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Analysis for nonextractable (bound) residues of pentachlorophenol in plant cells using a cell wall fractionation procedure
ECOTOXICOLOGY
AND
ENVIRONMENTAL
SAFETY
l&268-279
(1985)
Analysis for Nonextractable (Bound) Residues of Pentachlorophenol in Plant Cells Using a Cell Wall Fractionation Procedure’ C. LANGEBARTELS Institut Landwirtschaji
ftir Pjanzenerntihrung (FAL), Bundesallee
AND H. HARMS
und Bodenkunde, Bundesforschungsanstalt 50, Braunschweig D-3300, Federal Republic Received
fir of Germany
March 6, 1985
When plant cell cultures or aseptically grown wheat plants were treated with [‘Clpentachlorophenol (PCP) a major part of the label was found in a nonextractable or “bound” residue fraction. Soluble polar conjugates participated in the formation of these residues which were mainly located in the plant cell walls. By a sequential fractionation procedure using enzymatic and chemical methods, 90 to 95% of the bound radioactivity could be attributed to individual cell wall components. The 14C label from PCP was found mainly in hemicellulose, lignin, and protein fractions. Associations of hemicellulose with PCP derivatives with molecular weights up to 500,000 were purified to constant specific radioactivity. Hydrolysis of this fraction released 32% PCP and other unidentified products. o 1985 Academic PIW, LX. INTRODUCTION
Investigations of the metabolism of [ 14C]pentachlorophenol (PCP) in cell cultures of soybean, lupin, and wheat and aseptically grown wheat plants showed that a major part of the applied radioactivity was associated with fractions that could not be extracted with cold organic solvent mixtures (Langebartels and Harms, 1984). This nonextractable or bound radioactivity increased with time and was highest in wheat cultures where 35 to 50% of the applied radioactivity could be recovered in this fraction after 48 hr of incubation. Several components of the plant cell walls have been assumed to form complexes with xenobiotics (Kaufman et al., 1976; Huber and Otto, 1983; Sandermann et al., 1983). Association of PCP with cellulose and lignin fractions was reported for rice plants (Weiss et al., 1982) and with lignin in cell cultures of wheat (Scheel et al., 1984). This communication describes a sequential approach for fractionating bound residues in plants. Plant cells, as well as pelleted cell walls, were extracted with cold organic solvents and fractionated using a cell wall fractionation procedure. By this means individual fractions consisting of mainly starch, proteins, pectins, lignin, hemicellulose, or cellulose could be separated. Dark-grown cell cultures were mainly used for studying the formation of nonextractable residues to exclude PCP degradation by microorganisms and light; CO* refixation was not detected in these plant systems. MATERIALS
AND
METHODS
Plant Material Cell suspension cultures of Triticum aestivum L. and Glycine max L. were cultured as described by Langebartels and Harms (1984). Lupinus palyphyllus LINDL. SUS’ This communication was presented in part at the symposium “Bioavailability of Environmental Chemicals,” Schmallenberg, FRG, Sept. 12- 14, 1984. 0147-6513185 $3.00 Cop&h1 Q 1985 by Academic Press, Inc. All rights of rqxoduaion in any form reserved.
268
FRACTIONATION
OF NONEXTRACTABLE
PCP RESIDUES
269
pension cultures were propagated under white fluorescent light (8000 lx, 12 hr) at 28°C in two-tier vessels. COz concentration was kept at 1% by K&OJ/KHCO~ mixtures in the lower compartment. The culture medium was based on Murashige/Skoog with 2% sucrose, 2 mg/liter 2,4-D, and 0.25 mg/liter benzyladenine (C. Sator, Braunschweig, personal communication). Wheat plants were cultured in Knop’s nutrient solution under aseptic conditions according to Harms ( 198 1). Incubation
with [‘4C]PCP
Incubation times varied from 30 set to 48 hr under dark conditions for soybean and wheat cells, while photomixotrophic lupin cultures and wheat plants were grown under a 12-hr light/dark regime. Cell cultures were incubated with [u-“C]PCP (4 X 10e6 mol liter-‘, sp act 1.4 GBq/mmol; NEN, Dreieich, FRG) during the late stage of the logarithmic growth phase, while PCP was applied to wheat plants when they were 2-3 weeks old (15 to 20 cm). Plant cells and nutrient solutions were extracted with methanol:chloroform: water (2: 1:0.8) according to Ebing et al. (1984). Nonextractable residues were freeze-dried and then washed with 50 mM potassium phosphate buffer (pH 7.0), methanol:chloroform (2: 1, v/v), and acetone, successively. Alternatively, cells were separated from the medium by suction under mild vacuum and then homogenized in 50 mM potassium phosphate buffer, pH 7.0 (Fig. 1). Cell walls were pelleted by centrifugation at 6000g for 10 min and washed twice with buffer. Aqueous cell extracts and cell wall pellets were extracted with MeOH/CHC& (see Fig. 1). Differential centrifugation of the cell homogenates according to Ray (1977) was performed at 1000,2000,5000, 10,000, and 2O,OOOg( 10 min each) and at 40,OOOg X 30 min at 4°C. The 14C residue was determined by combustion in an oxidizer (OX 300, Zinsser, Frankfurt, FRG) and liquid scintillation counting. Cell Wall Fractionation Cell walls were fractionated into individual wall components by a modified extraction scheme according to Takeuchi and Komamine ( 1980). A flow diagram of the methods used is shown in Fig. 1. Extraction of water-soluble polysaccharides and proteins, extraction of lipids. One gram of cell wall or residue material was suspended in 100 ml of 50 mM potassium phosphate buffer (pH 7.0), sonificated for 5 min, and stirred for 4 hr at room temperature. The mixture was filtered through glass-fiber filters and the radioactivity of the filtrate was determined. The extracted residue was shaken for 2 hr with 100 ml methanol:chloroform (2: 1, v/v) and then acetone for 2 hr at room temperature. It was then washed with 20 ml of potassium phosphate buffer. All procedures in aqueous solutions were performed under an argon atmosphere and in the presence of toluene to prevent microbial infections. Slurry, after the individual steps, was filtered by suction on glass-fiber filters. Extraction ofstarch. Cell wall material on the filter, after washing with potassium phosphate buffer, was treated with 0.2 ml cu-amylase from porcine pancreas (Sigma, type IA) in 100 ml 50 mA4 potassium phosphate buffer (pH 7.0). Incubation was performed for 20 hr at 30°C. Extraction ofproteins. The material remaining after starch extraction was suspended in 100 ml Pronase E (3 mg/ml, Sigma) solution in 50 mM Tris-HCl buffer at pH 7.2 and incubated for 16 hr at 30°C.
270
LANGEBARTELS
AND HARMS
I
1
cells
culture
I homogenization centrifygation
medium
in PPB 6 000 g x 10 min
I supernatant wash!I'ifih chloroform
PPB , methanol/ (2 : 1), acetone
I
I
pelllet cell 1
walls
&
cell
extract
a-amylase starch pronase
pectinase
E
or EGTA 50 mM, 80 'C
dioxane/H20 dioxane/Z ~~~;,ase
9 : 1 N HCl 70 'C
t or KOH (1 N or
H2S04
dioxane-sol.
fract.
24 %)
72 % hydrolysate
residue CA
FIG. I. Flow diagram of the fractionation procedure for cell walls. Cells were homogenized and extracted either in MeOH/CHC13/H20 as described under Materials and Methods, or in aqueous buffer. Cell wags were collected by centrifugation and extracted. Cell wall residues or nonextractable residues were then fractionated by the same procedure starting with removal of contaminating starch.
Extraction of EGTA-soluble material (pectin fraction). Pectins were isolated by an incubation with 100 ml 50 rr&I EGTA (ethylene glycol bis-(Zaminoethyl ether)-N,N’tetraacetic acid) in 50 mJ4 sodium acetate buffer at pH 4.5. After 2 min of ultrasonic disintegration the suspension was heated to 80°C for 4 to 6 hr. A parallel incubation with pectinase from Aspergillus niger (Sigma) was also performed. An amount equal to 100 mg residue material was incubated with 10 ml of a pectinase solution (100 units, 16 mg protein) in 50 mM sodium acetate buffer at pH 4.0. The reaction was terminated by the addition of 200 ~1 glacial acetic acid. Extraction of lignin. Lignin was extracted in the sequential fractionation procedure by treatment with either DMSO or dioxane-water and dioxane:2 N HCl. Cell wall material collected by filtration after pectin extraction was suspended in 10 ml DMSO or dioxane-water (9: 1) for 48 to 96 hr at room temperature. During the first 30 min of the incubation period residues were sonificated with cooling on ice. The insoluble material was filtered off and further treated with 10 ml DMSO at 80°C for 16 hr or dioxane:2 N HCl(9: 1) at 70°C for 5 hr, respectively. After cooling to room temperature
FRACTIONATION
OF
NONEXTRACTABLE
PCP
RESIDUES
271
the mixture was passed through glass-fiber filters and the insoluble material was washed with DMSO or water. Extraction of KOH-soluble (hemicellulose) material. An extraction procedure using cold alkali is useful for the removal of noncellulosic polysaccharides of a neutral sugar composition. Cell wall material was suspended in 10 ml 1 N and 24% KOH at 27°C for 24 hr. The mixture was brought to pH 3-4 with 6 N acetic acid, diluted with water, and stirred for 1 hr. Precipitated material was pelleted by centrifugation ( 1OOOgX 10 min). PCP-hemicellulose associations were solubihzed by hemicellulase from A. niger (Sigma, 50 mg/ml) in 50 mM sodium acetate buffer, pH 5.5, for 4 hr at 27°C. H2S04 hydrolysate (cellulose fraction). The insoluble residue, after alkali treatment, was incubated in 72% sulfuric acid for 4 hr at room temperature. After neutralization with KOH the suspension was filtered off. The remaining insoluble material was combusted to COZ in an oxidizer and analyzed for radioactivity. Characterization
of Solubilized
Fractions and Cell Wall Polysaccharides
Polysaccharide and protein fractions were dialyzed against bidistilled water for 16 hr. The freeze-dried material was stored in a desiccator in the dark. Extracts were hydrolyzed with 2 N HCl (1 10°C) and separated on precoated silica gel (glass) plates (Merck) using (1) n-hexane:diethyl ether:formic acid (70:30:4) or (2) ethyl acetate: acetic acid:water (60:20:20) as solvents. Reference substances and HPLC separations used have been described by Langebartels and Harms ( 1984). The separated radioactive components were integrated with a TLC linear analyzer (Berthold, Wildbad, FRG). 14C radioactivity in the solutions was determined with a Philips liquid scintillation spectrometer. Total sugar was determined by the phenol-sulfuric acid method (Dubois et al., 1956) uranic acid content was measured using carbazol/sulfuric acid (Bitter and Muir, 1962). Hydrolysis of the polysaccharide fractions was performed by heating with 2 N trifluoroacetic acid at 120°C for 60 min or by enzymatic degradation. Monosaccharides were separated by HPLC on a Lichrosorb NH2 column (Merck) and with 80% acetonitrile as solvent using a Melz refractive index detector (LCD 201, Gynkotek, Mtinchen, FRG). Molecular
Sieve Chromatography
Pectic and hemicellulosic polysaccharides, associated with the 14C label from PCP, were subjected to gel filtration chromatography on Sepharose 4 B, Sephacryl S-300, and Bio-Gel P2 (all 90 X 1.5 cm, Pharmacia and BioRad). The lyophilized and dialyzed materials were dissolved in 50 mA4 sodium acetate buffer, pH 5.0, and a small amount of insoluble matter was removed by centrifugation (5 min at 5000 rpm) after heating the samples to 80°C for 5 min. Samples, containing 5- 12 mg of carbohydrate material, were applied to the columns and eluted with sodium acetate buffer at a flow rate of lo-15 ml/hr. Fractions of 2.5 to 4.5 ml were collected. Aliquots were taken for the determination of radioactivity ( 1 ml), total sugar content (0.2 ml), and monosaccharide composition (l-3 ml), Dextrans of defined molecular weight ranging from 10,000 to 500,000 (Pharmacia) were used as markers for molecular weight determinations.
272
LANGEBARTELS
RESULTS
AND
AND
HARMS
DISCUSSION
Adsorption of PCP by Soybean and Wheat Cells Previous time-course studies with soybean cells (Langebartels and Harms, 1984) indicated that the PCP concentration in the culture media decreased drastically after only 30 min of incubation. Figure 2 shows the time course of PCP decrease in the nutrient solutions of soybean and wheat suspension cultures for incubation periods ranging from 0.5 to 60 min. After 2-3 min 80 to 90% of the initial concentration was adsorbed by the cells in both cultures. Soybean cells, inactivated by a heat treatment ( 10 min at 100°C) before incubation, exhibited the same time course and extent of adsorption (data not shown). PCP is thus partitioned into the membrane structures of the cells very rapidly and remains associated with the cells long enough to allow separation from the medium by filtration. Pulse-Chase Experiments Formation of soluble, as well as nonextractable, metabolite fractions in wheat cells was studied after a 30-min pulse with radiolabeled PCP. Subsequently the cells were washed free from the nutrient solution and were further cultured in PCP-free media for up to 2 days. PCP at these concentrations was shown to have no inhibitory effects on the growth of the cells. At the times indicated in Fig. 3, cells and media were extracted and analyzed for metabolite formation. PCP decreased to 18% during the first 12 hr and radioactivity, found in the polar metabolite and cell wall-bound fraction, increased immediately after the pulse. 38% of the polar metabolites were localized within the cells and 2% (applied radioactivity) within the nutrient solution. They were shown to be mainly conjugates of PCP, of which PCP-P-D-glucoside (Langebartels and Harms, 1984) and the malonyl derivative of this glucoside (Schmitt et al., 1985) have been identified.
02
5
10
30
In,”
60
FIG. 2. Kinetics of adsorption of PCP by soybean and wheat suspension cells. PCP was applied to the medium and the cells were separated from the medium by suction under vacuum. After the times indicated the remaining radioactivity in the medium was measured for three parallel samples.
FRACTIONATION
OF 1
NONEXTRACTABLE I
80-
I
PCP I
30 O-48
RESIDUES
213
I
miffpulse -
hrs
chase
t
0
10
30
40
50 Ihrsl
FIG. 3. Pulse-chase experiment for metabolite formation in wheat cultures. Cultures were incubated with I mg/liter PCP for 30 min and then the cells were separated from the medium by 40-pm gauze filters. Cells were washed with medium and then cultured in PCP-free medium for the times indicated. Radioactivity in soluble metabolites, original substance, and cell wall-bound material was determined in three parahel cultures. Bars represent + standard error.
After a 24-hr chase the soluble polar fraction started to decrease. Further studies revealed that PCP-@-D-glucoside content was mainly responsible for this decrease. For the first 3 hr of incubation it was the only detectable metabolite, being 16% of the applied radioactivity. It then increased up to 39% within the first 12 hr. Afterward it decreased to 12% while other conjugates showed only minor changes during the second day of incubation (Langebartels and Harms, unpublished). It can be assumed that after 24 hr the soluble conjugates were partly converted to a nonextractable material as the radioactivity in the cell wall fraction increased to a higher extent than the PCP decreased. It is not known if polar conjugates of PCP derivatives are associated directly with the cell wall components or only after release of the aglycon. 14C label detected in the cell wall fraction increased with time and reached approximately 40% of the applied radioactivity after 48 hr, whereas in soybean cultures only 14% was found. In these cultures the amount of radioactivity in the polar metabolites increased for the first 24 hr and remained at the same level during the second day of incubation. Most of the polar metabolites were released into the nutrient solution immediately after their synthesis and were therefore not available for further conversion to nonextractable residues (data not shown). Comparison
of Cell Wall-Bound
and Organic Solvent-Extracted
Residues
Plant enzyme systems metabolize environmental chemicals mainly to water-soluble conjugates and “terminal” residues insoluble in the usual solvents. In this investigation the impact of the cell wall on the formation of these residues has been studied. Individual cell wall fractions were separated to determine to which cell structures or biopolymers 14C label from PCP was associated. As it has been suggested that the major part of the nonextractable activity is associated with cell wall components such as
274
LANGEBARTELS
AND HARMS
lignin or polysaccharides, experiments were performed in which cell walls were isolated after homogenization in buffer, collected by centrifugation and extracted with aqueous and organic solvents. A comparison is made in Table 1 of the metabolite patterns and radioactivity percentages determined by this method and those from the extraction of cell homogenates with MeOH/chloroform/water. No major differences were found in the soluble metabolite pattern when the homogenization process was performed either in buffer solutions or in organic solvents. Pronase E or sodium dodecylsulfate (SDS; 10 min at 100°C) released more radioactivity from cell residues which had been extracted with cold organic solvents. It is assumed that under these extraction conditions more 14Clabel associated with proteins is trapped. A differential centrifugation of cell homogenates from wheat suspensions was performed according to Ray (1977) in order to investigate the location of the radioactivity from [ 14C]PCP. Aqueous cell homogenates were centrifuged at increasing centrifugal forces and the resulting pellets were subjected to MeOH/CHC& extraction. The major part of the total nonextractable activity was found in the 1000 to 2000g X 10 min pellets (34%) representing the cell wall fraction whereas other membrane TABLE
1
COMPARISON OF METABOLITE PATTERNS AND RESIDUE FRACTIONS FOUND AFTER ISOLATION OF CELL WALL RESIDUES OR COLD ORGANIC SOLVENT-EXTRACTED RESIDUES~ Applied
radioactivity
Cell wall residues Nutrient solution Cell extract Buffer MeOH/CHC13/H20 MeOH/CHC& (2: 1) Cell walls/insoluble residue Total recovery of 14C
Formation
6.4
47.1 5.5 33.4 91.6
44.9 38.3 89.6
of the residue with
0.9 0.1 10.4 13.2 of soluble metabolites
Residues after cell extraction
5.6
Release of 14C label after treatment MeOH/CHC& Acetone Pronase E or SDS
(%)
in cells and nutrient
5.9 0.9 16.9 17.8 solution
PCP Polar metabolites Nonpolar metabolites
2.5 55.7 0.0
1.2 50.1 0.0
1 g Residue equal to
2.6 X lo6 dpm
3.0 X lo6 dpm
n Wheat suspension cells were incubated with PCP for 48 hr and then either the cells were homogenized in buffer and cell wall residues isolated according to Fig. I (four parallel samples) or cells were extracted in MeOH/CHCI,/H20 (six parallel samples).
FRACTIONATION
OF NONEXTRACTABLE
PCP RESIDUES
275
systems and organelles as well as the supernatant fraction contained only 2.5% (data not shown). Cell Wall Fractionation Procedure Bound radioactivity was analyzed by a sequential fractionation scheme adapted from cell wall fractionation procedures (Takeuchi and Komamine, 1980; Asamizu and Nishi, 1980). This procedure separated six fractions which were mainly composed of starch, proteins, pectin, lignin, hemicellulose, and cellulose, respectively. Fractionation of bound plant residues and their differentiation into several wall components was also reported for nitrofen (Honeycutt and Adler, 1975), buturon (Haque et al., 1976), and deltamethrin (Khan et al., 1984). The total polysaccharide distribution in the wheat cells investigated at the late stage of exponential growth was 8% pectin, 53% hemicellulose, and 39% cellulose. Figure 1 shows the flow chart of the fractionation process. Cells were homogenized in potassium phosphate buffer (PPB). Cell walls were pelleted by centrifugation and extracted with organic solvents. Cell walls or residues not extracted by organic solvents were enzymatically purified from starch and proteins and then polysaccharides and lignin were successively isolated by chemical degradation or enzymatic release of radioactivity. PCP was not degraded under the conditions used. The individual fractionation steps were performed for sufficient lengths of time such that reextraction under the same conditions gave no further release of 14C label. Figure 4 shows a comparison of methods for pectin isolation and their efficiency in releasing 14C label. EDTA (80°C) or pectinase were used for the isolation of pectins and released a comparable amount of PCP-radioactivity. Uranic acids were also released in higher amounts than with oxalate or other extraction methods. Extraction of pectic substances at 20 to 40°C was incomplete. EDTA was finally replaced by EGTA because of its specificity for Ca ions.
5’
FIG. 4. Progress of release of “‘C radioactivity during various pectin isolation procedures. Cell wall residues were extracted with MeOH/CHCI,, buffer, and Lu-amylase and freeze-dried. Aliquots were treated with several chemicals and the released radioactivity was measured at different times after filtration on glass-fiber filters or aRer centrifugation. Results are means of two experiments. 0, Pectinase from A niger, 50 units/ ml in sodium acetate buffer, pH 4.5, at 25°C. n , Na2EDTA (50 n04) in sodium acetate buffer, pH 4.5, at 80°C. 0, Pectinase 10 units/ml, sp act. A, 0.5% Ammonium oxalate/oxalic acid, pH 4.0, under reflux. 0, Sodium acetate buffer, pH 4.5, at 25°C (control).
276
LANGEBARTELS
AND HARMS
DMSO and dioxane-water were not effective for the release of lignin-bound radioactivity from cell walls under the conditions used, so dioxane-2 N HCl was routinely taken for 5 to 7 hr at 70°C. Hem&cellulose was extracted by 1 Nor 24% KOH and was characterized by its degradability with hemicellulase from A. niger and its monosaccharide composition. Mainly xylose, arabinose, and glucose were found, after hydrolysis and separation of sugars on NH2 columns by HPLC, and indicated the hemicellulosic nature of this fraction. The soluble H2S04 hydrolysate was referred to as the cellulose fraction. The final nonextractable residue was combusted in an oxidizer and [i4C]C02 radioactivity was counted. Distribution
of 14C Label in Cell Wall Fractions
Table 2 summarizes the distribution of i4C label from PCP in the individual cell wall fractions of wheat suspension cells, of photomixotrophic lupin cultures, and of aseptically grown wheat plants. Different amounts of radioactivity were found in all fractions and it is probable that PCP derivatives are bound by several mechanisms. 90 to 105% of the total radioactivity of the residue fractions could be recovered from the cell wall fractions. Only 3 to 5% radioactivity was still not extracted by this procedure. Isolated cell walls or residues after cold organic solvent extraction from wheat cultures showed a very similar pattern. Nonextracted residues exhibited more radioactivity in the protein fraction, as expected, as proteins of the cell fluid were also precipitated by this method. TABLE
2
DISTRIBUTION OF 14C LABEL IN CELL WALL FRACTIONS FROM [‘4C]PCP-~~~~~ SUSPENSION CULTURES AND PLANTS” Wheat suspension
cells
Lupin cells
Residues Residues Cell wall after cell after cell residues extraction extraction % Applied radioactivity in the Bound fraction:
33
38
8
CELL
Wheat plants Residues after cell extraction 16
Release(%) of 14Clabelafter successive Treatment MeOH/CHC13 Buffer cu-Amylase PronaseE EGTA Dioxane/HCl KOH HzS04
treatments with
Fraction
Starch Proteins Pectin Lignin Hemicellulose Cellulose Residue
3.3 15.2 2.2 15.8 6.5 18.9 33.6 1.6 2.9
2.9 17.8 2.3 19.4 5.8 18.1 29.8 1.6 2.3
11.0 10.9 3:: 16:5 5.9 10.8 0.4 4.7
8.8 7.1 9.2 28.0 14.7 10.8 13.6 4.7 3.1
a Suspension cultures from wheat and lupin, as well as aseptically grown wheat plants, were incubated with [‘4C]PCP and insoluble residues and cell walls of wheat were subjected to successivetreatments for the release of radioactivity from cell wall fractions.
FRACTIONATION
OF
NONEXTRACTABLE
PCP
RESIDUES
277
Buffer treatments of the lyophilized residue material released 10 to 20% of the total activity. Buffer solubilization of nonextracted residues has also been reported by Khan et al. (1984). Associations with proteins were found for the cell cultures investigated and for wheat plants. It could be shown that these associations in wheat cells are partially of high-molecular weight, as dialysis of the material and separation on Sephacryl S-300 gave molecular weights ranging from 10,000 to 100,000 (data not shown). As well as complex formation with lignins, which has been thoroughly studied by Scheel et al. ( 1984), [ 14C]pentachloropheno1 was also located in the pectin and hemicellulose fraction which amounted to 27-40% of the total nonextractable activity. The highest amounts of applied 14Clabel from PCP were associated with the hemicellulosic fraction from wheat suspension cultures (29.8 to 33.6%). Most of the experiments were carried out with heterotrophic cell cultures grown in the dark to exclude photodegradation of PCP. For comparison a photomixotrophic lupin culture (8% nonextractable residues), as well as hydroponic wheat plants (16%), were studied. In both plant systems 5 to 10% of the radioactivity was present in the starch and cellulose fractions. This may indicate that under light conditions the original compound is partially degraded to CO* and refixed in natural products. Characterization
of [‘4CJPCP/Hemicellulose
Associations
Associations of hemicellulose and PCP derivatives were further purified from a small amount of uv-absorbing material. After dialysis only material higher than mol wt 10,000 to 15,000 was used for further characterization (37-44% of the initial radioactivity of this fraction). The purified fraction was separated by molecular sieve chromatography on Sepharose 4 B (Fig. 5). Radioactivity from [‘4C]PCP and total sugar content showed a very similar elution pattern with two broad peaks at mol wt approx 40,000 and 500,000. These peaks were not due to an agglutination of molecules as they did not change
FIG. 5. Molecular sieve chromatography of the purified PCP-hemicellulose associations on Sepharose 4 B. Polysaccharide material (10 mg) was eluted with 50 mM sodium acetate buffer, pH 5.0, at 10 ml/min and 4.5-ml fractions were assayed for “C radioactivity and total sugar content. Arrows indicate peaks of dextran markers. V, = void volume.
278
LANGEBARTELS
AND HARMS
their elution position when they were rerun under the same conditions. Radioactivity from [ 14C]PCP is thus bound to a high-molecular-weight polysaccharide fraction with hemicellulosic character that in this purified form amounted to 6 to 8% of the original radioactivity in wheat cultures. High-molecular-weight [ “C]PCP/hemicellulose associations were further characterized by various treatments (Table 3). Chemicals, such as SDS and urea, that attack only noncovalent associations had little effect in releasing radioactive metabolites from the material, while HCl and sulfuric acid partially hydrolyzed these purified complexes. Hemicellulase from A. niger released completely the 14Clabel whereas pectinase from A. niger and purified @-1,3 and B- 1,Cglucanases showed little effect. Hemicellulase or HCl treatments released 18 or 32% of the radioactivity as the original test substance PCP. In addition a very hydrophilic fraction, and two other fractions of higher polarity than PCP, were detected. Conclusions The experiments reported here establish that PCP derivatives are present in cell wall fractions of wheat and lupin suspension cells as well as in wheat plants. A comparison of wheat cells homogenized in buffer and in cold organic solvents showed that most of the bound radioactivity could be attributed to the cell wall compartment of the plant cell. Soluble polar conjugates of PCP seem to be intermediates in the deposition of PCP or its metabolites into cell wall material. Using a sequential approach to yield more information on the nature of nonextractable residues in plants it could be shown that PCP or its derivatives are associated to polysaccharide fractions. Besides proteins and lignin [ 14C]PCP was localized in the pectic and mainly in the hemicellulose fraction. PCP and its metabolites were bound by strong bonds, presumably covalent linkages, to hemicellulose. As the original compound could be partially released from this material by enzymatic action the persistence and bioavailability of these polysaccharide-PCP associations should be investigated. Preliminary results in the use of this cell wall fractionation procedure for residues from 4-chloroaniline and 3,4-dichloTABLE
3
RELEASE OF 14C LABEL FROM PIJRIFIED PCPHEMICELLULOSE ASSOCIATIONS AVER DIFFERENT TREATMENTS
% Radioactivity released 4 M urea, 2 hr at 30°C 2% SDS, 10 min at 100°C 1 N HCI, 2 hr at 100°C Pectinase, 4 hr at 25°C p- 1.3~glucanase, 22 hr at 30°C /I- 1.Cglucanase, 22 hr at 30°C Hemicellulase, 4 hr at 27°C
10 16 39 15 11 16 98
a PCP-hemicellulose association was derived from the successive fractionation procedure and further purified as described. It was dialyzed against water and the fraction of mol wt 40,000 and higher was, after elution from Sepharose 4 B, treated with different chemicals.
FRACTIONATION
OF NONEXTRACTABLE
PCP RESIDUES
279
roaniline have shown that radioactivity in these residues could also be fractionated. The method released 80 to 95% of the bound radioactivity with significant amounts located in the polysaccharide fractions. ACKNOWLEDGMENTS We thank Dr. C. Sator for providing the lupin culture. The skillful technical assistance of M. Ellies is gratefully adknowledged. This work was supported by the German Federal Ministry of Research and Technology (BMFT).
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DUBOIS, M., GILLES, K. A., HAMILTON, J. K., BEBERS,P. A., ANDSMITH, F. (1956). Calorimetric method for determination of sugars and related substances. Anal. Chem. 28,350-356. EBING, W., HAQUE, A., SCHUPHAN, I., HARMS, H., LANGEBARTELS, C., SCHEEL, D., v. D. TRENCK, T., AND SANDERMANN, H. (1984). Ecochemical assessmentof environmental chemicals: Draft guideline of the test procedure to evaluate metabolism and degradation of chemicals by plant cell cultures. Chemosphere 13,947-957.
HAQUE, A., WEISGERBER,I., AND KLEIN, W. (1976). Buturon-“‘C bound residue complex in wheat plants. Chemosphere3,
161-112.
HARMS, H. (I 98 1). Aufnahme und Metabolismus polycyclischer aromatischer Kohlenwasserstoffe (PCKs) in aseptisch kultivierten Nahrungspflanzen und Zellsuspensionskulturen. Landbauforsch. Voelkenrode 31, 1-6.
HONEYCUTT, R. C., AND ADLER, I. L. (1975). Characterization of bound residues of nitrofen in rice and wheat straw. J. Agric. Food Chem. 23, 1097-l 101. HUBER, R., AND Oreo, S. (1983). Bound pesticide residues in plants. In Pesticide Chemistry (J. Miyamoto, and P. C. Keamey, eds.), Vol. 3, pp. 357-362. Pergamon, Oxford. KAUFMAN, D. D., STILL, G. G., PAULSON, G. D., AND BANDAL, S. K. (eds.) (1976). Bound and Conjugated Pesticide Residues. ACS Symp. Ser. 29, Amer. Chem. Sot., Washington, D.C. KHAN, S. U., ZHANG, L.-Z., AND AKHTAR, M. H. (1984). Bound residues of deltamethrin in bean plants. J. Agric. Food Chem. 32, 1141-l 144. LANGEBARTELS, C., AND HARMS, H. (1984). Metabolism of pentachlorophenol in cell suspension cultures of soybean and wheat: Pentachlorophenol glucoside formation. Z. Pflanzenphys. 113,20 l-2 1 I. RAY, P. M. (1977). Auxin-binding sites of maize coleoptiles are localized on membranes of the endoplasmic reticulum. Plant Physiol. 59,594-599. SANDERMANN, H., SCHEEL,D., AND v. D. TRENCK, T. (1983). Metabolism of environmental chemicals by plants-copolymerization into lignin. J. Appl. Polymer Sci. Appl. Polym. Symp. 37,407-420. SCHEEL,D., SCHAFER,W., AND SANDERMANN,H. (1984). Metabolism ofpentachlorophenol in cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.). General results and isolation of lignin metabolites. J. Agric. Food Chem. 32, l237- 124 I. SCHMITT, R., KAUL, J., v. D. TRENCK, T., SCHALLER, E., AND SANDERMANN, H. (1985). @-D-glucosyl and 0-malonyl-B-Bglucosyl conjugates of pentachlophenol in soybean and wheat. Identification and enzymatic synthesis. Pest. Biochem. Physiol. 24,11-85. TAKEUCHI, Y., AND KOMAMINE, A. (1980). Turnover of cell wall polysaccharides of Vinca rosea suspension culture. Physiol. Plant. 48, 27 I-277. WEISS, U. M., MOZA, P., SCHEUNERT,I., HAQUE, A., AND KORTE, F. (1982). Fate of pentachlorophenol14Cin rice plants under controlled conditions. J. Agric. Food Chem. 30, I 186- 1190.
AND
ENVIRONMENTAL
SAFETY
l&268-279
(1985)
Analysis for Nonextractable (Bound) Residues of Pentachlorophenol in Plant Cells Using a Cell Wall Fractionation Procedure’ C. LANGEBARTELS Institut Landwirtschaji
ftir Pjanzenerntihrung (FAL), Bundesallee
AND H. HARMS
und Bodenkunde, Bundesforschungsanstalt 50, Braunschweig D-3300, Federal Republic Received
fir of Germany
March 6, 1985
When plant cell cultures or aseptically grown wheat plants were treated with [‘Clpentachlorophenol (PCP) a major part of the label was found in a nonextractable or “bound” residue fraction. Soluble polar conjugates participated in the formation of these residues which were mainly located in the plant cell walls. By a sequential fractionation procedure using enzymatic and chemical methods, 90 to 95% of the bound radioactivity could be attributed to individual cell wall components. The 14C label from PCP was found mainly in hemicellulose, lignin, and protein fractions. Associations of hemicellulose with PCP derivatives with molecular weights up to 500,000 were purified to constant specific radioactivity. Hydrolysis of this fraction released 32% PCP and other unidentified products. o 1985 Academic PIW, LX. INTRODUCTION
Investigations of the metabolism of [ 14C]pentachlorophenol (PCP) in cell cultures of soybean, lupin, and wheat and aseptically grown wheat plants showed that a major part of the applied radioactivity was associated with fractions that could not be extracted with cold organic solvent mixtures (Langebartels and Harms, 1984). This nonextractable or bound radioactivity increased with time and was highest in wheat cultures where 35 to 50% of the applied radioactivity could be recovered in this fraction after 48 hr of incubation. Several components of the plant cell walls have been assumed to form complexes with xenobiotics (Kaufman et al., 1976; Huber and Otto, 1983; Sandermann et al., 1983). Association of PCP with cellulose and lignin fractions was reported for rice plants (Weiss et al., 1982) and with lignin in cell cultures of wheat (Scheel et al., 1984). This communication describes a sequential approach for fractionating bound residues in plants. Plant cells, as well as pelleted cell walls, were extracted with cold organic solvents and fractionated using a cell wall fractionation procedure. By this means individual fractions consisting of mainly starch, proteins, pectins, lignin, hemicellulose, or cellulose could be separated. Dark-grown cell cultures were mainly used for studying the formation of nonextractable residues to exclude PCP degradation by microorganisms and light; CO* refixation was not detected in these plant systems. MATERIALS
AND
METHODS
Plant Material Cell suspension cultures of Triticum aestivum L. and Glycine max L. were cultured as described by Langebartels and Harms (1984). Lupinus palyphyllus LINDL. SUS’ This communication was presented in part at the symposium “Bioavailability of Environmental Chemicals,” Schmallenberg, FRG, Sept. 12- 14, 1984. 0147-6513185 $3.00 Cop&h1 Q 1985 by Academic Press, Inc. All rights of rqxoduaion in any form reserved.
268
FRACTIONATION
OF NONEXTRACTABLE
PCP RESIDUES
269
pension cultures were propagated under white fluorescent light (8000 lx, 12 hr) at 28°C in two-tier vessels. COz concentration was kept at 1% by K&OJ/KHCO~ mixtures in the lower compartment. The culture medium was based on Murashige/Skoog with 2% sucrose, 2 mg/liter 2,4-D, and 0.25 mg/liter benzyladenine (C. Sator, Braunschweig, personal communication). Wheat plants were cultured in Knop’s nutrient solution under aseptic conditions according to Harms ( 198 1). Incubation
with [‘4C]PCP
Incubation times varied from 30 set to 48 hr under dark conditions for soybean and wheat cells, while photomixotrophic lupin cultures and wheat plants were grown under a 12-hr light/dark regime. Cell cultures were incubated with [u-“C]PCP (4 X 10e6 mol liter-‘, sp act 1.4 GBq/mmol; NEN, Dreieich, FRG) during the late stage of the logarithmic growth phase, while PCP was applied to wheat plants when they were 2-3 weeks old (15 to 20 cm). Plant cells and nutrient solutions were extracted with methanol:chloroform: water (2: 1:0.8) according to Ebing et al. (1984). Nonextractable residues were freeze-dried and then washed with 50 mM potassium phosphate buffer (pH 7.0), methanol:chloroform (2: 1, v/v), and acetone, successively. Alternatively, cells were separated from the medium by suction under mild vacuum and then homogenized in 50 mM potassium phosphate buffer, pH 7.0 (Fig. 1). Cell walls were pelleted by centrifugation at 6000g for 10 min and washed twice with buffer. Aqueous cell extracts and cell wall pellets were extracted with MeOH/CHC& (see Fig. 1). Differential centrifugation of the cell homogenates according to Ray (1977) was performed at 1000,2000,5000, 10,000, and 2O,OOOg( 10 min each) and at 40,OOOg X 30 min at 4°C. The 14C residue was determined by combustion in an oxidizer (OX 300, Zinsser, Frankfurt, FRG) and liquid scintillation counting. Cell Wall Fractionation Cell walls were fractionated into individual wall components by a modified extraction scheme according to Takeuchi and Komamine ( 1980). A flow diagram of the methods used is shown in Fig. 1. Extraction of water-soluble polysaccharides and proteins, extraction of lipids. One gram of cell wall or residue material was suspended in 100 ml of 50 mM potassium phosphate buffer (pH 7.0), sonificated for 5 min, and stirred for 4 hr at room temperature. The mixture was filtered through glass-fiber filters and the radioactivity of the filtrate was determined. The extracted residue was shaken for 2 hr with 100 ml methanol:chloroform (2: 1, v/v) and then acetone for 2 hr at room temperature. It was then washed with 20 ml of potassium phosphate buffer. All procedures in aqueous solutions were performed under an argon atmosphere and in the presence of toluene to prevent microbial infections. Slurry, after the individual steps, was filtered by suction on glass-fiber filters. Extraction ofstarch. Cell wall material on the filter, after washing with potassium phosphate buffer, was treated with 0.2 ml cu-amylase from porcine pancreas (Sigma, type IA) in 100 ml 50 mA4 potassium phosphate buffer (pH 7.0). Incubation was performed for 20 hr at 30°C. Extraction ofproteins. The material remaining after starch extraction was suspended in 100 ml Pronase E (3 mg/ml, Sigma) solution in 50 mM Tris-HCl buffer at pH 7.2 and incubated for 16 hr at 30°C.
270
LANGEBARTELS
AND HARMS
I
1
cells
culture
I homogenization centrifygation
medium
in PPB 6 000 g x 10 min
I supernatant wash!I'ifih chloroform
PPB , methanol/ (2 : 1), acetone
I
I
pelllet cell 1
walls
&
cell
extract
a-amylase starch pronase
pectinase
E
or EGTA 50 mM, 80 'C
dioxane/H20 dioxane/Z ~~~;,ase
9 : 1 N HCl 70 'C
t or KOH (1 N or
H2S04
dioxane-sol.
fract.
24 %)
72 % hydrolysate
residue CA
FIG. I. Flow diagram of the fractionation procedure for cell walls. Cells were homogenized and extracted either in MeOH/CHC13/H20 as described under Materials and Methods, or in aqueous buffer. Cell wags were collected by centrifugation and extracted. Cell wall residues or nonextractable residues were then fractionated by the same procedure starting with removal of contaminating starch.
Extraction of EGTA-soluble material (pectin fraction). Pectins were isolated by an incubation with 100 ml 50 rr&I EGTA (ethylene glycol bis-(Zaminoethyl ether)-N,N’tetraacetic acid) in 50 mJ4 sodium acetate buffer at pH 4.5. After 2 min of ultrasonic disintegration the suspension was heated to 80°C for 4 to 6 hr. A parallel incubation with pectinase from Aspergillus niger (Sigma) was also performed. An amount equal to 100 mg residue material was incubated with 10 ml of a pectinase solution (100 units, 16 mg protein) in 50 mM sodium acetate buffer at pH 4.0. The reaction was terminated by the addition of 200 ~1 glacial acetic acid. Extraction of lignin. Lignin was extracted in the sequential fractionation procedure by treatment with either DMSO or dioxane-water and dioxane:2 N HCl. Cell wall material collected by filtration after pectin extraction was suspended in 10 ml DMSO or dioxane-water (9: 1) for 48 to 96 hr at room temperature. During the first 30 min of the incubation period residues were sonificated with cooling on ice. The insoluble material was filtered off and further treated with 10 ml DMSO at 80°C for 16 hr or dioxane:2 N HCl(9: 1) at 70°C for 5 hr, respectively. After cooling to room temperature
FRACTIONATION
OF
NONEXTRACTABLE
PCP
RESIDUES
271
the mixture was passed through glass-fiber filters and the insoluble material was washed with DMSO or water. Extraction of KOH-soluble (hemicellulose) material. An extraction procedure using cold alkali is useful for the removal of noncellulosic polysaccharides of a neutral sugar composition. Cell wall material was suspended in 10 ml 1 N and 24% KOH at 27°C for 24 hr. The mixture was brought to pH 3-4 with 6 N acetic acid, diluted with water, and stirred for 1 hr. Precipitated material was pelleted by centrifugation ( 1OOOgX 10 min). PCP-hemicellulose associations were solubihzed by hemicellulase from A. niger (Sigma, 50 mg/ml) in 50 mM sodium acetate buffer, pH 5.5, for 4 hr at 27°C. H2S04 hydrolysate (cellulose fraction). The insoluble residue, after alkali treatment, was incubated in 72% sulfuric acid for 4 hr at room temperature. After neutralization with KOH the suspension was filtered off. The remaining insoluble material was combusted to COZ in an oxidizer and analyzed for radioactivity. Characterization
of Solubilized
Fractions and Cell Wall Polysaccharides
Polysaccharide and protein fractions were dialyzed against bidistilled water for 16 hr. The freeze-dried material was stored in a desiccator in the dark. Extracts were hydrolyzed with 2 N HCl (1 10°C) and separated on precoated silica gel (glass) plates (Merck) using (1) n-hexane:diethyl ether:formic acid (70:30:4) or (2) ethyl acetate: acetic acid:water (60:20:20) as solvents. Reference substances and HPLC separations used have been described by Langebartels and Harms ( 1984). The separated radioactive components were integrated with a TLC linear analyzer (Berthold, Wildbad, FRG). 14C radioactivity in the solutions was determined with a Philips liquid scintillation spectrometer. Total sugar was determined by the phenol-sulfuric acid method (Dubois et al., 1956) uranic acid content was measured using carbazol/sulfuric acid (Bitter and Muir, 1962). Hydrolysis of the polysaccharide fractions was performed by heating with 2 N trifluoroacetic acid at 120°C for 60 min or by enzymatic degradation. Monosaccharides were separated by HPLC on a Lichrosorb NH2 column (Merck) and with 80% acetonitrile as solvent using a Melz refractive index detector (LCD 201, Gynkotek, Mtinchen, FRG). Molecular
Sieve Chromatography
Pectic and hemicellulosic polysaccharides, associated with the 14C label from PCP, were subjected to gel filtration chromatography on Sepharose 4 B, Sephacryl S-300, and Bio-Gel P2 (all 90 X 1.5 cm, Pharmacia and BioRad). The lyophilized and dialyzed materials were dissolved in 50 mA4 sodium acetate buffer, pH 5.0, and a small amount of insoluble matter was removed by centrifugation (5 min at 5000 rpm) after heating the samples to 80°C for 5 min. Samples, containing 5- 12 mg of carbohydrate material, were applied to the columns and eluted with sodium acetate buffer at a flow rate of lo-15 ml/hr. Fractions of 2.5 to 4.5 ml were collected. Aliquots were taken for the determination of radioactivity ( 1 ml), total sugar content (0.2 ml), and monosaccharide composition (l-3 ml), Dextrans of defined molecular weight ranging from 10,000 to 500,000 (Pharmacia) were used as markers for molecular weight determinations.
272
LANGEBARTELS
RESULTS
AND
AND
HARMS
DISCUSSION
Adsorption of PCP by Soybean and Wheat Cells Previous time-course studies with soybean cells (Langebartels and Harms, 1984) indicated that the PCP concentration in the culture media decreased drastically after only 30 min of incubation. Figure 2 shows the time course of PCP decrease in the nutrient solutions of soybean and wheat suspension cultures for incubation periods ranging from 0.5 to 60 min. After 2-3 min 80 to 90% of the initial concentration was adsorbed by the cells in both cultures. Soybean cells, inactivated by a heat treatment ( 10 min at 100°C) before incubation, exhibited the same time course and extent of adsorption (data not shown). PCP is thus partitioned into the membrane structures of the cells very rapidly and remains associated with the cells long enough to allow separation from the medium by filtration. Pulse-Chase Experiments Formation of soluble, as well as nonextractable, metabolite fractions in wheat cells was studied after a 30-min pulse with radiolabeled PCP. Subsequently the cells were washed free from the nutrient solution and were further cultured in PCP-free media for up to 2 days. PCP at these concentrations was shown to have no inhibitory effects on the growth of the cells. At the times indicated in Fig. 3, cells and media were extracted and analyzed for metabolite formation. PCP decreased to 18% during the first 12 hr and radioactivity, found in the polar metabolite and cell wall-bound fraction, increased immediately after the pulse. 38% of the polar metabolites were localized within the cells and 2% (applied radioactivity) within the nutrient solution. They were shown to be mainly conjugates of PCP, of which PCP-P-D-glucoside (Langebartels and Harms, 1984) and the malonyl derivative of this glucoside (Schmitt et al., 1985) have been identified.
02
5
10
30
In,”
60
FIG. 2. Kinetics of adsorption of PCP by soybean and wheat suspension cells. PCP was applied to the medium and the cells were separated from the medium by suction under vacuum. After the times indicated the remaining radioactivity in the medium was measured for three parallel samples.
FRACTIONATION
OF 1
NONEXTRACTABLE I
80-
I
PCP I
30 O-48
RESIDUES
213
I
miffpulse -
hrs
chase
t
0
10
30
40
50 Ihrsl
FIG. 3. Pulse-chase experiment for metabolite formation in wheat cultures. Cultures were incubated with I mg/liter PCP for 30 min and then the cells were separated from the medium by 40-pm gauze filters. Cells were washed with medium and then cultured in PCP-free medium for the times indicated. Radioactivity in soluble metabolites, original substance, and cell wall-bound material was determined in three parahel cultures. Bars represent + standard error.
After a 24-hr chase the soluble polar fraction started to decrease. Further studies revealed that PCP-@-D-glucoside content was mainly responsible for this decrease. For the first 3 hr of incubation it was the only detectable metabolite, being 16% of the applied radioactivity. It then increased up to 39% within the first 12 hr. Afterward it decreased to 12% while other conjugates showed only minor changes during the second day of incubation (Langebartels and Harms, unpublished). It can be assumed that after 24 hr the soluble conjugates were partly converted to a nonextractable material as the radioactivity in the cell wall fraction increased to a higher extent than the PCP decreased. It is not known if polar conjugates of PCP derivatives are associated directly with the cell wall components or only after release of the aglycon. 14C label detected in the cell wall fraction increased with time and reached approximately 40% of the applied radioactivity after 48 hr, whereas in soybean cultures only 14% was found. In these cultures the amount of radioactivity in the polar metabolites increased for the first 24 hr and remained at the same level during the second day of incubation. Most of the polar metabolites were released into the nutrient solution immediately after their synthesis and were therefore not available for further conversion to nonextractable residues (data not shown). Comparison
of Cell Wall-Bound
and Organic Solvent-Extracted
Residues
Plant enzyme systems metabolize environmental chemicals mainly to water-soluble conjugates and “terminal” residues insoluble in the usual solvents. In this investigation the impact of the cell wall on the formation of these residues has been studied. Individual cell wall fractions were separated to determine to which cell structures or biopolymers 14C label from PCP was associated. As it has been suggested that the major part of the nonextractable activity is associated with cell wall components such as
274
LANGEBARTELS
AND HARMS
lignin or polysaccharides, experiments were performed in which cell walls were isolated after homogenization in buffer, collected by centrifugation and extracted with aqueous and organic solvents. A comparison is made in Table 1 of the metabolite patterns and radioactivity percentages determined by this method and those from the extraction of cell homogenates with MeOH/chloroform/water. No major differences were found in the soluble metabolite pattern when the homogenization process was performed either in buffer solutions or in organic solvents. Pronase E or sodium dodecylsulfate (SDS; 10 min at 100°C) released more radioactivity from cell residues which had been extracted with cold organic solvents. It is assumed that under these extraction conditions more 14Clabel associated with proteins is trapped. A differential centrifugation of cell homogenates from wheat suspensions was performed according to Ray (1977) in order to investigate the location of the radioactivity from [ 14C]PCP. Aqueous cell homogenates were centrifuged at increasing centrifugal forces and the resulting pellets were subjected to MeOH/CHC& extraction. The major part of the total nonextractable activity was found in the 1000 to 2000g X 10 min pellets (34%) representing the cell wall fraction whereas other membrane TABLE
1
COMPARISON OF METABOLITE PATTERNS AND RESIDUE FRACTIONS FOUND AFTER ISOLATION OF CELL WALL RESIDUES OR COLD ORGANIC SOLVENT-EXTRACTED RESIDUES~ Applied
radioactivity
Cell wall residues Nutrient solution Cell extract Buffer MeOH/CHC13/H20 MeOH/CHC& (2: 1) Cell walls/insoluble residue Total recovery of 14C
Formation
6.4
47.1 5.5 33.4 91.6
44.9 38.3 89.6
of the residue with
0.9 0.1 10.4 13.2 of soluble metabolites
Residues after cell extraction
5.6
Release of 14C label after treatment MeOH/CHC& Acetone Pronase E or SDS
(%)
in cells and nutrient
5.9 0.9 16.9 17.8 solution
PCP Polar metabolites Nonpolar metabolites
2.5 55.7 0.0
1.2 50.1 0.0
1 g Residue equal to
2.6 X lo6 dpm
3.0 X lo6 dpm
n Wheat suspension cells were incubated with PCP for 48 hr and then either the cells were homogenized in buffer and cell wall residues isolated according to Fig. I (four parallel samples) or cells were extracted in MeOH/CHCI,/H20 (six parallel samples).
FRACTIONATION
OF NONEXTRACTABLE
PCP RESIDUES
275
systems and organelles as well as the supernatant fraction contained only 2.5% (data not shown). Cell Wall Fractionation Procedure Bound radioactivity was analyzed by a sequential fractionation scheme adapted from cell wall fractionation procedures (Takeuchi and Komamine, 1980; Asamizu and Nishi, 1980). This procedure separated six fractions which were mainly composed of starch, proteins, pectin, lignin, hemicellulose, and cellulose, respectively. Fractionation of bound plant residues and their differentiation into several wall components was also reported for nitrofen (Honeycutt and Adler, 1975), buturon (Haque et al., 1976), and deltamethrin (Khan et al., 1984). The total polysaccharide distribution in the wheat cells investigated at the late stage of exponential growth was 8% pectin, 53% hemicellulose, and 39% cellulose. Figure 1 shows the flow chart of the fractionation process. Cells were homogenized in potassium phosphate buffer (PPB). Cell walls were pelleted by centrifugation and extracted with organic solvents. Cell walls or residues not extracted by organic solvents were enzymatically purified from starch and proteins and then polysaccharides and lignin were successively isolated by chemical degradation or enzymatic release of radioactivity. PCP was not degraded under the conditions used. The individual fractionation steps were performed for sufficient lengths of time such that reextraction under the same conditions gave no further release of 14C label. Figure 4 shows a comparison of methods for pectin isolation and their efficiency in releasing 14C label. EDTA (80°C) or pectinase were used for the isolation of pectins and released a comparable amount of PCP-radioactivity. Uranic acids were also released in higher amounts than with oxalate or other extraction methods. Extraction of pectic substances at 20 to 40°C was incomplete. EDTA was finally replaced by EGTA because of its specificity for Ca ions.
5’
FIG. 4. Progress of release of “‘C radioactivity during various pectin isolation procedures. Cell wall residues were extracted with MeOH/CHCI,, buffer, and Lu-amylase and freeze-dried. Aliquots were treated with several chemicals and the released radioactivity was measured at different times after filtration on glass-fiber filters or aRer centrifugation. Results are means of two experiments. 0, Pectinase from A niger, 50 units/ ml in sodium acetate buffer, pH 4.5, at 25°C. n , Na2EDTA (50 n04) in sodium acetate buffer, pH 4.5, at 80°C. 0, Pectinase 10 units/ml, sp act. A, 0.5% Ammonium oxalate/oxalic acid, pH 4.0, under reflux. 0, Sodium acetate buffer, pH 4.5, at 25°C (control).
276
LANGEBARTELS
AND HARMS
DMSO and dioxane-water were not effective for the release of lignin-bound radioactivity from cell walls under the conditions used, so dioxane-2 N HCl was routinely taken for 5 to 7 hr at 70°C. Hem&cellulose was extracted by 1 Nor 24% KOH and was characterized by its degradability with hemicellulase from A. niger and its monosaccharide composition. Mainly xylose, arabinose, and glucose were found, after hydrolysis and separation of sugars on NH2 columns by HPLC, and indicated the hemicellulosic nature of this fraction. The soluble H2S04 hydrolysate was referred to as the cellulose fraction. The final nonextractable residue was combusted in an oxidizer and [i4C]C02 radioactivity was counted. Distribution
of 14C Label in Cell Wall Fractions
Table 2 summarizes the distribution of i4C label from PCP in the individual cell wall fractions of wheat suspension cells, of photomixotrophic lupin cultures, and of aseptically grown wheat plants. Different amounts of radioactivity were found in all fractions and it is probable that PCP derivatives are bound by several mechanisms. 90 to 105% of the total radioactivity of the residue fractions could be recovered from the cell wall fractions. Only 3 to 5% radioactivity was still not extracted by this procedure. Isolated cell walls or residues after cold organic solvent extraction from wheat cultures showed a very similar pattern. Nonextracted residues exhibited more radioactivity in the protein fraction, as expected, as proteins of the cell fluid were also precipitated by this method. TABLE
2
DISTRIBUTION OF 14C LABEL IN CELL WALL FRACTIONS FROM [‘4C]PCP-~~~~~ SUSPENSION CULTURES AND PLANTS” Wheat suspension
cells
Lupin cells
Residues Residues Cell wall after cell after cell residues extraction extraction % Applied radioactivity in the Bound fraction:
33
38
8
CELL
Wheat plants Residues after cell extraction 16
Release(%) of 14Clabelafter successive Treatment MeOH/CHC13 Buffer cu-Amylase PronaseE EGTA Dioxane/HCl KOH HzS04
treatments with
Fraction
Starch Proteins Pectin Lignin Hemicellulose Cellulose Residue
3.3 15.2 2.2 15.8 6.5 18.9 33.6 1.6 2.9
2.9 17.8 2.3 19.4 5.8 18.1 29.8 1.6 2.3
11.0 10.9 3:: 16:5 5.9 10.8 0.4 4.7
8.8 7.1 9.2 28.0 14.7 10.8 13.6 4.7 3.1
a Suspension cultures from wheat and lupin, as well as aseptically grown wheat plants, were incubated with [‘4C]PCP and insoluble residues and cell walls of wheat were subjected to successivetreatments for the release of radioactivity from cell wall fractions.
FRACTIONATION
OF
NONEXTRACTABLE
PCP
RESIDUES
277
Buffer treatments of the lyophilized residue material released 10 to 20% of the total activity. Buffer solubilization of nonextracted residues has also been reported by Khan et al. (1984). Associations with proteins were found for the cell cultures investigated and for wheat plants. It could be shown that these associations in wheat cells are partially of high-molecular weight, as dialysis of the material and separation on Sephacryl S-300 gave molecular weights ranging from 10,000 to 100,000 (data not shown). As well as complex formation with lignins, which has been thoroughly studied by Scheel et al. ( 1984), [ 14C]pentachloropheno1 was also located in the pectin and hemicellulose fraction which amounted to 27-40% of the total nonextractable activity. The highest amounts of applied 14Clabel from PCP were associated with the hemicellulosic fraction from wheat suspension cultures (29.8 to 33.6%). Most of the experiments were carried out with heterotrophic cell cultures grown in the dark to exclude photodegradation of PCP. For comparison a photomixotrophic lupin culture (8% nonextractable residues), as well as hydroponic wheat plants (16%), were studied. In both plant systems 5 to 10% of the radioactivity was present in the starch and cellulose fractions. This may indicate that under light conditions the original compound is partially degraded to CO* and refixed in natural products. Characterization
of [‘4CJPCP/Hemicellulose
Associations
Associations of hemicellulose and PCP derivatives were further purified from a small amount of uv-absorbing material. After dialysis only material higher than mol wt 10,000 to 15,000 was used for further characterization (37-44% of the initial radioactivity of this fraction). The purified fraction was separated by molecular sieve chromatography on Sepharose 4 B (Fig. 5). Radioactivity from [‘4C]PCP and total sugar content showed a very similar elution pattern with two broad peaks at mol wt approx 40,000 and 500,000. These peaks were not due to an agglutination of molecules as they did not change
FIG. 5. Molecular sieve chromatography of the purified PCP-hemicellulose associations on Sepharose 4 B. Polysaccharide material (10 mg) was eluted with 50 mM sodium acetate buffer, pH 5.0, at 10 ml/min and 4.5-ml fractions were assayed for “C radioactivity and total sugar content. Arrows indicate peaks of dextran markers. V, = void volume.
278
LANGEBARTELS
AND HARMS
their elution position when they were rerun under the same conditions. Radioactivity from [ 14C]PCP is thus bound to a high-molecular-weight polysaccharide fraction with hemicellulosic character that in this purified form amounted to 6 to 8% of the original radioactivity in wheat cultures. High-molecular-weight [ “C]PCP/hemicellulose associations were further characterized by various treatments (Table 3). Chemicals, such as SDS and urea, that attack only noncovalent associations had little effect in releasing radioactive metabolites from the material, while HCl and sulfuric acid partially hydrolyzed these purified complexes. Hemicellulase from A. niger released completely the 14Clabel whereas pectinase from A. niger and purified @-1,3 and B- 1,Cglucanases showed little effect. Hemicellulase or HCl treatments released 18 or 32% of the radioactivity as the original test substance PCP. In addition a very hydrophilic fraction, and two other fractions of higher polarity than PCP, were detected. Conclusions The experiments reported here establish that PCP derivatives are present in cell wall fractions of wheat and lupin suspension cells as well as in wheat plants. A comparison of wheat cells homogenized in buffer and in cold organic solvents showed that most of the bound radioactivity could be attributed to the cell wall compartment of the plant cell. Soluble polar conjugates of PCP seem to be intermediates in the deposition of PCP or its metabolites into cell wall material. Using a sequential approach to yield more information on the nature of nonextractable residues in plants it could be shown that PCP or its derivatives are associated to polysaccharide fractions. Besides proteins and lignin [ 14C]PCP was localized in the pectic and mainly in the hemicellulose fraction. PCP and its metabolites were bound by strong bonds, presumably covalent linkages, to hemicellulose. As the original compound could be partially released from this material by enzymatic action the persistence and bioavailability of these polysaccharide-PCP associations should be investigated. Preliminary results in the use of this cell wall fractionation procedure for residues from 4-chloroaniline and 3,4-dichloTABLE
3
RELEASE OF 14C LABEL FROM PIJRIFIED PCPHEMICELLULOSE ASSOCIATIONS AVER DIFFERENT TREATMENTS
% Radioactivity released 4 M urea, 2 hr at 30°C 2% SDS, 10 min at 100°C 1 N HCI, 2 hr at 100°C Pectinase, 4 hr at 25°C p- 1.3~glucanase, 22 hr at 30°C /I- 1.Cglucanase, 22 hr at 30°C Hemicellulase, 4 hr at 27°C
10 16 39 15 11 16 98
a PCP-hemicellulose association was derived from the successive fractionation procedure and further purified as described. It was dialyzed against water and the fraction of mol wt 40,000 and higher was, after elution from Sepharose 4 B, treated with different chemicals.
FRACTIONATION
OF NONEXTRACTABLE
PCP RESIDUES
279
roaniline have shown that radioactivity in these residues could also be fractionated. The method released 80 to 95% of the bound radioactivity with significant amounts located in the polysaccharide fractions. ACKNOWLEDGMENTS We thank Dr. C. Sator for providing the lupin culture. The skillful technical assistance of M. Ellies is gratefully adknowledged. This work was supported by the German Federal Ministry of Research and Technology (BMFT).
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