 1/2 MS medium with or without the test compound under constant light at 22.5 °C. Error bars indicate the means ± SE of three independent replicates, n = 10. Student’s t test was used to determine the significance of differences relative to the mock treatment: *, P < 0.05; **, P < 0.01. (F) After stratification at 4 °C for 2 days, seeds were light-pulsed for 6 h and germinated and grown on 1/2 MS medium with or without the test compound under constant dark at 22.5 °C. Hypocotyl length was measured at 5 DAG. Error bars indicate the means ± SE of three independent replicates, n = 10. Student’s t test was used to determine the significance of differences relative to the mock treatment: *, P < 0.05; **, P < 0.01.

derivative 4 are partly dependent on their effects on GA signaling by interrupting GA-induced DELLA degradation (Figure 10). Recently, many studies of plant immunity to pathogens have indicated a cross talk between SA and other plant hormones.30 Tan et al. showed that the effector protein of the pathogenic bacterium Xanthomonas campestris, XopDXcc8004, initiates the disease defense mechanisms of plants by interacting with RGA and delaying its degradation.31 This confirms that the stabilization of the DELLA proteins affects the plant’s disease responses. It has been shown that DELLA increases the plant’s resistance to necrotrophs but reduces its resistance to biotrophs, thus demonstrating that the DELLA proteins control plant immune responses by modulating the balance between SA and JA signaling.13 This supports our results that PAMD and 4 suppress the expression of SA-responsive genes and also stabilize the DELLA proteins. We suggest that the activity of PAMD and its derivatives in repressing PR gene expression is partly dependent on the DELLA proteins, although further study is required to confirm this. It is noteworthy that the growth of the 5Δdella mutant line was still suppressed by both PAMD and compound 4 (Figure 10A), indicating that DELLA and DELLA-induced signaling are

Figure 9. Quantification of salicylic acid and abscisic acid in Arabidopsis. Seedlings (5 DAG) were transferred to 1/2 MS medium containing 25 μM of the test compound, as indicated, for further cultivation for 8 days, followed by being sampled and analyzed by LCMS/MS. Error bars indicate the means ± SD of three replicates. Statistical differences between the groups were calculated with ANOVA analysis (Posthoc: Duncan). Bars with different letters are significantly different: P < 0.05 (SA) or P < 0.01 (ABA).

selected as a better candidate for SAR studies. However, at present, no derivative has yet been found that suppresses PR gene expression but does not inhibit plant growth. The new PAMD derivatives, such as 4, are more selective inhibitors of SA signaling but still inhibit Arabidopsis growth. Here, we have demonstrated that the inhibitory effects of PAMD and its 7131


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Journal of Agricultural and Food Chemistry Funding

This work was supported, in part, by a grant from the Core Research for Evolutional Science and Technology (CREST) Program of the Japan Science and Technology Agency (JST) and a JSPS Grant-in-Aid for Scientific Research (C; grant number 26440132). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Miguel A. Blázquez (Instituto de Biologiá Moleculary Celular de Plantas, Spain) for kindly providing the 5Δdella mutant.

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Figure 10. Effects of PAMD and 4 on GA signaling. (A) Seedlings (3 DAG) of Ler and 5Δdella grown on 1/2 MS medium under constant light at 22.5 °C were transferred to 1/2 MS medium containing GA4 (10 μM), PAMD (25 μM), 4 (25 μM), or combinations of them as indicated, followed by several days’ cultivation under constant light at 22.5 °C. Hypocotyl length was measured 9 days after treatment. Error bars indicate the means ± SE of three independent replicates (n = 6). Statistical differences between the groups were calculated with ANOVA analysis (Posthoc: Duncan). Bars with different letters are significantly different with P < 0.01. (B) The primary roots of 2 DAG RGA:GFP-RGA seedlings grown on 1/2 MS medium under constant light at 22.5 °C were placed onto slides and immersed in 1/2 MS medium with or without GA4 (10 μM) combined with PAMD (25 μM) or 4 (25 μM), as indicated. Green fluorescence was observed with a confocal-laser scanning microscope and time-lapse photography. Scale = 100 μm.

not the only points at which these compounds function. Although the exact target(s) of PAMD and its derivatives is (are) still unclear, investigation of these targets and their modes of function will extend our understanding of SA signaling and/ or its cross talk with other plant hormones.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b01521. Anthocyanin level of Arabidopsis treated with PAMD or 4. (PDF)

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Development of Inhibitors of Salicylic Acid Signaling - Journal of

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