- Email: [email protected]
Are chloroplasts polyploid?
414
R. G. Herrmann
ARE CHLOROPLASTS
POLYPLOID?
amounts usually show a correlation with the size of the nucleus (or of the cell or of the number of R. G. HERRMANN, Max-Planck-Institut fiir Pflanzenchloroplasts per cell), made it possible to disgenetik, 6802 Ladenburg/Rosenhof, Deutschland-West tinguish striking differences in chloroplast size In the past six years electron microscopy has from one plant to another. There are such plants provided evidence of the localization of DNase- among special trisomic and euploid populations sensitive fibrils in one or several interlamellar of Beta vulgaris [18] (fig. 1, euploid; fig. 2, regions of the chloroplast [e.g. I-61. A compari- trisomic plant). We began by applying semison of the number of these areas in the photoquantitative autoradiography to tissues congraphs included by some authors lead one to taining chloroplasts of different sizes for comspeculate that the differences may be dependent parison of incorporation values and location of upon the developmental stage of the organelle 3H-thymidine label. Nearly 30 plants out of [2, 3, 5,6]. This raisesthe question of the genetic seven groups were examined, each group made valency of a chloroplast. up of sister plants. They were grown under Any attempt to solve this problem entails carefully controlled conditions. During growth experimental difficulties in connection with the the development of the chloroplasts in the accurate determination of the DNA content per individual leaves was checked repeatedly accordplastid [7] and of the quantity of the genetic unit ing to the method of Butterfass [18]. Small of the chloroplast. The exact electron-microsampleswere removed from a specific region of scopic representation and measurement of one or the first 6 to 10 leaves of each plant at the darkmore DNA molecules is difficult becauseof the light transition. One part of each sample was relatively high molecular weight of chloroplast fixed in formaldehyde for the determination of DNA [g-IO]; the problem is considerably simpler chloroplast sizes [18], the rest was immersed in in the case of mitochondrial DNA [e.g. 11-141. a sterilized diluted Benecke solution containing, In addition, the considered biochemical data can 10 ,&/ml 3H-thymidine (3H-6-thymidine, sp. act. be more accurately associated with the morpho5.0 C/m&‘; Radiochem. Centre, Amersham). logical picture in the case of mitochondria than After O?1,29,6,12,24,48 and 72 h of incubation in that of chloroplasts, sincethe former organelles small parts of each piece were processed by a usually have only one, or at least only a small modified technique based on that of Wollgiehn number of DNA-containing regions [2, 11, 13, and Mothes [19], in order to avoid any adsorp141. The experimental data so far available are tion of either cyto- or caryoplasm, or-in the thus indicative only of the fact that mitochondria case of longer incubation periods-of bacteria. can be either polyploid and/or polyenergid The sampleswere fixed with osmium tetroxide, [camp. 1I, 13-151. glutaraldehyde, formaldehyde or ethanol (in the The importance of knowing the number of cold), and prepared by the usual defatting and genetic units per chloroplast or the sum of the RNase and/or DNase or acid treatments, folgenetic units of all plastids per cell for physio- lowed by autoradiography (Kodak AR-10 logical and genetic problems has been stated by stripping film). While DNase treatment after several authors [16, 171. Though it is very fixation with 0~0, produced no conclusive difficult (well nigh impossible, with the methods results, we were able to reduce the label conavailable at present) to obtain an exact DNA siderable after treatment with formaldehyde, value for a given chloroplast, one should at least ethanol [2, 19,201 or even glutaraldehyde [21] by employ an experimental system that permits extensive washing, followed by long treatment relative comparisons between different types of with DNase and washings. plastids. Thus in the investigation reported here, From calculations of label activity, incubation we used a plant system which, in analogy to the periods and exposition time it was evident that established fact that multiple nuclear DNA the amount of incorporated label was small Exptl
Cell Res 55
Are chloroplasts
polyploid?
415
1, 2. Different sizes of chloroplats in comparable leaves of a euploid (fig. 1) and a trisomic plant (fig. 2) (one progeny). Chloroplasts from spongy mesophyll, prepared according to Butterfass [18]. Insets: Fig. I. Autoradiogram of two chloroplasts from the same tissue (size 4 EC);fig. 2, Autoradiograms (lightfield and phase contrast) of the same chloroplast (size 10 ,u). Fixed with glutaraldehyde, extensively washed in buffer, treated with preheated RNase (3O”C, 8 h). The clusters of grains are clearly visible. Figs
[cf 191. In properly fixed chloroplasts (OsO,, glutaraldehyde) the label appeared mostly in centers, each center consisting of a group of grains (up to 30), and rarely in a diffuse pattern (insets figs 1, 2). The centers show the same pattern in nearly all progenies and in chloroplasts of all sizes, except the smallest ones (i.e. -12 ,u). Each of these centers appears over at least one DNA region (as attested by electron microscopy); it can, however, correspond to more such regions, inasmuch as it is possiblethat they may be piled up. From each specimen prepared, 50-150 chloroplasts were chosen at random and analysed as follows: (1) The two lengths were measured across the surface, (2) the number of silver grains, and (3) the number of labeled centers over a chloroplast were counted. There are more silver grains, i.e. more labeled centers, clustered over larger chloroplasts than over smaller ones. For example, we found between 20 and 30 over chloroplasts measuring 3 ,u, between 40 and 80 over those measuring 5 ,u, and between 100 and 200 over those measuring 10 ,u in diameter. For a given chloroplast size the number of labeled centers conforms to a distribution which is nearly always independent of the labeling time; for example we found as
many as six centers for chloroplasts measuring 3 ,u, and as many as 17 for those measuring 8 ,LL in diameter. (Fully labeled chloroplasts can be observed after only 1 and 2$ h incubation, particularly in the most actively incorporating leaves.) This is valid for a variable chloroplast population from a small sample of a leaf (for example, the variation in chloroplast diameter in a 2 mm2 sample of leaf no. 5 of the euploid plant in fig. l-the leaf itself measuring 5 cm in length-ranged from 2 ,U to 7 ,M), from different leaves of a single plant as well as of different plants. Based on calculations of the absorption of tritium B-particles by the chloroplast [22], which depends on thickness and density of a specimen, relative DNA amounts for each chloroplast size can be estimated from the mean silver grain counts (both large and small chloroplasts have approximately the same thickness and show the same density distribution in an isopycnical gradient). As far as we know now, all variations in chloroplast size and in the number of centers seemto exist even in small tissue samples. Even if methods were available to determine an exact analytical DNA amount per chloroplast, the result would still be the calculation of a mean value in the tissue concerned. The problem of Exptl
Cell
R es 55
416
R. G. Herrmann
DNA amount per single chloroplast, therefore, still remains even more complicated. It can be solved only by a microcolorimetric method sensitive enough to measure accurately such small DNA differences between chloroplasts, or by a synchronous cell system containing only one genetic and developmental type of chloroplasts. At the present moment neither of these requirements can be fulfilled. Though the questions concerning the number of genetic units per chloroplast and their relation to the electron-microscopic picture remain unresolved, an attempt should be made to clarify the nomenclature in order to avoid possible misunderstandings. It is evident from the present experiments that the organization of the chloroplast is polyenergid, as has been described for blue-green algae [23-251. The term polyenergid implies that the extranuclear genetic units within the organelle are dispersed in several places, as, for example, the nuclei in the cells of Siphonales or Siphonocladiales. Whether chloroplasts are, in addition, also polyploid remains an open question. In a polyploid organelle, multiple DNA amounts would have to be localized at one point, just as they are in polyploid nuclei. If one begins to consider the possibility of mixoploidy of one or all chloroplasts of a cell, the situation becomeseven more complicated. A detailed account of the results will be published elsewhere. I should like to express my thanks to Dr T T Butterfass for supplying the plant material used and to Dr Karvita Walia for her help in the preparation of the manuscript.
REFERENCES Ris, H & Plaut, W, J cell biol 13 (1962) 383.
:: Kislev, N, Swift, H & Bogorad, L, J cell biol25 (1965) 337. 3. Gunning, B E S, Protoplasma 60 (1965) 111. 4. Wettstein, D v, Photosynthesis in plant life (ed San Pietro, Greer & Army) p. 153. Academic Press, New York (1967). 5. Sprey, B, Planta (Berlin) 78 (1968) 115. 6. Jacobson, A B, J cell biol 38 (1968) 238. 7. Loening, U E, Ann rev plant physiol 19 (1968) 37. 8. Woodcock, C L F & Fernandez-Moran, H, J molec biol 31 (1968) 627. 9. Werz, G & Kellner, 6, J ultrastruct res 24 (1968) 109. 10. Ray, D S & Hanawalt, P C, J molec biol 11 (1965) 760. Exptl
Cell Res 55
Nass, M M K, Proc natl acad sci US 56 (1966) 1215. 12. Borst, P, Biochem j 105 (1967) 37 P. 13. Avers, C J, Billheimer, F E, Hoffmann, H-P & Pauli, R M, Proc natl acad sci US 61 (1968) 90. 14. Suyama, Y & Miura, K, Proc natl acad sci US 60 (1968) 235. 15. Nass. M M K. Nass S & Afzelius. I B A.z*Exntl cell res 37 (1965) 516.’ 16. Renner, 0, Ber Verhandl SBchs Akad Wiss Leipzig. Math-phys K186 (1934) 241, 253. 17. Frandsen, N 0, Theoret appl genetics 38 (1968) 152. 18. Butterfass, Th, Planta (Berlin) 76 (1967) 75. 19. Wollgiehn, R & Mothes, K, Exptl cellres 35 (1964) 52. 20. Swift, H, Am Naturalist 99 (1965) 201. 21. Yokomura, E, Acta med Okayama 21 (1967) 1. 22. Maurer, W & Primbsch, E, Exptl cell res 33 (1964) 8. 23. Fuhs, G W, Arch Mikrobio128 (1958) 270. 24. Beck, S, Flora 153 (1963) 194. 25. Ris, H & Singh, R N, J biophys biochem cytol 9 (1961) 63. 11.
Received November 26, 1968 Revised version received March 10, 1969
THE PERMEABlLITY OF INTERCELLULAR JUNCTIONS IN THE EARLY EMBRYO OF XENOPUS LAEVZS, STUDIED WITH A FLUORESCENT TRACER CHRISTINE SLACK of Biology as Applied Medical Hospital
and J. F. PALMER,
to Medicine, Middlesex School, and Department of Physiology, Medical School, London WI, UK
Department Hospital Middlesex
Electrotonic coupling between cells has been the object of considerable interest in view of the possible role of low resistance junctions in the control of tissuefunction [l]. Although electrical coupling between embryonic cells has been demonstrated [2, 3, 41, the necessity during normal development for the presence of such junctions has not been proved. It has been suggestedthat they may function aspathways for the intercellular movement of developmentally significant molecules. The question as to whether information transfer between embryonic cells could involve the movement of large molecules as well as small ions may be approached by investigation of the permeability of the junctions to a variety of tracers. Experimental evidence for permeability of low resistance junctions to large molecules has already been obtained in nonembryonic systems. Electrotonically coupled fibres of the crayfish septate axon are permeable to fluorescein [5]
R. G. Herrmann
ARE CHLOROPLASTS
POLYPLOID?
amounts usually show a correlation with the size of the nucleus (or of the cell or of the number of R. G. HERRMANN, Max-Planck-Institut fiir Pflanzenchloroplasts per cell), made it possible to disgenetik, 6802 Ladenburg/Rosenhof, Deutschland-West tinguish striking differences in chloroplast size In the past six years electron microscopy has from one plant to another. There are such plants provided evidence of the localization of DNase- among special trisomic and euploid populations sensitive fibrils in one or several interlamellar of Beta vulgaris [18] (fig. 1, euploid; fig. 2, regions of the chloroplast [e.g. I-61. A compari- trisomic plant). We began by applying semison of the number of these areas in the photoquantitative autoradiography to tissues congraphs included by some authors lead one to taining chloroplasts of different sizes for comspeculate that the differences may be dependent parison of incorporation values and location of upon the developmental stage of the organelle 3H-thymidine label. Nearly 30 plants out of [2, 3, 5,6]. This raisesthe question of the genetic seven groups were examined, each group made valency of a chloroplast. up of sister plants. They were grown under Any attempt to solve this problem entails carefully controlled conditions. During growth experimental difficulties in connection with the the development of the chloroplasts in the accurate determination of the DNA content per individual leaves was checked repeatedly accordplastid [7] and of the quantity of the genetic unit ing to the method of Butterfass [18]. Small of the chloroplast. The exact electron-microsampleswere removed from a specific region of scopic representation and measurement of one or the first 6 to 10 leaves of each plant at the darkmore DNA molecules is difficult becauseof the light transition. One part of each sample was relatively high molecular weight of chloroplast fixed in formaldehyde for the determination of DNA [g-IO]; the problem is considerably simpler chloroplast sizes [18], the rest was immersed in in the case of mitochondrial DNA [e.g. 11-141. a sterilized diluted Benecke solution containing, In addition, the considered biochemical data can 10 ,&/ml 3H-thymidine (3H-6-thymidine, sp. act. be more accurately associated with the morpho5.0 C/m&‘; Radiochem. Centre, Amersham). logical picture in the case of mitochondria than After O?1,29,6,12,24,48 and 72 h of incubation in that of chloroplasts, sincethe former organelles small parts of each piece were processed by a usually have only one, or at least only a small modified technique based on that of Wollgiehn number of DNA-containing regions [2, 11, 13, and Mothes [19], in order to avoid any adsorp141. The experimental data so far available are tion of either cyto- or caryoplasm, or-in the thus indicative only of the fact that mitochondria case of longer incubation periods-of bacteria. can be either polyploid and/or polyenergid The sampleswere fixed with osmium tetroxide, [camp. 1I, 13-151. glutaraldehyde, formaldehyde or ethanol (in the The importance of knowing the number of cold), and prepared by the usual defatting and genetic units per chloroplast or the sum of the RNase and/or DNase or acid treatments, folgenetic units of all plastids per cell for physio- lowed by autoradiography (Kodak AR-10 logical and genetic problems has been stated by stripping film). While DNase treatment after several authors [16, 171. Though it is very fixation with 0~0, produced no conclusive difficult (well nigh impossible, with the methods results, we were able to reduce the label conavailable at present) to obtain an exact DNA siderable after treatment with formaldehyde, value for a given chloroplast, one should at least ethanol [2, 19,201 or even glutaraldehyde [21] by employ an experimental system that permits extensive washing, followed by long treatment relative comparisons between different types of with DNase and washings. plastids. Thus in the investigation reported here, From calculations of label activity, incubation we used a plant system which, in analogy to the periods and exposition time it was evident that established fact that multiple nuclear DNA the amount of incorporated label was small Exptl
Cell Res 55
Are chloroplasts
polyploid?
415
1, 2. Different sizes of chloroplats in comparable leaves of a euploid (fig. 1) and a trisomic plant (fig. 2) (one progeny). Chloroplasts from spongy mesophyll, prepared according to Butterfass [18]. Insets: Fig. I. Autoradiogram of two chloroplasts from the same tissue (size 4 EC);fig. 2, Autoradiograms (lightfield and phase contrast) of the same chloroplast (size 10 ,u). Fixed with glutaraldehyde, extensively washed in buffer, treated with preheated RNase (3O”C, 8 h). The clusters of grains are clearly visible. Figs
[cf 191. In properly fixed chloroplasts (OsO,, glutaraldehyde) the label appeared mostly in centers, each center consisting of a group of grains (up to 30), and rarely in a diffuse pattern (insets figs 1, 2). The centers show the same pattern in nearly all progenies and in chloroplasts of all sizes, except the smallest ones (i.e. -12 ,u). Each of these centers appears over at least one DNA region (as attested by electron microscopy); it can, however, correspond to more such regions, inasmuch as it is possiblethat they may be piled up. From each specimen prepared, 50-150 chloroplasts were chosen at random and analysed as follows: (1) The two lengths were measured across the surface, (2) the number of silver grains, and (3) the number of labeled centers over a chloroplast were counted. There are more silver grains, i.e. more labeled centers, clustered over larger chloroplasts than over smaller ones. For example, we found between 20 and 30 over chloroplasts measuring 3 ,u, between 40 and 80 over those measuring 5 ,u, and between 100 and 200 over those measuring 10 ,u in diameter. For a given chloroplast size the number of labeled centers conforms to a distribution which is nearly always independent of the labeling time; for example we found as
many as six centers for chloroplasts measuring 3 ,u, and as many as 17 for those measuring 8 ,LL in diameter. (Fully labeled chloroplasts can be observed after only 1 and 2$ h incubation, particularly in the most actively incorporating leaves.) This is valid for a variable chloroplast population from a small sample of a leaf (for example, the variation in chloroplast diameter in a 2 mm2 sample of leaf no. 5 of the euploid plant in fig. l-the leaf itself measuring 5 cm in length-ranged from 2 ,U to 7 ,M), from different leaves of a single plant as well as of different plants. Based on calculations of the absorption of tritium B-particles by the chloroplast [22], which depends on thickness and density of a specimen, relative DNA amounts for each chloroplast size can be estimated from the mean silver grain counts (both large and small chloroplasts have approximately the same thickness and show the same density distribution in an isopycnical gradient). As far as we know now, all variations in chloroplast size and in the number of centers seemto exist even in small tissue samples. Even if methods were available to determine an exact analytical DNA amount per chloroplast, the result would still be the calculation of a mean value in the tissue concerned. The problem of Exptl
Cell
R es 55
416
R. G. Herrmann
DNA amount per single chloroplast, therefore, still remains even more complicated. It can be solved only by a microcolorimetric method sensitive enough to measure accurately such small DNA differences between chloroplasts, or by a synchronous cell system containing only one genetic and developmental type of chloroplasts. At the present moment neither of these requirements can be fulfilled. Though the questions concerning the number of genetic units per chloroplast and their relation to the electron-microscopic picture remain unresolved, an attempt should be made to clarify the nomenclature in order to avoid possible misunderstandings. It is evident from the present experiments that the organization of the chloroplast is polyenergid, as has been described for blue-green algae [23-251. The term polyenergid implies that the extranuclear genetic units within the organelle are dispersed in several places, as, for example, the nuclei in the cells of Siphonales or Siphonocladiales. Whether chloroplasts are, in addition, also polyploid remains an open question. In a polyploid organelle, multiple DNA amounts would have to be localized at one point, just as they are in polyploid nuclei. If one begins to consider the possibility of mixoploidy of one or all chloroplasts of a cell, the situation becomeseven more complicated. A detailed account of the results will be published elsewhere. I should like to express my thanks to Dr T T Butterfass for supplying the plant material used and to Dr Karvita Walia for her help in the preparation of the manuscript.
REFERENCES Ris, H & Plaut, W, J cell biol 13 (1962) 383.
:: Kislev, N, Swift, H & Bogorad, L, J cell biol25 (1965) 337. 3. Gunning, B E S, Protoplasma 60 (1965) 111. 4. Wettstein, D v, Photosynthesis in plant life (ed San Pietro, Greer & Army) p. 153. Academic Press, New York (1967). 5. Sprey, B, Planta (Berlin) 78 (1968) 115. 6. Jacobson, A B, J cell biol 38 (1968) 238. 7. Loening, U E, Ann rev plant physiol 19 (1968) 37. 8. Woodcock, C L F & Fernandez-Moran, H, J molec biol 31 (1968) 627. 9. Werz, G & Kellner, 6, J ultrastruct res 24 (1968) 109. 10. Ray, D S & Hanawalt, P C, J molec biol 11 (1965) 760. Exptl
Cell Res 55
Nass, M M K, Proc natl acad sci US 56 (1966) 1215. 12. Borst, P, Biochem j 105 (1967) 37 P. 13. Avers, C J, Billheimer, F E, Hoffmann, H-P & Pauli, R M, Proc natl acad sci US 61 (1968) 90. 14. Suyama, Y & Miura, K, Proc natl acad sci US 60 (1968) 235. 15. Nass. M M K. Nass S & Afzelius. I B A.z*Exntl cell res 37 (1965) 516.’ 16. Renner, 0, Ber Verhandl SBchs Akad Wiss Leipzig. Math-phys K186 (1934) 241, 253. 17. Frandsen, N 0, Theoret appl genetics 38 (1968) 152. 18. Butterfass, Th, Planta (Berlin) 76 (1967) 75. 19. Wollgiehn, R & Mothes, K, Exptl cellres 35 (1964) 52. 20. Swift, H, Am Naturalist 99 (1965) 201. 21. Yokomura, E, Acta med Okayama 21 (1967) 1. 22. Maurer, W & Primbsch, E, Exptl cell res 33 (1964) 8. 23. Fuhs, G W, Arch Mikrobio128 (1958) 270. 24. Beck, S, Flora 153 (1963) 194. 25. Ris, H & Singh, R N, J biophys biochem cytol 9 (1961) 63. 11.
Received November 26, 1968 Revised version received March 10, 1969
THE PERMEABlLITY OF INTERCELLULAR JUNCTIONS IN THE EARLY EMBRYO OF XENOPUS LAEVZS, STUDIED WITH A FLUORESCENT TRACER CHRISTINE SLACK of Biology as Applied Medical Hospital
and J. F. PALMER,
to Medicine, Middlesex School, and Department of Physiology, Medical School, London WI, UK
Department Hospital Middlesex
Electrotonic coupling between cells has been the object of considerable interest in view of the possible role of low resistance junctions in the control of tissuefunction [l]. Although electrical coupling between embryonic cells has been demonstrated [2, 3, 41, the necessity during normal development for the presence of such junctions has not been proved. It has been suggestedthat they may function aspathways for the intercellular movement of developmentally significant molecules. The question as to whether information transfer between embryonic cells could involve the movement of large molecules as well as small ions may be approached by investigation of the permeability of the junctions to a variety of tracers. Experimental evidence for permeability of low resistance junctions to large molecules has already been obtained in nonembryonic systems. Electrotonically coupled fibres of the crayfish septate axon are permeable to fluorescein [5]