Purification and identification of naringenin 7-O-methyltransferase, a key enzyme in biosynthesis of flavonoid phytoalexin sakuranetin in rice

Article abstract of DOI:10.1074/jbc.M112.351270

Sakuranetin, the major flavonoid phytoalexin in rice, is induced by ultraviolet (UV) irradiation, CuCl₂ treatment, jasmonic acid treatment, and infection by phytopathogens. It was recently demonstrated that sakuranetin has anti-inflammatory activity, anti-mutagenic activity, anti-pathogenic activities against Helicobacter pylori, Leishmania, and Trypanosoma and contributes to the maintenance of glucose homeostasis in animals. Thus, sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin by naringenin 7-O-methyl-transferase (NOMT). In previous research, rice NOMT (OsNOMT) was purified to apparent homogeneity from UV-treated wild-type rice leaves, but the purified protein, named OsCOMT1, exhibited caffeic acid O-methyltransferase (COMT) activity and not NOMT activity. In this study, we found that OsCOMT1 does not contribute to sakuranetin production in rice in vivo, and we purified OsNOMT using the oscomt1 mutant. A crude protein preparation from UV-treated oscomt1 leaves was subjected to three sequential purification steps, resulting in a 400-fold purification from the crude enzyme preparation. Using SDS-PAGE, the purest enzyme preparation showed a minor band at an apparent molecular mass of 40 kDa.Two O-methyltransferase-like proteins, encoded by Os04g0175900 and Os12g0240900, were identified from the 40-kDa band by MALDI-TOF/TOF analysis. Recombinant Os12g0240900 protein showed NOMT activity, but the recombinant Os04g0175900 protein did not. Os12g0240900 expression was induced by jasmonic acid treatment in rice leaves prior to sakuranetin accumulation, and the Os12g0240900 protein showed reasonable kinetic properties toOsNOMT.Onthe basis of these results, we conclude that Os12g0240900 encodes an OsNOMT.

Full text of DOI:10.1074/jbc.M112.351270

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2012/04/09/M112.351270.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 23, pp. 19315–19325, June 1, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Purification and Identification of Naringenin
7-O-Methyltransferase, a Key Enzyme in Biosynthesis
□
S
of Flavonoid Phytoalexin Sakuranetin in Rice*
Received for publication, February 9, 2012, and in revised form, April 3, 2012 Published, JBC Papers in Press, April 9, 2012, DOI 10.1074/jbc.M112.351270
Takafumi Shimizu‡, Fengqiu Lin‡, Morifumi Hasegawa§, Kazunori Okada‡1, Hideaki Nojiri‡, and Hisakazu Yamane‡2
From the ‡Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo and the §College of Agriculture,
Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki, Japan
Background: Sakuranetin is a major rice phytoalexin and a potential pharmaceutical agent. Rice naringenin 7-O-methyl-
transferase (OsNOMT), the key enzyme for sakuranetin biosynthesis, was previously unknown.
Results: We isolated OsNOMT and identified Os12g0240900 as OsNOMT.
Conclusion: Stress-induced OsNOMT regulates sakuranetin biosynthesis in rice.
Significance: Identification of OsNOMT enables the production of large amounts of sakuranetin through transgenic rice and
microorganisms.
Sakuranetin, the major flavonoid phytoalexin in rice, is
induced by ultraviolet (UV) irradiation, CuCl2 treatment, jas-
monic acid treatment, and infection by phytopathogens. It was
recently demonstrated that sakuranetin has anti-inflammatory
activity, anti-mutagenic activity, anti-pathogenic activities
against Helicobacter pylori, Leishmania, and Trypanosoma and
contributes to the maintenance of glucose homeostasis in
animals. Thus, sakuranetin is a useful compound as a plant anti-
biotic and a potential pharmaceutical agent. Sakuranetin is
biosynthesized from naringenin by naringenin 7-O-methyl-
transferase (NOMT). In previous research, rice NOMT
(OsNOMT) was purified to apparent homogeneity from UV-
treated wild-type rice leaves, but the purified protein, named
OsCOMT1, exhibited caffeic acid O-methyltransferase (COMT)
activity and not NOMT activity. In this study, we found that
OsCOMT1 does not contribute to sakuranetin production in rice
in vivo, and we purified OsNOMT using the oscomt1 mutant. A
crude protein preparation from UV-treated oscomt1 leaves was
subjected to three sequential purification steps, resulting in a 400-
fold purification from the crude enzyme preparation. Using SDS-
PAGE, the purest enzyme preparation showed a minor band at an
apparent molecular mass of 40 kDa. Two O-methyltransferase-like
proteins, encoded by Os04g0175900 and Os12g0240900, were
identified from the 40-kDa band by MALDI-TOF/TOF analysis.
Recombinant Os12g0240900 protein showed NOMT activity, but
the recombinant Os04g0175900 protein did not. Os12g0240900
expression was induced by jasmonic acid treatment in rice leaves
prior to sakuranetin accumulation, and the Os12g0240900 protein
showed reasonable kinetic properties to OsNOMT. On the basis of
these results, we conclude that Os12g0240900 encodes an
OsNOMT.
When plants are attacked by pathogenic microorganisms,
they respond with a variety of defense reactions, including the
production of secondary metabolites called phytoalexins (1),
which serve as plant antibiotics. In rice, 15 phytoalexins have
been isolated and characterized, including 14 diterpenes,
namely momilactones A and B, phytocassanes A–E, oryzalexins
A–F, and oryzalexin S (2–10), and one flavonoid phytoalexin,
sakuranetin (Fig. 1) (11). Among them, sakuranetin is consid-
ered to be one of the most biologically important phytoalexins
in terms of its high antimicrobial activity and high accumula-
tion in rice leaves infected by Magnaporthe oryzae, one of the
major photopathogenic fungi (11). In addition, it was recently
reported that sakuranetin has anti-inflammatory activity by
inhibiting 5-lipoxygenase, which is involved in arachidonic acid
metabolism in animal cells (12), anti-mutagenic activity (13),
anti-Helicobacter pylori activity by inhibiting ␤-hydroxyacyl-
acyl carrier protein dehydratase (14), and antileishmanial and
antitrypanosomal activities (15), and that sakuranetin can
induce adipogenesis of 3T3-L1 cells through enhanced expres-
sion of peroxisome proliferator-activated receptor ␥2 to con-
tribute to the maintenance of glucose homeostasis in animals
(16). Thus, sakuranetin is a useful compound as a plant antibi-
otic and a potential pharmaceutical agent.
Sakuranetin is biosynthesized from naringenin by S-adenosyl-
L-methionine (AdoMet)3-dependent naringenin 7-O-methyl-
transferase (NOMT) (Fig. 1) (17). Naringenin is the first flavonoid
transformed from naringenin chalcone by chalcone isomerase and
is also a key biosynthetic intermediate to an isoflavone and a vari-
ety of flavones. Therefore, NOMT plays a key role in sakuranetin
biosynthesis at a branching point from a common flavonoid bio-
synthetic pathway.
Sakuranetin was first identified from the cortex of the bark of
a cherry tree (Prunus spp.) as an aglycone of sakuranin (18) and
* This work was supported by the Program for Promotion of Basic Research
Activities for Innovative Biosciences.
□S
This article contains supplemental Figs. S1–S4 and Table S1.
The nucleotide sequence(s) reported in this paper has been submitted to the
DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB692949.
1 To whom correspondence should be addressed. Tel.: 81-3-5841-3070; Fax:
81-3-5841-3070; E-mail: ukokada@mail.ecc.u-tokyo.ac.jp.
3 The abbreviations used are: AdoMet, S-adenosyl-L-methionine; NOMT, nar-
ingenin 7-O-methyltransferase; COMT, caffeic acid O-methyltransferase;
OMT, O-methyltransferase; JA, jasmonic acid; RACE, rapid amplification
of cDNA ends; qRT, quantitative RT; Rubisco, ribulose-bisphosphate
carboxylase/oxygenase.
2 Present address: Dept. of Biosciences, Teikyo University, Toyosatodai,
Utsunomiya, Tochigi, Japan.
JUNE 1, 2012•VOLUME 287•NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 19315

Purification and Identification of Rice Sakuranetin Synthase
FIGURE 1. Biosynthesis of sakuranetin from naringenin.
then found in rice and several other plant species, including ing and characterization of OsNOMT as a sakuranetin synthase
Artemisia campestris, Prunus spp., Baccharis spp., Betula spp., in rice.
and Juglans spp. (19). However, NOMT has not been identified
EXPERIMENTAL PROCEDURES
from such plant species. Although an O-methyltransferase
(OMT) from barley (F1-OMT) that mainly methylates apigenin to
Chemicals—Racemic naringenin, luteolin, caffeic acid, and
form genkwanin has weak NOMT activity, sakuranetin has not ferulic acid were purchased from Kanto Chemical, Co., Inc.
been identified in barley (20). It was also reported that another (Tokyo, Japan). Racemic daidzein, biochanin A, and sinapic
OMT isolated from Streptomyces avermitilis (SaOMT-2) is able to acid were purchased from Sigma. Apigenin, kaempferol, myric-
catalyze the NOMT reaction, but SaOMT-2 has broad substrate etin, and quercetin were purchased from Wako Pure Chemical
specificity against isoflavones, flavones, and a flavanone, and its Industries (Osaka, Japan). Racemic liquiritigenin was pur-
biological function remains unknown (21).
chased from Funakoshi Co., Ltd. (Tokyo, Japan). All chemicals
Although sakuranetin is not found in healthy rice leaves, its used in matrix-assisted laser desorption/ionization two-stage
biosynthesis is rapidly induced by both biotic and abiotic time-of-flight mass spectrometry (MALDI-TOF/TOF) ana-
stresses, such as infection with phytopathogens such as M. lyses were of analytical grade. In addition, 4-sulfophenyl iso-
oryzae, Xanthomonas oryzae, and Ditylenchus angustus, infesta- thiocyanate, ␣-cyano-4-hydroxycinnamic acid, sodium bicar-
tionwithSogatellafurcifera, ultraviolet(UV)irradiation, andtreat- bonate, and ammonium bicarbonate were purchased from
ment with CuCl2 or jasmonic acid (JA) (11, 17, 22–25). Previously, Sigma.
it was reported that NOMT was purified to apparent homogeneity
Plant Materials, Growth Conditions, and Elicitation—The
(ϳ985-fold) from crude extracts of UV-treated wild-type rice oscomt1 mutant (PFG-2B-50240, Oryza sativa L. cv. Dongjin)
leaves (22). However, the amino acid sequence of the purified was searched for in the Rice Functional Genomic Express Data-
protein was highly homologous to that of a caffeic acid 3-O- base as a tDNA insertion mutant of Os08g0157500 (TIGR ID,
methyltransferase (COMT) from maize. In fact, the recombi- LOC_Os08g06100) and purchased from Postech Biotech Cen-
nant protein expressed in Escherichia coli showed COMT but ter. Because the seeds of the oscomt1 mutant we obtained were
not NOMT activity (26), and this enzyme was named from the F1 generation, the F1 generation plants were analyzed
OsCOMT1. These results suggest at least two possibilities as by genomic PCR using specific primers for OsCOMT1 and
follows: one is that OsCOMT1 is a major component and the tDNA to separate oscomt1 homozygotes, heterozygotes,
rice NOMT (OsNOMT) is a minor one in the purified fraction. and wild-type rice plants, and the F2 generation seeds were
If this is the case, isolation of OsNOMT might be quite difficult obtained from the respective plants. We used these F2 genera-
because of the masking effect by OsCOMT1. Another is that tion seeds of homozygotes and wild type as “oscomt1” and “wild
OsCOMT1 is involved in NOMT enzymatic activity in rice in type” in the experiments on the characterization of the oscomt1
vivo but that a post-translational modification and/or the pres- mutant and purification of OsNOMT. Primers used for
ence of an interacting factor is needed to show NOMT activity. genomic PCR are described in Fig. 2A and supplemental Table
In the former case, if UV-irradiated leaves of the OsCOMT1 S1. Wild-type rice (O. sativa L. cv. Nipponbare) plants were
tDNA insertion mutant oscomt1 are used as plant material, used for 5Ј- and 3Ј-RACE of OsNOMT mRNA and for analysis
OsNOMT is expected to be purified quite efficiently without of the effects of JA treatment or inoculation of M. oryzae spores
masking by OsCOMT1. In the latter case, NOMT activity will on the expression of OsNOMT and quantification of accumu-
not be induced in oscomt1 mutant leaves even after elicitation. lated sakuranetin.
In this study, we first confirmed that NOMT activity is
To grow rice plants, seeds were sterilized with 2.5% sodium
induced by elicitor treatment in the oscomt1 mutant leaves. We hypochlorite solution (Kanto Chemical) for 30 min and then
then purified and identified OsNOMT from UV-irradiated washed with sterilized water. Surface-sterilized seeds were
leaves of the oscomt1 mutant. Kinetic analysis of recombinant incubated on 0.7% agar gel containing 0.1% of the liquid fertil-
OsNOMT and expression analysis of the OsNOMT gene in rice izer Hanakoujou (Sumika-Takeda Garden Products, Tokyo,
plants were also performed. This is the first report on the clon- Japan) for 10 days at 28 °C in the light. Germinated seeds were
19316 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 23•JUNE 1, 2012

Purification and Identification of Rice Sakuranetin Synthase
FIGURE 2. Genome structure and gene expression of OsCOMT1 in the oscomt1 mutant. A, tDNA sequence is inserted 374 bp downstream of the transcrip-
tion initiation site of OsCOMT1 in the oscomt1 mutant. Black arrowheads (#1, #2, and #3) indicate the locations of primers used for genomic PCR, and white
arrowheads indicate the locations of primers used for expression analysis. B, results of genomic PCR of the oscomt1 mutant allele. C, transcriptional expression
of OsCOMT1 in wild-type rice and the oscomt1 mutant. Ubiquitin expression was used as a control.
then transplanted into a mixture of vermiculite and artificial com- (27). Naringenin and sakuranetin levels were determined with
post, Bonsol (Sumitomo Chemical, Tokyo, Japan), to grow in a combinations of the precursor and product ions of m/z 273/153
greenhouse (12 h of light at 28 °C/12 h of dark at 25 °C). Three- for naringenin and m/z 287/167 for sakuranetin in the multiple
month-old plants were used for subsequent experiments.
reaction monitoring mode. The retention times of naringenin
For UV irradiation, excised rice leaves were floated on dis- and sakuranetin were 2.5 and 3.3 min, respectively.
tilled water and incubated for 20 min at 20 cm under a UV lamp Preparation of Crude Protein Extract from Rice Leaves—All
in a biological safety cabinet. Then the irradiated leaves were steps were carried out at 4 °C unless otherwise stated. Rice
incubated at 28 °C under continuous white light conditions. For leaves were homogenized by using Polytron (Kinematica AG,
JA or CuCl2 treatment, a leaf disk (6 mm in diameter) from the Lucerne, Switzerland) with a 3-fold (v/w) volume of buffer A
uppermost leaf blades of rice plants was floated on 100 ␮l of an (0.2 M Tris-HCl (pH 8.5), containing 10% (v/v) glycerol). The
assay solution (500 ␮M of JA or CuCl2) in each well of 96-well homogenate was filtered through four layers of Miracloth (Cal-
plastic plates.
biochem), and the filtrate was centrifuged at 10,000 ϫ g for 30
Expression Analysis—RT-PCR and qRT-PCR were used to min, with the resulting supernatant used as a crude protein
determine the level of gene expression. Total RNA extracted extract.
from plant tissue using Sepasol-RNA I Super (Nacalai Tesque,
NOMT and COMT Enzymatic Activity Assays—The stan-
Inc., Tokyo, Japan) was used to synthesize cDNA by RT reac- dard assay mixture consisted of enzyme preparation (ϳ500 ng
tion using a Quantitect RT kit (Qiagen K.K., Tokyo, Japan). of protein), 300 ␮M racemic naringenin or caffeic acid, 300 ␮M
With the cDNA, RT-PCR and qRT-PCR were performed to AdoMet, and 0.1 M Tris-HCl (pH 8.5) (containing 5 mM DTT
determine the level of gene expression. For qRT-PCR, SYBR and 1 mM EDTA) in a final volume of 50 ␮l. After an 18-h
Green technology on an ABI PRISM 7300 real time PCR system incubation at 28 °C, the reaction was terminated by adding 5 ␮l
(Applied Biosystems) was used. Raw data from qRT-PCR were of 1 M HCl. The resulting products were extracted three times
analyzed using the standard curve method, and the results were with 60 ␮l of ethyl acetate, evaporated to dryness, and finally
expressed as relative mRNA values normalized to the expression dissolved in 100 ␮l of the phytoalexin extraction solvent. The
level of ubiquitin (OsUBQ). Primers used for expression analysis content of sakuranetin or ferulic acid was quantified using an
are described in supplemental Table S1.
API-3000 LC-MS/MS system. Ferulic acid levels were deter-
Extraction and Quantification of Naringenin and Sakurane- mined with combinations of m/z 195/177 in the multiple reac-
tin from Rice Leaves—Plant tissue (40–200 mg fresh weight per tion monitoring mode.
sample) was homogenized by Multi Beads Shocker (Yasui Kikai,
Purification of OsNOMT—All steps were carried out at 4 °C
Osaka, Japan) and suspended in 2 ml of phytoalexin extraction unless otherwise stated. A crude protein extract (620 ml) from
solvent (ethanol/water/acetonitrile/acetic acid, 79:13.9:7:0.1, 200 g, fresh weight, of oscomt1 mutant rice plants 48 h after UV
v/v). The sample was centrifuged at 8,000 ϫ g for 15 min at 4 °C. irradiation was applied to the sequential three-step purification
The supernatant was collected to a vial and subjected to deter- described below.
mination of phytoalexins by liquid chromatography two-stage
Ammonium Sulfate Precipitation—Well ground ammonium
mass spectrometry (LC-MS/MS), which was composed of API- sulfate powder (108.7 g; Kanto Chemical) was added to the
3000 with an electrospray ion source (AB SCIEX) and an Agi- crude protein extract, and the mixture was gently agitated for
lent 1100 HPLC instrument (Agilent Technologies) equipped 2 h. The mixture was then centrifuged at 10,000 ϫ g for 30 min.
with a PEGASIL ODS SP100 column (150 mm long, 2.1 mm in The supernatant was collected to a clean beaker; 77.5 g of
diameter; Senshu Scientific, Tokyo, Japan). The analytical con- ammonium sulfate powder was added, and the mixture was
ditions were the same as the method described by Shimizu et al. gently agitated for 2 h. After that, the mixture was centrifuged
JUNE 1, 2012•VOLUME 287•NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 19317

Purification and Identification of Rice Sakuranetin Synthase
at 10,000 ϫ g for 30 min. The supernatant was removed, and 40 Sequence tag searches were performed with the program
ml of buffer A was added to the pellet. Next, 48 ml of the MASCOT.
obtained protein solution was ultrafiltered (10-kDa cutoff) and
For identification of components of the 45- and 52-kDa
finally resolved in 12 ml of buffer A, and the resultant solution bands in SDS-PAGE (Fig. 4A) by peptide mass fingerprinting
was subjected to the subsequent purification. with MALDI-TOF, trypsin-digested proteins were mixed with
Anion Exchange on DEAE Column—The protein solution (10 a saturated solution of ␣-cyano-4-hydroxycinnamic acid in 50%
ml) was loaded to an AKTA fast protein LC (FPLC) system (GE acetonitrile containing 0.1% TFA and subjected to MALDI-
Healthcare) equipped with a pre-equilibrated Hi PrepTM 16/10 TOF analysis (Ettan MALDI-ToF Pro, Amersham Biosciences)
DEAE FF column (GE Healthcare). Buffer A was used as a pre- as described previously (29). Spectra were collected from 350
equilibration buffer. The applied proteins were washed with 50 shots per spectrum over an m/z range of 600–3000 and cali-
ml of buffer A at a flow rate of 2 ml/min. After that, a 50-min brated by a two-point internal calibration using trypsin auto-di-
linear gradient (0–60% buffer B (buffer A containing 1 M KCl)) gestion peaks (m/z 842.5099 and 2211.1046). A peak list was gen-
was applied as an elution step with a flow rate of 2 ml/min and erated using the Ettan MALDI-TOF Pro Evaluation Module
then the column was washed with 100 ml of 100% buffer B. The (version 2.0.16). The threshold used for peak picking was as fol-
eluate was collected in steps of 5 ml each, checked for NOMT lows: 5,000 for minimum resolution of monoisotopic mass, 2.5 for
enzymatic activity, and the active fractions were pooled. The signal-to-noise ratio. The search program MASCOT, developed
pooled samples were mixed together, ultrafiltered (10-kDa cut- by Matrix Science, was used for protein identification by peptide
off), and finally resolved in 6 ml of buffer A, with the product mass fingerprinting. The following parameters were used for the
used for subsequent purification.
database search: trypsin as the cleaving enzyme, a maximum of
Affinity Chromatography—Affinity chromatography on aden- one missed cleavage, iodoacetamide (Cys) as a complete modifica-
osine-agarose was performed as described previously (22). tion, oxidation (Met) as a partial modification, monoisotopic
First, 5Ј-AMP-agarose (5 ml; Sigma) was washed with distilled masses, and a mass tolerance of Ϯ0.1 Da. Peptide mass fingerprint
water and incubated with 800 units of calf intestinal alkaline acceptance criteria is probability scoring.
phosphatase (Sigma) in calf intestinal alkaline phosphatase
5Ј- and 3Ј-RACE Analysis of Os12g0240900—Because expressed
buffer (pH 9.0) in a total volume of 10 ml. The gel was then mRNA of Os12g0240900 had not been previously identified, we
dephosphorylated for 24 h at 37 °C by gentle agitation, trans- used estimated ORF information in the rice genomic sequence
ferred to an open column (15 mm inner diameter), and washed of the locus for RACE analysis. Extraction of total RNA from
with 50 ml of distilled water and then with 50 ml of buffer B. UV-irradiated wild-type rice leaves (cv. Nipponbare) was con-
Finally, the column was equilibrated with 100 ml of buffer A. ducted using Sepasol-RNA I Super (Nakalai Tesque). and the
The DEAE-purified protein solution (1 ml, ϳ18.7 mg of pro- mRNA was then purified using the Absolutely mRNATM puri-
tein) was directly applied onto the adenosine-agarose gel col- fication kit (Agilent) from the total RNA. cDNA synthesis from
umn. The column was washed first with 50 ml of buffer A and the mRNA and the subsequent 5Ј- and 3Ј-RACE analyses were
then with 50 ml of buffer B with gravity flow. Elution was done performed using the SMARTerTM RACE cDNA amplification
with 25 ml of 4 mM AdoMet in buffer B. Fractions (5 ml each) kit (Clontech). Primers used in 5Ј- and 3Ј-RACE analyses are
were collected, and the NOMT enzymatic activity of each frac- described in supplemental Table S1. The 1,548-bp sequence of
tion was checked. Active fractions were combined, ultrafiltered the obtained cDNA of Os12g0240900 was deposited in the
(10-kDa cutoff), and finally dissolved in 1.2 ml of buffer A. The DNA Data Bank of Japan (DDBJ) under accession number
resultant solution was subjected to SDS-PAGE.
AB692949.
In-gel Protein Digestion—Protein bands on SDS-polyacryl-
Cloning, Expression, and Purification of Os04g0175900 and
amide gels were enzymatically digested in-gel as described pre- Os12g0240900—Full-length ORFs of Os04g0175900 and
viously (28) using modified porcine trypsin (Promega). The Os12g0240900 were amplified by RT-PCR using KOD-FX
resultant gel pieces were washed with 50% acetonitrile and vac- Neo (TOYOBO, Tokyo, Japan) with cDNA prepared from
uum dried, then rehydrated with trypsin solution (8–10 ng/␮l CuCl2-treated wild-type rice leaves (cv. Nipponbare) as tem-
50 mM ammonium bicarbonate (pH 8.7)), and incubated for plates. To construct a directional TOPO cloning transfer
8–10 h at 37 °C.
vector, the 5Ј-CACC-3Ј sequence was incorporated into the
Identification of Purified Protein by MALDI-TOF and PCR upstream primer at its 5Ј ends. Then the PCR products
MALDI-TOF/TOF Analysis—For identification of components were inserted into the pENTR/D-TOPO vector (Invitrogen),
of the 40-kDa band in SDS-PAGE (Fig. 4A) by peptide mass and the ligated products were transformed into TOP10 chem-
fingerprinting with MALDI-TOF/TOF, in-gel digested protein ically competent E. coli. The ORFs of Os04g0175900 and
samples were analyzed using the Applied Biosystems 4700 pro- Os12g0240900 inserted into pENTR/D-TOPO vector were
teomics analyzer with TOF/TOFTM ion optics. Both MS and fused into pDEST15 (Invitrogen) using LR Clonase II enzyme
MS/MS data were acquired with a Nd:YAG laser with a 200 Hz mix (Invitrogen). Finally, we obtained expression vectors of
repetition rate, and up to 4,000 shots were accumulated for N-terminal GST tag-fused OMTs, pDEST15-1759 and
each spectrum. The MS/MS mode was operated with 1 keV pDEST15-2409, respectively.
collision energy; air was used as the collision gas such that nom-
The resulting plasmids were transformed into E. coli Rosetta
inally single collision conditions were achieved. Both MS and II (DE3) (Novagen). These strains were pre-cultured for 24 h at
MS/MS data were acquired using the instrument default cali- 37 °C in LB medium containing carbenicillin (100 ␮g/ml) and
bration, without applying internal or external calibration. chloramphenicol (34 ␮g/ml) and then cultured for 48 h at 20 °C
19318 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 23•JUNE 1, 2012

Purification and Identification of Rice Sakuranetin Synthase
in Overnight ExpressTM instant TB Medium (Novagen) con- ing 5 ␮l of 1 M HCl. The resulting products were extracted with
taining carbenicillin (100 ␮g/ml) and chloramphenicol (34 150 ␮l of ethyl acetate, and 75 ␮l of the extract was applied to a
␮g/ml). The cells were collected by centrifugation, suspended liquid scintillation counter.
in buffer A, and disrupted by mild sonication on ice. After cen-
Preparation and Inoculation of Fungal Conidia—The rice
trifugation at 10,000 ϫ g for 15 min, the supernatant was col- blast fungus M. oryzae (race 001.0 P91-15B) was kindly sup-
lected and loaded to the AKTA FPLC (GE Healthcare) plied by Dr. E. Minami (National Institute of Agrobiological
equipped with a pre-equilibrated GSTrap HP column (bed vol- Sciences). The blast fungi were grown on oatmeal agar (60 g
ume 5 ml) (GE Healthcare). Buffer A was used as the pre-equil- of homogenized oatmeal and 12 g of agar per liter of water) in
ibration buffer. Applied protein was washed with 25 ml of Petri dishes incubated at 25 °C for 9 days. Conidial suspen-
buffer A with a flow rate of 2 ml/min. Next, 25 ml of buffer A sion was prepared as described previously (30) and then fil-
containing 10 mM reduced glutathione was applied for the elu- tered through two layers of Miracloth (Calbiochem) to
tion. The purified recombinant proteins were ultrafiltered (10- remove cell debris. Conidial suspension was subjected to an
kDa cutoff) and finally resolved in 1 ml of buffer A, and that inoculation test within 2 h after preparation. For inocula-
solution was used for kinetic analysis.
tion, the excised fourth leaf blades of rice plants at the 4.5-
MS/MS Analysis of Sakuranetin Produced by NOMT Enzymatic leaf stage were sprayed with conidial suspension at a concen-
Assays—To confirm that GST-2409 product was sakuranetin, tration of 105 spores/ml. Then the inoculated leaf blades
we compared product ion spectrum of the product and authen- were incubated for 120 h at 25 °C under light conditions, and
tic sakuranetin with LC-MS/MS system. MS/MS analysis was the samples were applied for gene expression analysis and
performed with AB SCIEX TripleTOFTM 5600 System (AB quantitative analysis of sakuranetin.
SCIEX) equipped with SHIMADZU Nexera UFLC system (Shi-
RESULTS
madzu, Kyoto, Japan). Four-min gradient elution with 10–90%
acetonitrile containing 0.1% formic acid was performed with a
Characterization of oscomt1 Mutant—We obtained PFG-
CAPCELL PAK C18 IF S2 column (Shiseido Co., Tokyo, Japan). 2B-50240 (cv. Dongjin) as a tDNA insertion mutant in the
Ionization was performed with electrospray ionization and pre- first exon of Os08g0157500 (TIGR ID, LOC_Os08g06100,
cursor ions (m/z ϭ 100–1,000) and product ions (m/z ϭ OsCOMT1) (Fig. 2A) from the Rice Functional Genomic
50–1,000) were scanned with positive ion mode. The retention Express Database and selected the oscomt1 homozygote
time of sakuranetin was 2.85 min. The product ion spectrum of mutant and wild-type rice from the F2 generation of seeds
precursor ion (m/z ϭ 287.1) at the retention time of sakurane- obtained by genomic PCR (Fig. 2B). After confirmation of
tin was observed.
impaired transcriptional expression of the OsCOMT1 gene
Chiral Analysis of Sakuranetin Produced in the NOMT Enzy- in leaves of the oscomt1 mutant by RT-PCR (Fig. 2C), we
matic Assays—For chiral analysis of sakuranetin, 70 min of iso- examined the ability of the oscomt1 mutant leaves to produce
cratic elution with 30% acetonitrile containing 0.1% acetic acid sakuranetin. Excised leaf blades of oscomt1 and wild-type
(v/v/v) was performed with a Sumichiral OA-7500 column (250 rice were treated with CuCl2, and the accumulated amounts
mm long, 2 mm in diameter; Sumika Chemical Analysis Ser- of sakuranetin in the leaf blades 72 h after treatment were
vice, Tokyo, Japan) and detected by using API-3000 LC-MS/MS determined by LC-MS/MS analysis. As shown in Fig. 3A, the
system as described above. The retention times of natural and accumulation of sakuranetin in oscomt1 was comparable
unnatural sakuranetin were 54 and 61 min, respectively.
with that in the wild type. We also compared NOMT activity
Kinetic Analysis—The standard assay mixture without the and COMT activity in leaf blades of oscomt1 and the wild
enzyme was incubated at 30 °C for 5 min. The reaction was type. Crude enzyme extracts derived from rice leaves of
initiated by adding 1 pmol of the enzyme, and the reaction oscomt1 and the wild type 48 h after CuCl2 treatment were
mixture was incubated at 30 °C for 5 min. The reaction was subjected to NOMT and COMT enzymatic assays in which
terminated by adding 5 ␮l of 1 M HCl. The resulting products naringenin and caffeic acid were used as substrates, respec-
were extracted three times with 60 ␮l of ethyl acetate, evapo- tively. To determine NOMT and COMT activity, the reac-
rated to dryness, and finally dissolved in 100 ␮l of phytoalexin tion products of these assays, namely sakuranetin and ferulic
extraction solvent. The content of sakuranetin was quantified acid, were measured by LC-MS/MS. As shown in Fig. 3, B
using an API-3000 LC-MS/MS system. The kinetic parameters and C, the specific NOMT activity of oscomt1 was compara-
were derived from a Hanes-Woolf plot. The effect of substrate ble with that of the wild type, whereas the specific COMT
concentration on reaction velocity was examined at various activity of oscomt1 was severely suppressed compared with
concentrations (30, 24, 18, 12, 6, and 0 ␮M) of racemic nar- that of the wild type. This indicates that UV-irradiated
ingenin, unnatural naringenin being not a substrate for oscomt1 leaf blades are suitable plant materials for purifica-
OsNOMT. The concentration of natural naringenin was calcu- tion of OsNOMT.
lated as half of racemic naringenin.
Purification of OsNOMT from oscomt1 and MALDI-TOF-
Substrate Specificity Assay—The standard assay mixture TOF Analysis of Purified Protein—Because NOMT activity in
without AdoMet was incubated at 30 °C for 5 min. The reaction oscomt1, as already described, was comparable with that in the
was initiated by adding 45.9 pmol (1 ␮l) of S-[methyl-3H] wild type, we performed purification of OsNOMT from
adenosyl-L-methionine (ϳ444 GBq/mmol, 20.4 MBq/ml, oscomt1 leaves using a series of chromatographic steps. A crude
PerkinElmer Life Sciences), and the reaction mixture was incu- enzyme extract prepared from oscomt1 leaves 48 h after UV
bated at 30 °C for 10 min. The reaction was terminated by add- treatment was purified through three steps of ammonium sul-
JUNE 1, 2012•VOLUME 287•NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 19319

Purification and Identification of Rice Sakuranetin Synthase
FIGURE3. AccumulationofsakuranetinandNOMTandCOMTenzymaticactivityinelicitedoscomt1andwild-typericeleaves. A, accumulatedsakurane-
tin in oscomt1 and wild-type rice leaves 48 h after CuCl2 treatment was determined by LC-MS/MS. B and C, specific NOMT (B) and COMT (C) activities of crude
enzyme preparations from oscomt1 and wild-type rice leaves 48 h after CuCl2 treatment. Amounts of sakuranetin and ferulic acid extracted from the enzymatic
reaction mixture were measured by LC-MS/MS, and the specific activities were determined. The data are means Ϯ S.D. of three replicates. An asterisk indicates
significant differences compared with wild-type rice in a t test at p Ͻ 0.05.
TABLE 1
Purification of OsNOMT from UV-irradiated oscomt1 rice leaves
Purification steps
Total activity
Total protein
Specific activity
Recovery
Purification
picokatal
104.0
43.6
mg
picokatal/mg
%
-fold
Crude extract
695
0.150
100
1
Ammonium sulfate precipitation
Anion exchange (DEAE)
288
0.152
42.0
25.4
5.6
1.01
26.4
112.2
0.096
0.235
1.57
Affinity chromatography (adenosine-agarose)
5.8
60.629
405.35
fate precipitation, DEAE anion exchange chromatography, and (1,548 bp) of Os12g0240900 was amplified by RT-PCR using
adenosine-agarose chromatography. The specific activity of primer sets constructed based on the sequences of the 5Ј and 3Ј
OsNOMT and the results of SDS-PAGE analysis at each step ends and deposited in the DNA Data Bank of Japan under
are given in Table 1 and Fig. 4A, respectively. The specific activ- accession number AB692949.
ity of OsNOMT was dramatically concentrated by adenosine-
The longest ORFs of Os04g0175900 (AK104764) and
agarose chromatography, and a 40-kDa band was detected on Os12g0240900 (AB692949) were amplified by RT-PCR, cloned
SDS-PAGE after adenosine-agarose chromatography. The using pENTR/D-TOPO vector (Invitrogen), incorporated into
40-kDa band was excised from the gel and treated with trypsin, the expression vector pDEST15 (Invitrogen), and overex-
and the products of tryptic digestion were analyzed by MALDI- pressed in E. coli as N-terminal GST-tagged proteins, desig-
TOF/TOF MS. The obtained data were entered into the nated as GST-1759 and GST-2409, respectively. The crude
MASCOT Database. As a result, two putative OMTs encoded extracts containing the recombinant proteins were subjected to
by loci Os04g0175900 (TIGR ID, LOC_Os04g09654) and FPLC with a glutathione-Sepharose column (GSTrap HP,
Os12g0240900 (TIGR ID, LOC_Os12g13800) were identified Amersham Biosciences) to obtain the pure GST-1759 and
as components of the 40-kDa band (Fig. 4, B and C). The mass GST-2409. NOMT enzymatic activities of the purified recom-
spectra of these OMTs are shown in supplemental Fig. S1. binant proteins (Fig. 5A) were examined, and the Michaelis-
Although proteins of 45 and 52 kDa were also detected as major Menten kinetic parameters (Km and kcat values) were deter-
bands in the fraction after adenosine-agarose chromatography mined. As shown in Fig. 5B and supplemental Fig. S2,
(Fig. 4A), they were identified by MALDI-TOF MS as Rubisco- sakuranetin was detected as a product of the enzymatic reac-
related proteins (45-kDa protein, Rubisco large chain precursor tion catalyzed by GST-2409 but not by GST-1759. As we used
(AK058623, 40.2 kDa); 52-kDa protein, Rubisco large chain racemic naringenin as a substrate, we confirmed by using chiral
(AK105600, 52.8 kDa)).
column chromatography that sakuranetin produced by the
cDNA Cloning and Functional Identification of Os04g0175900 and NOMT enzymatic assay with GST-2409 was a natural form
Os12g0240900—To investigate whether the Os04g0175900 (supplemental Fig. S3). Km and kcat values of GST-2409 for nat-
and Os12g0240900 gene products have NOMT enzymatic ural naringenin determined by the Hanes-Woolf plot (Fig. 5C)
activity, their cDNAs were expressed as recombinant proteins were 1.9 Ϯ 0.1 ␮M and 25 Ϯ 3/s, respectively. These results
in E. coli. For preparation of the Os04g0175900 cDNA, indicate that the gene product of Os12g0240900, but not that of
sequence information of AK104764 in the RAP-DB was used. Os04g0175900, can catalyze 7-O-methylation of naringenin.
Because a full-length cDNA clone of Os12g0240900 had not We therefore designated Os12g0240900 as OsNOMT.
been previously isolated, 5Ј- and 3Ј-RACE analysis was per-
Substrate Specificity of GST-OsNOMT—It was reported that
formed using predicted ORF information from rice genomic F1-OMT and SaOMT-2 catalyze the methylation of several fla-
sequence of this gene locus. Total RNA was extracted from vonoids, including naringenin (20, 21). Therefore, we deter-
wild-type leaf blades 48 h after UV irradiation. mRNA was puri- mined the substrate specificity of OsNOMT against several
fied from the total RNA and subjected to cDNA synthesis fol- types of phenolic compounds, including flavonoids and isofla-
lowed by 5Ј- and 3Ј-RACE PCR. Finally, the full-length cDNA vonoids. The relative activity of purified GST-OsNOMT was
19320 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 23•JUNE 1, 2012

Purification and Identification of Rice Sakuranetin Synthase
FIGURE 4. Identification of O-methyltransferase-like proteins by MALDI-TOF/TOF analysis. A, Coomassie-stained SDS-PAGE of purified enzyme prepara-
tions is shown. Lane 1, crude enzyme; lanes 2–4, ammonium sulfate precipitation-, DEAE-, and adenosine-agarose affinity-purified enzymes; lane M, protein
marker. Black arrowhead indicates the 40-kDa proteins, which include OsNOMT. White arrowheads indicate the major components of Rubisco-related proteins.
B and C, amino acid sequences of O-methyltransferase-like proteins encoded by gene loci Os04g0175900 (B) and Os12g0240900 (C) obtained from the 40-kDa
band by MALDI-TOF/TOF analysis are shown. Bold italic characters indicate the fragments identified by MALDI-TOF/TOF analysis.
tested on the methylation of two flavanones (naringenin and methylation activity with racemic naringenin (100%), followed by
liquiritigenin), five flavones (apigenin, luteolin, kaempferol, kaempferol(73%), apigenin(61%), luteolin(44%), racemicliquiriti-
quercetin, and myricetin), an isoflavanone (daidzein), an isofla- genin (34%), and quercetin (30%). The methylation activity of
vone (biochanin A), caffeic acid, and sinapic acid. A mixture of OsNOMT with myricetin, isoflavones, and other phenolics was
a substrate and the purified OsNOMT was incubated with 3H-la- similar to that of the negative control.
beled AdoMet for methylation quantification. The reaction prod-
Relationship between Expression of OsNOMT and Accumu-
uct was extracted with ethyl acetate, and its 3H radioactivity was lation of Sakuranetin in Rice Leaves after JA Treatment and M.
determined by liquid scintillation counter to evaluate the substrate oryzae Infection—Accumulation of sakuranetin is induced by
specificity of OsNOMT. As shown in Fig. 6, OsNOMT preferen- treatment with elicitors, such as JA and CuCl2 in rice leaves,
tially methylated flavanones and flavones, showing the highest and CuCl2 treatment induced JA prior to accumulation of
JUNE 1, 2012•VOLUME 287•NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 19321

Purification and Identification of Rice Sakuranetin Synthase
FIGURE 5. NOMT enzymatic activity of GST-fused recombinant proteins. Crude enzyme preparations from E. coli expressing either GST-2409 or GST-1759
were purified by affinity chromatography with GSTrap HP column, and the purified preparations were applied to SDS-PAGE and NOMT enzymatic activity
assays. A, Coomassie-stained SDS-PAGE of affinity purified GST-1759 and GST-2409. (C, crude; P, purified). B, LC-MS/MS chromatograms of reaction products in
the NOMT enzymatic activity assay using either GST-2409 or GST-1759 and an authentic standard of sakuranetin. C, Hanes-Woolf plots for GST-2409 with
natural naringenin. The concentration of natural naringenin was calculated as the half of racemic naringenin. The data are means Ϯ S.E. of three replicates.
flavanone
flavone
R1
OH
OH
R2
HO
O
HO
O
naringenin
kaempferol
apigenin
a
R3
b
R
O
OH
O
b
liquiritigenin: R=H
naringenin: R=OH
apigenin: R1=R2=R3=H
luteolin:
R1=OH, R2=R3=H
luteolin
c
kaempferol: R1=R2=H, R3=OH
quercetin: R1=R3=OH, R2=H
myricetin: R1=R2=R3=OH
liquiritigenin
quercetin
cd
d
isoflavanone
O
isoflavone
myricetin
e
HO
O
HO
biochanin A
daidzein
e
e
e
e
e
OH
O
O
OCH3
OH
caffeic acid
sinapic acid
negative control
daidzein
biochanin A
others
HO
HO
COOH
CH3
O
COOH
0
20
40
60
80
100
120
HO
relative methylation activity (%)
OCH3
caffeic acid
sinapic acid
FIGURE 6. Relative methylation activities of OsNOMT against flavonoids and other phenolic compounds. Purified GST-OsNOMT was incubated with 0.3
mM of phenolic substrates and a trace amount of S-[methyl-3H]adenosyl-L-methionine. The reaction was terminated by adding 1 M HCl. The reaction products
were extracted with ethyl acetate and the incorporated methyl-3H was measured. The data are means Ϯ S.E. of three replicates. Different characters indicate
significant differences in a Tukey’s test (p Ͻ 0.05) among the activities against the substrates.
sakuranetin (17). Therefore, we performed time course ana- sakuranetin were quantitated by LC-MS/MS. The mRNA
lyses of expression levels of OsNOMT and accumulation of level of OsNOMT was transiently induced at 6 h and then
sakuranetin to further support the involvement of OsNOMT gradually decreased until 72 h after JA treatment (Fig. 7A).
in sakuranetin production in rice leaves after JA treatment. Accumulation of sakuranetin started at 6 h and continuously
For JA treatment, rice leaf disks were floated on 100 ␮M JA. increased until at least 72 h after JA treatment, when the
Then the expression levels of OsNOMT in the leaf disks were accumulation level of sakuranetin reached ϳ4 ␮g/g FW (Fig.
determined by qRT-PCR, and the endogenous levels of 7B).
19322 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 23•JUNE 1, 2012

Purification and Identification of Rice Sakuranetin Synthase
DISCUSSION
Here, we present details of the purification of the rice narin-
genin 7-O-methyltransferase OsNOMT from rice oscomt1
mutant leaves and its detailed characterization. Although a
putative OsNOMT was previously partially purified from UV-
irradiated wild-type rice leaves, as described above, a major
protein in the purified fraction showed high homology to maize
COMT, and its recombinant protein expressed in E. coli
showed COMT, but not NOMT, activity and was named
OsCOMT1 (26).
Our study indicated that sakuranetin accumulation and
NOMT enzymatic activity were similarly induced after elicita-
tion in the oscomt1 mutant and wild-type rice leaves. This
strongly suggests that OsCOMT1 is not involved in sakuranetin
production in rice. A possible reason why OsNOMT was not
detected from the purified fraction with NOMT enzymatic
activity from crude extracts of UV-treated wild-type rice leaves
in the previous study (17) may be that the more abundantly
generated OsCOMT1 exhibited similar behavior to OsNOMT
in a series of purification steps and masked the presence of
OsNOMT. In this study, purification of OsNOMT was success-
fully performed without masking by OsCOMT1 by using elic-
ited oscomt1 leaves. Among the three purification steps of
OsNOMT, adenosine-agarose chromatography is most effec-
tive, leading to a more than 400-fold enrichment of the enzyme.
This chromatography is known to be very effective in purifying
AdoMet-dependent methyltransferases (31). After this purifi-
cation step, a 40-kDa band on SDS-PAGE was detected. It was
shown that molecular masses of type 1 OMTs, whose substrates
are caffeic acid, flavonoids, coumarin, and alkaloids, are about
38–43 kDa (32), and OsNOMT and OsCOMT1 were sug-
gested to have molecular masses of around 41 kDa (22). The
40-kDa band was excised from the gel and subjected to
MALDI-TOF/TOF MS analysis after treatment with trypsin,
resulting in identification of the two OMTs as candidate pro-
teins for OsNOMT. Preparation of GST-tagged gene products
of Os04g0175900 (AK104764) and Os12g0240900 (AB692949)
FIGURE 7. Time course analysis of OsNOMT mRNA and accumulation of
sakuranetin and naringenin in JA-treated wild-type rice leaves. A, rela-
tive expression levels of OsNOMT in rice leaves from 0 to 72 h after treat-
ment with 100 ␮M JA. The mRNA levels were determined using qRT-PCR.
Each value was normalized to the OsUBQ mRNA level. The data are
means Ϯ S.D. of three experiments. B and C, accumulated amounts of
sakuranetin (B) and naringenin (C) from 0 to 72 h after treatment with 100
␮M JA were quantified using LC-MS/MS. The data are means Ϯ S.E. of three
experiments.
in E. coli followed by in vitro NOMT enzymatic assays clearly
demonstrated that Os12g0240900 encodes OsNOMT.
Kinetic analysis of recombinant GST-OsNOMT revealed
that the Km of GST-OsNOMT for naringenin was ϳ1.9 ␮M.
Conversely, the endogenous level of naringenin accumulation
was ϳ1.8 ␮M in rice leaves 48 h after JA treatment (Fig. 7C).
Taken together, the Km value was quite reasonable in under-
standing the function of OsNOMT in sakuranetin production
in rice leaves.
In addition, we found that accumulation of naringenin, a
direct precursor of sakuranetin, was transiently induced
slightly prior to accumulation of sakuranetin, peaking at ϳ0.5
␮g/g FW 48 h after JA treatment (Fig. 7C). Thus, it was indi-
cated that when rice produces sakuranetin, both the expression
of the OsNOMT gene and the supply of the substrate nar-
ingenin are induced.
Besides, accumulation of sakuranetin is also induced by M.
oryzae infection. We confirmed up-regulation of OsNOMT
expression and increase in accumulation of both naringenin
and sakuranetin in rice leaves at 5 days post-inoculation with
M. oryzae spores (supplemental Fig. S4).
Substrate specificity of OsNOMT was also determined in
this study as described by Christensen et al. (20), and the results
revealed that GST-OsNOMT showed higher methylation
activity on naringenin than the flavanone liquiritigenin and fla-
vones and no methylation activity on other phenolics, including
isoflavonoids (Fig. 6). In the case of F1-OMT in barley, the
methylation activity on apigenin was three times higher than
that on naringenin (20). In contrast, SaOMT-2 has broad sub-
strate specificity and can catalyze the methylation of isofla-
vonoids as well as naringenin (21). Sakuranetin has not been
identified in either barley or S. avermitilis. These results indi-
JUNE 1, 2012•VOLUME 287•NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 19323

Purification and Identification of Rice Sakuranetin Synthase
Kato, Y., Kitahara, Y., and Takahashi, N. (1973) Momilactones, growth
cate that the actual biological functions of F1-OMT and
SaOMT-2 are still unknown.
inhibitors from rice, Oryza sativa L. Tetrahedron Lett. 14, 3861–3864
10. Tamogami, S., Mitani, M., Kodama, O., and Akatsuka, T. (1993) Oryza-
lexin S structure. A new stemarane-type rice plant phytoalexin and its
biogenesis. Tetrahedron 49, 2025–2032
Time course analyses of OsNOMT expression and of accu-
mulation levels of sakuranetin and its direct precursor narin-
genin in rice leaves treated with JA indicate that OsNOMT
expression was induced transiently prior to accumulation of
sakuranetin, and accumulation of naringenin was also tran-
siently induced slightly prior to that of sakuranetin (Fig. 7C).
Up-regulation of OsNOMT was confirmed in the pathogen M.
oryzae-infected rice leaves in which accumulation of sakurane-
tin and its precursor naringenin was increased (supplemental
Fig. S3). These results further support that OsNOMT functions
as a sakuranetin synthase and is involved in defense responses
through elicitor-induced production of sakuranetin in rice. The
success of identification of OsNOMT as a sakuranetin synthase
in this study will enable enhancement of pathogen resistance
through regulation of the endogenous content of sakuranetin in
rice. In addition, as described in Introduction, sakuranetin is a
useful compound showing various pharmaceutical activities
(12–16). Because the fermentative production of naringenin by
microorganisms carrying an artificially assembled phenylpro-
panoid pathway has been established (33), our success in clon-
ing OsNOMT will also enable the production of a large amount
of sakuranetin by microorganisms for medical research.
11. Kodama, O., Miyakawa, J., Akatsuka, T., and Kiyosawa, S. (1992)
Sakuranetin. A flavanone phytoalexin from ultraviolet-irradiated rice
leaves. Phytochemistry 31, 3807–3809
12. Zhang, X., Hung, T. M., Phuong, P. T., Ngoc, T. M., Min, B. S., Song, K. S.,
Seong, Y. H., and Bae, K. (2006) Anti-inflammatory activity of flavonoids
from Populus davidiana. Arch. Pharm. Res. 29, 1102–1108
13. Miyazawa, M., Kinoshita, H., and Okuno, Y. (2003) Antimutagenic activity
of sakuranetin from Prunus jamasakura. J. Food Sci. 68, 52–56
14. Zhang, L., Kong, Y., Wu, D., Zhang, H., Wu, J., Chen, J., Ding, J., Hu, L., Jiang,
H., and Shen, X. (2008) Three flavonoids targeting the ␤-hydroxyacyl-acyl
carrier protein dehydratase from Helicobacter pylori. Crystal structure char-
acterization with enzymatic inhibition assay. Protein Sci. 17, 1971–1978
15. Grecco Sdos, S., Reimão, J. Q., Tempone, A. G., Sartorelli, P., Cunha, R. L.,
Romoff, P., Ferreira, M. J., Fávero, O. A., and Lago, J. H. (2012) In vitro
antileishmanial and antitrypanosomal activities of flavanones from Bac-
charis retusa DC. (Asteraceae). Exp. Parasitol. 130, 141–145
16. Saito, T., Abe, D., and Sekiya, K. (2008) Sakuranetin induces adipogenesis
of 3T3-L1 cells through enhanced expression of PPAR␥2. Biochem. Bio-
phys. Res. Commun. 372, 835–839
17. Rakwal, R., Hasegawa, M., and Kodama, O. (1996) A methyltransferase for
synthesis of the flavanone phytoalexin sakuranetin in rice leaves. Biochem.
Biophys. Res. Commun. 222, 732–735
18. Asahina, Y.(1908) Ueber das sakuranin, ein neues glykosid der rinde von Prunus
pseudo-Cerasus lindl var. sieboldi Maxim. Arch. Pharm. 246, 259–272
19. Harborne, J. B., Baxter, H., and Moss, G. P. (eds) (1999)Phytochemical
Dictionary: A Handbook of Bioactive Compounds from Plants, 2nd Ed.,
p.976, Taylor & Francis, London
Acknowledgments—We thank Dr. H. Nagasawa, Dr. S. Nagata, and
M. Kuroiwa (Graduate School of Agricultural and Life Sciences, The
University of Tokyo) for their support with purification and identifi-
cation of OsNOMT. We thank Dr. T. Kuzuyama and T. Ozaki (Bio-
technology Research Center, The University of Tokyo) for their support
with product ion analysis with LS-MS/MS of sakuranetin. We thank
Dr. E. Minami (National Institute of Agrobiological Sciences) for
sharing the culture of M. oryzae used in this study. We thank Dr.
Gynheung An (Institution and Pohang University of Science and
Technology) for providing the oscomt1 mutant.
20. Christensen, A. B., Gregersen, P. L., Olsen, C. E., and Collinge, D. B. (1998)
A flavonoid 7-O-methyltransferase is expressed in barley leaves in re-
sponse to pathogen attack. Plant Mol. Biol. 36, 219–227
21. Kim, B. G., Jung, B. R., Lee, Y., Hur, H. G., Lim, Y., and Ahn, J. H. (2006)
Regiospecific flavonoid 7-O-methylation with Streptomyces avermitilis
O-methyltransferase expressed in Escherichia coli. J. Agric. Food Chem. 54,
823–828
22. Rakwal, R., Agrawal, G. K., Yonekura, M., and Kodama, O. (2000) Naringenin
7-O-methyltransferase involved in the biosynthesis of the flavanone phytoa-
lexin sakuranetin from rice (Oryza sativa L.). Plant Sci. 155, 213–221
23. Liu, H., Li, X., Xiao, J., and Wang, S. (2012) A convenient method for
simultaneous quantification of multiple phytohormones and metabolites.
Application in study of rice-bacterium interaction. Plant Methods 8, 2
24. Plowright, R. A., Grayer, R. J., Gill, J. R., Rahman, M. L., and Harborne, J. B.
(1996) The induction of phenolic compounds in rice after infection by the
stem nematode Ditylenchus angustus. Nematologica 42, 564–578
25. Kanno, H., Hasegawa, M., and Kodama, O. (2011) Accumulation of salicylic
acid, jasmonic acid, and phytoalexins in rice, Oryza sativa, infested by the
white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae).
Appl. Entomol. Zool. 47, 27–34
REFERENCES
1. VanEtten, H. D., Mansfield, J. W., Bailey, J. A., and Farmer, E. E. (1994)
Two classes of plant antibiotics. Phytoalexins versus “Phytoanticipins.”
Plant Cell 6, 1191–1192
2. Koga, J., Shimura, M., Oshima, K., Ogawa, N., Yamauchi, T., and
Ogasawara, N. (1995) Phytocassanes A, B, C, and D, novel diterpene phy-
toalexins from rice, Oryza sativa L. Tetrahedron 51, 7907–7918
3. Koga, J., Ogawa, N., Yamauchi, T., Kikuchi, M., Ogasawara, N., and Shimura,
M. (1997) Functional moiety for the antifungal activity of phytocassane E, a
diterpene phytoalexin from rice. Phytochemistry 44, 249–253
26. Lin, F., Yamano, G., Hasegawa, M., Anzai, H., Kawasaki, S., and Kodama,
O. (2006) Cloning and functional analysis of caffeic acid 3-O-methyltrans-
ferase from rice (Oryza sativa). J. Pestic. Sci. 31, 47–53
4. Yajima, A., and Mori, K. (2000) Synthesis and Absolute Configuration of
(Ϫ)-Phytocassane D, a diterpene phytoalexin isolated from the rice plant,
Oryza sativa. Eur. J. Org. Chem. 21, 4079–4091
27. Shimizu, T., Jikumaru, Y., Okada, A., Okada, K., Koga, J., Umemura, K.,
Minami, E., Shibuya, N., Hasegawa, M., Kodama, O., Nojiri, H., and Ya-
mane, H. (2008) Effects of a bile acid elicitor, cholic acid, on the biosyn-
thesis of diterpenoid phytoalexins in suspension-cultured rice cells. Phy-
tochemistry 69, 973–981
5. Akatsuka, T., Kodama, O., Sekido, H., Kono, Y., and Takeuchi, S. (1985)
Novel phytoalexins (oryzalexins A, B, and C) isolated from rice blast leaves
infected with Pyricularia oryzae. Part I: Isolation, characterization, and
biological activities of oryzalexins Agric. Biol. Chem. 49, 1689–1694
6. Kato, H., Kodama, O., and Akatsuka, T. (1993) Oryzalexin E. A diterpene 28. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectro-
phytoalexin from UV-irradiated rice leaves. Phytochemistry 33, 79–81
7. Kato, H., Kodama, O., and Akatsuka, T. (1994) Oryzalexin F. A diterpene
phytoalexin from UV-irradiated rice leaves. Phytochemistry 36, 299–301
8. Cartwright, D. W., Langcake, P., Pryce, R. J., Leworthy, D. P., and Ride, J. P.
(1981) Isolation and characterization of two phytoalexins from rice as
momilactones A and B. Phytochemistry 20, 535–537
metric sequencing of proteins silver-stained polyacrylamide gels. Anal.
Chem. 68, 850–858
29. Fernandez, J., Gharahdaghi, F., and Mische, S. M. (1998) Routine identifi-
cation of proteins from sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) gels or polyvinyl difluoride membranes using
matrix-assisted laser desorption/ionization-time of flight-mass spectrom-
etry (MALDI-TOF-MS). Electrophoresis 19, 1036–1045
9. Kato, T., Kabuto, C., Sasaki, N., Tsunagawa, M., Aizawa, H., Fujita, K.,
19324 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 23•JUNE 1, 2012

Purification and Identification of Rice Sakuranetin Synthase
30. Koga, H. (1994) Hypersensitive death, autofluorescence, and ultrastruc-
9250–9257
tural changes in cells of leaf sheaths of susceptible and resistant near- 32. Noel, J. P., Dixon, R. A., Pichersky, E., Zubieta, C., and Ferrer, J. L. (2003)
isogenic lines of rice (Pi-zt) in relation to penetration and growth of Py-
ricularia oryzae. Can. J. Bot. 72, 1463–1477
Structural, functional, and evolutionary basis for methylation of plant
small molecules. Recent Adv. Phytochem. 37, 37–58
31. Attieh, J. M., Hanson, A. D., and Saini, H. S. (1995) Purification and charac- 33. Horinouchi, S. (2009) Combinatorial biosynthesis of plant medicinal
terization of a novel methyltransferase responsible for biosynthesis of halo-
polyketides by microorganisms. Curr. Opin. Chem. Biol. 13, 197–
methanes and methanethiol in Brassica oleracea. J. Biol. Chem. 270,
204
JUNE 1, 2012•VOLUME 287•NUMBER 23
JOURNAL OF BIOLOGICAL CHEMISTRY 19325

FIGURE S1. MALDI-TOF/TOF analysis of triptic peptides from the 40-kDa band. The MS spectrum of
precursor ions generated by MALDI (A) and MS/MS spectra of product ions that were assigned by
MASCOT to peptide fragments from the gene products of Os04g0175900 (B, C, D, E) and
Os12g0240900 (F, G, H) are shown.
FIGURE S2. MS/MS analysis of produced compound by NOMT enzymatic assay using GST-2409.
Chromatograms of precursor ions detected on m/z = 287.1 (A, upper: authentic sakuranetin, lower:
produced compound by NOMT enzymatic assay using GST-2409) and mass spectrums of product ions
detected from the major peaks (B, upper: authentic sakuranetin, lower: produced compound by NOMT
enzymatic assay using GST-2409) are shown.
FIGURE S3. Chiral analysis of natural and unnatural sakuranetin. LC-MS/MS chromatograms of
sakuranetin produced by the NOMT enzymatic activity assay using GST-2409, sakuranetin extracted
from M. oryzae infected rice leaves and an authentic standard of racemic sakuranetin are shown.
FIGURE S4. OsNOMT mRNA levels and sakuranetin accumulation in M. oryzae-infected wild-type rice
leaves. (A) The relative mRNA levels of OsNOMT in rice leaves 5 days post-inoculation (dpi) with M.
oryzae. The mRNA levels were determined using qRT-PCR. Each value was normalized to the OsUBQ
mRNA level. The data are means ± standard deviations of three experiments. (B, C) The accumulated
amounts of sakuranetin (B) and naringenin (C) in rice leaves 5 dpi with M. oryzae were quantified using
LC-MS/MS. The data are means ± standard errors of three experiments.
TABLE S1
Primers used in this paper

Supplemental FIGURE S1
A
F
B
b2 b3
a1 a2
b6
a*6
b2
a1 a2 b3
L L A S F N V V R
L L Q F F R
a1
a1
y5 y*4 y3 y2 y1
y4
y*3y*2 y1
y3 y2
100
100
50
50
y1
y2
y1
y3
y*3
b3
a2
b6
y*2
a2
b2
b3
b2
y2
y4
y*4
y3
y5
a*6
100
200
300
400
500
600
400
m/z
600
700
800
100
200
300
500
m/z
G
C
b3
b4
b6 b7
G F D A G A G V D V L V D V G G G V G A T L R
A A I Q L G L I D A L T A A A D G R
y14
y10
y2 y1
y9 y8 y7 y6
y4 y3 y2 y1
y1
y10
100
100
50
y2
50
b3
y2
y9
y14
b7
y7
y6
b6
y1
b4
y3
y4
y8
800
0
200
400
600
800
1000
1200
1400
1600
1800
300
100
200
400
500
m/z
600
700
900
m/z
D
H
b2 b3
b4
A L T A G E L V A Q L P A V D D A E A A T S V D R
AAIELGLVDELLAAAGAAVTAEELAAR
b2
y1
y10
y7
y4
y1
y5 y4 y3
y1
100
50
100
50
y4
b4
y1
y10
y3
y5
y4
y7
b3
0
200
400
600
800
m/z
1000
1200
1400
0
400
800
1200
m/z
1600
2000
2400
E
b2 b3
b5b6
V I V V D I V L P E T P K P V P E A Q N P L R
y13y12
y10
y8
y6
b2
100
50
y6
b5
b3
y12
y13
y8
b6
y10
0
400
800
1200
m/z
1600
2000
2400

Supplemental FIGURE S2
A
B
6000
150
120
90
Authentic sakuranetin
Product ion spectrum of
authentic sakuranetin
5000
4000
3000
2000
1000
287.09
m/z = 287.1
167.03
147.04
119.05
60
91.05
30
50000
40000
30000
20000
10000
0
800
700
600
500
400
300
200
100
0
Produced compound
m/z = 287.1
Product ion spectrum of
produced compound
167.03
287.09
147.04
119.05
91.05
2.0
2.5
3.0
3.5
4.0
50
100
150
200
250
300
Time (min)
m/z

Supplemental FIGURE S3
Natural
Unnatural
sakuranetin sakuranetin
16000
Racemic sakuranetin
8000
0
10000
Sakuranetin
extracted from M. oryzae
inoculated rice leaves
5000
3200
1600
0
Sakuranetin
produced by NOMT enzymatic
assay using GST-2409
35
40
45
50
55
60
65
Time, min

Supplemental FIGURE S4
A
B
C
40
40
30
20
10
0
30
20
10
0
mock
inoculated
mock
mock
30
20
10
0
inoculated
inoculated
initial
5 dpi
initial
5 dpi
initial
5 dpi

Supplemental TABLE S1
Primer sequence
Used for
Primer name
COMT1T-DNA check F
COMT1T-DNA check R
T-DNA Seq2 RB outer R
OsCOMT1 F
5'-GACAAGCTGCCGTCCAAG-3'
Genomic PCR
5'-ATGTCAGCGAGAGAGGAGGA-3'
5'-TTGGGGTTTCTACAGGACGTAAC-3'
5'-AGGTGTTCGACCATCGTCTT-3'
5'-CACCGGAATTGAACATCAAA-3'
5'-CTAGCCGGATGCATGAAAGT-3'
5'-TGCACGTATAGGCACACACA-3'
5'-GGACTGGTTAAATCAATCGTCA-3'
5'-CCATATACCACGACCGTCAAAA-3'
5'-CATGGAGAACTGGTACTACCTGAAG-3'
5'-GGTAGTACCAGTTCTCCATGAACAC-3'
5'-CACCATGGCTTCGGGCATTAGCA-3'
5'-CTACTTGCATAGTTCAATTG-3'
OsCOMT1 R
OsNOMT F
Gene expression assay
OsNOMT R
OsUBQ F
OsUBQ R
2409-3RACE-F
2409-5RACE-R
1759 pENTR ORF F
1759 ORF R
5'- and 3'-RACE
DNA cloning
2409 pENTR ORF F
2409 ORF R
5'-CACCATGGGAGACATGGTGAGCCC-3'
5'-TTACTTTGTGAACTCGAGAG-3'