Development of Inhibitors of Salicylic Acid Signaling

Article abstract of DOI:10.1021/acs.jafc.5b01521

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.

Full text of DOI:10.1021/acs.jafc.5b01521

Article
pubs.acs.org/JAFC
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
itself is an SA receptor.9 In their SA-perception model, SA
binding to NPR1 induces a conformational change in NPR1
INTRODUCTION
■
Agricultural crops and stock are seriously damaged by various
pathogens, causing severe production losses.1 To avoid or
and relieves the repression of the transcription activation
domain of NPR1 by its autoinhibitory N-terminal domain.
attenuate this damage, the plants themselves have evolved
Several studies have suggested the existence of other SA
defense mechanisms, including physical defenses such as the
receptors,6,7 and further studies are required to comprehen-
sively 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 brassinoste-
roid, 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 biosyn-
thesis, 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)-1,1-
dioxide,15,16 benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-
methyl ester,17−19 and 2,6-dichloroisonicotinic acid,20,21 but
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 patho-
genesis 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
Received: March 27, 2015
Revised: July 30, 2015
Accepted: August 3, 2015
© XXXX American Chemical Societ
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Hypocotyl Growth. To determine hypocotyl growth during the
there is very limited information available on the inhibitors of
SA signaling.
1
constant dark treatment, the seeds were germinated on
/ MS
2
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
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) sup-
presses 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.
1
after germination (3 DAG) to /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)
1
germinated and grown on
/ MS medium at constant light were
2
1
transferred to treatment medium /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′- CCGCAGACCGCAATCTT-
3′ 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-d4) and deuterium-labeled ABA
(ABA-13C2) were purchased from Wako Pure Chemical Industries,
Ltd. (Japan).
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.
1
Growth Condition. Seedlings (5 DAG) grown on /2 MS medium
1
under constant light at 22.5 °C were transferred to /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.
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′-CGTCTTTGTAGCTCTTGT-
AGGTGC-3′ and 5′-TGCCTGGTTGTGAACCCTTAG-3′), PR2
(5′-CGTCTTTGTAGCTCTTGTAGGTGC-3′ and 5′-TGCCTG-
GTTGTGAACCCTTAG-3′), PR5 (5′-CGTCTTTGTAGC-
TCTTGTAGGTGC-3′ and 5′-TGCCTGGTTGTGAACCCTTAG-
3′), 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.
2, 2-((3-(Trifluoromethyl)phenylamino)methylene)cyclohexane-
1,3-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
B
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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
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-dimethylcyclohex-
ane-1,3-dione. 5,5-Dimethyl-1,3-cyclohexanedione (10 mmol), ortho-
acetic 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
1
hexane. The crystal was collected by suction filtration. H 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-Cyclo-
hexanedione (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
1
acetate and hexane. The crystal was collected by suction filtration. H
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 trifluorome-
thylaniline (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).
1
filtration. H 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-1,3-
dione. 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
10, 2-((2-Chlorophenylamino)methylene)-5,5-dimethylcyclohex-
ane-1,3-dione. 5,5-Dimethyl-1,3-cyclohexanedione (10 mmol), ortho-
acetic 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
1
was collected by suction filtration. H 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-dimethylcyclo-
hexane-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
1
acetate and hexane. The crystal was collected by suction filtration. H
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-dimethylcyclo-
hexane-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
1
with hexane. The crystal was collected by suction filtration. H 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-dimethylcyclohex-
ane-1,3-dione. 5,5-Dimethyl-1,3-cyclohexanedione (10 mmol), ortho-
acetic 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
1
acetate and hexane. The crystal was collected by suction filtration. H
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
1
acetate and hexane. The crystal was collected by suction filtration. H
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-dimethyl-
cyclohexane-1,3-dione. 5,5-Dimethyl-1,3-cyclohexanedione (10
mmol), orthoacetic acid triethyl ester (13 mmol), and 3-chrolo-2-
methylaniline (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
1
acetate and hexane. The crystal was collected by suction filtration. H
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
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1
Figure 1. Effects of PAMD on Arabidopsis growth. (A) At 12 days after germination (12 DAG), Arabidopsis thaliana Col-0 was grown on /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 <
1
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 /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.
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-signal-
inhibiting 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).
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
1
RGA:GFP-RGA line was germinated on
/ MS medium under
2
constant light at 22.5 °C. The primary roots of the seedlings (2 DAG)
1
were placed on a microscope slide with a drop of liquid-phase /2 MS
SA-Signal-Inhibiting Activities of PAMD Derivatives.
The effects of the derivatives described above on the
suppression of PR1 expression were determined with real-
time 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 cyclo-
hexadione 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 N-
attached 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
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.
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)-express-
ing 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 dose-
dependence 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-growth-
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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. Unexpect-
edly, 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 combina-
tions 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.
Figure 3. Determination of SA signal inhibition by PAMD derivatives.
Seedlings grown for 5 DAG on 1/2 MS medium under constant light at
1
22.5 °C were transferred to /2 MS medium containing the indicated
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
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,
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Figure 4. Estimation of the inhibitory effects of PAMD and its
1
derivatives. Seedlings (5 DAG) grown on
/ MS medium under
2
1
constant light at 22.5 °C were transferred to /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.
Figure 5. Suppressive effect of PAMD (1) and 4 on the expression of
1
PR2 and PR5. Seedlings (5 DAG) grown on /2 MS medium under
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 concentration-
dependent 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
1
constant light at 22.5 °C were transferred to
/ MS medium
2
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
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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 (At-
DISCUSSION
■
Actin7). Error bars indicate the means
SD of three replicates.
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.
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.
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
1
concentration of 10 μM. Seedlings (5 DAG) grown on
/ MS
2
1
medium under constant light at 22.5 °C were transferred to /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 wild-
type plants (Ler). To further investigate whether this restorative
effect of GA on hypocotyl growth is related to the DELLA
The side effects of PAMD on growth have also been
investigated here. PAMD severely impacted seedling establish-
ment 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
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1
Figure 8. Inhibitory effects of PAMD and 4 on Arabidopsis growth. (A) Seedlings (5 DAG) of Arabidopsis grown on /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.
Figure 9. Quantification of salicylic acid and abscisic acid in
Arabidopsis. Seedlings (5 DAG) were transferred to /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 LC-
1
MS/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
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
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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.
ACKNOWLEDGMENTS
■
́
We thank Miguel A. Blaz
Celular de Plantas, Spain) for kindly providing the 5Δdella
́
quez (Instituto de Biologia Moleculary
mutant.
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Corresponding Author
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