Post on 18-Jan-2023
Abstract Plants exude a variety of substances
through their roots, germinating seeds and aerial
parts. Some of these released compounds seem to
have an inhibitory effect against pathogens. The
aim of this work was to investigate and identify
antifungal proteins present in exudates from
imbibed cowpea seeds (Vigna unguiculata (L.)
Walp). The obtained exudation was analyzed in
regard to specific protein activities by enzymatic
or immunological assays for plant defense pro-
teins, from 4 h to 48 h of seed imbibition. Our
results show that cowpea seeds exudates present
several defense related proteins characterized as
b-1,3-glucanases, cystatins, vicilins and lipid
transfer proteins (LTPs), as well as a storage
vacuole membrane a-TIP protein, since the very
first hours of imbibition. These exudates also have
an ‘‘in vitro’’ inhibitory effect on the growth of
the fungus Fusarium oxysporum f. sp. phaseoli.
Our results suggest that seed exudates should
promote seed protection from soil pathogens.
Keywords LTP Æ b-1,3-glucanases Æ Vicilin ÆCystatin Æ Fusarium oxysporum f. sp. phaseoli ÆSeed exudates Æ Soil pathogen Æ Cowpea
Introduction
Cowpea (Vigna unguiculata) is a tropical legume
originated from Africa, which is cultivated in
most tropical regions of the world (Ehlers and
Hall 1997). In Brazil, cowpea is mainly produced
in the Northeastern part of the country and, in
this region, their grains are the main protein
source for the great majority of the population.
Cowpea seeds are severely damaged both in the
field and during storage by the cowpea weevil
(Callosobruchus maculatus), and by a great
number of pathogens such as virus, bacteria and
mainly fungi (Allen 1982; Rios 1988). Among the
fungal pathogens Fusarium oxysporum is an
important pathogen causing Fusarium wilt a
serious disease in Brazil (Ehler and Hall 1997). It
is thought that this high susceptibility of seeds of
T. L. Rose Æ L. A. Okorokov Æ A. O. Carvalho ÆV. M. Gomes (&)Laboratorio de Fisiologia e Bioquımica deMicrorganismos, Centro de Biociencias eBiotecnologia, Universidade Estadual do NorteFluminense, Av. Alberto Lamego 2000, 28013-600Campos dos Goytacazes, RJ, Brazile-mail: valmg@uenf.br
A. da S. Conceicao Æ F. Marty Æ D. Marty-MazarsLaboratoire de PhytoBiologie Cellulaire, Universitede Bourgogne, BP 47870, 21078 Dijon Cedex, France
J. Xavier-Filho Æ K. V. S. FernandesLaboratorio de Quımica e Funcao de Proteınas ePeptıdeos, Centro de Biociencias e Biotecnologia,Universidade Estadual do Norte Fluminense, Av.Alberto Lamego 2000, 28013-600 Campos dosGoytacazes, RJ, Brazil
Plant Soil (2006) 286:181–191
DOI 10.1007/s11104-006-9036-0
123
ORIGINAL PAPER
Defense proteins from Vigna unguiculataseed exudates: characterization and inhibitoryactivity against Fusarium oxysporum
Tatiana L. Rose Æ Alexandre da Silva Conceicao Æ Jose Xavier-Filho ÆLev A. Okorokov Æ Katia V. S. Fernandes Æ Francis Marty ÆDaniele Marty-Mazars Æ Andre O. Carvalho Æ Valdirene M. Gomes
Received: 16 November 2005 / Accepted: 12 May 2006 / Published online: 11 August 2006� Springer Science+Business Media B.V. 2006
cowpea to pests is associated with inadequate
defenses, either constitutive or induced (Singh
and Rachie 1985). Nevertheless, several cowpea
cultivars are able to express defense components
against many viruses, bacteria, fungi and insects
(Singh et al. 1985; Gomes and Xavier-Filho 1994)
and many of these defense compounds were
identified as proteins such as vicilins (7S globu-
lins), chitinases, b-1,3-glucanases, and the two
antimicrobial peptides, a defensin and a lipid
transfer protein (LTP) (Macedo et al. 1993;
Gomes et al. 1996; Fernandes and Xavier-Filho
1998; Carvalho et al. 2001). These proteins have
also been found in seeds of different plant species
(Kader 1996; Fernandes and Xavier Filho 1998;
Flores et al. 2001; Thomma et al. 2002).
Germinating seeds and roots release com-
pounds that may interfere with the development
of surrounding microorganisms (Nobrega et al.
2005; Okubara and Paulitz 2005). Several com-
pounds exuded from seeds were identified and
some of them have the ability to inhibit pathogen
growth, thus preventing seed infection, and others
have direct beneficial effects on the germination
itself (Barbour et al. 1991). The best-described
groups of defense compounds released from seeds
are soluble sugars, flavonoids, sterols and proteins
(Casey et al. 1998; Terras et al. 1995). Volatile
compounds are also among the well-known seed
exuded components and are chemically classified
as aldehydes, alcohols, ethylene, CO2 and fatty
acids (Gorecki et al. 1985; Duvick et al. 1992).
Emmert et al. (1998) revealed that the exudation
of the amino acid canavanine from alfalfa seeds
influenced negatively the growth of Bacillus cer-
eus, which is a beneficial microorganism known as
a suppressor of seedling damping off of alfalfa.
Terras et al. (1995) showed that exudates of rad-
ish seed contain as a principal component a pep-
tide identified as a plant defensin. These exuded
defensins are responsible for causing fungal
growth inhibition around the germinating seed,
thus providing a safe environment on seed
periphery during this vulnerable phase of plant
development.
In the last years, several proteins such as
trypsin and papain inhibitors (Xavier-Filho et al.
1989), variant vicilins (7S globulin storage pro-
teins) (Fernandes and Xavier-Filho 1998),
glucan hydrolases represented by chitinases and
b-1,3-glucanase (Gomes et al. 1996), antimicro-
bial peptides identified as lipid transfer proteins
(LTP) and defensins (Carvalho et al. 2001,
2004) were found in seeds of cowpea. These
proteins are commonly linked to plant defense
mechanisms. They are induced and accumulate
in the infected tissues systemically and also
exhibit antimicrobial activity (Van Loon and
Van Strien 1999). These proteins have different
described mechanisms of action. For example,
vicilins present affinity to chitin, binding to it
and interfering with fungal growth by an un-
known mechanism (Gomes et al. 1997, 1998).
Chitinases and b-1,3-glucanases are hydrolases
that break the b-1,3 linked glucans and b-1,4
linked N-acetyl-D-glucosamine, respectively,
which are the two major structural components
of the fungal cell wall, thus inhibiting their
development (Carlile et al. 2001). Defensins and
LTPs interact with fungal membrane causing its
permeabilization (Thevissen et al. 1999; Regente
et al. 2005).
The aim of this work was to identify defense
proteins in exudates from germinating cowpea
seeds and test the ability of such exudates for
inhibition of the fungal pathogen, Fusarium oxy-
sporum. Herein we report the identification,
partial characterization and antifungal activity of
several defense proteins exuded from imbibed
cowpea seeds. We consider such information as a
prerequisite to further understand the physiolog-
ical significance of their presence in the micro-
environment surrounding germinating seeds and
how microorganisms dwelling in it are affected by
them.
Materials and methods
Plant material
Cowpea (Vigna unguiculata L. Walp.) seeds of
the EPACE-10 cultivar, which are susceptible to
the bruchid insect Callosobruchus maculatus
(Xavier-Filho et al. 1989), were supplied by the
Centro de Ciencias Agrarias, Universidade Fed-
eral do Ceara, Fortaleza, Brazil where they were
developed.
182 Plant Soil (2006) 286:181–191
123
Fungi
The fungal isolate Fusarium oxysporum forma
specialis phaseoli, causing wilting of beans, were
kindly supplied by CNPAF/EMBRAPA, Goia-
nia, Goias, Brazil. The fungi were maintained on
Sabouraud agar (1.0% peptone, 2.0% glucose and
1.7% agar–agar).
Obtaining of seed exuded proteins and protein
determination
Seed exudate was obtained from cowpea using a
method similar to that described by Nelson and
Hsu (1994). To prepare the seed exudates, 50
seeds, with no cracks or other injuries on the pill,
were superficially disinfested for 5 min in 25 ml
0.5% sodium hypochloride containing one drop
of Tween-20 and finally rinsed three times with
sterile water. The disinfested seeds were added to
sterile flasks containing 50 ml of sodium acetate
buffer (50 mM, pH 4.5), water (pH 6.0), or
sodium phosphate buffer (100 mM, pH 7.5), at
27�C on a rotary shaker at 70 rpm for 4, 8, 16, 24,
32 or 48 h. At each time flask contents were fil-
tered, and the exuded proteins were recovered by
precipitation with ammonium sulfate (0–90%)
followed by dialysis against water and freeze-
drying. Protein content was determined as
described by Bradford (1976) using BSA as a
standard protein.
Fungal growth inhibition
Fungal growth inhibition was assayed as described
by Broekaert et al. (1990a) with some modifica-
tions. For the preparation of conidia of F. oxy-
sporum f. sp. phaseoli, fungal culture was
transferred to Petri dishes containing Sabouraud
agar for 12 days; after this period sterile distilled
water (10 ml) was added to the dish, and it was
gently shaken for 1 min for conidia liberation,
with the help of a Drigalski loop. Conidia were
quantified in a Neubauer chamber to ensure
appropriate dilutions in further inhibition assays.
Initially conidia suspension (20,000 in 1 ml of
Sabouraud broth) were incubated at 28�C with the
exudate solutions (200 ll at 100 mg ml–1), for
60 h, in microplates. Optical readings were taken
at 600 nm at each 6 h interval. All the experiments
were run in triplicate and the reading averages,
the standard errors and coefficients of variation
were calculated. A general control without addi-
tion of exudates was also utilized. After 60 h, fungi
cells were separated from the growth medium by
centrifugation, washed in 0.1 M Tris–HCl buffer
(pH 8.0) and plated for observation with a light
microscope (Axioplan Zeiss).
Gel electrophoresis
Sodium dodecyl sulfate electrophoresis (SDS-
PAGE) was carried out according to Laemmli
(1970). SDS-Tricine-gel electrophoresis was per-
formed according to Schagger and Von Jagow
(1987). Samples (1 mg of lyophilized exudate)
were solubilized in 0.1 ml Tris–HCl buffer
(100 mM, pH 8.0) containing 10% SDS, 5%
sucrose and 0.001% bromophenol blue, at 100�C
for 5 min and loaded (20 ll) on the gels.
Western blotting
Polyclonal antibodies specific against vicilins,
cystatins and LTPs from seeds of cowpea were
raised in New Zealand white rabbits according to
Macedo et al. (1993), Flores et al. (2001), and
Carvalho et al. (2001), respectively. Polyclonal
antibodies against a-TIP were prepared as
described by Johnson et al. (1989).
Western blottings were carried out using nitro-
cellulose membranes according to the method
described by Towbin et al. (1979). Blots were
incubated overnight with primary antiserum
against vicilins, LTPs and cystatins (at 1:2000) and
against a-TIP (at 1:3000) diluted in the blocking
buffer. Membranes were then washed (4 times,
during 10 min at each time) with 0.1 M phosphate
buffer containing 0.15 M NaCl, pH 7.3 (PBS). They
were incubated with peroxidase-conjugated goat
anti-rabbit secondary antibody (Sigma Immuno
Chemicals) (at 1:2000) diluted in blocking buffer,
for 2 h, at room temperature and then, washed as
above. Antibody binding was revealed using a
chemiluminescence detection Kit (ECL-Western
blotting detection reagents/RPN 2209) according
to the manufacturer’s instructions. The chemilu-
minescence signal was recorded on an X-ray film.
Plant Soil (2006) 286:181–191 183
123
Antibodies and ELISA test
Immunodetection ELISA assays were performed
on micro plates (96 wells; Nunc, Denmark), which
were initially coated with 100 ll of seed exudates
for 16 h. Antigen-coated plates were then washed
twice in PBS/0.05% Tween-20 and then blocked
with 1% BSA/PBS/0.05% Tween-20 for 1 h.
After one wash with PBS/0.05% Tween-20, vicilin
and LTP antibodies diluted at 1:5000 in 1% BSA/
PBS/0.05% Tween-20 were added. Plates were
incubated for 2 h at 37�C, and then washed four
times with PBS/0.05% Tween-20. Anti-IgG con-
jugated to alkaline phosphatase (at 1:2000 in 1%
BSA/PBS/0.05% Tween-20) was added and after
1 h of incubation at 37�C plates were washed four
times with PBS/0.05% Tween-20 and one last
time with PBS. The reaction was revealed using
ortho-phenylenediamine (OPD) as substrate.
Reaction intensity was measured using an ELISA
colorimetric plate reader at 410 nm. Negative
controls were performed by employing the IgG–
alkaline phosphatase complex without addition of
the anti-vicilin/anti-LTP IgGs and a blank well
with no added antibodies.
Enzymatic assays
b-1,3-glucanase was assayed by the method of
Fink et al. (1988), which employs laminarin
(Sigma Chemicals Co., St. Louis) as the substrate.
Exudates (50 ll) were incubated at 37�C, for
12 h, with 0.125 ml of a 2 mg ml–1 laminarin
solution in 0.05 M acetate buffer, pH 5.0 in a final
volume of 0.2 ml. Color was developed by the
copper–arsenomolybdate method (Nelson 1944;
Somogy 1952) and the absorbance was monitored
at 500 nm. One unit of activity was defined as the
concentration of enzyme that produces an
absorbance variation of 0.001, in relation to that
from the control (prepared by replacing the sub-
strate with buffer) reaction tube.
Chitinase activity was determined by incubat-
ing 50 ll of exudate with 0.25 M of the fluorogenic
substrate 4-methylumbelliferyl-N,N¢,N¢¢-tria-
cetylchitotrioside (Sigma Chemical Co.) in 2 ml of
25 mM phosphate buffer pH 5.0. The fluorescent
methylumbelliferyl (4-MU-sodium sat) released
during the reaction was measured using a Hitachi
F4500 fluorescence spectrophotometer, equipped
with a 320 nm primary filter and a 460 nm sec-
ondary filter. In order to relate fluorescence out-
put to the concentration of the released product a
calibration curve was plotted using 4-MU-sodium
salt. One unit of chitinase activity will liberate
1 nmol of methylumbeliferone (MU) per min.
Controls were made by replacing the substrate
with buffer. This methodology was described by
O’Brien and Colwell (1987).
Inhibitory activity assay
The inhibitory activity of the seed exuded cystatin,
against papain, was measured essentially as de-
scribed by Abe et al. (1994). The substrate for this
assay was the N-benzoyl-L-arginine-2-naphthyla-
mide (BANA, Sigma). The inhibitory activity was
detected as the reduction of BANA hydrolysis
activity of papain, per ml of inhibitor solution, and
expressed as the OD540 h–1 ml–1. Negative con-
trols were performed with no BANA or no seed
sample added to the reaction media. In a positive
control seed exudate was absent.
Results
Fungal growth inhibition
The incubation of F. oxysporum f. sp. phaseoli,
with the exudates obtained from cowpea seeds
imbibed in phosphate (pH 7.5), water (pH 6.0)
and acetate (pH 4.5) caused inhibition of 45.2,
82.1 and 75.7%, respectively, of fungal growth, in
comparison with the control (Fig. 1).
The micrographs taken from the fungal myc-
elia at the final time of the growth inhibition assay
demonstrated that fungal growth was inhibited by
all exudates in relation to the control and also
that the exudates were interfering with fungal
germination, germ tube elongation and formation
of hyphae (data not shown).
Characterization of proteins present on the
exudates
Analysis by SDS-PAGE electrophoresis showed
significant differences in the pattern of proteins
184 Plant Soil (2006) 286:181–191
123
exuded from seeds obtained with the different
media (Fig. 2A, B, C). In acetate buffer (pH 4.5),
the exuded proteins presented low molecular
masses, ranging from 6 kDa to 30 kDa (Fig. 2A).
With water (pH 6.0) as imbibition medium, the
predominant exuded proteins range from 30 kDa
to 97 kDa (Fig. 2B), i.e., with higher molecular
masses. The exudate obtained from seeds im-
bibed in phosphate buffer (pH 7.5) contained
protein masses in the range of 40–45 kDa
(Fig. 2C). Tricine gel electrophoresis, performed
for analyses of proteins of low molecular masses,
also showed different pattern of exuded proteins,
ranging from 6 kDa to 25 kDa (Fig. 2D, E, F).
With acetate buffer (pH 4.5) and water (pH 6.0)
media almost the same exudate protein profile
was observed. The major proteins had molecular
masses above 20 kDa (Fig. 2D, E). In phosphate
buffer (pH 7.5) medium, exudate showed proteins
ranging, mainly, from 6 kDa to 20 kDa (Fig. 2F).
The quantitative analysis of proteins
released during the imbibition of cowpea seeds
0
0,2
0,4
0,6
0,8
1
1,2
0 12 18 24 36 42 48 60
Time, h
Abs
orba
nce,
600
nm
Fig. 1 The effect ofexudates from cowpeaseeds on the growth ofFusarium oxysporumfungi. The absorbance at600 nm was taken as ameasure of fungal growth.Control (m); exudateobtained in phosphatebuffer, pH 7.5 (n); water,pH 6.0 (•) and acetatebuffer, pH 4.5 (e).Experiments were run intriplicate and the resultsare shown with the data ofthe standard deviations
4h 8h 16h 24h 32h 48h
16.914.410.6
8.1
6.2
4h 8h 16h 24h 32h 48h
6645
3121.5
14.5
pH 4.5
16.914.410.6
8.1
6.2
6645
3121.5
14.5
pH 6.0
16.914.410.6
8.1
6.2
6645
3121.5
14.5
pH 7.5
A D
E
F
B
C
Fig. 2 SDS-PAGEelectrophoresis (A, B, C)and Tricine-gelelectrophoresis (D, E, F)of different exudates fromimbibed seeds obtained inacetate buffer (A, D),water (B, E), phosphatebuffer (C, F) and indifferent time period. Sidenumbers in lane (M) referto molecular mass (kDa)markers
Plant Soil (2006) 286:181–191 185
123
demonstrated that their content increased with
time (from 4 h to 48 h). Results also demonstrate
that the highest levels of exuded proteins oc-
curred from 24 h to 48 h for all conditions studied
(Fig. 3).
Specific assay to determine the b-1,3-glucanase
activity revealed its presence at all exudates since
the very beginning of imbibition, at 4 h. The level
of b-1,3-glucanase activity increased significantly
and was highest at 48 h of imbibition. At this
imbibition stage it reached up to six times of the
initial value found for all three media (Fig. 4).
Chitinase activity was analyzed by a fluorogenic
technique but no chitinase activity was detected
in the different analyzed exudates (data not
shown).
The qualitative analysis of the exuded proteins
revealed the presence of proteins immunorelated
to vicilins (66 kDa) (Fig. 5A), cystatins (96 kDa)
(Fig. 5C) and the antimicrobial LTP peptide
(10 kDa) (Fig. 5B) by Western blotting technique
using specific antisera against these proteins.
In order to quantify the specific amount of
vicilins and LTPs in the exudates, ELISA tests
were performed. The results showed that vicilins
represented ca. 3–10% of the total exuded seed
proteins and quantification of LTPs revealed that
they correspond to ca. 0.7–5.6% of total exuded
seed proteins. Vicilins and LTPs concentrations
reached a maximum after 4–8 h of imbibition and
remained almost constant until 48 h (Fig. 5). The
quantity of cystatins was determined by papain
inhibition assay, which showed that there were no
significant differences in cystatin activities among
all studied exudates. Maximum levels of exuda-
tion were already present at the early beginning
of imbibition for all times (data not shown).
The presence of the a-TIP, an aquaporin mem-
ber protein, was investigated also by Western
blotting technique by the use of a specific antibody
against it. Results revealed a cross-reactive band
of 31 kDa in exudates obtained with the three
different media with different kinetics (Fig. 6).
In the acetate buffer and water exudates the
presence of a-TIP was detected after 8 h and 16 h
of imbibition, respectively (Figs. 6A, B) with the
strongest response obtained in water exudates
derived from water imbibition (Fig. 6B). In phos-
phate buffer, the immunodetection was observed
earlier, at 4 h after imbibition (Fig. 6C).
Discussion
Biological effects of exuded proteins on fungi
growth
Initial infections of seeds and roots by pathogens
may, at one time, be driven by attractive nutritive
exudate molecules, and at the same time, be
restrained by some other defense related exuded
molecules (Nelson 1990). In order to investigate
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
pH 4.5 pH 6.0 pH 7.5
To
tal p
rote
ins
(mg
/ml)
A AA
BC
D
A
BB
C
D
E
AB B
C
DE
4h 8h 16h 24h 32h 48h 4h 8h 16h 24h 32h 48h 4h 8h 16h 24h 32h 48h
pH 4.5 pH 6.0 pH 7.5
Fig. 3 Proteinconcentration of thecowpea seed exudatesfrom different media.Protein content wasdetermined as describedby Bradford, using BSAas protein standard. Thesignificance of the resultswas analyzed by theStudent’s t-test(P < 0.05). Similar lettersindicate non-significantlydifferent values
186 Plant Soil (2006) 286:181–191
123
the cowpea seeds exudates in respect to their
antifungal inhibitory function, growth curves of
the plant pathogen F. oxysporum f. sp. phaseoli in
the presence of the three exudates were done.
The strongest inhibitory effects, obtained at pH
6.0 and 4.5, were noticed as compared to the
control medium (Fig. 1). The inhibitory activity
of fungal growth by seed exudates was shown to
be influenced by pH and the buffer used, which
mimics the natural environmental conditions
found in soils of Brazil. This differential inhibition
might be due to these treatments, which influence
the exudation of different proteins, qualitatively
and quantitatively, as can be observed on the
Figs. 2, 3 and 5. Such exuded substances, as
demonstrated by us and Terras et al. (1995),
interfere with microorganisms and may do so in a
specific way, which could be triggered by specific
stimuli and environmental conditions.
Terras et al. (1995) have also shown that
defensins, a group of antimicrobial peptides, are
also exuded by radish seeds. The exudation of
defensins by radish seeds occurs when their teg-
uments are disrupted either by natural radicles
emergence or by mechanical incisions. Further-
more, radish seeds when germinating in the
presence of defensins (Rs-AFP1 or Rs-AFP2)
suppressed the growth of the fungus Pyrenophora
tritici-repentis. We have previously shown by
protein purification that cowpea seeds also accu-
mulate several types of antimicrobial proteins
(Gomes et al. 1996, 1997; Carvalho et al. 2001).
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
pH 4.5 pH 6.0 pH 7.5
β-1,
3 g
luca
nas
e ac
tivi
ty
(un
its.
mg
-1 p
rote
in)
4h 8h 16h 24h 32h 48h 4h 8h 16h 24h 32h 48h 4h 8h 16h 24h 32h 48h
AA
A A
B
C
A AA
A
B
C
A A
AB
C
D
pH 4.5 pH 6.0 pH 7.5
Fig. 4 b-1,3-glucanaseactivity of cowpea seedexudates of differentimbibition media. Thesignificance of the resultswas analyzed by theStudent’s t-test(P < 0.05). Similar lettersindicate non-significantlydifferent values
4h 8h 16h 24h 32h 48h 4h 8h 16h 24h 32h 48h4h 8h 16h 24h 32h 48h
pH 4.5 pH 7.5pH 6.0
10
96
66
kDa(A)
(B)
(C)
Fig. 5 Western blotting of different exudates fromimbibed seeds using antisera against vicilin (A), LTP (B)and cystatin (C) obtained in acetate buffer (pH 4.5—left),water (pH 6.0—middle), and phosphate buffer (pH7.5—right) at different time
kDa
31
4h 8h 16h 24h 32h 48h
pH 4.5
pH 6.0
pH 7.5
31
31
(A)
(B)
(C)
Fig. 6 Western blotting using antiserum against a-TIP forexudates obtained in acetate buffer (A), water (B), andphosphate buffer (C) after different time of seed imbibi-tion
Plant Soil (2006) 286:181–191 187
123
Identification of the exuded proteins by
electrophoresis
The quality and quantity of materials released
from germinating seeds and developing plant
roots interfere with plant physiology and may be
affected by a number of factors. Some of those,
such as the stage of plant development, the soil
pH, or temperature, have been shown to influence
the release of certain exudate components into
the rhizosphere (Nelson 1990). In order to iden-
tify proteins in exudates obtained from cowpea
seeds, which are differentially induced by specific
environmental conditions, different imbibition
medium were used. Three media were selected
for their ion composition and principally for their
pH values of 4.5, 6.0 and 7.5, which bear a
resemblance to some soil characteristics of the
Atlantic forest and the Northeastern parts of
Brazil where cowpea is produced (Abreu et al.
2003). The analyses showed that many proteins
were exuded from imbibed cowpea seeds (Fig. 2).
These results suggest the state of an intense out-
flow of proteins from the seeds during the very
beginning of germination. The release of antimi-
crobial proteins from seeds during germination
has already been described on the literature
(Terras et al. 1995; Diz et al. 2003). Furthermore
it has been described that some antimicrobial
peptides, such as LTP, are released by dipping the
plant tissues in extraction buffer (Molina and
Garcia-Olmedo 1993) and, in cell cultures are
also secreted to the growth medium (Sterk et al.
1991). We have previously characterized a LTP
exuded from imbibed cowpea seeds (Diz et al.
2003).
The electrophoretic results also suggest that
exuded proteins from cowpea seeds are distinc-
tively expressed and/or extractable according to
the environmental conditions of seed imbibition
and seem to be severely influenced by the pH and
ionic strength of the surrounding environment.
The results also showed significant quantitative
expression differences among exuded proteins
from 4 h to 48 h. These data were confirmed
when total exuded proteins were estimated by the
Bradford (1976) method. In all tested medium,
concentrations of exuded proteins increased with
time (Fig. 3).
Partial characterization of the exuded proteins
Seeds of many legumes contain abundant supplies
of reserve proteins stored in the parenchyma cells
of the cotyledons. These cells are characterized by
the presence of many large protein bodies con-
sisting of an amorphous protein matrix sur-
rounded by a limiting membrane. In Phaseolus
vulgaris the major reserve proteins are vicilins
and phytohemagglutinins, 50% and 10% of the
total protein content of the cotyledons at seed
maturity, respectively (Johnson et al. 1989).
During seedling growth, the reserve proteins are
catabolized within the protein bodies, which are
known as hydrolytic compartments, while their
surrounding membranes remain intact. Legume
seed exudates contain compounds that inhibit the
growth of rhizobia and other bacteria (Materson
and Weaver 1984), and antimicrobial peptides are
also found in those exudates from non-legumi-
nous seeds of Zea mays, Mirabilis jalapa and
Amaranthus caudatus (Broekaert et al. 1990b;
Cammue et al. 1992; Duvick et al. 1992).
To investigate the presence of defense proteins
such as vicilins, LTPs and cystatins in exudates
obtained from cowpea imbibed seeds, we used
Western blotting technique using specific anti-
sera. These proteins were present in all samples
since the first hours of imbibition. Characteriza-
tion and antifungal activities of vicilins (Gomes
et al. 1997), LTPs (Molina et al. 1993; Kader
1996; Diz et al. 2003) and cystatins (Pernas et al.
1999; Siqueira-Junior et al. 2002) have been pre-
viously reported. Molecular masses for the three
protein categories determined in the present work
are in accordance to the literature, stressing that,
in the case of cystatins, the high molecular mass
observed, despite not being compatible with the
most usual sizes for 1 and 2 cystatin families, is
well-matched to those of the multidomain kinin-
ogen type of plant cystatins. Among these are the
potato multicystatin (Walsh and Strickland 1993;
Waldron et al. 1993) and tomato multicystatin
(Siqueira-Junior et al. 2002). Our findings thus
indicate that antimicrobial proteins are released
from seeds during germination. Western blottings
for vicilins, LTPs and cystatins showed no signif-
icant quantitative differences between the three
analyzed exudates. However, pH was seen to
188 Plant Soil (2006) 286:181–191
123
affect the exudation kinetics of these proteins
(Fig. 5).
Plants also produce enzymes such as b-1,
3-glucanases and chitinases (Brunner et al. 1998;
Santos et al. 2004) that can break down pathogen
cell wall components. Given the importance of
these enzymes as potential determinants in the
resistance of plants to fungal diseases (Ji and Kuc
1996; Lawrence et al. 1996), the presence of
b-1,3-glucanases and chitinases in the three exu-
dates from cowpea seeds were also analyzed. The
b-1,3-glucanase activity was detected since the
beginning of the imbibition, at 4 h and increased
significantly and was highest after 48 h of imbi-
bition (Fig. 4). This enzyme is involved in defense
and also participates in physiological and devel-
opmental processes during seed germination
(Sela-Buurlage et al. 1993). Chitinase activity,
tested by using the fluorogenic substrate
4-methylumbelliferyl-N,N¢,N¢¢-triacetylchitotrio-
side, was not detected in any of the different
analyzed exudates.
a-TIP (28–31 kDa) is a protein specific of the
membrane limited storage vacuoles, involved in
the transport of water (Marty 1997). In our
results specific anti-a-TIP antibodies were
employed and revealed a cross-reactive band of
31 kDa in exudates obtained with the three
different media with different kinetic (Fig. 6).
However, further studies are necessary to
understand the origin and the function of this
protein, immunologically related to a-TIP, in
exudates of cowpea seeds.
The work presented here shows that cowpea
seeds exude, during germination, several proteins,
among those some defense related proteins, from
the very first hours of imbibition. These proteins
have an ‘‘in vitro’’ inhibitory effect on the growth
of the F. oxysporum f. sp. phaseoli fungus. The
findings reported in this paper on the presence of
defense proteins in exudates from cowpea seeds
suggest an important role for these proteins in
constitutive host defense mechanisms against
microbial pathogens during seed germination.
Microorganisms, such as soil-borne bacteria and
fungi, dwelling on rhizosphere, and the regulating
balance of such microbial populations is crucial to
plant survival because some microorganisms are
pathogens that can attack and destroy plant
tissues that come in contact with soil, like seeds and
roots, while others are beneficial organisms that
have an imperative role on normal plant develop-
ment such as mycorrhiza. Thus, understanding
what kind of substances are released by seeds and
also roots, in what developmental stage and also in
which environmental conditions these events take
place are the first steps in understanding how ex-
udates might affect the spermosphere and their
population microorganisms (Zhu et al. 1997;
Emmert et al. 1998; Nobrega et al. 2005).
This knowledge is also essential for developing
technologies on how to manipulate the spermo-
sphere and rhizosphere environment in order to
reduce or eliminate seed or root infection by
pathogenic fungi. From these analyses new in-
sights on how to manipulate the spermosphere
may be drawn. Further work will include the
usage of these mechanisms for the development
of biological control approaches of fungal patho-
gens typical for this crop.
Acknowledgements This study is a part of the MSc De-gree Thesis of TLR, carried out in the Universidade Es-tadual do Norte Fluminense through an internationalcollaboration between CAPES and COFECUB. Weacknowledge the financial support of the Brazilian agen-cies CAPES, CNPq and FAPERJ and the InternationalFoundation for Science (IFS), Stockholm, Sweden,through a Grant to C/2806-3F. We are grateful to M.T.Gobo for technical assistance.
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