JOURNAL OF ULTRASTRUCTURE RESEARCH 65, 30-35 (1978)
Thylakoid Membranes in Sunflower and in Other Plants GIORGIO CASADORO AND NICOLETTA RASCIO Institute of Botany and Plant Physiology, University of Padua, Italy Received February 22, 1978, and in revised form, April 25, 1978
In the early ontogenesis of the sunflower plastids, the thylakoids show a particular staining. They are characterized by a wide, electron-transparent central region and by a thin electron-dense line on the stroma's side to which, possibly, an analogous dark line corresponds on the intrathylakoidal side of the compartment. The latter line is difficult to see because of the electron density of the intrathylakoid compartment. The peculiar membrane appearance persists for a long time, and sometimes it is observed in plastids with well-arranged grana and abundant starch. Therefore we can assume that the photosystems can be inserted into such membranes. The presence of chlorophyllous pigments on "lightly stained membranes" also seems to be suggested by the cotyledonous etioplasts that show prolameUar bodies with a paracrystalline lattice. As the LS membranes are present in plants belonging to very different families, we may assume that they are not a peculiarity, but on the contrary, are widely distributed in nature. F o r a long t i m e t h e p r e s e n c e of t h y l a k o i d m e m b r a n e s w i t h a p a r t i c u l a r staining in plastids o f m e r i s t e m a t i c or v e r y y o u n g tissues h a s b e e n k n o w n {Israel a n d S t e w a r d , 1967; S r i v a s t a v a a n d P a u l s o n , 1968; Blackwell et al., 1969; Catesson, 1970; S a l e m a a n d A b r e u , 1972; S a l e m a et al., 1972; M a r t y , 1973; C r a n a n d P o s s i n g h a m , 1974; D a m s z a n d Mikulska, 1976; P l a t t - A l o i a a n d T h o m son, 1977). S u c h m e m b r a n e s , u p to n o w n a m e d "negative," h a v e r e c e n t l y b e e n c o n s i d e r e d as i n c o m p l e t e l y organized ones r a t h e r t h a n artifacts ( D a m s z a n d Mikulska, 1976; P l a t t Aloia a n d T h o m s o n , 1977). I n fact, w h e n t h e y are present, b o t h t h e plastid e n v e l o p e a n d t h e o t h e r cellular m e m b r a n e s a p p e a r n o r m a l l y stained; s u b s e q u e n t l y , t h e y also a s s u m e a n o r m a l staining c o n t e m p o r a n e o u s l y w i t h t h e d i s a p p e a r a n c e o f t h e elect r o n - d e n s e m a t e r i a l u s u a l l y c o n t a i n e d in t h e i n t r a m e m b r a n a l c o m p a r t m e n t s (PlattAloia a n d T h o m s o n , 1977). As s u c h m e m b r a n e s r e p r e s e n t a n o r m a l o n t o g e n e t i c stage for t h e plastids containing t h e m , a n d as t h e y h a v e b e e n r e p o r t e d in p l a n t s belonging to different families, we d e e m e d it of s o m e interest to investigate their fine s t r u c t u r e a n d to see if we could find t h e m in additional plants.
MATERIALS AND METHODS Open-air-grown plants of Helianthus annuus L. and of other species from the Botanic Gardens of the Padua University were used. Tissue smnples were obtained, during late spring, from successively older leaves. Seeds of Helianthus annuus L., soaked in water for 24 hr, were placed in sealed petri dishes in the dark for 7 days. Tissue from cotyledons was sampled and fixed in the dark. All the tissue segments were fixed for 2 hr in 6% glutaraldehyde in 0.1 M cacodylate buffer (pH 6.9), washed in buffer, then postfixed 2 hr in 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 a LKB Ultrotome III, were poststained with lead citrate and examined with a Hitachi HS 9 electron microscope operating at 75 kV. RESULTS AND DISCUSSION I n t h e y o u n g sunflower leaves t h e plastids s h o w a v e r y p a r t i c u l a r fine s t r u c t u r e . I n a r a t h e r d a r k s t r o m a t h e r e are large amounts of more strongly electron-dense m a t e r i a l enclosed in v a r i o u s l y dilated c o m p a r t m e n t s limited b y m e m b r a n e s . T h e s e m e m b r a n e s (Fig. la) look like electront r a n s p a r e n t lines defining t h e single c o m partments. However, where higher magnification reveals pairing o f two electrond e n s e c o m p a r t m e n t s , we c a n see t w o light stripes s e p a r a t e d b y a c e n t r a l d a r k line (Fig. 3o
0022-5320/78/0651-0030502.00/0 Copyright© 1978by AcademicPress, Inc. All rights of reproductionin any form reserved.
SUNFLOWER THYLAKOID MEMBRANES l b ) . T h e r e f o r e t h i s w h o l e r e g i o n looks like a "negatively" stained trilaminar unit memb r a n e . T h i s f e a t u r e of t h e p l a s t i d ' s i n t e r n a l m e m b r a n e s , o b s e r v e d also b y o t h e r a u t h o r s (see r e f e r e n c e s a t b e g i n n i n g of i n t r o d u c tion}, does n o t affect e i t h e r t h e p l a s t i d env e l o p e m e m b r a n e s or t h e o t h e r c e l l u l a r membranes which appear normally stained
31
(Fig. l a ) . T h e w h o l e t r i s t r a t i f i e d r e g i o n is a b o u t 11.5-13 n m thick, w h i l e e a c h of t h e elect r o n - t r a n s p a r e n t s t r i p e s is a b o u t 4-4.5 n m a n d t h e c e n t r a l d a r k l i n e is a b o u t 3.5-4 n m thick. W h e r e two p a i r e d t h y l a k o i d s s e p a r a t e (Fig. 2a, w h i t e arrow) we c a n see t h a t also t h e two e l e c t r o n - t r a n s p a r e n t s t r i p e s
FIG. 1. Plastid of a very young sunflower leaf. Electron-dense material is visible in all the intramembrane compartments which appear variously dilated (a). Both the plastid envelope and the other cellular membranes show a staining different from that of the plastid internal membranes. (b) shows a detail of (a). In the partition regions two electron-transparent stripes enclosing one central dark line are visible. (a), x 48 000; (b), x 90 000. The following abbreviations apply to all figures: LSL, light-staining lamella; LSM, lightly stained membrane; ME, mitochondrion envelope; NE, nuclear envelope; PB, prolamellar body; PE, plastid envelope; P1, plasm~lemma; RLP, ribosome-like particles; S, starch; T, tonoplast. FIG. 2. At a high magnification it is possible to see that when two paired compartments separate (a, white arrow) the two light stripes between them separate with the respective compartment. The central dark line also divides, and each of the resulting lines (arrows) bounds the two light stripes. In some places (b, arrows) it is possible to see a tristratified structure of the thylakoid membrane, when an electron-dense line is visible on the compartment side of the thylakoid. (a), x 200 000; (b), x 200 000. FiG. 3. Granum of a differentiated sunflower chloroplast with heavily stained thylakoid membranes. × 200 000. Fro. 4. Palisade plastid of a 6-cm-long sunflower leaf. LSM are present in thylakoids, which have dense contents in the compartments. Two starch granules occur in the stroma. × 45 000. Fro. 5. Etioplast of sunflower cotyledons. The prolamellar body shows a paracrystalline structure with ribosome-like particles (a, arrows). The tubule contents appear very electron-dense, and the tubular membranes forming the paracrystalline lattice are not discernible. In some places the membranes departing from the prolamellar body show a LSL (b, arrow). (a), × 50 000; (b), × 120 000.
32
CASADORO AND RASCIO
FIGS. 2 and 3
SUNFLOWER THYLAKOID MEMBRANES
Fins. 4 and 5
33
34
CASADORO AND RASCIO
separate and, singly, extend to bound the respective electron-dense compartments (Fig. 2a, arrows). The central dark line also divides, and each of the resulting electrondense lines bounds one of the two electrontransparent stripes. The whole complex of light stripe and dark line delimiting a compartment from the stroma is about 6.0 nm thick. In agreement with what was observed in cultured carrot cells by Israel and Steward (1967) and in Cattleya sp. leaves by Damsz and Mikulska (1976), the sunflower membranes limiting the compartments also appear to be formed by an electron-transparent stripe (about 4.0-4.5 nm) and by a thin electron-dense line (about 1.5-2.0 nm), the latter touching the stroma. Because of the great electron density of the intracompartmental material, the expected electrondense membranal line on the side of the compartment, which would allow us to think of a tristratified membrane, is usually not discernible. In some places, however, we can distinguish an electron-dense line next to the compartment (Fig. 2b), but its thickness cannot be evaluated with certainty. Assuming that an inner dark line has a thickness similar to that of the corresponding outer dark line on the stroma side, we obtain a total thickness of 7.0-8.5 nm. These values agree with those proposed for the normally stained thylakoid membranes. Furthermore, this range of sizes can be found also in the normally stained thylakoid membranes that are present in the same sunflower plastids at a more differentiated stage (Fig. 3). So the thylakoid membranes in the young sunflower leaves do not appear "negatively" stained but constitute tristratified membranes with a rather wide central electron-transparent zone bordered by two narrower electron-dense lines. The central zone could be named "light staining lamella" (LSL) and the membranes containing it "lightly stained membranes" (LSM) rather than "negative" membranes, a name that has often been used till now to indicate
such membranes. Some authors have considered the LSM as incompletely organized membranes rather than artifacts even when they continued to use the term "negative" (Damsz and Mikulska, 1976; Platt-Aloia and Thomson, 1977). The lack of staining observed by us in the LSL, suggests that the incomplete organization especially affects the lipidic matrix of the membrane. In some cases plastids retain the thylakoid membranes with LSL for a long time. In fact, in 6-cm-long sunflower leaves we observed palisade chloroplasts maintaining thylakoid membranes with LSL in well-developed grana {Fig. 4). There was abundant starch in these plastids which led us to assume that the plastids were photosynthetically active. Therefore, even if still incompletely organized, the thylakoid membranes with LSL would allow a steady insertion of photosystems and of photophosphorylation factors. The membrane structure in etioplasts also seems to suggest the possible insertion of the chlorophyllous pigments on the LS membranes. Studies of etiolated cotyledons have revealed the occurrence of etioplasts with well-developed prolamellar bodies and with an evident paracrystalline structure (Fig. 5a). However, while the electrondense material filling the tubules was clearly distinguishable, the tubular membranes forming the paracrystalline lattice were not discernible. The membranal outlines departing from the prolamellar body appear to be filled with electron-dense material. The membranes are not easily distinguishable, but in some places we could see the LSL (Fig. 5b) which suggests the presence of LS membranes also in etioplasts. It is generally accepted that one of the basic constituents of the prolamellar body membranes is the protochlorophyllide (Lafl~che et al., 1972) and that there is a close correlation between the presence of protochlorophyllide and the formation of welldeveloped prolamellar bodies (Boynton and Henningsen, 1967; Henningsen and Boynton, 1967; Shumway and Weier, 1967; Mil-
SUNFLOWER THYLAKOID M E M B R A N E S TABLE I SPECIES WITH LIGHTLY STAINED THYLAKOID MEMBRANES Plant species Family P o p u l u s tremuloides" Salicaceae P h y l l a n t h u s nivosus a Euphorbiaceae C i n n a m o m u m sp? Lauraceae Apple ~ Rosaceae D a u c u s carota ~ Umbelliferae A t r o p a belladonna b Solanaceae D a t u r a arborea b Nicotiana tabacum b Sesamum indicum a R u e l l i a strepens b Lippia citriodora ~ Coleus blumei" M e n t h a acquatica b Fraxinus americana b H e l i a n t h u s a n n u u s ~' L a c t u c a sativa" Cattleya sp2 Gongora sp2 V a n d a sp."
Pedaliaceae Acanthaceae Verbenaceae Labiatae Oleaceae Compositae Orchidaceae
" Reported in the literature. h Found by us.
lerd et al., 1969). Therefore the paracrystalline structure of the prolammelar bodies in sunflower etioplasts suggests that also in this case the LS membranes allow the insertion of chlorophyllous pigments. As things are we can assume that in the LS membranes their two external electrondense lines are mainly proteinaceous in nature and that they comprise the proteins binding the chlorophyllous pigments. In sunflower the stage with LS membranes is apparently an early one in the ontogenetic process leading to the formation of well-differentiated chloroplasts with normally stained thylakoids. In the proplastids the few internal membranes are often seen in continuity with the inner membrane of the plastid envelope and are always normally stained as is the plastid envelope (Casadoro and Rascio, unpublished data). In contrast, near these few membranes which are still in continuity with the plastid envelope in the young chloroplasts, real internal membranes begin to appear as LSM. From their appearance until their change into the normally stained membranes of quite differentiated chloroplasts, the LSM have not been seen in continuity with the
35
plastid envelope (Casadoro and Rascio, unpublished data). This leads us to assume that in the sunflower chloroplasts the internal membrane system, at least almost entirely, if not wholly, is not formed by invaginations of the inner membrane of the plastid envelope but rather directly as thylakoid membranes inside the chloroplast. A possible confirmation of this hypothesis may be inferred from the cotyledonous etioplast whose internal membrane system also appears to be stained in a different manner in comparison with the plastid envelope and has never been seen in continuity with the plastid envelope itself. As previously mentioned, the presence of anomalously stained membranes in the plastids of some plants is reported in the literature. A research carried out with leaves of different species has allowed us to increase the number of reports regarding LSM (Table I). The presence of plastids with LS membranes in species belonging to different families leads us to assume that this phenomenon does not represent a peculiarity but that, on the contrary, it is widely distributed in nature. REFERENCES BLACKWELL, S. J., LAETSCH, W. M., AND HYDE, B. B. (1969) A m e r . J. Bot. 56, 457. BOYNTON, J. E., AND HENNINGSEN, K. W. (1967) Stud. Biophys. 5, 85. CATESSON, A. M. (1970) J. Microsc. 9, 949. CRAN, D. G., AND POSSINGHAM, J. V. (1974) A n n . Bot. 38, 843. DAMSZ, B., AND MIKULSKA, E. (1976) Bioehem. Physiol. P f l a n z e n 169, 257. HENNINGSEN, K. W., AND BOYNTON, J. E. (1967) Stud. Biophys. 5, 89. ISRAEL, H. W., AND STEWARD, F. C. (1967) A n n . Bot. 31, 1. LAFLECHE, D., BOVE, J. M., AND DURANTON, J. (1972). J. Ultrastruct. Res. 40, 205. MARTY, D. (1973) C. R. H. A c a d . Sci. 277, 45. MILLERD, A., GOODCHILD, D. J., AND SPENCER, D. {1969) P l a n t Physiol. 44, 567. PLATT-ALoIA, K. A., AND THOMSON, W. W. (1977) N e w Phytol. 78, 599. SALEMA, R., AND ABREU, I. (1972) Broteria 41, 1. SALEMA, R., MESQUITA, J. F., AND ABREU, I. (1972) J. Submicrosc. Cytol. 4, 161. SHUMWAY, L. K., AND WEIER, T. E. (1967) A m e r . J. Bot. 54, 773. SRIVASTAVA, L. M., AND PAULSON, R. E. (1968) Canad. J. Bot. 46, 1447.
Thylakoid Membranes in Sunflower and in Other Plants GIORGIO CASADORO AND NICOLETTA RASCIO Institute of Botany and Plant Physiology, University of Padua, Italy Received February 22, 1978, and in revised form, April 25, 1978
In the early ontogenesis of the sunflower plastids, the thylakoids show a particular staining. They are characterized by a wide, electron-transparent central region and by a thin electron-dense line on the stroma's side to which, possibly, an analogous dark line corresponds on the intrathylakoidal side of the compartment. The latter line is difficult to see because of the electron density of the intrathylakoid compartment. The peculiar membrane appearance persists for a long time, and sometimes it is observed in plastids with well-arranged grana and abundant starch. Therefore we can assume that the photosystems can be inserted into such membranes. The presence of chlorophyllous pigments on "lightly stained membranes" also seems to be suggested by the cotyledonous etioplasts that show prolameUar bodies with a paracrystalline lattice. As the LS membranes are present in plants belonging to very different families, we may assume that they are not a peculiarity, but on the contrary, are widely distributed in nature. F o r a long t i m e t h e p r e s e n c e of t h y l a k o i d m e m b r a n e s w i t h a p a r t i c u l a r staining in plastids o f m e r i s t e m a t i c or v e r y y o u n g tissues h a s b e e n k n o w n {Israel a n d S t e w a r d , 1967; S r i v a s t a v a a n d P a u l s o n , 1968; Blackwell et al., 1969; Catesson, 1970; S a l e m a a n d A b r e u , 1972; S a l e m a et al., 1972; M a r t y , 1973; C r a n a n d P o s s i n g h a m , 1974; D a m s z a n d Mikulska, 1976; P l a t t - A l o i a a n d T h o m son, 1977). S u c h m e m b r a n e s , u p to n o w n a m e d "negative," h a v e r e c e n t l y b e e n c o n s i d e r e d as i n c o m p l e t e l y organized ones r a t h e r t h a n artifacts ( D a m s z a n d Mikulska, 1976; P l a t t Aloia a n d T h o m s o n , 1977). I n fact, w h e n t h e y are present, b o t h t h e plastid e n v e l o p e a n d t h e o t h e r cellular m e m b r a n e s a p p e a r n o r m a l l y stained; s u b s e q u e n t l y , t h e y also a s s u m e a n o r m a l staining c o n t e m p o r a n e o u s l y w i t h t h e d i s a p p e a r a n c e o f t h e elect r o n - d e n s e m a t e r i a l u s u a l l y c o n t a i n e d in t h e i n t r a m e m b r a n a l c o m p a r t m e n t s (PlattAloia a n d T h o m s o n , 1977). As s u c h m e m b r a n e s r e p r e s e n t a n o r m a l o n t o g e n e t i c stage for t h e plastids containing t h e m , a n d as t h e y h a v e b e e n r e p o r t e d in p l a n t s belonging to different families, we d e e m e d it of s o m e interest to investigate their fine s t r u c t u r e a n d to see if we could find t h e m in additional plants.
MATERIALS AND METHODS Open-air-grown plants of Helianthus annuus L. and of other species from the Botanic Gardens of the Padua University were used. Tissue smnples were obtained, during late spring, from successively older leaves. Seeds of Helianthus annuus L., soaked in water for 24 hr, were placed in sealed petri dishes in the dark for 7 days. Tissue from cotyledons was sampled and fixed in the dark. All the tissue segments were fixed for 2 hr in 6% glutaraldehyde in 0.1 M cacodylate buffer (pH 6.9), washed in buffer, then postfixed 2 hr in 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 a LKB Ultrotome III, were poststained with lead citrate and examined with a Hitachi HS 9 electron microscope operating at 75 kV. RESULTS AND DISCUSSION I n t h e y o u n g sunflower leaves t h e plastids s h o w a v e r y p a r t i c u l a r fine s t r u c t u r e . I n a r a t h e r d a r k s t r o m a t h e r e are large amounts of more strongly electron-dense m a t e r i a l enclosed in v a r i o u s l y dilated c o m p a r t m e n t s limited b y m e m b r a n e s . T h e s e m e m b r a n e s (Fig. la) look like electront r a n s p a r e n t lines defining t h e single c o m partments. However, where higher magnification reveals pairing o f two electrond e n s e c o m p a r t m e n t s , we c a n see t w o light stripes s e p a r a t e d b y a c e n t r a l d a r k line (Fig. 3o
0022-5320/78/0651-0030502.00/0 Copyright© 1978by AcademicPress, Inc. All rights of reproductionin any form reserved.
SUNFLOWER THYLAKOID MEMBRANES l b ) . T h e r e f o r e t h i s w h o l e r e g i o n looks like a "negatively" stained trilaminar unit memb r a n e . T h i s f e a t u r e of t h e p l a s t i d ' s i n t e r n a l m e m b r a n e s , o b s e r v e d also b y o t h e r a u t h o r s (see r e f e r e n c e s a t b e g i n n i n g of i n t r o d u c tion}, does n o t affect e i t h e r t h e p l a s t i d env e l o p e m e m b r a n e s or t h e o t h e r c e l l u l a r membranes which appear normally stained
31
(Fig. l a ) . T h e w h o l e t r i s t r a t i f i e d r e g i o n is a b o u t 11.5-13 n m thick, w h i l e e a c h of t h e elect r o n - t r a n s p a r e n t s t r i p e s is a b o u t 4-4.5 n m a n d t h e c e n t r a l d a r k l i n e is a b o u t 3.5-4 n m thick. W h e r e two p a i r e d t h y l a k o i d s s e p a r a t e (Fig. 2a, w h i t e arrow) we c a n see t h a t also t h e two e l e c t r o n - t r a n s p a r e n t s t r i p e s
FIG. 1. Plastid of a very young sunflower leaf. Electron-dense material is visible in all the intramembrane compartments which appear variously dilated (a). Both the plastid envelope and the other cellular membranes show a staining different from that of the plastid internal membranes. (b) shows a detail of (a). In the partition regions two electron-transparent stripes enclosing one central dark line are visible. (a), x 48 000; (b), x 90 000. The following abbreviations apply to all figures: LSL, light-staining lamella; LSM, lightly stained membrane; ME, mitochondrion envelope; NE, nuclear envelope; PB, prolamellar body; PE, plastid envelope; P1, plasm~lemma; RLP, ribosome-like particles; S, starch; T, tonoplast. FIG. 2. At a high magnification it is possible to see that when two paired compartments separate (a, white arrow) the two light stripes between them separate with the respective compartment. The central dark line also divides, and each of the resulting lines (arrows) bounds the two light stripes. In some places (b, arrows) it is possible to see a tristratified structure of the thylakoid membrane, when an electron-dense line is visible on the compartment side of the thylakoid. (a), x 200 000; (b), x 200 000. FiG. 3. Granum of a differentiated sunflower chloroplast with heavily stained thylakoid membranes. × 200 000. Fro. 4. Palisade plastid of a 6-cm-long sunflower leaf. LSM are present in thylakoids, which have dense contents in the compartments. Two starch granules occur in the stroma. × 45 000. Fro. 5. Etioplast of sunflower cotyledons. The prolamellar body shows a paracrystalline structure with ribosome-like particles (a, arrows). The tubule contents appear very electron-dense, and the tubular membranes forming the paracrystalline lattice are not discernible. In some places the membranes departing from the prolamellar body show a LSL (b, arrow). (a), × 50 000; (b), × 120 000.
32
CASADORO AND RASCIO
FIGS. 2 and 3
SUNFLOWER THYLAKOID MEMBRANES
Fins. 4 and 5
33
34
CASADORO AND RASCIO
separate and, singly, extend to bound the respective electron-dense compartments (Fig. 2a, arrows). The central dark line also divides, and each of the resulting electrondense lines bounds one of the two electrontransparent stripes. The whole complex of light stripe and dark line delimiting a compartment from the stroma is about 6.0 nm thick. In agreement with what was observed in cultured carrot cells by Israel and Steward (1967) and in Cattleya sp. leaves by Damsz and Mikulska (1976), the sunflower membranes limiting the compartments also appear to be formed by an electron-transparent stripe (about 4.0-4.5 nm) and by a thin electron-dense line (about 1.5-2.0 nm), the latter touching the stroma. Because of the great electron density of the intracompartmental material, the expected electrondense membranal line on the side of the compartment, which would allow us to think of a tristratified membrane, is usually not discernible. In some places, however, we can distinguish an electron-dense line next to the compartment (Fig. 2b), but its thickness cannot be evaluated with certainty. Assuming that an inner dark line has a thickness similar to that of the corresponding outer dark line on the stroma side, we obtain a total thickness of 7.0-8.5 nm. These values agree with those proposed for the normally stained thylakoid membranes. Furthermore, this range of sizes can be found also in the normally stained thylakoid membranes that are present in the same sunflower plastids at a more differentiated stage (Fig. 3). So the thylakoid membranes in the young sunflower leaves do not appear "negatively" stained but constitute tristratified membranes with a rather wide central electron-transparent zone bordered by two narrower electron-dense lines. The central zone could be named "light staining lamella" (LSL) and the membranes containing it "lightly stained membranes" (LSM) rather than "negative" membranes, a name that has often been used till now to indicate
such membranes. Some authors have considered the LSM as incompletely organized membranes rather than artifacts even when they continued to use the term "negative" (Damsz and Mikulska, 1976; Platt-Aloia and Thomson, 1977). The lack of staining observed by us in the LSL, suggests that the incomplete organization especially affects the lipidic matrix of the membrane. In some cases plastids retain the thylakoid membranes with LSL for a long time. In fact, in 6-cm-long sunflower leaves we observed palisade chloroplasts maintaining thylakoid membranes with LSL in well-developed grana {Fig. 4). There was abundant starch in these plastids which led us to assume that the plastids were photosynthetically active. Therefore, even if still incompletely organized, the thylakoid membranes with LSL would allow a steady insertion of photosystems and of photophosphorylation factors. The membrane structure in etioplasts also seems to suggest the possible insertion of the chlorophyllous pigments on the LS membranes. Studies of etiolated cotyledons have revealed the occurrence of etioplasts with well-developed prolamellar bodies and with an evident paracrystalline structure (Fig. 5a). However, while the electrondense material filling the tubules was clearly distinguishable, the tubular membranes forming the paracrystalline lattice were not discernible. The membranal outlines departing from the prolamellar body appear to be filled with electron-dense material. The membranes are not easily distinguishable, but in some places we could see the LSL (Fig. 5b) which suggests the presence of LS membranes also in etioplasts. It is generally accepted that one of the basic constituents of the prolamellar body membranes is the protochlorophyllide (Lafl~che et al., 1972) and that there is a close correlation between the presence of protochlorophyllide and the formation of welldeveloped prolamellar bodies (Boynton and Henningsen, 1967; Henningsen and Boynton, 1967; Shumway and Weier, 1967; Mil-
SUNFLOWER THYLAKOID M E M B R A N E S TABLE I SPECIES WITH LIGHTLY STAINED THYLAKOID MEMBRANES Plant species Family P o p u l u s tremuloides" Salicaceae P h y l l a n t h u s nivosus a Euphorbiaceae C i n n a m o m u m sp? Lauraceae Apple ~ Rosaceae D a u c u s carota ~ Umbelliferae A t r o p a belladonna b Solanaceae D a t u r a arborea b Nicotiana tabacum b Sesamum indicum a R u e l l i a strepens b Lippia citriodora ~ Coleus blumei" M e n t h a acquatica b Fraxinus americana b H e l i a n t h u s a n n u u s ~' L a c t u c a sativa" Cattleya sp2 Gongora sp2 V a n d a sp."
Pedaliaceae Acanthaceae Verbenaceae Labiatae Oleaceae Compositae Orchidaceae
" Reported in the literature. h Found by us.
lerd et al., 1969). Therefore the paracrystalline structure of the prolammelar bodies in sunflower etioplasts suggests that also in this case the LS membranes allow the insertion of chlorophyllous pigments. As things are we can assume that in the LS membranes their two external electrondense lines are mainly proteinaceous in nature and that they comprise the proteins binding the chlorophyllous pigments. In sunflower the stage with LS membranes is apparently an early one in the ontogenetic process leading to the formation of well-differentiated chloroplasts with normally stained thylakoids. In the proplastids the few internal membranes are often seen in continuity with the inner membrane of the plastid envelope and are always normally stained as is the plastid envelope (Casadoro and Rascio, unpublished data). In contrast, near these few membranes which are still in continuity with the plastid envelope in the young chloroplasts, real internal membranes begin to appear as LSM. From their appearance until their change into the normally stained membranes of quite differentiated chloroplasts, the LSM have not been seen in continuity with the
35
plastid envelope (Casadoro and Rascio, unpublished data). This leads us to assume that in the sunflower chloroplasts the internal membrane system, at least almost entirely, if not wholly, is not formed by invaginations of the inner membrane of the plastid envelope but rather directly as thylakoid membranes inside the chloroplast. A possible confirmation of this hypothesis may be inferred from the cotyledonous etioplast whose internal membrane system also appears to be stained in a different manner in comparison with the plastid envelope and has never been seen in continuity with the plastid envelope itself. As previously mentioned, the presence of anomalously stained membranes in the plastids of some plants is reported in the literature. A research carried out with leaves of different species has allowed us to increase the number of reports regarding LSM (Table I). The presence of plastids with LS membranes in species belonging to different families leads us to assume that this phenomenon does not represent a peculiarity but that, on the contrary, it is widely distributed in nature. REFERENCES BLACKWELL, S. J., LAETSCH, W. M., AND HYDE, B. B. (1969) A m e r . J. Bot. 56, 457. BOYNTON, J. E., AND HENNINGSEN, K. W. (1967) Stud. Biophys. 5, 85. CATESSON, A. M. (1970) J. Microsc. 9, 949. CRAN, D. G., AND POSSINGHAM, J. V. (1974) A n n . Bot. 38, 843. DAMSZ, B., AND MIKULSKA, E. (1976) Bioehem. Physiol. P f l a n z e n 169, 257. HENNINGSEN, K. W., AND BOYNTON, J. E. (1967) Stud. Biophys. 5, 89. ISRAEL, H. W., AND STEWARD, F. C. (1967) A n n . Bot. 31, 1. LAFLECHE, D., BOVE, J. M., AND DURANTON, J. (1972). J. Ultrastruct. Res. 40, 205. MARTY, D. (1973) C. R. H. A c a d . Sci. 277, 45. MILLERD, A., GOODCHILD, D. J., AND SPENCER, D. {1969) P l a n t Physiol. 44, 567. PLATT-ALoIA, K. A., AND THOMSON, W. W. (1977) N e w Phytol. 78, 599. SALEMA, R., AND ABREU, I. (1972) Broteria 41, 1. SALEMA, R., MESQUITA, J. F., AND ABREU, I. (1972) J. Submicrosc. Cytol. 4, 161. SHUMWAY, L. K., AND WEIER, T. E. (1967) A m e r . J. Bot. 54, 773. SRIVASTAVA, L. M., AND PAULSON, R. E. (1968) Canad. J. Bot. 46, 1447.
