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
3
.
1
. Accumulation of 6estitol in elicited L. japonicus seedlings
GSH is the most abundant thiol compound in living cells. In higher plants, GSH is assumed to
play a role in protection against oxidative damage. GSH stimulates the activation of defense re-
sponses and the production of phytoalexins [29]. In birdsfoot trefoil Lotus corniculatus, a te-
traploid relative of L. japonicus, isoflavan phy- toalexins, vestitol and sativan, are induced to
accumulate in response to inoculations with fungi [30]. After treatment with GSH, vestitol and sati-
van also accumulated in Agrobacterium rhizoge- nes-transformed L. corniculatus root cultures
[31,32]. Thus, in this study, GSH was used as an elicitor.
Fig. 2 shows the effect of GSH treatment on the composition of the materials released from L.
japonicus seedlings. The accumulation of vestitol was clearly observed in the exudate of seedlings 10
h after the GSH treatment. The amount of vestitol exuded 10 h after GSH-treatment was estimated to
be 37.7 ng per seedling 5.14 mgg fresh weight. Levels of vestitol accumulation 10 and 20 h after
GSH-treatment were constant data not shown. Other
isoflavonoid compounds
were not
identified.
3
.
2
. Cloning of cDNAs encoding isofla6onoid biosynthesis
To isolate P450 cDNAs, the PCR strategy was employed using GSH-treated L. japonicus seedling
Fig. 2. HPLC profile of exudates of L. japonicus seedlings. After 10 h elicitation with 10 mM GSH, an isoflavan vestitol,
which was not detected in the control H
2
O treatment, had accumulated.
roots. Six degenerate oligonucleotide primers were designed from highly conserved regions of known
plant P450s of the flavonoid pathway Fig. 3. Nested-PCR with 93S2 and 450AS1 primers us-
ing the product of the first PCR with 93S1 and 450AS2 primers as the template gave primer-spe-
cific fragments. Sequence analysis revealed that three clones were similar to known plant P450s.
These clones were named Lj-1, Lj-3 and Lj-4, and their sequences were about 78, 80 and 85 identi-
cal at the nucleotide level to CYP93C1, CYP73A trans-cinnamate 4-hydroxylase and CYP98A2,
respectively. PCR with 81S2 and 81AS2 primers amplified a 700 bp fragment of expected size.
Sequence analysis revealed that this fragment showed 73 identity to CYP81E1 at nucleotide
level. This fragment was named Lj-2.
Two sets of specific primers were designed based on the Lj-1 and Lj-2 sequences, and 3- and
5-RACE were performed. 3-RACE with Lj-1 and Lj-2 yielded fragments of about 830 and 700 bp,
respectively, each containing the stop codons and 3-non-coding sequences. Similarly, 5-RACE with
Lj-1 and Lj-2 yielded fragments of about 1000 and 730 bp containing initiation codons.
Using two sets of re-designed specific primers and the cDNA of the roots of GSH-treated
seedlings as the template, two full length P450 sequences, LjCYP-1 and LjCYP-2, were cloned.
LjCYP-1 contains an ORF consisting of 1554 bp encoding 518 amino acids, and showed 83.6
identity at the amino acid level to Glycyrrhiza echinata CYP93C2 [28] see Fig. 4A. The ORF of
LjCYP-2 consists of 1497 bp 499 amino acids with 82.2 identity at the amino acid level to G.
echinata CYP81E1 [24] see Fig. 4B. The deduced amino acid sequences of LjCYP-1 and LjCYP-2
displayed the consensus sequence FWSGN HXGDXRHPTXCLIVMFAPGA
D of P450s for the heme-binding region. LjCYP-1 and LjCYP-2 proteins were named CYP93C17
and CYP81E6, respectively, by P450 Nomencla- ture Committee.
Full length cDNA of putative IFR was also obtained from GSH-treated seedlings and named
LjIFR. The deduced amino acid sequence of LjIFR is 77.4, 79.6, 81.1 and 83.0 identical to
soybean, pea [33], chickpea [34] and alfalfa [35] IFR cDNAs, respectively.
3
.
3
. Catalytic functions of LjCYP-
1
and LjCYP-
2
proteins To examine the catalytic functions of LjCYP-1
and LjCYP-2 proteins, the cDNAs were expressed in yeast cells.
After induction, the microsome of the recombi- nant yeast expressing LjCYP-1 was incubated with
RS-liquiritigenin and NADPH, and the ethyl acetate extract of the reaction mixture was ana-
lyzed by reverse-phase HPLC. As shown in Fig. 5A, three peaks, P1, P2 and P3, were observed.
These peaks did not appear in the control using the vector without the insert. The Rt’s of P1 and
P3 were identical to those of 2,7,4-trihydroxyisofla- vanone and 7,4-dihydroxyisoflavone daidzein,
respectively, and P2 to putative 3-hydroxyliquiriti- genin. These compounds have recently been iden-
tified in the reaction with CYP93C2 protein IFS of G. echinata [28]. On acid treatment of the ethyl
acetate extract, the P1 peak decreased whereas the P3 peak increased. Furthermore, the chemical struc-
ture of P3 was confirmed to be daidzein by electron impact mass spectrometry data not shown. There-
fore, it was concluded that LjCYP-1 encodes IFS.
The microsome of yeast cells transformed with LjCYP-2 was incubated with formononetin and
NADPH, and the reaction product was analyzed by HPLC. As a result, a new peak that was not
observed in the control reaction appeared, and its retention time was the same to that of 2-hydroxy-
formononetin Fig. 5B. The identity of the reac- tion
product was
further confirmed
by co-chromatography. Therefore, LjCYP-2 protein
was shown to be I2H.
3
.
4
. Expression of genes encoding enzymes of the isofla6onoid pathway
mRNA was
isolated from
elicitor-treated seedlings harvested at different times after elicita-
tion. For RT-PCR, the quantity of each template was adjusted to give roughly equal amplification
of actin cDNA. As shown in Fig. 6, the transcript levels of IFS, I2H and IFR all increased 10 h after
elicitation. Only a low level of transcription was observed in untreated seedlings. The transcripts
had decreased to the control level by 20 h. The transient accumulation of mRNAs of these P450s
and IFR in the GSH-treated seedlings indicated a coordinated expression of essential enzymes of
vestitol biosynthesis upon elicitation.
3
.
5
. Genomic organization of isofla6onoid biosynthetic genes
Genomic DNA was digested with several re- striction enzymes, and Southern blot analysis was
performed using the P450 ORFs as probes Fig. 7. LjCYP-
1
has one restriction site for EcoRI and no restriction sites for DraI, HindIII or XbaI.
With this probe, one strong band from each EcoRI or XbaI-digested DNA, two bands from
Fig. 3. Cloning strategy of P450 cDNAs by PCR. Six degen- erate oligonucleotide primers were designed from conserved
regions of P450s of the flavonoid pathway.
Fig. 4. Amino acid sequence alignments of LjCYP-1 and LjCYP-2. Gaps - are inserted to optimize the alignments. Positions with identical amino acid residues in both sequences are in reverse type. A Comparison of the LjCYP-1 deduced amino acid sequence
with that of the G. echinata CYP93C2, AB023636 [28], G. max CYP93C1v2, AF135484 [37], mung bean AF195806 [39], pea AF195812 [39] and red clover AF195810 [39] IFS sequences. LjCYP-1 exhibited 80.9 and 83.6 identity to CYP93C1v2 and
CYP93C2, respectively. B Comparison of the LjCYP-2 deduced amino acid sequence with that of the G. echinata CYP81E1, P93147 I2H sequence [24]. 410 of the 499 amino acids are identical 82.2.
HindIII-digested DNA, respectively, were ob- served. In addition, 2 weak bands appeared with
EcoRI-digested DNA, and 1 weak band was ap- parent with XbaI-digested DNA.
On the other hand, using LjCYP-2 as a probe, only one strong band was observed in each lane
except for DraI-digested DNA. Because LjCYP- 2 has no restriction sites for these four enzymes,
two bands in DraI lane may be due to the pres- ence of a restriction site in the intron sequence.
These results
indicate that,
in the
L. japonicus genome, two or three copies of the IFS
gene are present while only one copy of I2H gene is.
4. Discussion
