The fungal leaf endophyte Paraconiothyrium variabile specifically metabolizes the host-plant metabolome for its own benefit

Article abstract of DOI:10.1016/j.phytochem.2014.09.021

Fungal endophytes live inside plant tissues and some have been found to provide benefits to their host. Nevertheless, their ecological impact is not adequately understood. Considering the fact that endophytes are continuously interacting with their hosts, it is conceivable that both partners have substantial influence on each other's metabolic processes. In this context, we have investigated the action of the endophytic fungus Paraconiothyrium variabile, isolated from the leaves of Cephalotaxus harringtonia, on the secondary metabolome of the host-plant. The alteration of the leaf compounds by the fungus was monitored through metabolomic approaches followed by structural characterization of the altered products. Out of more than a thousand molecules present in the crude extract of the plant leaf, we have observed a specific biotransformation of glycosylated flavonoids by the endophyte. In all cases it led to the production of the corresponding aglycone via deglycosylation. The deglycosylated flavonoids turned out to display significant beneficial effects on the hyphal growth of germinated spores. Our finding, along with the known allelopathic role of flavonoids, illustrates the chemical cooperation underlying the mutualistic relationship between the plant and the endophyte.

Full text of DOI:10.1016/j.phytochem.2014.09.021

Phytochemistry 108 (2014) 95–101
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
The fungal leaf endophyte Paraconiothyrium variabile specifically
metabolizes the host-plant metabolome for its own benefit
Yuan Tian a, Séverine Amand a, Didier Buisson a, Caroline Kunz a,b, François Hachette c, Joëlle Dupont d,
Bastien Nay a, Soizic Prado a,
⇑
a Molécules de Communication et Adaptation des Microorganismes (MCAM), UMR 7245 CNRS/Muséum National d’Histoire Naturelle, Paris, France
b Sorbonne Universités, UPMC University Paris 06, UFR 927, Paris, France
c Jardins Botaniques, Arboretum de Chèvreloup, USM 0802, Muséum National d’Histoire Naturelle, Rocquencourt, France
d Institut de Systématique, Evolution, Biodiversité (ISEB), UMR 7205 CNRS/Muséum National d’Histoire Naturelle, Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 May 2014
Received in revised form 15 September 2014
Available online 21 October 2014
Fungal endophytes live inside plant tissues and some have been found to provide benefits to their host.
Nevertheless, their ecological impact is not adequately understood. Considering the fact that endophytes
are continuously interacting with their hosts, it is conceivable that both partners have substantial
influence on each other’s metabolic processes. In this context, we have investigated the action of the
endophytic fungus Paraconiothyrium variabile, isolated from the leaves of Cephalotaxus harringtonia, on
the secondary metabolome of the host-plant. The alteration of the leaf compounds by the fungus was
monitored through metabolomic approaches followed by structural characterization of the altered prod-
ucts. Out of more than a thousand molecules present in the crude extract of the plant leaf, we have
observed a specific biotransformation of glycosylated flavonoids by the endophyte. In all cases it led to
the production of the corresponding aglycone via deglycosylation. The deglycosylated flavonoids turned
out to display significant beneficial effects on the hyphal growth of germinated spores. Our finding, along
with the known allelopathic role of flavonoids, illustrates the chemical cooperation underlying the mutu-
alistic relationship between the plant and the endophyte.
Keywords:
Endophyte
Plant secondary metabolome
Biotransformation
Metabolomics
Plant–endophyte interactions
Mutualism
Flavone
Signaling molecules
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
effect of plant allelopathic metabolites on fungi (mainly pathogens)
has been extensively studied. Indeed plants use an intricate defense
Endophytic fungi colonize intercellularly the living tissues of
plants without causing apparent damage and include a very
diverse group of fungi with diverse life history strategies including
latent pathogens. Mutualistic endophytic fungi have been the focus
of a growing interest worldwide notably because of their beneficial
role for plant fitness, such as tolerance to biotic and abiotic stres-
ses, nutrient acquisition and increased growth (Prado et al.,
2011). Nevertheless, the underlying mechanisms in this phenome-
non are still poorly known. Considering the fact that endophytes
are continuously interacting with their hosts, it is conceivable that
both partners have substantial influence on each other’s metabolic
processes. It was thus suggested that secondary metabolites play a
critical role in the interaction between endophytic fungi and their
host, especially for signaling purposes, defense, and regulation of
the symbiosis (Schulz and Boyle, 2005).
system against pests and pathogens including the production of
toxic compounds of low molecular weight such as induced phytoal-
exins and constitutive phytoanticipins. They are part of the plant
response repertoire and have been well described in crops such as
Brassicaceae, Fabaceae, Solanaceae, Vitaceae and Poacea (Ahuja
et al., 2012). Pathogenic fungi have developed diverse responses
to counteract this arsenal of allelopathic metabolites. These include
repression of the plant’s defense system, active efflux of antibiotics,
antibiotic resistance and more generally detoxification (Morrissey
and Osbourn, 1999). Indeed, numerous examples highlight the
ability of plant pathogens to significantly detoxify plant defensive
compounds (Pedras et al., 2011; Saunders and Kohn, 2008). In the
case of endophytes, few studies have focused on the role of the
host-plant metabolites, except in some cases of natural product bio-
transformation. Indeed, recent studies have shown the interest of
studying endophytic fungi for the production of new compounds
through biotransformation of natural products such as monoter-
penes (Bicas et al., 2009; Marostica and Pastore, 2009), alkaloids
(Zikmundova et al., 2002), taxoids (Zhang et al., 1998), naphthoqui-
nones (Prado et al., 2013) or flavonoids (Agusta et al., 2005; Shibuya
While there is a lack of knowledge concerning the impact of
secondary metabolites produced by endophytes on the host-plant,
⇑
Corresponding author. Tel.: +33 1 40 79 31 19.
E-mail address: sprado@mnhn.fr (S. Prado).
http://dx.doi.org/10.1016/j.phytochem.2014.09.021
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

96
Y. Tian et al. / Phytochemistry 108 (2014) 95–101
et al., 2005). Nevertheless, these are biotechnical and chemical
studies rather than attempts to unravel the ecological role of such
transformations.
fingerprints at 16, 20, 40, and 60 h post inoculation (hpi). Using
the XCMS software, the protonated ions of the biotransformed
metabolites at the different hpi were compared to the control
crude extract sample (t = 0 h).
Recent reports have also described the influence of fungal
endophyte colonization on the metabolite content of the
host-plant. It is notably the case for grasses in which infection by
Clavicipitaceae endophytes influences the phenol content of the
plant (Qawasmeh et al., 2012). Recently, Estrada et al. showed that
the presence of endophytes affects some of the leaf metabolites,
although none of these compounds were chemically identified
(Estrada et al., 2013).
From these analyses more than one hundred protonated ions
showed a decreasing intensity in the course of the biotransforma-
tion. To analyze the most abundant and relevant ions, we selected
only those with an intensity superior to 10,000 and a relative fold
change, as defined by XCMS, superior to 100. With these criteria,
five metabolites appeared to be transformed predominantly by
the fungus. Indeed, the pseudomolecular ions [M+H]+ at m/z
371.21, 373.22, 579.14, 621.18 and 651.19 underwent a significant
decrease of intensity 40 h after incubation with the mycelium of P.
variabile (Fig. 1). Among these metabolites, those at m/z 579.14,
621.18 and 651.19 were the most transformed.
In a previous study, we showcased the cultivable fungal diver-
sity present in the leaves of Cephalotaxus harringtonia (Knight ex J.
Forbes) K. Koch (Langenfeld et al., 2013), an Asian medicinal plant
rich in cytotoxic compounds, whose phytochemical content has
been particularly well described (Abdelkafi and Nay, 2012; Bocar
et al., 2003; Evanno et al., 2008). More than 640 isolates were iden-
tified by ITS rDNA sequencing (Langenfeld et al., 2013). Among
them, Paraconiothyrium variabile Riccioni, Damm, Verkley & Crous
(LCP5644) appeared to be relatively specific to C. harringtonia and
was used to carry out the biotransformation of juglone, a plant
metabolite, into isosclerone, an important aromatic fungal pentake-
tide (Prado et al., 2013). Furthermore, the chemical communication
between P. variabile and the phytopathogene Fusarium oxysporum
was dissected by metabolomic means, showing the importance of
fungal oxylipins during their competition (Combes et al., 2012).
In this context, we have investigated the effect of P. variabile on
the secondary metabolome of C. harringtonia by following the
alteration of leaf extracts in the presence of the fungus through
metabolomic profiling and analysis. Remarkably, this plant pro-
duces a highly diverse set of secondary metabolites, especially in
the alkaloid, diterpene and flavonoid series (Abdelkafi and Nay,
2012). Since the access to plant compounds by current phytochem-
ical methods can be time-consuming and technically cumbersome,
only allowing the isolation of the main components, we designed
an alternative approach to study the impact of the endophytic fun-
gus on the host-plant metabolic totum. The biotransformation of
the plant extract (referred to as the metabolome) by P. variabile
was analyzed using comparative metabolomics. The transforma-
tion of the main plant compounds present in the extracts was thus
monitored by mass spectrometry coupled to the XCMS analytical
tool (Smith et al., 2006). The chemical identification of these com-
pounds was achieved using the available databases and our knowl-
edge of the C. harringtonia phytochemistry (Bocar et al., 2003;
Evanno et al., 2008) (Abdelkafi and Nay, 2012). Their identity
was confirmed after isolation from the crude extract and rigorous
structural characterization.
2.2. Isolation and characterization of the main compounds
metabolized by P. variabile
We started by analyzing the compounds that had been most
transformed generating ions at m/z 579.14, 621.18 and 651.19.
We isolated them from the crude extract by semi-preparative HPLC
guided by MS detection, giving the tree main metabolites at m/z
579.14, 621.18 and 651.19. The analysis of 1D and 2D NMR spectra,
along with the MS/MS fragmentations and bibliographic data,
allowed unambiguous characterization of these compounds. The
collision-induced dissociation (CID) experiments on compound
(1), which displays a pseudomolecular protonated ion [M+H]+ at
m/z: 579.14, led to two main fragments at m/z 433 and 271, corre-
sponding to the successive loss of 146 u and 162 u. These were
attributed to the loss of a deoxyhexose and a hexose suggesting
that compound 1 is a diglycoside (Fig. 2).
The 1H MNR spectrum of compound 1 in CD3OD displayed an
olefinic signal at dH 6.51 (1H, s, H-3) characteristic of a flavone
structure. This compound appeared to be dihydroxylated at posi-
tion 40 and 7, as indicated by signals at dH 7.81 (2H, d, J = 8.7 Hz,
H-20, H-60), dH 6.92 (2H, d, J = 8.7 Hz, H-30, H-50) which are charac-
teristic of a p-hydroxyphenyl ring, and at dH 6.63 (1H, d, J = 2.2 Hz,
H-8) and dH 6.60 (1H, d, J = 2.2 Hz, H-6) which reveal the presence
of a phloroglucinol ring. Two anomeric protons at dH 5.37 (1H, d,
J = 1.5 Hz, H-1000) and dH 5.30 were also (1H, d, J = 6.6 Hz, H-100) pres-
ent, characteristic of the diglycoside part, and a methyl proton at
dH 1.12 (3H, d, J = 7.0 Hz, H-6000) showing the deoxyhexose nature
of one of the sugar unit. The interglycosidic linkage as well as
the attachment position of the diglycoside to the aglycone was
confirmed by rigorous analysis of the 1HA1H COSY and 1HA13C
HMBC correlations, both showing 2-bond and 3-bond scalar cou-
plings. The configuration of the glycosidic linkage of the glucopy-
Our work revealed that P. variabile specifically metabolizes the
glycosylated flavonoids of C. harringtonia, leading to the production
of the aglycone moiety. By analogy with the role of flavonoids in the
pre-symbiotic stage of two major plant–microbe symbioses, i.e.
legume–rhizobia and plant–arbuscular mycorrhizal fungi (Abdel-
Lateif et al., 2012), we investigated the impact of these aglycone
flavonoids on spore germination and hyphal elongation of P. vari-
able. We show that these flavonoids significantly increase of the
hyphal growth of germinated spores of the fungal endophyte.
ranoside and rhamnopyranoside were determined to be b and a,
3
respectively, on the basis of the J1 –2 (6.6 Hz) and 3J1
00
00
000–2000
(1.5 Hz) values of the anomeric protons. Thus, from these results
and from comparison with bibliographic data, the structure of this
compound was assigned as apigenin 5-O-a-L-rhamnopyranosyl-
(1?3)-b- -glucopyranoside (1) which was previously isolated
D
from a Cephalotaxus tree (Bae et al., 2007) (Fig. 2).
In the same way, we identified by one-and two-dimensional
NMR as well as MS/MS experiments, compound 2 which displayed
a pseudomolecular protonated ion [M+H]+ at m/z: 621.18 as apige-
2. Results
nin 5-O-a-L D-glucopy-
-rhamnopyranosyl-(1?2)-(600-O-acetyl)-b-
2.1. Metabolomics analysis of the C. harringtonia crude extract
transformed by the endophyte P. variabile
ranoside, also known as euryanoside (2) (Inada et al., 1989).
Compound 3 with an m/z 651.19 was assigned as chrysoeriol 5-O-
a-L D-glucopyranoside,
-rhamnopyranosyl-(1?4)-(600-O-acetyl)-b-
Biotransformations of the crude extract of C. harringtonia by the
endophyte P. variabile were monitored by standardized LC–MS
another flavone glycoside previously isolated from a Cephalotaxus
tree (Zhang et al., 2011) (Fig. 2).

Y. Tian et al. / Phytochemistry 108 (2014) 95–101
97
Fig. 1. Decrease of the relative intensity of the protonated ions [M+H]+ at m/z 651.19, 621.18, 579.14, 373.22 and 371.21 of the metabolites mainly biotransformed by P.
variabile in the crude extract of C. harringtonia at 16, 20, 40 and 60 h post-inoculation by comparison to control (t = 0) generated by XCMS software. Stars indicate significant
differences calculated by XCMS by comparison to control (t = 0) at t = 40 h and t = 60 h. ⁄⁄p < 0.01 (n = 3).
OH
4'
9
HO
7
O
O
2
1'
2'
HO
HO
10
(1)
5
4
O1"
OH
O
m/z : 271
O
m/z : 433
H3C
HO
O 1"'
HO
OH m/z : 579.14
OH
4'
4' OH
O
9
9
HO
7
O
O
2
HO
O
7
O
O
2
O
1'
1'
2'
2'
O
O
10
10
5
4
5
4
(2)
(3)
O1"
O1"
OH
HO
HO
O
O 1"'
O
O
HO
m/ z : 271
m/z : 301
O
H3C
HO
m/ z : 475
m/ z : 505
O1"'
H3C
HO
HO
OH
m/z : 651.19
HO
OH m/ z : 621.18
1H-13 C HMBC correlations
Fig. 2. Structures of the main compounds (1–3) isolated from the leaves of C. harringtonia (A) biotransformed by the endophytic fungus P. variabile and their fragments at m/z
provided by fragmentation of their respective protonated ion [M+H]+ at m/z 579.14, 621.18, 651.19 from CID experiments. Key NMR correlations (HMBC) are also represented
for localization of the acetate group of compound (2) and are identical for compound (3).
2.3. Preparative scale biotransformation of the isolated compounds 1–
3
from flavonoids 1 and 2, apigenin (4) was characterized while,
from flavonoid 3, the small amount of product (5) could be
assigned as chrysoeriol by comparison with a commercial standard
(Fig. 3).
Preparative scale biotransformations of the pure glycosylated
flavonoids 1–3 obtained from the crude extract of C. harringtonia
were performed. Compounds 1–3 were added to P. variabile in a
phosphate buffer. In all cases, large decreases of the substrate con-
centrations were observed by HPLC monitoring after 24 h of inoc-
ulation. The resulting products were isolated by chromatography
before NMR and MS analyses. From these, we saw deglycosylation
reactions in all cases, leading to two different aglycones. Indeed,
2.4. Flavones 4 and 5 increase the growth of P. variabile hyphae
We investigated the respective impact of glycosylated flavo-
noids (1–3) and aglycones (4,5) on the germination and hyphal
growth of the spores of P. variabile. While no effect was observed
on spore germination in any case (data not shown), we clearly

98
Y. Tian et al. / Phytochemistry 108 (2014) 95–101
observed (Fig. 4) that after 24 h, the two aglycones apigenin (4)
and, mostly, chrysoeriol (5) caused a significant improvement of
the hyphal growth compared to the corresponding glycosylated
compounds (1–3). No inhibition of spore germination and hyphal
growth of the spore of P. variabile was observed with either the gly-
cosylated flavonoids, or the corresponding flavonoids.
for understanding the endophytic effects, their impact on the
dynamics of communities and for the evaluation of their potential
as biological control tools (Herre et al., 2007). In contrast to the
mycorrhizal symbiosis and its well-known symbiotic relationship
regulated by molecular signaling (Parniske, 2008), the precise role
of fungal endophytes during their relationship with the host plant
is not known and their ecological role remains underexplored.
To address this issue we have studied the interaction of the
endophytic fungus P. variabile (Langenfeld et al., 2013) with its
host plant C. harringtonia. This was achieved by the analysis of
the fungal impact on the composition of the host-plant secondary
metabolites and their feed-back effect on the fungal growth. A
metabolomic approach, associated with the study of the biotrans-
formation of the isolated plant compounds by the endophyte,
allowed us to prove that three main metabolites out of the hundred
produced by the plant were specifically metabolized. Their chemi-
cal structures were assigned by spectroscopic analyses and
revealed that they all belong to the glycosylated flavonoid series.
In all cases, a significant deglycosylation was observed leading
to the corresponding aglycone. Such hydrolysis reactions have pre-
viously been reported for Aspergillus glycosidases (Pricelius et al.,
2009). The net result of these fungus-driven chemical reactions
may be a flavonoid accumulation in the endophyte environment.
Interestingly, flavonoids are known as signal molecules of the rhi-
zosphere (Weston and Mathesius, 2013). In particular, a flavonoid
effect on the rhizosphere was shown to involve the activation of
the nod genes of symbiotic rhizobia (Peters et al., 1986; Redmond
et al., 1986) and they are suggested to act on mycorrhization by
stimulating germination of spores and hyphal growth (Kikuchi
et al., 2007; Scervino et al., 2005, 2007; Siqueira et al., 1991) and
they could have a possible interaction with strigolactones of the
arbuscular mycorrhiza endosymbiosis (Abdel-Lateif et al., 2012).
Other studies reported the role of flavonoids on the germination
of propagules of fungal species such as Fusarium solani (Ruan
3. Discussion
Worldwide, endophytes have attracted a growing interest not
only for their enormous biological diversity but also for their capa-
bility to biosynthesize biologically interesting secondary metabo-
lites (Prado et al., 2011) (Tan and Zou, 2001). In addition,
endophytes play diverse and important functions in the plant
development, stress tolerance and adaptation to biotic stress of
their host plant. So far, endophytes have been essentially consid-
ered as a reservoir of potentially bioactive compounds and only a
few reports have described their interactions with host plants or
their role within the plant microbiome (Kusari et al., 2012). More-
over, the knowledge of plant–endophyte interactions has essen-
tially been based on the ‘‘balanced antagonism’’ hypothesis. This
hypothesis suggests that colonization of the plant by endophytes
results from an antagonism balance between the host and the
endophyte involving an equilibrium between fungal virulence
and plant defense and in which environmental parameters play a
major role (Schulz et al., 1999). Recently, it was demonstrated that
the plant–endophyte interaction involves a much more complex
and precisely controlled interaction process (Kusari et al., 2012).
Considering the fact that endophytes reside within plants and
are continuously interacting with their host, it is legitimate to sug-
gest that plants have a substantial influence on the in planta phys-
iology of the endophytes and vice versa. Moreover, elucidating the
mechanisms involved in plant–endophyte interactions is crucial
OH
HO
O
O
HO
HO
1
( )
O
O
OH
O
OH
HO
O
O
H3C
HO
O
P. variabile
HO
OH
HO
(4)
OH
OH
O
O
O
2
( )
O
HO
HO
O
O
O
H3C
HO
O
HO
OH
OH
O
OH
HO
O
O
HO
O
O
O
O
P. variabile
O
(5)
3
( )
O
OH
O
O
HO
H3C
HO
OH
O
HO
OH
Fig. 3. Biotransformations by P. variabile of the glycosylated flavonoids (1–3) and isolated from the crude extract of C. harringtonia and structures of the obtained aglycone
moieties (4,5) evidenced by metabolomics.

Y. Tian et al. / Phytochemistry 108 (2014) 95–101
99
Fig. 4. Hyphal length (mm) from the spores of P. variabile in presence of glycosylated flavonoids (1–3) and aglycones (4–5) after 24 h and compared to control DMSO.
⁄⁄⁄p < 0.001 (n = 3).
et al., 1995). Moreover, flavonoids are important for pigmentation
and UV light protection. They serve as signals for pollinators and
other beneficial organisms, participate in hormone signaling, and
function as phytoalexins. A number of biological processes, such
as transcriptional regulation, signal transduction, and cell-to-cell
communication, are influenced by flavonoids. Due to their
antioxidant activity, flavonoids appear to play an important role
in the regulation of reactive oxygen species (ROS) in plant cells.
(Ringli et al., 2008). They also display antibacterial and antifungal
properties and are thus part of the defensive arsenal produced by
the host-plant (e.g. phytoalexins and phytoanticipins) against
pathogens (Weston and Mathesius, 2013). On the host-plant side,
flavonoids are biosynthesized in the cytoplasm and are stored in
the vacuole as sugar-conjugated forms (glycosylated) leading to
better stability and water solubility. Usually, they need to be
released and then cleaved by b-glycosidases into their active form
to exert functions in the plant cytoplasm (Weston and Mathesius,
2013).
Accordingly, we checked for an effect of compounds 4 and 5 on
the growth of P. variabile. It turned out that compounds 4 and 5, in
contrast to the glycosylated parent compounds 1–3, have a positive
impact on the length of fungal hyphae but no effect on spore ger-
mination. Taken together, our results strongly suggest that the
endophyte P. variabile induces a deglycosylation of flavonoids,
probably involving the secretion of glycosidases and thereby pro-
moting its own growth in the host-plant. This up-regulated growth
of the fungus may thus improve the colonization of the plant by
the endophyte.
5. Experimental
5.1. General experimental procedures
Optical rotations were determined with a Perkin–Elmer Model
341 Polarimeter and the [a]
D values are given in deg.cm2.gꢀ1. Mass
spectra were recorded on an API Q-STAR PULSAR i of Applied Bio-
system. For the CID spectra, the collision energy was 40 eV and the
collision gas was nitrogen. All NMR experiments were recorded on
Bruker Avance III HD 400 MHz and 600 MHz spectrometers (Wiss-
embourg, France) equipped with a BBFO Plus Smartprobe and a tri-
ple resonance TCI cryoprobe, respectively.
5.2. Fungal material
The fungus was isolated in October 2008 as an endophyte from
a needle of C. harringtonia referenced under N° 2686 in the Arbore-
tum de Chèvreloup (MNHN) (Langenfeld et al., 2013). Both mor-
phological assessment and internal transcribed species (ITS)
sequencing were performed to characterize the isolate as Para-
coniothryrium variabile. It is maintained at the LCP culture collec-
tion (culture collection of the Museum National d’Histoire
Naturelle, Paris) under the number LCP 5644.
5.3. Plant material
C. harringtonia (N° E1/2692) were sampled in the area of the
‘‘Arboretum de Chèvreloup’’ (MNHN) (48° 490 4900 N, 02° 126 060
4200 E) in the southwestern suburb of Paris (Rocquencourt, Yvelines,
France).
4. Conclusion
5.4. Extraction of the secondary metabolites of C. harringtonia
Our work suggests that flavonoids, other than their well-known
importance in plant root endosymbiosis, may often play a role in
endophyte hyphal growth stimulation which may strengthen the
plant–endophyte relationship such as shown for the mutualistic
relationship between P. variabile and C. harringtonia. Moreover,
the release of flavonoid aglycones caused by the endophyte could
have a profound impact on the physiology and defense reactions
of the host-plant. Our work constitutes a first step in deciphering
one possible molecular mechanisms involved in the colonization
of plants by a non-pathogenic endophytic fungus, as it highlights
a chemical aspect of the role of endophytes in plants.
Needles of C. harringtonia (40 g) were cut and extracted by suc-
cessive maceration in methanol (MeOH) (3 ꢁ 500 mL) under agita-
tion at ambient temperature. After filtration and evaporation, 12 g
of crude extract were afforded.
5.5. Purification of the secondary metabolites of C. harringtonia
Apigenin-5-O-
side (1), euryanoside (2) and chrysoeriol 5-O-
syl-(1?4)-(600-O-acetyl)-b-
-glucopyranoside (3) were purified
a
-
L
-rhamnopyranosyl-(1?3)-b-
D-glucopyrano-
a
-L
-rhamnopyrano-
D

100
Y. Tian et al. / Phytochemistry 108 (2014) 95–101
by semi-preparative HPLC (Agilent Technologies 1260 Infinity)
coupled to a Diode Array Detector using the following gradient
pyranoside or euryanoside (2, available from purification) solubi-
lized in DMSO (1 g/mL) and added to the mycelium in phosphate
buffer (final concentration 0.3 mg/mL). Biotransformations were
performed at 27 °C in an orbital shaker and were monitored by
HPLC. After total disappearance of euryanoside (2) (T = 21 h), the
reaction was stopped as described above and the product obtained
(4.72 mg, 85% yield) was analyzed by LC/MS (10 mg/mL in MeOH)
and compared to the aglycone apigenin standard (4).
with a C18 Eclipse XDB column (21.2 ꢁ 150 mm, 5
lm) with a
flow of 10 mL/min.
Mobiles phases were (A): 95% water (0.05% trifluoroacetic acid),
5% acetonitrile and (B): 5% water (0.05% TFA), 95% acetonitrile.
The separation was achieved at a flow rate of 10 mL/min with
the following gradient: 10% B during 2 min, 10–40% B in 35 min,
40–80% B in 38 min, 80% B during 2 min, 80–90% B in 43 min,
90% B during 2 min, 90–10% B in 45 min. From 100 mg of crude
extract in MeOH (volume injected: 100
obtained respectively at 13.0 (4.8 mg), 18.5 (12.3 mg) and 19.1
(3.1 mg) minutes.
l
L), compounds (1–3) were
5.9. Biotransformation of chrysoeriol 5-O-
a-L-rhamnopyranosyl-
(1?4)-(600-O-acetyl)-b-
D-glucopyranoside (3) by P. variabile
The same protocol as described above was used from 3 mg of
chrysoeriol 5-O- D-
-rhamnopyranosyl-(1?4)-(600-O-acetyl)-b-
5.6. Biotransformation of the crude extract of C. harringtonia by P.
variabile
a
-L
glucopyranoside (3, available from purification) solubilized in
DMSO (1 g/mL) and added to the mycelium in phosphate buffer
(final concentration 0.5 mg/mL). Biotransformations were per-
formed at 27 °C in an orbital shaker and were monitored by HPLC.
After total disappearance of the product (3) (T = 24 h), the reaction
was stopped as described above and the product obtained was ana-
lyzed by LC/MS (10 mg/mL in MeOH) and compared to the agly-
cone chrysoeriol standard (5) (purchased from Extrasynthese,
France). Due to the low amount of (3), the reaction of biotransfor-
mation was only monitored by LC/MS with a comparison to the
commercial standard chrysoeriol. The product was not isolated.
Liquid culture media containing (g/L) glucose 16, yeast extract
4, malt extract 10 and soybean peptones 5 (YMS medium) were
sterilized without glucose at 120 °C for 20 min. The separately ster-
ilized glucose solution was added afterwards. Flasks (250 mL) con-
taining 125 mL of YMS-culture medium were inoculated with
150 lL glycerol spores suspension of P. variabile and incubated at
27 °C and 160 rpm (orbital shaker). After 96 h of culture, the bio-
mass was collected by filtration and cells (3 g) were suspended
in 10 mL of phosphate buffer (0.2 M, pH 6.5). The crude extract
of C. harringtonia (1 g/mL) was dissolved in dimethylsulfoxide
(DMSO) and was added to the buffer (final concentration: 10 mg/
mL). Biotransformations were performed at 27 °C in an orbital sha-
ker (160 rpm). 3 mL of samples (mycelium and supernatant) were
collected at 0, 16, 20, 40, and 60 h for a kinetic monitoring. At each
time the reaction was stopped by cooling to ꢀ4 °C and 2 mL of
MeOH were added. After sonication (30 min) and centrifugation
(4000 rpm, 15 min, 4 °C), the supernatant was sampled, dried
5.10. LC/MS and LC/MS/MS experiments on metabolite extracts
obtained from biotransformations
Samples from biotransformations were analyzed on a liquid
chromatography (UltiMate 3000Ò, Dionex, Germany) coupled to a
Quadrupole-Time of flight (Q-TOF) hybrid mass spectrometer
equipped with an electrospray ionization source. The injected vol-
and diluted in 600 lL of MeOH before analysis by LC/MS or LC/
MS/MS. Controls of the crude extract in phosphate buffer and of
the mycelium alone have also been made to check the stability
of the compounds in the buffer and the absence of interference
with the secondary metabolism of the fungus.
ume was 10
Eclipse XDB-phenyl column, 3.5
were milliQ water containing 0.1% (v/v) formic acid (A) and aceto-
nitrile containing 0.08% (v/v) formic acid (B). The separation was
lL. Extract separation was conducted on an C18AQ
l
m, 1.0 ꢁ 100 mm. Mobile phases
achieved at a flow rate of 40 lL/min with the following gradient:
5.7. Biotransformation of the apigenin 5-O-
a
-
L
-rhamnopyranosyl-
3% B during 5 min, 3–40% B in 35 min, 40–80% B in 37 min, 80–
95% B in 40 min, 95–5% B in 48 min. LC/MS and LC/MS–MS analyses
were carried out in positive electrospray ionization mode. For LC/
MS analyses, the capillary voltage was set to 2500 V, the ion spray
potential and declustering potential were 5200 V and 40 eV. Full
scan mass spectra were performed from 50 to 1300 m/z at 1 s/scan
in continuum mode. For LC/MS–MS analyses cone voltages of 42 V
and collision energies of 40 eV were applied.
(1?3)-b- -glucopyranoside (1) by P. variabile
D
5 mg of apigenin 5-O-a-L-rhamnopyranosyl-(1?3)-b-D-gluco-
pyranoside (1) purified by HPLC from the crude extract of C. har-
ringtonia was dissolved in DMSO (1 g/mL) and was added (final
concentration: 0.5 mg/mL) to 10 mL of mycelium in buffer
obtained as describe above.
Biotransformations were performed at 27 °C in an orbital shaker
(160 rpm). 0.5 mL of samples (mycelium and supernatant) were
collected at 0, 4, 8, 24, 32, 46 h for a kinetic monitoring. At each time
5.11. LC/MS data processing and analysis
the reaction was stopped by cooling to ꢀ4 °C and 200
lL of MeOH
were added. After sonication (30 min), the mycelium was removed
by filtration in vacuo after being thoroughly washed by ethyl ace-
tate. The resulting supernatant was extracted by ethyl acetate tree
times (30 mL). Organic phases were combined, dried over MgSO4
and evaporated under vacuum. The product (1.74 mg, 75% yield)
was then diluted in methanol at a concentration of 10 mg/mL
before analysis by LC/MS and comparison with the standard of
the aglycone apigenin (4) available in the laboratory.
Analyst QSÒ software (ABsciex) was used to convert the original
mass spectrometry data files (*.wiff) to a more exchangeable for-
mat (*.cdf) with the included File translator utility. The data were
then processed by the open-source XCMS software (Smith et al.,
2006) (XCMS parameters for the R language were implemented
in an automation script). Without using internal standards, the
method dynamically identified hundreds of endogenous metabo-
lites for use as standards, calculating a nonlinear retention time
correction profile for each sample. This tool allowed us to extract
ions statistically over- or under-represented between two groups
of samples (the software calculates t-test and p-value). The final
result data table was then imported to ExcelÒ. The analysis was
performed on LC/MS data issued from three independent culture
experiments made in duplicate.
5.8. Biotransformation of apigenin 5-O-
a-L-rhamnopyranosyl-(1?2)-
(600-O-acetyl)-b-
D
-glucopyranoside (2) by P. variabile
The same protocol as described above was used from 12.8 mg of
apigenin-O-
-L-rhamnopyranosyl-(1?2)-(600-O-acetyl)-b-D-gluco-
a

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This project was supported by the following grants: «Projets Inno-
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«Biodiversité des Microorganismes dans les Ecosystèmes Actuels
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