JOURNAL OF ULTRASTRUCTURE RESEARCH
61, 186-192 (1977)
Morphogenesis of Membrane-Bound Bodies in Belladonna (Atropa belladonna L.) Plastids GIORGIO CASADORO AND NICOLETTA RASCIO
Institute of Botany and Plant Physiology, University of Padua, Padua, Italy Received January 4, 1977, and in revised form, June 14, 1977 In the meristematic and adult tissues of belladonna roots and shoots, the plastids contain electron-dense, roundish bodies, limited by a single membrane at maturity. Their ontogenesis has been followed in the different tissues, and a model has been proposed, according to which membranes with electron-dense contents, grouped to form tubular complexes, and isolated membranes with electron-transparent contents may be involved in the biogenesis of membrane-bound bodies.
Plastid membrane-bound bodies, reported as early as 1960 (Gerola and Dassfl, 1960; Hohl, 1960) in a variety of tissues and plants, have been interpreted as having either a storage function (Marinos, 1967; Newcomb, 1967; Salema and Badenhuizen, 1967) or a role in the building up of the thylakoid system (Gerola and Dass/1, 1960; Srivastava, 1966; Israel and Steward, 1967; Jensen and Valdovinos, 1967; Blackwell et al., 1969; Pellegrini and Gerola, 1969; Stetler and Laetsch, 1969; Boasson et al., 1972; Salema et al., 1972; Marty, 1973; Ames and Pivorun, 1974; Cran and Possingham, 1974; Henry, 1975; Damsz and Mikulska, 1976; Casadoro et • al., 1977). However, there are only a few papers describing their morphogenesis (Newcomb, 1967; Blackwell et al., 1969; Salema et al., 1972). This is a report on the steps in the development of the membrane-bound bodies observed in root and shoot plastids of belladonna. MATERIALS AND METHODS Plants of Atropa belladonna L., open-air-grown in the Botanical Gardens of Padua University, were used. Tissue segments were obtained during late spring from the following: (1) the apical meristems, the differentiation region, the cortical primary tissues, and the secondary parenchyma tissues of roots; and (2) the apical meristems, subapical tissues and leaf primordia, the inner (0.5 mm long) and the outer (1 mm long) leaves of vegetative shoots. The tissue segments were fixed for 2 h in 6% glutaralde-
hyde in 0.1 M cacodylate buffer (pH 6.9), rinsed in buffer, then postfixed 2 hr in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 6.9), and dehydrated in a graded series of ethyl alcohol and propylene oxide. Staining with uranyl acetate was effected while dehydrating with 75% alcohol. Tissues were embedded in a Epon-Durcupan ACM mixture; the thin sections, cut with an LKB Ultrotome III, were poststained with lead citrate and examined with a Hitachi HS 9 electron microscope operating at 75 kV. OBSERVATIONS
(A) T h e R o o t T i s s u e s
In the promeristematic cells of the root tip, the plastids are fairly reduced in size (1-1.5 /~m) and have a poor lamellar system (Fig. 1). Two types of internal membranes are clearly distinguishable. One type forms vesicles and cisternae with electron-transparent contents (Figs. 1 and 2) usually localized in the plastid periphery. The membranes of the other type form tubule-like structures with electrondense contents more frequently in the central area of the stroma. They can appear as isolated outlines (Fig. 1) or form an entangled heap of tubules (Fig. 2) similar to the ~%ubular complexes" described by Newcomb (1967) and by Blackwell et al. (1969). The crosswise cut tubules show an electron-dense core surrounded by a dark membranal outline. Moreover, the diameter of the tubules is larger than the distance separating the two membranes of
186 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
Fins. 1 and 2. Root apical m e r i s t e m plastids. Vesicles w i t h e l e c t r o n - t r a n s p a r e n t contents and t u b u l e s w i t h electron-dense contents are visible in the stroma. F i g u r e 2 shows a plastid w i t h an electron-dense t u b u l a r complex and vesicles and cisternae w i t h e l e c t r o n - t r a n s p a r e n t contents r u n n i n g along the plastid periphery. Fig. 1 x 62 000; Fig. 2, x 90 000. Abbreviations are as follows in these and all other figures: DM = t u b u l a r m e m b r a n e w i t h electron-dense contents, LM = vesicles or cisternae w i t h electront r a n s p a r e n t contents, TC = t u b u l a r complex, OB = opaque body not limited by an evident m e m b r a n e , MBB = m e m b r a n e - b o u n d body, MM = m u l t i p l e m e m b r a n e , M = single m e m b r a n e , T - thylakoids, S = starch. Fro. 3. Plastid of the root d e t e r m i n a t i o n zone. It h a s a r o u n d i s h opaque body w i t h a not clearly d i s t i n g u i s h a b l e l i m i t i n g m e m b r a n e . In its interior, d a r k m e m b r a n e - l i k e outlines are visible (arrow). Some cisternae w i t h e l e c t r o n - t r a n s p a r e n t contents are visible n e a r the opaque body. x 90 000. Fro. 4. Plastid of the root n e a r the differentiation zone. It h a s an opaque body w i t h more h o m o g e n e o u s contents t h a n the one in Fig. 3. x 76 000. 187
188
CASADORO AND RASCIO
the cisternae with electron-transparent contents in the same plastid. In the determination tissues, the plastids include very electron-dense, smallsized, roundish bodies containing very opaque, linear, or vesicular membranelike outlines (Fig. 3). These inclusions are not limited by an evident membrane. Isolated tubules with electron-dense contents may also be present in the plastid stroma, but tubular complexes were not observed. Cisternae and vesicles with electron-transparent contents are always present, sometimes in continuity with the inner merebrane of the plastid envelope (Fig. 3) or near the electron-dense body surface. Proceeding to the differentiation region, the electron-dense inclusions of the plastids are larger, with more homogeneous contents, and appear rimmed by a very electron-dense outline (Fig. 4). A limiting membrane is not clearly visible. Vesicles or cisternae with electron-transparent contents are often visible near the surface of the included body and, together with short membranal outlines with electron-dense contents, in the stroma. In the root primary parenchyma, some plastids present vesicles and isolated cisternae with electron-transparent contents and a system of connected tubules organized to form a prolamellar body-like structure (Fig. 5). The tubules have very electron-dense contents in transverse section and appear linked similarly to the "cubic lattice" described for the etioplasts by Gunning and Steer (1975). In some plastids the membranes merge to form irregular, very enlarged outlines with electron-dense contents looking like irregular, flat thylakoids with large perforations (Fig. 6). Other plastids contain a roundish inclusion which is more or less electron-dense and nonhomogeneous and whose periphery presents superimposed membranal outlines, sometimes tightly appressed, sometimes Clearly distinguishable. Usually cisternae with electrontransparent contents are visible near the
inclusion surface (Fig. 7). Finally, in the secondary root parenchyma the plastids are represented only by well-differentiated amyloplasts, 8-10 /~m large, containing small, isolated thylakoids and large starch granules. Frequently they present an included body surrounded by a single membrane. This type of body has more homogeneous contents, and its volume is larger than that of the previously described inclusions, even if it occupies a fairly limited region of the amyloplast.
(B) The Shoot Tissues Proceeding from promeristematic to more and more differentiated cells, in the shoot apex tissues the steps leading from proplastids without any inclusions to plastids with an electron-dense body are similar to those shown in Figs. 1-4 for the root tissues. In very young leaves (0.5 mm) the opaque inclusion occupies most of the plastid volume. These inclusions, whose contents seem to be more homogeneous and less electron-dense than those of the inclusions in the previously described tissues, present a peripheral membrane which, in some places, breaks down into overlapped outlines (Fig. 9). Several flattened and overlapped cisternae with electron-transparent contents, similar to rudimentary thylakoids,, may appear inclined against the inclusion surface (Fig. 10). In the l-mm-long leaves, the plastid inclusion is surrounded by a well-defined single membrane (Fig. 11) and is identical to the plastid inclusions present in young belladonna leaves (Casadoro et al., 1977) and called "membrane-bound bodies" by the authors. Adhering to the limiting membrane and in the plastid stroma, small grana and intergrana thylakoids are present. CONCLUSIONS In belladonna tissues, the plastid fine structure may markedly vary according to
BELLADONNA PLASTID MEMBRANE-BOUND BODIES
189
Fios. 5-7. Primary root cortical parenchyma plastids. In Fig. 5 a prolamellae body-like tubular complex is formed by tubules with very electron-dense contents. A more irregular tubular complex with very enlarged tubules is shown in Fig. 6. Figure 7 illustrates a plastid with a nonhomogeneous, electron-dense inclusion showing more t h a n one membrane at its periphery. Fig. 5, x 45 000; Fig. 6, x 100 000; Fig. 7, x 62 000. Fio. 8. Amyloplast of the cortical parenchyma of a root in secondary structure. An included body is surrounded by a single membrane and has r a t h e r homogeneous contents, x 32 000.
190
CASADORO AND RASCIO
Fins. 9-11. Foliar primordia cell plastids. The included body contents are not very electron-dense and are rather homogeneous. The body is surrounded by a membrane that in some places appears multiple. Cisternae with electron-transparent contents are appressed to the bodyrsnrrounding membrane and form two little grana. In the plastid in Fig. 11, the included body is clearly limited by a single membrane. One can note also granal and intergranal thylakoids. Fig. 9, x 48 000; Fig. 10, x 44 000; Fig. 11, x 45 000.
BELLADONNA PLASTID MEMBRANE-BOUND BODIES
the stage of cell differentiation. The sequence of the plastid types, apparently strictly connected to the tissue developmental stages, suggests that they merely represent different ontogenetic stages of the plastids. A scheme of the possible sequence of plastid morphogenesis is presented in Fig. 12. Therefore, one could suppose that the material contained in the ~membrane-bound bodies" of the most differentiated tissue plastids of the 1-mmlong leaf and of the amyloplasts of the secondary root cortical parenchyma originated from the tubular complexes. This assumption is supported by the observation that the tubular complexes, present in the meristematic tissue plastids, are absent in the plastids of the most differentiated tissues in which, on the contrary, the electron-dense bodies are usually found. Moreover, the latter are initially nonhomogeneous and have internal membrane-like contents while, afterward, they are more homogeneous. On the basis of this developmental scheme, the building up of the inclusionsurrounding membrane probably derives from a melting of vesicles and cisternae with electron-transparent contents around the electron-dense inclusion surface. A subsequent rearrangement of such membranal material would lead to the formation of the single limiting membrane found in the included bodies of the plastids in the most differentiated tissues. The plastidial inclusion biogenesis model reported in the present paper (Fig. 12) is somewhat different from the ones previously proposed. Indeed, all the models (Newcomb, 1967; Blackwell et al., 1969; Salema et al., 1972) indicate a close mutual relation between included bodies and plastidial tubular complexes, and all emphasize the simultaneous presence of both the membrane-bound body and the tubular complexes. Moreover, Newcomb (1967) for plastids of bean root tips, Blackwell et al. (1969) for plastids in aspen tissues in culture, and Salema et al. (1972)
leaf mesophyll ÷
191
root parenchyma
Fro. 12. A proposed model of the possible sequence of the morphogenesis of plastid inclusions in belladonna.
for plastids of Gongora sp. and V a n d a sp. aerial roots postulate that the included bodies may arise by swelling of the peripheral tubules of the tubular complexes, with which they remain connected. In fact, their limiting membrane appeared continuous with the tubular complex membranes. Therefore, the main characteristic of these included bodies is the presence of a clearly defined limiting membrane, homologous to a primary lamella. These observations suggest the possibility of at least two types of membranebound bodies, one characterized by tile contemporary presence of tubular corm plexes, and always limited by a clearly defined membrane, and another, described in this paper, which is never present at the same time as tubular complexes, and which is limited by a clearly defined merabrane only at the latest stages. Its first thylakoid-like structures, grouped on the inclusion surface in the 0.5-mm-long leaf
192
CASADORO AND RASCIO
tissues, derived from cisternae with electron-transparent contents and hence, clearly, from the plastid envelope, suggest that they may have an origin different from the one described previously (Gerola and Dassfi, 1960; Pellegrini and Gerola, 1969; Salema et al., 1972; Marty, 1973; Damsz and Mikulska, 1976). However, in a previous paper Casadoro et al. (1977) reported that the belladonna plastid inclusions contain storage material utilized in thylakoid lamellae formation during the maturation of the plastids themselves. Such being the case, the presence of this inclusion in belladonna plastids acquires a significance analogous to that proposed for the ones in other plants. Therefore, the building up and the utilization of these plastid inclusions seem strictly related to the dynamics of membrane development in relation to cell differentiation and plant species. REFERENCES AMES, I. H., AND PIVORUN, J. P. (1974) Amer. J. Bot. 61, 794. BLACKWELL, S. J., LAETSCH, W. M., AND HYDE,.B. B. (1969) Amer. J. Bot. 56, 457.
BOASSON, R., LAETSCH, W. M., AND PRICE, I. (1972) Amer. J. Bot. 59, 217. CASADORO, G., RASCIO, N., AND PAGANELLI CAPPEL~ LETTI, E. M. (1977). Biol. Cell. (ex J. Microsc.) 29, 61. CRAN, D. G., AND POSSINGHAM, J. V. (1974). Ann. Bot. 38, 843. DAMSZ, B., AND MIKULSKA, E. (1976)Biochem. Physiol. Pflanz. 169, 257. GEROLA, F. M., AND DASSO, G. (1960) Nuovo G. Bot. Ital. 67, 63. GUNNING, B. E. S., AND STEER, M. W. (1975) Ultrastructure and the Biology of Plant Cells, p. 111, E. Arnold, London. HENRY, E. W. (1975)J. Microsc. 22, 109. HOHL, H. R. (1960) Ber. Schweiz. Bot. Ges. 70, 395. ISRAEL, H. W., AND STEWARD, F. C. (1967)Ann. Bot. 31, 1. JENSEN, T. E., AND VALDOVINOS,J. G. (1967)Planta 77, 298. MARINOS, N. G. (1967) J. Ultrastruct. Res. 17, 91. MARTY, D. (1973) C. R. Acad. Sci. Paris 277, 45. NEWCOMB, E. H. (1967) J. Cell Biol. 33, 143. PELLEGRINI, S., AND GEROLA, F. M. (1969) J. Submicrosc. Cytol. 1, 53. SALEMA, R., AND BADENHUIZEN, N. P. (1967) J . Ultrastruct. Res. 20, 383. SALEMA, R., MESQUITA, J. F., AND ABREU, I. (1972) J. Submicrosc. Cytol. 4, 161. SRIVASTAVA, L. M. (1966)J. Cell Biol. 31, 79. STETLER, D. A., AND LAETSCH, W. M. (1969) Amer. J. Bot. 56, 260.
61, 186-192 (1977)
Morphogenesis of Membrane-Bound Bodies in Belladonna (Atropa belladonna L.) Plastids GIORGIO CASADORO AND NICOLETTA RASCIO
Institute of Botany and Plant Physiology, University of Padua, Padua, Italy Received January 4, 1977, and in revised form, June 14, 1977 In the meristematic and adult tissues of belladonna roots and shoots, the plastids contain electron-dense, roundish bodies, limited by a single membrane at maturity. Their ontogenesis has been followed in the different tissues, and a model has been proposed, according to which membranes with electron-dense contents, grouped to form tubular complexes, and isolated membranes with electron-transparent contents may be involved in the biogenesis of membrane-bound bodies.
Plastid membrane-bound bodies, reported as early as 1960 (Gerola and Dassfl, 1960; Hohl, 1960) in a variety of tissues and plants, have been interpreted as having either a storage function (Marinos, 1967; Newcomb, 1967; Salema and Badenhuizen, 1967) or a role in the building up of the thylakoid system (Gerola and Dass/1, 1960; Srivastava, 1966; Israel and Steward, 1967; Jensen and Valdovinos, 1967; Blackwell et al., 1969; Pellegrini and Gerola, 1969; Stetler and Laetsch, 1969; Boasson et al., 1972; Salema et al., 1972; Marty, 1973; Ames and Pivorun, 1974; Cran and Possingham, 1974; Henry, 1975; Damsz and Mikulska, 1976; Casadoro et • al., 1977). However, there are only a few papers describing their morphogenesis (Newcomb, 1967; Blackwell et al., 1969; Salema et al., 1972). This is a report on the steps in the development of the membrane-bound bodies observed in root and shoot plastids of belladonna. MATERIALS AND METHODS Plants of Atropa belladonna L., open-air-grown in the Botanical Gardens of Padua University, were used. Tissue segments were obtained during late spring from the following: (1) the apical meristems, the differentiation region, the cortical primary tissues, and the secondary parenchyma tissues of roots; and (2) the apical meristems, subapical tissues and leaf primordia, the inner (0.5 mm long) and the outer (1 mm long) leaves of vegetative shoots. The tissue segments were fixed for 2 h in 6% glutaralde-
hyde in 0.1 M cacodylate buffer (pH 6.9), rinsed in buffer, then postfixed 2 hr in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 6.9), and dehydrated in a graded series of ethyl alcohol and propylene oxide. Staining with uranyl acetate was effected while dehydrating with 75% alcohol. Tissues were embedded in a Epon-Durcupan ACM mixture; the thin sections, cut with an LKB Ultrotome III, were poststained with lead citrate and examined with a Hitachi HS 9 electron microscope operating at 75 kV. OBSERVATIONS
(A) T h e R o o t T i s s u e s
In the promeristematic cells of the root tip, the plastids are fairly reduced in size (1-1.5 /~m) and have a poor lamellar system (Fig. 1). Two types of internal membranes are clearly distinguishable. One type forms vesicles and cisternae with electron-transparent contents (Figs. 1 and 2) usually localized in the plastid periphery. The membranes of the other type form tubule-like structures with electrondense contents more frequently in the central area of the stroma. They can appear as isolated outlines (Fig. 1) or form an entangled heap of tubules (Fig. 2) similar to the ~%ubular complexes" described by Newcomb (1967) and by Blackwell et al. (1969). The crosswise cut tubules show an electron-dense core surrounded by a dark membranal outline. Moreover, the diameter of the tubules is larger than the distance separating the two membranes of
186 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
Fins. 1 and 2. Root apical m e r i s t e m plastids. Vesicles w i t h e l e c t r o n - t r a n s p a r e n t contents and t u b u l e s w i t h electron-dense contents are visible in the stroma. F i g u r e 2 shows a plastid w i t h an electron-dense t u b u l a r complex and vesicles and cisternae w i t h e l e c t r o n - t r a n s p a r e n t contents r u n n i n g along the plastid periphery. Fig. 1 x 62 000; Fig. 2, x 90 000. Abbreviations are as follows in these and all other figures: DM = t u b u l a r m e m b r a n e w i t h electron-dense contents, LM = vesicles or cisternae w i t h electront r a n s p a r e n t contents, TC = t u b u l a r complex, OB = opaque body not limited by an evident m e m b r a n e , MBB = m e m b r a n e - b o u n d body, MM = m u l t i p l e m e m b r a n e , M = single m e m b r a n e , T - thylakoids, S = starch. Fro. 3. Plastid of the root d e t e r m i n a t i o n zone. It h a s a r o u n d i s h opaque body w i t h a not clearly d i s t i n g u i s h a b l e l i m i t i n g m e m b r a n e . In its interior, d a r k m e m b r a n e - l i k e outlines are visible (arrow). Some cisternae w i t h e l e c t r o n - t r a n s p a r e n t contents are visible n e a r the opaque body. x 90 000. Fro. 4. Plastid of the root n e a r the differentiation zone. It h a s an opaque body w i t h more h o m o g e n e o u s contents t h a n the one in Fig. 3. x 76 000. 187
188
CASADORO AND RASCIO
the cisternae with electron-transparent contents in the same plastid. In the determination tissues, the plastids include very electron-dense, smallsized, roundish bodies containing very opaque, linear, or vesicular membranelike outlines (Fig. 3). These inclusions are not limited by an evident membrane. Isolated tubules with electron-dense contents may also be present in the plastid stroma, but tubular complexes were not observed. Cisternae and vesicles with electron-transparent contents are always present, sometimes in continuity with the inner merebrane of the plastid envelope (Fig. 3) or near the electron-dense body surface. Proceeding to the differentiation region, the electron-dense inclusions of the plastids are larger, with more homogeneous contents, and appear rimmed by a very electron-dense outline (Fig. 4). A limiting membrane is not clearly visible. Vesicles or cisternae with electron-transparent contents are often visible near the surface of the included body and, together with short membranal outlines with electron-dense contents, in the stroma. In the root primary parenchyma, some plastids present vesicles and isolated cisternae with electron-transparent contents and a system of connected tubules organized to form a prolamellar body-like structure (Fig. 5). The tubules have very electron-dense contents in transverse section and appear linked similarly to the "cubic lattice" described for the etioplasts by Gunning and Steer (1975). In some plastids the membranes merge to form irregular, very enlarged outlines with electron-dense contents looking like irregular, flat thylakoids with large perforations (Fig. 6). Other plastids contain a roundish inclusion which is more or less electron-dense and nonhomogeneous and whose periphery presents superimposed membranal outlines, sometimes tightly appressed, sometimes Clearly distinguishable. Usually cisternae with electrontransparent contents are visible near the
inclusion surface (Fig. 7). Finally, in the secondary root parenchyma the plastids are represented only by well-differentiated amyloplasts, 8-10 /~m large, containing small, isolated thylakoids and large starch granules. Frequently they present an included body surrounded by a single membrane. This type of body has more homogeneous contents, and its volume is larger than that of the previously described inclusions, even if it occupies a fairly limited region of the amyloplast.
(B) The Shoot Tissues Proceeding from promeristematic to more and more differentiated cells, in the shoot apex tissues the steps leading from proplastids without any inclusions to plastids with an electron-dense body are similar to those shown in Figs. 1-4 for the root tissues. In very young leaves (0.5 mm) the opaque inclusion occupies most of the plastid volume. These inclusions, whose contents seem to be more homogeneous and less electron-dense than those of the inclusions in the previously described tissues, present a peripheral membrane which, in some places, breaks down into overlapped outlines (Fig. 9). Several flattened and overlapped cisternae with electron-transparent contents, similar to rudimentary thylakoids,, may appear inclined against the inclusion surface (Fig. 10). In the l-mm-long leaves, the plastid inclusion is surrounded by a well-defined single membrane (Fig. 11) and is identical to the plastid inclusions present in young belladonna leaves (Casadoro et al., 1977) and called "membrane-bound bodies" by the authors. Adhering to the limiting membrane and in the plastid stroma, small grana and intergrana thylakoids are present. CONCLUSIONS In belladonna tissues, the plastid fine structure may markedly vary according to
BELLADONNA PLASTID MEMBRANE-BOUND BODIES
189
Fios. 5-7. Primary root cortical parenchyma plastids. In Fig. 5 a prolamellae body-like tubular complex is formed by tubules with very electron-dense contents. A more irregular tubular complex with very enlarged tubules is shown in Fig. 6. Figure 7 illustrates a plastid with a nonhomogeneous, electron-dense inclusion showing more t h a n one membrane at its periphery. Fig. 5, x 45 000; Fig. 6, x 100 000; Fig. 7, x 62 000. Fio. 8. Amyloplast of the cortical parenchyma of a root in secondary structure. An included body is surrounded by a single membrane and has r a t h e r homogeneous contents, x 32 000.
190
CASADORO AND RASCIO
Fins. 9-11. Foliar primordia cell plastids. The included body contents are not very electron-dense and are rather homogeneous. The body is surrounded by a membrane that in some places appears multiple. Cisternae with electron-transparent contents are appressed to the bodyrsnrrounding membrane and form two little grana. In the plastid in Fig. 11, the included body is clearly limited by a single membrane. One can note also granal and intergranal thylakoids. Fig. 9, x 48 000; Fig. 10, x 44 000; Fig. 11, x 45 000.
BELLADONNA PLASTID MEMBRANE-BOUND BODIES
the stage of cell differentiation. The sequence of the plastid types, apparently strictly connected to the tissue developmental stages, suggests that they merely represent different ontogenetic stages of the plastids. A scheme of the possible sequence of plastid morphogenesis is presented in Fig. 12. Therefore, one could suppose that the material contained in the ~membrane-bound bodies" of the most differentiated tissue plastids of the 1-mmlong leaf and of the amyloplasts of the secondary root cortical parenchyma originated from the tubular complexes. This assumption is supported by the observation that the tubular complexes, present in the meristematic tissue plastids, are absent in the plastids of the most differentiated tissues in which, on the contrary, the electron-dense bodies are usually found. Moreover, the latter are initially nonhomogeneous and have internal membrane-like contents while, afterward, they are more homogeneous. On the basis of this developmental scheme, the building up of the inclusionsurrounding membrane probably derives from a melting of vesicles and cisternae with electron-transparent contents around the electron-dense inclusion surface. A subsequent rearrangement of such membranal material would lead to the formation of the single limiting membrane found in the included bodies of the plastids in the most differentiated tissues. The plastidial inclusion biogenesis model reported in the present paper (Fig. 12) is somewhat different from the ones previously proposed. Indeed, all the models (Newcomb, 1967; Blackwell et al., 1969; Salema et al., 1972) indicate a close mutual relation between included bodies and plastidial tubular complexes, and all emphasize the simultaneous presence of both the membrane-bound body and the tubular complexes. Moreover, Newcomb (1967) for plastids of bean root tips, Blackwell et al. (1969) for plastids in aspen tissues in culture, and Salema et al. (1972)
leaf mesophyll ÷
191
root parenchyma
Fro. 12. A proposed model of the possible sequence of the morphogenesis of plastid inclusions in belladonna.
for plastids of Gongora sp. and V a n d a sp. aerial roots postulate that the included bodies may arise by swelling of the peripheral tubules of the tubular complexes, with which they remain connected. In fact, their limiting membrane appeared continuous with the tubular complex membranes. Therefore, the main characteristic of these included bodies is the presence of a clearly defined limiting membrane, homologous to a primary lamella. These observations suggest the possibility of at least two types of membranebound bodies, one characterized by tile contemporary presence of tubular corm plexes, and always limited by a clearly defined membrane, and another, described in this paper, which is never present at the same time as tubular complexes, and which is limited by a clearly defined merabrane only at the latest stages. Its first thylakoid-like structures, grouped on the inclusion surface in the 0.5-mm-long leaf
192
CASADORO AND RASCIO
tissues, derived from cisternae with electron-transparent contents and hence, clearly, from the plastid envelope, suggest that they may have an origin different from the one described previously (Gerola and Dassfi, 1960; Pellegrini and Gerola, 1969; Salema et al., 1972; Marty, 1973; Damsz and Mikulska, 1976). However, in a previous paper Casadoro et al. (1977) reported that the belladonna plastid inclusions contain storage material utilized in thylakoid lamellae formation during the maturation of the plastids themselves. Such being the case, the presence of this inclusion in belladonna plastids acquires a significance analogous to that proposed for the ones in other plants. Therefore, the building up and the utilization of these plastid inclusions seem strictly related to the dynamics of membrane development in relation to cell differentiation and plant species. REFERENCES AMES, I. H., AND PIVORUN, J. P. (1974) Amer. J. Bot. 61, 794. BLACKWELL, S. J., LAETSCH, W. M., AND HYDE,.B. B. (1969) Amer. J. Bot. 56, 457.
BOASSON, R., LAETSCH, W. M., AND PRICE, I. (1972) Amer. J. Bot. 59, 217. CASADORO, G., RASCIO, N., AND PAGANELLI CAPPEL~ LETTI, E. M. (1977). Biol. Cell. (ex J. Microsc.) 29, 61. CRAN, D. G., AND POSSINGHAM, J. V. (1974). Ann. Bot. 38, 843. DAMSZ, B., AND MIKULSKA, E. (1976)Biochem. Physiol. Pflanz. 169, 257. GEROLA, F. M., AND DASSO, G. (1960) Nuovo G. Bot. Ital. 67, 63. GUNNING, B. E. S., AND STEER, M. W. (1975) Ultrastructure and the Biology of Plant Cells, p. 111, E. Arnold, London. HENRY, E. W. (1975)J. Microsc. 22, 109. HOHL, H. R. (1960) Ber. Schweiz. Bot. Ges. 70, 395. ISRAEL, H. W., AND STEWARD, F. C. (1967)Ann. Bot. 31, 1. JENSEN, T. E., AND VALDOVINOS,J. G. (1967)Planta 77, 298. MARINOS, N. G. (1967) J. Ultrastruct. Res. 17, 91. MARTY, D. (1973) C. R. Acad. Sci. Paris 277, 45. NEWCOMB, E. H. (1967) J. Cell Biol. 33, 143. PELLEGRINI, S., AND GEROLA, F. M. (1969) J. Submicrosc. Cytol. 1, 53. SALEMA, R., AND BADENHUIZEN, N. P. (1967) J . Ultrastruct. Res. 20, 383. SALEMA, R., MESQUITA, J. F., AND ABREU, I. (1972) J. Submicrosc. Cytol. 4, 161. SRIVASTAVA, L. M. (1966)J. Cell Biol. 31, 79. STETLER, D. A., AND LAETSCH, W. M. (1969) Amer. J. Bot. 56, 260.
