Subunits in the membranes of chloroplasts of Phaseolus vulgaris, Pisum sativum, and Aspidistra sp.

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J. ULTRASTRUCTURERESEARCH13, 92--111 (1965)

Subunits in the Membranes of Chloroplasts of Phaseolus vulgaris,

Pisum sativum, and Aspidistra sp. T. E. WEIER, A. H. P. ENGELBRECHT,A. HARRISON,AND E. B. RISLEY

Department of Botany, University of California, Davis, California Received December 10, 1964 The partitions and frets of chloroplasts of Aspidistra, Phaseolus vulgar&, and Pisum sativum may be resolved after successful fixation (KMnO4 glutaraldehyde followed by Dalton's or OsO4, acrolein followed by Dalton's or OsO4, and by OsO4 alone) into a series of subunits which are interpreted as globular proteins with associated lipids. The fret membrane, about 75 A wide consists of a single row of subunits. Two rows of subunits form the partitions, which are 135 wide. They are less than twice the width of the two membranes that form them. The subunits consist of a light core with an average diameter of 37 ~_ surrounded by dark rims about 28 I t wide. The arrangement of the subunits in the partitions gives the partitions a superficial appearance of being fivelayered membranes formed of three dark components about 40 A wide separated by light leaflets at 19 A in width. The central dark component (40 A) because of its relationship with the outer components of the fret and margin membranes should be twice the width of the two outer dark components, or 80 A. The structure of the plastid membrane thus resolved by electron microscopy is compared with its structure as derived from studies with polarized light, X-rays, chemical information, and electron microscopic studies of whole membranes. The concept of membranes formed of spherical subunits is considered in the light of the unit membrane as it relates to the permeability studies of Danielli and the myelin sheath of nerve cells.

W h i l e detailed high r e s o l u t i o n studies o f c h l o r o p l a s t m e m b r a n e s have n o t been made, it is generally accepted t h a t they c o n f o r m to the unit m e m b r a n e type. I n view o f great differences between (a) the function o f the m e m b r a n e s considered b y Danielli a n d D a v s o n (3), the function a n d f o r m a t i o n o f the myelin sheath (24) a n d (b) the function a n d m o d e o f f o r m a t i o n o f c h l o r o p l a s t m e m b r a n e s , c o u p l e d with the a p p a rent structural similarity o f myelin sheath a n d grana, it becomes i m p o r t a n t to briefly review the unit m e m b r a n e concept. The D a n i e l l i - D a v s o n (3) m o d e l o f m e m b r a n e structure is shown in Fig. 1A. This m o d e l is b a s e d directly on experiments carried o u t b y Danielli a n d H a r v e y (4) on the interfacial tension between m a c k e r e l egg oil a n d m a c k e r e l a q u e o u s egg ma-

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terial. This interracial tension was 0.8 dyne. The surface tension between egg oil and sea water was 7 dynes, and that between egg oil and bromobenzene slowly increased from 8 to 39 dynes. Experiments indicated that this increase was due to a change in surface protein that rendered the protein insoluble in water. Further evidence that the surfactant material was a protein came from its precipitation by ammonium sulfate between concentrations of 35 and 45 %. This protein had globulin characteristics. A similar water-soluble protein was found in the aqueous egg material of eggs of FunduIus, Arbacia, Asterias, and the chicken. Studies on water-soluble proteins had already indicated they were approximately spherical units. Also proteins occurring at interfaces with high surface tensions became denatured and spread out into thin films, some 5 A in diameter. Since the surface tension at the egg oil-egg aqueous material is low (0.8 dyne), Danielli and Harvey (4) postulated such a surface to consist of a layer of water-soluble globular proteins as the surfactant material bordering a thin layer of lipids, possibly of monomolecular dimensions. Danielli and Davson (3) placed two surfaces together oil-to-oil, to form a cytoplasmic membrane separating two aqueous phases (Fig. 1 A). A schematic diagram of the present concept of the unit membrane model is shown in Fig. 1 B. It is based largely on an analysis of the development of the myelin sheath (23, 24). Robertson limited the number of monomolecular layers of lipid to two and specified that the nonlipid layers are fully unfolded proteins rather than layers of globular protein because X-ray studies indicated that there is not sufficient room in the compact myelin structure for globular proteins. The myelin sheath is formed by the spiral growth of the Schwann cell around an axon (10, 23). As the cell membranes come into contact due to the spiral growth, the outer components (Fig. 1 C, dotted line) of the unit membranes unite to form the interperiod line (M, Fig. 1 C) of the myelin sheath. Later the Schwann cell cytoplasm is obliterated and the remaining inner components of plasmalemma unit membrane (Fig. 1 C) came together along their cytoplasmic surfaces to form the denser period line of the myelin sheath. Under certain conditions the interperiod line may be resolved in two components (6, 24). Since the granal partitions may also be considered as closely appressed membranes (Fig. 3f, m, p) (11, 31), comparisons between the partition and the myelin sheath may have considerable significance. Although both may be thought of as the union of unit membranes, it should not pass unnoticed that the membranes of the myelin sheath are derived by the elimination of a protoplast, followed by the appression of the two inner components of unit membranes (Fig. 1 C solid lines). Spiral growth results in the union of the outer components of the same membrane (Fig. 1 C, dotted lines). This compound membrane now serves largely as a protecting and insulating layer around a nerve cell. On the other hand, the membranes forming the partitions

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of the grana are internal membranes of a cytoplasmic organelle and very active in photosynthesis. In spite of striking morphological similarities of granal and myelin membranes, basic differences in molecular morphology may occur. Of immediate importance in this comparison is the realization that although the membranes of the myelin sheath have been studied extensively their ultrastructure is not yet completely understood (6). The period and interperiod lines differ in a number of aspects, and there is good evidence indicating that a carbohydrate may be incorporating during the formation of the interperiod line (6) so that this component in the myelin sheath may be more than the union of the outer components of two unit membranes. Globular proteins approximate 35 A in diameter; fibrous proteins are 5-10 A in thickness (Fox and Foster, 1957). A membrane with overall dimensions of 75-150 and resolvable into components more than 25 ]k in thickness might be expected to consist of globular proteins. Electron microscopes can easily resolve particles of these dimensions if processing procedures can preserve them and render them opaque to electrons. That globular proteins, or lipoproteins, form a basic component of plastid membranes has been suggested by workers in a variety of fields (2, 8). The first electron microscope pictures of isolated and shadowed plastid membranes (9) showed them to have a globular structure. Frey-Wyssling (8) later postulated that globular lipoproteins entered into the structure of the chloroplast lameltae, and he was puzzled by their absence from the profiles of plastids in electron micrographs. Park and his co-workers (21, 22) have greatly extended the observations of Frey-Wyssling to develop the concept of granular quantasomes as regular components of chloroplast lamellae. Recently, Miihlethaler and Moor (20) showed the presence of globular bodies in chloroplast membranes after the freeze-etching technique (19). On the basis of careful measurements of changes induced in polarized light by chloroplasts, Goedheer (12, 13) came to the conclusion that the distribution of chlorophyll as indicated by his studies could be best accounted for if 1he chlorophyll was associated with a globular protein. X-ray analysis (15-17) gives very strong evidence for the presence of globular proteins in the thylakoid membranes. And finally chemical considerations have led Benson (2) to the conclusion that globular lipoproteins should be present in chloroplast membranes. Our own experience with spherical subunits in chloroplast membranes dates from 1962, when one collection of Aspidistra leaves showed partition and fret membranes distinctly composed of such subunits (Fig. 6). This was an isolated case and there were persuasive reasons for not publishing these observations at that time. Granular partitions were, however, noted in 1962 (32). Intensive studies of fixation and embedding procedure resulted in such great improvement of resolution that we now have

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LIPOID

: " "i 'I'" ""("@ ,.,@...

"." "~" "," '" .,.@,,,.®,,.@,.

FIG. IA. Membrane structure as proposed by Danielli and Davson in 1935 (3) [see also~Robertson (24)]. The lipid portion is composed of two monomolecular layers sandwiched between protein layers about 35 • in width, which may be globular proteins.

VVVVVVVVVV~VV

iiiiiiiiiiiiiiiii FI~. lB. The current concept of the unit membrane as developed by Robertson (24). The outer layer is thought to be a sheet of fibrous protein, largely because measurements of the myelin sheath_do not allow room enough for globular proteins.

MEMBRANE

FIG. 1C. Formations of the myelin sheath. The unit membranes surround the Schwann cell protoplasm. The outer component is indicated by a dashed line. At M these two components unite to form the interperiod line of compact myelin: at Y the Schwann cell cytoplasm is obliterated and the inner components unite to form the period line of compact myelin. After Robertson (23, 24).

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WEIER, A. H. P. E N G E L B R E C H T , A. HARRISON~ A N D E. B. RISLEY

several t h o u s a n d micrographs showing the presence of spherical subunits in chloroplast m e m b r a n e s at initial magnifications f r o m 40,000 to 80,000 diameters. Sj6strand (25) has shown that m i t o c h o n d r i a l m e m b r a n e s have a globular structure, a n d the concept of the elemental particle as expressed by Stoeckenius (27) a n d F e r n / m d e z - M o r / m et al. (5) further indicates the particulate n a t u r e of the mitochondrial m e m b r a n e . R o b e r t s o n (23) p o i n t e d out in 1958 that if the term cell m e m b r a n e is to have a n y physiological m e a n i n g , the m e m b r a n e m u s t be c o n s t a n t l y present a n d consist of more t h a n a single layer of a m o n o m o l e c u l a r film. The u n i t m e m b r a n e filled these requirements. It seems n o w that we have reached a stage of i m p r o v e d resolution a n d increased sophistication in the knowledge of the chemistry of m e m b r a n e s so that we m a y look for, a n d find b o u n d i n g p r o t o p l a s t surfaces more elaborate m e m b r a n e s t h a n those of the myelin sheath a n d others. More elaborate m e m b r a n e s m a y indeed exist in the cellular energy transducers, the m i t o c h o n d r i a a n d chloroplasts. This paper reports on the presence of spherical subunits in the chloroplast membranes of higher plants. MATERIALS A N D METHODS Leaf tissue of Aspidistra sp., Phaseolus vulgaris, and Pisum sativum were fixed in one of the following fixatives: 1.5 % KMnO~ for 10-20 minutes; 1.0 % OsO4 in 0.15 M phosphate buffer at pH 7.0 for 3 hours; 10% acrolein in 0.15 M phosphate buffer at pH 7.0 for 30 minutes followed by 1% OsO~ in 0.15 M phosphate buffer for 2 hours; 6% glutaraldehyde in 0.15 M phosphate buffer at pH 7.0 for 40-60 minutes followed by Dalton's chromeosmium fixative or 1.5 % KMnO~ for 1-2 hours. The material was dehydrated in a graded series of acetone followed by propylene oxide, and embedded in a Maraglas/Cardolite mixture made up by weight with BDMA as initiator (Maraglas 17.5 g, Cardolite 6.0 g, BDMA 0.5 ml = volume of 25 ml resin). Castings were cured under vacuum for 24 hours at 60°C. Sections were cut with a DuPont diamond kriife on a Porter-Blum microtome, and some were stained with a 2% aqueous solution of barium permanganate or barium chloride. Sections were examined in a Hitachi H U 11 electron microscope. All measurements were made on the negative with a Nikon Shadowgraph, model 63.

FIG. 2. Close-to-focus micrograph of a portion of a granum of Aspidistra with associated frets. The partitions (p) are formed of two rows of globular subunits, each having a central electron transparent core surrounded by a electron dense rim. The margins (m) and fret membranes (f) are formed by a single row of similar subunits. The associated dark rims and light cores superficially render the partition a five-layered membrane consisting of three dark components or leaflets and two light components or leaflets. The dense leaflets average 40 /~ in width, the light components 17 ~. The diameter of the individual light cores average 37 ~_. The dark rims separating the light cores are about 28 /~ wide. Regular arrays of granules at pr, and light rims bordering the loculus at pL Glutaraldehyde followed by Dalton's. Part of a through-focus series. Poststain, Ba(MnO4),. × 300,000. (See Fig. 16 for diagram.)

SUBUNITS

7 - 051824 J . Ultrc~vtruclure Research

IN CHLOROPLAST

MEMBRANES

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98

x . E . WEIER, A. H. P, ENGELBRECHT, A. HARRISON AND E. B. RISLEY

FIG. 3. Three small grana in a chloroplast of Phaseolus vulgaris. The uppermost, cut obliquely to the partitions, shows globules, but not distinctly. The lower portion of this granum cut tangentially with the margins shows the typical slanting connection between frets and grana. The two lower grana show the globular structure distinctly; regular arrangements are particularly distinct at pr. Union of margin (m) and frets (f) to form the partitions (p) is well illustrated. Measurements were made at the points indicated on frets and partitions. Glutaraldehyde followed by Dalton's. Poststain, Ba(MnO4)2. x 120,000.

SUBUNITS IN CHLOROPLAST MEMBRANES

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F~o. 4. A n irregular g r a n u m f r o m t h e s a m e plastid as Fig. 3 (Phaseolus vulgaris). T h e globular nature of t h e m e m b r a n e s is a p p a r e n t a l t h o u g h t h e r e is less contrast. T h e central row of rims within the partition is s o m e w h a t denser t h a n the two o u t e r rows of rims. T h i s m i c r o g r a p h was selected to show the contrast in structure between the plastid m e m b r a n e s a n d the ectoplast (ec). M e a s u r e ments were m a d e at points indicated. F r o m a t h r o u g h - f o c u s series. G l u t a r a l d e h y d e followed by Dalton's. Poststain, Ba(MnO~)2. × 240,000.

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WEIER, A. H. P. E N G E L B R E C H T , A. HARRISON A N D E. B. RISLEY

OBSERVATIONS The spherical subunits in the plastid membranes are graphically illustrated in all plates. Three factors are involved in assessing the reality of these subunits: (a) the granularity must not be due to the precipitation of a small granule of permanganate with the subsequent accumulation of additional permanganate; (b) it must not be due to focus; (c) it must not be a fixation artifact. That problems (a) and (b) are not factors is indicated by the presence of the globules in over two hundred throughfocus series (Fig. 5; also Figs. 2, 4, 6, 8-10, 12, and 13) which were selected from through-focus series). That precipitation of KMnO~ is not a causal factor is indicated by the smooth staining of the dense components and by the presence of subunits in material prepared without using permanganate in any form (Figs. 11 and 12). Figs. 2-4 and 10 were fixed in solutions lacking K M n O 4 but were stained in B a ( M n Q ) 2. That the subunits are not artifacts of fixation is of course one of the general problems of electron microscopy. We have obtained similar fixation images after K M n Q alone (Figs. 5-9, 14, and 15) after osmium alone (Fig. 12), after glutaraldehyde followed by KMnO4 or by Dalton's (Figs. 2-4), after acrolein followed by OsO 4 (Figs. 10 and 11), as well as after other combinations. We have not prepared material by freezing methods.

Fretwork The fret membranes are formed of a single row of subunits, the centers, or cores, of which are electron transparent while the rims are electron dense ( f i n Figs. 2, 3, and 10). In favorable regions a fight gray rim completely surrounds the ligher core (pr in Figs. 2, 3, 5-10, 12-15). The dense rims are not uniform; they vary in width and intensity of staining, from extremes of black lines bordering light cores (pr in all figures) to situations where the rims are so thin and lightly stained (pl in Figs. 2, 11, and 13) that the cores seem to merge with the stroma, or fret channel. The width of the rims would further be expected to vary with the section, a median section giving the narrowest rim. In addition, the dense regions may be small, giving the appearance of heavily stained bodies in lighter stained rims. These two factors, variation in staining and level of the section, mean that all measurements have a high degree of arbitrariness that cannot be eliminated by sections at 90 degrees to the membrane. This structure of the fret membrane means that light and dark leaflets do not exist as such although the array of outer rims and light cores can be measured to give figures comparable to those for light and dark components. However, the irregularity of rims in the frets makes such a measurement rather unreal Overall measurements of the width of fret membranes range from 89 ~ for KMnO4 fixation and 76 ~ for OsO4 fixing mixtures.

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Fro. 5. Three steps of a t h r o u g h - f o c u s series of a g r a n u m of Phaseolus vulgaris. T h e globular n a t u r e of the m e m b r a n e s is i n d e p e n d e n t of focus. A n orderly a r r a y of globules at pr. 1.5 % K M n O 4 . F r o m a through-focus series. Poststain, none. x 450,000.

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T . E . WEIER, A. H. P. ENGELBRECHT, A. HARRISON AND E. B. RISLEY

We have not taken measurements of dark rims but have 10 measurements o f light cores in Aspidistra after K M n O 4 fixation. These averaged 33 A, a value that would indicate a rim width o f about 28 A (Fig. 16).

Partitions The partitions are formed by the juxtaposition of two globular membranes: fretfret (not illustrated), fret-margin (f, m, p in Figs. 2 and 3), or margin-margin (m, m, p Fig. 3). If we, for the moment, disregard the lateral rims of the spherical subunits and consider only those horizontal rims parallel with the loculi, we can consider the partition as a fivelayered m e m b r a n e consisting o f three dark rows o f rims separated by light cores (Figs. 4, 6, 7, 14, and 16). U n d e r certain conditions the central leaflet formed by the juxtaposition o f rims f r o m two appressed membranes is m u c h darker than the two outer single rows o f rims (Figs. 4 and 14). We m a y now measure each row o f rims and the light cores, which m a y be measured individually or as a leaflet (Fig. 16). Reasonably accurate measurements m a y be obtained for the overall widths o f the fret membranes and partitions. After KMnO~ fixation, 32 fret membranes, selected f r o m micrographs o f Aspidistra, Phaseolus, and Pisum, had an average width o f 89 A; and after fixatives containing osmium, 13 fret membranes averaged 76 A in width. After K M n Q fixation 152 partitions bad an average width of 159 A; and after osmium fixing fluids the average width was 136 A for 35 partitions. While it seems likely that membranes are b r o a d e r after KMnO~ fixation, species differences m a y also have some influence since we had a preponderance of Aspidistra fixed in K M n O 4. F o r making comparisons between the widths o f fret membranes and partitions, variations induced by preparatory techniques and p h o t o g r a p h y make it necessary to average measurements made on the same micrograph. We have made comparisons between fret membranes and the partitions on 12 micrographs after KMnO~ fixation and on 10 micrographs after OsO~ fixation. A total of 50 membranes were measured. In all but five cases, the partition was less than twice the width o f the comparable fret membranes. Measurements of KMnO~-fixed material averaged 159 A for partitions and 89 A for a simple fret membrane. The partition width is thus 1.77 times that FIG. 6. A portion of a granum from Aspidistra. Area in unsupported section (pr) shows distinct globular nature of the partition. 1.5 % KMnO~. From a through-focus series. Poststain, none. x 450,000. FIG. 7. A portion of a granum of Aspidistra from the same collection as Fig. 6, but cut from a different block. Globules are very regular (pr). 1.5 % KMnO4. Poststain, none. × 300,000. FIG. 8. Portion of a granum from Phaseolus vulgaris. Globular nature of membranes is distinct, being quite regular at pr. From a through-focus series. 1.5 % KMnO~. Poststain, none. x 300,000. Fro. 9. From the s/~me collectidn as Fig. 8, but a different block. The section shows less contrast, but the globular subunits are present, being quite regular at pr. 1.5 % KMnO4. Poststain, none. × 300,000.

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T . E . WEIER, A. H. P. ENGELBRECHT, A. HARRISON AND E. B. RISLEY

of a simple fret membrane. After fixatives involving osmium, the comparable figures were 136 A for partitions and 76 A for the fret membranes. Here the partition width is 1.78 times that of a fret membrane. In neither case is the partition equal to the sum of the widths of the two membranes joining it. Partitions may be further analyzed, but with considerable arbitrariness because the irregularities induced by spherical subunits allows only a rough estimate of the relative widths of the light and dark components. We have measured these components in 176 partitions, obtaining an average width of 40 A for each dense component and of 17 A for each of the two light components. Average diameters of 37 A were obtained from the measurement of 230 individual light cores. Thirty-six of these light cores fixed in osmium fluids averaged 33 A, and the remaining KMnO4-fixed light cores averaged 43 A. Dark granules distributed over the membranes averaged only 56 A in a diameter. This is less than the 70 A that might be expected for the full diameter of the subunit. However, these dark granules are areas within the dense rims, and they could be a second category of granule (20) or surface views of rims of globules showing between adjacent light cores. Although, in general, the dark rims show variation in width and there is some irregularity in the placement of the light cores, sections of membranes do occur in which the light cores are parallel and opposite each other and the dark rims quite uniform in thickness (pr in Figs. 3, and 5-8). The difference between the 37 A measurement for the diameter of the light cores and the 17 A width of the light leaflets arises from the impossibility of placing the measuring lines of the Nikon Shadowgraph exactly tangent to an irregular row of subunits, with a resulting increase in the value of the figures obtained for the average width of the dark components. If we take the figure of 159 A obtained from the K M n Q material for the widths of the partitions and 37 A for the diameter of the light cores, this would leave 28 A for the thickness of the dark rim. This is also an arbitrary figure, for the rims are not of uniform thickness, being thicker in the corners and thinner laterally between the cores where they may be as thick as or about half as thick as the horizontal rims. They average about 25 A in thickness. These relationships are shown in Fig. 16.

FIG. 10. Portion of a granum from Phaseolus vulgaris to show globular nature of membranes after a prefixation in acrolein, followed by OsOa and poststained in Ba(MnO4)2. Globular fret membranes at f,, regular array of partition globules at pr. x 300,000. Fro. 11. Portion of a granum from Phaseolus vulgaris to show the globular nature of the membranes after prefixation in acrolein followed by OsO~ and poststained with Ba(C1)~. A clear example of clear cores seemingly opening on the material of the loculus'is shown at pl. x 300,000. Fro. 12. Portion of a granum from Pisum sativum fixed in OsO4 and unstained. The globular nature of the membranes is not so distinct, but it is present. A regular array is shown at pr. Part of a through-focus series, x 300,000.

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T . E . WEIER, A. H. P. ENGELBRECHT, A. HARRISON AND E. B. RISLEY

GranaI expansion Previous workers have reported that the partitions were separated by regular distances (14, 16, 26). It has been our experience that the loculus is a variable component of the plastid, a situation giving rise to a great variation in the distance between two partitions (31, 32). While such variation may be due to fixation or pretreatments, it may also arise because of physiological factors (1, 28). At best, the width of the loculus is the most sensitive of all plastid components to external conditions, and we hesitate to designate a regular periodicity separating the partitions. Figs. 2-12 illustrate the most general widths of the loculi in what may be considered well preserved chloroplasts. The loculi are distinct in Figs. 2-4, 7, 8, and 10-12. They are less distinct in Figs. 5 and 9. Considerable locular swelling has occurred in the plastid shown in Fig. 13. The loculus of Fig. 14 is variable in width, and to all purposes it is lacking in Fig. 15. The partitions maintain a width ranging from 140 to 175 ~ under all conditions of locular swelling. Even swelling the loculus as in Fig. 13 has no effect on partition width or subunit structure. In Fig. 15 the darkest line probably represents the line of fusion between the inner rims of two rows of subunits. The smaller rows of dense granules (Figs. 15 and 16) are derived from the dense margins of subunits bordering the hydrophilic loculi. The loculi themselves are almost obliterated, being represented by an occasional light component (1 in Figs. 15 and 16). Locations where such an arrangement of granules can be seen are indicated by arrows. In many places (arrows) the borders of these subunits seem to be in contact, thus completely obliterating the loculi. Light spaces probably representing loculi are shown (l, Fig. 5).

Other membranes We have not made a study of other membranes, except as we were able to show them in direct relation to the internal membranes of the plastid. The plastid envelope appears to be composed of spherical subunits while the ectoplast appears to be of the unit membrane type (ee, Fig. 3).

DISCUSSION In 1957 Frey-Wyssling noted (8) the presence of globules in surface views of grahal membranes but was unable to account for the absence of globules in sections of plastid membranes. As the result of extensive experimentation with processing procedures we have been able to show that the chloroplast membranes, at least of Phaseolus vulgaris, Pisum sativum, and Aspidistra sp. have a structure composed of spherical subunits. C. Crisp, a graduate student, working in our laboratory on cuticle of Chenopodium has recently obtained a number of excellent views of globular plastid

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F~o. 13. Portion of a chloroplast of an expanded granum of Phaseolus vulgar&. Partitions maintain their globular structure and dimensions in swollen grana. Points indicated were measured. End partition at ep; regular array of globules at pr. Part of a through-focus series. 1.5 % KMnO~. Poststain, KMnO4. x 240,000.

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membranes in this plant with spherical subunits--quite as a sideline f r o m his main project. Membranes c o m p o s e d of similar subunits also occur in the membranes o f the c h r o m a t o p h o r e of Scenedesmus quadricauda (30). The nature of these membranes makes accurate measurements difficult. Furthermore, as pointed out by Robertson (24), the juxtaposition of two membranes, as in the partition, introduces further complications. The union or fusion of two membranes, for instance, p r o b a b l y differs f r o m intimate apposition in the sense that fusion implies a reduction in the total n u m b e r o f layers o f the two constituent membranes. Intimate apposition implies only close juxtaposition and perhaps some degree o f overlap with some interpenetration o f side-chain groupings of the closely applied membranes. Two unit membranes of mesaxons o f myelinated nerve fibers measure a b o u t 150 A across when closely appressed, but 100-130 A within c o m p a c t myelin. While the difference could be accounted for by fusion, Robertson gives reasons for not considering it so and further points out a definitive answer to the problem of why such measurements cannot be given. We must agree with his conclusion that measurements of overall thickness are subject to a considerable degree o f arbitrariness, which is c o m p o u n d e d if, as we believe, we are measuring subunits o f irregular lipoprotein units; for here the thickness will be related to that particular sector o f a sphere that is measured, the largest measurement presumably representing the greatest diameter o f the particle. It is questionable under these circumstances whether a statistical analysis of our measurements would be meaningful. We have measured the thickness o f a b o u t 2000 membranes and spherical subunits. It seems reasonable to draw the following conclusions f r o m these measurements: 1. The overall width o f the fret m e m b r a n e is a b o u t 75 A and the partitions about 135 A. Since the sum o f two fret membranes is 150 A (Fig. 16), the partition is narrower than two combined fret membranes. 2. The spherical subunits composing the partitions have a light central core of approximately 37 A, and the average width o f the dense rim is about 20 A (Fig. 16). 3. The dense rims and the light cores f o r m lines dividing the partition superficially into a five-part membrane. The dark components so formed averaged 40 A, and the light components 17 A. The central dark c o m p o n e n t was the same width as each of

FIG. 14. Portion of a granum from Aspidistra showing the dark central component (pd). A regular array of globules is present at pr. There has been some locular swelling. 1% KMnO4. Poststain, none. x 300,000. FIG. 15. Portion of a granum from Aspidistra. This granum is particularly compact. The heavy dark line (pd) is interpreted as the dense central component of the partition. It is accompanied on each side by single, less dense, lines. These three lines, plus the light areas between them represent the five-layered partitions of other grana. The light line (l) between the five-layered partition represents loculus. That these membranes are composed of globular subunits with light cores and darkened rims is apparent at pr. 1% KMnO~. Poststain, none. x 300,000.

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T . E . WEIER, A. H. P. ENGELBRECHT, A. HARRISON AND E. B. RISLEY

19Ao 40A°

I

4QA°

19A°

I 19A~

40A°

I 40A°

FIG. 16. Diagram of the partition and fret membranes as they appear in the chloroplasts of Aspidistra sp, Phaseolus vulgaris, and Pisum sativum after a variety of fixations followed by embedment in Maraglas. The membranes are drawn to scale. The divergence in measurements between the diameters of individual clear cores and the so-called light component arises because of the globular nature of the light cores and the subsequent impossible task of placing a measuring line exactly tangential to each light core.

the outer components, even though the central component is associated with two components of two other membranes, fret-margin or margin-margin (Fig. 16). 4. Either fixation, or species, or both, may have an influence on membrane width. Membranes fixed in KMnO~ including a large number of Aspidistra collections were wider than membranes fixed in OsO4 fluids. While a detailed model of chloroplast membranes will appear elsewhere, it may be noted here that these measurements compare favorably with those derived by Kreutz (16) from X-ray defraction studies. Kreutz postulates a unit 71 N wide which he and Menke (17) interpret as a thylakoid membrane; it would correspond to a fret membrane, which we found to average 76 A in width after OsO4 fixation. Two of Kreutz's 71 A units, or 142 A, would correspond to our partition which averages 136 A after osmium fixation and 159 A after permanganate fixation. Kreutz postulates a protein particle 37 A in diameter; this matches almost too well the 37 A diameter we found for the light cores. Kreutz further postulates a lipid zone measuring 34 A in width in living chloroplasts. This may be divided into an aliphatic zone 23 A wide and an porphyrin ring zone 11 A wide. In air-dried chloroplasts the corresponding measurements are a 41 A overall width, with 20 A for the width of the aliphatic zone and 21 A for the porphyrin ring zone. This may be compared with our average measurements for the horizontal dark rims as they form dark components of the membrane. These dark components measured 40 A overall.

SUBUNITS IN CHLOROPLASTMEMBRANES

111

Although the correspondence between these two sets of measurements is encouraging, it should be pointed out that we envisage a dark rim surrounding a light core whereas Menke and Kreutz diagram two lipid zones between rows of 37 ~ protein globules. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

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