Phytochemistry 65 (2004) 813–820
www.elsevier.com/locate/phytochem
Tuber borchii fruit body: 2-dimensional profile and protein
identification
Raffaella Pierleonia, Michele Buffalinia, Luciana Vallorania, Chiara Guidia,
Sabrina Zeppaa, Cinzia Sacconia, Piero Puccib, Angela Amoresanob,
Annarita Casbarrab, Vilberto Stocchia,*
a
Istituto di Chimica Biologica ‘‘Giorgio Fornaini’’, Università degli Studi di Urbino ‘‘Carlo Bo’’, Via A. Saffi, 2, I-61029 Urbino (PU), Italy
Dipartimento di Chimica Organica e Biochimica, Università degli Studi di Napoli ‘‘Federico II’’, Complesso Universitario Monte S. Angelo,
Via Cynthia, 8, I-80126 Napoli, Italy
b
Received 14 November 2003; received in revised form 30 January 2004
Abstract
The formation of the fruit body represents the final phase of the ectomycorrhizal fungus T. borchii life cycle. Very little is known
concerning the molecular and biochemical processes involved in the fructification phase. 2-DE maps of unripe and ripe ascocarps
revealed different protein expression levels and the comparison of the electropherograms led to the identification of specific proteins
for each developmental phase. Associating micropreparative 2-DE to microchemical approaches, such as N-terminal sequencing
and 2-D gel-electrophoresis mass-spectrometry, proteins playing pivotal roles in truffle physiology were identified.
# 2004 Elsevier Ltd. All rights reserved.
Keywords: Tuber borchii; Ectomycorrhizal fungi; Fruit body maturation; Edman degradation; Mass spectrometry
1. Introduction
Truffles are hypogeous ascomycetous fungi belonging
to the genus Tuber which give origin to highly specialised
symbiotic associations with the fine roots of gymnosperms and angiosperms, named ectomycorrhizae
(Trappe, 1979). They are in great demand due to the
organoleptic properties of their fruit bodies (white
truffles), having a distinct flavour and aroma, while also
of considerable interest in the fields of forestry and
agronomy. The ontogenetic cycle of these fungi consists
in a limited initial phase of mycelial growth during
which hyphae proliferate; successively, they come into
contact with the roots of host plants leading to the
development of the ectomycorrhiza. In the final developmental phase the fungus organises a fruit body the
role of which is to produce sexual spores that are dispersed in the soil when the fruit body ripens. From these
* Corresponding author. Tel.: +39-0722-305262; fax: +39-0722320188.
E-mail address: v.stocchi@uniurb.it (V. Stocchi).
0031-9422/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.02.012
spores, vegetative mycelia develop giving rise to a new
extraradical phase thus closing the truffle life cycle.
Tuber hypogeous fruit bodies have a globular structure and their development begins with the aggregation
of different types of hyphae which give origin to an
external tissue, the peridium, and an internal tissue,
named gleba. Within this last tissue the ascogonium
forms and from this the reproductive hyphal element,
the ascus containing the ascospores, originates (Pegler,
1993; Read and Beckett, 1996). Although the succession
of events leading to fructification has been investigated,
little is known about the molecular and biochemical
processes on which hyphal differentiation, fruit body
development and the subsequent maturation, are based
(Balestrini et al., 1996). Studies on truffle biology are
difficult because, unlike other filamentous fungi such as
Pisolithus tinctorius (Malajezuk et al., 1990) or Hebeloma cylindrosporum (Debaud and Gay, 1987), these
mycelia grow very slowly in vitro and it is difficult to
obtain mycorrhizae or fruit bodies under axenic and
controlled conditions. Using an untargeted technique
such as the mRNA differential display we recently
identified several cDNAs differentially expressed during
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R. Pierleoni et al. / Phytochemistry 65 (2004) 813–820
the fructification phase of T. borchii (Zeppa et al., 2002).
By this approach a global vision of the gene expression
profile related to the maturation of fruit bodies, was
achieved. The identification of several putative genes
involved in the development of T. borchii ascocarps
represents a starting point in understanding the molecular mechanisms of cellular differentiation leading to
the fructification process. In the present study we performed the analysis of the ascocarp’s proteome using
two-dimensional electrophoresis (2-DE) in order to
obtain more information about events occurring during
this phase of the truffle life cycle. This electrophoretic
technique possesses high potentiality since it permits the
separation of most of the proteins of an organism or
tissue on a single gel and reveals possible modifications
in protein expression related to disease, stress conditions
or ambiental factors. 2-DE maps of both T. borchii ripe
and unripe fruit bodies were produced and analysed in
this study in order to verify the presence of proteins
specifically expressed in the two developmental stages.
Successively, in order to unequivocally identify some of
the ascocarp’s proteins micropreparative 2-DE was
associated with microchemical approaches, such as Nterminal sequencing and mass spectrometry, and the
data obtained were subsequently utilised to perform
cross-species matching (Wilkins and Williams, 1997).
2. Results and discussion
Three analytical two dimensional electrophoresis runs
on both unripe (stage 0) and ripe (stage 5) T. borchii
fruit bodies were performed. As shown in Fig. 1a and b
the electropherograms evidenced an appreciably different
protein expression level related to the maturation stage.
In particular, the careful comparison of the 2-DE maps
and the evaluation of different parameters carried out
using the Melanie 3 software, revealed that, in the ripe
fruit body, a decrease in the expression level of many
30–150 kDa proteins occurs. Concurrently other proteins
resulted to be more expressed in the mature tissues
(Table 1). Moreover, the overlapping and the further
matching of the electropherograms revealed specific
spots for each developmental stage (circled white or
black in the two electropherograms). All these results
might be explained by an involvement of these proteins
in different phases of the fructification process and with
a higher metabolic activity in the unripe ascocarps. It
stands to reason that in the initial phase of fructification
a higher energy supply is necessary to meet the cell
demand since biosynthetic pathways are strongly
induced. In fact, for the differentiation of the specialised
hyphae in asci and the subsequent formation of the
ascospores, new membranes and cell walls are required
(Kues, 2000). Moreover, meiotic and mitotic processes are
particularly active during spore maturation (Balestrini et
al., 2000). Therefore, a large quantity of carbohydrates,
polypeptides, lipids and nucleotides are necessary and
most metabolic pathways must be stimulated for their
production.
These preliminary electrophoretic data are in agreement with the molecular evidence obtained in a previous
study according to which ascocarp maturation is strictly
associated with differential gene expression, and most
of the differentially expressed identified genes show a
much higher expression level in the unripe fruit body
compared to the ripe one (Zeppa et al., 2002).
In order to gain more information regarding the
events leading to ascocarp development, the identification of proteins specific to one or both differentiation
phase has been carried out. About 20 spots excised from
2-DE gels were used for protein identification through
cross-species matching (Wilkins and Williams, 1997)
utilising both amino terminal sequence and mass spectrometry data.
The N-terminal sequencing was performed on
Coomassie-blue stained proteins excised from 2-DE gels
and then electroeluted as described in the ‘‘Experimental’’
Fig. 1. 2-DE electropherograms of T. borchii unripe (a) and ripe (b) fruit bodies. Specific proteins of the two fructification phases are circled; the
spots subjected to Edman degradation or mass spectrometry analysis are indicated also by an alphabetical letter.
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R. Pierleoni et al. / Phytochemistry 65 (2004) 813–820
Table 1
Characteristics of T. borchii constitutive protein spots
Spot
Status
Mol. mass (Da)a
pI a
A
downregulated
47,0001320
4.94 0.04
B1
upregulated
B2
upregulated
5.50 0.01
5.65 0.03
37,5001560
B3
upregulated
5.80 0.02
B4
upregulated
5.95 0.04
C1
upregulated
5.55 0.01
C2
no change
5.70 0.03
35,0002450
C3
upregulated
5.85 0.06
C4
upregulated
5.98 0.01
D1
no change
5.30 0.04
30,0002980
D2
no change
5.45 0.02
E
downregulated
26,6002100
5.35 0.02
G
upregulated
25,5001540
5.45 0.03
I
upregulated
12,5001100
6.50 0.01
L
no change
15,5001340
5.00 0.05
M
no change
11,0001120
5.10 0.04
N
no change
10,8001090
5.05 0.01
P
downregulated
20,6601560
6.50 0.03
R
no change
10,2701250
6.52 0.09
Sample
Normalized volumeb
Normalized O.D.b
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
unripe fruit body
ripe fruit body
1.580.12
0.060.01
0.350.08
1.140.21
0.570.08
1.40.32
0.370.07
0.660.09
0.370.07
0.840.06
0.150.01
0.320.05
0.160.02
0.120.01
0.30.07
0.780.09
0.180.03
0.710.08
0.160.02
0.140.05
0.210.04
0.20.03
0.240.08
0.060.01
0.040.009
0.110.02
0.070.002
0.140.02
0.310.08
0.40.07
0.190.06
0.230.05
0.130.05
0.120.03
0.540.09
0.260.03
0.650.1
0.780.05
1.090.15
0.790.05
1.050.2
1.770.5
1.070.25
1.750.41
1.040.24
1.670.33
1.080.18
1.770.23
0.850.03
1.480.36
0.960.11
1.60.24
1.060.11
1.720.3
0.980.1
1.720.22
1.010.18
0.820.13
0.990.24
0.930.11
0.980.13
1.170.31
0.550.02
0.930.1
0.920.15
1.590.13
1.010.17
1.570.2
1.080.13
1.50.1
0.940.11
1.510.23
1.120.24
1.80.31
1.120.17
1.80.27
a
Molecular masses and isoelectric points were calculated with Melanie 3 (GeneBio) using human plasma proteins as internal standards; the
reported values are the mean of three independent experimentsS.E.
b
Spot normalised volumes and optical density (O.D.) were calculated with Melanie 3 (GeneBio).
section. The possible interference due to the presence of
glycine in the electroelution buffer was avoided by extensive dialysis against HPLC-grade water.
Unfortunately, none of the phase-specific spots analysed provided information when subjected to Edman
degradation, thus suggesting that their N-terminals were
blocked. In the case of the immature ascocarp specific
spots, this could be due to the scarce quantity of
material available. In fact, it is very difficult to find
unripe fruit bodies and a large quantity of material is
required for the micropreparative electrophoretic runs
and the following analyses. Therefore, in order to avoid
the possible problems due to sensitivity our attention
was focused on proteins expressed in both maturation
stages. Only in few cases the N-terminal sequence analysis was successful (Table 2). The N-terminal tags and
the apparent pI and Mr values obtained from the 2-DE
gel’s calibration were used for matching against SWISSPROT (http://www.expasy.org), FASTA (http://www.ebi.
ac.uk) and NCBI (http://www.ncbi.nlm.nih.gov, using
BLASTP algorithm) database entries to find homology
with known proteins. Significant similarity to known
proteins of interest possibly playing an important role in
the T. borchii life cycle was found.
For instance, the N-terminal tag of the spot named R
(Table 2) showed significant homology with metallothioneins obtained from different sources including
fungi. This family of proteins is responsible for metal
ion chelation through the formation of tetrahedrically
coordinated metal-thiolate clusters, and is therefore of
primary importance in buffering the intracellular concentration of free thiophilic metal ions (Clemens, 2001;
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R. Pierleoni et al. / Phytochemistry 65 (2004) 813–820
Table 2
Edman degradation sequences analysis of T. borchii constitutive proteins
Spot
pI a
Mol. mass
(Da)a
Sequence tagb
Database
hitc
Homologies
S/E/I d
B4
5.950.04
375001560
SFDHKVAGYDCCLLE
P45699
P46239
P46236
CAD58875
AAM77702
P22669
P57661
P25528
Q51383
P00852
Endoglucanase (Fusarium oxysporum)
41/0.0096/44.4
21/0.21/66.7
32/0.049/50
20/0.12/60
24/0.084/33.3
20/0.15/40
45/4.9/62.5
41/18/50
41/18/50
24/0.088/60
L
5.000.05
155001340
EQDCACXSNPLQG
P
6.500.03
206601560
RTVIHMAIACLI
NP039504
R
6.520.09
102701250
DPLPQCFTLESCCSQC
P58280
P02802
CAD13456
AAF78959
CAB85689
CAA06385
NP593743
Endoglucanase (Agaricus bisporus)
Endoglucanase (Emericella desertorum)
Endoglucanase (Apergillus aculeatus)
Ferredoxin, 2Fe-2S (Buchnera aphidicola)
Ferredoxin, 2Fe-2S (Escherichia coli)
Ferredoxin, 2Fe-2S (Pseudomonas aeruginosa)
ATP-synthetase A chain (protein 6)
(Emericella nidulans)
ATP-synthetase A chain (protein 6)
(Schizosaccharomyces pombe)
Metallothionein (Bovin)
Metallothionein (Mouse)
Metallothionein (Gigaspora margarita)
Metallothionein (Candida albicans)
Metallothionein (Agaricus bisporus)
Metallothionein (Podospora anserina)
Metallothionein (Schizosaccharomyces pombe)
19/0.18/50
53/31/54
48/25/46
33/0.0096/25
29/0.014/30
34/0.005/50
34/0.0005/42
31/0.0033/30
a
Molecular masses and isoelectric points were calculated with Melanie 3 (GeneBio) using human plasma proteins as internal standards; the
reported values are the mean of three independent experimentsS.E.
b
N-terminal sequences were obtained utilising the automated Edman sequencer LF3000 Protein Sequencer (Beckman).
c
Database entries.
d
S, score; E, expected value; I, percent identity.
Prasad and Stzalka, 2002). Several studies have evidenced high levels of mycorrhization in soils containing
different kinds of heavy metal contamination, indicating
that in mycorrhizal fungi a heavy metal tolerance has
evolved. Because of their function metallothioneins may
play a crucial role in the fungal process of metal tolerance.
Therefore, the presence of these proteins in the tissues
of T. borchii ascocarps could suggest an involvement of
this fungus in the phytoremediation of the soil i.e. use
of the plants to remove non-volatile and immisible soil
contents (Weissenborn et al., 1995; Schützendübel and
Polle, 2002).
Comparing the two-dimensional electropherograms
of both ripe and unripe T. borchii fruit bodies no significant differences were detected regarding the expression
level of spot R, thus suggesting a constitutive role of
metallothioneins during the fructification phase of this
fungus (Table 1). At present further analyses are in progress to clone the complete gene coding these proteins.
The subsequent evaluation of its expression levels
during the different phases of truffle maturation as well
as in the mycelial and symbiotic tissues will allow a
better understanding of the physiological role of metallothioneins in the ontogenetic cycle of our fungus.
The N-terminal sequence of spot B4 showed the highest similarity with endoglucanases from fungi belonging
to Fusarium, Aspergillus, Agaricus and Emericella
species, respectively, as well as from other organisms
(Table 2). Endoglucanases (E.C. 3.2.1.4, endo-1,4-b-d-
glucanases) belong to the cellulose family which
includes all those enzymes able to degrade crystalline
cellulose to glucose and are produced by a broad range
of organisms. The first step in the colonisation process
of plant roots by mychorrizal fungi involves physical
contact of the outer layers of the cell walls and the
extracellular matrix of the two partners, and some
specialised molecules (proteins, carbohydrates etc.)
probably play essential roles. Then, hyphae progress to
the surface or penetrate the host tissues. In the case of
colonisation of tree roots by ectomycorrhizal fungi,
such as T. borchii, the plants accept a limited penetration
of the mycelium (Podila and Douds, 2000), whereas the
colonisation of plant roots by arbuscolar mycorrhizal
fungi requires the complete penetration of the host cell
by the fungus (Garcia-Garrido et al., 1996). It stands to
reason that cell wall-hydrolysing enzymes such as cellulases must be involved in the establishment of these
symbioses (Garcia-Romera et al., 1990). The capacity of
fungi to produce endoglucanases also allows them to
grow and fruit on particular substrates, thus conferring
to this class of enzymes a pivotal role even in the process
producing energetic substrate (Cai et al., 1999; Buswell
et al., 1996). Biochemical and molecular investigations
carried out with the edible mushrooms Lentinula edodes
(Berk) and Agaricus bisporus showed that the activities
of endoglucanases are strongly regulated during fruit
body development (Ohga and Royse, 2001; Manning
and Wood, 1983). In particular, enzymatic activity, as
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R. Pierleoni et al. / Phytochemistry 65 (2004) 813–820
well as mRNA level, peaked at the veil break stage of
fruit body development. All these results support the
presence of these proteins in both ripe and unripe T.
borchii fruit bodies and suggest an involvement of
endoglucanases even in truffle formation and maturation
processes. Moreover, the comparison of the two electropherograms revealed that this protein is markedly more
expressed in the advanced phase of the fructification
process than in the initial phase as reported above for
other fungi (Table 1). Regarding the N-terminal tags of
spots L and P homology was found with two interesting
proteins that play a fundamental ‘‘energetic’’ role. In
particular, the 13 amino acid N-terminal tag from spot
L showed homology with ferredoxin (2Fe–2S) (Table 2).
These iron-sulfur proteins transfer electrons in a variety
of metabolic reactions and play a crucial function in
ATP-synthesis being one of the components of electron
transport chain complexes. During truffle formation
and maturation processes, most of the biosynthetic
metabolisms are strongly induced and a large amount of
energy is required for spore formation as well as mitosis/meiosis processes (Balestrini et al., 2000), therefore a
deep involvement of ferredoxin in these developmental
phases it is very likely. No substantial changes in the
expression level of this protein were detected suggesting
that it is not influenced by the maturation stage of
ascocarps (Table 1).
Finally, the N-terminal sequence obtained for spot P
matches with various fungal ATP-synthetase A chains
(protein 6) such as those from Schizosaccharomyces
pombe and Emericella nidulans (Table 2). This protein is
an integral membrane protein and a key component of
the proton channel constituting ATP-synthase the
activity of which is of primary importance in this phase
of the T. borchii ontogenetic cycle. The comparison of
2-DE maps revealed a decrease in the expression level of
this protein during truffle maturation (Table 1). These
data were in agreement with the higher energy supply
required in the initial phase of fructification for the
formation of membranes, cell walls and other processes
(Balestrini et al., 2000) and supported the molecular data
concerning T. borchii fruit body maturation previously
obtained (Zeppa et al., 2002).
The evidence obtained regarding the expression levels
of all the analysed spots supports the hypothesis that
the ripe fruit bodies are still metabolically active structures even if to a lesser extent when compared to those
which are not yet ripe.
In order to identify some of the N-terminal blocked
proteins, mass-spectrometry experiments were carried
out, also. Spots from micropreparative 2-DE gels of ripe
and unripe fruit bodies were excised, digested overnight
with trypsin, and analysed by MALDI/TOF. The peptide mass fingerprinting was limited to search for
homologous proteins that might show conserved tryptic
peptides in databases of fungi or other similar species,
but no entries were found. It should be underlined that
this analysis represented just an attempt to find some
homologous sequence; in fact it is well known that
MALDI MS approach can be successfully used with
fully-sequenced genomes or when enough ESTs are
available. Therefore, the peptides digested from the
most abundant spots were fractionated by HPLC. Peptide sequencing by tandem mass spectrometry was
undertaken on all abundant peaks from each spot (1–5
sequences were obtained per spot), leading to sequences
ranging from 5 to 10 residues. The variability in the
number and length of the sequence fragments from each
spot was typical of this kind of analysis (Table 3).
The MALDI analyses performed permitted us to
observe the same peptide pattern in the mass spectra for
spot C1, C2, C3 and C4, thus we combined the peptide
mixtures before LC/MS/MS analyses (named spot C in
Table 3); a similar procedure was applied for D1 and
D2 samples (named spot D). The occurrence of very
similar proteolytic patterns for different spots sharing
the same molecular mass but differing in the isoelectric
point is probably due to post translational modifications such as glycosylation or phosphorylation. Spots
C and D gave us the best results, in spite of this it was
rarely possible to obtain a complete sequence for the
ions isolated in the ion trap and fragmented. Some
interesting data were also obtained for the spots A, I, M
and N. The peptide sequences obtained from each different spots were submitted to the FASTA database but
no certain identification was possible. Due to the limited
amount of proteins, the intensity of the peaks in the
mass spectra of the spots E, G, H and L was not sufficient to furnish relevant MS/MS results.
Table 3
Tandem mass spectrometry analysis of T. borchii constitutive proteins
Spot
Peptide sequence obtained by tandem
mass spectrometrya
Parent ion
A
NH2-FD(L/I)(L/I)(L/I)SR-COOH
NH2-Y(L/I)(L/I)SYPDV(L/I)K-COOH
-(L/I)AGVR-COOH
NH2-(Q/K)P(L/I)(L/I)SDHVPK-COOH
-DS(L/I)(L/I)PK-COOH
-VSV(L/I)PA(Q/K)K-COOH
862.7 (MH+)
1210.8 (MH+)
716.9 (MH+)
1134.4 (MH+)
1167.4 (MH+)
1547.7 (MH+)
nh2-(L/I)EG(L/I)VSGK-COOH
-TSAV(L/I)STGR-COOH
-A(L/I)(L/I)SDPP-COOH
-G(L/I)VFGSGWSVG(L/I)PH-COOH
NH2-WSMTWTVPVPR-COOH
-EP(L/I)(L/I)SDF-CCGFVTFW-DNS(L/I)(L/I)-GSGWSVHEK-COOH
802.6 (MH+)
577.6 (MH2+
2 )
615.9 (MH2+
2 )
822.36 (MH2+
2 )
1359.8 (MH+)
643.8 (MH2+
2 )
859.4 (MH2+
2 )
539.2 (MH2+
2 )
745.9 (MH2+
2 )
C
D
I
M
N
a
The sequences obtained by tandem mass spectrometry contain
ambiguous residues represented within parentheses.
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R. Pierleoni et al. / Phytochemistry 65 (2004) 813–820
In light of the data reported above, assuming that
enough protein material was available and the obtained
amino acid sequences were long enough, the tandem
mass-spectrometry approach resulted to be unsuitable
to T. borchii protein identification certainly due to the
limited availability of information concerning its genome
and proteome.
basis of the percentage of asci containing mature spores:
stage 0=0%, stage 1=1–5%, stage 2=6–25%, stage 3=
26–50%, stage 4=51–75% and stage 5=76–100%. The
maturation stage of the spores was defined by morphological method: the mature spores presented a reddishyellow brown colour and a reticulate ornamentation.
The ascocarps were analysed by morphological and
molecular methods (Pegler et al., 1993; Bertini et al.,
1998) to confirm them as Tuber species.
3. Conclusions
4.3. Two-dimensional electrophoresis
This study represents a useful step toward the comprehension of events responsible for the formation and
maturation of the T. borchii fruit body. The comparison
of 2-DE maps of ripe and unripe ascocarps evidenced a
different protein expression suggesting that the initial
phase of the fructification is associated with higher
metabolic activity compared to that of more advanced
stages of fruit body maturation. Moreover, maturation
is characterised by the appearance and disappearance of
a certain number of specific proteins. Even though there
is a lack of information regarding the T. borchii genome, some of the proteins present in both maturation
stages were identified by Edman degradation analysis.
This provided some new evidence concerning the cellular
processes that might take place during this phase of T.
borchii life cycle. Other proteins were subjected to massspectrometry analysis, but no suitable matches were
obtained from these data. In order to identify the processes leading to fructification, new sequencing and
mass spectrometry experiments are in progress to gain
de novo sequences for all those proteins specific to
either the ripe or unripe T. borchii fruit body.
4. Experimental
4.1. General experimental procedures
All the chemicals and solvents used for Edman
degradation analysis were sequence- or HPLC-grade and
supplied by Beckman (Beckman Coulter, Fullerton-USA).
Trypsin, dithiothreitol and a-cyano-4-hydroxycinnamic acid were purchased from Sigma. HPLCgrade trifluoroacetic acid (TFA) was obtained from
Carlo Erba (Italy). All other reagents and solvents for
the MS analyses were of the highest purity available
from Baker (USA).
4.2. Fruit body collection
T. borchii fruit bodies were collected in an experimental truffle orchard, located near Marina di Ravenna,
in Northern Italy (Zambonelli et al., 2000).
The degree of maturation of the ascocarps was
defined using the following categorised stages on the
For two-dimensional analyses unripe (stage 0) and
ripe (stage 5) T. borchii ascocarps were homogenised
using a Potter homogenizer with a lysis buffer containing
8 M urea, 4% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 40 mM Trisbase and 65 mM dithiothreitol (DTT). To prevent
protease and phosphatase interference 1 mM orthovanadate was added to the buffer and pH adjusted to the
value of 9.0. The suspension obtained was centrifuged at
14,000 rpm for 10 min and the supernatant used for the
electrophoretic runs. For the analytical and micropreparative 2-DEs, 45 mg and 1 mg of total protein
respectively, were loaded onto 18 cm nonlinear Immobiline strips, pH range 3–10 (Amersham), previously
rehydratated (Vallorani et al., 2000). The protein content
was determined according to the Bradford method
(1976), using bovine serum albumin as standard. For
isoelectrofocusing (IEF) a constant voltage of 200 V
was applied for 1–3 hours and increased from 300 to 3500
V over 4 h and stabilised at 5000 V for 22 h. The seconddimensional run was carried out on 9–16% polyacrylamide linear gradient gels at 40 mA/gel constant
current at 9 C. The pI and Mr calibration was performed
by co-electrophoresis of fruit body proteins with 40 mg of
human serum proteins as internal standard (Bjellqvist et
al., 1993). Analytical gels were stained with silver salt as
described in Oakley (1981), while micropreparative gels
were stained with 0.25% (w/v) Coomassie Brilliant Blue
R-250 in 50% methanol/ 20% acetic acid. Silver-stained
2-D gels were scanned with a Hewlett Packard Scanjet 4c
scanner and then processed using the Melanie 3 software
package (Bio-Rad Laboratories, Hercules, USA).
4.4. Electroelution and N-terminal sequencing
Protein spots were excised from micropreparative gels
and electroelution was performed using a Centrilutor
Micro-Electroeluter (Amicon, Millipore-USA) with a
buffer consisting of 3% (w/v) Tris, 14.4% (w/v) glycine
and 1% (w/v) sodium dodecyl sulfate (SDS). A costant
voltage of 200 V was applied for 2 h and then the samples
were dialysed against HPLC-grade water using suitable
cut-off Amicon Centricon (Amicon, Millipore-USA),
then concentrated and dried under vacuum.
R. Pierleoni et al. / Phytochemistry 65 (2004) 813–820
819
Protein samples obtained as above were resuspended
in 30 ml of 30% acetonitrile (v/v) (ACN) and the Nterminal sequence determined using an automatic LF
3000 Protein Sequencer equipped with an on-line Gold
HPLC system (Beckman Instruments, Fullerton-USA)
for the detection of the phenylthiohydantoin (PTH)
amino acids. The PTH amino acids were separated on
an Ultrasphere ODS column (25 cm2.0 mm, 5 mm
particle size).
means of a linear gradient from 5 to 65% solvent B for
60 min at a flow rate of 0.2 ml/min. The effluent was
directly inserted into the ion source through the electrospray probe and both ES/MS and ES/MS/MS spectra
were acquired throughout the entire analysis by using
the software provided by the manufacturers.
4.5. In situ digestion
We thank Dr. Alessandra Zambonelli of the University
of Bologna, Dipartimento di Protezione e Valorizzazione
Agroalimentare, for providing and analysing the truffle
samples. This work was supported by the PRIN: Cofin
2003.
The analysis was performed on the Coomassie-blue
stained proteins excised from 2-D gels. The excised
spots were washed first with ACN and then with 0.1 M
ammonium bicarbonate. Protein samples were reduced
by incubation in 10 mM dithiothreitol (DTT) for 45 min
at 56 C. The cysteines were alkylated by incubation in
5 mM iodoacetamide for 15 min at room temperature
in the dark. The gel particles were then washed with
ammonium bicarbonate and ACN.
Enzymatic digestion was carried out with trypsin
(12.5 ng/ml) in 50 mM ammonium bicarbonate pH 8.5
at 4 C for 4 h. The buffer solution was then removed
and a new aliquot of the enzyme/buffer solution was
added for 18 h at 37 C. A minimum reaction volume,
enough for the complete rehydratation of the gel was used.
Peptides were then extracted washing the gel particles with
20 mM ammonium bicarbonate and 0.1% TFA in 50%
ACN at room temperature and then lyophilized.
4.6. MALDI analyses
MALDI-TOF mass spectra were recorded using an
Applied Biosystem Voyager DE-PRO reflector instrument
on a new MALDI/TOF mass spectrometer. A mixture of
analyte solution and alfa-cyano-hydroxycinnamic acid
(10 mg/ml in ACN/0.1% TFA, 2.5:1, v/v,) was applied
to the metallic sample plate and dried under vacuum.
Mass calibration was performed using external standards.
Raw data were analysed by using the computer software
provided by the manifactures and reported as monoisotopic masses.
4.7. Liquid chromatography-electrospray tandem mass
spectrometry (LC/ES/MS/MS)
Tryptic peptide mixtures obtained as previously
described were analysed by LC/ES/MS/MS ‘‘on-line’’
using an LCQ ion trap instrument (Finnigan Corp., San
Josè, CA). Proteolytic digest was fractionated on an HP
1100 HPLC apparatus (Hewlett-Packard, Palo Alto,
CA) using a narrowbore Phenomenex Jupiter C18
column (2502.1 mm, 300 Å) (Torrance, CA) using
0.05% TFA, 5% formic acid in H2O (solvent A) and
0.05% TFA, 5% formic acid in ACN (solvent B) by
Acknowledgements
References
Balestrini, R., Hahn, M.G., Bonfante, P., 1996. Location of cell-wall
components in ectomycorrhizae of Corylus avellana and Tuber
magnatum. Protoplasma 191, 55–69.
Balestrini, R., Mainieri, D., Soragni, E., Garnero, L., Rollino, S.,
Viotti, A., Ottonello, S., Bonfante, P., 2000. Differential expression
of chitin synthase III and IV mRNAs in ascomata of Tuber borchii
Vittad. Fungal Genetics and Biology 23, 219–232.
Bertini, L., Agostini, D., Potenza, L., Rossi, I., Zeppa, S., Zambonelli,
A., Stocchi, V., 1998. Molecular markers for the identification of the
ectomycorrhizal fungus Tuber borchii. New Phytologist 139, 565–570.
Bjellqvist, B., Hughes, G.J., Pasquali, C., Paquet, N., Ravier, F., Sanchez,
J.-C., Frutiger, S., Hochstrasser, D.F., 1993. A nonlinear wide-range
immobilized pH gradient for two-dimensional elctrophoresis and its
definition in a relevant pH scale. Electrophoresis 14, 1357–1365.
Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of
protein–dye binding. Analytical Biochemistry 72, 248–254.
Buswell, J.A., Cai, Y.J., Chang, S.T., Peberdy, J.F., Fu, S.Y., Yu, H.-S.,
1996. Lignocellulolytic enzyme profiles of edible mushroom fungi.
World Journal of Microbiological Biotechnology 12, 537–542.
Cai, Y.J., Chapman, J., Buswell, J.A., Chang, S.-T., 1999. Production
and distribution of endoglucanase, cellobiohydrolase, and b-glucosidase components of the cellulolytic system of Volvoriella volvacea,
the edible straw mushroom. Applied and Environmental Microbiology 65, 553–559.
Clemens, S., 2001. Molecular mechanisms of plant metal tolerance and
homeostasis. Planta 212, 475–486.
Debaud, J.C., Gay, G., 1987. In vitro fruiting under controlled conditions of the ectomycorrhizal fungus Hebeloma cylindrosporum
associated with Pinus pinaster. New Phytologist 105, 429–435.
Garcia-Romera, I., Garcia-Garrido, J.M., Martinez-Molina, E.,
Ocampo, J.A., 1990. Possibile influence of hydrolytic enzymes on
vesicular arbuscolar mycorrhizal infection of alfalfa. Soil Biology &
Biochemistry 22, 148–152.
Garcia-Garrido, J.M., Garcia-Romera, I., Parra-Garcia, M.D.,
Ocampo, J.A., 1996. Purification of an arbuscolar mycorrhizal
endoglucanase from onion roots colonized by Glomus mosseae. Soil
Biology and Biochemistry 28, 1443–1449.
Kues, U., 2000. Life history and developmental processes in the basidiomycete Coprinus cinereus. Microbiology and Molecular Biology
Reviews 64, 316–353.
Malajezuk, N., Garbaye, J., Lapeyrie, F., 1990. Infectivity of pine and
eucalypt isolates of Pisolithus tinctorius on roots of Eucalyptus
urophylla ‘‘in vitro’’. I. Mycorrhiza formation in model systems.
New Phytologist 114, 627–631.
820
R. Pierleoni et al. / Phytochemistry 65 (2004) 813–820
Manning, K., Wood, D.A., 1983. Production and regulation of cellulase of Agaricus bisporus. Journal of General Microbiology 129,
1839–1847.
Oakley, B.R., Kirsch, D.R., Morris, R., 1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels.
Analytical Biochemistry 105 (2), 361–363.
Ohga, S., Royse, D.J., 2001. Transcriptional regulation of laccase and
cellulase genes during growth and fruiting of Lentinula edodes on
supplemented sawdust. FEMS Microbiology Letters 201, 111–115.
Pegler, D.N., Spooner, B.M., Young, T.W.K., 1993. British Truffles.
A Revision of British Hypogeous Fungi. Royal Botanic Gardens,
Kew, UK.
Podila, G.K., Douds Jr., D.D., 2000. Current Advances in Mycorrhizae Research. American Phytopathological Society, Minnesota,
pp. 69–71.
Prasad, M.N.V., Stzalka, K., 2002. Physiology and Biochemistry of
Metal Toxicity and Tolerance in Plants. Kluwer Academic Publishers,
Dordrecht, The Netherlands, pp. 59–94.
Read, N.D., Beckett, A., 1996. Ascus and ascospores morphogenesis.
Mycological Research 100, 1281–1314.
Schützendübel, A., Polle, A., 2002. Plant response to abiotic stresses:
heavy metal-induced oxidative stress and protection by mycorrhization. Journal of Experimental Botany 53, 1351–1365.
Trappe, J.M., 1979. The order, families, and genera of hypogeous
ascomycotina (truffles and their relatives). Mycotaxon 9, 297–340.
Vallorani, L., Bernardini, F., Sacconi, C., Pierleoni, R., Pieretti, B.,
Piccoli, G., Buffalini, M., Stocchi, V., 2000. Identification of Tuber
borchii Vittad mycelium proteins separated by two-dimensional
polyacrilamide gel electrophoresis using amino acid analysis and
sequence tagging. Electrophoresis 21, 3710–3716.
Weissenborn, I., Leyval, C., Berthelin, J., 1995. Bioavailability of
heavy metals and abundance of arbuscolar mycorrhiza in soil
polluted by atmospheric deposition from a smelter. Biology and
Fertility of Soils 19, 22–28.
Wilkins, M.R., Williams, K.L., 1997. Cross-species protein identification using amino acid composition, peptide mass fingerprinting,
isoelectric point and molecular mass: a theoretical evaluation.
Journal of Theoretical Biology 186, 7–15.
Zambonelli, A., Iotti, M., Rossi, I., Hall, I., 2000. Interaction between
Tuber borchii and other ectomycorrhizal fungi in a field plantation.
Mycological Research 104, 698–702.
Zeppa, S., Guidi, C., Zambonelli, A., Potenza, L., Vallorani, L., Pierleoni, R., Sacconi, C., Stocchi, V., 2002. Identification of putative
genes involved in the development of Tuber borchii fruit body by
mRNA differential display in agarose gel. Current Genetics 42, 161–
168.