Jul 18, 1988 - zyme were found in cell walls whereas intercellular spaces, cytoplasm, .... vant were obtained from Difco laboratories, Detroit, MI, USA.
Plant Physiol. (I1988) 88, 13 17-1322 0032-0889/88/88/131 7/06/$0 1.00/0
Immunocytochemical Localization of a Wheat Germ Lysozyme in Wheat Embryo and Coleoptile Cells and Cytochemical Study of Its Interaction with the Cell Wall1 Received for publication May 10, 1988 and in revised form July 18, 1988
PATRICE AUDY, NICOLE BENHAMOU*, JEAN TRUDEL, AND ALAIN ASSELIN
Departement de phytologie, Faculte des sciences de l'agriculture et de l'alimentation, Universite Laval, Quebec, Quebec GIK 7P4, Canada enzymic activity toward various substrates (22). To our knowledge, no attempt has ever been made to study plant lysozymes Among several wheat (Triticum aestiyum L.) germ proteins able to in situ. lyse Micrococcus lysodeikticus, one lysozyme (WI A) was purified by ionThis report describes the use of an indirect immunocytochemexchange chromatography, gel filtration, and preparative polyacrylamide ical technique (5, 6) for localizing one lysozyme (W1A) purified gel electrophoresis. Polyclonal antibodies against this lysozyme were from wheat germ (2). The use of polyclonal antibodies against raised in rabbits. The in situ localization of WlA lysozyme was achieved this lysozyme in conjunction with the protein A-gold technique by the indirect protein A-gold technique. Large amounts of WIA lyso- has allowed localization of this enzyme. Furthermore, in an zyme were found in cell walls whereas intercellular spaces, cytoplasm, attempt to detect the presence of a putative endogenous substrate, and organelles were nearly free of labeling. Specificity of labeling was WIA lysozyme was conjugated to gold and applied to tissue assessed with several controls. In an attempt to detect the presence of sections. Results suggest that WIA lysozyme can interact with binding sites, WlA lysozyme was complexed to colloidal gold. Particles some polysaccharide(s) in the cell wall. ABSTRACT
were specifically distributed in large amounts over wheat embryo and coleoptile cell walls. The absence of labeling over isolated coleoptile cell walls treated with 0.1 and 0.4 molar potassium hydroxide for hemicellulose extraction indicated that WlA lysozyme binding sites were probably of hemicellulosic nature.
MATERIALS AND METHODS Plant Material. Wheat seeds (Triticum aestivum L. cv Laval 19) were germinated in a commercial Biosta sprouter in the dark at 22°C and provided daily with tap water. Embryos were excised from mature seeds and processed for electron microscope studies. Coleoptiles were harvested when they were about 2.5 cm long. Isolation of Wheat Coleoptile Cell Walls and Sequential Removal of Polysaccharides. Twenty-five g (fresh weight) of etiolated coleoptiles were homogenized in a mortar using 50 mL Ever since the first report on plant lysozyme activity in turnip of 0.05 M Tes buffer (pH 7.2) at 4°C. Walls and cell debris were (17), lysozymes (EC 3.2.1.17) have been studied in various plant collected by vacuum filtration on a Whatman GF/C filter and tissues and latices (for review see 10, 22). In the last 20 years, successively washed with 50 mL of the following solutions: 0.5 several plant lysozymes have been purified from dicotyledons M potassium phosphate (pH 7.0) (2x); bi-distilled water (4x); (10, 15, 19, 21, 29, 30). Plant lysozymes exhibit strong chitino- chloroform:methanol (1: 1, v/v) at 45°C for 30 min (2x); acetone lytic activity in addition to hydrolysis of N-acetyl-glucosaminyl- (lx); methanol (2x); bi-distilled water (2x). The recovered maN-acetylmuramic linkages in bacterial cell walls. Since endog- terial was frozen at -80°C and lyophilized. enous substrates have not been found, plant lysozymes Removal of polysaccharides was performed according to Carare assumed to be involved in defense or resistance against pita (12) with slight modifications. For extraction of starch, 250 infection (1 1). mg of lyophilized walls was suspended in 100 mL of DMSO and Several histochemical and cytochemical methods have been was shaken overnight. Wall material was then collected by vacdeveloped for identification of lysozymes in human and animal uum filtration on a Whatman No. 1 filter paper. For extraction cell compartments (27). Most of these techniques have been of pectic substances, wall material collected by filtration was based on the hydrolytic properties of the enzyme using Micro- incubated with gentle shaking in 100 mL of 0.5% (w/v) ammococcus lysodeikticus (syn: luteus) or chitin as substrates. Immu- nium oxalate/HCl (pH 4.5) at 100°C with two changes at 1 h nofluorescence and immunoenzymatic labeling have also been intervals. Insoluble cellulose and hemicelluloses were recovered used (1, 23, 24). More recently, studies concerning the localiza- by filtration and were frozen and lyophilized. This remaining tion of intracellular lysozymes in human tissues involved im- material was successively suspended in 100 mL of potassium munoelectron microscopic techniques combining the use of hydroxide at 0.01 M, 0.1 M, and 0.4 M containing 3 mg/mL of specific antibodies and colloidal gold as an electron-dense sodium borohydride and was agitated with magnetic stirring marker (13). under nitrogen for 1 h. Samples collected between each potasSo far, the literature on plant lysozymes has been restricted to sium hydroxide treatment were washed with bi-distilled water, enzyme purification procedures and determination of in vitro frozen, and lyophilized. Tissue Processing. Wheat embryos, coleoptiles, untreated, and ' Supported by the Natural Sciences and Engineering Research Coun- treated coleoptile cell walls embedded in 1% aqueous agarose, cil of Canada. were sliced with a double-edge razor blade into small pieces (1 1317
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AUDY ETAL.
mm3) in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) and incubated for 2 h at 4°C. Some samples were postfixed in 1% osmium tetroxide in sodium cacodylate buffer for 1 h at 4C. All samples were dehydrated in a graded ethanol series and embedded in Epon 812. Ultrathin sections were collected on nickel grids (200 mesh). Purification of Wheat Germ Lysozyme WlA and Preparation of Polyclonal Antibodies. Lysozyme purification was performed as described by Audy et al. (2). The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography, gel filtration, and preparative PAGE under native conditions. Antiserum against WIA lysozyme was raised in rabbits by four intramuscular injections (1 week apart) of purified W1A lysozyme (0.25 mg) emulsified in complete (first injection) or incomplete (subsequent injections) Freund's adjuvant. One week after the last intramuscular injection, rabbits were boosted with an intravenous injection. Animals were bled from the marginal ear vein 9 d after the intravenous injection. Antiserum was separated from red cells by centrifugation at 10,000g for 20 min at room temperature. Aliquots (1 mL) were stored at -20C. Western Blotting. Analytical PAGE under denaturing conditions was performed as described by Audy et al. (2). The electrophoretic transfer of proteins from SDS polyacrylamide gels to nitrocellulose was performed using a transfer cell (Trans-blot cell, Bio-Rad) in 50 mm Tris, 384 mM glycine, and 0.1% (w/v) SDS. Proteins were transferred at room temperature for 3 h at 375 mamp. Immunodetection (28) after western blotting was performed according to Fortin et al. (16). Preparation of Protein A-Gold Complex. Colloidal gold with a mean particle diameter of 15 nm was prepared according to Frens (18). Preparation of the protein A-gold complex was adapted from Bendayan (3). The minimal amount of protein A for full stabilization of the gold solution at pH 6.9 was estimated to be 30 Ug. A 10-fold excess of protein (300 ,ug) was dissolved in 100 ,L of distilled water and mixed with 10 mL of colloidal gold at pH 6.9. After centrifugation, the red pellet was resuspended in 0.5 mL of PBS (pH 7.4) containing 0.02% PEG 20,000. This was the stock solution. Immunocytochemical Labeling. Labeling was carried out at room temperature. Ultrathin tissue sections were incubated for 5 min on a drop of PBS (pH 7.4) containing 1% ovalbumin and transferred onto a drop of the diluted anti-WIA antiserum (1:40 with PBS-ovalbumin) for 60 min in a moist chamber. Sections were thoroughly rinsed with PBS and incubated with the protein A-gold complex diluted 1:30 in PBS-PEG for 30 min. Sections were washed with PBS, rinsed with distilled water and dried. Staining with uranyl acetate and lead citrate was performed before examination in a Siemens Elmiskope 102 electron microscope.
Plant Physiol. Vol. 88,1988
For preparation of the W lA lysozyme-gold complex, 0.10 mg of WIA lysozyme was dissolved in 100 1iL of distilled water and mixed with 10 mL of colloidal gold at pH 9.3. After centrifugation, the dark-red sediment was resuspended in 0.5 mL of PBSPEG (pH 6.0). This was the stock solution. Cytochemical Labeling. Ultrathin tissue sections were floated on a drop of PBS-PEG (pH 7.2) and transferred onto a drop of the WIA lysozyme-gold complex at 1:2 dilution in PBS-PEG (pH 7.2) for 30 min at room temperature in a moist chamber. Sections were rinsed, dried, and contrasted as described above. The following controls were carried out: (a) incubation of sections with the uncomplexed WlA lysozyme followed by incubation with the W1A lysozyme-gold complex, (b) incubation of sections with a nonenzymic protein-gold complex: bovine serum albumin (BSA)-gold complex, (c) incubation of sections with stabilized or unstabilized gold suspensions. Reagents. All chemicals for electrophoresis and protein assays were purchased from Bio-Rad Co, Mississauga, Ontario, Canada. Lyophilized cells of Micrococcus lysodeikticus, protein A, BSA, and sodium citrate were purchased from Sigma Chemical Co, St. Louis, MO, USA. Complete and incomplete Freund's adjuvant were obtained from Difco laboratories, Detroit, MI, USA. Tetrachloroauric acid was purchased from BDH Chemicals, Montreal, Canada, and PEG 20,000 from Fisher Scientific, Quebec, Canada. RESULTS Immunocytochemical Localization of WlA Lysozyme. The basic requirements for reliable immunocytochemical labeling are the high specificity of the antibodies and the preservation of antigenicity and ultrastructure (3). Antibodies were produced against the electrophoretically purified WIA lysozyme which exhibited only one band (25,400 Mr) after Coomassie blue staining in SDS-PAGE (2) and Figure
lA.
Immunodetection of purified WIA lysozyme (Fig. 1B) and
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Specificity of labeling was assessed with five control experi20.1ments: (a) incubation of sections with the protein A-gold complex, without the antiserum step; (b) incubation of sections with 14.2the antiserum previously absorbed with an excess of the corresponding antigen; (c) incubation of sections with the antiserum, followed by 30 min incubations with nonlabeled protein A and then with the protein A-gold complex; (d) incubation of sections with preimmune serum; (e) incubation of sections with the FIG. 1. Electrophoretogram and Western blot of purified WIA lysocolloidal gold suspension alone. zyme. Purified WIA lysozyme was separated in a linear gradient (10Preparation of WIA Lysozyme-Gold Complex. The optimal 15%) denaturing polyacrylamide gel and stained with Coomassie blue pH value for complexing colloidal gold with purified W1A R-250 (A), and transferred to nitrocellulose (B). Commercial purified lysozyme was 9.3 (isoelectric point of this enzyme). The minimal hen egg white lysozyme (HEWL) (C) was also transferred as a reference amount of WIA lysozyme needed to stabilize the gold solution lysozyme. Antigen-antibody complexes were detected using goat-antiat pH 9.3 was determined by adding1 mL of colloidal gold to rabbit IgG labeled with peroxidase as previously described (16). One 0.1 mL of serial dilutions of W1A lysozyme. After a few minutes, reacting band (B) could be observed with WIA lysozyme. No reaction t00mL of 10% NaCl solution was added, and flocculation was was noted with HEWL (C). Numbers on the left refer to molecular determined visually.
weights 103) of protein markers. (X
LOCALIZATION OF WHEAT GERM LYSOZYME AND ITS INTERACTION IN SITU
1319
FIGS. 2-4. Immunocytochemical localization of wheat germ WIA lysozyme using the postembedding protein A-gold
technique. FIG. 2. Endosperm cell walls of wheat embryos are intensely labeled. A few gold particles, likely corresponding to nonspecific labeling, are occasionally seen over the cytoplasm. x31,000. Bar = 0.5 Mm. FIG. 3. Gold particles are present over wheat coleoptile cell walls whereas intercellular spaces are free of labeling (x48,000; bar = 0.25 MAm). FIG. 4. Control test. Gold labeling is negligible over sections incubated with the anti-WlA lysozyme antiserum, previously absorbed with purified W1A lysozyme (x48,000; bar = 0.25 Am). IS, intercellular space; CW, cell wall; Cy, cytoplasm.
commercial hen egg white lysozyme (HEWL) (Fig. IC), blotted on nitrocellulose revealed that antibodies reacted with a single band (Fig. IB) with mol wt corresponding to the purified protein (Fig. IA). No reaction was observed with HEWL (Fig. IC). Two other isozymes of W1A lysozyme also reacted with the anti-WlA antiserum when analyzed in PAGE under native conditions (data not shown). Good preservation of antigenicity and cellular structure was obtained by fixation with glutaraldehyde followed by embedding in Epon. Antigenicity was not maintained when tissues were postfixed with osmium tetroxide prior to embedding. As previously reported (4, 31), osmication of tissues was found to significantly reduce the accessibility of antibodies toward antigens. Since cytoplasmic structures and antigenicity were satisfactorily preserved when tissues were fixed with glutaraldehyde only, this procedure was adopted for further immunocytochemical tests.
Pretreating sections with 1% ovalbumin in PBS was essential to avoid nonspecific labeling. Omission of this step resulted in an intense background signal. Incubation of thin sections from wheat embryos and coleoptiles with anti-WIA lysozyme antibody (1:40 in PBS-ovalbumin), followed by protein A-gold complex (1:30 in PBS-PEG) yielded an intense and specific labeling of cell walls (Figs. 2 and 3). A few randomly distributed gold particles were occasionally seen over the cytoplasm. As these particles were not consistently found from one experiment to another, they were probably related to nonspecific labeling. When sections were incubated with the antiserum previously absorbed with purified W1A lysozyme (1 mg/mL), gold deposition was negligible (Fig. 4). Similarly, sections were nearly free of labeling when (a) preimmune rabbit immunoglobulins were used instead of antiserum, (b) antiserum was omitted, (c) incubation with antiserum was followed by
1320
AUDY ETAL. *
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Plant Physiol. Vol. 88,1988
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are nearly free of labeling (Fig. 6). Intercellular spaces (Fig. 7) are unlabeled. Numerous gold particles are present over starch grains found in amyloplasts (Fig. 7). (Fig. 6-x40,000; bar = 0.25 um. Fig. 7-x31,000; bar = 0.5 um). FIG. 8. Control. Gold labeling is almost absent over sections incubated with the uncomplexed WIA lysozyme and then with the enzyme-gold complex. x40,000. Bar = 0.25 Mum. A, amyloplast; M, mitochondrion; N, nucleus; S, starch grain; ER, endoplasmic reticulum; other abbreviations as in Figures 2-4.
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incubation with unlabeled protein A followed by protein A-gold complex, and (d) incubation was performed with the gold suspension alone. Examination of sections from wheat embryos revealed that cell walls of the endosperm were specifically labeled (Fig. 2). Gold particles did not show any preferential localization over the wall and appeared unevenly distributed. Sections of wheat coleoptiles also exhibited an intense labeling of cell walls (Figs. 3 and 4). Areas corresponding to intercellular spaces were almost devoid of labeling whereas numerous gold particles were present over primary walls. In this case, gold deposition was also irregular. Cytochemical Localization of Interaction between WlA Lysozyme and Cell Wall. The optimal pH value for tagging WIA lysozyme to 15 nm gold particles was 9.3 and the minimal
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amount of enzyme necessary to fully stabilize the gold suspension was 20 ,g/mL. Fixation procedures influenced the labeling pattern obtained with the gold-complexed W1A lysozyme. Postfixation with osmium tetroxide had to be avoided since it was found to impede the accessibility of the enzyme toward the cell wall. Treatment of thin section from wheat embryos and coleoptiles with the WI A lysozyme-gold complex resulted in specific label-
ing of cell walls (Figs. 5-7). Cytoplasm, nuclei, mitochondria, and endoplasmic reticulum were nearly free of labeling (Figs. 6 and 7). In contrast, a few gold particles were reproducibly observed over amyloplasts especially over starch grains (Fig. 7). The specificity of the enzyme-wall interactions was supported by the low level of labeling over sections incubated with the uncomplexed WI A lysozyme and then with the enzyme-gold complex (Fig. 8). Similarly, when sections were incubated with a BSA-
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gold complex or with the gold suspension alone, labeling was negligible. An intense and specific gold labeling was always observed over cell walls, whereas intercellular spaces appeared unlabeled (Fig. 7). Over starch grains of amyloplasts, gold particles were randomly distributed (Fig. 7). Labeling of these structures was considered significant because it was observed from one experiment to another and absent in all controls. Following incubation with W1A lysozyme-gold complex, sections from isolated wheat coleoptile cell walls displayed an intense labeling (Fig. 9). Depending on the plane of sectioning, gold particles appeared randomly distributed or mostly localized over one side of the walls (Fig. 9, arrow). A similar labeling pattern was observed over cell walls treated with DMSO for starch removal (Fig. 10). In this case also, preferential deposition
1321
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FIGS. 9-13. Cytochemical localization of WIA lysozyme over isolated wheat coleoptile cell walls (CW). FIG. 9. Untreated wheat coleoptile cell walls exhibit an intense and specific labeling. Depending on the plane of sectioning, gold particles may appear mostly localized over one side of the walls (arrow) (x40,000; bar = 0.25 Am). FIG. 10. Gold particles are present over (Tfi isolated walls treated with DMSO for starch removal. A preferential deposition of gold particles over one part of the walls is observed (arrows). x30,000. Bar = 0.5
Am.
FIG. 11. Wheat coleoptile cell walls are still intensely labeled after treatment with DMSO and oxalate/HCl for pectin extraction (x40,000; bar = 0.25 Mm). FIG. 12. Gold particles are present over coleoptile cell walls treated with DMSO, oxalate/HCl, and 0.01 M potassium hydroxide for the first hemicellulosic fraction removal (x40,000; bar = 0.25 Mm). FIG. 13. Further treatment with 0.1 M potassium hydroxide for sequential extraction of hemicefluloses results in a loss ofgold labeling (x40,000; bar = 0.25 ,m).
of gold particles over one part of the walls was noticeable (Fig. 10, arrows). Sections of wheat coleoptile cell walls, treated with DMSO and ammonium oxalate/HCl for pectin extraction exhibited intense and specific labeling (Fig. 1 1). Similarly, gold particles were present over sections of isolated cell walls treated with DMSO, ammonium oxalate/HCl, and 0.01 M potassium hydroxide used for the first hemicellulosic fraction removal (Fig. 12). Further treatments of isolated walls with 0.1 or 0.4 M potassium hydroxyde for extraction of the remaining hemicelluloses resulted in loss of gold labeling (Fig. 13). Control experiments performed on untreated and treated coleoptile cell walls yielded negative results. DISCUSSION AND CONCLUSIONS The present results demonstrate that the use of glutaraldehyde for fixation and of Epon for embedding provides good morpho-
1322
AUDY ETAL.
logical integrity and satisfactory preservation of cyto- and immunocytochemical reactivity. As stated by Cramer and BretonGorius (13) who studied the localization of lysozyme in human neutrophils, postembedding colloidal gold techniques are highly suitable for accurate in situ localization of such macromolecules. The introduction of colloidal gold in plant biology as an alternative to markers such as peroxidase or fluorescein has greatly enhanced the resolution and specificity of labeling (7, 8). This is the first report dealing with the ultrastructural localization of a plant lysozyme and of its interaction with the cell wall. In the past, plant lysozymes have received less attention than animal lysozymes (22). In recent years, our knowledge has been mostly concerned with their structure and catalytic properties (9-11, 29). However, the physiological role of plant lysozymes is still open to question. Plant lysozymes are generally assumed to be defensive or protective enzymes (22). Because plant polysaccharides susceptible to enzymic cleavages by lysozymes have not been characterized, the possibility that such enzymes may play a physiological role did not receive much attention (26). Our results revealed that one wheat germ lysozyme (W1A) was associated with cell walls of wheat embryos and coleoptiles. This provides good evidence that wheat cells synthesize at least one lysozyme accumulating in the wall matrix. Cytochemical experiments conducted on isolated wheat coleoptile cell walls disclosed that the cell wall-lysozyme interaction did not involve starch or pectic substances but rather hemicellulose. Since hemicellulose of higher plant cell walls (14) and peptidoglycan of bacteria (20) differ in structure, the question arises whether W1A lysozyme can display multiple enzymic activities. In this regard, further work is needed to study the hydrolytic activity of this lysozyme toward purified hemicellulosic fractions. However, the possibility of nonenzymic binding to wall components due to the cationic properties of this enzyme (pI of 9.3) has also to be evaluated. Formation of complexes between human lysozyme and anionic glycoproteins has already been described (25). Acknowledgments-The authors are grateful to A. Goulet and C. Baillargeon for excellent technical assistance and to L. Belanger for typing this manuscript. LITERATURE CITED 1. ASAMER H, F SCHMALZL, H BRAUNSTEINER 1969 Der immunozytologishe lysozymnachweis in menschlichen blutzellen. Acta Haematol 41: 49-52
2. AUDY P, J TRUDEL, A ASSELIN 1988 Purification and characterization of a lysozyme from wheat germ. Plant Sci (in press) 3. BENDAYAN M 1984 Protein A-gold electron microscopic immunocytochemistry: methods, applications and limitations. J Electron Microsc Techn 1: 243270 4. BENDAYAN M, E PUVION 1984 Ultrastructural localization of nucleic acids through several cytochemical techniques on osmium-fixed tissues. Comparative evaluation of the different labellings. J Histochem Cytochem 32: 11851191 5.
BENHAMOU N, JG LAFONTAINE, JR JOLY, GB OUELLETTE 1985 Ultrastructural localization in host tissues of a toxic glycopeptide produced by Ophiostoma
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Plant Physiol. Vol. 88,1988
chemical localization of antigen-binding sites in the cell surface of two ascomycete fungi using antibodies produced against fimbriae from Ustilago
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