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Cytochemical characterization of plastidal inclusions in Abutilon mosaic-infected Malva parviflora mesophyll cells
VIROLOGY
106,155-
158 (1980)
Cytochemical Characterization of Plastidal Inclusions in Abutilon Mosaic-Infected Malva parviflora Mesophyll Cells
H. JESKE AND G. WERZ Department of Ultrastructure Research and Electron Microscopy, Division of Biology, Kiinigin-Luise-Strasse 12-16a, D-1000 Berlin 33, Germany
Free University,
Accepted June 12, 1980
Electron microscopy of plastids from mesophyll cells of Malva pa&jZora infected with the Abutilon mosaic virus revealed elongated “chains of pearls” with subunits of 7.5 nm in diameter. Paracrystalline inclusions of the chains of pearls studied by means of cytochemical techniques gave evidence of the presence of DNA.
Abutilon mosaic virus (AbMV) has been suggested to belong to the gemini-virus group because of its transmission by Bemisia tabaci and through comparative symptomatology (1). Electron microscopy of diseased Abutilon sehwianum REG. supported this view by the observation of geminated particles of 16-18 nm in diameter within the nuclei of companion cells (2) (Fig. la). Using Malta parmi$ora as host system, virus-like structures have been isolated and purified, which were infective after mechanical inoculation (3 ). Contrary to the morphology of the intranuclear AbMV in Abutilon sellowianum REG., the isolated virus-like structures appeared as “chains of pearls” (Fig. lb) 8-10 nm in diameter. In electron microscopic preparations comparable structures were detected within the Malva plastids. Here they appeared as characteristic paracrystalloidal inclusions of different shape (Figs. lc and 2 (arrows)). They were never detected in healthy plants. Biochemical analysis gave evidence of the presence of DNA-like nucleic acid (3). The similarity of the aggregation patterns of the particles in the paracrystalloids in situ and that of the “chains of pearls” in vitro strengthens the assumption that they are identical (Figs. lb and c). On the other hand, since it had been shown that the “chains of pearls” contain DNA, the DNA component should be demonstrable within the inclusions. Therefore, we tested by light and 155
electron microscopic staining techniques whether the plastidal inclusions of Maha mesophyll cells contain DNA. For light microscopy, samples of leaf tissue were infiltrated under vacuum for 2 hr with a 0.3 M sucrose solution in 0.1 M Na-phosphate buffer (pH 7) containing 1% pectinase (SERVA 31660) and 0.01 M NaN, as bacteriostaticum at room temperature. Fixation was performed in alcohohglacial acetic acid (3:l v/v), in 70% alcohol, or in glutaraldehyde (6% in 0.15 M Na-phosphate buffer, pH 7.0). For the detection of DNAcontaining components the Feulgen reaction (4) was applied for transmission light microscopy. For fluorescence microscopy the acridine orange technique (5) or the DAPI method (4’,6’-diamino-2-phenylindone: SERVA 18860) (6) were employed. The stained samples were squeezed on a glass slide and examined in a Laborlux light microscope (LEITZ) immediately. Electron microscopy was performed according to (2), freeze-etching as in (7). Additionally, samples were fixed in 6% glutaraldehyde in 0.15 M Na-phosphate buffer, pH 7.5, at room temperature, only. They were processed as described previously (2). The demonstration and differentiation of nucleic acids were carried out by the following procedures: (a) low uranyl ionconcentration technique (8), (b) EDTA bleaching technique (9), and (c) alkaline hydrolysis according to (10). When needed osmium tetroxid-6xed sections were bleached 0042~6822/80/130155-04$02.00/O Copyright All rights
0 1980 by Academic Press. Inc. of reproduction in any form reserved.
FIG. 1. (a) Virus-like structures in companion cell nuclei of AbMV-infected Abutilon sellowkznum. Note the geminated appearance of the particles. (b) Isolated “chains of pearls,” which are associated with infectivity. Negative staining: PTA pH ‘7. (c, d) “Chains of pearls” in paracrystalline inclusions in mesophyll plastids of infected Maha pa,aluifZmu plants in longitudinal (c) and cross-section (d). Note the characteristic triangular shape of the cross-section. Positive staining; uranyl acetate/lead citrate. Bar = 106 nm. 156
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FIG. 2. Cytochemical characterization of the crystalline inclusions showing their DNA content. (a) Standard staining of the inclusion (arrow) inside the plastid: uranyl acetate/lead citrate. (b) Omitting the poststaining shows the lack of primary contrast of the inclusions after osmium fixation. (c) Freezeetch preparation exhibits the prismatic shape of the inclusion. (d) After bleaching the osmium by H,O, the triangles are selectively stained by low uranyl nitrate concentration (8) indicating the presence of nucleic acid. (e) Additional treatment with EDTA of sections stained as(d) leads to removal of the selective stain. ( f) Alkaline hydrolysis of the section does not affect the standard staining capacity of the triangles. (g) Sections through glutaraldehyde-fixed tissue. The paracrystalloid inclusions are visible (arrow). Standard poststaining. (h) Glutaraldehyde-fixed tissue stained with uranyl acetate/ EDTAilead citrate (9). EDTA does destain the triangles indicating the presence of DNA rather than RNA. (i) Light micrograph of Feulgen-stained plastids. The triangles reveal red stain. Bar = 1 pm.
with 3% H,O, for 30 min before the various treatments. Figure 2a shows a section of a plastid after staining with m-any1 acetate/lead citrate (2 >, in which the triangular inclusion
is clearly visible. Also freeze-etched preparations (Fig. 2~) of the plastidal inclusions are found with the comparable structures. Omitting poststaining, the plastidal inclusions appear nearly electron
158
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transparent (Fig. Zb), indicating that they neither contain lipid compounds nor represent aggregation of phytoferritin. However, if bleached sections were stained with 10e5M many1 nitrate the plastidal triangles became selectively electron dense (Fig. Zd). This behavior indicates the presence of nucleic acid in the plastidal inclusions (8) and allows their distinction from other crystals of pure protein, as they occur frequently in microbodies in the same section (2 ) and as they have been described by other authors (11) to be present in plastids. This selective stain is removed by treatment with EDTA (9) (Fig. 2e), while alkaline hydrolysis (10) does not affect the electron density of the inclusion but diminishes that of the cytoplasm and stroma (Fig. 2f). The two results indicate that the nucleic acid contained in the triangles is DNA rather than RNA. For control, the EDTA-bleaching technique was applied to glutaraldehyde-fixed tissue (Figs. 2g and h). The electron density of the inclusions is considerably decreased. The view that the plastidal inclusions contain DNA has been further substantiated by the application of the Feulgen technique, which gave a positive staining reaction as is expected for DNA (Fig. 2i). Acridineorange and DAPI-fluorescence also gave positive results, which will be discussed elsewhere. In summarizing the results obtained from electron and light microscopy we tend to conclude that the paracrystalloidal inclusions in the plastids of AbMV-infected Malva purui~oru contain DNA. This would be in accordance with the results obtained from isolated “chains of pearls” for which evidence indicated that they consist of DNA and protein (3). These results can be taken as further hints that the “chains of pearls”
reported to be associated with infectivity (3) are neither of exogenous origin nor aggregations of phytoferritin, which could be contaminated by viral nucleic acid by chance. It seems that the nucleic acid is an inherent constituent of the “chains of pearls.” Consequently the question is raised if gemini particles and “chains of pearls” could be dimorphic variants of the virus in viva and in vitro (for discussion, see (3)). ACKNOWLEDGMENTS The authors are indebted to Mrs. H. Zuther and Mr. M. Wehner for skillful technical assistance and to Miss E. Nielsen for help in the preparation and typing of the English text. This work was supported by a grant from the Deutsche Forschungsgemeinschaft We 47217. REFERENCES S., Ann. Rev. Phytopathol. 16, 429-449 (1976). 2. JESKE, H., MENZEL, D., and WERZ, G., PhytoI. COSTA, A.
path. 2. 89, 289-295 (197’7). 3. JESKE, H., and WERZ, G., Phytopath.
2. 97,
43-55 (1980). 4. GERLACH, D., “Botanische Mikrotechnik.” Georg Thieme Verlag, Stuttgart, 19’77. 5. CHAYEN, J., BITENSKY, L., and BUTCHER, R. G., “Histochemie.” Verlag Chemie, Weinheim, 1975. 6. WILLIAMSON, D. H., and FENELL, D. J., In “Methods in Cell Biology” (D. M. Prescott, ed.), Vol. XII, pp. 335-351. Academic Press, New York, 1975. 7. JESKE, H., and WERZ, G., Phytopath. 2. 91, l-13 (19’78). 8. ZOBEL, C. R., and BEER, M., J. Biophys. Biochem. Cyt. 10, 335-346 (1961). 9. BERNHARD, W., J.
Ultra&n.&.
Res. 27, 250-
265 (1969). 10. ROELS, F., and GOLDFISCHER, S., J. Histochem. Cytochem. 19, 713-714 (1971). 11. ESAU, K., J. Ultrastruct. Res. 53,235-243(1975).
106,155-
158 (1980)
Cytochemical Characterization of Plastidal Inclusions in Abutilon Mosaic-Infected Malva parviflora Mesophyll Cells
H. JESKE AND G. WERZ Department of Ultrastructure Research and Electron Microscopy, Division of Biology, Kiinigin-Luise-Strasse 12-16a, D-1000 Berlin 33, Germany
Free University,
Accepted June 12, 1980
Electron microscopy of plastids from mesophyll cells of Malva pa&jZora infected with the Abutilon mosaic virus revealed elongated “chains of pearls” with subunits of 7.5 nm in diameter. Paracrystalline inclusions of the chains of pearls studied by means of cytochemical techniques gave evidence of the presence of DNA.
Abutilon mosaic virus (AbMV) has been suggested to belong to the gemini-virus group because of its transmission by Bemisia tabaci and through comparative symptomatology (1). Electron microscopy of diseased Abutilon sehwianum REG. supported this view by the observation of geminated particles of 16-18 nm in diameter within the nuclei of companion cells (2) (Fig. la). Using Malta parmi$ora as host system, virus-like structures have been isolated and purified, which were infective after mechanical inoculation (3 ). Contrary to the morphology of the intranuclear AbMV in Abutilon sellowianum REG., the isolated virus-like structures appeared as “chains of pearls” (Fig. lb) 8-10 nm in diameter. In electron microscopic preparations comparable structures were detected within the Malva plastids. Here they appeared as characteristic paracrystalloidal inclusions of different shape (Figs. lc and 2 (arrows)). They were never detected in healthy plants. Biochemical analysis gave evidence of the presence of DNA-like nucleic acid (3). The similarity of the aggregation patterns of the particles in the paracrystalloids in situ and that of the “chains of pearls” in vitro strengthens the assumption that they are identical (Figs. lb and c). On the other hand, since it had been shown that the “chains of pearls” contain DNA, the DNA component should be demonstrable within the inclusions. Therefore, we tested by light and 155
electron microscopic staining techniques whether the plastidal inclusions of Maha mesophyll cells contain DNA. For light microscopy, samples of leaf tissue were infiltrated under vacuum for 2 hr with a 0.3 M sucrose solution in 0.1 M Na-phosphate buffer (pH 7) containing 1% pectinase (SERVA 31660) and 0.01 M NaN, as bacteriostaticum at room temperature. Fixation was performed in alcohohglacial acetic acid (3:l v/v), in 70% alcohol, or in glutaraldehyde (6% in 0.15 M Na-phosphate buffer, pH 7.0). For the detection of DNAcontaining components the Feulgen reaction (4) was applied for transmission light microscopy. For fluorescence microscopy the acridine orange technique (5) or the DAPI method (4’,6’-diamino-2-phenylindone: SERVA 18860) (6) were employed. The stained samples were squeezed on a glass slide and examined in a Laborlux light microscope (LEITZ) immediately. Electron microscopy was performed according to (2), freeze-etching as in (7). Additionally, samples were fixed in 6% glutaraldehyde in 0.15 M Na-phosphate buffer, pH 7.5, at room temperature, only. They were processed as described previously (2). The demonstration and differentiation of nucleic acids were carried out by the following procedures: (a) low uranyl ionconcentration technique (8), (b) EDTA bleaching technique (9), and (c) alkaline hydrolysis according to (10). When needed osmium tetroxid-6xed sections were bleached 0042~6822/80/130155-04$02.00/O Copyright All rights
0 1980 by Academic Press. Inc. of reproduction in any form reserved.
FIG. 1. (a) Virus-like structures in companion cell nuclei of AbMV-infected Abutilon sellowkznum. Note the geminated appearance of the particles. (b) Isolated “chains of pearls,” which are associated with infectivity. Negative staining: PTA pH ‘7. (c, d) “Chains of pearls” in paracrystalline inclusions in mesophyll plastids of infected Maha pa,aluifZmu plants in longitudinal (c) and cross-section (d). Note the characteristic triangular shape of the cross-section. Positive staining; uranyl acetate/lead citrate. Bar = 106 nm. 156
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FIG. 2. Cytochemical characterization of the crystalline inclusions showing their DNA content. (a) Standard staining of the inclusion (arrow) inside the plastid: uranyl acetate/lead citrate. (b) Omitting the poststaining shows the lack of primary contrast of the inclusions after osmium fixation. (c) Freezeetch preparation exhibits the prismatic shape of the inclusion. (d) After bleaching the osmium by H,O, the triangles are selectively stained by low uranyl nitrate concentration (8) indicating the presence of nucleic acid. (e) Additional treatment with EDTA of sections stained as(d) leads to removal of the selective stain. ( f) Alkaline hydrolysis of the section does not affect the standard staining capacity of the triangles. (g) Sections through glutaraldehyde-fixed tissue. The paracrystalloid inclusions are visible (arrow). Standard poststaining. (h) Glutaraldehyde-fixed tissue stained with uranyl acetate/ EDTAilead citrate (9). EDTA does destain the triangles indicating the presence of DNA rather than RNA. (i) Light micrograph of Feulgen-stained plastids. The triangles reveal red stain. Bar = 1 pm.
with 3% H,O, for 30 min before the various treatments. Figure 2a shows a section of a plastid after staining with m-any1 acetate/lead citrate (2 >, in which the triangular inclusion
is clearly visible. Also freeze-etched preparations (Fig. 2~) of the plastidal inclusions are found with the comparable structures. Omitting poststaining, the plastidal inclusions appear nearly electron
158
SHORT COMMUNICATIONS
transparent (Fig. Zb), indicating that they neither contain lipid compounds nor represent aggregation of phytoferritin. However, if bleached sections were stained with 10e5M many1 nitrate the plastidal triangles became selectively electron dense (Fig. Zd). This behavior indicates the presence of nucleic acid in the plastidal inclusions (8) and allows their distinction from other crystals of pure protein, as they occur frequently in microbodies in the same section (2 ) and as they have been described by other authors (11) to be present in plastids. This selective stain is removed by treatment with EDTA (9) (Fig. 2e), while alkaline hydrolysis (10) does not affect the electron density of the inclusion but diminishes that of the cytoplasm and stroma (Fig. 2f). The two results indicate that the nucleic acid contained in the triangles is DNA rather than RNA. For control, the EDTA-bleaching technique was applied to glutaraldehyde-fixed tissue (Figs. 2g and h). The electron density of the inclusions is considerably decreased. The view that the plastidal inclusions contain DNA has been further substantiated by the application of the Feulgen technique, which gave a positive staining reaction as is expected for DNA (Fig. 2i). Acridineorange and DAPI-fluorescence also gave positive results, which will be discussed elsewhere. In summarizing the results obtained from electron and light microscopy we tend to conclude that the paracrystalloidal inclusions in the plastids of AbMV-infected Malva purui~oru contain DNA. This would be in accordance with the results obtained from isolated “chains of pearls” for which evidence indicated that they consist of DNA and protein (3). These results can be taken as further hints that the “chains of pearls”
reported to be associated with infectivity (3) are neither of exogenous origin nor aggregations of phytoferritin, which could be contaminated by viral nucleic acid by chance. It seems that the nucleic acid is an inherent constituent of the “chains of pearls.” Consequently the question is raised if gemini particles and “chains of pearls” could be dimorphic variants of the virus in viva and in vitro (for discussion, see (3)). ACKNOWLEDGMENTS The authors are indebted to Mrs. H. Zuther and Mr. M. Wehner for skillful technical assistance and to Miss E. Nielsen for help in the preparation and typing of the English text. This work was supported by a grant from the Deutsche Forschungsgemeinschaft We 47217. REFERENCES S., Ann. Rev. Phytopathol. 16, 429-449 (1976). 2. JESKE, H., MENZEL, D., and WERZ, G., PhytoI. COSTA, A.
path. 2. 89, 289-295 (197’7). 3. JESKE, H., and WERZ, G., Phytopath.
2. 97,
43-55 (1980). 4. GERLACH, D., “Botanische Mikrotechnik.” Georg Thieme Verlag, Stuttgart, 19’77. 5. CHAYEN, J., BITENSKY, L., and BUTCHER, R. G., “Histochemie.” Verlag Chemie, Weinheim, 1975. 6. WILLIAMSON, D. H., and FENELL, D. J., In “Methods in Cell Biology” (D. M. Prescott, ed.), Vol. XII, pp. 335-351. Academic Press, New York, 1975. 7. JESKE, H., and WERZ, G., Phytopath. 2. 91, l-13 (19’78). 8. ZOBEL, C. R., and BEER, M., J. Biophys. Biochem. Cyt. 10, 335-346 (1961). 9. BERNHARD, W., J.
Ultra&n.&.
Res. 27, 250-
265 (1969). 10. ROELS, F., and GOLDFISCHER, S., J. Histochem. Cytochem. 19, 713-714 (1971). 11. ESAU, K., J. Ultrastruct. Res. 53,235-243(1975).