Abscinazole-E1, a novel chemical tool for exploring the role of ABA 8′-hydroxylase CYP707A

Article abstract of DOI:10.1016/j.bmc.2010.11.011

We developed abscinazole-E1 (Abz-E1), a specific inhibitor of abscisic acid (ABA) 8′-hydroxylase (CYP707A). This inhibitor was designed and synthesized as an enlarged analogue of uniconazole (UNI), a well-known plant growth retardant, which inhibits a gib

Full text of DOI:10.1016/j.bmc.2010.11.011

Bioorganic & Medicinal Chemistry 19 (2011) 406–413
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Abscinazole-E1, a novel chemical tool for exploring the role
of ABA 80-hydroxylase CYP707A
Mariko Okazaki a, , Hataitip Nimitkeatkai b, , Taku Muramatsu a, , Hikaru Aoyama a, Kotomi Ueno c,
Masaharu Mizutani c, Nobuhiro Hirai d, Satoru Kondo b, Toshiyuki Ohnishi e, Yasushi Todoroki a,
⇑
a Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan
b Division of Bioresource Science, Graduate School of Horticulture, Chiba University, Matsudo 271-8510, Japan
c Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan
d Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8501, Japan
e Division of Global Research Leaders, Shizuoka University, Shizuoka 422-8529, Japan
a r t i c l e i n f o
a b s t r a c t
We developed abscinazole-E1 (Abz-E1), a specific inhibitor of abscisic acid (ABA) 80-hydroxylase
(CYP707A). This inhibitor was designed and synthesized as an enlarged analogue of uniconazole (UNI),
a well-known plant growth retardant, which inhibits a gibberellin biosynthetic enzyme (ent-kaurene oxi-
dase, CYP701A) as well as CYP707A. Our results showed that Abz-E1 functions as a potent inhibitor of
CYP707A and a poor inhibitor of CYP701A both in vitro and in vivo. Abz-E1 application to plants resulted
in improved desiccation tolerance and an increase in endogenous ABA.
Article history:
Received 12 October 2010
Revised 4 November 2010
Accepted 5 November 2010
Available online 10 November 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Plant growth regulator
P450 inhibitor
ABA
1. Introduction
concentration during water deficit stress or dormancy maintenance
and breaking.13,14
Abscisic acid (ABA) is a plant hormone involved in stress toler-
ance, stomatal closure, seed dormancy, and other physiological
events.1–4 The endogenous levels of ABA in plants are cooperatively
controlled by biosynthesis, transport, and catabolic inactivation in
response to environmental changes.1–4 A natural or artificial chem-
ical that perturbs this highly controlled system is promising not
only as a chemical probe for the mechanism of ABA action,5 but
also because of its potential use in agriculture and horticulture.
Although ABA is registered asa farm chemical (plant growth regu-
lator), its practical use has been limited, mainly due to its weak ef-
fect in field trials,6 considered to be due to its rapid inactivation
through biodegradation. Catabolic inactivation of ABA is mainly
controlled by ABA 80-hydroxylase, which is the cytochrome P450
catalyzing the C80-hydroxylation of ABA to 80-hydroxy-ABA and
its more stable tautomer, phaseic acid (PA), which has much lower
hormonal activity than ABA (Fig. 1).4 ABA 80-hydroxylase was iden-
tified as CYP707A1-4 in the model plant Arabidopsis thaliana in
2004,7,8 and since then many CYP707A isozymes have been found
in plants.9–12 Gene knockdown and overexpression studies suggest
that ABA 80-hydroxylase is a key enzyme for controlling ABA
S-Uniconazole (UNI), a plant growth retardant developed in the
1980s,15,16 functions as an inhibitor of CYP707A17 in addition to
ent-kaurene oxidase (CYP701A), which catalyzes the three-step
oxidation of ent-kaurene to ent-kaurenoic acid (KA),18 a biosyn-
thetic precursor of the plant hormone gibberellin (GA). S-Dinico-
nazole, the R enantiomer of which is a fungicide with a structure
very similar to UNI, also inhibits CYP707A,19 although it arrests
plant growth.20 The broad inhibition spectrum of some azole com-
pounds against P450 enzymes has apparently resulted from the
heme coordination of an azole nitrogen, which is a common mech-
anism of azole P450 inhibitors. In other words, their specificity for
individual P450 enzymes depends on structural properties other
than the azole group.
In early work,21 we focused on the small and flexible structure
of UNI to develop a more specific inhibitor against CYP707A by
three approaches: (1) molecular enlargement;22 (2) conforma-
tional restriction;23 and (3) modification of the azole ring.24 All ap-
proaches resulted in an increase in specificity for CYP707A.
Representative inhibitors are summarized in Table 1. Although
these inhibitors are more specific than UNI, they have drawbacks:
a low synthetic yield for Abz-F1 and DSI-505M, and a low aqueous
solubility for UT4. In the present study, we focused on the molec-
ular enlargement approach to develop a novel UT derivative with
higher water solubility than UT4 and with high extensibility to
be transformed into diverse functional probes. This paper describes
⇑
Corresponding author. Tel./fax: +81 542384871.
E-mail addresses: aytodor@ipc.shizuoka.ac.jp, aytodor@agr.shizuoka.ac.jp
(Y. Todoroki).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.011

M. Okazaki et al. / Bioorg. Med. Chem. 19 (2011) 406–413
407
ABA catabolism
HO
O
ABA 8'-hydroxylase
(CYP707A)
OH
OH
CO2H
CO2H
O
S-(+)-ABA
8'-hydroxy-ABA
Abz-F1
S-(+)-UNI
UT4
Abz-E1
DSI-505ME
GA
ent-kaurene oxidase
(CYP701A)
CO2H
ent-kaurene
KA
GA biosynthesis
Figure 1. Uniconazole (UNI) is a potent inhibitor of ABA 80-hydroxylase and ent-kaurene oxidase.
Table 1
Azole inhibitors of CYP707A
Name
Structure
CYP707A inhibitiond
Growth inhibitione
KI (nM)
IC50 (lM)
HO
Cl
N
UNIa
10
0.18
N
N
N
N
N
HO
UT4a
195
42
N
N
N
HO
O
N
Cl
3
Abz-F1b
>100
>100
420 (3R)
970 (3S)
N
H3CO2C
HO
N
Cl
DSI-505MEc
120
N
a
Ref. 22.
Ref. 23.
Ref. 24.
b
c
d
Arabidopsis recombinant CYP707A3 coexpressed with Arabidopsis P450 reductase (ATR2) in E. coli.
Rice seedlings.
e
the design and preparation of UT1-E2Ts, which we named absci-
nazole-E1 (Abz-E1), and its in vitro and in vivo inhibition of
CYP707A and CYP701A.
at the C40 side chain is poor compared with that of UT with an alkyl
side chain, although the 1,2,3-triazolyl moiety had no effect on the
inhibitory potency.22 This finding suggests that the CYP707A active
site is relatively, but nor entirely, hydrophobic, like P450 active
sites generally. Therefore, we selected ethylene glycol ether, which
is a hydrophilic moiety with no protic group. Considering also
extensibility to be transformed into diverse functional probes, we
designed UT1-E2Ts with the tosylate of diethylene glycol as a novel
UT derivative. UT1-E2Ts was easily prepared from the 40-azide
derivative of UNI, compound 2, using click chemistry,25 like other
2. Results and discussion
2.1. Design, preparation, and water solubility
In a previous study, we found that the CYP707A inhibitory po-
tency of UT with protic groups such as hydroxy and carboxy groups

408
M. Okazaki et al. / Bioorg. Med. Chem. 19 (2011) 406–413
UT derivatives22 (Scheme 1). Water solubility of UT1-E2Ts was
compared with UNI and UT4. Table 2 shows that UT1-E2Ts had
higher water solubility than UT4, but slightly lower solubility than
UNI.
Table 2
Water solubility of UT1-E2Ts (Abz-E1)
Compound
Solubility (%) in water (2 mL)a
20 nmol
79.4
40.7
68.9
200 nmol
39.5
—
21.3
UNI
UT4
2.2. Inhibition of Arabidopsis CYP707A3 and rice CYP701A6 in
an in vitro enzyme assay
b
UT1-E2Ts (Abz-E1)
a
Calculated based on the in water/in MeOH ratio of HPLC peak area.
Not tested.
Inhibitory activity of UT1-E2Ts against ABA 80-hydroxylase was
examined using recombinant Arabidopsis CYP707A3 co-expressed
with Arabidopsis P450 reductase (ATR2) in Escherichia coli.22 The
activity was evaluated based on the decrease in the amount of
the enzymatic product, PA. The mode of inhibition was competitive
(Fig. 2) and the inhibition constant (KI) was 27 8 nM, which was
almost equal to that of UNI (10 nM) (Table 3). This means that UT1-
E2Ts is the most potent CYP707A inhibitor, at least in vitro, among
all the UT derivatives as well as Abz-F1 and DSI-505M.
To construct an assay system for evaluating inhibitory activity
against ent-kaurene oxidase, CYP701A, we expressed recombinant
rice (Oryza sativa L. cv. Nipponbare) CYP701A6 in insect cells using
a baculovirus vector. This enzyme catalyzes all three steps to KA
via the corresponding alcohol and aldehyde,18 meaning that kinetic
analysis is very complicated. Therefore, instead of the KI value, we
evaluated the IC50 value for formation of the end product, KA
(Fig. 2b and Table 3). The IC50 value of UNI was 45 nM when
b
suggest that Abz-E1 does not function as a potent inhibitor of
P450 enzymes (e.g., CYP701A) involved in seed germination and
seedling growth, whereas it may inhibit the catabolism of ABA
in vivo.
2.4. Effect on stomatal closure, drought tolerance, and
endogenous ABA and PA
To determine whether in vivo inhibition of Abz-E1 against
CYP707A is sufficient to intensify ABA-induced physiological re-
sponses, we tested the effect of Abz-E1 on stomatal aperture,
drought tolerance, and endogenous amount of ABA and its catabo-
lite PA. We tested 90-day-old apple seedlings by spraying them
with an aqueous solution containing Abz-E1 at 10 lM. The seed-
1
l
M ent-kaurene was used as the substrate, whereas that of
lings were water-stressed for 12 days beginning 24 h after treat-
ment with the inhibitor, and then rehydrated on the 13th day.
Application of Abz-E1 before dehydration induced drought toler-
ance (Fig. 6a) and stomatal closure (Fig. 6b) during dehydration.
The endogenous amount of ABA during dehydration increased sig-
nificantly after treatment with Abz-E1 (Fig. 6c), whereas the
amount of PA decreased (Fig. 6d). This indicates that Abz-E1 inhib-
ited the hydroxylation of ABA to PA by CYP707A in apple seedlings.
UT1-E2Ts was 1 M under the same assay conditions. This means
l
that the inhibitory activity of UT1-E2Ts against CYP701A6 was sub-
stantially reduced compared with UNI. We renamed UT1-E2Ts as
abscinazole-E1 (Abz-E1) before testing it in bioassays.
2.3. Effect on seed germination and early growth
We tested the effect of Abz-E1 on germination of Arabidopsis
and lettuce seeds. Although Abz-E1 exhibited no effect on rooting
of Arabidopsis and lettuce seeds, it affected leaf emergence of Ara-
3. Conclusion
bidopsis slightly at 100
E1 enhanced the effect of ABA on early growth of lettuce upon
simultaneous application of 100 M of Abz-E1 (Fig. 4). We also
tested the effect of Abz-E1 on early growth of rice seedlings and
found it had little effect on seedling growth (Fig. 5). These results
lM, similarly to UNI at 10 lM (Fig. 3). Abz-
UNI, which was developed as an inhibitor of a GA biosynthetic
enzyme (CYP701A), is a low selectivity P450 inhibitor that inhibits
multiple P450 enzymes including ABA 80-hydroxylase (CYP707A).
Based on our speculation that the low selectivity of UNI may have
resulted from its small size, we earlier developed enlarged UNI
l
Scheme 1. Synthesis of UT1-E2Ts (Abz-E1). Reagents and conditions: (i) NaH, dry DMF, rt, 78%; (ii) CuSO4, sodium ascorbate, THF, rt, 96%.

M. Okazaki et al. / Bioorg. Med. Chem. 19 (2011) 406–413
4. Experimental
409
a
7
6
5
4
3
2
1
0
4.1. General
(+)-ABA was a gift from Toray Industries Inc., Tokyo, Japan. ent-
Kaurene and ent-kaurenoic acid were a gift from Professor Tomo-
nobu Toyomasu (Yamagata University) and Dr. Shinjiro Yamaguchi
(RIKEN Plant Science Center), respectively. ent-Kaurenoic acid was
also purchased from Olchemlm Ltd, Czech Republic. 1H NMR spec-
tra were recorded with tetramethylsilane as the internal standard
using JEOL JNM-EX270 (270 MHz) and JNM-LA500 (500 MHz) NMR
spectrometers. 13C NMR and 2D-correlation NMR experiments
were recorded using a JNM-LA500 (500 MHz) NMR spectrometer.
High resolution mass spectra were obtained with a JEOL JMS-
T100LC ‘AccuTOF’. Column chromatography was performed on sil-
ica gel (Wakogel C-200).
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1/ABA (µM)
0
50
100
150
200
250
300
ABA (µM)
4.2. Synthesis of UT1-E2Ts (Abz-E1)
b
4.2.1. 2-(2-(Prop-2-ynyloxy)ethoxy)ethyl 4-
methylbenzenesulfonate (1)
100
80
60
40
20
0
To a stirred solution of propargyl alcohol (100 mg, 1.8 mmol) in
dry DMF (30 mL) was added NaH (60% in oil, 900 mg, 22 mmol) at
0 °C under Ar. After stirring for 30 min at room temperature, dieth-
ylene glycol bis(p-toluenesulfonate) (2.8 g, 6.7 mmol) was added to
the mixture at 0 °C. The mixture was stirred for 1 h at room tem-
perature. After quenching with sat. NH4Cl at 0 °C, the resulting
mixture was extracted with EtOAc (250 mL ꢀ 3). The organic layer
was washed with brine, dried over Na2SO4, and concentrated in va-
cuo. The residual oil was purified by silica gel column chromatog-
raphy with 20% EtOAc in hexane to obtain 1 (410 mg, 78%) as a
colorless oil. 1H NMR (270 MHz, CDCl3): d 2.43 (1H, t, J = 2.6 Hz,
H-1), 2.45 (3H, s, CH3 in tosylate), 3.59–3.66 (4H, m, –OCH2-
CH2O–), 3.70 (2H, m, –OCH2CH2OSO2–), 4.16 (2H, m, HCCCH2O–),
4.17 (2H, m, –OCH2CH2OSO2–), 7.34 and 7.80 (each 2H, m, tosyl-
ate); HRMS (ESI-TOF, positive mode): calcd for C14H18O5SNa
[M+Na]+ 321.0773, found 321.0771.
-8
-7
-6
log [M]
-5
-4
Figure 2. (a) Competitive inhibition of CYP707A3 by UT1-E2Ts (Abz-E1). Assays
contained S-ABA (d) or S-ABA and 40 nM Abz-E1 (s). The inset is a double-
reciprocal plot of the same data. (b) Dose response in inhibition of CYP701A6 by UNI
(d) and UT1-E2Ts (Abz-E1) (s).
4.2.2. (E)-2-(2-((1-(4-(3-Hydroxy-4,4-dimethyl-2-(1H-1,2,4-
triazol-1-yl)pent-1-en-1-yl)phenyl)-1H-1,2,3-triazol-4-
yl)methoxy)ethoxy)ethyl 4-methylbenzenesulfonate (Abz-E1)
To a stirred solution of 1 (2.5 g, 8.5 mmol) and 222 (1.4 g,
4.6 mmol) in THF (280 mL) was added aqueous CuSO4
(10 mg mLꢁ1, 380 mL) and aqueous sodium ascorbate (10 mg
mLꢁ1, 340 mL) at room temperature. The mixture was stirred for
1 h before extraction with CH2Cl2 (600 mL ꢀ 3). The organic layer
was washed with brine, dried over Na2SO4, and concentrated in va-
cuo. The residual oil was purified by silica gel column chromatog-
raphy with 20% EtOAc in hexane to obtain Abz-E1 (2.6 g, 96%) as a
pale yellow oil. 1H NMR (500 MHz, CDCl3): d 0.68 (9H, s, t-butyl),
2.44 (3H, s, CH3 in tosylate), 3.64–3.67 (2H, m, –OCH2CH2O–),
3.69–3.72 (4H, m, –OCH2CH2OCH2CH2OSO2–), 4.18 (2H, m, –OCH2-
CH2OSO2–), 4.42 (1H, d, J = 8.5 Hz, HO-3), 4.62 (1H, d, J = 8.5 Hz, H-
3), 4.77 (2H, s, 1,2,3-triazole-CH2O–), 7.01 (1H, s, H-1), 7.33 (2H, m,
tosylate), 7.55 (2H, m, 1-phenyl), 7.78 (2H, m, tosylate), 7.83 (2H,
m, 1-phenyl), 8.07 (1H, s, 1,2,3-triazole), 8.12 and 8.60 (each 1H,
s, 1,2,4-triazole); 13C NMR (125 MHz, CDCl3): d 21.6 (methyl in tos-
ylate), 26.1 (methyl in t-butyl), 36.2 (tertiary carbon in t-butyl),
64.7, 68.7, 69.2, 69.8, and 70.7 (diethylene glycol linker), 75.6
(C3), 120.6 (C30 and C50), 120.7 (C300 and 1,2,3-triazole), 127.6
(C1), 127.9 (tosylate), 129.8 (tosylate), 130.3 (phenyl), 132.9 (tosyl-
ate), 134.4 (phenyl), 136.6 (phenyl), 137.9 (C2), 144.9 (tosylate),
Table 3
UNI and UT1-E2Ts (Abz-E1) KI values for Arabidopsis CYP707A3 and IC50 values for
rice CYP701A6
Compound
KI for CYP707A3 (nM)
IC50 for CYP701A6 (nM)
UNI
10a
45
970
UT1-E2Ts (Abz-E1)
27
8
a
Ref. 22 and 23.
analogues, UT compounds, that have a 1,2,3-triazolyl alkyl chain.
Although UT compounds showed strong CYP707A inhibition, the
biological activity of most of UT compounds was not tested be-
cause of their water insolubility. Because the introduction of protic
functional groups in the alkyl chain diminished the inhibitory
activity, in the present study we developed Abz-E1, which has a
diethylene glycol chain with a terminal tosylate. Abz-E1 showed
good water solubility and strong inhibitory activity against
CYP707A in vitro and in vivo, equivalent to that of UNI. On the
other hand, against CYP701A, Abz-E1 was a poorer inhibitor than
UNI. Abz-E1 is expected to be a useful tool in chemical biology
and chemical genetics as well as a novel plant growth regulator.
Moreover, because Abz-E1 has a terminal tosylate, it is expected
to be a useful precursor for synthesis of multifunctional chemical
probes.
146.1 (phenyl and 1,2,3-triazole); UV kmax (MeOH) nm (e): 271.2
(20,000); HRMS (ESI-TOF, positive mode): calcd for C29H36N6O6SNa
[M+Na]+ 619.2315, found 619.2312.

410
M. Okazaki et al. / Bioorg. Med. Chem. 19 (2011) 406–413
Figure 3. Effect of Abz-E1 on leaf emergence in Arabidopsis 54 h after imbibition: (a) control, (b) 10 lM UNI, (c) 30 lM Abz-E1, and (d) 100 lM Abz-E1.
4.3. Water-solubility test
MeOH solutions of test samples were put in a glass vial and con-
centrated in vacuo. Distilled water (2 mL) or MeOH (2 mL) was
added to the vial. After shaking several times, 10 lL of the solution
was subjected to HPLC. HPLC conditions were: ODS column, Hydro-
sphere C18 (150 ꢀ 6.0 mm, YMC); solvent, 65% MeOH in H2O; flow
rate, 1.0 mL minꢁ1; detection, 254 nm. Solubility (%) in water was
calculated based on the in water/in MeOH ratio of the HPLC peak
area.
4.4. Preparation of recombinant enzymes
4.4.1. Coexpression of recombinant Arabidopsis CYP707A3 and
Arabidopsis P450 reductase (ATR2) in E. coli
A truncated Arabidopsis CYP707A3 (707A3d28), which lacked
the putative membrane-spanning segment of the N-terminus, res-
idues 3–28, was constructed. Cells of E. coli strain BL21 were trans-
formed with the constructs pCW-CYP707A3d28 and pACYC-AR2.
Cultures (3 mL) were grown overnight in Luria-Bertani medium
Figure 4. Enhancement of Abz-E1 on ABA activity in early growth (14 days after
germination) of lettuce.
supplemented with ampicillin (50
(100
g mLꢁ1). Then, 50 mL of Terrific Broth medium supple-
mented with ampicillin (50 chloramphenicol
g mLꢁ1),
(100
g mLꢁ1), and aminolevulic acid (0.5 mM) was inoculated
l
g mLꢁ1) and chloramphenicol
l
l
l
with an aliquot of the overnight culture (0.5 mL). The culture
was incubated with gentle shaking (225 rpm) at 37 °C until A600
reached 0.6 (at 2.5–3 h), and expression of the P450 enzyme was
induced by the addition of isopropyl b-D-thiogalactopyranoside
(0.1 mM). The culture was shaken continuously (150 rpm) at
25 °C and cells were harvested 48 h later by centrifugation at
2330g for 20 min at 4 °C. Pelleted cells were suspended in 2.5 mL
of buffer A (50 mM potassium phosphate buffer, pH 7.25, 20% glyc-
erol, 1 mM EDTA, 0.1 mM dithiothreitol). The suspension was son-
icated for 30 s, and the enzyme solution in the supernatant was
collected by centrifugation at 23,470g for 30 min at 4 °C. The
P450 content was determined by spectrophotometric analysis,
using the extinction coefficient of a reduced CO difference spec-
trum (91.1 mMꢁ1 cmꢁ1).
Figure 5. Comparison of effect of Abz-E1 and UNI on rice seedling growth on day 7
after treatment.

M. Okazaki et al. / Bioorg. Med. Chem. 19 (2011) 406–413
411
dehydration
a
b
at day 13
4
3
2
1
0
0
10
50
Abz-E1 (µM)
100
0
2
4
6
8
10 12 14 16
days
dehydration
dehydration
c
d
5
4
3
2
1
0
5
4
3
2
1
0
0
2
4
6
8
10 12 14 16
0
2
4
6
8
10 12 14 16
days
days
Figure 6. Effect of Abz-E1 on drought tolerance (a), stomatal aperture (b), ABA content (c), and PA content (d) during dehydration: control (d) and 10
lM Abz-E1 (s). Arrows
show dehydration period (day 1 to day 13).
4.4.2. Cloning and heterologous expression of OsCYP701A6 with
a baculovirus-insect cell system
OsCYP701A6, Sf9 cells infected by baculovirus containing OsCYP701A6
cDNA were incubated in Grace’s insect cell medium, 0.1% (w/v)
cDNA containing the entire open-reading frame of OsCYP701A6
was amplified by RT-PCR. The full-length OsCYP701A6 (AK066285)
was obtained from the Rice Genome Resource Center (the National
Institute of Agrobiological Sciences, Japan), and the OsCYP701A6
ORF was amplified by PCR with the full-length cDNA as template.
Nucleotide sequences of gene-specific primers were as follows:
TO-35: 50-AAAACTAGTATGGAGGCGTTCGTGC-30 (SpeI site in italics
and start codon underlined) and TO-36: 50-AAAATTCGAATCA-
CATCCTTCCTCTGCG-30 (HindIII site in italics and stop codon under-
lined). A 1-ng aliquot of the full-length cDNA was used as a
Pluronic F-68 (Invitrogen), 10% (v/v) fetal bovine serum, 100
lM
5-aminolevulinic acid and 100 M ferrous citrate on a rotary sha-
l
ker at 27 °C and 150 rpm for 96 h. Insect cells were then harvested
by centrifugation at 3000g for 10 min and washed with PBS buffer
three times. After centrifuging again, insect cells were sonicated in
buffer A containing 50 mM potassium phosphate (pH 7.3), 20% (v/
v) glycerol, 1 mM EDTA, 0.1 mM dithiothreitol and centrifuged at
8000g for 10 min. The supernatant was further centrifuged at
100,000g for 1 h and the resulting pellet (microsomal fraction)
was homogenized with buffer A. The concentration of active
P450 was estimated from the carbon monoxide difference
spectrum.26
template for the PCR reaction in a 20-
l
L reaction mixture contain-
L), PrimeSTAR HS DNA
Polymerase (0.5 U), 200 lM dNTP mixture, and 0.2 lM of the
ing 5 ꢀ PrimeSTAR Buffer (Mg2+ plus) (4
l
gene-specific primers described above. The PCR product was gel-
purified and cloned into pJET1.2/bluntusing a CloneJET PCR Cloning
Kit (Fermentas, Canada). The cloned inserts were sequenced with
pJET1.2 forward and reverse sequencing primers to confirm the ab-
sence of PCR errors in the inserts. Full-length cDNA of OsCYP701A6
in pJET1.2/blunt plasmid vector was excised with the restriction
enzymes SpeI and HindIII (TaKaRa Bio) and purified by 1% (w/v)
agarose gel electrophoresis. This cDNA was ligated into pFastBac1
vector (Invitrogen, USA) digested with the same set of restriction
enzymes. The pFastBac1-OsCYP701A6 constructs were used for
the preparation of recombinant bacmid DNA by transformation
of E. coli strain DH10Bac (Invitrogen). Preparation of recombinant
baculovirus DNA containing OsCYP701A6 cDNA and transfection
of Sf9 (Spodoptera frugiperda 9) cells were performed according
to the manufacturer’s protocol (Invitrogen). For expression of
4.5. Enzyme assay
4.5.1. CYP707A3
A reaction mixture containing 25
somes coexpressed AR2 in E. coli, (+)-ABA (final conc. 0.5–
128 M), inhibitors (0 for control, 2–1000 nM in 5 L DMF) and
50 M NADPH in 50 mM potassium phosphate buffer (pH 7.25)
were incubated for 10 min at 30 °C. Reactions were initiated by
adding NADPH, and stopped by addition of 50 L of 1 M NaOH.
Each reaction mixture was acidified with 100 L of 1 M HCl. To ex-
l
g mLꢁ1 of CYP707A3 micro-
l
l
l
l
l
tract the reaction products, the mixture was loaded onto an Oasis
HLB cartridge (1 mL, 30 mg; Waters) and washed with 1 mL of 10%
MeOH in H2O containing 0.5% AcOH. The enzyme products were
then eluted with 1 mL of MeOH containing 0.5% AcOH, and the elu-
ate was concentrated in vacuo. The dried sample was dissolved in

412
M. Okazaki et al. / Bioorg. Med. Chem. 19 (2011) 406–413
50
lL of MeOH, and 10
lL was subjected to HPLC. HPLC conditions
4.6.4. Drought tolerance assay
were: ODS column, Hydrosphere C18 (150 ꢀ 6.0 mm, YMC); sol-
vent, 35% MeOH or 20% MeCN in H2O containing 0.1% AcOH; flow
rate, 1.0 mL minꢁ1; detection, 254 nm. Enzyme activity was con-
firmed by determining the amount of PA in control experiments
before each set of measurements. Inhibition constants were deter-
mined using the Enzyme Kinetics module of SigmaPlot 10 soft-
ware27 after determining the mode of inhibition by plotting the
reaction velocity in the presence and absence of inhibitor on a dou-
ble-reciprocal plot. For the uninhibited enzymatic reaction, the KM
Apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]
cultivar Fuji seeds were soaked overnight in water, then sown in
moist vermiculite and grown in a greenhouse. Seedlings (90 days
old) were sprayed uniformly with 2 mL of a test solution (10, 50,
or 100 lM) containing Abz-E1 or distilled water (control) 24 h be-
fore water-stressing them for 12 days. Stomatal aperture was mea-
sured as follows: the extent of opening of 10 stomatal apertures
per leaf was observed using a TM-1000 microscope (Hitachi
High-Technologies Co., Tokyo, Japan); three leaves from three ran-
domly selected seedlings (one leaf per seedling) were used for a to-
tal of 30 stomata per treatment.
for (+)-ABA was calculated to be 3.4 0.6 lM, based on five sepa-
rate experiments. All assays were conducted at least three times.
4.5.2. CYP701A6
4.6.5. ABA and PA analysis and quantification in apple seedlings
30,50,50,70,70,70-Hexadeuterated ABA (ABA-d6) was purchased
from Shoko Co. (Tokyo, Japan). PA was received as described by
Hirai et al.29 An internal standard of PA, 70,70,70-trideuterated
PA(PA-d3), was prepared according to a previously reported meth-
od.30 The extraction and quantification of ABA and PA in apple
seedlings were performed according to a previously reported
method31 using HPLC and gas chromatography–mass spectrome-
try–selective ion monitoring (GC–MS–SIM) (Shimadzu QP5000).
ABA was calculated by the ratio of peak areas for m/z 190(d0)/
194(d6). PA was determined from m/z 276(d0)/279(d3).
The microsomal fractions of OsCYP701A6 were combined with
purified NADPH-P450 reductase.28 Assays of 400
lL contained
50 mM potassium phosphate (pH 7.25), 25 pmol mL of OsCY-
P701A6 microsomal fraction, purified NADPH-P450 reductase
(0.1 U), ent-kaurene (10
10 M). Reactions were initiated by addition of NADPH, and were
carried out at 30 °C for 30 min. After termination by adding 1 M
HCl (100 L) and EtOAc (200 L), 5 L of 1 mM abietic acid was
lM) as substrate, and inhibitor (0.01–
l
l
l
l
added as an internal standard. The reaction products were ex-
tracted three times with an equal volume of ethyl acetate and
the organic phase was collected. Anhydrous Na2SO4 was added to
remove residual water. To derivatize the reaction products, meth-
Acknowledgments
anol (100 lL) and TMS-CHN2 (100 lL) (2.0 M in Et2O) were added
and the reaction mixture was incubated at room temperature for
15 min. Organic solvent was removed under Ar and samples were
We thank Toray Industries Inc., Tokyo, Japan, for a gift of (+)-
ABA. This research was supported by a Grant-in-Aid for Scientific
Research (No. 22580118) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.
adjusted to 200
Shimadzu Corp., Japan). GC conditions were: column, DB-5 ms
m film thickness; J&W Scientific); car-
lL with Et2O before GC–MS analysis (QP2010-plus,
(0.25 mm id ꢀ 15 m; 0.25
l
rier gas, He; flow rate, 1.84 mL minꢁ1; injection port temperature,
280 °C; splitless injection; column oven temperature, 80 °C
(1 min), 80–200 °C (18 °C minꢁ1), 200–210 °C (2 °C minꢁ1), 210–
280 °C (30 °C minꢁ1), 280 °C (3 min). The content of KA was calcu-
lated from the area ratio of molecular and fragment ions of methyl
KA (m/z 316, 273, and 257) to those of methyl abietate (m/z 316
and 256).
Supplementary data
Supplementary data (1H NMR spectra of new compounds) asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2010.11.011.
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