JXB Advance Access originally published online on July 28, 2003
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Journal of Experimental Botany, Vol. 54, No. 390, pp. 2045-2052,
September 1, 2003
© 2003 Oxford University Press
Multiple forms of endo-1,4-ß-glucanases in the endosperm of Euphorbia heterophylla L.
Received 20 March 2003; Accepted 28 May 2003
1 Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, 14049-900, Brazil
2 Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, 14040-901, Brazil
* To whom correspondence should be addressed. Fax: +55 16 602 3666. E-mail: jarbas{at}ffclrp.usp.br
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Abstract |
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Germinating seeds of Euphorbia heterophylla L. contain endo-1,4-ß-glucanases which degrade carboxymethylcellulose (CMC). The activity decreased approximately 66% in extracts of endosperm containing isopropanol or ethanol. The endoglucanases were isolated from endosperm extracts using ammonium sulphate fractionation followed by Sephacryl S-100-HR chromatography resulting in two main peaks: I and II. Peak I endoglucanase was further purified about 15-fold on DEAE-Sephadex A50 and then by affinity chromatography (CF11-cellulose). Peak II endoglucanases were further purified 10-fold on CM-cellulose chromatography. The results indicated the occurrence of a 66 kDa endoglucanase (fractionated by SDS-PAGE and visualized by activity staining using Congo Red). Several acidic (pI 3.0 to 5.7) and basic (pI 8.5 to 10.0) forms from both peaks which differed in their capacities for degrading CMC or xyloglucans from Copaifera langsdorffii or Hymenaea courbaril were detected.
Key words: Endoglucanase, Euphorbia heterophylla, Euphorbiaceae, seed germination.
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Introduction |
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Endoglucanases (EC 3.2.1.4 [EC] ), commonly referred to as cellulases, are usually assayed by their capacity to degrade the artificial substrate carboxymethylcellulose (CMC) (Maclachlan and Carrington, 1991). However, with few exceptions, every CMC-degrading endo-1,4-ß-glucanase from micro-organisms or plants that has been tested is capable of hydrolysing xyloglucan suggesting that this hemicellulose is a likely substrate for endoglucanases in vivo (Maclachlan and Brady, 1992).
Xyloglucans are the major hemicellulosic polysaccharide in the primary cell wall of dicotyledons and were first found as amyloids in cell walls of seeds (Kooiman, 1960; Hayashi, 1989). In the case of seeds, xyloglucan-specific endo-1,4-ß-glucanase (later namely xyloglucan endotransglycosylase (XET); Fry et al., 1992) has been purified from cotyledons of nasturtium and lentils (Edwards et al., 1986; Steele et al., 2001) and have also been detected in cotyledons from tamarind, pea, bean, and Hymenaea courbaril (Sulová et al., 1995; Tiné et al., 2000).
The activity of either xyloglucanases or CMC-degrading endoglucanases from seeds increases following germination in nasturtium (Edwards et al., 1985), H. courbaril (Tiné et al., 2000), coffee-bean (Takaki and Dietrich, 1980; Giorgini, 1992) and cold-tolerant tomato lines (Leviatov et al., 1995). In nasturtium and H. courbaril cotyledons the physiological role of XET has been correlated with the mobilization of storage xyloglucan from the cell walls following germination (Edwards et al., 1985; Tiné et al., 2000). However, in the other cases above as well as in Phaseolus vulgaris cotyledons (Lew and Lewis, 1974) the precise physiological role of endoglucanase remains to be elucidated.
In seeds of Euphorbia heterophylla L. (Euphorbiaceae), a troublesome annual weed widespread in the tropics (Wilson, 1981), the endosperm contains high amounts of lipids and proteins which comprise together about 86% of seed dry mass and starch is not found (Suda and Giorgini, 2000). Therefore, this species does not store high amounts of polysaccharides in their seeds. E. heterophylla endosperm surrounds the embryo and their cotyledons. The role of endoglucanases in this species is unclear, however, it is possible that endoglucanases facilitates cotyledon expansion by lowering endosperm resistance and, at the same time, the diffusion of degradation products into cotyledons.
The aim of this study was to isolate and characterize endo-1,4-ß-glucanases from E. heterophylla endosperm. Evidence was found for the presence of several forms of endo-1,4-ß-glucanase which differed in their capacities for degrading CMC and/or xyloglucans. To the authors’ knowledge no detailed investigation has been carried out on xyloglucan-degrading enzymes in seed endosperms.
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Materials
Seeds of Euphorbia heterophylla L. were collected from local plants and stored in air-tight flasks at 5 °C until use. Under these conditions seeds remain viable for several months (Bannon et al., 1978; Suda and Pereira, 1997). Seeds were germinated in Petri dishes on two sheets of filter papers moistened with distilled water. The dishes were placed in a growth chamber at 30 °C under darkness. Germinating seeds were collected at the 3rd d from the start of imbibition, except when otherwise stated, and the endosperm was isolated by removing the seed coat and embryo.
The enzyme substrates used were carboxymethylcellulose (CMC type 7H3SF, Hercules Incorporated), xylan from oat spelt (Sigma), lichenan from Cetraria islandica (Sigma), xyloglucan purified from Hymenaea courbaril kindly provided by Dr Carem G Vargas-Rechia (Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo), and xyloglucan purified from Copaifera langsdorffii, provided by Dr MS Buckeridge (Instituto de Botânica, São Paulo).
Partial purification of endoglucanases
All procedures were performed at 4 °C. Usually 100 isolated endosperms (approximately 0.8 g) were homogenized with 10.0 ml 0.05 M sodium acetate buffer, pH 5.0, containing 0.4 M NaCl and 0.02% NaN3 in a Polytron-type homogenizer operated at maximum speed for 15 s. The homogenate was centrifuged at 10 000 g for 10 min and the supernatant filtered through glass wool to obtain the crude extract. Solid ammonium sulphate was added to the crude extract to 30% saturation. The protein pellet obtained after centrifugation (10 000 g, 15 min) was discarded and solid ammonium sulphate added to the supernatant to 85% saturation and then centrifuged. The precipitate was dissolved in a minimum volume of 0.05 M sodium acetate buffer, pH 5.0, containing 0.1 M NaCl and 0.02% NaN3 and applied to a Sephacryl S-100-HR column (1.8x116.0 cm) that had been equilibrated with the same buffer. Two peaks (I and II) containing activity on CMC were obtained (Fig. 1), the active fractions of each peak were combined and stored at –15 °C with 10% final concentration of glycerol.
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The fractions corresponding to peak I were dialysed against pure water. The precipitate which formed inside the dialysis sac was collected by centrifugation (15 000 g, 2 min), dissolved in 0.02 M TRIS-HCl buffer, pH 8.0 and applied to a DEAE-Sephadex A50 (Sigma) column (1.7x9.0 cm) which was washed with the same TRIS-HCl buffer. When the absorbance at 280 nm of the eluate had fallen to a constant value, the column was eluted with a NaCl gradient (0–0.7 M in the same buffer, over 20 bed volumes). Fractions containing endo-ß-glucanase activity were pooled, dialysed against water and lyophilized. The resulting powder obtained was dissolved in TRIS-HCl 0.05 M, pH 8.1, containing 0.1 M NaCl, and applied to a CF11-cellulose (Whatman) affinity column equilibrated with the above buffer. The column was successively eluted with: the buffer alone (2 bed volumes), the buffer containing 1.0 M NaCl (3 bed volumes) and the buffer containing 1.0 M NaCl+0.1 M cellobiose (5 bed volumes). The fractions containing activity were pooled, concentrated and precipitated at –15 °C by the addition of acetone (3 vols) for isoeletric focusing.
Solid ammonium sulphate to 85% saturation was added to the fractions corresponding to peak II and the precipitate collected by centrifugation (10 000 g, 15 min). The pellet was dissolved in 0.05 M ammonium bicarbonate buffer, pH 7.8 and dialysed against the same buffer for desalting. After dialysis the sample was lyophilized, dissolved in 0.02 M sodium acetate buffer, pH 5.0 and applied to a CM-cellulose (Sigma) column (1.7x9.0 cm) equilibrated with the same buffer. The column was successively eluted with: the same buffer alone (5 bed vols), the buffer containing 2.0 M NaCl (5 bed volumes) and the buffer containing 0.1 M NaCl+0.1 M cellobiose (5 bed volumes).
Protein was quantified using BSA as the standard (Bradford, 1976).
Viscometric assay
The assay mixture consisted of 1.5 ml of enzyme extract and 13.5 ml of 0.3% CMC in sodium acetate buffer 0.05 M, pH 5.0, containing 0.1 M NaCl and 0.02% NaN3. Viscosity changes were measured using a glass Ostwald viscometer. Initial flow time of the assay mixture was about 130 s; flow time of distilled water was 29 s. Flow times were converted to relative units of endoglucanase activity using –3.66 as the empirical substrate specific constant (Almin et al., 1967; Durbin and Lewis, 1988). In preliminary experiments, the linear relationships ‘enzyme activity versus enzyme concentration’ and ‘intrinsic viscosity (N–3.66) versus incubation time’ were investigated. In assays for the relationship ‘specific viscosity versus incubation time’, activity was also determined by the increase in reducing power by the method of Nelson-Somogyi (Somogyi, 1952). These determinations required the prior purification of the crude extract on a Biogel P-6DG (0.9x9.0 cm) to remove interfering substances.
Endo-ß-glucanase in individual column chromatography fractions was determined in terms of percentage reduction in flow-time of the assay mixture (0.2 ml of enzyme extract and 0.4 ml 1.2% CMC in 0.05 M sodium acetate buffer, pH 5.0, containing 0.1 M NaCl and 0.02% NaN3). The flow-time was measured using a calibrated 0.1 ml pipette.
The specific viscosity was also determined by using xyloglucan as substrate. Assay mixture contained 0.1 ml of enzyme extract and 0.2 ml of substrate solution (2.0% xyloglucan from the seed of Hymenaea courbaril in 0.05 M sodium acetate buffer). The flow-time was measured using a calibrated 0.1 ml pipette.
The assay mixture was always maintained at 30 °C.
Overall xyloglucan-degrading activities
Activities were determined according to Sulová et al. (1995). The assay mixture contained 0.1 ml enzyme extract, 0.1 ml substrate solution (0.04% xyloglucan from the seeds of either Hymenaea courbaril or Copaifera langsdorffii, in 0.05 M sodium acetate buffer containing 0.02% NaN3). The mixtures were incubated at 30 °C for 30 min, and the reaction was stopped by addition of 0.1 ml 1.0 M 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.
Polyacrylamide gel electrophoresis (PAGE)
Native PAGE was carried out by the procedure described by Davis (1964). SDS-PAGE was performed as described by Laemmli (1970), however, in the assays carried out to determine enzyme activity after SDS-PAGE, boiling of the protein sample prior to electrophoresis was avoided. Instead, the protein sample (containing SDS and ß-mercaptoethanol) was maintained for 30 min at room temperature and then loaded onto the gel. Proteins were visualized by Coomassie Blue-staining.
Isoeletric focusing (IEF)
Native IEF was carried out in a vertical polyacrylamide minigel system (10x8 cm cell format) according to Robertson et al. (1987). Ampholites pH 3–10 (Pharmalyte, Sigma) were used. Runs were at 4 °C for 1.5 h at a constant 180 V and then for an additional 1.5 h at a constant 400 V. The pIs were determined using pI protein markers (Isoeletric Focusing Calibration kit, pH 3–10, Pharmacia).
Activity staining
Endo-ß-glucanase activity after native PAGE, SDS-PAGE or IEF was detected by the gel overlay technique (Bertheau et al., 1984).
After electrophoresis the polyacrylamide gels were briefly rinsed with water, and preincubated with gentle shaking in appropriate buffers. Gels from native PAGE were preincubated for 15 min in 0.1 M sodium acetate buffer, pH 5.0, containing 0.02% NaN3 (three buffer changes). Gels from SDS-PAGE were preincubated for 15 min in 0.0625 M TRIS-HCl buffer, pH 6.8 (three changes) and then in the sodium acetate buffer for 10 min (two changes) (Lamed et al., 1983). Gels from IEF were immersed thoroughly for 15 min in 0.1 M KCl (three changes) for the removal of ampholites, briefly rinsed with water and preincubated in the sodium acetate buffer for 10 min (two changes).
Polyacrylamide gels were then carefully layered onto agar gel (1.5% agar in 0.1 M sodium acetate buffer, pH 5.0, containing 0.02% NaN3 and one of the following substrates: 0.5% CMC, 0.5% xylan, 0.5% lichenan, 0.5% xyloglucan from Copaifera langsdorffii or 0.4% xyloglucan from Hymenaea courbaril) and incubated at 30 °C. The incubation time varied from 1.5 h to 22 h, depending on the amount of protein initially loaded for separation. After incubation, the agar gels were immersed in 1.0% Congo Red solution for 40 min. Agar gels containing CMC were destained in 1.0 M NaCl until clear bands in the red background were obtained. In the case of agar gels containing other substrates, the best results were obtained when gels were destained in distilled water. For photographic purposes (better contrast), the agar overlay was rinsed in 0.5% or 5.0% acetic acid which turned the red background into dark blue (Kanellis and Kalaitzis, 1992).
Proteinase inhibitor, EDTA and ß-mercaptoethanol on endoglucanase activity
To verify the effect of proteinase inhibitors, 50 endosperms were homogenized with 10.0 ml of sodium acetate buffer as described above to obtain the crude extract, except that the buffer contained one of following inhibitors: 0.1 mM or 1.0 mM phenylmethylsulphonyl fluoride (PMSF), 0.01 mM antipain, 0.01 mM pepstatin, 0.01 mM chymostatin, 1.0 mM p-chloromercuribenzoic acid (pCMB) or 1.0 mM EDTA. The PMSF stock solution was prepared by dissolving 0.01 g PMSF in 0.4 ml isopropanol or ethanol, the final concentration of these solvents in the crude extract being 0.7%.
To verify the effect of ß-mercaptoethanol, E. heterophylla seeds were placed to germinate for 2 d, then 10 endosperms were isolated and homogenized with 15.0 ml of sodium acetate buffer containing 5 mM ß-mercaptoethanol.
Activities of ß-glucosidase, 
- and ß-galactosidase and ß-xylosidase
Activities were determined according to Alcântara et al. (1999) by using p-nitrophenyl (pNP) glycosides.
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Endohydrolytic action
For a mixture of the crude extract from the E. heterophylla endosperm and CMC there was a linear relationship between intrinsic viscosity (N–3.66) and incubation time and between relative activity and enzyme concentration. As shown in Fig. 2A the enzyme very rapidly decreased the specific viscosity of the CMC solution during the early stages of the reaction, whereas the reducing power of the incubation mixture increased at a low but constant rate indicating endohydrolysis of CMC. When xyloglucan from H. courbaril was used as the substrate, the specific viscosity also decreased (Fig. 2B), indicating the occurrence of xyloglucan-degrading enzymes in the E. heterophylla endosperm.
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Inhibition of endoglucanases activity
Crude extracts with relatively high activities of endoglucanase from the E. heterophylla endosperm could be obtained at pH 5.0 when 0.4 M NaCl was added to the buffer, and these conditions were routinely used in the extraction procedures. Addition of proteinase inhibitors had no effect on the specific activity of enzymes in the extract, however, isopropanol and ethanol used to dissolve PMSF inhibited enzyme activity after 24 h storage of crude extract at –15 °C (Table 1). The addition of 5.0 mM ß-mercaptoethanol to the extraction buffer decreased the specific activity of the enzyme by about 50% enzyme specific activity.
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Purification and properties of endoglucanases
The crude extract containing endoglucanases was initially fractionated using ammonium sulphate precipitation. The enzyme activity was mainly distributed into the protein fractions precipitated between 30% and 85% ammonium sulphate saturation, although the highest enzyme activity was detected between 45% and 65% saturation. Therefore, the fraction precipitated between 30% to 85% was routinely collected. The fraction was further fractionated on Sephacryl-S100-HR and exhibited two main peaks: I and II that corresponded to activity (Fig. 1). Determinations of the apparent molecular weight of peak I and II endoglucanases by calibrated Sephacryl column gave values around 27 kDa and 8 kDa, respectively.
The fractions from peak I were pooled and further purified by the steps summarized in Table 2. The fraction which precipitated upon dialysis against water represented a 5-fold purification. At the DEAE-Sephadex step almost 15-fold purification was obtained. The fraction containing CMC- and xyloglucan-degrading enzymes was eluted from DEAE-Sephadex column at a range around 0.25 M NaCl (Fig. 3). This same fraction was analysed by native PAGE and SDS-PAGE. Native PAGE presented three protein bands stained with Coomassie-blue, however, only one band was active against CMC. SDS-PAGE revealed several polypeptide bands, however, activity on CMC was associated with only one polypeptide of about 66 kDa. A similar result was obtained when xyloglucan from H. courbaril was used as the substrate in the agar gel. At this purification step the activities of ß-glucosidase, - and ß-galactosidase, ß-xylosidase, as well as on xylan and lichenan, were not detected, indicating that the fraction could be free of these enzymes. This same protein fraction was rechromatographed on the Sephacryl S-100-HR column and analysed by SDS-PAGE, the protein bands exhibited similar migrations as those obtained in the previous step. Thus, gel-permeation chromatography following DEAE-Sephadex proved to be an unnecessary step. Instead, a CF11-cellulose affinity column was used as an additional purification step and, in this case, CMC- and xyloglucan-degrading enzymes eluted as multiple peaks. Isoeletric focusing of the protein of each peak, followed by activity staining by the gel overlay technique, showed the occurrence of many acidic (pI 3.0 to 5.7) and basic (pI 8.5 to 10.0) endoglucanases (Table 3). As shown in Table 3, substrate specificity varied among the isoforms: acidic isoforms (pI 3.0, 3.3 and 3.8), which were active against CMC and xyloglucan from C. langsdorffii, but not against xyloglucan from H. courbaril; the enzymes of pI 4.4, 4.9 and 5.7, which were active only on CMC, and of pI 5.4, which was active only on xyloglucan from H. courbaril. The basic isoforms were active against both CMC and xyloglucan from H. courbaril.
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Endoglucanases from peak II were partially purified about 10-fold by the steps summarized in Table 4. After fractionation with ammonium sulphate the sample was dialysed against ammonium bicarbonate buffer for desalting and lyophilization. This protocol was preferred because dialysis against water or acidic buffers, or the use of desalting columns such as Biogel P6-DG or Sephadex G-25 eluted with water, resulted in low recoveries of peak II endoglucanases. The major endoglucanase fraction adsorbed on CM-cellulose was eluted only with the increase in NaCl concentration to 2.0 M. Probably the eluate with more than 0.5 M NaCl is due to the affinity for cellulose rather than for anion. In fact, further addition of cellobiose increased enzyme activity in the eluted fractions, suggesting that cellobiose improved elution. Native PAGE followed by Coomassie blue staining of fractions eluted with cellobiose showed three protein bands (Fig. 4); activity against CMC was detected in band 2 and in the uppermost region of the gel which was not stained with Coomassie blue. Activity on xyloglucan from H. courbaril was also associated with band 2. These protein fractions were also analysed by IEF followed by activity staining by gel overlay. The results showed the occurrence of acidic (pIs 3.2 and 4.2) and basic (pIs 8.5, 9.3 and 10.0) endoglucanases. The pI 3.2 enzyme was active against CMC and xyloglucans from H. courbaril and C. langsdorffii and the pI 4.2 enzyme was active against H. courbaril xyloglucan, but not against C. langsdorffii xyloglucan. The basic endoglucanases degrade CMC, but were not active against both xyloglucans. The protein fraction eluted in the presence of cellobiose was also analysed by mild denaturating SDS-PAGE followed by activity staining, however, no endoglucanase activity was detected (not shown).
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Endo-1,4-ß-glucanase activity from 3 d germinated seeds was inhibited in extracts containing isopropanol or ethanol. Organic solvents may affect the activity of some transglycosylases and endohydrolases such as ß-glucosidases from bacteria, fungi and animal (Watt et al., 1998; Christakopoulos et al., 1994; Gopalan et al., 1989) and endo-ß-N-acetylglucosaminidase from bacteria (Fan et al., 1995). Interference on activity of some polysaccharide synthases such as arabinan synthase, callose synthase, xylan synthase, and endo-1,4-ß-glucan synthase have also been verified (Kerry et al., 2001). The reason for this inhibition in E. heterophylla was not investigated in the present work, however, since the activity of transglycosylases may be affected by alcohols it is possible that some endoglucanase in E. heterophylla may also have transglycosylating activity.
On SDS-PAGE followed by activity staining, endoglucanases from peak I which had an apparent molecular weight of 66 kDa were detected. However, on gel-permeation chromatography, the apparent molecular weight of the undenatured enzyme from peak I was much lower (27 kDa). This difference may be due to delayed elution caused by partial adsorption of enzyme to the Sephacryl bed. In XET of nasturtium seeds (Edwards et al., 1986) and xyloglucanase from pea stems (Matsumoto et al., 1997) the apparent molecular weights of undenatured enzymes, determined by gel permeation chromatography, were also lower than by SDS-PAGE. In both cases compact tertiary structures have been suggested.
The fractionation of enzymes from peak I on the CF11-celulose column revealed the occurrence of several endoglucanases which were eluted as multiple peaks. The peak A was composed by acidic endoglucanases (pI 3.0, 3.3 and 3.8) which were not adsorbed on CF11-celulose, whereas peaks F to K contained endoglucanases which adsorbed on this cellulose. Although accurate pI determination was not possible by using the gel overlay technique, these results suggests that there are several endoglucanases from peak I with similar pIs but with different affinity for CF11-cellulose. The activity of endoglucanases from peaks B to E could not be detected by the gel overlay technique.
Both basic endoglucanases from peak I and peak II degraded CMC, however, those from peak I degraded xyloglucan from H. courbaril whereas those from peak II did not. Nevertheless, the possibility that these peak II enzymes degrade xyloglucan from E. heterophylla endosperm should not be discarded. A fraction of CMC-degrading endoglucanases from peak II exhibited small mobility on native PAGE (detected in the uppermost region of gel), but these enzymes were not stained by Coomassie blue. Polysaccharides tightly associated with cell wall hydrolase may hinder the demonstration of the enzyme by electrophoretic methods (Messner et al., 1990). By contrast with peak I endoglucanases, peak II enzymes were not active after mild denaturating SDS-PAGE. This could be due to the cleavage of disulphide bonds and/or subunits dissociation which changed enzymatically active conformation of these endoglucanases.
The proportion of monosaccharides (ratio Glu:Xyl:Gal) of xyloglucans from seeds of H. courbaril is 4:3.4:1.2 and from C. langsdorffi is 4:3.0:1.8 (Buckeridge et al., 1997). Although both xyloglucans have a similar monosaccharide composition, there are differences between them in relation to the fine-structural details. After hydrolysis with pure fungal cellulase, the limit digest of xyloglucan from seeds of C. langsdorfii is composed of a mixture of oligosaccharide XXXG, XLXG, XXLG, and XLLG, whereas hydrolysate of xyloglucan from H. courbaril is composed of about 50% of these oligosaccharides and 50% of XXXXG itself plus its ß-galactosyl substituted subunits (Buckeridge et al., 1997). Some endoglucanases of E. heterophylla hydrolyse H. courbaril xyloglucan, but do not hydrolyse C. langsdorffii xyloglucan or vice-versa. It is known that the pattern of substitution of the glucan backbone around the cleavage site can prevent chain-cleavages by xyloglucan-degrading enzymes (Fanutti et al., 1996; Vincken et al., 1996). Perhaps some E. heterophylla endoglucanases recognize highly specific cleavage sites being active on one of the xyloglucans but not on the other xyloglucan. Alternatively, the activity of these enzymes may depend on the previous action of -xylosidases and/or ß-galactosidase which remove xylose and galactosyl substituents of glucan backbone, respectively.
The analysis of monosaccharide composition of water-soluble polysaccharides from the endosperm of this species indicated the occurrence of glucose (11.0%), galactose (36.9%), xylose (47.9%), and arabinose (4.2%); moreover, in the germinating seeds, the activities of endoglucanases and other xyloglucan-degrading enzymes such as ß-galactosidase, -xylosidase and ß-glucosidase overlap (CNK Suda, MS Buckeridge, JF Giorgini, unpublished data). These results strongly suggest xyloglucan as a cell wall component of E. heterophylla endosperm, although the occurrence of xyloglucan remains to be confirmed in the seeds of this species. The majority of endoglucanases from E. heterophylla were able to hydrolyse xyloglucans from H. courbaril and/or from C. langsdorffii. The physiological role of these enzymes in E. heterophylla seeds could involve the degradation of xyloglucan.
The activity of endoglucanases (cellulases) from seeds have been detected in cotyledons of germinating kidney beans (Lew and Lewis, 1974) and after germination of both tomato (Leviatov et al., 1995) and coffee bean (Takaki and Dietrich, 1980; Giorgini, 1992). In these cases, the enzymes hydrolysed CMC, but the activity on xyloglucans was not reported. Otherwise, xyloglucan-specific endo-1,4-ß-glucanase (or XET) of nasturtium seeds only hydrolyses xyloglucans, with no action on cellulose, other soluble cellulose derivatives, cello-oligosaccharides or other substrates with 1,4-ß-D-glucosyl linkages (Edwards et al., 1986). It is possible that, in the seeds of E. heterophylla, xyloglucan is depolimerized by CMCases which can act as xyloglucanases, by xyloglucan-specific endoglucanases which can not hydrolyse CMC, and by glycosidases that remove xyloglucan side chains.
Seed xyloglucanases have been investigated only in species that have stored xyloglucan in the cotyledon (Edwards et al., 1985; Sulová et al., 1995; Tiné et al., 2000; Steele et al., 2001). The present work is one of the first to investigate the properties of xyloglucan-degrading endoglucanases in seed endosperm.
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Acknowledgements |
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We thank Mr Jayme L Zeotti for technical assistance and Professor João A Jorge for helpful suggestions during the course of this work. This work was supported by grants 92/3176-4 and 96/8069-2 from FAPESP to JFG. CNKS is thankful to CAPES for a doctoral fellowship.
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