Plant Science 155 2000 213 – 221
Naringenin 7-O-methyltransferase involved in the biosynthesis of the flavanone phytoalexin sakuranetin from rice Oryza sati6a L.
Randeep Rakwal
a,
, Ganesh Kumar Agrawal
a
, Masami Yonekura
b
, Osamu Kodama
c
a
United Graduate School, Tokyo Uni6ersity of Agriculture and Technology, Tokyo, Japan
b
Food Function Laboratory, School of Agriculture, Ibaraki Uni6ersity, Ibaraki, Japan
c
Laboratory of Phytochemical Ecology, School of Agriculture, Ibaraki Uni6ersity, Ibaraki, Japan Received 23 August 1999; received in revised form 8 February 2000; accepted 10 February 2000
Abstract
An inducible S-adenosyl-
L
-methionine:naringenin 7-O-methyltransferase NOMT catalyzing the methylation of naringenin to sakuranetin, a major rice phytoalexin was purified approximately 985-fold from ultraviolet UV-irradiated rice leaves. The
enzyme is not found in healthy tissues and was purified to a nearly homogeneous preparation in one step using adenosine-agarose affinity chromatography, with 1 g rice leaves UV-irradiated as starting material. Gel filtration chromatography resulted in an
almost pure enzyme, as evidenced by a major band migrating to a position corresponding to a molecular mass of approximately 41 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified NOMT was strongly inhibited by Mn
2 +
, Ni
2 +
, Cu
2 +
, Zn
2 +
, Hg
2 +
, and Cd
2 +
, and to a low degree by Co
2 +
, Mg
2 +
, Ba
2 +
, Ca
2 +
and ethylenediamine tetraacetic acid. The amino acid sequence of a NOMT cyanogen bromide CNBr-cleavage peptide was highly homologous to that of a caffeic acid
3-O-methyltransferase from maize, and about 70 of the amino acid sequence was obtained after sequencing the peptides generated by CNBr andor formic acid hydrolysis. NOMT was also shown to be induced in a time-dependent manner, and
purified from rice leaves treated with jasmonic acid and copper chloride. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Naringenin 7-O-methyltransferase; Oryza sati6a; Sakuranetin; Phytoalexin; Affinity chromatography; Jasmonic acid www.elsevier.comlocateplantsci
1. Introduction
Plants possess inducible defence mechanisms against pathogen attack that include the produc-
tion of phytoalexins. These are low molecular weight, antimicrobial compounds that are both
synthesized by, and accumulate in, plants after their exposure to microorganisms, as well as to
chemical and physical stress [1 – 4]. Sakuranetin 5,4-dihydroxy-7-methoxyflavanone is a major
rice phytoalexin accumulating both in ultraviolet UV irradiated [5] and blast infected [5], as well as
in copper chloride CuCl
2
or jasmonic acid JA, treated rice leaves [6]. It has been suggested that
flavonoids, in general, have a function in the pro- tection of plants against UV light and other
stresses [7]. Numerous flavonoids possess methoxy groups at different positions of the A and B rings,
as well as at position 3 of the heterocycle. O- Methyltransferases that are involved in the biosyn-
thesis of flavonoids have been described previously [8,9]. Recently, it was reported that a methyltrans-
ferase
naringenin 7-O-methyltransferase
NOMT catalyzing the methylation of narin-
Abbre6iations
:
CuCl
2
, copper chloride; CNBr, cyanogen bromide; JA, jasmonic acid; NOMT, naringenin 7-O-methyltransferase; SDS-
PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; UV, ultraviolet.
Corresponding author. Present address: Department of Molecu- lar Genetics, National Institute of Agrobiological Resources, Kan-
nondai 2-1-2,
Tsukuba 305-8602,
Ibaraki, Japan.
Tel.: +
81-298-387490; fax: + 81-298-387032. E-mail address
:
ranabr.affrc.go.jp R. Rakwal 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 2 2 3 - 5
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
