The Ultrastructure of the Chloroplast in Spinacia oleracea

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1 The Ultrastructure of the Chloroplast in Spinacia oleracea Katsumi Ueda Botanical Institute, Nara Women's University, Nara, Japan Received March 16, 1964 Studies of the ultrastructure of the chloroplasts have been carried out previously with non-sectioned isolated chloroplasts, and recently with thin sectioned chloroplasts. Several interpretations of their structure have been published. For instance, Steinmann and Sjostrand (1955) assumed that the grana of the chloroplast are formed by double membrane discs and these discs are connected with the stroma lamellae. Wettstein (1957, 1958) con sidered that the chloroplasts consist of a lamellar system; two adjacent lamellae form a disc, and the grana region on the disc contains strongly e1-ectron dense materials. Hodge (1955, 1959) thought that the grana and intergrana lamellae are formed by unit membranes. Weier et al. (1961, 1963) considered that the grana consist of compartments with common partitions, and the grana are interconnected by a fretwork system. These different opinions seem to be mainly derived from different interpretations of the relation between the "grana lamellae" and the "stroma lamellae". This report deals with the ultrastructure of the chloroplasts in Spinacia with special reference to the "lamellar system" and the particles attached to it. Material and methods Fully expanded leaves of Spinacia oleracea, grown under a sufficient intensity of light were used for the present investigation. The epidermal cells of the leaves were stripped off, and the small pieces of mesophyll were fixed in 2% aqueous solution of potassium permanganate for about 2 hours. The mesophyll, after fixation, was dehydrated with acetone and embedded in Epon. Sections were cut with glass knives on a ultramicrotome, and examined with an HU-11 electron microscope. Micrographs were taken at a magnification of 20,000 times, except for those shown in Fig. 6 which were taken at a magnification of 40,000 times. Observations Three components are distinguished in the chloroplast surrounded by the chloroplast membrane. These are the grana, the stroma and the so called stroma lamellae (Fig. 1). These are fundamental constituents of the chloroplasts. However, they have three different appearances in electron micrographs even when preparations are prepared by the same method.

2 416 K. Ueda Cytologia 29 Figs , portion of a chloroplast showing grana (G), stroma (S), and so called stroma lamellae (SL) within the chloroplast membrane (CM). ~100,000. 2, portion of a granum. An enlargement of the area in the rectangle in Fig. 1. Rectangular (R) and triangular (T) particles can be seen. ~220,000.

3 1964 The Ultrastructure of the Chloroplast in Spinacia oleracea 417 Fig. 3. Portion of a chloroplast. Some membranes of the grana extend into the stroma from the points indicated by arrows. These membranes in the stroma have been called "stroma lamellae". Arrows (E) show the ends of "stroma lamellae" at the edges of the grana. A starch grain (ST) is seen. ~120,000.

4 418 K. Ueda Cytologia 29 In the first appearance, grana are apparently composed of many lamellae. The outermost lamellae are about 35A in thickness, and thiner than the inner ones of A thickness. The inner lamellae or the thick lamellae are usually formed of two lamellae, each of which has the same thickness as that of the outermost lamellae. The outermost lamella continues to the lamella which is a member of the thick lamella lying just beneath the outer most lamella, thus forming a flat sac (Fig. 3). Another member of the thick lamella continues to the member of the neigh bouring thick lamella. Thus, the grana consist of many flat sacs. The membrane of the sacs is about 35A in thickness. Fig. 4. Grana with fork-shaped sacs. The sacs in the stroma bifurcate at the points showed by arrows and the bifur cated sacs extend into the granum. ~ 130,000. The lumens of the sacs are about 40A in thickness, but the outermost sacs sometimes have wider lumens. The distance between two membranes of adjacent sacs is about 30A. The number of the sacs in a granum is variable. Some of the flat sacs in the grana are folded back on themselves and often have a fork-shaped with some of the folded membrane extruding into the strome as shown by arrows in Fig. 4. So called "stroma lamellae" always appear in pairs, and the thickness of each lamella is about 35A. Usually, the paired lamellae are inserted into a granum, where they do not differ in appearance from the membranes of the grana sacs. The paired lamellae are joined at the ends to form a flat sac as in the case of the grana sacs. In Fig. 3 arrows E show the terminal edges of the sacs. The other ends of the paired lamellae are also closed either in the stroma or at the edge of a neighbouring granum. There seems to be no rule

5 1964 The Ultrastructure of the Chloroplast in S pinacia oleracea 419 about the ratio of the numbers of sacs in the gr anum and in the stroma. S acs not inserted into the granum are found in the st roma as isolated sacs. Fig. 5. Portion of a chloroplast. Small particles are seen not only on the sacs in the grana but also on the sacs in the stroma. ~165,000. Cytologia 29,

6 420 K. Ueda Cytologia 29 Fig. 6. Portion of a chloroplast and a mitochondrion. Triple layers are seen in the membranes of each sac both in the grana and in the stroma (SL). The chloroplast memb rane (CM) appears as a triple layered double membrane. At the top right, there is a portion of a mitochondrion in which the inner membrane (IM) of the mitochondrion seems to be a triple layered double membrane. ~210,000.

7 1954 The Ultrastructure of the Chloroplast in Spinacia oleracea 421 In the second appearance, the grana also seem to consist of sacs, but the membranes of the sacs are very thin and hardly detectable. Many small particles are observed where membranes of 35A thickness are seen. The distance between two sacs is, in this case, about 90A, and spaces between sacs are denser than not only the lumens of the sacs but also the stroma (Fig. 2). The particles on the sacs are not always round but often rectan gular, trapezoid or triangular (Fig. 2). The average length of the particles in contact with the sacs is 55A. The height of the particles measured between two sacs varies. For instance, when the particle is rectangular, the length on the sacs is about 55A, but the height varies from 20 to 40A. The distance between the centers of the particles is about 80A. Small particles can also be observed on the sacs of the stroma (Fig. 5). In the third appearance, the membranes of the sacs, both of the grana and stroma, are about 45A in thickness. Each membrane is seen as triple layers or double membranes (Fig. 6). Each of the double membranes is about 15A in thickness. Therefore, each of the "thick lamellae" appears as four dense lines, and each of the "stroma lamellae" as two lines. The small particles seen in the second appearance are not apparent. The chlo roplast membrane appears to be two double membranes (Fig. 6). In Fig. 6, a portion of a mitochondrion is seen at the top right. Cristae, or folded inner membranes of the mitochondrion also show the two double membranes, each of which may be less than 10A in thickness. Discussion and conclusion Three different appearances of the structure of chloroplasts were observed in electron micrographs. The common feature of these three appearances is that the chloroplasts have a saccular structure rather than a lamellar structure. Most previous authors have recognized two kinds of lamellae as consti tuents of the chloroplast, namely grana lamellae and stroma lamellae, which mainly differ in thickness. The results obtained in the present investigation did not indicate any fundamental difference in structure between the sacs of the grana and those of the stroma. However, there is a difference in their diameter. In sacs with a diameter larger than that of the grana, the portions of sacs protruding from the grana may have been described by some authors as "stroma lamellae". Therefore, the term "stroma lamellae" should be used only for convenience. The same is true of the term "grana lamellae". Hodge (1959) considered that the lamellar system of the chloroplast constructed with the unit membranes of about 70A in thickness, and that the unit membranes of both the grana and intergrana regions combine to form the compound lamellae. However, the present results indicate that the 28* is

8 422 K. Ueda Cytologia 29 membranes of the 35A thickness in the first appearance must be called as the unit membranes if the term of the unit membranes is used. Moreover it is the outside of the membranes that have a tendency to pair with each other, in contrary to Hodge's interpretation which implied that the pairing tendency was between the inner sides of the compound lamellae. Heitz (1961), Gelora et al. (1960) and Weier (1961) reported that the outermost membranes of the grana are thinner than the inner ones. This is understandable when we assume that each of the inner membranes is formed of two membranes of adjacent sacs, as described above. The portions between the sacs are certainly more electron dense than the stroma. This suggests that the grana may store different substances from the stroma. Little attention has been given to the small particles on the membrane of the sacs in sectioned chloroplasts. That these particles appear triangular, rectangular, trape zoid or spherical in electron micro graphs, they may be cylindrical in three dimensions. The length of these cylinders is about 55A and their diameter seems to be 40A at most. Since the mem branes of sacs on Fig. 7. Schematic drawing of the grana with the stroma sacs. which the particles The grana are composed of many sacs to which small particles are are arranged are attached. Some of these sacs are folded back on themselves. Some extended into the stroma, and these have been called "stroma hardly detectable lamellae" by previous authors. The spaces between two adjacent and the dis sacs are more electron dense than the regions of the stroma or tance between two the lumens of the sacs. sacs is about 90A in this case, the particles themselves may be the main component of the 35A thick membrane of the sac. If certain factors during the fixation of the chloroplasts prevent the appearance of these particles, then the 35A thick membrane of the sacs will be seen. Weier et al. (1963) have reported protuberances on the partitions and considered them to correspond to the quantisomes described by Calvin (1962). Neither protuberances completely crossing the partitions nor quantisomes as large as 200 ~100A (Sauer and Calvin 1962) could be seen in the present photographs. Particles similar to those on the sacs of the chloroplast have been

9 1964 The Ultrastructure of the Chloroplast in Spinacia oleracea 423 observed on the outer and inner membranes of mitochondria by Parsons (1963) and Pease (1962). However, Sjostrand (1962) observed two double membranes on the cristae of mitochondria, though he prefered figures showing three dense lines. As Fig. 5 shows, when the inner membranes of the mitochondria appear as two double membranes, the membranes of sacs in the chloroplast also appear as two double membranes without particles. There must be many enzymes associated with the sacs of chloroplasts. The sizes of these enzymes are not yet known, but they may not be widely different from those of the particles on the sacs, for the diameter of a molecule of cytochromes is considered to be 60A by Estabrook et al. (1961). The figures with particle covered sacs, therefore, may have more significance than those without particles. From the present observations and considerations, a model of the chlo roplast with special reference to the saccular system is represented in Fig. 7. Summary The ultrastructure of the chloroplasts in the leaves of Spinacia oleracea was examined. Three different appearances of the structure of the chloroplast were observed. In the first appearance, the grana seemed to be formed by sacs, the membranes of which were about 35A thick. In the second appearance, the grana seemed to be formed by sacs, the membranes of which were very thin and hardly detectable. On the outside of the membrane small particles were observed. In the third appearance, the grana seemed to be formed by sacs with double membrane. The figures with the second appearance were assumed to be more significant than those with the other two appearances. References Buvat, R Ann. sci. nat. Botan. et biol. vegetale 19: 121. Calvin, M Science 135: 879. Estabrook, T. W. and Holowinsky, A J. Biophys. Biochem. Cytol. 9: 19. Gerola, F. M. and Dassu, G Caryologia 13: , Cristofori, F. and Dassu, G Caryologia 13: 352. Heitz, E Z. Zellforsch. u. microskop. Anat. 53: 444. Hodge, A. J Rev. Modern Physics 31: , Mc Lean, J. E. and Mercer, G. V J. Biophys. Biochem. Cytol. 1: 605. Kahn, A. and von Wettstein, D J. Ultrastruc. Research 5: 557. Levon, H Exptl. Cell Research 7: 265. Menke, W Z. Naturforsch. 15b: 479. Muhlethaler, K. and Frey-Wyssling, A J. Biophys. Biochem. Cytol. 6: 507. Parsons, D. F Science 140: 958. Pease, D. C J. Cell Biol. 15: 385. Sauer, K. and Calvin, M J. Mol. Biol. 4: 451. Sjostrand, F. S Interpretation of Ultrastructure, New York. Steinmann, E. and Sjostrand, F. S Exptl. Cell Research 8: 15. Weier, T. E Am. J. Botany 48: , Stocking, C. R., Thomson, W. W. and Drever, H J. Ultrastruc. Research 8: 122. Von Wettstein, D Hereditas 43: , Brookhaven Symposia Biol. No. 11: 138.