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
2
.
1
. Elicitation of L. japonicus seedlings Seeds 30 seeds per culture pot of L. japonicus
accession B-129 ‘Gifu’ were sown on sterilized filter paper and grown in the dark at 25°C. Four
days after sowing, 1 ml of aqueous GSH 10 mM was added directly to seedlings under sterile condi-
tions. After elicitation, seedlings were harvested, frozen in liquid nitrogen and stored at − 80°C
until use for isolation of mRNA. Seedling exu- dates were extracted from the filter paper for 2 – 3
min in 2 ml of 99.5 ethanol at room temperature three times. The extracts were evaporated under
vacuum and dissolved in 1 ml of methanol, passed through an Ekicrodisc
®
3 filter Gelman Sciences, Japan, dried and stored at − 20°C. HPLC analy-
sis was performed using a Shim-pack CLC – ODS column 6.0 × 150 mm; Shimadzu with 55 vv
methanol and 3 vv acetic acid in H
2
O at a flow rate 1 mlmin at 40°C. The eluent was moni-
tored at 280 nm.
2
.
2
. Isolation of P
450
cDNA fragments by PCR The template cDNA was prepared from the
roots of
GSH-treated seedlings
by Dr
M. Kawaguchi Graduate School of Arts and Sci-
ences, The University of Tokyo. Degenerate oligonucleotide primers were designed from highly
conserved amino acid regions of CYP75A1, CYP93A1 and CYP93B1. The primers, 93S1 = 5-
NGCHGGNACNGAYAC-3,
93S2 = 5-TGG- GCHHTNGYNGA-3,
450AS1 = 5-YCTYTC- NGGRYKRAAYTC-3 and 450AS2 = 5-TDCC-
NGMNCCRAANG-3, were used to amplify CYP93 fragments. In addition, the primers, 81
S2 = 5-AARAGRTAYTAYGGNGRNGA-3 and 81AS2 = 5-RAANCKYTCNGGYTTRAA-3,
were used to amplify CYP81 fragments. For the amplification of CYP93 fragments, the first PCR
was performed with 93S1 and 450AS2 primers 2.5 pmol each using Taq DNA polymerase
Takara in a final volume of 20 ml according to the manufacturer’s protocol. The PCR was per-
formed at 94°C for 3 min followed by 30 cycles of 94°C for 30 s, 37°C for 30 s, 72°C for 1 min and
final extension at 72°C for 5 min. 93S2 and 450AS1 primers and 0.5 ml of the first PCR
product solution as the template were used for the second PCR nested-PCR under the same condi-
tions except that the annealing temperature was 43°C. The cDNA fragments obtained were named
Lj-1, Lj-3 and Lj-4. For the amplification of CYP81 fragments, PCR was performed with 81S2
and 81AS2 primers under the same conditions as the second PCR, and Lj-2 was obtained. PCR
products were ligated into a plasmid vector pT7Blue T-Vector Novagen and sequenced using
a LIC-4000 DNA sequencer Aloka.
2
.
3
. Rapid amplification of cDNA ends
3
- and
5
-RACE For 3- and 5-RACE, an adaptor sequence was
ligated to the double-strand cDNA derived from
GSH-treated seedling roots using a Marathon™ cDNA Amplification Kit Clontech. Four specific
primers were designed based on the Lj-1 and Lj-2 sequences,
93RS1 = 5-AGGCTTGTTGACGA- ATCAGATA-3, 93RAS1 = 5-TTCCAGTATT-
TTGGGTCTCTTTGC-3, 81rS1 = 5-GCCACA- ACTTTCAAGCCAGAGA-3 and 81rAS1 = 5-
ACTGCAGCAACTCGGTCACCAT-3. Primer
sets 0.5 pmol each for 3-RACE were 93RS1 or 81rS1 and adaptor primer AP1 5-CCATC-
CTAATACGACTCACTATAGGGC-3, Mara-
thon™ cDNA Amplification Kit, Clontech. 93R AS1 or 81rAS1 and AP1 were used for 5-RACE.
Reaction conditions were as described in the man- ufacturer’s manual.
2
.
4
. Cloning of full length P
450
cDNAs Two sets of specific primers containing BamHI,
XhoI, KpnI or XbaI sites shown in bold type were designed to obtain full length DNAs,
93BamHI = 5-AATTCAAGGATCCAGCAAAC- ACCA-3 and 93Xho = 5-AAACCCTCGAGCA-
CAAAGCAACAT-3 for Lj-1; 81 + Kpn = 5- AATGGTACCATGGATATCATCTCCTTCCTT-
3 and 81 − Xba = 5-CTCACTCTAGAAACAT- GTCCCCGATTCAAA-3 for Lj-2. PCR was
performed using KOD DNA polymerase Toy- obo in a final volume of 20 ml according to the
manufacturer’s protocol. The protocol for the am- plification of full length Lj-1 was 98°C for 1 min
followed by 25 cycles of 98°C for 15 s, 55°C for 10 s, 74°C for 30 s and final extension at 74°C for
10 min. The amplification of full length Lj-2 was performed as described above except that anneal-
ing temperature was altered to 60°C. The 5-ends of PCR products ca.1.5 kbp were phosphory-
lated with T4 polynucleotide kinase Takara and ligated into a dephosphorylated EcoRV site of
pBluescript II SK- Stratagene. These clones were named LjCYP-
1
and LjCYP-2, respectively.
2
.
5
. Cloning of LjIFR cDNA Degenerate PCR was carried out with primers
designed from known IFR amino acid sequences, IFRS1 = 5-AARGCHATHAARGARGC-3 and
IFR AS1 = 5 - ATCXCCYTTDATYTGYTG - 3, and subsequently 3- and 5-RACE were per-
formed using IFRRS1 = 5-TGTTGCCATGC- CTTCACTGGTT-3 and IFRRAS1 = 5-CCA-
ATCTTCTTCTTCCCACAATGC-3. PCR condi- tions were the same as described in ‘Isolation of
P
450
cDNA fragments by PCR’ and ‘Rapid am- plification of cDNA ends’. Annealing temperatures
were altered to 43 and 68°C, respectively. Based on their sequences, specific primers containing
BamHI or XhoI sites shown in bold type, were designed to amplify full length IFR cDNA:
IFRBam2 = 5-GTTCTGGATCCTGATGGCAC- CACAAGACAG-3, IFRXhoI = 5-TCACGAG-
AGGACAGGCTCGAGTAACAAACA-3.
PCR was carried out with the same procedure as de-
scribed in ‘Cloning of full length P
450
cDNAs’. Annealing temperature was altered to 57°C for
LjIFR amplification.
2
.
6
. Heterologous expression in yeast cells, preparation of microsomes and enzyme assay
The P450 cDNAs were digested with BamHI and XhoI or KpnI and XbaI and ligated into
corresponding sites of pYES2 Invitrogen to con- struct the expression plasmid vector pYESLjCYP-
1 and pYESLjCYP-2. Introduction of these plasmids into Saccharomyces cere6isiae strain
BJ2168, selection of transformants, induction of P450 proteins and preparation of microsomes were
performed as described [23]. The assays of LjCYP- 1 and LjCYP-2 proteins were performed as de-
scribed [24,28].
2
.
7
. mRNA preparation and re6erse transcription RT
-PCR analysis mRNA
was isolated
from elicitor-treated
seedlings fresh weight 0.2 g using Straight A’s mRNA isolation system Novagen, and 500 ng
each of mRNAs were used to synthesize single- strand cDNAs. For RT-PCR, the same primers as
described in ‘Cloning of full length cDNAs’ were used for amplification of IFS, I
2
H and IFR. RT-PCR was carried out with 0.5 pmol each of
specific primers using Taq DNA polymerase in a final volume of 20 ml according to manufacturer’s
protocol. The reaction was performed at 95°C for 1 min followed by 24 cycles of 95°C for 30 s,
57°C for 30 s, 72°C for 1 min and a final exten- sion at 72°C for 5 min. The products 5 ml were
subjected to electrophoresis on 1.2 agarose gel and stained with ethidium bromide.
2
.
8
. Southern blot analysis Genome DNAs were isolated from seedlings of
L. japonicus accession B-129 ‘Gifu’ using the cetyltrimethylammonium
bromide CTAB
method. Genome DNAs 10 mg each were di- gested with DraI, EcoRI, HindIII or XbaI. The
restricted fragments were separated by elec- trophoresis on 0.7 agarose gel and transferred to
Nylon membranes, positively charged Roche. Hybridization probes were prepared by a random
priming method using DIG-11-dUTP Roche. Hybridization and wash were done according to
the DIG hybridization protocol Roche.
3. Results
