Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia cell cultures treated with Venturia inaequalis

Article abstract of DOI:10.1016/j.phytochem.2013.09.003

Aucuparin is the most widely distributed biphenyl phytoalexin in the rosaceous subtribe Pyrinae, which includes the economically important fruit trees apple and pear. The biphenyl scaffold is formed by biphenyl synthase, which catalyzes biosynthesis of 3,5-dihydroxybiphenyl. Conversion of this precursor to aucuparin (3,5-dimethoxy-4-hydroxybiphenyl) was studied in cell cultures of Sorbus aucuparia after treatment with an elicitor preparation from the scab-causing fungus Venturia inaequalis. The sequence of the biosynthetic steps detected was O-methylation - 4-hydroxylation - O-methylation. The two alkylation reactions were catalyzed by distinct methyltransferases, which differed in pH and temperature optima as well as stability. Biphenyl 4-hydroxylase was a microsomal cytochrome P450 monooxygenase, whose activity was appreciably decreased by the addition of established P450 inhibitors. When fed to V. inaequalis-treated S. aucuparia cell cultures, radioactively labeled 3,5-dihydroxybiphenyl was not only incorporated into aucuparin but also into the dibenzofuran eriobofuran, the accumulation of which paralleled that of aucuparin. However, biphenyl 2′-hydroxylase activity proposed to be involved in dibenzofuran formation was detected in neither microsomes nor cell-free extracts in the presence of NADPH and 2-oxoglutarate, respectively. Nevertheless, a basis for studying biphenyl biosynthesis at the gene level is provided.

Full text of DOI:10.1016/j.phytochem.2013.09.003

Phytochemistry xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia
cell cultures treated with Venturia inaequalis
Mohammed N.A. Khalil a, Till Beuerle a, Andreas Müller a, Ludger Ernst b, Vijaya B.R. Bhavanam a,
Benye Liu a, Ludger Beerhues a,
⇑
a Institute of Pharmaceutical Biology, Technische Universität Braunschweig, Mendelssohnstr. 1, 38106 Braunschweig, Germany
b Department of Chemistry, Central NMR Laboratory, Technische Universität Braunschweig, Hagenring 30, 38106 Braunschweig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Aucuparin is the most widely distributed biphenyl phytoalexin in the rosaceous subtribe Pyrinae, which
includes the economically important fruit trees apple and pear. The biphenyl scaffold is formed by biphe-
nyl synthase, which catalyzes biosynthesis of 3,5-dihydroxybiphenyl. Conversion of this precursor to
aucuparin (3,5-dimethoxy-4-hydroxybiphenyl) was studied in cell cultures of Sorbus aucuparia after
treatment with an elicitor preparation from the scab-causing fungus Venturia inaequalis. The sequence
of the biosynthetic steps detected was O-methylation – 4-hydroxylation – O-methylation. The two alkyl-
ation reactions were catalyzed by distinct methyltransferases, which differed in pH and temperature
optima as well as stability. Biphenyl 4-hydroxylase was a microsomal cytochrome P450 monooxygenase,
whose activity was appreciably decreased by the addition of established P450 inhibitors. When fed to V.
inaequalis-treated S. aucuparia cell cultures, radioactively labeled 3,5-dihydroxybiphenyl was not only
incorporated into aucuparin but also into the dibenzofuran eriobofuran, the accumulation of which par-
alleled that of aucuparin. However, biphenyl 20-hydroxylase activity proposed to be involved in dibenzo-
furan formation was detected in neither microsomes nor cell-free extracts in the presence of NADPH and
2-oxoglutarate, respectively. Nevertheless, a basis for studying biphenyl biosynthesis at the gene level is
provided.
Received 12 June 2013
Received in revised form 25 August 2013
Available online xxxx
Keywords:
Sorbus aucuparia
Venturia inaequalis
Phytoalexins
Biphenyls
Dibenzofurans
Biosynthesis
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
intensely studied to enhance the disease resistance potential of
the economically valuable cultivars (Norelli et al., 2003).
The rosaceous subtribe Pyrinae, formerly known as subfamily
Maloideae, includes a number of economically valuable fruit trees,
such as apple (Malus ꢀ domestica) and pear (Pyrus communis)
(Campbell et al., 2007). In 2010, the world-wide production of ap-
ple and pear was valued 64 and 13 milliard US$, respectively (FAO,
2010). However, the numerous cultivars of the fruit crops are af-
flicted by many diseases, leading to dramatic losses of both trees
and fruits. The most devastating diseases include fire blight, caused
by the bacterium Erwinia amylovora, and scab, triggered by the fun-
gus Venturia inaequalis (MacHardy, 1996; Vanneste, 2000). The an-
nual losses due to the fire blight disease and the costs of control are
valued at over US$ 100 million in the USA alone (Norelli et al.,
2003). In terms of control expenditure, apple scab is the most
costly apple disease (Carisse and Bernier, 2002). Due to the great
economic impact of these diseases on fruit production, the interac-
tions between the fruit trees and the devastating pathogens are
One plant defense strategy is formation of phytoalexins (Dixon,
2001). Within the Rosaceae, the Pyrinae are the only taxon that
produces biphenyls and dibenzofurans as de novo formed defense
compounds when subjected to biotic and abiotic stresses (Fig. 1;
Kokubun and Harborne, 1995). So far, 10 biphenyls and 17 dib-
enzofurans were isolated from 14 of the 30 Pyrinae genera and
demonstrated to be induced by infection or elicitation (Chizzali
and Beerhues, 2012). Their antibacterial and antifungal activities
against pathogens were established, however, the underlying
mechanisms of antimicrobial action are not yet elucidated (Chizz-
ali et al., 2012b; Kokubun et al., 1995). Biphenyls and dibenzofu-
rans were also found in species outside the Pyrinae, however,
their function here is to contribute to the constitutive barrier
against microorganisms and herbivores rather than being infec-
tion-induced phytoalexins (Hüttner et al., 2010).
In infected plants of the Pyrinae, no single species was found to
simultaneously accumulate both classes of phytoalexins in the
same tissue, provoking the conclusion that the biosynthetic path-
ways of biphenyls and dibenzofurans are parallel rather than
sequential (Kokubun and Harborne, 1995). Only recently,
⇑
Corresponding author. Tel.: +49 531 391 5689; fax: +49 531 391 8104.
E-mail address: l.beerhues@tu-bs.de (L. Beerhues).
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.09.003
Please cite this article in press as: Khalil, M.N.A., et al. Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia cell cultures treated with
Venturia inaequalis. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.003

2
M.N.A. Khalil et al. / Phytochemistry xxx (2013) xxx–xxx
Fig. 1. Survey of biphenyl and dibenzofuran biosynthesis. BIS, biphenyl synthase; OMT, O-methyltransferase. Solid lines, established reactions; dashed lines, hypothetic
reactions.
fire-blight-infected shoots of the apple cultivar ‘Holsteiner Cox’
and the pear cultivar ‘Conference’ have been reported to form both
biphenyls and dibenzofurans in the transition zone, which is lo-
cated between the healthy and necrotic parts of the infected stem
(Chizzali et al., 2012a,b, 2013). Before, in vitro systems had already
been shown to produce the two classes of compounds simulta-
neously. Cell cultures of the scab-resistant apple cultivar ‘Liberty’
accumulated three biphenyls and one dibenzofuran upon elicita-
tion with yeast extract (Borejsza-Wysocki et al., 1999; Hrazdina
et al., 1997). Cell cultures of Sorbus aucuparia formed various pat-
terns of phytoalexins in response to the type of elicitor added
(Hüttner et al., 2010). The biphenyl aucuparin was the major phy-
toalexin after the addition of yeast extract, whereas the dibenzofu-
ran eriobofuran was the major inducible compound when E.
amylovora and V. inaequalis preparations were added. In addition,
the two latter elicitors induced maximum phytoalexin levels.
These recent observations provided the basis for postulating
sequential rather than parallel biosynthetic pathways of biphenyl
and dibenzofuran formation (Chizzali and Beerhues, 2012; Hrazdi-
na and Borejsza-Wysocki, 2003; Hüttner et al., 2010).
The carbon skeleton of the compounds is formed by biphenyl
synthase (BIS), a type III polyketide synthase, which catalyzes iter-
ative condensation of benzoyl-CoA with three molecules of malo-
nyl-CoA to give a tetraketide intermediate which then undergoes
aldol condensation and decarboxylation to yield 3,5-dihydroxybi-
phenyl (Fig. 1; Liu et al., 2004, 2007, 2010). The conversion of this
precursor to aucuparin may proceed via three alternative routes,
which differ in the sequence of the methylation and hydroxylation
steps (Fig. 2). Either two O-methylation steps are followed by
hydroxylation or vice versa. As a third alternative, the methylation
steps frame the hydroxylation reaction. Furthermore, all metabo-
lites involved are potential candidates to be converted to the
corresponding dibenzofurans by 20-hydroxylation and subsequent
intramolecular cyclization (Fig. 1).
Here we report biochemical elucidation of the biosynthetic
pathway leading from 3,5-dihydroxybiphenyl to aucuparin in S.
aucuparia cell cultures treated with V. inaequalis extract. Further-
more, tracer experiments resulted in incorporation of radioactively
labeled 3,5-dihydroxybiphenyl not only in aucuparin but also in
the dibenzofuran eriobofuran.
2. Results
2.1. Accumulation of phytoalexins after treatment with V. inaequalis
The biphenyl aucuparin and the dibenzofuran eriobofuran are
the major phytoalexins present in S. aucuparia cell cultures after
treatment with V. inaequalis extract, as previously observed (Hütt-
ner et al., 2010). Here, we have studied the time course of their
accumulation. Formation of aucuparin and eriobofuran started 9
and 12 h, respectively, after the onset of the treatment and the
phytoalexin levels increased up to 96 and 120 h post-elicitation,
respectively (Fig. 3). Control cell cultures which received only an
equal volume of water failed to form the defense compounds.
2.2. Synthesis and feeding of radioactive 3,5-dihydroxybiphenyl
To study if aucuparin and eriobofuran are derived from 3,5-
dihydroxybiphenyl, as proposed by Hüttner et al. (2010), the latter
compound was enzymatically synthesized as radioactive tracer.
Recombinant BIS1 of S. aucuparia (Liu et al., 2007) was incubated
with benzoyl-CoA and [2-14C]malonyl-CoA, resulting in the forma-
tion of 14C-labeled 3,5-dihydroxybiphenyl (Fig. 1). This tracer was
Please cite this article in press as: Khalil, M.N.A., et al. Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia cell cultures treated with
Venturia inaequalis. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.003

M.N.A. Khalil et al. / Phytochemistry xxx (2013) xxx–xxx
3
Fig. 2. Proposed alternative routes of aucuparin formation starting from 3,5-dihydroxybiphenyl.
0.4
0.3
0.2
0.1
0
Aucuparin
Aucuparin
2
1.5
1
Eriobofuran
0.5
0
0
20
40
60
80
100
120
140
Time (h)
Fig. 3. Accumulation of aucuparin and eriobofuran in S. aucuparia cell cultures after the addition of V. inaequalis extract. Quantification was relative to 4-phenylphenol as
internal standard. SDs are indicated (n = 3).
fed to S. aucuparia cell cultures after treatment with V. inaequalis
extract. Using radiodetector-coupled HPLC, accumulation of both
labeled aucuparin and labeled eriobofuran was detected. After 3
d of tracer feeding, the incorporation rates of 3,5-dihydroxybi-
phenyl into aucuparin and eriobofuran were 0.6% and 3.3%, respec-
tively, reflecting the ratio of the aucuparin and eriobofuran levels
(Fig. 3). Feeding of [U–14C]benzoic acid in a parallel experiment
achieved incorporation rates of 1.1% and 4.6%. No glycosylated
compounds are detectable after elicitor treatment, as demon-
strated previously by enzymatic and acidic hydrolyses of extracts
(Hüttner et al., 2010).
cell cultures treated with V. inaequalis for 15 h. While the micro-
somal fraction failed to convert 3,5-dihydroxybiphenyl, cell-free
extract was capable of forming a new compound, as demonstrated
by HPLC-DAD analysis. This product was absent from assays that
either lacked SAM or contained heat-denatured protein. The enzy-
matic product was identified as 3-hydroxy-5-methoxybiphenyl by
co-chromatography with chemically synthesized reference com-
pound. When 3-hydroxy-5-methoxybiphenyl itself was incubated
with desalted cell-free extract and SAM, no further O-methylation
to give 3,5-dimethoxybiphenyl was observed (Table 1). Among the
dibenzofurans tested, 2,4-dihydroxydibenzofuran was O-methyl-
ated to give 2-hydroxy-4-methoxydibenzofuran, which in turn
did not undergo
a second methylation to yield 2,4-dim-
2.3. Detection of O-methyltransferase (OMT1) activity
ethoxydibenzofuran. Similarly, 4-hydroxy-2-methoxydibenzofu-
ran did not experience O-methylation. All dibenzofurans used
were chemically synthesized and their structures were confirmed
by mass spectrometry and NMR spectroscopy (1H, 13C including
DEPT-135, HSQC, HMBC, NOESY or NOEDIFF). While the Km values
for 3,5-dihydroxybiphenyl and 2,4-dihydroxydibenzofuran were
similar, the Vmax values differed by factor 5 (Table 2). The pH and
To study if either O-methylation or hydroxylation is the first
biosynthetic step, non-radioactive 3,5-dihydroxybiphenyl was
chemically synthesized according to Nilsson and Norin (1963)
and incubated with both desalted cell-free extract in the presence
of S-adenosyl-
L-methionine (SAM) and microsomes in the presence
of NADPH. The protein fractions were prepared from S. aucuparia
Please cite this article in press as: Khalil, M.N.A., et al. Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia cell cultures treated with
Venturia inaequalis. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.003

4
M.N.A. Khalil et al. / Phytochemistry xxx (2013) xxx–xxx
Table 1
Table 3
Substrate specificity of OMT1 activity catalyzing the first methylation reaction.
Cofactor dependence of B4H and effect of inhibitors.
Substrate
Relative activity (%)
100a
Conditions
Relative activity (%)
Standard assay
ꢁNADPH
100a
1.1
119
43.9
1.4
OH
3
+NADH
+0.5
+10
+10
+50
lM miconazole
lM miconazole
lM cytochrome c
lM cytochrome c
5
6.9
1.3
OH
3,5-dihydroxybiphenyl
a
0.44 nkat.
n.d.
OH
3
Table 4
Effect of freeze–thaw on OMT activity in cell-free extracts. SDs are indicated (n = 3).
5
OMe
Storage of cell-free
Relative OMT activity (% of max. each)
3-hydroxy-5-methoxybiphenyl
extract at ꢁ80 °C
3,5-Dihydroxybiphenyl
Noraucuparin
20.4
OH
1
With glycerol (20%, v/v)
Without glycerol
100
32.3 4.4
97.5 2.5
100
7
2
9b
9a
4
5
O
OH
and NADPH, leading to formation of noraucuparin (Fig. 4). No prod-
uct formation was found in assays that either lacked NADPH or
contained heat-denatured microsomes. Identification of the enzy-
matic product was carried out by co-chromatography with a chem-
ically synthesized and structurally confirmed reference compound
(Hüttner et al., 2010). 3,5-Dihydroxybiphenyl and 3,5-dimethoxy-
biphenyl failed to be 4-hydroxylated when tested under identical
conditions. Nor did 2,4-dihydroxydibenzofuran, 2-hydroxy-4-
methoxydibenzofuran, and 4-hydroxy-2-methoxydibenzofuran
serve as substrates. Formation of noraucuparin was hardly detect-
able in the absence of NADPH but was stimulated by the addition
of NADH (Table 3). Well known inhibitors of cytochrome P450 en-
zymes, such as miconazole and cytochrome c (Ortiz de Montellano,
2005), led to inhibition of the hydroxylase activity in a concentra-
tion-dependent manner. The pH and temperature optima of B4H
were 8.5 and 20 °C, respectively. Enzyme activity was linear for
2,4-dihydroxydibenzofuran
n.d.
n.d.
OH
7
9a
2
9b
4
5
O
OMe
2-hydroxy-4-methoxydibenzofuran
OMe
1
7
2
9b
9a
4
5
O
OH
4-hydroxy-2-methoxydibenzofuran
a
0.49 nkat; n.d., not detectable.
up to 90 min with protein concentrations up to 300
l
g/assay. The
Km value for 3-hydroxy-5-methoxybiphenyl was 0.6 0.1
lM and
the Vmax value was 0.38 nkat.
Table 2
Unlike 4-hydroxylase activity, 20-hydroxylase activity was not
detectable. Microsomes failed to convert 3,5-dihydroxybiphenyl,
3-hydroxy-5-methoxybiphenyl, noraucuparin, and aucuparin
(Fig. 2) into the corresponding 20-hydroxylated products. Nor were
the potential substrates hydroxylated by cell free-extracts in the
presence of 2-oxoglutarate, ferrous, and ascorbate, which soluble
dioxygenases rely on.
Kinetic parameters of OMT activity in cell-free extracts.
Substrate
Km
(l
M)
Vmax (nkat)
3,5-Dihydroxybiphenyl
2,4-Dihydroxydibenzofuran
Noraucuparin
0.85
1.17
4.46
0.49
0.10
0.12
temperature optima found for the OMT activity were 8.5 and 40 °C,
respectively.
2.5. Detection of OMT2 activity
Noraucuparin was incubated with desalted cell-free extract and
SAM, resulting in formation of a single product, which was identi-
fied as aucuparin by co-chromatography with an authentic refer-
ence compound (Hüttner et al., 2010). Incubation of aucuparin
did not lead to further methylation at the 4-hydroxyl group to give
2.4. Detection of biphenyl 4-hydroxylase (B4H) activity
3-Hydroxy-5-methoxybiphenyl was incubated with the micro-
somal fraction from V. inaequalis-treated S. aucuparia cell cultures
Fig. 4. Established sequence of biosynthetic reactions leading from 3,5-dihydroxybiphenyl to aucuparin in V. inaequalis-treated S. aucuparia cell cultures. OMT, O-
methyltransferase; B4H, biphenyl 4-hydroxylase.
Please cite this article in press as: Khalil, M.N.A., et al. Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia cell cultures treated with
Venturia inaequalis. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.003

M.N.A. Khalil et al. / Phytochemistry xxx (2013) xxx–xxx
5
3,4,5-trimethoxybiphenyl. The pH and temperature optima of the
noraucuparin methylation reaction were 7.5 and 45 °C, respec-
tively, and thus differed from the values observed with OMT1
(8.5 and 40 °C). OMT2 activity was linear for up to 35 min with
protein concentrations up to 80 lg/assay. When either heat-dena-
tured protein was incubated or SAM was omitted from the assay,
was found to be the only substrate for B4H which formed norau-
cuparin. Microsomal localization, NADPH dependency, and repres-
sion by CYP inhibitors indicated that B4H is a cytochrome P450
monooxygenase. Subsequently, a second OMT activity (OMT2) cat-
alyzed methylation of the 3-hydroxyl group of noraucuparin to
yield aucuparin. The presence of two distinct OMTs in S. aucuparia
cell cultures was indicated by different pH and temperature opti-
ma and differential sensitivity to freeze–thaw in the absence of
glycerol. In contrast to the strict regiospecificities of the S. aucupa-
ria OMTs, some plant OMTs perform two sequential methylation
steps on a single substrate, e.g. flavonoid OMTs (Cacace et al.,
2003; Zhou et al., 2006) and resveratrol OMT (Schmidlin et al.,
2008). In roses, two consecutively acting OMTs were detected
and catalyze formation of 3,5-dimethoxytoluene from orcinol (La-
vid et al., 2002; Scalliet et al., 2002, 2008). Both enzymes were able
to methylate both intermediates but the catalytic efficiencies dif-
fered. Thorough studies of the S. aucuparia OMTs at the molecular
level are needed to gain detailed insight into their properties.
In contrast to aucuparin biosynthesis, the conversion of biphe-
nyls to dibenzofurans is not yet understood. Unlike 4-hydroxylase
activity involved in aucuparin formation, 20-hydroxylase activity
proposed to participate in dibenzofuran biosynthesis (Fig. 1) could
not be detected. Notably, 20-hydroxyaucuparin has previously been
isolated from elicitor-treated cell cultures of S. aucuparia and M.
no product formation was observed. The Km value for noraucuparin
was 4.5 0.1 lM and the Vmax value was 0.12 nkat (Table 2).
Upon freeze–thaw of desalted cell-free extracts in the presence
of glycerol, both the 3,5-dihydroxybiphenyl- and the noraucupa-
rin-converting OMT activities were stable (Table 4). However,
when glycerol was omitted, the 3,5-dihydroxybiphenyl-metaboliz-
ing OMT1 activity was strongly decreased, whereas the noraucup-
arin-converting OMT2 activity remained unchanged.
3. Discussion
Our observation that 3,5-dihydoxybiphenyl is the precursor of
both aucuparin and eriobofuran in V. inaequalis-treated S. aucupa-
ria cell cultures indicates that biphenyls and dibenzofurans are
formed via sequential rather than parallel pathways. Thus, BIS cat-
alyzes the biosynthesis of the carbon skeletons of both classes of
phytoalexins present in the Pyrinae. Three BIS cDNAs were previ-
ously cloned from elicitor-treated S. aucuparia cell cultures (Liu
et al., 2007, 2010). Unlike BIS2 mRNA, the BIS1 and BIS3 transcript
levels rapidly and strongly increased after elicitation, indicating
that these two isoenzymes are involved in phytoalexin formation.
So far, no 3,5-dihydoxybiphenyl has been isolated from any in-
fected plant species although it exhibits pronounced antibacterial
activity against E. amylovora (Chizzali et al., 2012b). Variable arrays
of derivatives of this precursor may exhibit synergistic activities
against a broad range of pathogens. While biphenyls have some-
what stronger antibacterial activity than structurally related dib-
enzofurans, the opposite tendency was observed for antifungal
activity (Chizzali and Beerhues, 2012). Transgenic approaches aim-
ing to introduce a single new phytoalexin into a susceptible plant
resulted in neutralizing the severe symptoms associated with the
infection but did not endow plant resistance (He and Dixon,
2000; Hipskind and Paiva, 2000). More than one phytoalexin com-
pound or class appears to be essential to provide plants with resis-
tance (Dixon, 2001).
The absence of detectable quantities of 3,5-dihydroxybiphenyl
from infection sites and elicitor-treated cells indicates rapid con-
version of this precursor to downstream products. The major prod-
ucts of this metabolism in V. inaequalis-treated S. aucuparia cell
cultures are the biphenyl aucuparin and the dibenzofuran eri-
obofuran. Both compounds accumulate in parallel over 4–5 d
post-elicitation. Therefore, S. aucuparia cell cultures provide a valu-
able system for studying biphenyl and dibenzofuran biosynthesis.
Accumulation over several days was previously observed with
malusfuran, the O-glucoside of 9-hydroxyeriobofuran, in yeast-ex-
tract treated cell cultures of the scab-resistant apple cultivar ‘Lib-
erty’ (Hrazdina et al., 1997). Unlike cell culture systems, intact
shoots contain only a limited number of phytoalexin-producing
cells, as indicated by the exclusive phytoalexin accumulation in
the transition zones of fire blight-infected stems of the apple culti-
var ‘Holsteiner Cox’ and the pear cultivar ‘Conference’ (Chizzali
et al., 2012b, 2013).
domestica, the latter cell culture also producing 20-O-b-
D-glucopyr-
anosylaucuparin (Borejsza-Wysocki et al., 1999; Hüttner et al.,
2010). In addition, 20-hydroxyaucuparin was detected in the tran-
sition zones of fire-blight-infected shoots of the apple cultivar ‘Hol-
steiner Cox’ and the pear cultivar ‘Conference’ (Chizzali et al.,
2012b, 2013). There are two classes of enzymes known to catalyze
aromatic hydroxylation, namely microsomal cytochrome P450
monooxygenases and soluble 2-oxoglutarate-dependent dioxygen-
ases. These enzymes, both of which use molecular oxygen, can re-
place each other in different species (Frey et al., 2003; Lukacin
et al., 2001). However, neither enzyme activity could be detected
in V. inaequalis-treated S. aucuparia cell cultures using cell-free ex-
tract together with 2-oxoglutarate, ferrous, and ascorbate and
microsomes together with NADPH.
As an alternative to 20-hydroxylation at the biphenyl level, the
phenolic 20-hydroxyl group might be introduced at an earlier bio-
synthetic stage. Utilization by BIS of ortho-hydroxybenzoyl-CoA
(salicoyl-CoA) rather than benzoyl-CoA was previously believed
to directly yield 20,3,5-trihydroxybiphenyl. However, all recombi-
nant BISs so far incubated with salicoyl-CoA formed 4-hydroxy-
coumarin after
a single decarboxylative condensation with
malonyl-CoA rather than 20,3,5-trihydroxybiphenyl. This even
holds true for BIS3 from S. aucuparia which preferred salicoyl-
CoA over benzoyl-CoA as a starter molecule (Chizzali et al.,
2012a; Liu et al., 2010). Furthermore, benzoic acid and cinnamic
acid rather than salicylic acid were reported to be incorporated
into aucuparin and malusfuran in elicitor-treated apple cell cul-
tures (Hrazdina and Borejsza-Wysocki, 2003). These findings are
in accord with the high incorporation of radioactively labeled
3,5-dihydroxybiphenyl into the dibenzofuran eriobofuran, indicat-
ing 20-hydroxylation at the biphenyl level which, however, needs
to be detected. Once formed, the 20-hydroxylated biphenyl may
be converted to the corresponding dibenzofuran via oxidative phe-
nol coupling in analogy to the intramolecular cyclization of benz-
ophenones to xanthones, catalyzed by cytochrome P450 enzymes
(Peters et al., 1997).
In our study, the pathway leading from 3,5-dihydroxybiphenyl
to aucuparin in V. inaequalis-treated S. aucuparia cell cultures was
elucidated at the enzyme level. Interestingly, the elucidated se-
quence of reactions was O-methylation – 4-hydroxylation – O-
methylation (Fig. 4). The first methylation step yields 3-hydroxy-
5-methoxybiphenyl, which failed to undergo further methylation
at the 3-hydroxyl group. Instead, 3-hydroxy-5-methoxybiphenyl
4. Concluding remarks
Both classes of phytoalexins present in the Pyrinae are derived
from 3,5-dihydroxybiphenyl, which in turn is formed from
Please cite this article in press as: Khalil, M.N.A., et al. Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia cell cultures treated with
Venturia inaequalis. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.003

6
M.N.A. Khalil et al. / Phytochemistry xxx (2013) xxx–xxx
benzoyl-CoA and malonyl-CoA by BIS, the only enzyme of the bio-
synthetic pathway that has so far been studied at the biochemical
and molecular levels. An interesting sequence of biosynthetic steps
resulting in formation of the best known biphenyl phytoalexin,
aucuparin, was elucidated. However, the branch point at which a
biphenyl intermediate is channelled into dibenzofuran metabolism
is still obscure. Regarding aucuparin biosynthesis, the way for fur-
ther investigations at the molecular genetic level is now paved.
Exploitation of biphenyl metabolism may provide new control
strategies to improve the disease resistance of valuable apple and
pear cultivars.
5.2.3. Synthesis of 2,4-dihydroxydibenzofuran, 2-hydroxy-4-
methoxydibenzofuran, and 4-hydroxy-2-methoxydibenzofuran
Demethylations were achieved using a modified method of Bao
et al. (2009). A CH2Cl2 solution of 2,4-dimethoxydibenzofuran
(5.2.2; 545 mg, 2.39 mmol) was mixed with freshly prepared mag-
nesium iodide etherate solution (7.17 mmol). After evaporation
under reduced pressure, the residue was heated at 80 °C for 10 h
under a stream of argon gas. The reaction was stopped by the addi-
tion of a saturated solution of NH4Cl (50 ml; Anioł et al., 2008; as
alternative to Na2S2O3 and washing with NaHCO3). The resulting
solution was acidified using aqueous HCl (5%). The aqueous phase
was extracted with CH2Cl2 (3 ꢀ 50 ml) and dried over anhydrous
Na2SO4. The reaction products were fractionated on a silica gel col-
umn using petroleum ether/EtOAc (65:35, v/v) to yield fractions A
and B with Rf values 0.8 and 0.24, respectively.
5. Experimental
5.1. Chemicals
Fraction A (323 mg) was further purified on a silica gel column
using petroleum ether/EtOAc (8:2, v/v) to yield 4-hydroxy-2-meth-
oxydibenzofuran (267 mg, 1.24 mmol, 52.2% yield, Rf = 0.64) and 2-
hydroxy-4-methoxydibenzofuran (12 mg, 0.056 mmol, 2.3% yield,
Rf = 0.4).
MSTFA and benzoyl-CoA were purchased from ABCR (Karlsruhe,
Germany) and Sigma–Aldrich (Steinheim, Germany). [2-14C]malo-
nyl CoA (55.2 mCi/mmol; 0.1 mCi/ml) was supplied by Hartmann
Analytic (Braunschweig, Germany). Solvents were of HPLC grade
and used without further purification. 3,5-Dihydroxybiphenyl,
3,4,5-trihydroxybiphenyl, 3,4,5-trimethoxybiphenyl, and 20-
hydroxyaucuparin were prepared, as described by Hüttner et al.
(2010) and Chizzali et al. (2012b). The identities of the products
were confirmed by spectroscopic analyses and were in accordance
with published data (Song et al., 2006).
5.2.3.1. 4-Hydroxy-2-methoxydibenzofuran
MS of mono-TMS derivative (70 eV), m/z (% rel abundance) 286
(100, [M]+), 271 (53, [M]+-15), 256 (62, [M]+-30), 240 (9), 228 (9),
185 (10), 126 (10), 73 (18); RI (ZB5-MS) 2086. 1H NMR
(400 MHz, CDCl3, TMS) 7.89 (ddd, J = 7.7, 1.3, 0.6 Hz, 1 H, H-9),
7.54 (ddd, J = 8.7, 1.0, 0.7 Hz, 1 H, H-6), 7.44 (ddd, J = 8.4, 7.2,
1.3 Hz, 1 H, H-7), 7.32 (td, J = 7.5, 1.0 Hz, 1 H, H-8), 7.00 (d,
J = 2.4 Hz, 1 H, H-1), 6.66 (d, J = 2.4 Hz, 1 H, H-3), 5.5 (br. s, OH),
3.88 (s, 3 H, 2-OMe); 13C NMR (101 MHz, CDCl3, d = 77.01 ppm)
156.76 (C-2), 156.54 (C-5a), 141.31 (C-4), 139.14 (C-4a), 127.23
(C-7), 125.49 (C-9b), 124.79 (C-9a), 122.70 (C-8), 120.88 (C-9),
111.84 (C-6), 102.26 (C-3), 95.89 (C-1), 56.07 (2-OMe).
5.2. Syntheses
5.2.1. Synthesis of 2,4-dimethoxy-1-phenoxybenzene
Ullmann condensation of 1-bromo-2,4-dimethoxybenzene and
phenol was based on a method published by Oliveira et al.
(2003), except that additional amounts of phenol (0.14 mmol
equivalents) were added 1, 2.5, 4, and 5.5 h after the onset of the
reaction. The total reaction time was 7 h. The product was purified
on a silica gel column using CH2Cl2/petroleum ether (40:60, v/v).
The yield was 31% (5.36 g, 23.3 mmol).
5.2.3.2. 2-Hydroxy-4-methoxydibenzofuran
MS of mono-TMS derivative (70 eV), m/z (% rel abundance) 286
(100, [M]+), 271 (64, [M]+-15), 256 (4, [M]+-30), 126 (12), 73 (20);
RI (ZB5-MS) 2133. 1H NMR (400 MHz, CDCl3, TMS) 7.86 (ddd,
J = 7.7, 1.3, 0.7 Hz, 1 H, H-9), 7.59 (dt, J = 8.3, 0.8 Hz, 1 H, H-6),
7.45 (ddd, J = 8.2, 7.3, 1.3 Hz, 1 H, H-7), 7.32 (ca. td, J ꢂ 8, 1 Hz, 1
H, H-8), 6.95 (d, J = 2.3 Hz, 1 H, H-1), 6.58 (d, J = 2.3 Hz, 1 H, H-3),
4.03 (s, 3 H, 4-OMe); 13C NMR (101 MHz, CDCl3, d = 77.01 ppm)
156.66 (C-5a), 152.21 (C-2), 145.87 (C-4), 140.16 (C-4a), 127.26
(C-7), 125.6 (C-9b), 124.29 (C-9a), 122.55 (C-8), 120.74 (C-9),
112.03 (C-6), 99.16 (C-1), 97.63 (C-3), 56.27 (4-OMe).
MS (70 eV), m/z (% rel abundance) 230 (100, [M]+), 215 (15,
[M]+-15), 200 (2, [M]+-30), 153 (11), 125 (10), 77 (14, [Ph]+); RI
(VF5-MS) 1866. 1H NMR (400 MHz, CDCl3, TMS) 7.26 (m, 2 H, H-
30, 50), 6.99 (m, 1 H, H-40), 6.95 (d, J = 8.7 Hz, 1 H, H-6), 6.89 (m, 2
H, H-20, 60), 6.58 (d, J = 2.8 Hz, 1 H, H-3), 6.45 (dd, J = 8.7, 2.8 Hz,
1 H, H-5), 3.81 (s, 3 H, 4-OMe), 3.78 (s, 3 H, 2-OMe); 13C NMR
(101 MHz, CDCl3, d = 77.01 ppm) 158.83 (C-10), 157.28 (C-4),
152.54 (C-2), 138.18 (C-1), 129.38 (C-30, 50), 122.32 (C-6), 121.81
(C-40), 116.09 (C-20, 60), 104.19 (C-5), 100.66 (C-3), 55.94 (2-
OMe), 55.62 (4-OMe).
Fraction B (120 mg) was purified on a silica gel column using
CH2Cl2/MeOH/HCOOH (5.0:0.15:0.05, v/v/v) to yield 2,4-
dihydroxydibenzofuran (70 mg, 0.35 mmol, 13.68% yield).
5.2.2. Synthesis of 2,4-dimethoxydibenzofuran
5.2.3.3. 2,4-Dihydroxydibenzofuran
2,4-Dimethoxy-1-phenoxybenzene was subjected to oxidative
coupling using Pd(OAc)2 and acetic acid (Oliveira et al., 2003).
The crude product was purified on a silica gel column using CH2Cl2/
petroleum ether (25:75, v/v). The yield was 12% (810 mg,
3.55 mmol).
MS of di-TMS derivative (70 eV), m/z (% rel abundance) 344
(100, [M]+), 329 (40, [M]+-15), 73 (75); RI (ZB5-MS) 2136. 1H
NMR (400 MHz, CDCl3, TMS) 7.86 (ddd, J = 7.4, 1.3, 0.6 Hz, 1 H, H-
9), 7.54 (dt, J = 8.3, 0.4 Hz, 1 H, H-6), 7.45 (ddd, J = 8.4, 7.2,
1.3 Hz, 1 H, H-7), 7.32 (ddd, J = 7.7, 7.3, 1.0 Hz, 1 H, H-8), 6.95 (d,
J = 2.3 Hz, 1 H, H-1), 6.58 (d, J = 2.3 Hz, 1 H, H-3), 5.47 (br. s, OH),
4.81 (br. s, OH); 13C NMR (101 MHz, CDCl3, d = 77.01 ppm) 156.6
(C-5a), 152.27 (C-2), 141.32 (C-4), 139.05 (C-4a), 127.41 (C-7),
125.8 (C-9b), 124.53 (C-9a), 122.76 (C-8), 121.02 (C-9), 111.85
(C-6), 102.57 (C-3), 98.26 (C-1).
MS (70 eV), m/z (% rel abundance) 228 (100, [M]+), 213 (26,
[M]+-15), 199 (7, [M]+-29), 185 (40), 170 (26), 142 (11), 126 (10),
114 (16); (RI (VF5-MS) 2079. 1H NMR (600 MHz, CDCl3, TMS)
7.89 (ddd, J = 7.7, 1.3, 0.7 Hz, 1 H, H-9), 7.59 (dt, J = 8.3, 1.6 Hz, 1
H, H-6), 7.44 (ddd, J = 8.3, 7.3, 1.3 Hz, 1 H, H-7), 7.32 (ddd, J = 7.7,
7.3, 1.0 Hz, 1 H, H-8), 6.99 (d, J = 2.3 Hz, 1 H, H-1), 6.62 (d,
J = 2.3 Hz, 1 H, H-3), 4.03 (s, 3 H, 4-OMe), 3.91 (s, 3 H, 2-OMe);
13C NMR (151 MHz, CDCl3, d = 77.01 ppm) 156.71 (C-2), 156.54
(C-5a), 145.83 (C-4), 140.22 (C-4a), 127.04 (C-7), 125.17 (C-9b),
124.59 (C-9a), 122.51 (C-8), 120.58 (C-9), 112.02 (C-6), 99.36 (C-
3), 94.31 (C-1), 56.17 (4-OMe), 55.99 (2-OMe).
5.2.4. Synthesis of eriobofuran
The procedure resembled that described for the dioxygenated
dibenzofurans (5.2.1–5.2.3). The intermediates 1,2,3-trimethoxy-
4-phenoxybenzene and 2,3,4-dimethoxydibenzofuran were pre-
pared by Ullmann synthesis and oxidative coupling, respectively.
Please cite this article in press as: Khalil, M.N.A., et al. Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia cell cultures treated with
Venturia inaequalis. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.003

M.N.A. Khalil et al. / Phytochemistry xxx (2013) xxx–xxx
7
The MS and NMR data of both reaction products were identical to
those published by Kemp et al. (1983). Demethylation of 2,3,4-
trimethoxydibenzofuran was carried out using magnesium iodide
etherate. MS and NMR data of eriobofuran were identical to those
reported by Hüttner et al. (2010).
redissolved in a volume of methanol (ꢃ150
l
l), its identity was
confirmed by co-chromatography (HPLC) with a sample of authen-
tic reference compound, and its radioactivity was measured by
scintillation counting (Beckman LS 6500). Due to the relatively
low yield and the moderate stability of the product, further purifi-
cation was omitted and the tracer was fed to the cell cultures right
away.
5.2.5. Synthesis of aucuparin
Demethylation
of
3,4,5-trimethoxybiphenyl
(400 mg,
1.64 mmol) was achieved using magnesium iodide etherate solu-
tion (30 equivalents). The solutions of both reactants in diethyl
ether were mixed and evaporated to dryness. The residue was
heated on an oil bath at 80 °C for 11 h under a stream of argon
gas and protected from light. The residue was dissolved in water,
and Na2S2O3 was added to remove I2. The solution was acidified
and extracted with CH2Cl2 (2 ꢀ 50 ml). The organic layer was
washed with NaHCO3 solution (100 ml) and brine solution
(100 ml) and dried over anhydrous Na2SO4. The concentrated resi-
due was purified on a silica gel column using CH2Cl2. The yield was
76.9% (290 mg, 1.26 mmol). The spectroscopic properties were in
accordance with published data (Hüttner et al., 2010).
5.5. Feeding of radiolabeled 3,5-dihydroxybiphenyl
Cultured cells (0.3 g) were inoculated into 5 ml LS medium in
25 ml Erlenmeyer flasks. On day 5, the extract of V. inaequalis
was added and, after 4.5 h, the methanolic solution of radiolabeled
3,5-dihydroxybiphenyl (117 ll; 0.52 lCi) was fed under sterile
conditions. Cells were harvested by centrifugation 72 h after the
onset of elicitation and homogenized in methanol (7 ml). After cen-
trifugation, the residue was extracted twice with methanol (4 ml
each) by vortexing for 1 min. The combined methanol extract
was evaporated to dryness and the residue was redissolved in
100 ll methanol (HPLC grade). Aliquots were used for scintillation
counting and HPLC analysis.
5.2.6. Synthesis of noraucuparin
Aucuparin (240 mg, 1.04 mmol) was dissolved in 10 ml CH2Cl2.
Cold (4 °C) BBr3 solution (1 ml, 1 M in CH2Cl2) was added (Bao
et al., 2009). The reaction mixture was stirred at room temperature
for 3 h under moisture-free atmosphere. Then additional BBr3 solu-
tion (0.5 ml, 1 M in CH2Cl2) was added and the reaction was con-
tinued for another 3 h. The reaction was stopped by dropwise
addition of water. The resulting mixture was acidified with aque-
ous HCl (5%). The organic layer was separated and the aqueous
layer was further extracted with CH2Cl2 (2 ꢀ 10 ml). The organic
layer was evaporated and noraucuparin was purified on a silica
gel column using CH2Cl2 (100 mg; 0.46 mmol, 45% yield). The spec-
troscopic properties were identical to published data (Hüttner
et al., 2010).
5.6. Radiodetector-coupled HPLC
The HPLC apparatus (Agilent 1200 series) was coupled with
both a UV detector (Agilent 1200 Variable Wavelength Detector)
and a radiodetector (RAMONA star 2; Raytest, Germany). A Lichro-
spher C18 column (250 ꢀ 4.6 mm i.d., 5
lm; Wicom, Germany) was
used. The solvent for isocratic analysis consisted of 55% (v/v) water
containing o-phosphoric acid (0,1%, v/v) and 45% (v/v) methanol.
Data acquisition and analysis were carried out using the GINA star
4 software (Raytest, Germany).
5.7. Protein extraction
Cultured cells (30 g) were harvested 15 h after elicitation and
homogenized in 6 ml 0.1 M Tris–HCl buffer (pH 7) containing
5.3. Cell cultures, elicitation, and analysis of phytoalexin accumulation
10 mM DTT and 10 lM PMSF in a mortar for 15 min. The homoge-
Cell cultures of S. aucuparia were grown in the dark at 25 °C, as
described before (Liu et al., 2004). Mycelium of V. inaequalis was
extracted with water (pH 2) for 1 h at 100 °C, as described by
Zhang et al. (2000). The resulting elicitor solution, whose pH was
readjusted to 5, was added to 5-day-old cell cultures (50 ml) at a
final concentration of 50 mg/l. Aliquots (10 ml) were taken at var-
ious times under sterile conditions, and cells were collected by
centrifugation and lyophilized. Phytoalexins were previously found
to accumulate intracellularly (Hüttner et al., 2010). After determi-
nation of the dry weight, cells were extracted three times with
methanol (5 ml each). The combined methanol extract was evapo-
rated to dryness. Preparation of samples for GC–MS analysis and
quantitation of compounds were carried out as described previ-
ously considering the real response factors of the compounds
(Hüttner et al., 2010). Three biological samples and up to three
technical replicates each were studied.
nate was centrifuged at 9.000g for 25 min and the supernatant was
passed through a PD10 column (GE Healthcare). The resulting cell-
free protein extract was used for testing OMT and hydroxylase
activities. To prepare the microsomal fraction, the crude extract
was centrifuged at 100.000g for 90 min. The microsomal pellet
was resuspended in 0.1 M Tris–HCl buffer (pH 7) containing 14%
(w/v) sucrose, 3.5 mM b-mercaptoethanol and 10 lM PMSF. The
microsomal pellet could be stored at ꢁ80 °C without appreciable
decrease in enzyme activity. Protein contents were determined
colorimetrically using Bradford’s reagent and a bovine serum albu-
min calibration curve (Bradford, 1976).
5.8. Enzyme assays
To optimize the assay conditions, the incubation temperature
was varied between 25 and 60 °C, except for B4H: 7–60 °C. The
pH values tested ranged from 6 to 11 using potassium phosphate
buffer (6–7), Tris–HCl buffer (7–8.5), and glycine–NaOH buffer
(9–11). The optimum incubation time and protein concentration
were also determined. To study kinetic parameters, first a wide
range of substrate concentrations were used for rough determina-
tion. Subsequently, concentrations of 0.2–5 ꢀ Km were used for
5.4. Enzymatic preparation of radiolabeled 3,5-dihydroxybiphenyl
BIS1 of S. aucuparia was heterologously produced in Escherichia
coli and isolated by affinity chromatography, as described earlier
(Liu et al., 2007). The enzyme assay (200
C]malonyl CoA (1.27 nmol, 0.07 Ci), 1.15 nmol malonyl-CoA,
7.4 M benzoyl-CoA, and 3 g BIS1 in 0.1 M potassium phosphate
buffer (pH 7). After incubation for 30 min at 35 °C, the reaction was
stopped by adding 40 l HCl (10%) and the product was extracted
twice with 200 l ethylacetate each. The extracts from 20 incuba-
tions were combined and evaporated to dryness. The product was
ll) consisted of 7 l
l [2-14-
l
accurate measurement (0.62–50
B4H substrate). The standard assays were as follows:
The B4H assay (200 l) consisted of 3-hydroxy-5-
methoxybiphenyl and 1 mM NADPH in 0.1 M Tris–HCl buffer (pH
7). The reaction was initiated by the addition of 80 g microsomal
protein. After incubation for 30 min at 22 °C, the reaction was
lM OMT substrates; 0.12–6 lM
l
l
l
6 lM
l
l
l
Please cite this article in press as: Khalil, M.N.A., et al. Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia cell cultures treated with
Venturia inaequalis. Phytochemistry (2013), http://dx.doi.org/10.1016/j.phytochem.2013.09.003

8
M.N.A. Khalil et al. / Phytochemistry xxx (2013) xxx–xxx
Campbell, C.S., Evans, R.C., Morgan, D.R., Dickinson, T.A., Arsenault, M.P., 2007.
stopped by the addition of 40
was extracted twice with ethyl acetate (200
l
l HCl (10%). The reaction product
l each) and the com-
bined organic phase was evaporated to dryness. The residue was
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l
dissolved in 60
under 5.9.
ll methanol and analyzed by HPLC, as described
The OMT assay (200
droxybiphenyl, noraucuparin), 50
acid in 0.1 M Tris–HCl buffer (pH 8.5). The reaction was initiated
by the addition of 40 g protein. After incubation for 20 min at
l
l) consisted of 15
lM substrate (3,5-dihy-
lM SAM, and 1.5 mM ascorbic
l
37 °C, the reaction was stopped and the product was extracted
and analyzed by HPLC, as described under 5.9. To test the stability
of OMT activities, aliquots of the cell-free protein extract, prepared
in 0.1 M Tris–HCl buffer pH 7 (5.7), were stored with and without
glycerol (20%, v/v) at ꢁ80 °C. After 6 months, the aliquots were
thawed on ice and their OMT activities were determined under
standard assay conditions.
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sisted of 5 mM 2-oxoglutarate, 0.5 mM ferrous sulphate and 5 mM
ascorbic acid in 0.1 M Tris–HCl buffer (pH 7). In addition, it con-
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enex). Data acquisition and analysis were carried out using the Ez-
Chrome Elite software (Agilent). The solvents consisted of water
containing 0.1% o-phosphoric acid (A) and methanol (B). The gradi-
ent was 50–80% B in 14 min, 80% B for 10 min, 80–100% B in 2 min,
and 100% B for 2 min at a flow rate of 0.3 ml/min. The detection
wavelengths were 254 and 269 nm for biphenyls and 285 and
300 nm for dibenzofurans and 20-hydroxylated biphenyls. For the
analysis of 2,4-dihydroxydibenzofuran and its methylated product,
2-hydroxy-4-methoxydibenzofuran, the following gradient was
used: 45% B for 2 min, 45–65% B in 8 min, 65–76% B in 10 min,
76–100% B in 2 min, and 100% B for 3 min at a flow rate of
0.3 ml/min. The detection wavelengths were 252 and 285 nm.
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Acknowledgements
Mohammed N.A. Khalil is recipient of a German Egyptian Re-
search Long-Term Scholarship (GERLS). This work was supported
by the Deutsche Forschungsgemeinschaft (DFG).
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