Plant Science 160 2000 37 – 47
Induction of isoflavonoid pathway in the model legume Lotus japonicus: molecular characterization of enzymes involved in
phytoalexin biosynthesis
Norimoto Shimada, Tomoyoshi Akashi, Toshio Aoki, Shin-ichi Ayabe
Department of Applied Biological Sciences, Nihon Uni6ersity, Fujisawa, Kanagawa
252
-
8510
, Japan Received 3 May 2000; received in revised form 1 August 2000; accepted 1 August 2000
Abstract
Treatment of the seedlings of Lotus japonicus, a model legume for molecular genetic studies, with reduced glutathione GSH resulted in the accumulation of an isoflavan phytoalexin, vestitol. Using PCR strategies based on the conserved amino acid
sequences, full length P450 cDNAs were obtained from GSH-treated seedling roots. When the clones, LjCYP-1 CYP93C family and LjCYP-2 CYP81E family, were heterologously expressed in yeast, the proteins exhibited 2-hydroxyisoflavanone synthase
IFS and isoflavone 2-hydroxylase I2H activities, respectively. The transcription levels of LjCYP-1, LjCYP-2 and isoflavone reductase, which are all involved in vestitol biosynthesis, coordinately increased upon elicitation. Genomic Southern blot analysis
indicated that the IFS gene forms a small gene family and a single copy of the I2H gene is present in the L. japonicus genome. Molecular biological aspects of P450s involved in the isoflavonoid pathway and the genomic approach to flavonoid metabolism
in this unique plant are discussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Lotus japonicus; Fabaceae; Cytochrome P450; Isoflavonoid; Vestitol www.elsevier.comlocateplantsci
1. Introduction
Flavonoids possess a basic C
6
C
3
C
6
skeleton derived from a phenylpropanoid C
6
C
3
and three C
2
units. Extensive biosynthetic modifications bring about remarkably diverse structures to
which multiple physiological functions are at- tributed. Like the widely occurring anthocyanin
pigments in vascular plants, some flavonoids act as screens from harmful UV-light and scavengers of
reactive oxygen species [1]. Major ‘polyphenols’, which are alleged to be beneficial to human health,
are flavonoids [2,3]. Leguminous flavonoids are very important in
the interactions of the producer plants with envi- ronmental organisms [4,5]. Isoflavonoids, a dis-
tinct class of flavonoid with rearranged C
3
parts, are distributed almost exclusively in this family;
they are typical legume phytoalexins active in the defense against phytopathogenic organisms [6].
Flavonoids are also important in the symbiotic relationship of leguminous plants with soil bacte-
ria to establish nitrogen-fixing root nodules. In the initial
stage of
nodulation, recognition
of flavonoids by NodD proteins of specific Rhizobia
leads to activation of other nod genes [7,8]. Ex- pression of genes encoding enzymes of the
flavonoidisoflavonoid pathway in developing al- falfa root nodules has also been reported [9]. The
functions of flavonoids in the symbiosis with ar-
Abbre6iations
:
GSH, reduced glutathione; IFS, 2-hydroxyisofla- vanone synthase; I2H, isoflavone 2-hydroxylase; IFR, isoflavone
reductase; P450, cytochrome P450; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription-polymerase chain reac-
tion. The nucleotide sequence data reported in this paper have been
deposited in the DDBJ, EMBL and GenBank databases under the following
accession numbers:
LjCYP-1, AB024931;
LjCYP-2, AB025016
Corresponding author. Tel.: + 81-466-843703; fax: + 81-466- 801141.
E-mail address
:
ayabebrs.nihon-u.ac.jp S.-i. Ayabe. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0168-94520000355-1
bascular fungi [10] and in the defense against the herbivores have also been suggested [5]. There is,
thus, a strong interest in the mechanism of the discrimination between pathogenic and symbiotic
interactions with environmental organisms by the host plant cells and their responses, in which
flavonoids may play crucial roles.
The biosynthetic scheme of flavonoids as well as their physiological functions described above is as
complicated as other pathways of secondary metabolism. A possible regulation network in
flavonoid metabolism may exist for the maximal adaptation of the plant to its environment. Thus,
there should be organ-specific and temporally reg- ulated expression of the genes required for the
construction of specific structural classes of flavonoids in response to pathogen attack and
during the establishment of symbiotic relation- ships with microorganisms. Such a total view of
secondary metabolism would be explored best in model plants in which genome organization has
been clarified and a genetic approach through, for example, positional cloning and functional charac-
terization of genes by sense- and antisense-sup- pression is possible. Genetic studies by searching
mutants and identifying responsible genes would also be beneficial. In Arabidopsis thaliana, a non-
legume model plant, several mutants of flavonoid biosynthesis have been described, and genetic
analysis of these has greatly contributed to the understanding of flavonoid physiology and bio-
chemistry [11]. Of course, A. thaliana cannot be the model plant for the investigation of legume-
specific phenomena, i.e. symbiotic nitrogen-fixa- tion and isoflavonoid biosynthesis.
In 1992, Lotus japonicus Regel Larsen, a non- agricultural species, was proposed as a model
plant that has beneficial traits for classical and molecular genetics of the legume family [12]. It is
a diploid with the chromosome number n = 6 and has a small genome size and a short genera-
tion time. Also, it is self-fertile, and transforma- tion by Agrobacterium infection is possible. The
utility of this plant for fundamental studies has been demonstrated [13]. So far, for investigation of
the mechanisms that underlie the nitrogen fixation and nodulation, analysis of the symbiotic mutants
that were induced by the chemical mutagen ethyl- methane sulfonate, T-DNA insertion and transpo-
son tagging has been performed [14 – 17]. Genes specifically expressed during nodule development
were also identified [18,19]. However, only few studies have been made of flavonoids and their
biosynthesis in L. japonicus.
As shown in Fig. 1, a cytochrome P450 P450, 2-hydroxyisoflavanone synthase IFS, catalyzes
the hydroxylation associated with aryl migration of flavanone to form the isoflavonoid skeleton
[20 – 22]. Another P450, isoflavone 2-hydroxylase I2H, is essential in the biosynthesis of biologi-
cally active compounds such as isoflavan and pte- rocarpan
phytoalexins. Therefore,
for the
Fig. 1. Biosynthesis of isoflavan phytoalexins, vestitol and sativan. P450 enzymes involved are emphasized in bold letters. The final steps of vestitol biosynthesis are still unknown. Abbre6iations
:
CHI, chalcone isomerase; CHS, chalcone synthase; IFD, 2-hydroxyisoflavanone dehydratase; IFS, 2-hydroxyisoflavanone synthase; I2H, isoflavone 2-hydroxylase; IFR, isoflavone
reductase; IOMT, isoflavonoid-O-methyltransferase; PKR, polyketide reductase.
investigations of the leguminous plant responses to various environmental factors, focusing attention
on the P450s that catalyze critical steps of isoflavonoid biosynthesis would be a promising
avenue.
Recently, cDNAs
of P450s
of the
flavonoid pathway have been cloned, e.g. 2S- flavanone 2-hydroxylase F2H [23], I2H [24] and
dihydroxypterocarpan 6a-hydroxylase
D6aH [25], in addition to flavonoid 3- and 3,5-hydroxy-
lase F3H and F35H [26,27]. These P450s are classified as aromatic hydroxylases F3H, F35H
and I2H and non-aromatic hydroxylases F2H and D6aH. F2H and D6aH belong to the CYP93
family. Because IFS catalyzes the hydroxylation of non-aromatic carbon and its substrate is flavanone
as well as F2H, the cloning of IFS cDNA by PCR based on amino acid sequences conserved in the
CYP93 family was feasible. In addition to P450s, isoflavone reductase IFR a NADPH-dependent
reductase is also critical in isoflavonoid biosyn- thesis, and its cDNA has been cloned from several
leguminous plant species. IFR catalyzes the reduc- tion of the heterocyclic ring of isoflavone to form
isoflavanone, which is an intermediate of ptero- carpan and isoflavan synthesis.
In this paper, we first describe the examination of the exudate from L. japonicus seedlings elicited
with reduced glutathione GSH and the finding of the induction of isoflavonoid biosynthesis. Then,
we present the molecular cloning of cDNAs in- volved in isoflavonoid biosynthesis in L. japonicus,
their genomic organization, and the effect of GSH on transcription of these genes.
2. Materials and methods
