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Brazilian Journal of Plant Physiology

On-line version ISSN 1677-9452

Braz. J. Plant Physiol. vol.15 no.3 Londrina Sep./Dec. 2003 



Cell wall hydrolases in the seeds of Euphorbia heterophylla L. during germination and early seedling development


Hidrolases da parede celular em sementes de Euphorbia heterophylla L. durante a germinação e desenvolvimento inicial da plântula



Cecilia N. K. SudaI; Marcos S. BuckeridgeII; Jarbas F. GiorginiIII, *

IDepartamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, CEP 14049-900, Ribeirão Preto, SP, Brasil
IISeção de Fisiologia e Bioquímica de Plantas, Instituto de Botânica, CP 4005, CEP 01061-970, São Paulo, SP, Brasil
IIIDepartamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, CEP 14040-901, Ribeirão Preto, SP, Brasil




Activities of cell wall hydrolases of Euphorbia heterophylla L. (wild poinsettia) endosperm were investigated during pre- and post-emergence periods, defined as the time interval before and after 2.2 days from the start of imbibition, respectively. The activities of endo-b-mannanase and b-mannosidase are higher over the pre-emergence when compared to the post-emergence period and they may be involved in the process of germination in E. heterophylla. On the other hand, the activities of b-galactosidase, b-glucosidase, a-xylosidase, b-xylosidase and glucanases, which hydrolyse CMC, xyloglucans from Hymenaea courbaril or Copaifera langsdorffii, xylan, Avicel and lichenan, are higher over the post-emergence period. Activity on laminarin occurs over both periods. The activity of xyloglucanases was promoted in the presence of oligosaccharide XXLG. E. heterophylla endosperm surrounds the embryo and their cotyledons, which increases in area after 1 day from the start of imbibition. Rather than the mobilization of cell wall reserves the activity of hydrolases over the post-emergence period may be related to facilitation of cotyledon expansion by lowering endosperm resistance. The fraction of water-soluble polysaccharides extracted from the seed coat is composed of mannose (15.9 %), galactose (20.5 %), and glucose (63.6 %) whereas the fraction from decoated seed is composed of glucose (11.0 %), galactose (36.9 %) and xylose (47.9 %).

Key words: cotyledon expansion, endosperm, endoglucanase, endomannanase, Euphorbiaceae, xylanase.


Foram investigadas atividades de hidrolases da parede celular no endosperma de Euphorbia heterophylla L. (amendoim-bravo) durante os períodos de pré- e de pós-emergência, definidos, respectivamente, antes e após 2,2 dias desde o início da embebição. Atividades de endo-b-mananase e b-manosidase são mais elevadas na pré-emergência e podem estar envolvidas no processo de germinação. Por outro lado, atividades de b-galactosidase, b-glucosidase, a-xilosidase, b-xilosidase e de glucanases que hidrolisam CMC, xiloglucanos de Hymenaea courbaril ou Copaifera langsdorffii, xilano, Avicel e liquenano são maiores na pós-emergência. Atividade sobre laminarina ocorre em ambos os períodos. A atividade de xiloglucanases foi promovida na presença do oligossacarídeo XXLG. O endosperma de E. heterophylla envolve o embrião e os cotilédones. Estes aumentam em área a partir do 1º dia do início da embebição. As atividades das hidrolases na pós-emergência podem estar mais relacionadas com o decréscimo da resistência endospérmica para a expansão dos cotilédones do que com a mobilização de reservas da parede celular. A fração de polissacarídeos hidrossolúveis extraída do tegumento é composta de manose (15,9 %), galactose (20,5 %) e glicose (63,6 %), e do restante da semente de glicose (11,0 %), galactose (36,9 %) e xilose (47,9 %).

Palavras-chave: expansão cotiledonar, endosperma, endoglucanase, endomananase, Euphorbiaceae, xilanase.




In most seeds radicle emergence characterizes the end of germination and the beginning of seedling development. The activity of cell wall hydrolases such as endo-b-mannanases and b-1,3-glucanases during the pre-emergence stage may be related to the softening of the tissue in the micropylar region, where the protrusion of the radicle occurs (Leubner-Metzger et al., 1995; Nonogaki and Morohashi, 1996; Sánchez and de Miguel, 1997; Nonogaki et al., 2000; de Miguel et al., 2000; Leubner-Metzger and Meins Jr., 2000). The activity of some mannan- and xyloglucan- degrading enzymes following radicle emergence has been related to degradation of cell wall reserve polysaccharides mobilized to supply the seedling with sugars before it becomes autotrophic (Buckeridge et al., 2000b).

Euphorbia heterophylla L. (wild poinsettia), an Euphorbiaceae, is a native plant of tropical and subtropical America (Hutchinson and Dalziel, 1958) and now widely spread. It is an important weed in at least 28 tropical countries and is present in 37 more (Wilson, 1981). In Brazil it has become a major weed, found mainly in soybean fields (Lorenzi, 2000).

The occurrence of storage cell wall polysaccharides in E. heterophylla seeds has not been studied yet. Apart from a report by Zeier et al. (1999) who investigated cell wall composition of the root endodermis of Ricinus communis, there is little information in the literature related to cell wall composition and cell wall hydrolases in Euphorbiaceae. E. heterophylla endosperm contains as major seed reserves lipids and proteins which comprise 59 % and 27 % of seed dry mass, respectively; but starch has not been detected in the endosperm (Suda and Giorgini, 2000). Therefore, this species does not store large amounts of polysaccharides in the seeds and the physiological role of its cell wall hydrolases following emergence is unclear. In a previous report (Suda and Giorgini, 2003), endo-1,4-b-1,3-glucanases from E. heterophylla endosperm were isolated and characterized.

In the present work an investigation was carried out of: a) the monosaccharide composition of water-extractable polysaccharide from the endosperm and the seed coat of E. heterophylla, and b) the activities of cell wall hydrolases from the endosperm in relation to germination and the process of endosperm weakening that facilitates cotyledon expansion inside the endosperm.



Plant material: Mature fruits of Euphorbia heterophylla L. were collected from native plants and dried at 30 ºC until dehiscence; seeds were stored in air-tight flasks at 5 ºC. At this temperature seeds remain viable for several months (Bannon et al., 1978; Suda and Pereira, 1997).

Germination: Seeds were germinated in Petri dishes on two sheets of filter paper moistened with distilled water. The dishes were kept at 30 ºC in the dark. For enzyme extraction, material was sampled from this population of seeds. For germination tests, four 9 cm Petri dishes were used, each containing 50 seeds.

Average germination time: The average germination time ± confidence interval (95 %) was calculated according to Labouriau and Osborn (1984).

Extraction of cell wall hydrolases: At various time intervals after imbibition, the seeds or seedlings were harvested and their seed coats manually removed. Decoated seeds were dissected and the embryo (cotyledon plus embryo-axis) was separated from the endosperm. One hundred and fifty isolated endosperms were homogenized for 15 sec in a Polytron-type homogenizer with 15 mL of 0.05 mol.L-1 sodium acetate buffer (pH 5.0), containing 0.4 mol.L-1 NaCl and 0.02 % NaN3. The homogenate was centrifuged at 10,000 gn for 10 min. The supernatant was collected and stored at –15 ºC prior to use. The enzymes extractions were carried out at 4 ºC.

Assay of nitrophenylglycosidases: The reaction mixture contained 15 mL of enzyme extract, 15 mL of 0.1 mol.L-1 sodium acetate buffer (pH 5.0), and 15 mL of 0.05 mol.L-1 (in H2O) of the following p-nitrophenyl (pNP) glycosides (Sigma): pNP-a-D-galactopyranoside or pNP-b-D-galactopyranoside for a- and b-galactosidases, respectively; pNP-b-D-glucopyranoside for b-glucosidase; and pNP-b-D-mannopiranoside for b-mannosidase. Thirty mL of p-nitrophenyl-b-D-xylopyranoside (7 mmol.L-1 in McIlvane buffer, pH 5.0) were added to 15 mL of enzyme extract for b-xylosidase activity. The reaction mixture was incubated at 35 ºC during 20 min for b-galactosidase and b-glucosidase activities, and 60 min for the other enzymes. The reaction was stopped by the addition of 1.5 mL of 0.05 mol.L-1 Na2CO3. The p-nitrophenol released was determined by the absorbance at 405 nm and the enzyme activities calculated using a molar extintion of 18,400 for p-nitrophenol in 0.05 mol.L-1 Na2CO3 (Alcântara et al., 1999). Controls were prepared by adding 1.5 mL of 0.05 mol.L-1 Na2CO3 to the reaction mixture prior to incubation (time 0). Control values were subtracted from all sample values. Enzyme activity was expressed as Katal (Kat).

Assay of endo-b-mannanase activities: The reaction mixture contained 100 mL of enzyme extract and 200 mL of substrate solution (either 0.8 % guar gum or 0.8 % locust bean galactomannan, both from Sigma, in 0.05 mol.L-1 sodium acetate buffer, pH 5.0, containing 0.02 % NaN3). The changes in drainage time of the mixture through a calibrated upper portion of a 0.1 mL pipette at 30 ºC were used as a measure of viscosity. Drainage times were converted to relative units of enzymic activity (Almin et al., 1967) using -1.68 as the value of the empirical substrate specific constant (Eriksson and Winnel, 1968). In preliminary experiments the linear relationships "enzyme activity vs. enzyme concentration" and "intrinsic viscosity (N-1.68) vs. incubation time" were checked.

Assay of endoglucanase by the viscometric method: The reaction mixture contained 1.5 mL of the enzyme extract and 13.5 mL of 0.3 % carboxymethylcellulose (CMC type 7H3SF, Hercules Incorporated) in 0.05 mol.L-1 sodium acetate buffer, pH 5.0, containing 0.1 mol.L-1 NaCl and 0.02 % NaN3. Viscosity changes were measured using a glass Ostwald viscometer maintained at 30 ºC by a circulating water-bath. Initial flow time of the assay mixture was about 130 s; flow time of distilled water was 29 sec. These times were converted to relative units of endoglucanase activity using -3.66 as the value of the empirical substrate specific constant (Almin et al., 1967; Durbin and Lewis, 1988).

Assay of glucanases and xylanases: The reaction mixture contained 100 mL of enzyme extract and 500 mL of substrate (1.2 % CMC type 7H3SF (Hercules Incorporated), 1.2 % xylan from oat spelt (Sigma), 0.25 % laminarin from Laminaria digitata (Sigma), 0.5 % Avicel (Merck) or 0.25 % lichenan from Cetraria islandica (Sigma)) in 0.05 mol.L-1 sodium acetate buffer, pH 5.0, containing 0.1 mol.L-1 NaCl and 0.02 % NaN3. The Avicel was dissolved in the above buffer without NaCl. The mixture was incubated at 30 ºC during 60 min and the reducing power produced was determined by the Nelson-Somogyi method (Somogyi, 1952).

Assays of overall xyloglucan-degrading activity: The activities were determined according to Sulová et al. (1995). For overall xyloglucan-degrading activity the reaction mixture contained 100 mL of enzyme extract, 100 mL of substrate (either 0.04 % xyloglucan of Hymenaea courbaril or Copaifera langsdorffii in 0.05 mol.L-1 sodium acetate buffer containing 0.02 % NaN3) and 15 mL of oligosaccharide XXLG (10-6 kg.mL-1 in water) or 15 mL of water (without XXLG). The blank sample contained no xyloglucan and the control sample contained no enzyme. The mixtures were incubated at 30 ºC for 30 min and the reaction stopped by addition of 0.1 mL of 1.0 mol.L-1 HCl. To each sample, 1.0 mL of 20 % Na2SO4 and 0.2 mL of iodine solution (0.5 % I2 + 1.0 % KI) were added and the tubes were allowed to stand for 1 h in the dark. The optical density was measured at 620 nm against the blank. The enzyme activity was expressed in arbitrary units and corresponds to the percentage of substrate (xyloglucan) degraded to molecular species with Mr < 10 kDa during the assay.

The decrease in xyloglucan-iodine complex was calculated subtracting absorbance values of assays without XXLG from values of assays containing XXLG.

Hymenaea courbaril xyloglucan and purified XXLG were kindly provided by Dr. Carem G. Vargas-Rechia (Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo). Copaifera langsdorffii xyloglucan was prepared according to Buckeridge and Dietrich (1990) and Alcântara et al. (1999).

Assay of a-xylosidase: The activities were determined by assaying the free pentose released according to Roe and Rice (1948) as modified by Edwards et al. (1985). The assay mixture consisted of 20 mL enzyme extract and 50 mL substrate (either 0.2 % xyloglucan from Hymenaea courbaril or 0.2 % xyloglucan from Copaifera langsdorffii in 0.1 mol.L-1 sodium acetate buffer, pH 5.0, containing 0.02 % NaN3). The mixture was incubated at 30 ºC for 24 h and free pentose determined using xylose as standard.

Isolation of the polysaccharides: The seed coat of quiescent seeds was removed and polysaccharides were extracted from both seed coats and decoated seeds. Decoated seeds (6.0 × 10-3 kg) were homogenized for 30 sec in a Polytron-type homogenizer in chloroform-methanol (2:1, v/v) to remove lipids. Raw extract volume was adjusted to 60 mL with chloroform-methanol (2:1, v/v) and stirred. Following centrifugation at 2,000 gn for 10 min the supernatant was discarded and the pellet re-extracted with 60 mL chloroform-methanol. After 3 extractions followed by centrifugation the pellet was dried at 30 ºC for 48 h.

Polysaccharides extraction was performed according to Buckeridge and Dietrich (1990). Seed coats (2.5 x 10-3 kg) or defatted seed powder (2.7 x 10-3 kg) as obtained above were stirred in 250 or 270 mL distilled water (80 ºC), respectively, for 8 h. The resulting extract was filtered through nylon cloth to remove remaining insoluble residues. Polysaccharides were precipitated from the extract with 3 volumes of ethanol. The precipitate was left overnight at 5 ºC, collected by filtration through nylon cloth and dried at 60 ºC. The dried material was resuspended in hot water and centrifuged (13,000 gn, 15 min). The supernatant was collected and freeze dried.

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD): Acid hydrolysis of the polysaccharides was performed by a pre-incubation of 10 mg of the freeze-dried material in 0.1 mL of 72 % H2SO4 at 30 ºC for 30 min followed by dilution to a concentration of 3 % H2SO4 with distilled water and hydrolysis in an autoclave for 1 h at 120 ºC (Saeman et al., 1954). After hydrolysis, the solution was neutralized with NaOH (Dionex). The resulting monosaccharides were analysed by HPAEC-PAD (Dionex PD40 Diode Array Detector) on a CarboPac PA-1 columm eluted by pure water (flow rate 1.0 mL.min-1) and post-columm detection with 0.50 mol.L-1 NaOH. The relative proportions of monosaccharides were corrected according to detector sensitivity to each monosaccharide, calculated by using equimolar standards.

Assay of protein: Protein content was determined by the method of Bradford (1976), using bovine serum albumin as standard.

Determination of cotyledon area: At various time intervals after the start of imbibition seeds were collected, cotyledons excised and their maximum length (a) and width (b) measured. Since the shape of E. heterophylla cotyledons is approximately elliptic, the area was estimated using the equation for an ellipse: Area = p.(a/2).(b/2)



When seeds of E. heterophylla are soaked in water, a transparent, gel-like coating of mucilage is extruded and envelops the seeds. During polysaccharide extraction only a part of the mucilage could be dissolved in hot water (80 ºC) indicating that it may be composed of water-soluble and water-insoluble substances.

As shown in the table 1, water-extractable polysaccharides from E. heterophylla seed coat are composed of glucose, galactose and mannose whereas from decoated seeds they are composed of xylose, galactose, glucose and arabinose. Mannose was not detected in decoated seeds of E. heterophylla.



The average germination time of E. heterophylla seed was 2.2 ± 0.7 days. In the present work, pre- and post-emergence periods were respectively defined as the time interval before and after 2.2 days from the start of imbibition.

As shown in figure 1, cotyledon area increased 4.4 fold between 0 and 2 days (pre-emergence), and 1.7 fold between 2 and 4 days from the start of imbibition (post-emergence).



As shown in figure 2A, endo-b-mannanase ativity was already present in quiescent seeds (time 0) and its level decreased during pre-emergence, remaining low during the post-emergence period. The activity on locust bean galactomannan was higher than the activity on guar galactomannan.

b-mannosidase activity was also present in the quiescent seed, however its level decreased approximately 78 % up to 24 h and remained constant thereafter (figure 2B). Unlike endo-b-mannanase and b-mannosidade, the levels of a-galactosidase increased during the post-emergence period (figure 2B).

As shown in figure 2C, there was b-1,3-glucanase activity on laminarin during both pre-emergence and post-emergence periods. The xylanase and b-xylosidase activities in E. heterophylla endosperm remained essentially constant during pre-emergence and increased during the post-emergence period (figure 2D).

The xyloglucan-degrading activity during pre-emergence was slightly higher in the presence of xyloglucan from H. courbaril than in the presence of the xyloglucan from C. langsdorffii (figure 3A). The activity was low during pre-emergence increasing after radicle emergence in both assays: with XXLG (not shown) or without XXLG (figure 3A). However, the activity was always higher in the presence of XXLG than without it, indicating that XXLG promotes xyloglucanase activity in E. heterophylla. The difference between these two assays is showed in figure 3B. In the mixture containing xyloglucan, XXLG and extract from quiescent seeds (time 0), the amount of xyloglucan-iodine complex decreased 52.8 % or 27.3 % in relation to a corresponding mixture without XXLG (figure 3B). The color intensity of the mixture decreased since xyloglucan was cleaved and the colorization of fragments generated below 10 kDa does not occur (Sulová et al., 1995). Nevertheless, the difference between the two assays decreased during pre-emergence becoming undetectable during post-emergence (figure 3B). b-galactosidase activity of E. heterophylla seeds slightly decreased during 24 h after seed imbibition, increasing continuously thereafter (figure 3C). b-glucosidase activities were relatively constant during pre-emergence increasing slightly during the post-emergence period (figure 3C).

The activity of a-xylosidase on both H. courbaril and C. langsdorffii xyloglucans increased from the 1st day after imbibition (figure 3D). However, while the activity on H. courbaril xyloglucan increased continuously up to the 3rd day decreasing thereafter, the activity on C. langsdorffii xyloglucan remained high during the post-emergence period. A minor fraction of H. courbaril xyloglucan is composed of arabinose (Tiné et al., 2000), thus the activity of E. heterophylla a-xylosidase activity on H. courbaril xyloglucan may be the sum of arabinosidase plus a-xylosidase activities because the method assayed released free pentose.

The hydrolytic activities on CMC (figure 3E), Avicel or lichenan (figure 3F) were very low or not detected during pre-emergence and increased only in the post-emergence period. The activity profile on CMC determined by viscometry was similar to that of activity determined by reducing power production (figure 3E).



The occurrence of galactose and mannose suggests the presence of galactomannan in the seed coat. Moreover, the high level of glucose also suggests the presence of a glucan in E. heterophylla seed mucilage. Glucans have been detected in seed mucilages, such as from Sinapis alba (mustard) which contains a water-soluble heterogeneous polysaccharide of a 1,4-linked b-D-glucan (similar to cellulose but more branched) and a pectic material (Balke and Diosady, 2000). Seeds of Cydonia oblonga (quince) release a water-extractable mucilage containing cellulose microfibrils tightly associated with a highly acidic glucuronoxylan (Reis et al., 1991). Activities of endo-b-mannanase, a-galactosidase and b-mannosidase have been associated with galactomannan mobilization (Buckeridge et al., 2000a). However, water-soluble galactomannans probably do not occur in E. heterophylla endosperm because mannose was not detected in the endosperm of E. heterophylla. It is possible that endo-b-mannanase activity in E. heterophylla was related to degradation of structural mannan of the cell wall, not extractable by hot water. Since the activity of E. heterophylla endo-b-mannanase on locust bean galactomannan was higher than the activity on guar galactomannan, it is possible that the enzyme preferably degrades galactomannans with fewer galactosyl branch-points such as pure mannans, which are insoluble in water (Buckeridge et al., 2000a,b). The mannose:galactose ratio of galactomannans from locust bean and guar are approximately 3.5 and 1.5, respectively (Daas et al., 2000). The activity of E. heterophylla endo-b-mannanase was high during pre-emergence suggesting a possible role in endosperm weakening in order to allow radicle emergence.

The substrate of a-galactosidase in E. heterophylla endosperm is uncertain. The a-galactosidase is also involved in raffinose degradation (McCleary and Matheson, 1974; Buckeridge and Dietrich, 1996), however this oligosaccharide was not detected in E. heterophylla endosperm (Suda and Giorgini, 2000). Other Euphorbiaceae such as Ricinus communis (castor bean) or Hevea brasiliensis (rubber) do not have raffinose in their seeds either (Amuti and Pollard, 1977; Achinewhu, 1986).

The activity of b-1,3-glucanase from E. heterophylla endosperm during pre-emergence may be related to hydrolysis of b-1,3-glucan to facilitate radicle protrusion. The activity during post-emergence may be related to completion of endosperm cell wall degradation.

The presence of xylan-degrading enzymes and the high level of xylose in the water-soluble polysaccharide from endosperm strongly suggest the occurrence of xylan compounds as cell wall reserve, probably mobilized after seed germination. To the authors' knowledge, only one study has attempted to measure xylanase activity of dicotyledonous seeds during germination (Wankhede et al., 1977). In contrast to E. heterophylla xylanases, in Arachis hypogea (groundnut) the activity of pentosanase (xylanase) increases prior to radicle emergence (Wankhede et al., 1977).

Endo-b-1,4-glucanase or xyloglucan endo-transgly-cosylase (XET), b-galactosidase, b-glucosidase and a-xylosidase are considered the main enzymes associated with the mobilization of xyloglucans (Edwards et al., 1985; Crombie et al., 1998; Tiné et al., 2000). In E. heterophylla seeds the overall xyloglucan-degrading activity, imputed to endo-b-1,4-glucanase activity, increased during the post-emergence period together with b-galactosidase and b-glucosidase activities. The increase in a-xylosidase activity started before these other enzymes but the activity was present during the post-emergence period. These results and the occurrence of xylose, galactose and glucose in water-extractable polysaccharide from E. heterophylla endosperm strongly suggest xyloglucan as the cell wall component degraded, mainly during post-emergence in this species.

We have evidence for the existence of endo-1,4-b-1,3-glucanases which degrade CMC and xyloglucans in E. heterophylla endosperm (Suda and Giorgini, 2003), since profiles of CMC- and xyloglucan-degrading activities were similar over the experimental period. Besides xyloglucan another potential target for E. heterophylla endoglucanases are accessible sites in the peripheral and integral noncrystalline regions of cellulose microfibril as proposed for avocado Cx cellulase (O'Donoghue et al., 1994). It is also possible that E. heterophylla endoglucanases depolymerize xyloglucans and noncrystalline regions of cellulose for further attack of Avicelase on crystalline cellulose. In fact, the increase of Avicelase activity occurred after the increase of other endoglucanases in E. heterophylla endosperm. It is noteworthy that few plant cellulases are able to degrade crystalline cellulose (O'Donoghue et al., 1994; Rose and Bennet, 1999).

E. heterophylla endosperm contains high amounts of lipids and proteins which together comprise about 86 % of seed dry mass (Suda and Giorgini, 2000) and its degradation is paralleled by cotyledon expansion. E. heterophylla endosperm surrounds the embryo and their cotyledons, which expand continuously inside the seed during germination and early seedling development. Rather than mobilization of cell wall reserves, the activity of hydrolases during post-emergence in E. heterophylla probably facilitates cotyledon expansion by degrading endosperm cell walls and lowering endosperm resistance and at the same time facilitates the diffusion of degradation products into cotyledons.

Xyloglucanases have been investigated only in the seeds that store xyloglucan in the cotyledons (Edwards et al., 1985; Sulová et al., 1995; Tiné et al., 2000; Steele et al., 2001) but little information is available on this enzyme in endospermic seeds.

Acknowledgments: We thank Ms. Liliana Martim and Mr. Clovis O. Silva for assistance with polysaccharide isolation and analysis of monosaccarides by HPAEC-PAD and Mr. Jaime L. Zeotti for general technical assistance. We are grateful for helpful advice and suggestions given by Dr. Rita C.L.F. Ribeiro. This work was supported by grants 92/3176-4 and 96/8069-2 from FAPESP to J.F.G. C.N.K.S. acknowledges the receipt of a doctoral fellowship from CAPES.



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Received: 13/07/2003, Accepted: 23/08/2003



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