JXB Advance Access originally published online on May 31, 2005
Journal of Experimental Botany 2005 56(417):1923-1931; doi:10.1093/jxb/eri187
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RESEARCH PAPER |
Lygodium japonicum fern accumulates copper in the cell wall pectin
1Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan
2Shionogi Research Laboratories, Shionogi & Co., Ltd., Osaka 553-0002, Japan
* To whom correspondence should be addressed. Fax: +81 86 434 1249. E-mail: hakonno{at}rib.okayama-u.ac.jp
Received 19 November 2004; Accepted 7 April 2005
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Abstract |
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The present work reports the results of a study on the growth kinetics and characterization of matrix polysaccharides in the cell walls of Lygodium japonicum prothallium grown in the presence of copper (Cu). When the prothallium was cultured in the media containing 0.2 mM or 0.4 mM CuSO4, it showed a rapid accumulation of Cu with a maximum uptake of Cu measured in the cells up to 20 d of culture. The maximum rate of Cu uptake into the prothallium was greater for 0.4 mM Cu-treated cells (17.2 µmol g–1 DW) than for 0.2 mM Cu-treated cells (3.2 µmol g–1 DW). Cell walls were isolated from both untreated control and Cu-treated cells and then extracted sequentially with cyclohexane-trans-1,2-diaminetetra-acetate (CDTA), Na2CO3, 1 M KOH, and 4 M KOH. The amount of pectin solubilized from 0.4 mM Cu-treated cell walls decreased to 53% of its level in the control, whereas the amount of hemicellulose solubilized from the Cu-treated cell walls represented 82% of that from control cell walls. When the polysaccharides were fractionated by anion-exchange chromatography into four carbohydrate components, considerable increases in fractions PI-3 and PII-3 eluted with 0.5 M NaCl were observed in CDTA-soluble (PI) and Na2CO3-soluble (PII) pectic polymers from Cu-treated cell walls. Fractions PI-3 and PII-3 were composed predominantly of uronic acid (more than 71% of total sugars). Approximately 66% of Cu within the cell walls was released from the 0.4 mM Cu-treated cells with the endo-pectate-lyase treatment, suggesting that most of the Cu that accumulated into the Lygodium prothallium is tightly bound to the homogalacturonan of the cell wall pectin.
Key words: Copper accumulation, endo-pectate lyase, fern (pteridophyte), homogalacturonan, Lygodium japonicum, pectin structure, phytoremediation, prothallium
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Heavy metals such as copper (Cu) and zinc are required by biological systems as catalytic and structural components of proteins and as cofactors essential to plant growth and development. However, since many heavy metals can bind easily to sulphydryl groups in the active sites of enzymes and structural proteins, a high heavy metal concentration interferes with the metabolism for normal growth, cell proliferation, and differentiation of most plant cells. The pollution of soil and water with Cu, iron, zinc, and lead is widespread; therefore, high levels of tolerance to these metals are required by plants growing in these contaminated sites. It has been reported to date that a small number of wild plants are capable of the extraction of heavy metals from contaminated soil and water by concentrating the metals in their roots and shoots (Kumar et al., 1995
). Currently, environmental remediation using the ability of the endemic plants to accumulate heavy metals has been proposed as ‘phytoremediation’ (Reeves and Baker, 2000).
Various mechanisms have been suggested for tolerance and detoxification of potentially toxic heavy metals in plants (Hall, 2002). It is generally well demonstrated that small cysteine-containing peptides capable of binding heavy metals, termed phytochelatins and metallothioneins, are rapidly synthesized within several plant cells in response to toxic levels of heavy metals (Steffens, 1990). The roots of some plant species also excrete specific organic materials, such as citrate, malate, and histidine, that can bind to Cu, nickel, and zinc, and, consequently, the formation of metal-chelating compounds could reduce the uptake of toxic free metal ions into the plant cells (Rauser, 1999). On the other hand, it has been thought that cell walls are implicated in playing the role of a storage compartment for heavy metals in tolerant plants. In Silene vulgaris grown in Cu-rich soil, as well as in Armeria maritima (Neumann et al., 1995), Cu is tightly bound to the cell wall proteins, and one of these proteins isolated from the wall preparations has been identified as oxalate oxidase by homology search and cross-reaction with an antibody (Bringezu et al., 1999). However, since most of the heavy metals in cell walls are tightly bound and cannot be removed, the subcellular localization of binding metals in the cell walls and their role as a mechanism of metal tolerance are still controversial ideas. Therefore, the interest in how cell walls participate in plant metal tolerance has led to attempts to elucidate the binding site of heavy metals within the cell walls of tolerant plants.
The gregarious mosses, such as Scopelophila cataractae and Pohlia bulbifera, accumulate a large amount of Cu and zinc in their cell wall materials (Oda and Honjo, 1995), and Mielichhoferia elongata and Scopelophila cataractae growing in Cu-enriched environments are known as the so-called ‘copper mosses’. High quantities of Cu, cadmium, and uranium are also accumulated within the cell walls of the roots and shoots of an aquatic fern Azolla filiculoides grown in the presence of high concentrations of these metals, and most of the cadmium is found in aggregates with phosphate or calcium in the xylem cells of the shoot bundle (Sela et al., 1988). Evidently, some species of bryophytes (liverworts and mosses) and pteridophytes (ferns) have been found in areas polluted with high concentrations of heavy metals (Shaw, 1994). A recent report shows that Pteris vittata (brake fern) growing on a site contaminated with chromated copper arsenate can take up large amounts of arsenic into its fronds in a short time (Ma et al., 2001), but the localization of arsenic in the fronds is still unknown.
Cell walls play a critical role in the mechanisms for growth, cell proliferation and differentiation. Thus, the structural features and characteristics of cell walls from seed plants, in particular, dicotyledons, have been studied extensively, and the new structural models of cell walls are documented (Carpita and Gibeaut, 1993). However, the cell walls of cryptogamous plants, such as bryophytes and pteridophytes, have received little attention (Bailey and Pain, 1971; Bremner and Wilkie, 1971; Konno et al., 1987; Popper et al., 2001), and so the peculiarities of the wall-polysaccharides in these plants have yet to be fully understood (O'Neill and York, 2003). Therefore, it is of keen interest to specify the cell wall components of cryptogamous plants grown in extreme environments. A climbing fern Lygodium japonicum is classified into the Filicales and is distributed throughout the warm temperate areas in Japan. The purpose of the present work was to elucidate whether Cu is accumulated within the cell walls when L. japonicum is grown under Cu-rich conditions and to characterize the matrix polysaccharides in the cell walls of the untreated control and Cu-treated cells. The localization of Cu accumulated in the walls of Lygodium cells grown in the presence of 0.4 mM CuSO4 was also investigated.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Plant material and growth conditions
The culture medium of Lygodium japonicum Sw. prothallium was based on the MSK-3 medium (Katoh, 1983
) used for Marchantia polymorpha culture, free from 2,4-dichlorophenoxyacetic acid and kinetin, supplemented with 30 g l–1 sucrose and solidified with 10 g l–1 agar. The prothallia developed from the aseptic spores were cultured onto fresh medium at 22 °C under constant irradiance of approximately 98 µmol m–2 s–1 and subcultured about every 2 months. Copper-treated cells were cultured by transferring a few pieces (approximately 0.5 g fresh weight) of prothallium to 100 ml Erlenmeyer flasks containing 50 ml of the medium, adding either 0.2 mM or 0.4 mM CuSO4 under the same environmental conditions. After 2 months of culture, the untreated control and Cu-treated cells were carefully removed from the medium, briefly rinsed with 1 mM EDTA, washed with distilled water, and stored at –80 °C until required. To establish the kinetics of L. japonicum growth, at least three flasks were randomly selected, and the untreated control and Cu-treated cells were harvested after 10 d of growth, briefly rinsed with 1 mM EDTA, washed with distilled water, dried with filter paper, and weighed. The prothallium was then dried for 3 d at 80 °C for further measurements of dry weight and Cu concentration.
Preparation of cell walls and sequential chemical extraction of the matrix polysaccharides
The frozen prothallium was homogenized in 100 mM K-phosphate (pH 7.0) containing 10 mM 2-mercaptoethanol using a Warring blender (PH91: SMT Corp, Tokyo, Japan) for 5 min at 0 °C. The homogenates were filtered through glass-fibre filters under suction at 2–4 °C; the soluble fraction was removed. The residues were washed with distilled water, resuspended in 50 mM Na-acetate (pH 5.2) containing 3 M LiCl, and stirred overnight at 4 °C. The suspension was filtered as described above, and the residues were treated with 90% (v/v) ethanol at 80 °C to inactivate wall-bound enzyme. The alcohol-insoluble residues were subsequently treated with 
-amylase (pancreas Type I-A; Sigma Chemical Corp., St Louis, MO, USA) and protease (Actinase E; Kaken Chemical Corp., Tokyo, Japan), as described previously (Konno et al., 2002b), washed with distilled water and ethanol, and then air-dried.
The procedure used to extract cell-wall polysaccharides is slightly modified from the method as described by Redgwell and Selvendran (1986). In brief, the ethanol-insoluble residues were treated sequentially with 50 mM cyclohexane-trans-1,2-diaminetetra-acetate (CDTA, pH 6.5) at 20 °C for 8 h and 50 mM Na2CO3 at 1 °C for 20 h to solubilize pectin. The depectinated cell walls were treated sequentially with 1 M KOH at 20 °C for 2 h under N2 and 4 M KOH at 20 °C for 2 h under N2 to solubilize hemicellulose. The extracting solutions of Na2CO3, 1 M KOH, and 4 M KOH contained 20 mM NaBH4 to prevent any possible ß-oxidation of the cell wall polysaccharides. All extracts were dialysed exhaustively against distilled water; Na2CO3- and KOH-extracts were neutralized with glacial acetic acid prior to dialysis.
Chromatography of extracted polysaccharides
Extracted polysaccharides were fractionated by anion-exchange chromatography, as described previously (Konno et al., 2002a). In brief, the extracted polysaccharides were concentrated by evaporation and dialysed against 20 mM K-phosphate (pH 6.0). An aliquot (containing 30–100 mg sugar) was applied to a DEAE-Sepharose CL-6B (Amersham Biosciences, Uppsala, Sweden) column (2.0x20 cm) previously equilibrated with 20 mM K-phosphate (pH 6.0), and eluted sequentially with the same buffer and with buffer containing 125, 250, or 500 mM NaCl, and finally with 200 mM NaOH. The eluate was collected as 4 ml fractions and assayed for total sugars.
Enzyme treatment of cell walls
The cell walls (200 mg dry weight) isolated from 0.4 mM Cu-treated cells were incubated in 40 ml reaction mixture containing 150 mg of endo-pectate lyase (EC 4.2.2.2
[EC]
; Pectolyase Y23 from Aspergillus japonicus; Seishin Corp., Tokyo, Japan), 0.5 mM CaCl2, and 50 mM TRIS–HCl (pH 8.6), with constant shaking for 48 h at 30 °C in the presence of 0.02% (w/v) NaN3 acting as a bacteriostatic. After the enzyme treatment, the wall residues were separated from the digestion products by centrifugation at 12 000 g for 20 min, washed with distilled water and ethanol, and then air-dried. The digestion product was adjusted to pH 5.0 with glacial acetic acid and heated in a boiling water bath for 3 min. The product was concentrated by evaporation and applied to a Bio-Gel A-5m (Bio-Rad Laboratories, Hercules, CA, USA) column (1.5x90 cm) equilibrated with 50 mM K-phosphate (pH 7.0) containing 20 mM EDTA, and eluted with the same buffer. The eluate was collected as 2 ml fractions, and assayed for total sugars and Cu concentration. For endo-polygalacturonase (EC 3.2.1.15
[EC]
) treatment, the Cu-treated cell walls were incubated in 40 ml reaction mixture containing 250 mg of Pectinase P-2401 (from Rhizopus sp; Sigma Chemical Corp.) and 50 mM Na-acetate (pH 4.0). The reaction was carried out as described above.
Carbohydrate analyses
Total sugars and uronic acid in each sample were estimated by the phenol–H2SO4 (Dubois et al., 1956) and m-hydroxydiphenyl (Blumenkranz and Asboe-Hansen, 1973) methods, respectively, using glucose and galacturonic acid as the standards. Uronic acid content in intact cell walls and the wall residues was determined after complete hydrolysis with concentrated H2SO4 (Ahmed and Labavitch, 1977). Neutral carbohydrate composition of non-cellulosic polysaccharides was determined by gas-liquid chromatography of the alditol acetate derivatives following hydrolysis in 2 M trifluoroacetic acid for 1 h at 121 °C (Albersheim et al., 1967).
Copper and calcium determinations
Copper and calcium concentrations in the intact cells and cell wall preparations were determined by atomic absorption spectrometry after acid digestion in boiling HNO3/HClO4 mixture (6:1 v/v).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Plant growth and copper accumulation
The growth of L. japonicum prothallium under normal and Cu-enriched conditions was examined (Fig. 1A). Cell mass (g fresh weight) increased from the start up to 90 d, with pre-exponential (0–30 d of culture) and exponential (30–90 d) phases, irrespective of whether the cells were cultured in normal or Cu-enriched medium. Copper led to a reduction in cell growth; the maximum mass of cells grown in the presence of Cu decreased to 40–50% of the control level at 90 d. However, the cell mass continued to increase from the start up to 90 d in culture and appeared in similar amounts in the media containing 0.2 mM or 0.4 mM CuSO4 throughout the culture period of 90 d. Total Cu content accumulated in the cells also increased linearly from the start up to 90 d in culture on the media containing 0.2 mM or 0.4 mM CuSO4, as shown in Fig. 1B. In the control cells, Cu was only detected in trace amounts or was not detected during cell growth. The prothallium was tinged with bright green and no change in pigmentation was observed in both untreated control and Cu-treated cells up to 90 d of culture. Although the Cu-treated cells became slightly more firm compared with control cells, necrotic areas were insignificant at the end of the culture period (data not shown).
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When the cells were grown in the presence of 0.2 mM or 0.4 mM CuSO4, a rapid uptake of Cu was shown with high Cu concentration in the cells up to 20 d of culture (Fig. 1C). The maximum rate of Cu uptake into the cells was greater for 0.4 mM Cu-treated cells (17.2 µmol Cu g–1 DW) than for 0.2 mM Cu-treated cells (3.2 µmol Cu g–1 DW); the uptake of Cu increased 5.4-fold in 0.4 mM Cu-treated cells over the level in 0.2 mM Cu-treated cells throughout the cell culture period of 90 d. After 20 d of culture, the Cu uptake in 0.4 mM Cu-treated cells continued to decrease up to 60 d, whereas the Cu uptake in 0.2 mM Cu-treated cells remained more or less constant at approximately 3.7 µmol g–1 DW of cells from 20 d up to 90 d of culture. Perhaps the decrease of Cu uptake in 0.4 mM Cu-treated cells results in the reduction in the rate of Cu uptake into the cells rather than the exudation of accumulated Cu during the growth process.
Characteristics and carbohydrate content of Lygodium cell walls
The contaminating starch present in the cell wall residues was removed by the treatment with pancreatic -amylase. After an exhaustive reaction time, the iodo-starch reaction (I2-KI solution) of the cell walls was negative. Thus starch was scarcely contained in the cell wall preparation. Copper concentrations in cell wall preparations (g dry weight) from the control, 0.2 mM Cu-treated, and 0.4 mM Cu-treated cells were 0.1 µmol, 2.2 µmol, and 8.1 µmol (mean of two replicates), respectively, after 60 d of culture. A considerably higher Cu content was observed in 0.4 mM Cu-treated cell walls compared with the control. Calcium concentrations in cell wall preparations from the control and Cu-treated cells were 58.1 µmol and 134.4 µmol, respectively. High concentrations of apoplastic calcium stimulate the deposition of pectic polysaccharides in cell walls (Eklund and Eliasson, 1990; Konno et al., 2002a) and the acidification of pectic molecules (His et al., 1997) of some plants. It is interesting to note, therefore, that calcium concentration in Cu-treated cell walls was 2.3-fold higher than that of the control cell walls.
A comparison was made of the carbohydrate composition of non-cellulosic polysaccharides in cell walls from the control and Cu-treated cells (Table 1). Fucose could only be detected in trace amounts in the cell walls from both the control and Cu-treated cells (data not shown). Uronic acid appeared in similar amounts in the control and Cu-treated cells, whereas arabinose in 0.4 mM Cu-treated cell walls decreased to a value of 61% of that found in the control cell walls. The suggestion has been made that the side chains (e.g. arabinan and galactan) of pectic polysaccharides are regulated in relation to the proliferation and differentiation of plant cell development (Willats et al., 1999; Ermel et al., 2000; McCartney and Knox, 2002). Recently, it has been shown that the leaf cell walls of the lycophyte Selaginella apoda contained unique 3-O-methyl-D-galactose, in addition to the usual glycosyl residues detected in cell walls of seed plants (Popper et al., 2001). Thus, the change in the neutral sugar contents of cell walls from the growth-limited cells of L. japonicum under Cu enrichment is an intriguing problem, which is still under investigation.
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Analysis of pectic and hemicellulosic polysaccharides from Lygodium cell walls
To elucidate the variation in the amount of matrix polysaccharides, cell walls isolated from untreated control and Cu-treated cells of L. japonicum were exposed to a sequential extraction using the chemical extractants CDTA, Na2CO3, 1 M KOH, and 4 M KOH (Table 2). The pectic fraction (PI) held in the cell walls by calcium was solubilized with CDTA as a chelating agent, and the CDTA-insoluble pectic fraction (PII) was extracted with Na2CO3 at 1 °C (Jarvis et al., 1981
). Pectic fractions (PI and PII) solubilized from the control, 0.2 mM Cu-treated, and 0.4 mM Cu-treated cell walls with both CDTA and Na2CO3 treatment accounted for 10.3%, 7.7%, and 5.5%, respectively, of cell wall dry weight; the amounts of pectic fractions from 0.2 mM Cu-treated cell walls and 0.4 mM Cu-treated cell walls decreased to 74% and 53%, respectively, of the level in the control cell walls. Subsequent extraction with 1 M KOH (HI) and 4 M KOH (HII) following extraction of pectic polysaccharides solubilized the hemicellulosic polysaccharides. The hemicellulosic fractions (HI and HII) were 14.2%, 12.1%, and 11.6% from the depectinated cell walls of the control, 0.2 mM Cu-treated, and 0.4 mM Cu-treated cells, respectively. Thus, the extracted matrix polysaccharides accounted for 24.5%, 19.8%, and 17.0% of the cell walls from the control, 0.2 mM Cu-treated, and 0.4 mM Cu-treated cells, respectively. It is clear that the walls of Cu-treated cells contained a much lower proportion of pectic polysaccharides soluble in CDTA and Na2CO3 than the control cell walls and a somewhat lower amount of hemicellulosic polysaccharides, that could be solubilized.
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The carbohydrate composition of non-cellulosic polysaccharides in the wall residues remaining after extraction of pectic and hemicelllulosic fractions was also analysed for the control and Cu-treated cells (Table 1). The wall residues still contained the neutral sugars and uronic acid detected in native cell walls, suggesting that some of matrix polysaccharides were extracted incompletely by all of the methods used in this study. Some of the matrix polysaccharides in L. japonicum cell walls might be tightly linked to other wall components.
Carbohydrate composition of the extracted pectic and hemicellulosic polysaccharides
The carbohydrate composition of pectic and hemicellulosic polysaccharides solubilized from cell walls was compared for the control and Cu-treated cells (Table 3). The pectic fractions (PI and PII) of both control and Cu-treated cell walls were composed mainly of uronic acid (more than 60% of total sugars) followed by galactose, arabinose, rhamnose, and glucose. Uronic acid in fraction PI of Cu-treated cell walls was present in higher amounts than in the control cell walls. By contrast, fraction PI from the Cu-treated cell walls showed a lower content of galactose compared with control cell walls. In hemicellulosic polysaccharides, a considerable increase in uronic acid content was observed in fraction HI of the Cu-treated cell walls. Since neither CDTA nor Na2CO3 are completely efficient in solubilizing pectic polysaccharides, some of the pectic polymers are probably extracted later with the far more efficient 1 M KOH. These results clearly show that Cu complexation with uronic acid-rich pectic polymers leads to reduced solubility and better binding to the other wall-components. Concentrated alkali (4 M KOH) is usually required to solubilize xyloglucan from the cellulose/xyloglucan complex in cell walls (Pauly et al., 1999). Xylose and glucose were the predominant sugars present (more than 52% of total sugars) in fraction HII from both the control and Cu-treated cell walls, presumably from xyloglucan that is a ubiquitous constituent of hemicellulose found in the cell walls of many seed plants.
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Fractionation of pectic and hemicellulosic polysaccharides
The pectic polysaccharides (PI and PII) solubilized from the control and Cu-treated cell walls were chromatographed on a DEAE-Sepharose CL-6B column into four carbohydrate polymer fractions using a stepwise increase in NaCl concentration and alkaline solution. The chromatographic recoveries of each sample were 67–72% and 60–94% for the polysaccharides from the control and Cu-treated cell walls, respectively. Most of the pectic fractions (PI-1 and PII-1) solubilized from the control cell walls were eluted in the void volume of the column, and were distinctly different from those of the corresponding fractions from the Cu-treated cell walls, as shown in Fig. 2. The yields were approximately 55% of fractions PI-1 and PII-1 from the polysaccharides of control cell walls, and were 4% of fraction PI-1 and 2% of fraction PII-1 from the polysaccharides of 0.4 mM Cu-treated cell walls. While the yields were approximately 2% of fractions PI-3 and PII-3 from the polysaccharides of the control cell walls, and were 23% of fraction PI-3 and 27% of fraction PII-3 from the polysaccharides of 0.4 mM Cu-treated cell walls. An increase of more than 20% was observed in fractions PI-3 and PII-3 from Cu-treated cell walls compared with their amounts in control cell walls. When hemicellulosic fractions (HI and HII) were fractionated by DEAE-Sepharose CL-6B chromatography as well, the profiles for the polysaccharides of Cu-treated cell walls were very similar to those of the control cell walls (data not shown).
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The carbohydrate composition of major fractions eluted with buffer, buffer containing 250 and 500 mM NaCl, and 200 mM NaOH was compared with those of the control and Cu-treated cells (Tables 4, 5). The major fractions (PI-1 and PII-1) from the control cell walls contained similar amounts of uronic acid and neutral sugars, mainly galactose, arabinose, and rhamnose. Fractions PI-1 and PII-1 were not retained on the DEAE-Sepharose column despite the high levels of uronic acid. This could be due to the structural complexity of the components or the aggregation of pectic molecules. Fraction PI-3 from 0.4 mM Cu-treated cell walls was composed mostly of uronic acid (76% of total sugars), very similar to the compositions of fraction PII-3 from 0.4 mM Cu-treated cell walls. Thus, the variation in the elution patterns of CDTA-soluble and Na2CO3-soluble polymers between the control and Cu-treated cell walls would reflect the increase in the amount of uronic acid in the pectic polysaccharides of the Cu-treated cell walls. Pectin is a very complex molecule made up of acidic and neutral domains and is instrumental in the formation of cell wall characteristics, cell-to-cell adhesion, and cell differentiation (Ridley et al., 2001
). In suspension-cultured tobacco cells, cells adapted to water and saline stress increased the proportion of their uronic acids derived from the acidic domains, such as homogalacturonan and rhamnogalacturonan, compared with unadapted cells (Iraki et al., 1989). The walls of adapted tomato cells growing in a medium containing a herbicide (2,6-dichlorobenzonitrile) also contained a substantially higher proportion of homogalacturonan and rhamnogalacturonan-like polymers (Shedletzky et al., 1990). Thus, the change in the acidic domains in Cu-treated cells could be considered as the principal mechanism of tolerance against environmental stress. After the extraction with chelating agents, dilute alkali (Na2CO3) is effective in solubilizing galactan-rich pectic polymer from the potato cell walls (Jarvis et al., 1981). However, it is difficult to explain a considerably higher amount of galactose in fraction PII-2 from the control cell walls and fractions PII-2 and PII-4 from Cu-treated cell walls.
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Localization of copper in the cell walls of Lygodium cells grown in the presence of copper
Finally, it is of keen interest to specify the cell wall components (e.g. pectin, hemicellulose, and cellulose) to which heavy metals bind. Copper could scarcely be detected in the matrix polysaccharides extracted with chemical agents from 0.4 mM Cu-treated cell walls, but the Cu content in the wall residues had decreased to 20–23% of the level of 0.4 mM Cu-treated cell walls. This suggests that the majority of Cu bound to the matrix polysaccharides of Cu-treated cell walls, and Cu might be removed from the solubilized polysaccharides during the chemical extraction procedure. The possibility of Cu being associated with pectic polysaccharides was tested by pectic enzyme-extracting Cu-treated cell walls from Lygodium cells grown in the presence of 0.4 mM CuSO4. Pectate lyase and polygalacturonase are able to attack the intact cell walls directly and effectively solubilize homogalacturonan as the enzymic digestion proceeds under mild conditions (Konno et al., 1986
). When the Cu-treated cell walls were incubated with purified endo-pectate lyase or endo-polygalacturonase, these enzymes released more than 66% and 35% of Cu, respectively, from Cu-treated cell walls (Table 6). The difference in extraction of Cu from Cu-treated cell walls could reflect a difference in the action pattern of two enzymes on cell wall polysaccharides. The incubation with TRIS–HCl buffer (pH 8.6) alone, free from enzyme, also released 21% of Cu from the cell walls, which is possibly due to the ß-eliminative cleavage of homogalacturonan during incubation with the alkali medium (Brett and Waldron, 1996). The reaction product obtained after treatment with endo-pectate lyase was further chromatographed by size-exclusion on a Bio-Gel A-5m column and was fractionated into four carbohydrate fractions (F-1, F-2, F-3, and F-4; Fig. 3). Each fraction was pooled, concentrated, and measurements taken of total sugar and Cu contents. The sugar content yields were 1.4 mg of fraction F-1, 6.9 mg of fraction F-2, 11.9 mg of fraction F-3, and 125 mg of fraction F-4 from 171 mg of the soluble sugar released from Cu-treated cell walls, and the Cu content was 0.06 µmol of fraction F-1, 0.03 µmol of fraction F-2, 0.03 µmol of fraction F-3, and 0.70 µmol of fraction F-4 from 0.87 µmol of Cu released from the Cu-treated cell walls. Thus, chromatographic recoveries were 85% of the sugar and 94% of the Cu from the Cu-treated cell walls. Fraction F-4 obtained at the exclusion limit of the Bio-Gel A-5m gel was co-eluted with the majority of total Cu content solubilized from the Cu-treated cell walls. These results show clearly that a significant part of the Cu in Cu-treated cells is tightly bound to galacturonic acids of homogalacturonan in the cell wall pectin.
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Since Cu was also detected in the cell wall residues remaining after the treatment with endo-pectate lyase, the carbohydrate composition of non-cellulosic polysaccharides of the wall residues was analysed (Table 1). Although the wall residues still contained similar amounts of neutral sugars in the native Cu-treated cell walls, the content of uronic acid in the wall residues decreased markedly after the enzyme treatment. The wall residues contained arabinose, galactose, and approximately equal amounts of rhamnose and uronic acid, suggesting a composition typical for rhamnogalactuonan I with several neutral side-chains. The cell wall preparation used in this experiment was treated with LiCl and protease, and so ionically and tightly bound proteins and structural proteins present in the cell walls had already been removed. It seems likely that the remainder of the Cu in the wall residues would bind tightly to the galacturonic acids of rhamnogalacturonans in Cu-treated cells.
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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A significant finding in the present study is that the amount of acidic pectic domains solubilized from the cell walls of L. japonicum prothallium grown in the present of Cu was very different from that from control cells, and Cu taken up into the cells was tightly bound to homogalacturonan in the cell wall pectin. To the best of current knowledge, this is the first report of the characteristics of pectic polysaccharides of cell walls from pteridophytes (ferns) grown under metal stress conditions. The variation in the carbohydrate composition of matrix polysaccharides could be a consequence of an alteration of the polysaccharide synthesis and the linkage breakdown caused by the glycosyl-hydrolytic enzymes (Fry, 1995
; Konno et al., 2002b). Many glycohydrolase activities can be detected in the protein fraction from active cell walls of Cu-treated cells of L. japonicum (H Konno, unpublished data). The study of specific cell wall-modifying enzymes of Cu-treated cells compared with those of control cells will be of considerable interest. The endemic plants that tolerate unusually high concentrations of heavy metals accumulate heavy metals in their vegetative organs having primary and secondary walls of different composition. The cell walls of the Lygodium prothallium examined in the present work are probably similar to the characteristics of the primary cell wall and may be somewhat different from those of fronds of the whole plant. In the light of these considerations, a further study is planned to investigate the difference in the pectic polysaccharides between the two. Finally, it must be considered whether high levels of Cu are detected in the cell wall pectin of Lygodium fronds growing in the metal-enriched conditions. |
Acknowledgements |
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The authors would like to thank Dr Tomoyuki Yamaya (Tohoku University, Sendai, Japan) for his helpful discussion. This research was supported in part by grants from the Foundation of Sanyo Broadcasting and the Oohara Foundation for Agricultural Sciences.
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References |
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