Flavone synthase II (CYP93B16) from soybean (Glycine max L.)

Article abstract of DOI:10.1016/j.phytochem.2010.01.007

Flavonoids are a very diverse group of plant secondary metabolites with a wide array of activities in plants, as well as in nutrition and health. All flavonoids are derived from a limited number of flavanone intermediates, which serve as substrates for a variety of enzyme activities, enabling the generation of diversity in flavonoid structures. Flavonoids can be characteristic metabolites, like isoflavonoids for legumes. Others, like flavones, occur in nearly all plants. Interestingly, there exist two fundamentally different enzymatic systems able to directly generate flavones from flavanones, flavone synthase (FNS) I and II. We describe an inducible flavone synthase activity from soybean (Glycine max) cell cultures, generating 7,4′-dihydroxyflavone (DHF), which we classified as FNS II. The corresponding full-length cDNA (CYP93B16) was isolated using known FNS II sequences from other plants. Functional expression in yeast allowed the detailed biochemical characterization of the catalytic activity of FNS II. A direct conversion of flavanones such as liquiritigenin, naringenin, and eriodictyol into the corresponding flavones DHF, apigenin and luteolin, respectively, was demonstrated. The enzymatic reaction of FNS II was stereoselective, favouring the (S)- over the (R)-enantiomer. Phylogenetic analyses of the subfamily of plant CYP93B enzymes indicate the evolution of a gene encoding a flavone synthase which originally catalyzed the direct conversion of flavanones into flavones, via early gene duplication into a less efficient enzyme with an altered catalytic mechanism. Ultimately, this allowed the evolution of the legume-specific isoflavonoid synthase activity.

Full text of DOI:10.1016/j.phytochem.2010.01.007

Phytochemistry 71 (2010) 508–514
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Flavone synthase II (CYP93B16) from soybean (Glycine max L.)
Judith Fliegmann a,1, Katarina Furtwängler a,
Georg Malterer a, Corrado Cantarello a, Göde Schüler b, Jürgen Ebel a, Axel Mithöfer b,
*
a Department Biology I, Ludwig-Maximilians University, Botany, Menzinger Str. 67, 80638 München, Germany
b Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, 07745 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Flavonoids are a very diverse group of plant secondary metabolites with a wide array of activities in
plants, as well as in nutrition and health. All flavonoids are derived from a limited number of flavanone
intermediates, which serve as substrates for a variety of enzyme activities, enabling the generation of
diversity in flavonoid structures. Flavonoids can be characteristic metabolites, like isoflavonoids for
legumes. Others, like flavones, occur in nearly all plants. Interestingly, there exist two fundamentally dif-
ferent enzymatic systems able to directly generate flavones from flavanones, flavone synthase (FNS) I and
II. We describe an inducible flavone synthase activity from soybean (Glycine max) cell cultures, generating
7,40-dihydroxyflavone (DHF), which we classified as FNS II. The corresponding full-length cDNA
(CYP93B16) was isolated using known FNS II sequences from other plants. Functional expression in yeast
allowed the detailed biochemical characterization of the catalytic activity of FNS II. A direct conversion of
flavanones such as liquiritigenin, naringenin, and eriodictyol into the corresponding flavones DHF, apige-
nin and luteolin, respectively, was demonstrated. The enzymatic reaction of FNS II was stereoselective,
favouring the (S)- over the (R)-enantiomer. Phylogenetic analyses of the subfamily of plant CYP93B
enzymes indicate the evolution of a gene encoding a flavone synthase which originally catalyzed the
direct conversion of flavanones into flavones, via early gene duplication into a less efficient enzyme with
an altered catalytic mechanism. Ultimately, this allowed the evolution of the legume-specific isoflavonoid
synthase activity.
Received 6 November 2009
Received in revised form 22 December 2009
Accepted 13 January 2010
Available online 3 February 2010
Keywords:
Glycine max L.
Leguminosae
Enzyme characterization
Enantiomer selectivity
Flavonoids
Flavone synthase II
7,40-Dihydroxyflavone
Liquiritigenin
CYP93B16
Coronalon
12-Oxo-phytodienoic acid
JA, Jasmonic acid
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
The crucial reaction in the biosynthesis of flavonoids is the conden-
sation of one molecule of p-coumaroyl-CoA with three molecules
Flavonoids represent a huge and diverse group of secondary
plant metabolites which can be found in all vascular plants and
in various tissues (Harborne and Baxter, 1999). In plants, these
compounds have various functions in physiology, biochemistry,
and ecology. For example, flavonoids are involved in UV-protec-
tion, flower coloration, interspecies interaction, and plant defence.
In addition, flavonoids are useful tools in phylogenetic studies. Due
to the antioxidant or putative anticancer activities of certain flavo-
noids, they also have nutritional values and medicinal benefits to
humans (Dixon and Steele, 1999; Martens and Mithöfer, 2005).
of malonyl-CoA to a chalcone intermediate. Flavonoids are derived
from this intermediate after conversion of the chalcone to the cor-
responding flavanone. Flavanones display the basic structures for
all other flavonoid-classes, including flavanols, anthocyanidines,
flavonols, flavones, and isoflavones (Harborne and Baxter, 1999;
Martens and Mithöfer, 2005). The biosynthesis of flavones from
flavanones involves the introduction of a double bond between
C2 and C3 in the heterocyclic ring of the flavanone skeleton
(Fig. 1). Double bond formation can be catalyzed by two different
flavone synthase proteins (FNS I and FNS II), a unique feature with-
in the flavonoid biosynthesis. FNS I, a soluble 2-oxoglutarate- and
Fe2+-dependent dioxygenase (Britsch, 1990), is mainly described
for Apiaceae, and has only recently been identified from monocot-
yledonous plants as well (Kim et al., 2008). In contrast, FNS II, a
membrane-bound cytochrome P450 monooxygenase, which re-
quires NADPH and molecular oxygen (Heller and Forkmann,
1993), is widespread among higher plants (Martens and Mithöfer,
2005). All FNS II proteins known belong to the plant cytochrome
P450 subfamily CYP93B. However, their catalytic mechanisms
Abbreviations: FNS, flavone synthase; 12-OPDA, 12-oxo-phytodienoic acid; DHF,
7,40-dihydroxyflavone; RACE, rapid amplification of cDNA ends; JA, jasmonic acid;
F2H, flavanone 2-hydroxylase; IFS, 2-hydroxyisoflavanone synthase (isoflavonoid
synthase).
* Corresponding author. Tel.: +49 (0)3641 571263; fax: +49 (0)3641 571256.
E-mail address: amithoefer@ice.mpg.de (A. Mithöfer).
1
Present address: UMR 5546 CNRS/Université Paul Sabatier Toulouse III, Pôle de
Biotechnologie Végétale, 24 Chemin de Borde Rouge, BP42617 Auzeville, 31326
Castanet-Tolosan, France.
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.01.007

J. Fliegmann et al. / Phytochemistry 71 (2010) 508–514
509
Table 1
R2
FNS I and FNS II activities in soybean cell cultures treated with coronalon. Soybean
cell cultures were treated for 96 h with 2.5 M coronalon. Soluble and microsomal
OH
4‘
OH
l
B
proteins were extracted and used for FNS I and FNS II specific enzyme assays with
liquiritigenin as substrate. DHF as product was determined and quantified by HPLC to
calculate the specific enzyme activities. For both, control and induced cell cultures,
three independently treated cell samples were pooled for analysis.
HO
7
2
HO
O
O
O
A
5
C
3
R1
O
Enzyme
Enzyme activity (pkat mg proteinꢀ1
)
Flavanone
R1
R2
Flavone
Control
Induced
FNS I
FNS II
n.d.
0.12
n.d.
1.83
Liquiritigenin
Naringenin
Eriodictyol
H
H
H
7,4’-Dihydroxyflavone
Apigenin
OH
OH
FNS, flavone synthase; n.d., not detected.
OH
Luteolin
Fig. 1. Flavone formation catalyzed by flavone synthase II.
(control showed 0.13 nmol DHF g FWꢀ1), 12-OPDA at a concentra-
tion of 34 M induced the production of up to 85 nmol and 10
l
lM
coronalon (Schüler et al., 2004) induced about 40 nmol DHF g
might differ from each other. On the one hand, various recombi-
nant proteins heterologously expressed from cloned FNS II-encod-
ing genes showed a direct conversion of flavanones into the
corresponding flavones, such as the FNS II from Gerbera hybrids
(CYP93B2) (Martens and Forkmann, 1999), Antirrhinum majus
(CYP93B3), Torenia hybrida (CYP93B4) (Akashi et al., 1999), and
Perilla frutescens (CYP93B6) (Kitada et al., 2001). On the other hand,
for CYP93B1 from Glycyrrhiza echinata (Akashi et al., 1998) and
CYP93B10 and 11 from Medicago truncatula (Zhang et al., 2007), a
2-OH-flavanone intermediate was suggested that might be further
converted, enzymatically or chemically, into flavones. Thus, the
latter enzymes can be classified as flavanone 2-hydroxylases.
Here, we show the isolation and heterologous expression of a
novel, oxylipin-induced FNS II from soybean (Glycine max) as well
as the accumulation of its main product, 7,40-Dihydroxyflavone, in
oxylipin-treated soybean tissues. The biochemical characterization
indicates a direct and stereoselective formation of the flavone from
the flavanone substrate. CYP93B16 from soybean represents there-
fore the first FNS II known from a leguminous plant which is capa-
ble to catalyze the conversion of flavanones directly into flavones.
FWꢀ1, respectively. Subsequent dose–response analyses revealed
EC50 values of 7
respectively.
lM for 12-OPDA and of 0.33 lM for coronalon,
2.2. Identification of GmFNS II cDNA from soybean cell cultures
In order to analyse the properties of the enzyme responsible for
the inducible accumulation of DHF in soybean cells, a cDNA encod-
ing a putative FNSII was isolated as described in Section 4.4. The
cDNA showed an open reading frame of 1581 bp corresponding
to 527 amino acid residues (Fig. S2). Northern blotting experiments
were used to monitor the time course of transcript accumulation
for the putative FNS II in soybean cell cultures in comparison to
genes encoding ‘‘early” and ‘‘late” enzymes of the inducible phe-
nylpropanoid metabolism. Both, the ‘‘early” chalcone synthase, as
well as the ‘‘late” dihydropterocarpan 6a-hydroxylase (CYP93A1),
were transiently induced by coronalon at 12 and 24 h, respectively.
Transcripts cross-hybridizing with the isolated putative cDNA for
FNS II showed to accumulate at even later time points, which coin-
cided with the observed late accumulation of DHF (Fig. S1). The de-
duced amino acid sequence of the cDNA was 56.6% and 49.7%
identical to CYP93B2 from Gerbera hybrids and CYP93B1 from G.
echinata, respectively. The encoded protein was therefore classified
as a new member of the CYP93B subfamily, CYP93B16 (D. Nelson,
pers. communication).
2. Results and discussion
2.1. Flavone synthesis in soybean cell cultures
The recent release of the first chromosome-scale assembly of
the soybean genome (http://www.phytozome.net/soybean; Soy-
bean Genome Project, DoE Joint Genome Institute) allowed a first
glance on the genomic organization of the gene encoding
CYP93B16. A transcript corresponding to the GmFNS II cDNA is pre-
dicted on chromosome 12 and was named Glyma12g07190.1
(Fig. S2). A duplication of this gene with 95% identical nucleotide
positions in the protein coding sequence is located 6200 base pairs
further downstream on chromosome 12 (Glyma12g07200.1). Both
genes contain one intron each, highly divergent in sequence and
size (Fig. S2). Interestingly, only the gene corresponding to the
cloned cDNA encoding CYP93B16 seems to be expressed, since
no transcripts corresponding to the second gene can be retrieved
from the databases (Fig. S2, and data not shown).
Flavones are a large class of plant secondary metabolites which
exhibit many different biological functions, such as, for example,
antioxidants or UV-protectants, or in plant–microbe interactions
(Martens and Mithöfer, 2005). They are synthesized from flavanon-
es, either directly via the action of flavone synthases (either type I
or type II), or indirectly via C-glycosylated intermediates (Brazier-
Hicks et al., 2009; Martens and Mithöfer, 2005). Soybean cell
cultures were reported to accumulate the flavone 7,40-dihydroxyf-
lavone (DHF) after treatment with coronalon, a synthetic jasmo-
nate analogon (Schüler et al., 2004; Fig. S1). For osmotic stress,
the induction of a FNS II has already been described (Kochs and
Grisebach, 1987; Kochs et al., 1987). In order to identify the under-
lying enzymatic activity for the generation of DHF by coronalon, in-
duced soybean cell cultures were analyzed for both flavone
synthase activities known in plants, FNS I and FNS II. As reported
in Table 1, only FNS II activity was detected, which was shown to
be enhanced 15-fold by coronalon treatment. FNS I activity could
not be found.
2.3. Heterologous expression and biochemical characterization of
CYP93B16
As coronalon might mimic both jasmonic acid (JA) as well as its
biosynthetic precursor 12-oxo-phytodienoic acid (12-OPDA)
(Mithöfer et al., 2005), we compared the efficiency of these three
compounds to induce DHF accumulation after 48 h in soybean cell
To determine and prove the enzymatic activity of the putative
FNS II from soybean, the open reading frame encoding CYP93B16
was inserted into the pYeDP60 vector (Urban et al., 1990). The re-
combinant protein was expressed in a yeast strain that co-ex-
presses the yeast NADPH-cytochrome P450 reductase gene
(Saccharomyces cerevisiae W(R)) (Latunde-Dada et al., 2001; Truan
cultures. Whereas JA at a concentration of 100
lM was a compar-
atively weak inductor, resulting in only about 8 nmol DHF g FWꢀ1

510
J. Fliegmann et al. / Phytochemistry 71 (2010) 508–514
et al., 1993). To characterize the metabolite formed by CYP93B16,
microsome suspensions of recombinant yeast cells were incubated
with the flavanone liquiritigenin and NADPH. The HOAc/EtOAc ex-
tract of the reaction mixture was analyzed by RP-HPLC (Fig. 2) and
the only product formed was identified as DHF with authentic
samples and by mass spectroscopy. No product was detected from
microsomes isolated from yeast cells transformed with the empty
vector. The enzymatic reaction was dependent on the presence of
NADPH, with a pH optimum at pH 7.9 and a temperature optimum
at 25 °C. Using these optimized conditions, the substrate efficiency
of GmFNS II for three different flavanones, liquiritigenin, naringe-
nin, and eriodictyol was determined by quantifying the production
of their corresponding flavones, DHF, apigenin, and luteolin,
respectively. The enzyme was able to accept all substrates with
Recombinant CYP93B6 from P. frutescens exhibited Km values of
8.8 M for naringenin and 11.9 M for eriodictyol (Kitada et al.,
2001). In all cases, the enzymes converted naringenin more effi-
l
l
ciently than the other tested flavanones.
Within the CYP93B subfamily, two groups of enzymes have
been characterized: one group that directly converts the flavanone
substrates into the corresponding flavone products without any
free intermediate; and a second group, where the enzymes act as
flavanone 2-hydroxylases (CYP93B1 from G. echinata and
CYP93B10 and 11 from M. truncatula). In this latter group, the
mechanism of the final conversion of the 2-hydroxylated flavanon-
es into flavones is still unknown. In the case of heterologously ex-
pressed CYP93B1 from G. echinata, the 2-hydroxyflavanone
products were converted to flavones in vitro only on acid treat-
ment, suggesting the involvement of an additional plant enzyme,
a dehydratase, for the formation of the flavones in vivo (Akashi
et al., 1998). Zhang et al. (2007) assumed that an endogenous yeast
dehydratase was responsible to form flavones from 2-hydroxyflav-
anones when FNS II-1 (CYP93B10) and FNS II-2 (CYP93B11) from
M. truncatula were expressed in yeast cells. The novel CYP93B16
isolated from soybean directly converted the substrates into the
corresponding flavones. Acid treatment was not necessary for the
formation of the flavone products nor could any by-product, repre-
senting a 2-hydroxyflavanone, be detected in the experiments
performed.
affinities in the low
lM range (Km between 1.8 lM (eriodictyol)
and 4.2 M (liquiritigenin); Fig. S3; Table 2). The efficiency to con-
l
vert the different flavanones into the corresponding flavones (rel.
Vmax Kꢀm1) was about two times higher for naringenin than for
liquiritigenin and eriodictyol, respectively (Table 2). These values
in the low lM range are similar to those available for the few re-
combinant FNS II enzymes, for which quantitative assays have
been described up to now. Recombinant FNS II from Gentiana trifl-
ora (CYP93B13) exhibited Km values of 8.9 and 19.1 lM with
naringenin and eriodictyol, respectively (Nakatsuka et al., 2006).
LIQ
(A)
(R)- LIQ
DHF
(S)- LIQ
(R)- LIQ
(B)
1
2
3
4
5
6
7
8
9
10
DHF
Time (min)
(S) -LIQ
Fig. 2. HPLC analysis of liquiritigenin conversion in the FNS II assay. Upper part:
standards of liquiritigenin (LIQ) and 7,40-dihydroxyflavone (DHF). Lower part:
extracts of reaction mixtures after incubation of liquiritigenin and NADPH with a
microsomal fraction from yeast cells expressing CYP93B16.
8
9
10
11
12
13
Time (min)
Table 2
Substrate specificity of GmFNS II.
Fig. 3. HPLC analysis of (R)- and (S)-liquiritigenin enantiomers after conversion in
the FNS II assay. (A) Separation of (R)- and (S)-liquiritigenin (LIQ) on a chiral
resolution column. (B) Separation of the reaction mixture after incubation of (R)-
and (S)-liquiritigenin and NADPH with a microsomal fraction from yeast cells
expressing CYP93B16. In (A) and (B), the same initial amounts of (R)- and (S)-
liquiritigenin were used.
ꢀ1
Mꢀ1
)
Flavanone
rel. Vmax
Km (lM)
rel. V
K
(l
max
m
Liquiritigenin
Naringenin
Eriodictyol
100
116
47
4.2
2.5
1.8
23.8
46.4
26.1

J. Fliegmann et al. / Phytochemistry 71 (2010) 508–514
511
To investigate the enantiomer selectivity of the GmFNS II-cata-
2.4. Phylogeny of the CYP93B subfamily
lyzed reaction, a chiral resolution column, Chiralpak IA, was em-
ployed in HPLC analysis. This column was able to separate the
(S)- and (R)-enantiomers of liquiritigenin (Fig. 3A). Only the (S)-
enantiomer was accepted as substrate (Fig. 3B; Table 3). Interest-
ingly, CYP93B1 from G. echinata, which in fact is a flavanone 2-
hydroxylase, showed the same selectivity for the (S)-enantiomer
(Akashi et al., 1998). Increasing concentrations of (R)-liquiritigenin,
compared to (S)-liquiritigenin, in mixtures of both forms inhibited
the conversion rate of the soybean enzyme for the (S)-form (Table
3). This result suggested the competition of both enantiomers at
the same binding site of FNS II. Taken together, these results clearly
indicate that the isolated cDNA from soybean encodes a functional
FNS II.
CYP93B16 from soybean represents the first FNS II character-
ized from a leguminous plant which is capable to directly form
the flavone from a flavanone precursor. This direct mode of conver-
sion was up to now only reported from Asterales, Lamiales, and
Gentianales, whereas the studies on enzymes from the Fabales
(G. echinata CYP93B1 and M. truncatula CYP93B10 and 11) identi-
fied them to be flavanone 2-hydroxylases (F2H) rather than flavone
synthases. The biochemical diversity is mirrored by the phyloge-
netic analysis of the subfamily CYP93B (Fig. 4). A clear bifurcation
separates the flavanone 2-hydroxylases (CYP93B1, 10, and 11)
from all other members of the CYP93B subfamily, for all of which
the direct conversion can be predicted, if not experimentally
shown already. A CYP93 protein containing flavone synthase activ-
ity has been considered to be the ancestor of the present day CYP93
family (Sawada and Ayabe, 2005), which, in addition to FNS and
F2H, includes 2-hydroxyisoflavanone synthase (IFS, CYP93C) and
pterocarpan 6a-hydroxylase (CYP93A) (Sawada and Ayabe, 2005;
Schopfer et al., 1998). Strikingly, a deletion of eight amino acid res-
idues correlates with the disability of the F2H enzymes to intro-
duce the double bond between C2 and C3 of the 2-hydroxylated
flavanone to form flavones directly (Fig. S4); however, this finding
needs to be confirmed experimentally. A gene duplication event
early in the evolution of Leguminosae was postulated (Sawada
and Ayabe, 2005), which resulted in the generation of enzymes
Table 3
Enantiomer selectivity of GmFNS II.
Liquiritigenin
FNS II activity (
l
kat kgꢀ1
)
Conversion (%)
(R)
(S)
n.d.
n.d.
9.3
8.9
8.4
4.6
14.2 1.1
13.6 0.3
12.8 0.2
7.01 0.8
(R) + (S) (1:1)
(R) + (S) (2:1)
(R) + (S) (6:1)
FNS, flavone synthase; n.d., not detected.
Fig. 4. Phylogenetic analysis of the CYP93B subfamily. The DNA parsimony method was applied on a multiple sequence alignment after exclusion of unaligned overhanging
positions (see Fig. S4). Bootstrap values for 1000 replicates are indicated on the consensus tree shown. The boxes group those species for which the enzymatic activity was
reported according to the final product of the enzymatic conversion of flavanones to either 2-hydroxyflavanone or flavone. For the description of the species included in the
analyses, see Table S2. CYP93A1 (D6aH) from Glycine max served as an outgroup.

512
J. Fliegmann et al. / Phytochemistry 71 (2010) 508–514
capable to hydroxylate C2, which is one precondition for the rather
unusual aryl migration forming isoflavonoids, a branch of flavo-
noids present predominantly in leguminous plants, as shown so
far. However, the parent was preserved during evolution as well,
as shown by the ancient, FNS-type reaction of CYP93B16 in
soybean.
(NADPH-cytochrome P450 reductase) upon galactose induction
(Truan et al., 1993) and was provided by Rhône-Poulenc Agro
(Lyon, France).
4.3. HPLC analysis
Interestingly, M. truncatula is up to now the only leguminous
plant for which ancient (FNS) as well as modern (F2H) CYP93B
activities can be predicted. CYP93B10 and 11 have been shown
to be flavanone 2-hydroxylases (Zhang et al., 2007), whereas
CYP93B12 clusters unambiguously with FNS in the phylogenetic
reconstruction (Fig. 4). Moreover, according to the Medicago Gene
Expression Atlas (Benedito et al., 2008), the expression pattern of
CYP93B10/11 versus CYP93B12 seems to be mutually exclusive,
with the former being expressed in roots and nodules and the lat-
ter being expressed mainly in the aerial parts of the plant. These
data might indicate that, in a given tissue, always only one type
of FNS II reaction will be performed, the reason for which would
be interesting to study in the model legume M. truncatula.
Organic residues were dissolved in 200 ll EtOH and analyzed by
HPLC (Nova-Pak C18, 4
l
m, 4.6 ꢁ 150 mm (Waters); flow rate
1 ml minꢀ1; linear gradient either 40–75% (v/v) MeOH in 7 min
for the separation of liquiritigenin and DHF, or 25–75% (v/v) MeOH
in 18 min) and monitored at 330 nm.
4.4. Cloning of CYP93B16 cDNA, expression in yeast, and phylogenetic
analysis
The isolation of a cDNA encoding a putative FNS II was per-
formed in multiple steps. First, a fragment of 352 bp (pos. 411–
763, Fig. S2) was amplified from genomic DNA of soybean using
degenerated oligonucleotides cons93b-f1/r1 (provided by S. Mar-
tens; see supplemental Table S1 for all oligonucleotides used). By
comparing the cDNAs encoding CYP93B enzymes from G. echinata,
Gerbera hybrids, A. majus, T. hybrida, Callistephus chinensis, and P.
frutescens, versus those encoding CYP93A enzymes from soybean
(see Table S2 for all accessions used), a degenerated oligonucleo-
tide (cons93b-r2) was designed which enabled the distinction of
the closely related gene families in soybean. It was employed, in
conjunction with the oligonucleotide gm93b-f2, derived from the
first partial soybean DNA sequence, in a RT-PCR reaction using
RNA isolated from soybean cell cultures treated for 60 h with
3. Concluding remarks
Recently, flavone synthases were shown to be involved in
mutualistic as well as parasitic interactions of M. truncatula
(Uppalapati et al., 2009; Zhang et al., 2007, 2009). RNAi-mediated
silencing of CYP93B10 and 11 strongly reduced both, the amount of
7,40-dihydroxyflavone and the level of nodulation in M. truncatula
roots, which could be restored by exogenous application of DHF.
This effect was independent of a pretreatment of the rhizobia with
nod-gene inducing flavones, which suggested a dual role of flav-
ones in the rhizobial symbiosis of M. truncatula (Zhang et al.,
2007, 2009). DHF might play a role during the infection of M. trun-
catula roots with the necrotrophic fungus Phymatotrichopsis omni-
vora as well since flavone synthase II (CYP93B10/11) was found to
be highly induced early during the interaction (Uppalapati et al.,
2009). Interestingly, ethylene and jasmonic acid signalling path-
ways were activated during the interaction as well, emphasizing
our observation that JA-related compounds are potent elicitors of
DHF production in soybean cell cultures. Whether, in soybean, flav-
ones are involved in disease and symbiosis, as it is obviously the
case in M. truncatula, needs to be investigated.
2.5 lM coronalon. The resulting fragment of 919 bp overlapped
over 97 bp with the first fragment (Fig. S2). RACE (rapid amplifica-
tion of cDNA ends; (Frohman et al., 1988)) was used to isolate the
missing 50 and 30 regions of the cDNA, by using RNA isolated from
soybean cell cultures, elicited with coronalon for 96 h. For 50RACE
(Mithöfer et al., 2000) the gene-specific primers GSP1 (for reverse
transcription), GSP2, and adaptor primers bsh–t17 and bsh were
used (Fliegmann and Sandermann, 1997). 30RACE was performed
accordingly using the gene-specific oligonucleotide GSP3 in combi-
nation with bsh after reverse-transcription with bsh-t17. The open
reading frame encoding CYP93B16 was amplified using the oligo-
nucleotide pair gm93b-f3/r3 and proof-reading Pfu-polymerase
(Promega), subcloned after adenylation into pGEM-T (Promega),
and sequenced. Using the restriction sites inserted at the 50 and
the 30-ends, the open reading frame was released and inserted into
appropriately prepared pYeDP60. Yeast was transformed, grown
and induced for protein synthesis at 30 °C for 2 days in SC/gal Uraꢀ
medium as described (Fliegmann et al., 2005).
4. Experimental
4.1. General experimental procedures
12-Oxo-phytodienoic acid (12-OPDA) was purchased from Cay-
man Chemicals, Hornby, Canada. 6-Ethyl-1-oxo-indanoyl isoleu-
cine methyl ester (coronalon) was synthesized as described
(Schüler et al., 2001). Naringenin (5,7,40-trihydroxyflavanone), eri-
odictyol (5,7,3040-tetrahydroxyflavanone), apigenin (5,7,40-trihydr-
oxyflavone), and luteolin (5,7,30,40-tetrahydroxyflavone) were
from the lab collection, liquiritigenin (7,40-dihydroxyflavanone)
and 7,40-dihydroxyflavone (DHF) from Extrasynthèse, Genay,
France.
An alignment of all 15 open reading frames encoding CYP93Bs
(including the corrected version of CYP93B12 of M. truncatula,
see supplemental Fig. S5) and one encoding CYP93A1 from soybean
was generated by using the Webinterface Multalin (Corpet, 1988).
50 and 30-non overlapping regions of the alignment were removed,
guided by an amino acid sequence alignment (Fig. S4). The trun-
cated nucleic acid sequence alignment was fed into the phylogeny
interference package version 3.66 (Felsenstein, 2004) and was used
to estimate the phylogeny by running seqboot (1000 replicates,
random number seed 4533), dnapars (random number seed
4533, 33 jumbles, 1818 trees generated), and consense. The con-
sense tree was drawn by using TreeView 1.6.6 (Page, 1996) and
the bootstrap values were added manually according to the output
file from consense.
4.2. Biological materials
Soybean (G. max L. cv. Harosoy 63) cell suspension cultures were
grown as described (Hille et al., 1982). Five-day-old cultures were
used for induction studies, extracted and the samples prepared as
described (Fliegmann et al., 2003). The plasmid pYeDP60 (provided
by D. Pompon, Gif-sur-Yvette, France) (Urban et al., 1990) was used
for recombinant protein synthesis in S. cerevisiae strain W(R). This
strain had previously been engineered to express the yeast CPR
4.5. Enzyme assays
For protein extraction, treated and non-treated cells were
harvested at the time indicated and separated from the medium.

J. Fliegmann et al. / Phytochemistry 71 (2010) 508–514
513
Bradford, M.M., 1976.
A
rapid and sensitive method for the quantitation of
Soybean cells (8 g), frozen in liquid nitrogen, were homogenized by
mortar and pestle and resuspended in 10 ml 0.1 M Tris/HCl, pH 7.5,
0.1% 2-mercaptoethanol in the presence of 2 g Dowex 1 ꢁ 2. After
centrifugation at 9000g at 4 °C for 10 min, the microsomal fraction
was collected from the supernatant by adding MgCl2 (final concen-
tration 30 mM) followed by a second centrifugation step at
150,000g and 4 °C for 25 min. The pellet obtained was suspended
microgram quantities of protein utilizing the principle of protein–dye binding.
Anal. Biochem. 72, 248–254.
Brazier-Hicks, M., Evans, K.M., Gershater, M.C., Puschmann, H., Steel, P.G., Edwards,
R., 2009. The C-glycosylation of flavonoids in cereals. J. Biol. Chem. 284, 17926–
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Britsch, L., 1990. Purification and characterization of flavone synthase I,
a 2-
oxoglutarate-dependent desaturase. Arch. Biochem. Biophys. 276, 348–354.
Corpet, F., 1988. Multiple sequence alignment with hierarchical clustering. Nuceic
Acids Res. 16, 10881–10890.
Dixon, R.A., Steele, C.L., 1999. Flavonoids and isoflavonoids – a gold mine for
metabolic engineering. Trends Plant Sci. 4, 394–400.
Felsenstein, J., 2004. PHYLIP (Phylogeny Inference Package) version 3.6. Department
of Genome Sciences, University of Washington, Seattle (Distributed by the
author).
Fliegmann, J., Sandermann Jr., H., 1997. Maize glutathione-dependent formaldehyde
dehydrogenase cDNA: a novel plant gene of detoxification. Plant Mol. Biol. 34,
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in 500 ll of 0.1 M Tris/HCl, pH 7.5, 0.02% 2-mercaptoethanol and
used for enzyme assays as well as the supernatant of the second
centrifugation. Microsomes from induced yeast cells were pre-
pared according to Urban et al. (1994) by mechanical disruption
using glass beads and recovered by centrifugation for 1 h at
106,000g. The pellet was suspended in 50 mM Tris/HCl, pH 7.5,
2 mM EDTA, 20% (v/v) glycerol and stored at ꢀ80 °C. Protein was
determined as described (Bradford, 1976), using bovine serum
albumin as standard.
Fliegmann, J., Montel, E., Djulic, A., Cottaz, S., Driguez, H., Ebel, J., 2005. Catalytic
properties of the bifunctional soybean b-glucan-binding protein, a member of
family 81 glycoside hydrolases. FEBS Lett. 579, 6647–6652.
Frohman, M.A., Dush, M.K., Martin, G.R., 1988. Rapid production of full-length
The enzyme assay for FNS II was performed according to Mar-
tens and Forkmann (1998). Briefly, the incubation mixture (final
volume 200
liquiritigenin, 0.5 mM reduced glutathione, 1 mM NADPH, and
50 g of crude protein fraction. The enzyme assay for FNS I was
ll) contained: 0.1 M Tricine/KOH, pH 7.9, 0.1 mM
cDNAs from rare transcripts: amplification using
a single gene-specific
oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002.
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cultured soybean cells by an elicitor from Phytophthora megasperma f.sp.
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l
carried out according to (Britsch, 1990) in 1 ml final volume. The
incubation mixture contained: 0.1 M Tricine/KOH, pH 7.5,
0.1 mM liquiritigenin, 0.5 mM reduced glutathione, 0.25 mM 2-
oxo-glutarate, 5 mM ascorbic acid, 15 lM Fe(II)SO4, and 50 lg of
crude protein fraction. After 30 min at 25 °C, each reaction was
stopped and the mixture extracted twice by an equal volume of
EtOAc. For the separation of liquiritigenin and DHF, the pooled or-
ganic solvent was evaporated and the remaining compounds used
for HPLC analysis as described above. When naringenin and eri-
odictyol were assayed as substrates, products were analyzed using
linear gradients comprising 1% HOAc in MeOH–acetonitrile (1:1,
solvent A) and 1% HOAc in H2O (solvent B; 25–75% of solvent A
within 14 min) (Latunde-Dada et al., 2001). For the separation of
(S)-and (R)-forms of liquiritigenin, chiral resolution HPLC with
Chiralpak IA (4.6 ꢁ 150 mm; Chiral Technologies Europe, Ilkirch
Cedex, France) was used. The eluent consisted of 100% MeOH at
a flow rate of 0.5 ml minꢀ1 at 25 °C.
Kitada, C., Gong, Z., Tanaka, Y., Yamazaki, M., Saito, K., 2001. Differential expression
of two cytochrome P450s involved in the biosynthesis of flavones and
anthocyanins in chemovarietal forms of Perilla frutescens. Plant Cell Physiol.
42, 1338–1344.
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a NADPH-
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cell cultures by elicitor or osmotic stress. Planta 171, 519–524.
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Hertkorn, N., Werck-Reichhart, D., Ebel, J., 2001. Flavonoid 6-hydroxylase from
soybean (Glycine max L.), a novel plant P-450 monooxygenase. J. Biol. Chem.
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Martens, S., Forkmann, G., 1998. Genetic control of flavone synthase II activity in
flowers of Gerbera hybrids. Phytochemistry 49, 1953–1958.
Martens, S., Forkmann, G., 1999. Cloning and expression of flavone synthase II from
Gerbera hybrids. Plant J. 20, 611–618.
Martens, S., Mithöfer, A., 2005. Flavones and flavone synthases. Phytochemistry 66,
2399–2407.
Mithöfer, A., Fliegmann, J., Neuhaus-Url, G., Schwarz, H., Ebel, J., 2000. The hepta-b-
glucoside elicitor-binding proteins from legumes represent a putative receptor
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Mithöfer, A., Maitrejean, M., Boland, W., 2005. Structural and biological diversity of
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Acknowledgements
We thank Dr. Stefan Martens for oligonucleotides cons93b-f1/r1
and Edda Kock for expert technical assistance. This work was sup-
ported by the Max Planck Society, the European Community’s Hu-
man Potential Programme under contract HPRN-CT-2002-00251,
SACC-SIG-NET, and the Virtual Institute of Biotic Interactions
(VIBI).
Nakatsuka, T., Nishihara, M., Mishiba, K., Yamamura, S., 2006. Heterologous
expression of two gentian cytochrome P450 genes can modulate the intensity
of flower pigmentation in transgenic tobacco plants. Mol. Breeding 17, 91–99.
Page, R.D.M., 1996. TREEVIEW: an application to display phylogenetic trees on
personal computers. Comput. Appl. Biosci. 12, 357–358.
Sawada, Y., Ayabe, S., 2005. Multiple mutagenesis of P450 isoflavonoid synthase
reveals a key active-site residue. Biochem. Biophys. Res. Commun. 330, 907–
913.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2010.01.007.
Schopfer, C.R., Kochs, G., Lottspeich, F., Ebel, J., 1998. Molecular characterization and
functional expression of dihydropterocarpan 6a-hydroxylase, an enzyme
specific for pterocarpanoid phytoalexin biosynthesis in soybean Glycine max L.
FEBS Lett. 432, 182–186.
Schüler, G., Goerls, H., Boland, W., 2001. 6-Substituted indanoyl isoleucine
conjugates mimic the biological activity of coronatine; elicitors of plant
secondary metabolism. Eur. J. Org. Chem., 1663–1668.
Schüler, G., Mithöfer, A., Baldwin, I.T., Berger, S., Ebel, J., Santos, J.G., Herrmann, G.,
Hölscher, D., Kramell, R., Kutchan, T.M., Maucher, H., Schneider, B., Stenzel, I.,
Wasternack, C., Boland, W., 2004. Coronalon: a powerful tool in plant stress
physiology. FEBS Lett. 563, 17–22.
Truan, G., Cullin, C., Reisdorf, P., Urban, P., Pompon, D., 1993. Enhanced in vivo
monooxygenase activities of mammalian P450s in engineered yeast cells
producing high levels of NADPH-P450 reductase and human cytochrome-B5.
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