Preparation and biological activity of molecular probes to identify and analyze jasmonic acid-binding proteins

Article abstract of DOI:10.1271/bbb.68.1461

Several types of jasomonic acid (JA) derivatives, including JA-amino acid conjugates, a JA-biotin conjugate, a JA-dexamethasone heterodimer, and a JA-fluoresceine conjugate, were prepared as candidates for molecular probes to identify JA-binding proteins. These JA derivatives, excepting the JA-fluoresceine conjugate, exhibited significant biological activities in a rice seedling assay, a rice phytoalexin-inducing assay, and/or a soybean phenylalanine ammonia-lyase-inducing assay. These JA derivatives could therefore be useful probes for identifying JA-binding proteins. The activity spectra of the prepared compounds were different from each other, suggesting that different types of JA receptors were involved in the perception of JA derivatives in the respective bioassays.

Full text of DOI:10.1271/bbb.68.1461

This article was downloaded by: [Ams/Girona*barri Lib]
On: 19 November 2014, At: 02:24
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Bioscience, Biotechnology, and Biochemistry
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tbbb20
Preparation and Biological Activity of Molecular
Probes to Identify and Analyze Jasmonic Acid-
binding Proteins
Yusuke JIKUMARUa, Tadao ASAMIb, Hideharu SETOb, Shigeo YOSHIDAb, Tadashi
YOKOYAMAc, Naomi OBARAd, Morifumi HASEGAWAd, Osamu KODAMAd, Makoto
NISHIYAMAa, Kazunori OKADAa, Hideaki NOJIRIa & Hisakazu YAMANEa
a Biotechnology Research Center, The University of Tokyo
b The Institute of Physical and Chemical Research (RIKEN)
c Tokyo University of Agriculture and Technology
d College of Agriculture, Ibaraki University
Published online: 22 May 2014.
To cite this article: Yusuke JIKUMARU, Tadao ASAMI, Hideharu SETO, Shigeo YOSHIDA, Tadashi YOKOYAMA, Naomi OBARA,
Morifumi HASEGAWA, Osamu KODAMA, Makoto NISHIYAMA, Kazunori OKADA, Hideaki NOJIRI & Hisakazu YAMANE (2004)
Preparation and Biological Activity of Molecular Probes to Identify and Analyze Jasmonic Acid-binding Proteins, Bioscience,
Biotechnology, and Biochemistry, 68:7, 1461-1466, DOI: 10.1271/bbb.68.1461
To link to this article: http://dx.doi.org/10.1271/bbb.68.1461
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Biosci. Biotechnol. Biochem., 68 (7), 1461–1466, 2004
Preparation and Biological Activity of Molecular Probes to Identify
and Analyze Jasmonic Acid-binding Proteins
Yusuke JIKUMARU,1 Tadao ASAMI,2 Hideharu SETO,2 Shigeo YOSHIDA,2 Tadashi YOKOYAMA,3
Naomi OBARA,4 Morifumi HASEGAWA,4 Osamu KODAMA,4 Makoto NISHIYAMA,1
y
1;
Kazunori OKADA,1 Hideaki NOJIRI,1 and Hisakazu YAMANE
1Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
2The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
3Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan
4College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japan
Received January 14, 2004; Accepted March 18, 2004
Several types of jasomonic acid (JA) derivatives,
including JA–amino acid conjugates, a JA–biotin con-
jugate, a JA–dexamethasone heterodimer, and a JA–
fluoresceine conjugate, were prepared as candidates for
molecular probes to identify JA-binding proteins. These
JA derivatives, excepting the JA–fluoresceine conjugate,
exhibited significant biological activities in a rice seed-
ling assay, a rice phytoalexin-inducing assay, and/or a
soybean phenylalanine ammonia-lyase-inducing assay.
These JA derivatives could therefore be useful probes
for identifying JA-binding proteins. The activity spectra
of the prepared compounds were different from each
other, suggesting that different types of JA receptors
were involved in the perception of JA derivatives in the
respective bioassays.
asone (Dex) heterodimer, a JA–biotin conjugate, and a
JA–fluoresceine isothiocyanate (FITC) conjugate.
JA–amino acid conjugates are candidates for useful
molecular probes, because it is possible to prepare
[3H]JA–amino acid conjugates with high specific radio-
activity in few steps by using commercially available
[3H]amino acids.3) These radioactive probes would be
useful to monitor JA-binding proteins and determine
their JA-binding activity. A JA–Dex heterodimer would
be useful as bait in a yeast three-hybrid system for
detecting JA-binding proteins. The feasibility of the
yeast three-hybrid system for detecting small ligand–
protein receptor interaction has been demonstrated by
Licitra and Liu.4) A JA–biotin conjugate would be a
useful tool for the purification of JA-binding proteins,
because a convenient purification system can be con-
structed by utilizing the interaction between the biotin
moiety of a JA–biotin conjugate and avidine.5) A JA–
FITC conjugate would also be useful to identify and
analyze the function of JA-binding proteins.6) This will
make it possible to observe the binding between this
type of compound and JA-binding proteins.
If the prepared compounds were to exhibit JA-like
activity in a bioassay system, they would possibly
function as mimics of JA in the system. This means that
biologically active synthesized compounds could be
useful molecular probes for isolating and/or character-
izing JA-binding proteins. We report here the prepara-
tion and biological activities of potential molecular
probes for JA-binding proteins. Of the JA derivatives
synthesized in this study, (ꢀ)-JA–leucine and (ꢀ)-JA–
isoleucine conjugates have been reported to be bioactive
in the induction of sakuranetin, one of major rice
phytoalexins, and in the inhibition of rice shoot
Key words: jasmonic acid derivative; jasmonic acid-
binding protein; rice seedling assay; phenyl-
alanine ammonia-lyase-inducing assay; rice
phytoalexin-inducing assay
Jasmonic acid (JA) has been shown to be involved in
the plant responses to many types of biotic and abiotic
stress.1) JA also affects such diverse processes as fruit
ripening, production of viable pollens, tuber formation,
and root growth.2) These JA actions are considered to
start with the recognition of JA by specific receptors
which are likely to be proteins. However, there is little
information on JA receptors. To identify JA receptors,
we need to isolate the JA-binding proteins as candidates
for JA receptors, and to analyze their functions in detail.
Since it is useful to develop molecular probes for JA-
binding proteins, we prepared several types of JA
derivatives: JA–amino acid conjugates, a JA–dexameth-
y
To whom correspondence should be addressed. Fax: +81-3-5841-8030; E-mail: ayamane@mail.ecc.u-tokyo.ac.jp
Abbreviations: JA, jasmonic acid; Dex, dexamethasone; FITC, fluoresceine isothyocyanate; CD, circular dichroism; JA-Me, methyl jasmonate;
MeCN, acetonitrile; MeOH, methanol; DCC, N,N0-dicyclohexylcarbodiimide; DMF, N,N0-dimethylformamide; PAL, phenylalanine ammonia-lyase;
EtOH, ethanol

1462
Y. JIKUMARU et al.
growth.7) However, no extensive studies on the bio-
logical activities of JA derivatives have been reported.
dissolved in 0.1 M aqueous HCl (50 ml) and extracted
with ethyl acetate (50 ml, three times). The combined
ethyl acetate layers were dried over anhydrous sodium
sulfate and concentrated in vacuo. The residue was
chromatographed in a column of silica gel (10 g of
Wako C-300 gel), using a mixture of ethyl acetate–
acetic acid (99:1, v/v) as an eluent, to give the [(ꢁ)-
JA]–L-amino acid conjugate. The reaction of the [(ꢁ)-
JA]–N-hydroxysuccinimide ester with L-alanine, L-va-
line, L-leucine and L-isoleucine afforded a mixture of
diastereoisomeric N-jasmonoyl–L-amino acid conju-
gates, [(þ)-JA–L-amino acid and (ꢀ)-JA–L-amino acid].
The respective diastereomers were separated by high-
performance liquid chromatography (HPLC) in an ODS
4253D column (250 mm long, 10 mm internal diameter;
Senshu Scientific, Inc., Tokyo Japan), elution being
performed with 42.5% (JA–alanine), 50% (JA–valine),
or 57.5% (JA–leucine and JA–isoleucine) aqueous
MeOH containing 0.1% acetic acid at a flow rate of
3 ml minꢀ1. The products were monitored at 210 nm,
their retention times being as follows: [(þ)-JA]–L-
alanine, 28.0 min; [(ꢀ)-JA]–L-alanine, 34.5 min; [(þ)-
JA]–L-valine, 28.8 min; [(ꢀ)-JA]–L-valine, 39.2 min;
[(þ)-JA]–L-leucine, 20.0 min; [(ꢀ)-JA]–L-leucine, 25.3
min; [(þ)-JA]–L-isoleucine, 19.9 min; and [(ꢀ)-JA]–L-
isoleucine, 24.8 min. The stereochemistry of the JA–L-
amino acid conjugates was determined by analyzing the
CD data shown next. The [(þ)-JA]– and [(ꢀ)-JA]–L-
amino acid conjugates showed positive and negative
Cotton effects, respectively.3) The yields and analytical
data for the reaction products were as follows. [(ꢁ)-JA]–
glycine (182 mg, 0.45 mmol, 69% yield): 1H-NMR
(CDCl3, 300 MHz) ꢁ: 6.49 (1H, m), 5.49 (1H, m), 5.30
(1H, m), 4.10 (2H, d, J ¼ 3:9 Hz), 0.95 (3H, t,
J ¼ 7:5 Hz); MS (API 3000) m=z: 266.1 ½M ꢀ Hꢂꢀ.
[(ꢁ)-JA]–ꢀ-alanine (132 mg, 0.31 mmol, 47% yield):
1H-NMR (CDCl3, 300 MHz) ꢁ: 5.49 (1H, m), 5.30 (1H,
m), 3.49 (2H, d, J ¼ 3:6 Hz), 1.59 (3H, t, J ¼ 7:5 Hz)
0.95 (3H, t, J ¼ 7:5 Hz); MS (API 3000) m=z: 280.4
½M ꢀ Hꢂꢀ. [(þ)-JA]–L-alanine (155 mg, 0.36 mmol,
Materials and Methods
Instruments. NMR spectra were determined by using
tetramethylsilane as an internal standard with an AC-P
300 spectrometer (300 MHz, Bruker). Mass spectra of
the synthesized compounds were determined with an
API 3000 mass detector (Applied Biosystems Instru-
ments, Foster City, CA, USA), a JMS SX 102 EI MS
detector (JEOL, Tokyo, Japan), or a Voyager DE-STR
MALDI-TOF mass detector (Applied Biosystems In-
struments). Circular dichroism (CD) spectra of the
synthesized compounds were determined with a J-720
spectropolarimeter (JASCO, Tokyo, Japan).
Preparation of (ꢁ)-JA. To a solution of (ꢁ)-methyl
jasmonate (JA-Me; 5.2 g, 24 mmol) in methanol
(MeOH; 50 ml), a 5 M KOH aqueous solution (7.5 ml)
was added while stirring. Stirring was continued at room
temperature for 8 h, before the reaction mixture was
neutralized with 6 M aqueous HCl and concentrated in
vacuo. The residue was dissolved in H2O (50 ml), and
the solution adjusted to pH 2–3 with 6 M aqueous HCl,
before being extracted with ethyl acetate (50 ml, 3
times). The combined organic layers were dried over
anhydrous sodium sulfate and concentrated in vacuo.
The residue was chromatographed in a column of silica
gel (150 g of Wako C-300 gel; Wako Pure Chemical
Industries Ltd., Tokyo, Japan), using a mixture of n-
hexane–ethyl acetate–acetic acid (14:6:1, v/v/v) as an
eluent, to give (ꢁ)-JA (3.8 g, 18 mmol, 75% yield).
Preparation of a (ꢁ)-JA–N-hydroxysuccinimide ester.
To a mixture of (ꢁ)-JA (1 g, 4.8 mmol) in acetonitrile
(MeCN; 10 ml) and N-hydroxysuccinimide (1.5 g,
13 mmol) in N,N0-dimethylformamide (DMF; 7.5 ml),
dicyclohexylcarbodiimide (DCC; 1.25 g, 6.1 mmol) in
MeCN (5 ml) was added while stirring. Stirring was
continued at room temperature for 48 h, before water
(20 ml) was added to decompose the excess DCC, and
the reaction mixture was filtered to remove the dicyclo-
hexylurea. The filtrate was concentrated in vacuo and
purified in a column of silica gel (100 g of Wako C-300
gel), using a mixture of n-hexane–ethyl acetate (1:3,
v/v) as an eluent, to give the [(ꢁ)-JA]–N-hydroxy-
succinimide ester (1.2 g, 3.9 mmol, 81% yield).
1
55% yield): H-NMR (CDCl3, 300 MHz) ꢁ: 6.18 (1H,
m), 5.48 (1H, m), 5.29 (1H, m), 4.63 (1H, m), 1.59 (3H,
d, J ¼ 7:9 Hz), 0.95 (3H, t, J ¼ 7:5 Hz); MS (API 3000)
m=z: 280.4 ½M ꢀ Hꢂꢀ; CD (c 0.0281, MeOH) ꢀ"max
(nm): þ2:28 (296). [(ꢀ)-JA]–L-alanine (169 mg, 0.39
1
mmol, 60% yield): H-NMR (CDCl3, 300 MHz) ꢁ: 6.35
(1H, m), 5.46 (1H, m), 5.28 (1H, m), 4.63 (1H, m), 1.59
(3H, d, J ¼ 7:9 Hz), 0.95 (3H, t, J ¼ 7:5 Hz); MS (API
3000) m=z: 280.4 ½M ꢀ Hꢂꢀ; CD (c 0.0281, MeOH)
ꢀ"max (nm): ꢀ1:78 (299). [(þ)-JA]–L-valine (223 mg,
General procedure for preparing JA–L-amino acid
conjugates. [(ꢁ)-JA]–N-hydroxysuccinimide ester (200
mg, 0.65 mmol) in MeCN (10 ml) was mixed with a
solution of 1 mmol amino acid (glycine, ꢀ-alanine, L-
alanine, L-valine, L-leucine, or L-isoleucine) in H2O
(10 ml). To this mixture, triethylamine (1 ml, 8.5 mmol)
was added while stirring. Stirring was continued over-
night at room temperature, the resulting reaction mixture
then being concentrated in vacuo. The concentrate was
1
0.47 mmol, 72% yield): H-NMR (CDCl3, 300 MHz) ꢁ:
6.10 (1H, m), 5.47 (1H, m), 5.26 (1H, m), 4.62 (1H, m),
1.60 (1H, m), 0.88–1.09 (9H); MS (API 3000) m=z:
308.4 ½M ꢀ Hꢂꢀ; CD (c 0.0309, MeOH) ꢀ"max (nm):
þ2:39 (298). [(ꢀ)-JA]–L-valine (158 mg, 0.33 mmol,
1
51% yield): H-NMR (CDCl3, 300 MHz) ꢁ: 6.08 (1H,
m), 5.51 (1H, m), 5.40 (1H, m), 4.63 (1H, m), 1.63 (1H,
m), 0.91–1.07 (9H); MS (API 3000) m=z: 308.4

Molecular Probes for Jasmonic Acid-binding Proteins
1463
½M ꢀ Hꢂꢀ; CD (c 0.0309, MeOH) ꢀ"max (nm): ꢀ2:53
(296). [(þ)-JA]–L-leucine (171 mg, 0.34 mmol, 53%
yield): 1H-NMR (CDCl3, 300 MHz) ꢁ: 5.95 (1H, m),
5.47 (1H, m), 5.29 (1H, m), 4.69 (1H, m), 0.95–1.08
(9H); MS (API 3000) m=z: 322.4 ½M ꢀ Hꢂꢀ; CD (c
0.0323, MeOH) ꢀ"max (nm): þ2:49 (297). [(ꢀ)-JA]–L-
leucine (168 mg, 0.34 mmol, 52% yield): 1H-NMR
(CDCl3, 300 MHz) ꢁ: 5.86 (1H, m), 5.47 (1H, m), 5.35
(1H, m), 4.64 (1H, m), 0.91–1.03 (9H); MS (API 3000)
m=z: 322.4 ½M ꢀ Hꢂꢀ; CD (c 0.0323, MeOH) ꢀ"max
(nm): ꢀ2:03 (297). [(þ)-JA]–L-isoleucine (178 mg,
4.48 (1H, m), 0.99 (3H, t, J ¼ 7:5 Hz); MS (MALDI-
TOF) m=z: 564.3 ½M þ Hꢂþ.
Preparation of the JA–FITC conjugate. FITC
(312 mg, 0.80 mmol) and 1,5-diaminopentane (106 mg,
1.5 mmol) were dissolved in dry DMF (20 ml). To this
solution, triethylamine (1 ml, 8.5 mmol) was added
while stirring, stirring being continued at room temper-
ature for 20 min. The resulting reaction mixture was
concentrated in vacuo, and purified in a column of silica
gel (15 g of Wako C-300 gel), using a mixture of
chloroform–MeOH–acetic acid (50:49:1, v/v/v) as an
eluent, to give 1-amino-5-(N-fluoresceine thiocarbamo-
yl)-pentane (362 mg, 0.87 mmol). 1-Amino-5-(N-fluo-
resceine thiocarbamoyl)-pentane (180 mg, 0.47 mmol)
and [(ꢁ)-JA]–N-hydroxysuccinimide ester (150 mg,
0.48 mmol) were dissolved in MeOH (10 ml). To this
solution, triethylamine (1 ml, 8.5 mmol) was added
while stirring, stirring being continued at room temper-
ature for 48 h. The resulting reaction mixture was
concentrated in vacuo and purified on a column of
silica gel (30 g of Wako C-300 gel), using a mixture of
chloroform–MeOH–acetic acid (80:19:1, v/v/v) as an
eluent, to give the [(ꢁ)-JA]–FITC conjugate (285 mg,
1
0.36mmol, 55% yield): H-NMR (CDCl3, 300 MHz) ꢁ:
6.13 (1H, m), 5.46 (1H, m), 5.29 (1H, m), 4.66 (1H, m),
0.90–1.05 (9H); MS (API 3000) m=z: 322.4 ½M ꢀ Hꢂꢀ;
CD (c 0.0323, MeOH) ꢀ"max (nm): þ0:19 (298). [(ꢀ)-
1
JA]–L-isoleucine (184 mg, 0.37 mmol, 57% yield): H-
NMR (CDCl3, 300 MHz) ꢁ: 6.13 (1H, m), 5.47 (1H, m),
5.30 (1H, m), 4.66 (1H, m), 0.91–1.01 (9H); MS (API
3000) m=z: 322.4 ½M ꢀ Hꢂꢀ; CD (c 0.0323, MeOH)
ꢀ"max (nm): ꢀ0:43 (299).
Preparation of the JA–Dex heterodimer. Dex primary
amine (8.5 mg, 0.016 mmol) prepared as described
previously4) and [(ꢁ)-JA]–N-hydroxysuccinimide ester
(5 mg, 0.016 mmol) were dissolved in ethanol (EtOH;
10 ml). To this solution, triethylamine (0.1 ml, 0.85
mmol) was added while stirring, stirring being continued
at room temperature for 48 h. The resulting reaction
mixture was concentrated in vacuo and purified in a
column of silica gel (1 g of Wako C-300 gel), using a
mixture of n-hexane–ethyl acetate (1:2, v/v) as the
eluent, to give a mixture of diastereomeric JA–Dex
heterodimers (7.8 mg, 0.011 mmol, 67% yield), [(þ)-
JA]–Dex and [(ꢀ)-JA]–Dex. Since separation of these
diastereomers by reverse-phase HPLC was not success-
ful, the mixture of diastereomeric heterodimers was used
in the subsequent bioassays without further purification.
1H-NMR (CDCl3, 300 MHz) ꢁ: 6.16 (1H, m), 6.11 (1H,
br. s), 5.43 (1H, m), 5.24 (1H, m), 1.05 (3H, t,
J ¼ 7:5 Hz); MS (API 3000) m=z: 728.3 ½M þ Hꢂþ.
1
0.44 mmol, 95% yield). H-NMR (CDCl3, 300 MHz) ꢁ:
7.88 (1H, s), 6.35 (1H, m), 5.19 (1H, m) 5.06 (1H, m)
0.73 (3H, t, J ¼ 7:5 Hz). MS (MALDI-TOF) m=z: 684.0
½M þ Hꢂꢀ.
Rice seedling assay. Dwarf rice seedlings (Oryza.
sativa L. cv. Tan-ginbouzu) were used for this assay
which was carried out according to the micro-drop
method at 30 ꢃC under continuous white light (5 W mꢀ2
)
as reported by Murakami.8) The second leaf sheath
length was measured 4 days after a sample application.
Six seedlings were used for each sample.
Momilactone A-inducing assay. This assay was car-
ried out according to the method reported by Rakwal
et al.9) with slight modifications. Rice plants (O. sativa
L. cv. Nipponbare) were grown in a greenhouse, and the
immature top leaves of the plants at the fifth or sixth-leaf
stage were collected. Leaf disks (6 mm internal diam-
eter) were prepared from these leaves by using a cork
borer. Each leaf disk was floated on a solution contain-
ing a test sample in distilled water (200 ꢂl) in a well of a
96-well micro-plate (Iwaki, Tokyo, Japan) and incubat-
ed at 25 ꢃC under continuous white light (5 W mꢀ2) for
72 h. Five leaf disks were used to estimate the
momilactone A-inducing activity of a test sample. Leaf
disks that had been treated with the test sample were
boiled with 70% aqueous MeOH for 20 min. Each 70%
aqueous MeOH extract was directly analyzed by LC-
MS-MS with an HP 1100 HPLC instrument (Hewlett
Packard, Palo Alto, CA, USA) fitted with a Pegasil ODS
column (150 mm long, 4.6 mm internal diameter; Senshu
Scientific, Inc.), elution being performed with 80%
aqueous MeCN containing 0.1% formic acid at a flow
Preparation of the JA–biotin conjugate. 6-(biotinoyl-
amino)hexanoic acid hydrazide (102 mg, 0.20 mmol)
and [(ꢁ)-JA]–N-hydroxysuccinimide ester (108 mg,
0.30 mmol) were dissolved in dry DMF (10 ml). To this
solution, triethylamine (0.1 ml, 0.85 mmol) was added
while stirring, stirring being continued at room temper-
ature for 48 h. The resulting reaction mixture was
concentrated in vacuo and purified in a column of silica
gel (600 mg of Wako C-300 gel), using a mixture of
chloroform–MeOH (15:1, v/v) as an eluent, to give a
mixture of diastereomeric JA–biotin conjugates (89 mg,
0.1 mmol, 49% yield), [(þ)-JA]–biotin and [(ꢀ)-JA]–
biotin. Since separation of the diastereomers by reverse-
phase HPLC was not successful, this mixture of the
diastereomeric isomers was used in the subsequent
bioassays without further purification. 1H-NMR (CDCl3,
300 MHz) ꢁ: 5.45 (1H, m) 5.57 (1H, m), 4.27 (1H, m),

1464
Y. JIKUMARU et al.
rate of 1 ml minꢀ1. An API-3000 instrument with APCI
inlet system was also used for the LC-MS-MS analysis,
momilactone A being analyzed in the positive-ion mode
with nitrogen as a collision gas. Momilactone A was
detected in combination at m=z 315/271 in the multiple-
reaction monitoring mode.
ester with Dex primary amine which had been prepared
from Dex-acid and 1,10-diaminodecane. The JA–biotin
conjugate was prepared by the base-catalyzed reaction
of [(ꢁ)-JA]–N-hydroxysuccinimide ester with 6-(bio-
tinoylamino)hexanoic acid hydrazide. The JA–FITC
conjugate was prepared by the base-catalyzed reaction
of [(ꢁ)-JA]–N-hydroxysuccinimide ester with 1-amino-
5-(N-fluoresceine thiocarbamoyl)-pentane which had
been prepared by the reaction of FITC with 1,5-
diaminopentane in the presence of triethylamine. The
structures of these synthesized JA-derivatives are shown
in Fig. 1.
PAL-inducing assay. This assay was carried out by
the method reported by Recourt et al.10) with several
modifications. Approximately 5-ml aliquots of calli of
Glycine max were transferred to 300-ml Erlenmeyer
flasks containing 90 ml each of a Gamborg B5 medi-
um11) and cultured on a rotary shaker (79 rpm) at 25 ꢃC
in the dark for 1 week. The cell suspension 3 days after
being transferred to a fresh medium was used for the
subsequent experiments. One-ml aliquots of the cell
suspension were transferred to the respective wells of a
24-well micro-plate (Iwaki, Tokyo, Japan) and incubat-
ed on a rotary shaker (79 rpm) at 25 ꢃC in the dark. After
48 h, a 10-ꢂl aliquot of the stock solution of a test
compound (10 mM in a 10% aqueous MeOH solution)
was added to each well. Each reaction mixture was
incubated for 72 h under the same conditions. The
reaction mixture was then filtered off and the resulting
cells were sonicated in 5 ml of a borate buffer (0.1 M
borate and 20 mM 2-mercaptoethanol at pH 8.8) on ice.
After centrifugation (3000 g, 5 min), the supernatant was
assayed for PAL activity. To 1.5 ml of the supernatant,
0.5 ml of 7.25 mM of an L-phenylalanine solution was
added, and the reaction mixture was incubated at 37 ꢃC
for 36 h. The reaction was stopped by adding 100 ꢂl of
2 M aqueous HCl, and 1-ml aliquots of the reaction
mixture were extracted with ethyl acetate (0.5 ml, three
times). After concentrating the combined ethyl acetate
layers in vacuo, the residue was analyzed to quantify
trans-cinnamic acid by HPLC in a Pegasil ODS column
(250 mm long, 4.6 mm internal diameter; Senshu Scien-
tific, Inc.), eluting with 62% aqueous MeOH containing
0.1% acetic acid at a flow rate of 1 ml minꢀ1. Trans-
cinnamic acid was monitored at 280 nm.
Biological activities of the synthesized JA derivatives
The activities of the JA derivatives were examined by
three bioassays, all the results being shown in Table 1.
The JA–amino acid conjugates in the rice seedling assay
generally showed a clear inhibitory effect on the second
leaf sheath elongation at a dosage of 10 nmol plantꢀ1
.
[(ꢁ)-JA]–glycine, [(ꢁ)-JA]–ꢀ-alanine, and [(þ)-JA]–
and [(ꢀ)-JA]–L-valine exhibited almost the same inhib-
itory effect as that of (ꢁ)-JA. [(þ)-JA]– and [(ꢀ)-JA]–L-
alanine, [(ꢀ)-JA]–L-alanine, [(þ)-JA]– and [(ꢀ)-JA]–L-
leucine, and [(þ)-JA]– and [(ꢀ)-JA]–L-isoleucine were
slightly less active than (ꢁ)-JA. The [(ꢁ)-JA]–Dex
heterodimer and [(ꢁ)-JA]–biotin conjugate revealed
slight but significant inhibitory activity at a dosage of
10 nmol plantꢀ1. The [(ꢁ)-JA]–FITC conjugate did not
show any significant inhibitory activity. It is noteworthy
that the [(þ)-JA]– and [(ꢀ)-JA]–L-amino acid conju-
gates revealed almost the same activity, suggesting that
the JA receptor involved in JA-induced growth inhib-
ition similarly perceived both the [(þ)-JA]– and [(ꢀ)-
JA]–L-amino acid conjugates. It was also found that both
(þ)- and (ꢀ)-JA-Me exhibited an inhibitory effect on
the second leaf sheath elongation of rice seedlings, the
(þ)-form being slightly less active than the (ꢀ)-form.12)
The effects of the JA derivatives on the production of
the major phytoalexin in rice, momilactone A, were
examined by using the immature top leaves of rice
plants at the fifth- or sixth-leaf stage. [(ꢀ)-JA]–
L-alanine, [(ꢀ)-JA]–L-valine, and [(ꢀ)-JA]–L-leucine
were fairly active in the momilactone A-inducing assay,
their respective activity being 25%, 70%, and 75% of
that of (ꢁ)-JA at a concentration of 5 ꢄ 10ꢀ4 M. The
other conjugates did not show any significant momilac-
tone A-inducing activity at a dosage of 5 ꢄ 10ꢀ4 M. It
has been reported that [(ꢀ)-JA]–L-valine and [(ꢀ)-JA]–
L-leucine were identified as natural compounds from
rice plants.13) These results strongly suggest that the JA
receptor involved in momilactone A production recog-
nized the stereochemical difference between the (ꢀ)-
and (þ)-jasmonoyl moieties in the JA–L-amino acid
conjugates, showing that it preferred the [(ꢀ)-JA]–L-
amino acid conjugates to the corresponding diastereo-
meric [(þ)-JA]–L-amino acid conjugates.
Results and Discussion
Preparation of the JA derivatives
JA–amino acid conjugates were prepared by reacting
[(ꢁ)-JA]–N-hydroxysuccinimide ester with the amino
acids, glycine, ꢀ-alanine, L-alanine, L-valine, L-leucine,
and L-isoleucine, in the presence of triethylamine. The
reactions of [(ꢁ)-JA]–N-hydroxysuccinimide ester with
L-alanine, L-valine, L-leucine and L-isoleuscine afforded
a diastereomeric mixture of each amino acid conjugate.
This diastereomeric mixture was subjected to ODS-
HPLC to separate the [(þ)-JA]– and [(ꢀ)-JA]–amino
acid conjugates. Under these conditions, the respective
[(þ)-JA]–L-amino acid diastereomers were eluted before
the corresponding [(ꢀ)-JA]–L-amino acid–diastereom-
ers. The JA–Dex heterodimer was prepared by the base-
catalyzed reaction of [(ꢁ)-JA]–N-hydroxysuccinimide
The flavonoid compound, sakuranetin, is another
major phytoalexin in rice. Tamogami et al.7) have

Molecular Probes for Jasmonic Acid-binding Proteins
1465
Fig. 1. Structures of the Synthesized JA Derivatives.
Table 1. Effects of the Synthesized JA Derivatives and Their Related Compounds on the Growth of Rice Seedlings, Production of Momilactone A
in Immature Rice Leaves, and Production of trans-cinnamic Acid in Suspension-cultured G. max Cells
Length of 2nd leaf sheath
of rice seedlings (mm)a
Production of momilactone A
in rice leaves (ng diskꢀ1
Production of trans-cinnamic acid
in G. max cells [ng (mg fr wt)ꢀ1
]
Compound
b
c
)
Control
12:0 ꢁ 0:4
5:0 ꢁ 0:3
5:5 ꢁ 0:6
6:4 ꢁ 0:3
6:7 ꢁ 0:8
6:5 ꢁ 0:4
4:8 ꢁ 0:5
4:8 ꢁ 0:9
6:5 ꢁ 0:4
6:0 ꢁ 0:8
6:5 ꢁ 0:4
6:0 ꢁ 0:6
12:0 ꢁ 0:4
10:0 ꢁ 0:3
13:0 ꢁ 0:5
9:0 ꢁ 0:5
10:0 ꢁ 0:4
12:0 ꢁ 0:7
1:5 ꢁ 0:3
60:7 ꢁ 2:2
1:1 ꢁ 0:4
n.d.
0:4 ꢁ 0:1
153:7 ꢁ 20:9
109:9 ꢁ 1:6
58:8 ꢁ 2:0
143:5 ꢁ 10:4
99:3 ꢁ 9:2
48:8 ꢁ 4:6
231:4 ꢁ 43:1
35:1 ꢁ 2:5
100:2 ꢁ 17:1
33:2 ꢁ 7:8
47:3 ꢁ 7:9
8:7 ꢁ 2:3
(ꢁ)-JA
[(ꢁ)-JA]–glycine
[(þ)-JA]–L-alanine
[(ꢀ)-JA]–L-alanine
[(ꢁ)-JA]–ꢀ-alanine
[(þ)-JA]–L-valine
[(ꢀ)-JA]–L-valine
[(þ)-JA]–L-leucine
[(ꢀ)-JA]–L-leucine
[(þ)-JA]–L-isoleucine
[(ꢀ)-JA]–L-isoleucine
Dex
32:7 ꢁ 4:4
n.d.
0:2 ꢁ 0:0
42:7 ꢁ 1:1
0:1 ꢁ 0:0
44:2 ꢁ 5:5
n.d.
n.d.
n.d.
[(ꢁ)-JA]–dex
Biotin
n.d.
n.d.
20:7 ꢁ 2:5
1:6 ꢁ 0:4
[(ꢁ)-JA]–biotin
FITC
n.d.
4:7 ꢁ 1:0
n.d.
16:0 ꢁ 2:0
34:6 ꢁ 9:2
4:8 ꢁ 0:4
[(ꢁ)-JA]–FITC
a
The dose of each compound was 10 nmol plantꢀ1. Each value represents the mean ꢁ standard error from 6 replicates.
The dose of each compound was 5 ꢄ 10ꢀ4 M. Each value represents the mean ꢁ standard error from 5 replicates.
n.d.: not detected.
The concentration of each compound was 10ꢀ4 M. Each value represents the mean ꢁ standard error from 4 replicates.
b
c
reported that, in a sakuranetin-inducing assay with rice
recognized the stereochemical difference between the
(ꢀ)- and (þ)-jasmonoyl moieties in the JA–L-amino
acid conjugates.
The effects of the JA derivatives on the PAL-inducing
activity in suspension-cultured soybean cells were
determined. Such JA–amino acid conjugates as [(ꢁ)-
JA]–glycine, [(ꢁ)-JA]–ꢀ-alanine, [(ꢀ)-JA]–L-alanine,
leaves, [(ꢀ)-JA]–L-leucine, [(ꢀ)-JA]–L-isoleucine, and
[(ꢀ)-JA]–L-phenylalanine were more active than (ꢀ)-
JA, while the corresponding diastereomeric [(þ)-JA]–L-
amino acid conjugates were inactive. It was thus
suggested that the receptors involved in the production
of sakuranetin as well as momilactone A strictly

1466
Y. JIKUMARU et al.
[(ꢀ)-JA]–L-valine, and [(ꢀ)-JA]–L-leucine were highly
active, their activity being 64.5–150.7% of that of (ꢁ)-
JA. On the other hand, the activity of [(þ)-JA]–L-
alanine, [(þ)-JA]–L-valine, [(þ)-JA]–L-leucine, and
[(þ)-JA]– and [(ꢀ)-JA]–L-isoleucine was 21.4–38.8%
of that of (ꢁ)-JA. The [(ꢁ)-JA]–Dex heterodimer and
[(ꢁ)-JA]–biotin conjugate were slightly but significantly
active, their respective activity being 10.2% and 13.2%
of that of (ꢁ)-JA. The [(ꢁ)-JA]–FITC conjugate was
almost inactive. It should be noted that the [(ꢀ)-JA]–L-
amino acid conjugates were more active than the
corresponding [(þ)-JA]–L-amino acid conjugates, al-
though the [(þ)-JA]–L-amino acid conjugates were still
active in the PAL-inducing assay. These results suggest
that the receptor involved in JA-induced PAL induction
in G. max perceived a wide range of JA derivatives, but
recognized the stereochemical difference between the
(ꢀ)- and (þ)-jasmonoyl moieties in the JA–L-amino
acid conjugates.
It is noteworthy that the [(ꢀ)-JA]–L-amino acid
conjugates were highly active in these three bioassays.
Tamogami et al.7) have also reported that some [(ꢀ)-
JA]–L-amino acid conjugates were more polar than (ꢀ)-
JA and therefore seemed to permeate less into plant
cells, although the former were more active than (ꢀ)-JA
in the sakuranetin-inducing assay in rice. These facts
suggest that the [(ꢀ)-JA]–L-amino acid conjugates were
active per se in the plant cells; in other words, the
biological activity of the [(ꢀ)-JA]–L-amino acid con-
jugate was not due to (ꢀ)-JA released by hydrolytic
enzymes.
We prepared in this study JA–L-amino acid conju-
gates, a JA–biotin conjugate, a JA–Dex heterodimer,
and a JA–FITC conjugate as candidates for molecular
probes to identify JA-binding proteins. These JA
derivatives, except the JA–FITC conjugate, exhibited
significant biological activities in the rice seedling assay,
rice momilactone A-inducing assay, and/or soybean
PAL-inducing assay. The activity spectra of the pre-
pared JA derivatives were different from each other,
suggesting that different types of JA receptor functioned
in the respective bioassays. The prepared compounds
with JA-like activity could be useful molecular probes
for isolating and analyzing JA-binding proteins.
References
1) Turner, J. G., Ellis, C., and Devote, A., The jasmonate
signal pathway. Plant Cell, Supplement, 153–164
(2002).
2) Creelman, R. A., and Mullet, J. E., Biosynthesis and
action of jasmonates in plants. Ann. Rev. Plant Physiol.
Plant Mol. Biol., 48, 365–381 (1997).
3) Knofel, H.-D., Bruckner, C., Kramell, R., Sembdner, G.,
¨
¨
and Schreiber, K., Radioimmunoassay for the natural
plant growth regulator by (ꢀ)-jasmonic acid. Biochem.
Physiol. Pflanzen., 186, 387–394 (1990).
4) Licitra, E. J., and Liu, J. O., A three-hybrid system for
detecting small ligand–protein receptor interactions.
Proc. Natl. Acad. Sci. USA, 93, 12817–12821 (1996).
5) Leblond-Francillard, M., Areyfus, M., and Rougeon, F.,
Isolation of DNA–protein complex based on streptavi-
dine and biotin interaction. Eur. J. Biochem., 166, 351–
355 (1987).
6) Asami, T., Tao, L., Yamamoto, S., Robertson, S., Min,
Y., Murofushi, N., and Yoshida, S., Fluorescence-
labeled abscisic acid possessing abscisic acid-like
activity in barley aleurone protoplast. Biosci. Biotechnol.
Biochem., 61, 1198–1199 (1997).
7) Tamogami, S., Rakwal, R., and Kodama, O., Phytoalexin
production by amino acid conjugates of jasmonic acid
through induction of naringenin-7-O-methyltransferase,
a key enzyme on phytoalexin biosynthesis in rice (Oryza
sativa L.). FEBS Lett., 401, 239–242 (1997).
8) Murakami, Y., A new rice seedling test for gibberellins,
microdrop method and its use for testing extracts of rice
and morning glory. Bot. Mag., 79, 33–34 (1968).
9) Rakwal, R., Tamogami, S., and Kodama, O., Role of
jasmonic acid as a signaling molecule in copper
chloride-elicited rice phytoalexin production. Biosci.
Biotechnol. Biochem., 60, 1046–1048 (1996).
10) Recourt, K., Tunen, A. J. V., Mur, A. L., Brussel, A. A.
N. V., Lugtenberg, B. J. J., and Kijne, W., Activation of
flavonoid biosynthesis in root of Vicia sativa subsp.
Nigra plants by inoculation with Rhizobium lugumino-
sarum biovar. viciae. Plant Mol. Biol., 19, 411–420
(1992).
11) Gamborg, O. L., Miller, R. A., and Ojima, K., Nutrient
requirement of suspension cultures of soybean root cells.
Exp. Cell Res., 50, 151–158 (1992).
12) Yamane, H., Takahashi, N., Ueda, J., and Kato, J.,
Resolution of (ꢁ)-methyl jasmonate by high perform-
ance liquid chromatography and the inhibitory effect of
(þ)-enantiomer on the growth of rice seedlings. Agric.
Biol. Chem., 45, 1709–1711 (1981).
Acknowledgments
13) Tamogami, S., and Kodama, O., Quantification of amino
acid conjugates of jasmonic acid in rice leaves by high-
performance liquid chromatography-turboionspray tan-
dem mass spectrometry. J. Chromatogr. A, 822, 310–315
(1998).
This work was supported in part by Grants-in-Aid for
Scientific Research (No. 12460051 and No. 80090520)
to H. Y. from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.