genin 5,7,4-trihydroxyflavanone to sakuranetin is induced in UV-irradiated rice leaves Fig. 1
[10]. Furthermore, it has been shown that not only JA, but also some JA amino acid conjugates cause
the induction of NOMT and subsequent accumu- lation of sakuranetin [11].
Numerous O-methyltransferases
have been
purified from plant tissues and cell suspension cultures [12,13]. However, most of the purification
protocols are time consuming and require large amounts of starting materials. In this study, the
purification and partial characterization of NOMT from UV-irradiated rice leaves is reported. More-
over, a rapid microscale purification procedure is used to obtain the purified enzyme efficiently, us-
ing affinity and gel filtration chromatography. The enzyme was also purified from JA- and CuCl
2
- treated rice leaves, and the induction of NOMT
shown to be time dependent, providing a good correlation with sakuranetin accumulation. This is
the first report of the purification of a phytoalexin- specific O-methyltransferase from rice, and may be
of particular interest in the context of elucidating the mechanism of resistance against the devastat-
ing rice blast disease caused by the blast fungus Magnaporthe
grisea anamorph,
Pyricularia oryzae.
2. Materials and methods
2
.
1
. Plant material Rice plants Oryza sati6a L. Hitomebore were
cultivated and UV-irradiated as previously de- scribed [5]. Leaves were stored at − 80°C until
analysis for NOMT activity and purification. For treatment with JA and CuCl
2
, rice leaves were wounded as described previously [6] and floated in
sterile Petri dishes on solutions of JA and CuCl
2
0.5 mM, respectively.
2
.
2
. Chemicals Sakuranetin was purchased from Roth Karl-
sruhe, Germany.
Naringenin, apigenin,
kaempferol, caffeic
acid and
polyvinyl- polypyrrolidone PVPP high molecular weight,
Product Number P 6755 were obtained from Sigma St. Louis, USA. Daidzein, luteolin and
genistein were
from Extrasynthe`se
Genay, France. S-[
14
C]adenosyl-
L
-methionine 48 mCi mmol was purchased from Moravek Biochemicals
Inc. Brea, USA. Adenosine 5-monophosphate AMP agarose cross-linked 4 beaded agarose
with C-8 attachment and a nine-atom spacer, Product Number A 1271 and S-adenosyl-
L
-me- thionine SAM were from Sigma, and Superdex
75 from Pharmacia Biotech Uppsala, Sweden. Calf intestinal alkaline phosphatase was purchased
from Takara Shuzo Co. Ltd. Tokyo, Japan. The protein dye reagent, TEMED, ammonium persul-
fate, and bisacrylamide were all from Bio-Rad Richmond, USA. All chemicals were of reagent
grade.
2
.
3
. Preparation of adenosine-agarose affinity gel Essentially, the protocol of Attieh et al. [14] was
followed for preparing the affinity matrix with the following modifications. 5-AMP-agarose 5 ml
was washed with distilled water and incubated with 800 U calf intestinal alkaline phosphatase
and 1 ml calf intestinal alkaline phosphatase buffer 10 × pH 9.0 in a total volume of 10 ml.
The gel was dephosphorylated for 24 h at 37°C in a continuously rotating reaction vial, transferred
to a column, and washed first with distilled water and then with 10 ml buffer B 0.02 M Tris – HCl
pH 7.8, 10 glycerol, 1 mM ethylenediamine tetraacetic acid EDTA, and 14 mM 2-mercap-
toethanol containing 2 M NaCl, finally being equilibrated with 100 ml buffer B only.
Fig. 1. Biosynthesis of sakuranetin from naringenin.
2
.
4
. Extraction of plant material All steps were carried out at 4°C unless other-
wise stated. UV-Irradiated rice leaves 1 g were homogenized in a mortar and pestle with 4 ml
buffer A 0.2 M Tris – HCl buffer pH 7.8, con- taining 14 mM 2-mercaptoethanol, 5 mM EDTA,
10 wv glycerol and 10 wv PVPP in the presence of 0.1 g sea sand. The homogenates were
centrifuged at 18 500 × g for 5 min, and the result- ing supernatant filtered through a 50 mM nylon
mesh. The filtrate was assayed for NOMT enzyme activity and protein concentration, and subse-
quently used for NOMT purification.
2
.
5
. Affinity chromatography on adenosine-agarose The filtrate was directly applied onto an
adenosine-agarose column 0.7 × 15 cm that had been previously equilibrated with buffer B. The
column was washed with 50 ml buffer B at a constant flow rate of 18 mlh, then with 50 ml of
the same buffer containing 0.2 M KCl. The NOMT was selectively eluted with 25 ml of 4 mM
SAM in buffer B 0.2 M KCl. Fractions 1.75 ml were collected and assayed for NOMT activity and
protein content.
2
.
6
. Gel filtration on Superdex
75
The fraction from the affinity column contain- ing most of the NOMT activity was loaded on a
Superdex 75 gel filtration column 3 × 30 cm pre- viously equilibrated with buffer B. Proteins were
eluted in the same buffer at a flow rate of 18 mlh, and 3.5 ml fractions were collected, assayed for
NOMT enzyme activity and protein content. The purified enzyme from this step was used for partial
characterization and amino acid sequencing.
2
.
7
. Molecular mass estimation The native molecular mass of NOMT was esti-
mated by gel filtration chromatography by high- performance liquid chromatography HPLC on a
TSKGEL SW G 3000 XL TOSOH gel filtration column 7.8 × 30 cm calibrated with the following
markers: glutamate dehydrogenase 290 000, lac- tate dehydrogenase 142 000, enolase 67 000,
adenylate kinase 32 000, and cytochrome C 12 400, and by 13.5 native-polyacrylamide gel
electrophoresis PAGE under nondenaturing con- ditions. The protein was eluted with the following
buffer: 0.1 M sodium phosphate containing 0.1 M sodium sulphate pH 7.0 at a flow rate of 1.0ml.
The molecular mass was estimated by sodium dodecyl sulfate SDS-PAGE under denaturing
conditions [15] using 13.5 polyacrylamide gels calibrated with molecular mass standards in the
range 14.4 – 94 kDa LMW Electrophoresis cali- bration kit; Pharmacia Biotech. Gels were fixed in
20 TCA for 1 h and stained with Coomassie Brilliant Blue R 250 for 1 h. Destaining occurred
in acetic acid:methanol:distilled water 1:1.5:2 overnight or until background staining was no
longer visible.
2
.
8
. N-Terminal sequencing and microsequencing For N-terminal amino acid sequencing, the
purified NOMT was subjected to N-terminal amino acid sequencing on an Applied Biosystems
494 protein sequencer Perkin Elmer, Foster City, USA. Edman degradation was performed accord-
ing to the standard program supplied by Applied Biosystems. For microsequencing analysis, NOMT
purified by gel filtration was concentrated using Centricon-30 Amicon, Beverly, USA, desalted
2 × 500 ml Milli Q water; Nihon Millipore Ltd., Tokyo, Japan and dried in a centrifugal vacuum
concentrator. The dried NOMT approximately 100 mg was dissolved in 200 ml of 1 CNBr in
70 formic acid. After incubation for 20 h in the dark at ambient temperature [16], the sample was
dried as already described, run on a Tricine-SDS- PAGE 16.5 T, 3 C gel [17] and electroblotted
onto a PVDF membrane Fluorotrans, Pall, Tokyo, Japan. The stained peptides were cut out
and placed directly into the cartridge for sequenc- ing on an Applied Biosystems 494 protein se-
quencer.
Selective hydrolysis
of NOMT
approximately 100 mg was performed using formic acid, which cleaves at the aspartic acid
residue. Briefly, a 75 formic acid solution was prepared in Milli Q water, and 200 ml of this
solution was added to the dried protein in an Eppendorf tube. The sample was mixed on a
vortex mixer, incubated for 48 h at 37°C in a heating block, dried as already described and
stored at 4°C until use for separation of peptides by Tricine-SDS-PAGE.
Table 1 Purification of naringenin 7-O-methyltransferase NOMT from UV-irradiated rice leaves
Purification steps Total activity
Total protein Purification
Specific activity Recovery
mkatmg x-fold
mkat mg
5.03 8.9
0.56 100
Crude 1
homogenate 1.84
0.011 Adenosine-
167.3 36.6
296.1 agarose
0.006 556.7
Gel filtration 66.4
3.34 985.2
2
.
9
. Other assays Methyltransferase activity was assayed as previ-
ously described [10]. Protein was determined ac- cording to the method of Bradford [18] using
Bio-Rad protein assay dye reagent and bovine serum albumin as standard.
3. Results and discussion
