Biosynthesis of Rice Phytoalexin
567
the sixth-leaf stage, the fourth- and ˆfth-stage leaves
were detached for use as experimental material.
These detached rice leaves were UV-irradiated and
incubated as described previously.4)
was concentrated by ultraˆltration with a Centricon
YM-30 membrane (Millipore). This fraction was
desalted in a PD-10 or NAP-10 column (Amersham
Pharmacia Biotech) that had been equilibrated with
the assay buŠer or by dialysis with the 0.2
M
Tris-HCl
Identiˆcation of 3
19,6 -olide ( ). Rice leaves (1.2 g), which had been
incubated for 2 d after UV irradiation, were boiled in
6 ml of 70 aqueous MeOH for 20 min. After 3 ml
b
-hydroxy-9
b
-pimara-7,15-dien-
buŠer at pH 8.0 before the assay. The protein con-
centrations of the crude extract, soluble protein frac-
tion and microsome fraction were 100–200, 60–500
b
1
z
and 430 mg ml, respectively, in the assay.
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of brine had been added to the extract, the mixture
was partitioned against 9 ml of EtOAc. The EtOAc
layer was concentrated to dryness and adsorbed to a
Enzyme assay for dehydrogenase. The reaction
mixture containing 150 l of the enzyme solution
and 150 l of the assay buŠer consisting of 0.2
Tris-HCl at pH 8.0 and 10 m 2-mercaptoethanol
was pre-incubated for 5 min at 30 C. To determine
the optimum pH value, 0.2 Hepes-NaOH, Tris-
m
Sep-Pak Light Silica cartridge (Waters) with
n
-
m
M
hexane. After the cartridge had been washed with
M
20
50
z
z
EtOAc in
EtOAc in
n
-hexane, the fraction eluted with
9
n
-hexane was collected and evaporat-
M
ed to dryness. The concentrate was dissolved in 10
of acetone. A 1- l aliquot of the synthetic compound
1 solution (10 ppm in acetone) and 1 l of the extract
obtained from UV-irradiated rice leaves were each
directly injected into the GC MS instrument (Saturn
m
l
HCl and 3-(cyclohexylamino)-1-propanesulfonic acid
(CAPS)-NaOH were used as the assay buŠer for the
pH ranges of 6–8, 7–9 and 9–10, respectively. The
m
m
reaction was initiated by adding 83
pound 1 in the assay buŠer containing 0.5
100 l of 5 m
NAD+ in the assay buŠer and 17
distilled water. After incubating at 30 C for 40 min,
m
l of 600
m
M
com-
DMSO,
l of
z
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2000R, Varian) which was equipped with an ion-trap
mass spectrometer. Separation was carried out with a
m
M
m
9
×
CP-Sil 8 CB low-bleed MS column (0.25 mm 30 m,
the reaction was stopped by the addition of 0.5 ml
of MeOH, before the solution was centrifuged
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0.25
mm ˆlm thickness, Varian). Helium was used as
×
the carrier gas at a ‰ow rate of 1.5 ml min, the injec-
(10,000
g for 10 min). A 10-ml aliquot obtained
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tor temperature was 250
gram of a column oven was 60
heating to 300 C at 10 C min. The conditions used
9
C, and the temperature pro-
from the assay was subjected to LC MS MS
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9
C for 1 min, before
(Perkin-Elmer SCIEX API-300, Applied Bio-
systems), the instrument being equipped with an
APCI inlet system, to determine the amount of
momilactone A by the method described previous-
ly.10) Momilactone A was detected at a combination
9
9
W
for the mass spectrometer were as follows: ionization
9
mode, EI (70 eV); ion trap temperature, 220 C; scan
range, m z 70-600; scan rate, 1 s scan.
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of m z 315 271 in the multiple reaction monitoring
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Protein assay. The protein concentration was
measured by the method of Bradford et al.,9) using
bovine serum albumin as a standard.
(MRM) mode.
Quantiˆcation of momilactone A and 3b-hydroxy-
9b-pimara-7,15-dien-19,6b-olide (1). UV-irradiated
Enzyme preparation. Rice leaves were collected at
2 d after UV irradiation or at the times indicated in
the time-course experiment. The leaves were cut into
small pieces and then homogenized with a mortar
and pestle on ice in 5 ml per gram fresh tissue weight
rice leaves were collected at the time indicated. These
leaves were cut into small pieces and then boiled in
70
weight) for 20 min. Ten
ed to an LC MS MS analysis by the method
z
aqueous MeOH (5 ml per gram fresh tissue
m
l of the extract was subject-
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of a buŠer consisting of 0.2
10 m 2-mercaptoethanol, 1 m
fonyl ‰uoride, and 1 (w v) polyvinylpolypyrroli-
done. The subsequent extraction was performed at
C. The resulting homogenate was ˆltered through
four layers of cheesecloth, and the ˆltrate centrifuged
M
Tris-HCl at pH 8.5,
described previously.10) Compound 1 was detected at
a combination of m z 317 299 in the MRM mode.
M
M
of phenylmethylsul-
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z
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Results and Discussion
4
9
Identiˆcation of 3
b-hydroxy-9b-pimara-7,15-dien-
×
at 10,000
g
for 40 min to remove the dense mem-
19,6 -olide in UV-irradiated rice leaves
b
branes and other cellular debris. The resulting super-
natant, which is referred to as the crude extract, was
We had speculated that momilactone A would be
biosynthesized from 1 by dehydrogenation at the 3-
position. Since 1 had not been previously reported as
a natural product prior to this investigation, it was
necessary to clarify whether this compound was
present in rice plants. The retention time and mass
×
subjected to ultracentrifugation at 100,000
g for
60 min to provide a supernatant (termed the soluble
protein fraction) and pellet (microsome fraction).
This pellet was dissolved in the small amount of the
extraction buŠer. The microsome fraction and crude
spectrum of synthetic 1 was determined by GC MS
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extract were dialyzed with a 0.2
M
Tris-HCl buŠer at
with a capillary column. The peak at the same reten-
tion time had an identical mass spectrum when the
pH 8.0 before the assay. The soluble protein fraction
Atawong, Anotai
