JOURNAL OF ULTRASTRUCTURE RESEARCH 60, 3 2 8 - 3 3 4
(1977)
Unusual Lipid Bodies in the Red Alga Palmaria palmata (= Rhodymenia palmata) CURT M.
PUESCHEL
Section of Genetics, Development and Physiology, Cornell University, Ithaca, New York 14853 Received February 18, 1977, and in revised form, April 19, 1977 Large inclusions in the outer cortex cells of the marine red alga Palmaria palmata (= Rhodymenia palmata) are concluded to be lipid bodies based on their reaction to osmium and Sudan B. Study by thin-section electron microscopy reveals t h a t many such bodies are surrounded by an apparent reticulum of bifurcating membranes. Freeze etching indicates that the chambers of the reticulate region are filled with material similar in fracturing texture to the lipid core. Apparently most of this reticulum substance is extracted during thin-section preparation, leaving only the bounding membranes intact. The bounding membranes are likely half-membranes, that is, phospholipid monolayers, similar to postulated membranes of spherosomes. When half-membranes of adjacent lipid vesicles are appressed, a unit membrane appearance is produced. Tight packing of many such units results in the appearance of bifurcating unit membranes. Particles, presumably proteins, are abundant on at least one face of the vesicle membrane.
Although most lipid bodies in both plants and animals apparently lack a detectable bounding membrane, several authors have reported membrane-bounded, lipid-containing structures in plant cells (e.g., Jacks et al., 1967; Mollenhauer and Totten, 1971; and Yatsu et al., 1971). Most of these reports deal with structures identified as spherosomes. Spherosomes, as the term was applied by light microscopists, are spherical structures which have a diameter of about 1 # m , are highly refractile, and can be stained by fat dyes. Some authors (Frey-Wyssling et al., 1963; Schwarzenbach, 1971) have proposed that reserve oil bodies are derived from spherosomes, but other workers (Smith, 1974; Sorokin, 1967) report that these two structures have separate, unrelated origins. Hydrolytic enzymes have been demonstrated to be present in some spherosomes (Balz, 1966; Matile and Spichiger, 1968), which implies the existence of a bounding membrane. Membranes recovered from isolated spherosomes are most unusual in that they apparently consist of monolayers of phospholipid molecules (Yatsu and Jacks,
1972). Presumably, the hydrophobic, nonpolar end of each phospholipid molecule is adjacent to the surface of the lipid droplet and the polar end of the molecule is in contact with the cytoplasm. These peculiar spherosome half-membranes have been reported to have a structural protein component similar to that of normal unit membranes (Yatsu and Jacks, 1972). This paper describes unusual lipid bodies, composed in part of an extensive array of membranes, in the marine red alga Palmaria palmata (L.) O. Kuntze = Rhodymenia palmata (L.) Greville (Guiry, 1975). On the basis of thin-section and freeze-etch electron microscopic study, it is proposed that the membranous portion of these structures is composed of half-membrane units, similar to individual spherosome membranes. Particles, presumably proteins, are revealed on one half-membrane face by freeze etching. The value of freezeetch technique is obvious, since it does not require treatments which might extract lipids. MATERIALS AND METHODS Specimens of Palmaria palmata were fixed in the field with glutaraldehyde-acrolein (Hess, 1966) in a
328 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
LIPID BODIES IN A RED ALGA 0.1 M cacodylate buffer at pH 7.0. NaC1 and CaC12 were added to the fixative solution according to the procedure of McDonald (1972). Material was then washed in buffer-salt solutions, postfixed in 1% OsO~, soaked overnight in 2% aqueous uranyl acetate, dehydrated in acetone, and embedded in Spurr medium (Spurr, 1969). Sections were stained with uranyl acetate and lead citrate. Histochemistry was performed on 0.5- to 1-t~m sections of material prepared as above. Sudan black B (Bronner, 1975) produced very intense staining of the inclusions under investigation. Specimens to be freeze etched were returned to the laboratory where they were maintained in culture at 15°Cin a 12-hr light-12-hr dark cycle at 3,4 x 104 erg/cm~ for 2 to 3 weeks. Pieces of tissue, 2 mm2, from proliferations and young thalli were pretreated for 2 hr with 2% glutaraldehyde in seawater, and subsequently for 3 hr with 25% aqueous glycerol. They were then freeze etched with a Balzers 360 freeze-etch apparatus following the method of Moor and Mfihlethaler (1963). Replicas and sections were examined and photographed with a Philips EM 300. The direction of shadowing is indicated by a circled arrow in each freeze-etch micrograph. OBSERVATIONS AND RESULTS Large, usually a m o r p h o u s ergastic inclusions were observed in Palmaria palmata. T h e a b u n d a n c e of these s t r u c t u r e s and their distribution within the t h a l l u s varied in different collections. Those illust r a t e d here were found only in cortex cells and were m u c h more c o m m o n in the stipe t h a n in the blade. Similar bodies in another collection were present t h r o u g h o u t the thallus, in both the m e d u l l a and cortex of the stipe and of the blade. In u n s t a i n e d sections examined with the light microscope, these inclusions appear to have a distinctive green color. Polysaccharide and protein stains failed to alter this color, b u t t r e a t m e n t with S u d a n black B (Bronner, 1975) stained these inclusions intensely (Fig. 1), indicating t h a t t h e y contain substantial a m o u n t s of lipid. The size and shape of these lipid bodies are variable: up to 5 tLm in the largest dimension and circular to very i r r e g u l a r and a n g u l a r in outline. In some cases an intensely stained lipid body appears to be s u r r o u n d e d by a w e a k l y sudanophili c region (Fig. 2). Similarities in shape, size, and distribu-
329
tion w i t h i n the thallus suggest t h a t the bodies which stain as lipids in thick sections are the same as large inclusions viewed in thin section and in freeze etch. Thin-sectioned lipid bodies are e x t r e m e l y electron-dense as a result of intense staining by o s m i u m tetroxide. Most lipid bodies are h o m o g e n e o u s l y dense and have few or no m e m b r a n o u s associations (Fig. 3). Some have lone unit m e m b r a n e s or several concentric ones a t t a c h e d to the surface of the body. Still other lipid bodies are composed of a homogeneous, dense core s u r r o u n d e d by a s h e a t h of a p p a r e n t l y a n a s t o m o s i n g and bifurcating unit membranes (Figs. 4 and 5). This r e t i c u l u m appears as a h o n e y c o m b in which the chambers are delineated by a n g u l a r , m e m b r a nous partitions, each 8- to 10-nm thick. Small particles can be seen within the chambers, often several per chamber, per section (Fig. 5). The entire lipid body, tog e t h e r with its reticulate sheath, appears to be s u r r o u n d e d by a unit m e m b r a n e . This m e m b r a n o u s s h e a t h is probably responsible for the w e a k l y Sudan-positive halo seen a r o u n d some lipid bodies with the light microscope (Fig. 2). Some lipid bodies observed in freezeetched specimens show differentiation into an a m o r p h o u s core and a honeycomb-like s h e a t h (Fig. 6). The face of the cross-fract u r e d core is m a r k e d by i r r e g u l a r bumps, ridges, and striations. The s t r i a t i o n s m a y reflect the presence of m e m b r a n e s within the core. F r a c t u r e s t h r o u g h the reticulate s h e a t h m a y either cross-fracture the c h a m b e r s or follow the m e m b r a n e s . In the former case the contents of the c h a m b e r s are exposed. The fine-grained fracture t e x t u r e of the substance in the r e t i c u l u m c h a m b e r s is indistinguishable from t h a t of the lipid core and quite unlike t h a t of the coarsely g r a n u l a t e cytoplasm (Fig. 6). F r o m this information it is concluded t h a t the s h e a t h contains lipids which are extracted by chemical fixation, leaving only the particles seen in Fig. 5. Thus it appears t h a t the
J~
!~,ii!i :
@
@
FIG. 1. Irregularly shaped lipid bodies in the stipe cortex of Palmaria palmata, stained with Sudan black B. x 3000. Fia. 2. Lipid body with an intensely sudanophilic core surrounded by a less densely stained region. × 6000. FIG. 3. Fine structure of amorphous lipid bodies similar to those in Fig. 1. C, Chloroplast. x 30 000. FIG. 4. Some lipid bodies are composed of an amorphous core surrounded by a honeycomb-like membranous layer which corresponds to the lightly stained halo shown in Fig. 2. x 54 000. FIG. 5. The honeycomb-like reticulum appears to be composed of bifurcating unit membranes (arrows) but, more likely, is a collection of half-membrane-bounded vesicles whose lipid contents have been extracted. Small particles, presumably remnants of the extracted lipid, are present in some of these vesicles. × 130 000. FIG. 6. Freeze-etched lipid body. The fracture texture of the reticulum contents is similar to t h a t of the lipid core, suggesting that the reticulate portion is actually a collection of lipid vesicles. The small arrow marks a possible membrane within the amorphous core. Direction of shadowing is indicated l~y a circled arrow. S, Starch grain, x 65 000. Fro. 7. The faceted nature of the reticulum subunits is the result of close packing of half-membranebounded lipid vesicles. After freeze etching, some vesicle facets bear an abundance of particles; others lack them entirely, x 110 000. 33O
332
CURT M. PUESCHEL
sheath reticulum is composed of the bounding membranes of individually packaged lipid droplets. These lipid vesicle membranes could be half-membranes, the adnation of which gives the appearance of unit membranes. Where contact between two half-membranes is lost and a third is interposed, the appearance of a unit membrane bifurcation is produced. The faceted outline of the individual lipid vesicles observed in thin-sectioned material (Fig. 5) is present in freeze-etched specimens as well (Fig. 7). Tight packing of the lipid vesicles is probably responsible for this angularity. The size of the lipid vesicles appears to be variable when viewed in the partly three-dimensional freeze-etched preparations (Fig. 7). When the fracture plane follows the course of the vesicle half-membranes in the sheath, 10-nm particles are revealed on some of the exposed surfaces (Fig. 7). Usually only a single facet of a vesicle bears abundant particles; the other facets lack them entirely. DISCUSSION In chemically fixed and dehydrated specimens, preservation of lipids is unpredictable, and interpretations based exclusively on such material must be considered questionable. In freeze-etched specimens the possibility of extraction of lipids seems low, despite glutaraldehyde and glycerol pretreatments. The discovery of finegrained substance within the seemingly ~empty" reticulate portion of the lipid bodies after freeze-etch preparation attests t o this. In combination, the two electron microscopic techniques give a clearer notion of the structure of the lipid bodies in Palmaria. The homogeneous core region may consist of some, but relatively few, membranes embedded in lipid material. Surrounding the core, amorphous lipids are packaged into small units by vesicle halfmembranes. The two portions of the body, the core and the sheath, may represent two chemically distinct types of lipids, or a
single type in two phases; that within the sheath may be more easily mobilized; it certainly appears to be more easily extracted. In thin section the sheath appears to consist of bifurcating unit membranes, but the fact that the chambers are normally filled with lipids allows a second interpretation. The spherosome membrane in higher plants is thought to consist of a single layer of phospholipid molecules, essentially a half-membrane (Yatsu and Jacks, 1972). The hydrophobic fatty acid end of the phospholipid molecule is likely to be adjacent to the hydrophobic spherosome substance, while the phosphate or polar end of each molecule would be exposed to the hydrophilic hyaloplasm. Such might also be the case in Palmaria, but many lipid droplets, each bounded by a half-membrane, would be involved in producing a single sheath. One objection that might be raised to the above interpretation is that the adnation of the polar faces of two half-membranes would not produce the appearance of a single unit membrane. It has been demonstrated that the dark-light-dark unit membrane appearance seen in thin section is the result of binding of both uranyl acetate and osmium at the polar surfaces, probably to the phosphate moiety, of the unit membrane (Stoekenius, 1960). The unstained fatty acid chains provide the relatively electron-transparent region between the stained polar portions. If the polar surfaces of two spherosome halfmembranes come into contact, a single dense line would be expected. Such a configuration has been reported for isolated spherosome ghosts of peanuts (Yatsu and Jacks, 1972). In Palmaria, the electron-transparent space between adjacent half-membranes may be the result of a sphere of hydration around each lipid vesicle, which maintains the integrity of the individual units. It is equally possible that this space is an artifact of chemical fixation.
LIPID BODIES IN A RED ALGA
Particles, presumed to be proteins, are a nearly universal constituent of unit membranes observed after freeze etching (Pinto da Silva and Miller, 1975). Fractures through the sheath of the lipid bodies in P a l m a r i a sometimes expose such particles, though half-membranes constitute the sheath. Yatsu and Jacks (1972) have isolated what they believe to be a structural protein component from spherosome half-membranes. The particles associated with the lipid vesicles in P a l m a r i a are either present in abundance or are absent entirely, and most often the latter is the case (Fig. 7). This dichotomy probably reflects whether the fracture separated the adjacent faces of the two half-membranes or whether it split the nonpolar face of the half-membrahe from the surface of the lipid droplet. It might be anticipated that fractures along the polar face of the half-membrane would not occur or would occur at a very low frequency, because fracture planes do not normally cleave the polar unit membrahe surface from the cytoplasm (Branton, 1966). Instead the cleavage plane might be expected to pass preferentially along the hydrophobic face, i.e., the interior of a typical unit membrane or, in the case of half-membrane-bounded lipids, between the surface of the lipid droplet and the nonpolar face of the half-membrane. It is reasonable to assume that the protein is associated with the polar end of the phospholipid molecule, and because of this, the protein particles are not commonly exposed. The reticulum facets lacking particles might be either the hydrophobic surface of the half-membrane or the surface of the lipid droplet. Electron-dense inclusions, possibly of lipoid nature, are commonly reported in ultrastructural studies of red algae. One type of massive, electron-dense body with some similarities to the lipid bodies of Palm a r i a has been studied by several authors (Bodard, 1968; Feldman and Feldman, 1950; Godin, 1970). This is the so-called
333
"corps en cerise" found in cortical cells of several species o f L a u r e n c i a . The "corps en cerise" is surrounded by a 5-nm-thick membrane (Bodard, 1968), which is suggestive of a spherosome half-membrane. After osmium fixation a honeycomb appearance was discernible throughout most of the body (Bodard, 1968). Fixation with potassium permanganate resulted in very poor preservation of these inclusions; most of the amorphous substance was extracted, leaving only the bounding membrane and a few internal membranes (Bodard, 1968). The composition of the "corps en cerise" has been reported to be part lipid and part aldehyde (Feldman and Feldman, 1950). Lipid bodies which are composed of an amorphous core surrounded by a reticulure have been reported in a few higher plants. Esau (1968) found such structural differentiation in plastoglobuli of B e t a vulg a r i s which was infected with beet yellows virus. Plastoglobuli of similar structure have also been reported in chlorotic peas (Silaeva and Shiryaev, 1966). It is unclear whether the membranous associations of the lipid bodies in P a l m a r i a are a manifestation of synthesis, accretion, or mobilization of the lipids. Similarly, it cannot be determined from this work whether the particles associated with these membranes are enzymatic and are involved in lipid metabolism, or whether they are structural. This study was supported by the Lester Sharp Fund and the Section of Genetics, Developmentand Physiology, Cornell University. The author wishes to express his appreciation to Drs. C. H. Uhl, M. V. Parthasarathy, and D. J. Paolillo for their helpful discussions and critical reviews ofthe manuscript. REFERENCES BALZ,H. P. (1966). Planta (Berl.) 70, 207-236. BODARD,M. (1968), C. R. Acad. Sci., Ser. D 266, 2393-2396. BRANTON,D. (1966). Proc. Nat. Acad. Sci. USA 55, 1048-1056. BRONNER,R. (1975). Stain Technol. 50, 1-4. ESAV,K. (1968). Viruses in Plant Hosts. University of Wisconsin Press, Madison.
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FELDMAN,J., ANDFELDMAN,G. (1950). C. R. Acad.
PINTO DA SILVA, P., AND MILLER, R. G. (1975). Proc. Sci. 231, 1335-1337. Nat. Acad. Sci. USA 72, 4046-4050. FREY-WYssLING, A., GRIESHABER, E., AND M(~HLE- SCHWARZENBACH,A. M. (1971). Cytobiologie 4, 145THALER, K. (1963). J. Ultrastruct. Res. 8, 506-516. 147. GODIN, H. (1970). C. R. Acad. Sci. Ser. D 271, 2290- SILAEVA, A. M., AND SHIRYAEV, A. I. (1966). Dokl. 2292. Akad. Nauk S S S R 170, 433-434 [English translaGUIRY, M. D. (1975). Syesis 8, 245-261. tion 595-597]. HEss, W. M. (1966). Stain Technol. 41, 27-35. SMITH, C. G. (1974). Planta (Berl.) 119, 125-142. JACKS, T. J., YATSU, L. Y., AND ALTSHUL, A. M. SOROKIN, H. P. (1967). Amer. J. Bot. 54, 1008-1016. (1967). Plant Physiol. 42, 585-597. SPURR, A. (1969). J. Ultrastruct. Res. 26, 31-43. MATILE, P., AND SPICHIGER, J. (1968). Z. PflanzenSTOEKENIUS, W. (1960) in Proceedings, European physiol. 58, 277-280. Reg. Conference Electron Microscopy, Delft, Vol. McDoNALD, K. (1972). J. Phycol. 8, 156-166. 2, pp. 716-720. MOLLENHAUER, H. H., AND TOTTEN, C. (1971). J. YATSC, L. Y., ANDJACKS, T. J. (1972). Plant Physiol. Cell Biol. 48, 533-541. 49, 937-943. MOOR, H., AND MOHLETHALER, K. (1963). J. Cell YATSU, L. Y., JACKS, T. J., AND HENSARLING,T. P. Biol. 17, 609-628. (1971). Plant Physiol. 48, 675-682.
(1977)
Unusual Lipid Bodies in the Red Alga Palmaria palmata (= Rhodymenia palmata) CURT M.
PUESCHEL
Section of Genetics, Development and Physiology, Cornell University, Ithaca, New York 14853 Received February 18, 1977, and in revised form, April 19, 1977 Large inclusions in the outer cortex cells of the marine red alga Palmaria palmata (= Rhodymenia palmata) are concluded to be lipid bodies based on their reaction to osmium and Sudan B. Study by thin-section electron microscopy reveals t h a t many such bodies are surrounded by an apparent reticulum of bifurcating membranes. Freeze etching indicates that the chambers of the reticulate region are filled with material similar in fracturing texture to the lipid core. Apparently most of this reticulum substance is extracted during thin-section preparation, leaving only the bounding membranes intact. The bounding membranes are likely half-membranes, that is, phospholipid monolayers, similar to postulated membranes of spherosomes. When half-membranes of adjacent lipid vesicles are appressed, a unit membrane appearance is produced. Tight packing of many such units results in the appearance of bifurcating unit membranes. Particles, presumably proteins, are abundant on at least one face of the vesicle membrane.
Although most lipid bodies in both plants and animals apparently lack a detectable bounding membrane, several authors have reported membrane-bounded, lipid-containing structures in plant cells (e.g., Jacks et al., 1967; Mollenhauer and Totten, 1971; and Yatsu et al., 1971). Most of these reports deal with structures identified as spherosomes. Spherosomes, as the term was applied by light microscopists, are spherical structures which have a diameter of about 1 # m , are highly refractile, and can be stained by fat dyes. Some authors (Frey-Wyssling et al., 1963; Schwarzenbach, 1971) have proposed that reserve oil bodies are derived from spherosomes, but other workers (Smith, 1974; Sorokin, 1967) report that these two structures have separate, unrelated origins. Hydrolytic enzymes have been demonstrated to be present in some spherosomes (Balz, 1966; Matile and Spichiger, 1968), which implies the existence of a bounding membrane. Membranes recovered from isolated spherosomes are most unusual in that they apparently consist of monolayers of phospholipid molecules (Yatsu and Jacks,
1972). Presumably, the hydrophobic, nonpolar end of each phospholipid molecule is adjacent to the surface of the lipid droplet and the polar end of the molecule is in contact with the cytoplasm. These peculiar spherosome half-membranes have been reported to have a structural protein component similar to that of normal unit membranes (Yatsu and Jacks, 1972). This paper describes unusual lipid bodies, composed in part of an extensive array of membranes, in the marine red alga Palmaria palmata (L.) O. Kuntze = Rhodymenia palmata (L.) Greville (Guiry, 1975). On the basis of thin-section and freeze-etch electron microscopic study, it is proposed that the membranous portion of these structures is composed of half-membrane units, similar to individual spherosome membranes. Particles, presumably proteins, are revealed on one half-membrane face by freeze etching. The value of freezeetch technique is obvious, since it does not require treatments which might extract lipids. MATERIALS AND METHODS Specimens of Palmaria palmata were fixed in the field with glutaraldehyde-acrolein (Hess, 1966) in a
328 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-5320
LIPID BODIES IN A RED ALGA 0.1 M cacodylate buffer at pH 7.0. NaC1 and CaC12 were added to the fixative solution according to the procedure of McDonald (1972). Material was then washed in buffer-salt solutions, postfixed in 1% OsO~, soaked overnight in 2% aqueous uranyl acetate, dehydrated in acetone, and embedded in Spurr medium (Spurr, 1969). Sections were stained with uranyl acetate and lead citrate. Histochemistry was performed on 0.5- to 1-t~m sections of material prepared as above. Sudan black B (Bronner, 1975) produced very intense staining of the inclusions under investigation. Specimens to be freeze etched were returned to the laboratory where they were maintained in culture at 15°Cin a 12-hr light-12-hr dark cycle at 3,4 x 104 erg/cm~ for 2 to 3 weeks. Pieces of tissue, 2 mm2, from proliferations and young thalli were pretreated for 2 hr with 2% glutaraldehyde in seawater, and subsequently for 3 hr with 25% aqueous glycerol. They were then freeze etched with a Balzers 360 freeze-etch apparatus following the method of Moor and Mfihlethaler (1963). Replicas and sections were examined and photographed with a Philips EM 300. The direction of shadowing is indicated by a circled arrow in each freeze-etch micrograph. OBSERVATIONS AND RESULTS Large, usually a m o r p h o u s ergastic inclusions were observed in Palmaria palmata. T h e a b u n d a n c e of these s t r u c t u r e s and their distribution within the t h a l l u s varied in different collections. Those illust r a t e d here were found only in cortex cells and were m u c h more c o m m o n in the stipe t h a n in the blade. Similar bodies in another collection were present t h r o u g h o u t the thallus, in both the m e d u l l a and cortex of the stipe and of the blade. In u n s t a i n e d sections examined with the light microscope, these inclusions appear to have a distinctive green color. Polysaccharide and protein stains failed to alter this color, b u t t r e a t m e n t with S u d a n black B (Bronner, 1975) stained these inclusions intensely (Fig. 1), indicating t h a t t h e y contain substantial a m o u n t s of lipid. The size and shape of these lipid bodies are variable: up to 5 tLm in the largest dimension and circular to very i r r e g u l a r and a n g u l a r in outline. In some cases an intensely stained lipid body appears to be s u r r o u n d e d by a w e a k l y sudanophili c region (Fig. 2). Similarities in shape, size, and distribu-
329
tion w i t h i n the thallus suggest t h a t the bodies which stain as lipids in thick sections are the same as large inclusions viewed in thin section and in freeze etch. Thin-sectioned lipid bodies are e x t r e m e l y electron-dense as a result of intense staining by o s m i u m tetroxide. Most lipid bodies are h o m o g e n e o u s l y dense and have few or no m e m b r a n o u s associations (Fig. 3). Some have lone unit m e m b r a n e s or several concentric ones a t t a c h e d to the surface of the body. Still other lipid bodies are composed of a homogeneous, dense core s u r r o u n d e d by a s h e a t h of a p p a r e n t l y a n a s t o m o s i n g and bifurcating unit membranes (Figs. 4 and 5). This r e t i c u l u m appears as a h o n e y c o m b in which the chambers are delineated by a n g u l a r , m e m b r a nous partitions, each 8- to 10-nm thick. Small particles can be seen within the chambers, often several per chamber, per section (Fig. 5). The entire lipid body, tog e t h e r with its reticulate sheath, appears to be s u r r o u n d e d by a unit m e m b r a n e . This m e m b r a n o u s s h e a t h is probably responsible for the w e a k l y Sudan-positive halo seen a r o u n d some lipid bodies with the light microscope (Fig. 2). Some lipid bodies observed in freezeetched specimens show differentiation into an a m o r p h o u s core and a honeycomb-like s h e a t h (Fig. 6). The face of the cross-fract u r e d core is m a r k e d by i r r e g u l a r bumps, ridges, and striations. The s t r i a t i o n s m a y reflect the presence of m e m b r a n e s within the core. F r a c t u r e s t h r o u g h the reticulate s h e a t h m a y either cross-fracture the c h a m b e r s or follow the m e m b r a n e s . In the former case the contents of the c h a m b e r s are exposed. The fine-grained fracture t e x t u r e of the substance in the r e t i c u l u m c h a m b e r s is indistinguishable from t h a t of the lipid core and quite unlike t h a t of the coarsely g r a n u l a t e cytoplasm (Fig. 6). F r o m this information it is concluded t h a t the s h e a t h contains lipids which are extracted by chemical fixation, leaving only the particles seen in Fig. 5. Thus it appears t h a t the
J~
!~,ii!i :
@
@
FIG. 1. Irregularly shaped lipid bodies in the stipe cortex of Palmaria palmata, stained with Sudan black B. x 3000. Fia. 2. Lipid body with an intensely sudanophilic core surrounded by a less densely stained region. × 6000. FIG. 3. Fine structure of amorphous lipid bodies similar to those in Fig. 1. C, Chloroplast. x 30 000. FIG. 4. Some lipid bodies are composed of an amorphous core surrounded by a honeycomb-like membranous layer which corresponds to the lightly stained halo shown in Fig. 2. x 54 000. FIG. 5. The honeycomb-like reticulum appears to be composed of bifurcating unit membranes (arrows) but, more likely, is a collection of half-membrane-bounded vesicles whose lipid contents have been extracted. Small particles, presumably remnants of the extracted lipid, are present in some of these vesicles. × 130 000. FIG. 6. Freeze-etched lipid body. The fracture texture of the reticulum contents is similar to t h a t of the lipid core, suggesting that the reticulate portion is actually a collection of lipid vesicles. The small arrow marks a possible membrane within the amorphous core. Direction of shadowing is indicated l~y a circled arrow. S, Starch grain, x 65 000. Fro. 7. The faceted nature of the reticulum subunits is the result of close packing of half-membranebounded lipid vesicles. After freeze etching, some vesicle facets bear an abundance of particles; others lack them entirely, x 110 000. 33O
332
CURT M. PUESCHEL
sheath reticulum is composed of the bounding membranes of individually packaged lipid droplets. These lipid vesicle membranes could be half-membranes, the adnation of which gives the appearance of unit membranes. Where contact between two half-membranes is lost and a third is interposed, the appearance of a unit membrane bifurcation is produced. The faceted outline of the individual lipid vesicles observed in thin-sectioned material (Fig. 5) is present in freeze-etched specimens as well (Fig. 7). Tight packing of the lipid vesicles is probably responsible for this angularity. The size of the lipid vesicles appears to be variable when viewed in the partly three-dimensional freeze-etched preparations (Fig. 7). When the fracture plane follows the course of the vesicle half-membranes in the sheath, 10-nm particles are revealed on some of the exposed surfaces (Fig. 7). Usually only a single facet of a vesicle bears abundant particles; the other facets lack them entirely. DISCUSSION In chemically fixed and dehydrated specimens, preservation of lipids is unpredictable, and interpretations based exclusively on such material must be considered questionable. In freeze-etched specimens the possibility of extraction of lipids seems low, despite glutaraldehyde and glycerol pretreatments. The discovery of finegrained substance within the seemingly ~empty" reticulate portion of the lipid bodies after freeze-etch preparation attests t o this. In combination, the two electron microscopic techniques give a clearer notion of the structure of the lipid bodies in Palmaria. The homogeneous core region may consist of some, but relatively few, membranes embedded in lipid material. Surrounding the core, amorphous lipids are packaged into small units by vesicle halfmembranes. The two portions of the body, the core and the sheath, may represent two chemically distinct types of lipids, or a
single type in two phases; that within the sheath may be more easily mobilized; it certainly appears to be more easily extracted. In thin section the sheath appears to consist of bifurcating unit membranes, but the fact that the chambers are normally filled with lipids allows a second interpretation. The spherosome membrane in higher plants is thought to consist of a single layer of phospholipid molecules, essentially a half-membrane (Yatsu and Jacks, 1972). The hydrophobic fatty acid end of the phospholipid molecule is likely to be adjacent to the hydrophobic spherosome substance, while the phosphate or polar end of each molecule would be exposed to the hydrophilic hyaloplasm. Such might also be the case in Palmaria, but many lipid droplets, each bounded by a half-membrane, would be involved in producing a single sheath. One objection that might be raised to the above interpretation is that the adnation of the polar faces of two half-membranes would not produce the appearance of a single unit membrane. It has been demonstrated that the dark-light-dark unit membrane appearance seen in thin section is the result of binding of both uranyl acetate and osmium at the polar surfaces, probably to the phosphate moiety, of the unit membrane (Stoekenius, 1960). The unstained fatty acid chains provide the relatively electron-transparent region between the stained polar portions. If the polar surfaces of two spherosome halfmembranes come into contact, a single dense line would be expected. Such a configuration has been reported for isolated spherosome ghosts of peanuts (Yatsu and Jacks, 1972). In Palmaria, the electron-transparent space between adjacent half-membranes may be the result of a sphere of hydration around each lipid vesicle, which maintains the integrity of the individual units. It is equally possible that this space is an artifact of chemical fixation.
LIPID BODIES IN A RED ALGA
Particles, presumed to be proteins, are a nearly universal constituent of unit membranes observed after freeze etching (Pinto da Silva and Miller, 1975). Fractures through the sheath of the lipid bodies in P a l m a r i a sometimes expose such particles, though half-membranes constitute the sheath. Yatsu and Jacks (1972) have isolated what they believe to be a structural protein component from spherosome half-membranes. The particles associated with the lipid vesicles in P a l m a r i a are either present in abundance or are absent entirely, and most often the latter is the case (Fig. 7). This dichotomy probably reflects whether the fracture separated the adjacent faces of the two half-membranes or whether it split the nonpolar face of the half-membrahe from the surface of the lipid droplet. It might be anticipated that fractures along the polar face of the half-membrane would not occur or would occur at a very low frequency, because fracture planes do not normally cleave the polar unit membrahe surface from the cytoplasm (Branton, 1966). Instead the cleavage plane might be expected to pass preferentially along the hydrophobic face, i.e., the interior of a typical unit membrane or, in the case of half-membrane-bounded lipids, between the surface of the lipid droplet and the nonpolar face of the half-membrane. It is reasonable to assume that the protein is associated with the polar end of the phospholipid molecule, and because of this, the protein particles are not commonly exposed. The reticulum facets lacking particles might be either the hydrophobic surface of the half-membrane or the surface of the lipid droplet. Electron-dense inclusions, possibly of lipoid nature, are commonly reported in ultrastructural studies of red algae. One type of massive, electron-dense body with some similarities to the lipid bodies of Palm a r i a has been studied by several authors (Bodard, 1968; Feldman and Feldman, 1950; Godin, 1970). This is the so-called
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"corps en cerise" found in cortical cells of several species o f L a u r e n c i a . The "corps en cerise" is surrounded by a 5-nm-thick membrane (Bodard, 1968), which is suggestive of a spherosome half-membrane. After osmium fixation a honeycomb appearance was discernible throughout most of the body (Bodard, 1968). Fixation with potassium permanganate resulted in very poor preservation of these inclusions; most of the amorphous substance was extracted, leaving only the bounding membrane and a few internal membranes (Bodard, 1968). The composition of the "corps en cerise" has been reported to be part lipid and part aldehyde (Feldman and Feldman, 1950). Lipid bodies which are composed of an amorphous core surrounded by a reticulure have been reported in a few higher plants. Esau (1968) found such structural differentiation in plastoglobuli of B e t a vulg a r i s which was infected with beet yellows virus. Plastoglobuli of similar structure have also been reported in chlorotic peas (Silaeva and Shiryaev, 1966). It is unclear whether the membranous associations of the lipid bodies in P a l m a r i a are a manifestation of synthesis, accretion, or mobilization of the lipids. Similarly, it cannot be determined from this work whether the particles associated with these membranes are enzymatic and are involved in lipid metabolism, or whether they are structural. This study was supported by the Lester Sharp Fund and the Section of Genetics, Developmentand Physiology, Cornell University. The author wishes to express his appreciation to Drs. C. H. Uhl, M. V. Parthasarathy, and D. J. Paolillo for their helpful discussions and critical reviews ofthe manuscript. REFERENCES BALZ,H. P. (1966). Planta (Berl.) 70, 207-236. BODARD,M. (1968), C. R. Acad. Sci., Ser. D 266, 2393-2396. BRANTON,D. (1966). Proc. Nat. Acad. Sci. USA 55, 1048-1056. BRONNER,R. (1975). Stain Technol. 50, 1-4. ESAV,K. (1968). Viruses in Plant Hosts. University of Wisconsin Press, Madison.
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FELDMAN,J., ANDFELDMAN,G. (1950). C. R. Acad.
PINTO DA SILVA, P., AND MILLER, R. G. (1975). Proc. Sci. 231, 1335-1337. Nat. Acad. Sci. USA 72, 4046-4050. FREY-WYssLING, A., GRIESHABER, E., AND M(~HLE- SCHWARZENBACH,A. M. (1971). Cytobiologie 4, 145THALER, K. (1963). J. Ultrastruct. Res. 8, 506-516. 147. GODIN, H. (1970). C. R. Acad. Sci. Ser. D 271, 2290- SILAEVA, A. M., AND SHIRYAEV, A. I. (1966). Dokl. 2292. Akad. Nauk S S S R 170, 433-434 [English translaGUIRY, M. D. (1975). Syesis 8, 245-261. tion 595-597]. HEss, W. M. (1966). Stain Technol. 41, 27-35. SMITH, C. G. (1974). Planta (Berl.) 119, 125-142. JACKS, T. J., YATSU, L. Y., AND ALTSHUL, A. M. SOROKIN, H. P. (1967). Amer. J. Bot. 54, 1008-1016. (1967). Plant Physiol. 42, 585-597. SPURR, A. (1969). J. Ultrastruct. Res. 26, 31-43. MATILE, P., AND SPICHIGER, J. (1968). Z. PflanzenSTOEKENIUS, W. (1960) in Proceedings, European physiol. 58, 277-280. Reg. Conference Electron Microscopy, Delft, Vol. McDoNALD, K. (1972). J. Phycol. 8, 156-166. 2, pp. 716-720. MOLLENHAUER, H. H., AND TOTTEN, C. (1971). J. YATSC, L. Y., ANDJACKS, T. J. (1972). Plant Physiol. Cell Biol. 48, 533-541. 49, 937-943. MOOR, H., AND MOHLETHALER, K. (1963). J. Cell YATSU, L. Y., JACKS, T. J., AND HENSARLING,T. P. Biol. 17, 609-628. (1971). Plant Physiol. 48, 675-682.
