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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