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Ultrastructural features of the parenchyma of young leaves of Datura stramonium L. systemically infected with potato virus X
VIROLOGY
96, 265-269 (19’79)
SHORT COMMUNICATIONS Ultrastructural
Features of the Parenchyma Systemically Infected
of Young Leaves of Datufa stramonium with Potato Virus X
L.
A. V. REUNOV Institute
of Biology
and Pedology, Far East Science Centre, USSR Academy of Sciences, Vladivostok 690022, USSR Accepted January
4, 1979
Mosaic symptoms were observed on the tip parts of upper leaves of Datura stramonium L. (2-3 cm in length), systemically infected with potato virus X (PVX); symptoms were absent on basal parts of the same leaves. Virus-like particles were not observed in the parenchyma cells of the basal parts lacking symptoms. Considerable retraction of the plasmalemma from the cell wall and the appearance of vesicles and tubules between them were the most characteristic features of such cells. In the tip parts of the leaves with mosaic symptoms, numerous PVX particles were observed in many parencliyma cells, where they were dispersed in the cytoplasm or grouped in aggregates. As a rule the dispersed PVX virions were associated with specific laminated inclusions. Sometimes virions appeared in areas of the cytoplasm subjected to local autolysis. The tonoplast was often disturbed in infected cells, and virus particles were released into a central vacuole.
There is evidence (1,2) that in very young plant tissues viruses accumulate to a lesser degree than in more mature tissues. Nevertheless, the mechanisms limiting virus accumulation in tissues of various ages remain unclear. It seems to us that in studying these mechanisms substantial attention should be paid to the peculiarities of interaction of viruses with the cells of tissues of various ages on the ultrastructural level. The present work presents some data on the ultrastructure of parenchyma cells of various-aged parts of upper leaves of Datura stramonium L., systemically infected with potato virus X (PVX). Studies were carried out on 6-week-old plants of D. stramonium L. grown in a greenhouse. Two lower leaves of 4-week-old plants intended for infection were inoculated. The inoculum used was sap from leaves of D. stramonium infected with a severe PVX strain “Taezhny” (3). Very young upper leaves (2-3 cm in length) were studied. The basal (zone I) and tip (zone II) parts of the leaves were studied separately. Marked mosaic symptoms were observed on
265
the tip parts of systemically infected leaves, while on the basal parts of the same leaves the symptoms were absent. For comparison, upper leaves of similar healthy plants were examined. Small pieces of leaves were fixed for 3 hr in 6.5% glutaraldehyde prepared in phosphate buffer, pH 7.4. Postfixation was for 2 hr in 1% osmium tetroxide; the material was dehydrated in a graded alcohol and acetone series and embedded in Epon epoxy resin. Sections were stained with uranyl acetate and lead citrate and observed on an EMV-100L (USSR) electron microscope. In several experiments, a comparative determination of infectivity was carried out in the two zones on half-leaves of G. globosa L. and the content of virus particles in these tissues was evaluated by examination under an electron microscope of crude sap preparations negatively stained with 2% phosphotungstate, pH 7.0. The results show that the infectivity from zone I of infected D. stramonium plants was 15- to 20-fold lower as compared to that
0042~6822/79/09026505$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
266
SHORT COMMUNICATIONS
of zone II. In negatively stained preparations of zone I, PVX particles were rarely observed, whereas in preparations from zone II a great many virions were observed. In examining parenchyma cell sections from zone I of infected plants we found neither virus-like particles nor typical laminated structures which may be induced by PVX (4-6). A characteristic of these cells was a considerable plasmalemma retraction from the cell wall and formation of broad zones containing vesicular and tubular structures between them (Fig. 1). In the process of retraction of the plasmalemma from the cell wall, intercellular cytoplasmic connections through the plasmodesmata may be broken. Retraction of the plasmalemma from the cell wall may sometimes be observed in healthy control cells, however in this case they are not as pronounced. In many parenchyma cells of zone II showing PVX symptoms, a great number filamentous virus-like particles were observed, which were dispersed in the cytoplasm or grouped in more or less dense aggregates. Like particles were never found in healthy control cells. The particles observed in longitudinal sections were much smaller in diameter than PVX virions. It should be noted, however, that on ultrathin sections the real diameter of virions may not be revealed (7). Capsid proteins may stain poorly with uranyl acetate and lead, yielding an image of the stained nucleic acid. Apart from the presence of virus-like particles, a representative feature of cells of zone II showing mosaic symptoms was an active formation of laminar inclusions (4 -6). All this testifies to the fact that the filamentous particles that we observed are PVX. The laminar inclusions were often twisted and PVX particles might be located in them (Fig. 2). Another peculiar character of the infected cells from zone II, was the activation of autolytic processes. In cytoplasmic areas which showed local autolysis not infrequently it was possible to observe PVX particles (Fig. 3). The tonoplast was quite often disrupted in infected cells, and PVX
particles were released into the central vacuole (Fig. 4). In control cells local autolysis was rarely seen and damaged tonoplasts were seldom observed. Retraction of the plasmalemma from the cell wall in cells of zone II of infected plants was observed less frequently and was less pronounced than in cells of zone I. Invaginations of the plasmalemma in healthy cells of zone II, as in zone I, were rarely seen. The results obtained show great differences in the interaction of PVX with the parenchyma cells of various ages from tip and basal parts of the upper leaves of D. stramonium L. Especially interesting is the fact that we were unable to detect virus in sections of zone I of infected plants, while many cells from zone II of the same leaves contained numerous virus particles. Taking into account the large number of cells investigated, it seemsunlikely that we might have overlooked PVX particle aggregates. However, considering the difficulty of identifying individual filamentous viruses in sections, we must not exclude the possibility that we were not able to identify single PVX virions in the matrix of the cytoplasm of cells in zone I. The data obtained at estimating the infectivity and in examining negatively stained preparations indicate that a certain, though a very small quantity of virus, is present in zone I. In which cells of zone I this virus is localized remains obscure. In any case, we may conclude that if PVX (strain “Taezhny”) accumulates in parenchyma cells of the very young tissue of zone I, it accumulates insignificantly. Accumulation of PVX in zone II seems to be partially the result of virus transport with photosynthate in the period when the leaf develops in the shoot apex. The latter is known (8) to possess a high demand for assimilates. It should be noted, however, that the leaf in seed plants, unlike the stem, is characterized by early cessation of apical growth (9) and in the course of further growth the tip part of the upper leaf matures much faster than the basal part (10). In what way these circumstances regulate the mechanisms determining virus accumu-
FIGS. 1 AND 2. Parts of parenchyma cells of upper leaves of Datura
stramonium
L. infected with
PVX. FIG. 1. Tubules and vesicular structures in the zones between the plasmalemma (Pl) and cell wall(W) in the cells of the basal leaf portion. M, mitochondrion; ER, endoplasmic reticulum; Pd, plasmodesmata. Bar = 500 nm. FIG. 2. Twisted forms of PVX-induced laminated inclusions (LI) in the cell of the tip portion of the leaf. V, virus particles; C, chloroplast; ER, endoplasmic reticulum. Bar = 250 nm. 267
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FIGS. 3 AND 4. Parenchyma cells in the tip part of D. stramonium leaves infected with PVX. FIG. 3. Local autolysis. Pl, plasmalemma; W, cell wall; V, virus particles; C, chloroplast; M,
mitochondrion; D, dictyosome. Bar = 250 nm. FIG. 4. PVX particles in the central vacuole (CV). V, virus particles; W, cell wall; Pl, plaamalemma; T, tonoplast; C, chloroplast; M, mitochondrion. Bar = 500 nm.
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lation in zones I and II and what kind of mechanisms they are remain to be investigated. Keeping in mind that in the spread of plant viruses from cell to cell and in particular of filamentous viruses, apparently an important part is played by the plasmodesmata (11) and we can assume that one of the mechanisms preventing the systemic penetration of PVX into cells of zone I in our experiments, as well as the intercellular spread of TMV in the research of Spencer and Kimmins (12), may have been the rupture of the plasmodesmata, which takes place during considerable retraction of the plasmalemma from the cell wall. In the more mature cells of zone II, this mechanism, if it existed at all, evidently took place much less often and thus could not limit systemic penetration of PVX into the cells. The biological function of the laminated bodies which formed so actively in our experiments in the PVX-infected cells of zone II, often taking a twisted position (Fig. 2), is still obscure. Localization of PVX particles in cytoplasmic areas which have been subjected to autolysis (Fig. 3) and their appearance together with the lysing cell material in the central vacuole (Fig. 4) which, according to current opinion, may function as a lysosome (13. 11). gives rise to a auestion as
to whether virus particles undergo any destruction in these cases. To answer this question, it is necessary to do further research. REFERENCES 1. SOLBERG, R. A., and BALD, J. G., Virology 17, 359-361 (1962). 2. FAED, E. M., and MATTHEWS, R. E. F., Virology 48, 546-554 (1972). 3. REIFMAN, V. G., and KOLESNIKOVA, S. A., In “Virus Diseases in Far East Plants,” Trudy Biologo-pochvennogo Inst. Vol. 14, No. 11’7, pp. 82-98. Vladivostok (USSR), 1973. 4. KOZAR, F. E., and SHELUDKO, Y. M., Virology 38, 220-229 (1969). 5. STOLS, A., HILL-VAN DER MEULEN, G. W., and TOEN, M. K. I., Virology 40, 168-1’70 (1970). 6. SHALLA, T. A., and SHEPARD, J. F., Virology 49, 654-667 (1972). 7. ESAU, K., “Viruses in Plant Hosts. Form, Distribution, and Pathologic Effects.” Univ. Wisconsin Press, Madison, Wis., 1968. 8. WARDLAW, I. F., Bat. Rev. 34, 79-105 (1968). Van 9. FOSTER, A. S., “Practical Plant Anatomy.” Nostrand, New York, 1942. 10. MAKSYMOWYCH, R., Amer. J. Bot. 46, 635-644 (1959). Il. WEINTRAUB, M., RAGETLI, H. W. J., and LEUNG, E., J. Ultrastruct. Res. 56, 351-364 (1976). 12. SPENCER, D. E., and KIMMINS, W. C., Canad. J. Bot. 49, 417-421 (1971). 13. MATILE, P., Biochem. J. 111, 26P-27P (1969). (USSR) 14, 1309a. BELIZER, N. V., Tsitologiya 1313 (1972).
96, 265-269 (19’79)
SHORT COMMUNICATIONS Ultrastructural
Features of the Parenchyma Systemically Infected
of Young Leaves of Datufa stramonium with Potato Virus X
L.
A. V. REUNOV Institute
of Biology
and Pedology, Far East Science Centre, USSR Academy of Sciences, Vladivostok 690022, USSR Accepted January
4, 1979
Mosaic symptoms were observed on the tip parts of upper leaves of Datura stramonium L. (2-3 cm in length), systemically infected with potato virus X (PVX); symptoms were absent on basal parts of the same leaves. Virus-like particles were not observed in the parenchyma cells of the basal parts lacking symptoms. Considerable retraction of the plasmalemma from the cell wall and the appearance of vesicles and tubules between them were the most characteristic features of such cells. In the tip parts of the leaves with mosaic symptoms, numerous PVX particles were observed in many parencliyma cells, where they were dispersed in the cytoplasm or grouped in aggregates. As a rule the dispersed PVX virions were associated with specific laminated inclusions. Sometimes virions appeared in areas of the cytoplasm subjected to local autolysis. The tonoplast was often disturbed in infected cells, and virus particles were released into a central vacuole.
There is evidence (1,2) that in very young plant tissues viruses accumulate to a lesser degree than in more mature tissues. Nevertheless, the mechanisms limiting virus accumulation in tissues of various ages remain unclear. It seems to us that in studying these mechanisms substantial attention should be paid to the peculiarities of interaction of viruses with the cells of tissues of various ages on the ultrastructural level. The present work presents some data on the ultrastructure of parenchyma cells of various-aged parts of upper leaves of Datura stramonium L., systemically infected with potato virus X (PVX). Studies were carried out on 6-week-old plants of D. stramonium L. grown in a greenhouse. Two lower leaves of 4-week-old plants intended for infection were inoculated. The inoculum used was sap from leaves of D. stramonium infected with a severe PVX strain “Taezhny” (3). Very young upper leaves (2-3 cm in length) were studied. The basal (zone I) and tip (zone II) parts of the leaves were studied separately. Marked mosaic symptoms were observed on
265
the tip parts of systemically infected leaves, while on the basal parts of the same leaves the symptoms were absent. For comparison, upper leaves of similar healthy plants were examined. Small pieces of leaves were fixed for 3 hr in 6.5% glutaraldehyde prepared in phosphate buffer, pH 7.4. Postfixation was for 2 hr in 1% osmium tetroxide; the material was dehydrated in a graded alcohol and acetone series and embedded in Epon epoxy resin. Sections were stained with uranyl acetate and lead citrate and observed on an EMV-100L (USSR) electron microscope. In several experiments, a comparative determination of infectivity was carried out in the two zones on half-leaves of G. globosa L. and the content of virus particles in these tissues was evaluated by examination under an electron microscope of crude sap preparations negatively stained with 2% phosphotungstate, pH 7.0. The results show that the infectivity from zone I of infected D. stramonium plants was 15- to 20-fold lower as compared to that
0042~6822/79/09026505$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
266
SHORT COMMUNICATIONS
of zone II. In negatively stained preparations of zone I, PVX particles were rarely observed, whereas in preparations from zone II a great many virions were observed. In examining parenchyma cell sections from zone I of infected plants we found neither virus-like particles nor typical laminated structures which may be induced by PVX (4-6). A characteristic of these cells was a considerable plasmalemma retraction from the cell wall and formation of broad zones containing vesicular and tubular structures between them (Fig. 1). In the process of retraction of the plasmalemma from the cell wall, intercellular cytoplasmic connections through the plasmodesmata may be broken. Retraction of the plasmalemma from the cell wall may sometimes be observed in healthy control cells, however in this case they are not as pronounced. In many parenchyma cells of zone II showing PVX symptoms, a great number filamentous virus-like particles were observed, which were dispersed in the cytoplasm or grouped in more or less dense aggregates. Like particles were never found in healthy control cells. The particles observed in longitudinal sections were much smaller in diameter than PVX virions. It should be noted, however, that on ultrathin sections the real diameter of virions may not be revealed (7). Capsid proteins may stain poorly with uranyl acetate and lead, yielding an image of the stained nucleic acid. Apart from the presence of virus-like particles, a representative feature of cells of zone II showing mosaic symptoms was an active formation of laminar inclusions (4 -6). All this testifies to the fact that the filamentous particles that we observed are PVX. The laminar inclusions were often twisted and PVX particles might be located in them (Fig. 2). Another peculiar character of the infected cells from zone II, was the activation of autolytic processes. In cytoplasmic areas which showed local autolysis not infrequently it was possible to observe PVX particles (Fig. 3). The tonoplast was quite often disrupted in infected cells, and PVX
particles were released into the central vacuole (Fig. 4). In control cells local autolysis was rarely seen and damaged tonoplasts were seldom observed. Retraction of the plasmalemma from the cell wall in cells of zone II of infected plants was observed less frequently and was less pronounced than in cells of zone I. Invaginations of the plasmalemma in healthy cells of zone II, as in zone I, were rarely seen. The results obtained show great differences in the interaction of PVX with the parenchyma cells of various ages from tip and basal parts of the upper leaves of D. stramonium L. Especially interesting is the fact that we were unable to detect virus in sections of zone I of infected plants, while many cells from zone II of the same leaves contained numerous virus particles. Taking into account the large number of cells investigated, it seemsunlikely that we might have overlooked PVX particle aggregates. However, considering the difficulty of identifying individual filamentous viruses in sections, we must not exclude the possibility that we were not able to identify single PVX virions in the matrix of the cytoplasm of cells in zone I. The data obtained at estimating the infectivity and in examining negatively stained preparations indicate that a certain, though a very small quantity of virus, is present in zone I. In which cells of zone I this virus is localized remains obscure. In any case, we may conclude that if PVX (strain “Taezhny”) accumulates in parenchyma cells of the very young tissue of zone I, it accumulates insignificantly. Accumulation of PVX in zone II seems to be partially the result of virus transport with photosynthate in the period when the leaf develops in the shoot apex. The latter is known (8) to possess a high demand for assimilates. It should be noted, however, that the leaf in seed plants, unlike the stem, is characterized by early cessation of apical growth (9) and in the course of further growth the tip part of the upper leaf matures much faster than the basal part (10). In what way these circumstances regulate the mechanisms determining virus accumu-
FIGS. 1 AND 2. Parts of parenchyma cells of upper leaves of Datura
stramonium
L. infected with
PVX. FIG. 1. Tubules and vesicular structures in the zones between the plasmalemma (Pl) and cell wall(W) in the cells of the basal leaf portion. M, mitochondrion; ER, endoplasmic reticulum; Pd, plasmodesmata. Bar = 500 nm. FIG. 2. Twisted forms of PVX-induced laminated inclusions (LI) in the cell of the tip portion of the leaf. V, virus particles; C, chloroplast; ER, endoplasmic reticulum. Bar = 250 nm. 267
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FIGS. 3 AND 4. Parenchyma cells in the tip part of D. stramonium leaves infected with PVX. FIG. 3. Local autolysis. Pl, plasmalemma; W, cell wall; V, virus particles; C, chloroplast; M,
mitochondrion; D, dictyosome. Bar = 250 nm. FIG. 4. PVX particles in the central vacuole (CV). V, virus particles; W, cell wall; Pl, plaamalemma; T, tonoplast; C, chloroplast; M, mitochondrion. Bar = 500 nm.
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lation in zones I and II and what kind of mechanisms they are remain to be investigated. Keeping in mind that in the spread of plant viruses from cell to cell and in particular of filamentous viruses, apparently an important part is played by the plasmodesmata (11) and we can assume that one of the mechanisms preventing the systemic penetration of PVX into cells of zone I in our experiments, as well as the intercellular spread of TMV in the research of Spencer and Kimmins (12), may have been the rupture of the plasmodesmata, which takes place during considerable retraction of the plasmalemma from the cell wall. In the more mature cells of zone II, this mechanism, if it existed at all, evidently took place much less often and thus could not limit systemic penetration of PVX into the cells. The biological function of the laminated bodies which formed so actively in our experiments in the PVX-infected cells of zone II, often taking a twisted position (Fig. 2), is still obscure. Localization of PVX particles in cytoplasmic areas which have been subjected to autolysis (Fig. 3) and their appearance together with the lysing cell material in the central vacuole (Fig. 4) which, according to current opinion, may function as a lysosome (13. 11). gives rise to a auestion as
to whether virus particles undergo any destruction in these cases. To answer this question, it is necessary to do further research. REFERENCES 1. SOLBERG, R. A., and BALD, J. G., Virology 17, 359-361 (1962). 2. FAED, E. M., and MATTHEWS, R. E. F., Virology 48, 546-554 (1972). 3. REIFMAN, V. G., and KOLESNIKOVA, S. A., In “Virus Diseases in Far East Plants,” Trudy Biologo-pochvennogo Inst. Vol. 14, No. 11’7, pp. 82-98. Vladivostok (USSR), 1973. 4. KOZAR, F. E., and SHELUDKO, Y. M., Virology 38, 220-229 (1969). 5. STOLS, A., HILL-VAN DER MEULEN, G. W., and TOEN, M. K. I., Virology 40, 168-1’70 (1970). 6. SHALLA, T. A., and SHEPARD, J. F., Virology 49, 654-667 (1972). 7. ESAU, K., “Viruses in Plant Hosts. Form, Distribution, and Pathologic Effects.” Univ. Wisconsin Press, Madison, Wis., 1968. 8. WARDLAW, I. F., Bat. Rev. 34, 79-105 (1968). Van 9. FOSTER, A. S., “Practical Plant Anatomy.” Nostrand, New York, 1942. 10. MAKSYMOWYCH, R., Amer. J. Bot. 46, 635-644 (1959). Il. WEINTRAUB, M., RAGETLI, H. W. J., and LEUNG, E., J. Ultrastruct. Res. 56, 351-364 (1976). 12. SPENCER, D. E., and KIMMINS, W. C., Canad. J. Bot. 49, 417-421 (1971). 13. MATILE, P., Biochem. J. 111, 26P-27P (1969). (USSR) 14, 1309a. BELIZER, N. V., Tsitologiya 1313 (1972).