Design and synthesis of biotin-tagged photoaffinity probes of jasmonates

Article abstract of DOI:10.1016/j.bmc.2010.03.059

Jasmonates (JAs) are a class of oxylipin compounds that play diverse roles in plant defense and development. The F-box protein coronatine insensitive1 (COI1) plays a crucial role in the JA signaling pathway. To determine whether COI1 binds directly to jasmonates, three biotin-tagged photoaffinity probes for JAs, a jasmonic acid photoaffinity probe (PAJA), a JAIle photoaffinity probe (PAJAIle), and a coronatine photoaffinity probe (PACOR), were designed and synthesized based on analysis of JA structure-activity relationships and molecular modeling of the interaction between COI1 and JAs. Among them, PACOR exhibited the most significant biological activity in inhibiting root growth, promoting accumulation of JA-responsive proteins, and triggering COI1-JAZ1 interaction in Arabidopsis seedlings. PACOR is an effective tool for elucidating the interaction between COI1 and JA.

Full text of DOI:10.1016/j.bmc.2010.03.059

Bioorganic & Medicinal Chemistry 18 (2010) 3012–3019
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of biotin-tagged photoaffinity probes of jasmonates
Min Gu a,b, , Jianbin Yan c, , Zhiyan Bai c, Yue-Ting Chen b, Wei Lu a, Jie Tang a, Liusheng Duan c, Daoxin Xie c,
,
*
Fa-Jun Nan b,
*
a Department of Chemistry and Institute of Medicinal Chemistry, East China Normal University, 3663 North Zhong Shan Road, Shanghai 200062, China
b National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes of Biological Sciences,
Chinese Academy of Sciences, 189, Guo Shou Jing Road, Shanghai 201203, China
c MOE Key Laboratory of Bioinformatics, School of life Sciences, Tsinghua University, Beijing 100084, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Jasmonates (JAs) are a class of oxylipin compounds that play diverse roles in plant defense and develop-
ment. The F-box protein coronatine insensitive1 (COI1) plays a crucial role in the JA signaling pathway. To
determine whether COI1 binds directly to jasmonates, three biotin-tagged photoaffinity probes for JAs, a
jasmonic acid photoaffinity probe (PAJA), a JAIle photoaffinity probe (PAJAIle), and a coronatine photo-
affinity probe (PACOR), were designed and synthesized based on analysis of JA structure–activity rela-
tionships and molecular modeling of the interaction between COI1 and JAs. Among them, PACOR
exhibited the most significant biological activity in inhibiting root growth, promoting accumulation of
JA-responsive proteins, and triggering COI1–JAZ1 interaction in Arabidopsis seedlings. PACOR is an effec-
tive tool for elucidating the interaction between COI1 and JA.
Received 4 February 2010
Revised 23 March 2010
Accepted 24 March 2010
Available online 27 March 2010
Keywords:
Jasmonate
Coronatine
Photoaffinity probes
Synthesis
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
results suggest that the SCFCOI1–JAZ complex is the site of JA-Ile/
COR perception.21
Plant hormones regulate numerous responses in plant growth
and development. Elucidation of the mechanisms responsible for
hormone perception is important for understanding the effects of
plant hormones. Although many hormone receptors, such as the
auxin receptor,1,2 the gibberellin receptor,3 and the brassinosteroid
receptor4 have been identified, little is known about jasmonate (JA)
receptors.
JAs are indispensable in plant defense responses and aspects of
plant growth and development.5–13 An Arabidopsis coronatine
insensitive1 (COI1) gene encodes an F-box protein required for
JA-regulated plant fertility and defense.14 We previously demon-
strated that COI1 interacts with multiple proteins to form the
SCFCOI1 E3 ubiquitin ligase complex, which is required for all JA
responses.15–17
To determine whether COI1 binds directly with JA-Ile/COR, we
used photoaffinity labeling activity-based probe technology, which
is very useful for identifying specific receptors for small-molecule
ligands and for localizing the ligand binding site within the recep-
tor. In our previous study,24 we reported a photoaffinity probe PA-
COR and demonstrated that PACOR physically binds with COI1
protein, which accelerates researches on the mechanism of JA per-
ception. Here, we explain chemical, analytical and biological detail
on design, synthesis and action of PACOR and two other similar
probes PAJA and PAJAIle, and comparison of these compounds in
terms of bioactivity and binding.
To get active photoaffinity probes for JAs, we firstly analyzed
the interaction between COI1 and several common jasmonates
through molecular modeling. We found that the jasmonate moiety
in JA-Ile could bind to the P1 and P2 pockets and the bottle neck
region of COI1, which suggests that the jasmonate moiety is
Recent studies have shown that the SCFCOI1 complex recognizes
JA transcriptional repressors called jasmonate ZIM-domain (JAZ)
proteins and targets them for subsequent degradation by the 26S
proteasome in the presence of the hormone.18–20 In addition, phys-
ical interaction of SCFCOI1 with the JAZ proteins is promoted by a
bioactive isoleucine-conjugated form of jasmonic acid (JA-Ile) or
its stereochemical mimic, coronatine (COR)21–23 (Fig. 1). These
O
O
O
8
2
11
10
9
12
6
7
3
5
4
COOH
NH
NH
O
O
1
HOOC
HOOC
* Corresponding authors. Tel./fax: +86 10 62781575 (D.X.); tel./fax:+86 21
50800954 (F.-J.N.).
E-mail addresses: daoxinlab@mail.tsinghua.edu.cn (D. Xie), fjnan@mail.shcnc.ac.
cn (F.-J. Nan).
(+)-7-iso-JA
(+)-7-iso-JA-Ile
Coronatine
These authors contributed equally to this work.
Figure 1. Structures of JA, JA-Ile, coronatine.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.059

M. Gu et al. / Bioorg. Med. Chem. 18 (2010) 3012–3019
3013
O
N
N
F3C
O
O
NH
O
O
N
O
O
O
N
O
H
HN
N
NH
H
H
H
N
H
N
S
O
PAJA
O
N
F3
C
O
N
F3C
N
N
NH
HN
NH
HN
H
H
H
H
H
N
H
N
H
H
O
O
O
O
O
O
S
O
O
S
O
O
N
O
O
N
H
H
HN
COOH
HN
COOH
HN
O
HN
O
O
O
PACOR
PAJAIle
Figure 2. Structures of jasmonate probes.
essential to stabilize its binding with COI1 and it is difficult to
modify at these positions for an active probe. However, the isoleu-
cine moiety of JA-Ile extends outward from the COI1 surface pock-
et, which makes the free carboxyl group become a possible site to
modify. Then, we designed and synthesized three biotin-tagged
photoaffinity probes based on different jasmonates including PAJA,
PAJAIle, and PACOR (Fig. 2), in the latter two probes, a free carbox-
ylic acid group was introduced through a lysine linker to mimic the
carboxylic acid group existed in JAIle and coronatine.
By comparing the bioactivities of PAJA, PAJAIle, and PACOR, PA-
COR was found to exhibit the highest biological activity in respect
of inhibiting root growth, accumulating JA-responsive proteins,
and triggering the COI1–JAZ1 interaction, while PAJAIle. PAJA just
showed weak biological activity. Therefore, PACOR was selected
as an useful tool for studying the interaction between COI1 and JAs.
der basic conditions to produce 10. Compound 10 was rapidly re-
acted with piperidine in DMF, removing the Fmoc group to
produce 11. Compound 11 was condensed with JA-Ile/coronatine
using DCC, HOBT, and Et3N in dichloromethane to obtain 12 and
13. Removal of the tert-butyloxycarbonyl protecting groups of 12
and 13 with 2 N HCl–EtOAc and subsequent incorporation of the
biotin tag in dichloromethane produced probes 14 and 15. Depro-
tection of the 2-(trimethylsilyl)ethyl groups of 14 and 15 using tet-
rabutylammonium fluoride in THF yielded the photoaffinity
probes, PAJAIle and PACOR.
3. Biological activities of the probes
The biological activities of the three JA photoaffinity probes
were examined in Arabidopsis using three bioassays: root growth
inhibition, accumulation of JA-responsive proteins, and promotion
of the COI1–JAZ1 interaction.
2. Preparation of the probes
One of the first biological activities observed for JAs was the
root growth inhibition.25,26 JAs supplemented to the growth med-
ium cause a significant inhibition of seedling root growth. There-
fore, we first investigated whether the effect of the probes on the
root growth of Arabidopsis seedlings was similar to that of the
Scheme 1 shows the synthesis of the PAJA probe. Preparation of
the probe was commenced with compound 1, to which tert-butyl
2-(2-(2-bromoethoxy)ethoxy)ethylcarbamate was attached, result-
ing in 2. Oxidization of 2 with (C4H9)4NMnO4 in pyridine resulted in
3, which was then condensed with succinimide using EDC in dichlo-
romethane to produce 4. Compound 4 was condensed with 2-(2-(2-
azidoethoxy)ethoxy)ethylcarbamate to produce 5, and removal of
the tert-butyloxycarbonyl protecting group of 5 and subsequent
condensation with JA-OSu resulted in 7. Click reaction of 7 with alky-
nylethylamide biotin produced the PAJA probe.
Scheme 2 shows the synthesis of the PAJAIle and PACOR probes.
First, Fmoc-Lys(Z)-OH was condensed with 2-(trimethylsilyl)etha-
nol using DCC and DMAP in dichloromethane to produce 8. Then 9
was obtained by reducing 8 with H2/Pd in methanol to remove the
CBZ group. At the same time, a few drops of acetic acid were added
to protect the Fmoc group. Compound 9 was condensed with 4 un-
JAs. Seedlings were grown on MS medium containing 100 lM of
PAJA, PAJAIle, PACOR, or MeJA for nine days, after which the root
lengths of the seedlings were measured. Treatment without JAs
or with BP (a negative control consisting of a photoreactive group
and a biotin tag) alone, did not inhibit root growth (Fig. 3). In con-
trast, treatment with MeJA had the strongest effect on root growth,
followed by PACOR, which indicates that PACOR had the best bio-
activity for JAs of the three probes developed (Fig. 3).
JAs are thought to play an important role in the defense reaction
of plants against phytopathogens. One of important ways that help
protect plants from fungal pathogens damage is the activation of
the expression of antifungal proteins, such as thionin, osmotin

3014
M. Gu et al. / Bioorg. Med. Chem. 18 (2010) 3012–3019
N
N
N
N
F3C
F3C
Br
O
O
NHBoc
(Bu4)NMnO4
O
NHBoc
O
O
K2CO3, Bu4NI
OH
rt, pyridine,12h,97%
CHO
2
CHO
1
65ºC, DMF,12h,57%
N
F3C
N
N
N
F3C
O
NHBoc
O
O
N
HOSu,EDC
rt, DCM,12h, 90%
O
O
O
N
O
NHBoc
O
COOH
O
O
4
3
F3C
N
N
F3C
N
O
NH2
N3
O
HCl-EtOAc
O
NH2
HCl
O
NHBoc
O
O
O
O
Et N, rt, CHCl , 85%
3
3
rt, 2h
O
N3
O
N3
O
N
O
O
N
H
O
H
5
6
O
N
N
F3C
O
JA-OSu,CHCl3,Et3N
rt, 12h, 73%
O
NH
O
O
O
O
N3
O
N
H
7
O
N
N
F3C
O
NH
HN
H
O
H
N
H
H
S
O
O
NH
O
O
O
O
CuSO4,sodium Vc,H2O,MeOH
rt,12h, 77%
O
N
O
H
HN
N
NH
H
N
N
H
H
N
H
PAJA
S
O
Scheme 1. Synthesis of PAJA.
and defensin. Arabidopsis thionin gene Thi2.1 can be used as a
molecular marker to detect the biological activity of jasmonate.27
To determine whether the probes induced expression of Thi2.1
proteins, we generated plants transgenic for a thionin 2.1-GUS (b-
glucuronidase) reporter construct,28 in which the GUS gene was
driven by the promoter of thionin 2.1 that is induced by JA treat-
ment. Seedlings carrying the thionin 2.1-GUS reporter construct
accumulation. The PACOR probe had the strongest promoting ef-
fect, followed by PAJAIle. The PAJA probe was inactive.
4. Discussion
The activity-based protein profiling (ABPP) strategy was devel-
oped in recent years for protein discovery. Typical ABPP probes
comprise a bifunctional moiety that binds to and covalently mod-
ifies the active site of a specific protein target. This enables efficient
and direct detection of the target protein through specific binding,
physical attachment of a chemical probe to the active site of the
target protein, and formation of a permanent covalent bond be-
tween the probe and its receptor via photolysis.29 For subsequent
retrieval of the bound complex, the chemical probe often contains
another moiety that can either be pre-attached or react with a re-
porter group such as a fluorescent tag or a biotin tag.
To develop a biotin-tagged photoaffinity probe for JA that has
biological activity, we analyzed the structure–activity relationship
between COI1 and JAs30–32 and performed molecular modeling of
the interaction between COI1 and JAs.24 We found that the JA
moiety of JA-Ile and the CFA moiety of COR are very important
for biological activity, which suggests that modification of the car-
boxylic groups of JAs and COR would be tolerated.24 Therefore,
were treated with 100 lM PAJA, PAJAIle, PACOR, or MeJA and were
then subjected to histochemical staining. As expected, the seed-
lings treated with MeJA showed strong blue staining, whereas
there were no blue staining in the seedlings without treatment
(Mock). The PAJA probe did not show any significant blue staining.
The PAJAIle probe induced slight GUS expression. In contrast, the
PACOR probe showed clear GUS activity, indicating that the PACOR
probe was the most active of the three probes (Fig. 4).
Finally, we examined whether the probes facilitated formation
of the COI1–JAZ1 interaction. An in vitro pull-down assay was per-
formed using recombinant Arabidopsis JAZ1–His and supernatants
of plant leaf extracts prepared from coi1–1 mutant plants
with transgenic expression of the HA-tagged COI1 protein
(COI1-HA).15 As shown in Figure 5, the ranking of the three probes
in promoting the interaction between COI1 and JAZ1 was the same
as those for root growth inhibition and thionin 2.1-GUS protein

M. Gu et al. / Bioorg. Med. Chem. 18 (2010) 3012–3019
3015
O
H
N
H
O
COOH
Fmoc
Si
O
N
N
Si
HO
O
O
HN
O
HN
DCC,DMAP,rt,DCM,2h,
67%
Fmoc
Fmoc-Lys(Z)-OH
8
N
F3C
O
H2N
Si
O
O
NHBoc
O
O
4, NaHCO3
H2/Pd, AcOH
rt, MeOH, 2h,80%
HN
Fmoc
rt, CHCl3,
12h,85%
O
N
H
O
FmocHN
Si
9
N
N
F3C
O
10
N
F3C
N
piperidine
DMF
O
NHBoc
DCC,HoBT
O
O
O
Et N,DCM
3
O
NHBoc
rt, 20min
80%
O
O
N
O
JA-Ile/Coronatine
H
O
N
H2N
Si
H
O
RHN
Si
O
O
11
12: R=JA-Ile, 13: R=Coronatine
O
N
N
F3C
N
H
HN
H
H
HCl-EtOAc, rt, 2h
Biotin-OSu,NaHCO3
rt, DCM, 12h
H
H
O
N
O
O
S
O
O
N
H
O
RHN
Si
O
14: R=JA-Ile, 15: R=Coronatine
O
N
N
F3C
NH
HN
H
H
(C4H9)4NF,THF
rt, 3h
H
N
H
O
O
O
S
O
O
N
H
RHN
COOH
PAJAIle: R=JA-Ile, PACOR: R=Coronatine
O
N
F3C
N
NH
HN
H
H
H
N
H
O
O
O
S
COOH
O
BP
Scheme 2. Synthesis of the PAJAIle and PACOR probes.
the photoaffinity group was introduced in the carboxylic groups
of JAs and COR. A polyethylene glycol linker with rational length
was introduced to link the photoaffinity group and the biotin tag,
ensuring a large degree of conformational freedom. Based on
these analyses, the probe PAJA was designed and synthesized
first. Unfortunately, PAJA just showed very weak activity, one pos-
sible reason is lack of a free carboxylic acid group, which also
indicated that jasmonic acid itself is not the endogenous bioactive
form of JAs.
According to the proposed model of JA-Ile binding to COI1, the
P3 pocket mainly contains hydrophilic residues,24 whereas the P4
pocket contains hydrophobic residues. These features of the P3
and P4 pockets may imply that both hydrophobic and electrostatic
interactions are involved in the interaction with JAZs. This infor-
mation indicated that the carboxylic acid group might be impor-
tant due to form electrostatic interaction. Therefore, a lysine link
was introduced between the JAs/COR motifs and the photoaffinity
group. The two amino groups of lysine were used to connected the
JAs/COR motifs and the photoaffinity group, therefore provided a
free carboxyl acid group near the JAs/COR motifs. The probe PAJAI-
le was found to have much better activity than PAJA, but still was
not satisfied for further mechanism studies. Finally, we chose COR
to replace the initial JA-Ile moiety. The new probe PACOR was
proved to has strong biological activity in several bioassays, which
is consistent with previous results that COR has stronger biological
activity than JA-Ile.22

3016
M. Gu et al. / Bioorg. Med. Chem. 18 (2010) 3012–3019
Figure 5. Comparison of the three probes in terms of promotion of the interaction
between COI1 and JAZ1. Pull-down reactions were carried out using recombinant
JAZ1-His and total plant extracts prepared from coi1–1[COI1-HA] seedlings. JAZ1-
His containing pull-down assays were supplemented with buffer (indicated by 0) or
JA-Ile, PAJA, PAJAIle, or PACOR at the indicated concentrations. Recovery of COI1
was detected using an anti-HA antibody (upper panel). The PVDF membrane was
stained to visualize the recovery of JAZ1 by the Ni-NTA affinity resin (lower panel).
of CDCl3 (1H NMR) as internal standards. Low-resolution mass data
were obtained on an Agilent 6110 Single Quadrupole LC/MS Sys-
tem. High-resolution mass data were obtained on a MICROMASS
Q-Tof UltimaTM spectrometer. HPLC analyses data were performed
on an HP 1100 series LC system (HP ChemStation A.06.03), system
A: column, Zorbax SB-C18, 4.6 mm ꢀ 150 mm, 5
CH3CN/H2O; flow rate, 1.0 ml/min; UV wavelength, 220 nm; tem-
perature, ambient temperature; injection volume, 10 l. System
; mo-
bile phase, CH3OH/H2O (pure H2O or H2O with 0.1% TFA); flow rate,
1.0 ml/min; UV wavelength, 220 nm; temperature, ambient tem-
l; mobile phase,
Figure 3. Comparison of the three probes in terms of root growth inhibition of
Arabidopsis seedlings. Wild-type (Col-0) Arabidopsis seedlings were grown on MS
medium without JAs (control) or containing BP, MeJA, PAJA, PAJAIle, or PACOR at
100 lM for nine days. (A) Photographic image of the seedlings. (B) Primary root
lengths of 9-day-old seedlings. Error Bars indicate SE (n >30).
l
B: column, phenomenex Gemini C18, 4.6 mm ꢀ 150 mm, 5
l
perature; injection volume, 10
5.2. Synthesis of probes
5.2.1. Compound 5
ll.
The PACOR probe was useful for revealing the direct binding of
JA to COI1.24 Studies are under way to elucidate the mechanism of
JA perception in greater detail using photoaffinity labeling
technology.
2-[2-(2-Azido-ethoxy)-ethoxy]-ethylamine (44 mg, 0.253 mmol)
and 4 (85 mg, 0.148 mmol) were dissolved in chloroform (5 ml) at
0 °C, then TEA (62 ll, 0.444 mmol) was added. The reaction was stir-
5. Experimental section
red for 12 h at room temperature. The solvent was concentrated and
HCl (1 N) was added to acidify the mixture to pH 2–3, the reaction
mixturewasextractedwith EtOAc(3 ꢀ 20 ml). Thecombinedorgan-
ic extracts were washed with saturated brine, dried over anhydrous
Na2SO4. The solvent was concentrated and the residue was purified
withsilica gel chromatography (CH3OH/CHCl3, 1:100) to afford5 as a
paleyellow gum (80 mg, 85%). 1HNMR (300 MHz, CDCl3): d = 8.21 (s,
1H), 8.15 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.68 (s, 1H), 5.02
(s, 1H), 4.24 (t, J = 4.5 Hz, 2H), 3.88 (t, J = 4.5 Hz, 2H), 3.61 (m, 12H),
3.49 (t, J = 5.1 Hz, 2H), 3.32 (m, 6H), 1.39 (s, 9H); ESI-LRMS (m/z):
C26H38F3N7NaO8: 656.2 [M+Na]+.
5.1. Synthetic materials and methods
Unless otherwise noted, all reagents were purchased from com-
mercial suppliers and used without further purification. ( )-Jas-
monic acid (J2500) were purchased from Sigma. JA-Ile was
ordered commercially from BioDuro Beijing and used as a mixture
of isomers. Coronatine (C8115) was purchased from Sigma. Com-
pounds 2, 3 and 4 were synthesized as reported.33 Dichlorometh-
ane (DCM) and tetrahydrofuran (THF) were distilled from
sodium, DMF was distilled in a vacuum, triethylamine (TEA) was
distilled under normal pressure. The reaction flasks containing all
diazirine derivatives were protected from light by wrapping with
aluminum foil. 1H (300 MHz) NMR spectra were recorded on a
Varian Mercury-Vx 300 M Fourier transform spectrometer. The
chemical shifts were reported in d (ppm) using the d 7.26 signal
5.2.2. Compound 7
Compound 5 (60 mg, 0.095 mmol) was dissolved in dichloro-
methane (5 ml), 2 ml HCl–EtOAc (2 N) was added to the solution
at 0 °C. The reaction was stirred for 2 h at room temperature. The
Figure 4. Comparison of the three probes in terms of induction of the Thionin2.1::GUS reporter construct in Arabidopsis seedlings. Eight-day-old Thionin2.1::GUS Arabidopsis
seedlings were treated with 100 M BP, PAJA, PAJAIle, PACOR, or MeJA or were untreated (Mock), and were then stained for GUS activity.
l

M. Gu et al. / Bioorg. Med. Chem. 18 (2010) 3012–3019
3017
solvent was removed to afford 6, 6 was dissolved in chloroform
(3 ml) at 0 °C, then JA-OSu (35 mg, 0.114 mmol) and TEA (68 l,
2H), 1.25 (m, 3H), 0.98 (m, 2H), 0.02 (s, 9H); ESI-LRMS (m/z):
C
26H36N2NaO4Si: 491.2 [M+Na]+.
l
0.50 mmol) was added. The reaction was stirred for 12 h at room
temperature. The solvent was concentrated and HCl (1 N) was
added to acidify the mixture to pH 2–3, the reaction mixture was
extracted with EtOAc (3 ꢀ 20 ml). The combined organic extracts
were washed with saturated brine, dried over anhydrous Na2SO4.
The solvent was concentrated and the residue was purified with
silica gel chromatography (CH3OH/CHCl3, 1:50) to afford 7 as a pale
yellow gum (50 mg, 73%). 1H NMR (300 MHz, CDCl3): d = 8.18 (s,
1H), 8.14 (d, J = 8.1 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 6.70 (s, 1H),
6.18 (s, 1H), 5.41 (m, 1H), 5.26 (m, 1H), 4.26 (t, J = 4.5 Hz, 2H),
3.88 (t, J = 4.5 Hz, 2H), 3.67 (m, 12H), 3.51 (t, J = 4.8 Hz, 2H), 3.42
(q, J = 5.1 Hz, 2H), 3.33 (t, J = 4.5 Hz, 2H), 2.50 (m, 1H), 2.31 (m,
4H), 2.05 (m, 7H), 1.82 (m, 1H), 1.36 (m, 1H), 0.93 (t, J = 7.8 Hz,
3H); ESI-LRMS (m/z): C33H46F3N7NaO8: 748.3 [M+Na]+.
5.2.6. Compound 10
Let 9 (25 mg, 0.05 mmol) and 4 (30 mg, 0.05 mmol) dissolved in
chloroform (2 ml) at 0 °C, then added NaHCO3 (10 mg, 0.12 mmol).
The reaction mixture was stirred for 12 h at room temperature. The
solvent was concentrated and HCl (1 N) was added to acidify the
mixture to pH 2–3, the reaction mixture was extracted with EtOAc
(3 ꢀ 20 ml). The combined organic extracts were washed with
saturated brine, dried over anhydrous Na2SO4. The solvent was
concentrated and the residue was purified with silica gel chroma-
tography (CH3OH/CHCl3, 1:80) to afford 10 as a pale yellow gum
(42 mg, 85%). 1H NMR (300 MHz, CDCl3): d = 8.19 (d, J = 8.4 Hz,
1H), 8.03 (t, J = 5.1 Hz, 1H), 7.76 (d, J = 7.5 Hz, 2H), 7.59 (t,
J = 6.3 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.29 (t, J = 7.2 Hz, 2H), 6.75
(d, J = 7.8 Hz, 1H), 6.64 (s, 1H), 5.67 (d, J = 7.8 Hz, 1H), 4.92 (s,
1H), 4.42 (t, J = 7.5 Hz, 2H), 4.18 (m, 7H), 3.76 (m, 2H), 3.58 (m,
2H), 3.43 (m, 4H), 3.23 (m, 2H), 1.84 (m, 1H), 1.75 (m, 1H), 1.60
(m, 2H), 1.41 (s, 9H), 1.25 (m, 3H), 0.99 (m, 2H), 0.03 (s, 9H);
ESI-LRMS (m/z): C46H60F3N5NaO10Si: 950.4 [M+Na]+.
5.2.3. PAJA
MeOH (3 ml) was added to a reaction bottle containing 7
(28 mg, 0.039 mmol), then prop-2-ynylamide-biotin (12 mg,
0.041 mmol) and 1 ml 10 mM CuSO4 water solution (0.01 mmol)
were added in. Under the protection of nitrogen, 30 mg/ml sodium
ascorbic solution (280
l
l, 0.042 mmol) was added at last. The reac-
5.2.7. Compound 11
tion was stirred for 12 h at room temperature. After MeOH was re-
moved, the mixture was extracted with EtOAc (3 ꢀ 20 ml). The
combined organic extracts were washed with saturated brine,
dried over anhydrous Na2SO4. The solvent was concentrated and
the residue was purified with silica gel chromatography (CH3OH/
CHCl3, 1:20) to afford probe PAJA as a pale yellow gum (30 mg,
77%). 1H NMR (300 MHz, CDCl3): d = 8.23 (s, 1H), 8.15 (d,
J = 8.1 Hz, 1H), 7.72 (s, 1H), 7.34 (s, 1H), 6.90 (d, J = 7.5 Hz, 1H),
6.71 (s, 1H), 6.50 (m, 1H), 6.39 (s, 1H), 5.73 (s, 1H), 5.42 (m, 1H),
5.28 (m, 1H), 4.48 (m, 2H), 4.26 (m, 2H), 3.84 (m, 2H), 3.60 (m,
8H), 3.48 (m, 2H), 3.42 (m, 2H), 3.12 (m, 1H), 2.90 (m, 1H), 2.74
(m, 1H), 2.52 (m, 1H), 2.33 (m, 4H), 2.22 (m, 2H), 2.13 (m, 2H),
2.06 (m, 2H), 1.87 (m, 1H), 1.64 (m, 4H), 1.47 (m, 4H), 1.25 (m,
8H), 0.95 (m, 5H); ESI-HRMS (m/z): calcd for C46H65F3N10NaO10S:
1029.4456 [M+Na]+; found 1029.4460. HPLC purity system A:
95.25%; system B: 95.12%.
Compound 10 (42 mg, 0.05 mmol) was dissolved in DMF(0.5 ml),
piperidine (25 ll, 0.25 mmol)wasadded to thesolution, thereaction
mixturewasstirredfor0.5 hat roomtemperature. Thereactionsolu-
tion was diluted by water, mixture was extracted with EtOAc
(3 ꢀ 20 ml). The combined organic extracts were washed with
water, saturated brine, dried over anhydrous Na2SO4. The solvent
was concentrated and the residue was purified with silica gel chro-
matography (CH3OH/CHCl3, 1:20) to afford 11 as a pale yellow
gum (26 mg, 80%). 1H NMR (300 MHz, CDCl3): d = 8.16 (d,
J = 8.1 Hz, 1H), 8.02 (t, J = 5.4 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.65
(s, 1H), 5.13 (s, 1H), 4.23 (m, 2H), 4.15 (m, 2H), 3.88 (m, 2H), 3.67
(m, 2H), 3.58 (m, 2H), 3.45 (m, 2H), 3.37 (m, 3H), 3.26 (m, 2H), 1.73
(m, 2H), 1.63 (m, 6H), 1.39 (s, 9H), 0.95 (m, 2H), 0.02 (s, 9H); ESI-
LRMS (m/z): C31H50F3N5NaO8Si: 728.3 [M+Na]+.
5.2.8. Compound 12
Compound 11 (24 mg, 0.034 mmol), JA-Ile (10 mg, 0.031 mmol),
DCC (20 mg, 0.097 mmol) and 1-hydroxybenzotriazole (HOBt)
(10 mg, 0.074 mmol) were dissolved in DCM (5 ml), then TEA
5.2.4. Compound 8
Fmoc-Lys(Z)-OH (502 mg, 1 mmol), 2-trimethylsilanyl-ethanol
(180 mg, 1.53 mmol), N0,N0-dicyclohexylcarbodiimide (DCC)
(310 mg, 1.50 mmol) and 4-dimethylamino-pyridine (DMAP)
(100 mg, 0.82 mmol) were dissolved in DCM (10 ml), The reaction
mixture was stirred for 3 h at room temperature. The solvent was
concentrated and HCl (1 N) was added to acidify the mixture to pH
2–3, the reaction mixture was extracted with EtOAc (3 ꢀ 50 ml).
The combined organic extracts were washed with saturated brine,
dried over anhydrous Na2SO4. The solvent was concentrated and
the residue was purified with silica gel chromatography (EtOAc/
petroleum ether, 1:3) to afford 8 as a pale yellow gum (395 mg,
67%). 1H NMR (300 MHz, CDCl3): d = 7.76 (d, J = 7.2 Hz, 2H), 7.60
(d, J = 6.9 Hz, 2H), 7.33 (m, 9H), 5.37 (m, 1H), 5.08 (s, 1H), 4.78
(m, 1H), 4.39 (m, 3H), 4.22 (m, 3H), 3.20 (d, J = 6.3 Hz, 2H), 1.85
(m, 1H), 1.65 (m, 2H), 1.56 (m, 2H), 1.39 (m, 2H), 1.01 (m, 2H),
0.06 (s, 9H); ESI-LRMS (m/z): C34H42N2NaO6Si: 625.3 [M+Na]+.
(10 ll, 0.08 mmol) was added, The reaction mixture was stirred
for 12 h at room temperature. The solvent was concentrated and
HCl (1 N) was added to acidify the mixture to pH 2–3, the reaction
mixture was extracted with EtOAc (3 ꢀ 20 ml). The combined or-
ganic extracts were washed with saturated brine, dried over anhy-
drous Na2SO4. The solvent was concentrated and the residue was
purified with silica gel chromatography (CH3OH/CHCl3, 1:100) to
afford 12 as a pale yellow gum (19 mg, 61%). 1H NMR (300 MHz,
CDCl3): d = 8.21 (d, J = 8.1 Hz, 1H), 8.13 (t, J = 4.8 Hz, 1H), 6.91 (d,
J = 8.4 Hz, 1H), 6.86 (m, 1H), 6.69 (s, 1H), 6.35 (m, 1H), 5.42 (m,
1H), 5.28 (m, 1H), 4.95 (s, 1H), 4.43 (m, 2H), 4.25 (m, 2H), 4.19
(m, 2H), 3.90 (m, 2H), 3.68 (m, 2H), 3.51 (m, 2H), 3.39 (m, 2H),
3.28 (m, 2H), 2.62 (m, 1H), 2.37 (m, 5H), 2.17 (m, 3H), 2.07 (m,
4H), 1.87 (m, 4H), 1.60 (m, 4H), 1.47 (s, 9H), 1.25 (m, 2H), 0.91
(m, 11H), 0.03 (s, 9H); ESI-LRMS (m/z): C49H77F3N6NaO11Si:
1033.4 [M+Na]+.
5.2.5. Compound 9
Compound 8 (80 mg, 0.13 mmol) was dissolved in methanol
(5 ml), Pd/C (8 mg) and a few drops of acetic acid were added in
the solution, the air in the reaction bottle were replaced by hydro-
gen. The reaction was stirred for 3 h at room temperature. The sol-
vent was removed to afford 9 as a gum (50 mg, 80%). 1H NMR
(300 MHz, CDCl3): d = 7.72 (d, J = 7.2 Hz, 2H), 7.53 (d, J = 7.2 Hz,
2H), 7.35 (m, 4H), 4.27 (m, 6H), 2.98 (s, 2H), 1.78 (s, 4H), 1.46 (s,
5.2.9. Compound 13
Compound 11 (15 mg, 0.021 mmol), coronatine (5 mg,
0.016 mmol), DCC (20 mg, 0.097 mmol) and 1-hydroxybenzotria-
zole(HOBt) (10 mg, 0.074 mmol) were dissolved in DCM (5 ml),
then TEA (10 ll, 0.08 mmol) was added, The reaction mixture
was stirred for 12 h at room temperature. The solvent was concen-
trated and HCl (1 N) was added to acidify the mixture to pH 2–3,

3018
M. Gu et al. / Bioorg. Med. Chem. 18 (2010) 3012–3019
the reaction mixture was extracted with EtOAc (3 ꢀ 20 ml). The
combined organic extracts were washed with saturated brine,
dried over anhydrous Na2SO4. The solvent was concentrated and
the residue was purified with silica gel chromatography (CH3OH/
CHCl3, 1:100) to afford 13 as a pale yellow gum (10 mg, 63%). 1H
NMR (300 MHz, CDCl3): d = 8.16 (d, J = 8.1 Hz, 1H), 8.00 (t,
J = 5.7 Hz, 1H), 7.39 (d, J = 5.1 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H),
6.70 (s, 1H), 6.65 (d, J = 5.1 Hz, 1H), 6.32 (s, 1H), 4.97 (t,
J = 4.8 Hz, 1H), 4.51 (m, 1H), 4.26 (m, 2H), 4.18 (m, 2H), 3.88 (m,
2H), 3.68 (m, 2H), 3.61 (m, 2H), 3.51 (m, 2H), 3.41 (m, 2H), 3.28
(m, 2H), 3.18 (m, 2H), 2.37 (m, 4H), 1.93 (m, 2H), 1.72 (m, 2H),
1.62 (m, 4H), 1.56 (m, 3H), 1.42 (s, 9H), 1.23 (m, 7H), 0.94 (m,
6H), 0.01 (s, 9H); ESI-LRMS (m/z): C49H73F3N6NaO11Si: 1029.4
[M+Na]+.
Reversed-Phase C18 chromatography (H2O/CH3OH, 50:50) to afford
PAJAIle as a pale yellow gum (6 mg, 82%). 1H NMR (300 MHz,
CDCl3): d = 8.17 (d, J = 8.1 Hz, 1H), 7.95 (t, J = 5.1 Hz, 1H), 7.53 (d,
J = 5.7 Hz, 1H), 7.43 (d, J = 6.3 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H),
6.69 (s, 1H), 6.58 (s, 1H), 5.41 (m, 1H), 5.32 (m, 1H), 5.07 (s, 1H),
4.50 (m, 2H), 4.31 (m, 3H), 4.23 (m, 2H), 3.88 (m, 2H), 3.78 (m,
2H), 3.68 (m, 2H), 3.64 (m, 2H), 3.53 (m, 2H), 3.38 (m, 3H), 3.15
(m, 1H), 2.90 (m, 1H), 2.64 (m, 2H), 2.33 (m, 4H), 2.12 (m, 7H),
1.79 (m, 2H), 1.66 (m, 4H), 1.44 (m, 4H), 1.24 (m, 6H), 0.92 (m,
10H); ESI-HRMS (m/z): calcd for C49H71F3N8NaO11S: 1059.4813
[M+Na]+; found 1059.4805. HPLC purity system A: 95.69%; system
B: 96.03%.
5.2.13. PACOR
Compound 15 (6 mg, 0.005 mmol) was dissolved in THF (1 ml),
a few drops of tetrabutylammonium fluoride was added to the
solution. The reaction was stirred for 3 h at room temperature.
The solvent was concentrated and the residue was purified with
Reversed-Phase C18 chromatography (H2O/CH3OH, 50:50) to afford
PACOR as a pale yellow gum (4 mg, 73%). 1H NMR (300 MHz,
CDCl3): d = 8.16 (d, J = 8.1 Hz, 1H), 8.02 (t, J = 5.1 Hz, 1H), 7.62 (s,
1H), 7.45 (d, J = 5.7 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 6.69 (s, 1H),
6.62 (s, 1H), 6.42 (s, 1H), 6.13 (s, 1H), 4.88 (s, 1H), 4.48 (m, 2H),
4.29 (m, 3H), 3.89 (t, 2H), 3.68 (m, 2H), 3.63 (m, 2H), 3.53 (m,
2H), 3.41 (m, 4H), 3.13 (m, 2H), 2.88 (m, 1H), 2.67 (m, 1H), 2.36
(m, 3H), 2.13 (m, 3H), 1.79 (m, 5H), 1.61 (m, 7H), 1.41 (m, 6H),
1.24 (m, 6H), 0.93 (m, 6H); ESI-HRMS (m/z): calcd for
C49H67F3N8NaO11S: 1055.4484 [M+Na]+; found 1055.4500. HPLC
purity system A: 96.81%; system B: 97.20%.
5.2.10. Compound 14
Compound 12 (9 mg, 0.009 mmol) was dissolved in dichloro-
methane (3 ml), 2 ml HCl–EtOAc (2 N) was added to the solution
at 0 °C. The reaction was stirred for 2 h at room temperature. The
solvent was removed, then the residue was dissolved in dichloro-
methane (2 ml) at 0 °C, then Biotin-OSu (6 mg, 0.018 mmol) and
NaHCO3 (10 mg, 0.12 mmol) was added. The reaction was stirred
for 12 h at room temperature. The solvent was concentrated and
HCl (1 N) was added to acidify the mixture to pH 2–3, the reaction
mixture was extracted with EtOAc (3 ꢀ 20 ml). The combined
organic extracts were washed with saturated brine, dried over anhy-
drous Na2SO4. The solvent was concentrated and the residue was
purified with silica gel chromatography (CH3OH/CHCl3, 1:50) to af-
ford 14 as a pale yellow gum (8 mg, 80%). 1H NMR (300 MHz, CDCl3):
d = 8.17 (d, J = 8.1 Hz, 1H), 8.10 (t, J = 5.1 Hz, 1H), 7.55 (d, J = 5.7 Hz,
1H), 7.33 (d, J = 5.7 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H), 6.69 (s, 1H),
6.58 (s, 1H), 5.40 (m, 1H), 5.32 (m, 1H), 5.07 (s, 1H), 4.50 (m, 2H),
4.31 (m, 3H), 4.23 (m, 2H), 4.18 (m, 3H), 3.88 (m, 2H), 3.78 (m, 2H),
3.68 (m, 2H), 3.64 (m, 2H), 3.53 (m, 2H), 3.38 (m, 3H), 3.15 (m, 1H),
2.90 (m, 1H), 2 .65 (m, 2H), 2.32 (m, 4H), 2.11 (m, 7H), 1.80 (m,
2H), 1.64 (m, 4H), 1.45 (m, 4H), 1.25 (m, 6H), 0.94 (m, 10H), 0.02 (s,
9H); ESI-LRMS (m/z): C54H83F3N8NaO11SSi: 1159.4 [M+Na]+.
5.3. Root length assay
Arabidopsis seeds (Col-0) were plated on MS medium or MS
containing 100 lM MeJA, PAJA, PAJAIle, PACOR and BP, respec-
tively. They were incubated for 3 d at 4 °C and then grown for
9 d at 22 °C. For each treatment, about 30 seedlings were grown
on a small plate (U35 mm) containing 2 ml of medium. The exper-
iment was repeated three times.
5.2.11. Compound 15
Compound 13 (9 mg, 0.009 mmol) was dissolved in dichloro-
methane (3 ml), 2 ml HCl–EtOAc (2 N) was added to the solution
at 0 °C. The reaction was stirred for 2 h at room temperature. The
solvent was removed, then the residue was dissolved in dichloro-
methane (2 ml) at 0 °C, then Biotin-OSu (6 mg, 0.018 mmol) and
NaHCO3 (10 mg, 0.12 mmol) was added. The reaction was stirred
for 12 h at room temperature. The solvent was concentrated and
HCl (1 N) was added to acidify the mixture to pH 2–3, the reaction
mixture was extracted with EtOAc (3 ꢀ 20 ml). The combined or-
ganic extracts were washed with saturated brine, dried over anhy-
drous Na2SO4. The solvent was concentrated and the residue was
purified with silica gel chromatography (CH3OH/CHCl3, 1:50) to af-
ford 15 as a pale yellow gum (7 mg, 70%). 1H NMR (300 MHz,
CDCl3): d = 8.17 (d, J = 8.1 Hz, 1H), 8.03 (t, J = 4.8 Hz, 1H), 7.67 (s,
1H), 7.45 (d, J = 5.7 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 6.69 (s, 1H),
6.63 (s, 1H), 6.44 (s, 1H), 6.09 (s, 1H), 4.76 (s, 1H), 4.45 (m, 2H),
4.26 (m, 3H), 4.15 (m, 3H), 3.89 (m, 2H), 3.68 (m, 2H), 3.63 (m,
2H), 3.52 (m, 2H), 3.41 (m, 4H), 3.13 (m, 2H), 2.92 (m, 1H), 2.72
(m, 1H), 2.36 (m, 3H), 2.15 (m, 3H), 1.83 (m, 5H), 1.70 (m, 7H),
1.44 (m, 6H), 1.22 (m, 6H), 0.93 (m, 6H), 0.01 (s, 9H); ESI-LRMS
(m/z): C54H79F3N8NaO11SSi: 1155.4 [M+Na]+.
5.4. Induction of JA-responsive gene in Arabidopsis seedlings
Arabidopsis seedlings (Col-0) carrying Thionin 2.1-GUS (b-glucu-
ronidase) reporter construct,28 in which the GUS gene was driven
by the promoter of thionin 2.1 that is induced by JA treatment, were
grown on MS plate for eight days, transferred to a 1.5 ml tube con-
taining liquid MS medium (Mock) or MS with 100 lM of BP, PAJA,
PAJAIle, PACOR, or MeJA, and incubated for 8 h in dark at 22 °C.
These seedlings then were stained in staining solution (0.05 M so-
dium phosphate buffer pH 7.2, 0.2% Triton X-100, 2 mM potassium
ferrocyanide, 2 mM potassium ferricyanide, and 2 mM X-Gluc) for
16 h in dark at 37 °C. The seedlings were finally washed and fixed
in ethanol.
5.5. Pull-down assays
Protein pull-down assays were performed as described.34 The
seedlings with transgenic expression HA-tagged COI1 protein were
ground in liquid nitrogen to fine powder. Soluble proteins was ex-
tracted from seedlings in a buffer containing 50 mM Tris-Cl (pH
7.8), 100 mM NaCl, 25 mM imidazole, 10% (v/v) glycerol, 0.1% (v/
v) Tween 20, 20 mM 2-mercaptoethanol, 10 mM MG132 and the
EDTA-free complete miniprotease inhibitor cocktail according to
manufacturer’s instructions (Roche). Recombinant JAZ1-His was
expressed in Escherichia coli and purified by Ni affinity chromatog-
raphy. Fifty micrograms JAZ1-His were incubated with 5 mg of to-
tal plant extract, supplemented where indicated with the probes
5.2.12. PAJAIle
Compound 14 (8 mg, 0.007 mmol) was dissolved in THF (1 ml),
a few drops of tetrabutylammonium fluoride was added to the
solution. The reaction was stirred for 3 h at room temperature.
The solvent was concentrated and the residue was purified with

M. Gu et al. / Bioorg. Med. Chem. 18 (2010) 3012–3019
13. Wasternack, C.; Hause, B. Prog. Nucleic Acid Res. Mol. Biol. 2002, 72, 165.
3019
and incubated for 1 h at 4 °C. After washing, pull-down mixtures
were loaded on SDS–polyacrylamide gel electrophoresis (PAGE),
transferred to PVDF membrane and detected with an anti-HA anti-
body (Sigma).
14. Xie, D.; Feys, B. F.; James, S.; Nieto-Rostro, M.; Turner, J. G. Science 1998, 280,
1091.
15. Xu, L.; Liu, F.; Lechner, E.; Genschik, P.; Crosby, W. L.; Ma, H.; Peng, W.; Huang,
D.; Xie, D. Plant Cell 2002, 14, 1919.
16. Liu, F.; Ni, W.; Griffith, M.; Huang, Z.; Chang, C.; Peng, W.; Ma, H.; Xie, D. Plant
Cell 2004, 16, 5.
17. Ren, C.; Pan, J.; Peng, W.; Genschik, P.; Hobbie, L.; Hellmann, H.; Estelle, M.;
Gao, B.; Peng, J.; Sun, C.; Xie, D. Plant J. 2005, 42, 514.
18. Thines, B.; Katsir, L.; Melotto, M.; Niu, Y.; Mandaokar, A.; Liu, G. H.; Nomura, K.;
He, S. Y.; Howe, G. A.; Browse, J. Nature 2007, 448, 661.
19. Chini, A.; Fonseca, S.; Fernandez, G.; Adie, B.; Chico, J. M.; Lorenzo, O.; Garcia-
Casado, G.; Lopez-Vidriero, I.; Lozano, F. M.; Ponce, M. R.; Micol, J. L.; Solano, R.
Nature 2007, 448, 666.
Acknowledgments
National Natural Science Foundation of China (Grants
30630044, 30725049, 30800593, 90717103, and 90813037) are
appreciated for their financial support.
20. Yan, Y. X.; Stolz, S.; Chetelat, A.; Reymond, P.; Pagni, M.; Dubugnon, L.; Farmer,
E. E. Plant Cell 2007, 19, 2470.
Supplementary data
21. Katsir, L.; Schilmiller, A. L.; Staswick, P. E.; He, S. Y.; Howe, G. A. Proc. Natl. Acad.
Sci. U.S.A. 2008, 105, 7100.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.03.059.
22. Melotto, M.; Mecey, C.; Niu, Y. J.; Chung, H. S.; Katsir, L.; Yao, J.; Zeng, W. Q.;
Thines, B.; Staswick, P.; Browse, J.; Howe, G. A.; He, S. Y. Plant J. 2008, 55, 979.
23. Fonseca, S.; Chini, A.; Hamberg, M.; Adie, B.; Porzel, A.; Kramell, R.; Miersch, O.;
Wasternack, O.; Solano, R. Nat. Chem. Biol. 2009, 5, 344.
24. Yan, J.; Zhang, C.; Gu, M.; Bai, Z.; Zhang, W.; Qi, T.; Cheng, Z.; Peng, W.; Luo, H.;
Nan, F.; Wang, Z.; Xie, D. Plant Cell 2009, 21, 2220.
25. Dathe, W.; Rönsch, H.; Preiss, A.; Schade, W.; Sembdner, G.; Schreiber, K. Planta
1981, 155, 530.
26. Staswick, P. E.; Su, W.; Howell, S. H. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 6837.
27. Bohlmann, H.; Vignutelli, A.; Hilpert, B.; Miersch, O.; Wasternack, C.; Apel, K.
FEBS Lett. 1998, 437, 281.
28. Wang, Z.; Dai, L.; Jiang, Z.; Peng, W.; Zhang, L.; Wang, G.; Xie, D. MPMI 2005, 18,
1285.
29. Jessani, N.; Cravatt, B. F. Curr. Opin. Chem. Biol. 2004, 8, 54.
30. Blechert, S.; Bockelmann, C.; Fusslein, M.; Von Schrader, T.; Stelmach, B. Planta
1999, 207, 470.
31. Mithofer, A.; Maitrejean, M.; Boland, W. J. Plant Growth Regul. 2004, 23, 170.
32. Miersch, O.; Kramell, R.; Parthier, B.; Wasternack, C. Phytochemistry 1999, 50,
353.
33. Hashimoto, M.; Hatanaka, Y.; Yang, J.; Dhesi, J.; Holman, G. D. Carbohydr. Res.
2001, 331.
References and notes
1. Dharmasiri, N.; Dharmasiri, S.; Estelle, M. Nature 2005, 435, 441.
2. Kepinski, S.; Leyser, O. Nature 2005, 435, 446.
3. Ueguchi-Tanaka, M.; Ashikari, M.; Nakajima, M.; Itoh, H.; Katoh, E.; Kobayashi,
M.; Chow, T.; Hsing, Y. C.; Kitano, H.; Yamaguchi, I.; Matsuoka, M. Nature 2005,
437, 693.
4. Kinoshita, T.; Cano-Delgado, A.; Seto, H.; Hiranuma, S.; Fujioka, S.; Yoshida, S.;
Chory, J. Nature 2005, 433, 167.
5. Cheng, H.; Song, S.; Xiao, L.; Soo, H. M.; Cheng, Z.; Xie, D.; Peng, J. PLoS Genet.
2009, 5, e1000440.
6. Farmer, E. E.; Almeras, E.; Krishnamurthy, V. Curr. Opin. Plant Biol. 2003, 6, 372.
7. McConn, M.; Browse, J. Plant Cell 1996, 8, 403.
8. McConn, M.; Creelman, R. A.; Bell, E.; Mullet, J. E.; Browse, J. Proc. Natl. Acad. Sci.
U.S.A. 1997, 94, 5473.
9. Schommer, C.; Palatnik, J. F.; Aggarwal, P.; Chetelat, A.; Cubas, P.; Farmer, E. E.;
Nath, U.; Weigel, D. PLoS Biol. 2008, 6, 1991.
10. Shan, X.; Zhang, Y.; Peng, W.; Wang, Z.; Xie, D. J. Exp. Bot. 2009, 60, 3849.
11. Staswick, P. E.; Su, W.; Howell, S. H. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 6837.
12. Ueda, J.; Kato, J. Plant Physiol. 1980, 66, 246.
34. Wasternack, C. Ann. Bot. (London) 2007, 100, 681.