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J. Cell Sri. 3, 41-48 (1968) 41 Printed in Great Britain

THE FINE STRUCTURE OF AND PYRENOIDS IN SOME MARINE

J. D. DODGE Department of Botany, Birkbeck College, London, W.C. 1

SUMMARY The chloroplasts of some members of the Dinophyceae are bounded by an envelope consist- ing of three membranes and having a mean thickness of 230 A. Within the are arranged, in a more or less parallel manner, many lamellae normally composed of three apposed , although the number of thylakoids often varies and may reach 30 in a single stack. By study of disintegrated chloroplasts it was found that the thylakoids are circular in shape with a diameter of 0*15-3-6 /* and a mean thickness of 240 A. Ribosomes, lipid droplets and DNA areas are present in the chloroplast stroma. No connexions were seen between the chloroplasts and any other organelles, nor did the chloroplasts contain girdle lamellae. Stalked pyrenoids, which are found in some dinoflagellates, are shown to arise from the inner face of the chloroplasts, to contain a finely granular material and to be frequently surrounded by an electron-transparent area. These findings are discussed in relation to the fine structure of the chloroplasts and pyrenoids of other algal classes.

INTRODUCTION Although the chloroplasts and pyrenoids of many have been extensively studied by electron microscopy over the past few years, those of dinoflagellates have received little attention. The first published electron micrograph of a sectioned was of Amphidinium elegans (Grell & Wohlfarth-Botterman, 1957). This showed the chloroplast to be lamellate with each lamella consisting of a number of parallel membranes. Ueda (1961) reported that the chloroplasts of Ceratium and were four-lamellate. In the present terminology this meant that the lamellae each consisted of three thylakoids. Later, Gibbs (1962 b, c), in a survey of the chloroplasts and pyrenoids of several algal classes, found that in Amphidinium carteri the lamellae (or bands) consisted of three or four apposed thylakoids (or discs). Occasional lamellae split into two, and the lamellae were normally so close together that there was little space for chloroplast matrix. This result may have been caused by the particular osmium tetroxide fixative used. Gibbs also found that A. carteri had one single, central, starch-sheathed pyrenoid with a number of chloroplast lamellae penetrating its dense ground substance. Bouck & Sweeney (1966), in a study of dinoflagellate , incidentally showed sections of the radially orientated chloroplasts of Gonyaulax polyedra. These appeared to contain numerous two or three lamellae arranged parallel to the long axis of the chloroplast. A somewhat similar arrangement of lamellae was found in Woloszynskia micro. (Leadbeater & Dodge, 3-2 42 J. D. Dodge 1966). Here the lamellae normally consisted of three thylakoids and some branching of lamellae was observed. In this organism elongated or flattened pyrenoids were found between the lamellae of the chloroplast. In the present paper the detailed structure of the chloroplasts of some small marine dinoflagellates will be described in detail for the first time and compared with the structure of chloroplasts in other algae. The single-stalked pyrenoids found in a number of dinoflagellates will also be described.

MATERIAL AND METHODS The main description relates to Aureodinium pigmentosum Dodge (Dodge, 1967) (Plymouth cultures 208 and 389 supplied by Dr M. Parke) and Glenodinium sp. (supplied from Florida, U.S.A. by Dr W. B. Wilson). Several other organisms, representing various genera, have also been examined; Woloszynskia micro. Leadbeater & Dodge (Plymouth 207) was mainly used for the work on extracted chloroplasts. Unialgal cultures were grown in Erdschreiber medium under various light condi- tions. Fixation was carried out using 3 % (v/v) cacodylate-buffered glutaraldehyde at pH 7-0 with sucrose added to give a molarity of 0-2 M. This fixative was used either cold for 1-2 h or at 20 °C for 5 min to i\ h and followed, after several washings in buffer, by post-fixation in 1 % (w/v) osmium tetroxide in either cacodylate or phos- phate buffer. After dehydration in ethanol the material was embedded in Araldite or Epon, sectioned with an LKB microtome and examined in a Zeiss EM 9 electron microscope. Whole mounts of broken chloroplasts were prepared by the following method. A dense suspension of cells was transferred to o-8 M sucrose in TRIS buffer at pH 7-8 and treated either with ultrasonics for 1-3 min or in a Vertis homogenizer for 4 min. The resulting material was layered on to a sucrose density gradient (i-6 M, 1-3 M, O-8M) and after centrifugation (3500 rev/min for 20 min) a coloured band which contained mostly chloroplast material was separated. Portions of this were transferred to water, dried on to grids and shadowed with gold palladium or negatively stained with 2 % (w/v) potassium phosphotungstate.

OBSERVATIONS Chloroplasts The form of the chloroplasts is rather variable. In small dinoflagellates such as Aureodinium pigmentosum they are probably saucer-shaped and are peripheral in position (Fig. 2). In larger organisms such as Gonyaulax tamarensis they are frequently lens-shaped and radial in position. A peripheral reticulate arrangement is seen in Exuviaella and Prorocentrum. The number of chloroplasts seems to be variable even within a species. The chloroplasts are surrounded by a distinct bounding membrane. With certain fixations this appears as a heavy dark line (Figs. 3, 4), whereas the nuclei and mito- chondria in the same cells can be seen to have distinct double membranes. On further Dinoflagellate chloroplasts and pyrenoids 43 investigation it was found that the chloroplast envelope consists of three membranes, normally of equal thickness (Figs. 1, 6, 7). Sometimes these membranes are very wrinkled, thus making determination of their number difficult. As with the thylakoids (see below), the thickness of the chloroplast envelope has proved very variable, ranging, in the photographs used in this paper, from 140 to 380 A (mean 230 A). However, in spite of this considerable variation, the width generally appears less than the width of a single thylakoid in the same micrograph, suggesting that whatever swelling or contraction may have happened during fixation had affected both equally. No connexions have been observed between the chloroplast envelope and endo- plasmic reticulum or any other organelle, nor have ribosomes been seen attached to the outer surface of the envelope. I 230 A

240 A

Fig. 1. Diagrammatic representation of part of a dinoflagellate chloroplast. A. The structure of the chloroplast envelope with its three membranes. B. Part of a lamella consisting of three apposed thylakoids.

The chloroplasts contain numerous lamellae which are oriented parallel to the longer axis of the organelle (Figs. 3-5). The lamellae do not connect with the chloro- plast envelope but normally terminate ju9t short of it. Except in Woloszynskia, where branching has been found, there are normally no interconnexions between lamellae. Girdle lamellae have not been seen in any of the dinoflagellates examined. Each normal lamella consists of a number of apposed thylakoids (or 'discs' in the older terminology) giving in cross-section the appearance: thin dark line, clear space, thick dark line, and so on, where the thick dark lines correspond to two apposed thylakoid membranes. Most frequently each lamella consists of three thylakoids, but four and two (Fig. 3) are often seen and occasionally deep stacks of up to 30 have been encoun- tered (Fig. 5). These may have been induced by abnormal growth conditions as they have generally been found in cells from old cultures. The thickness of the thylakoids, as seen in section, shows a considerable variation which is probably due to the state of the cells as well as to the method of preparation. In the tightly packed deep stacks of Fig. 5, for example, the thylakoids average only 190 A in width, but in the separate pairs of thylakoids of Fig. 6 (both Figs. 5 and 6 are from the same fixation) the width averages 380 A. The mean value for all measurements made is 240 A (Fig. 1), of which the outer membranes account for about 60 A each and the central space about 120 A. 44 J- D. Dodge From sectioned cells it is difficult to ascertain the shape of individual lamellae and thylakoids. However, by breaking cells and separating the chloroplast fraction it was possible to examine the form of the thylakoids. They are seen (Figs. 8, 9) almost always to be circular discs which exhibit a considerable variation in diameter. In Woloszynskia the size ranged from 0-15 to 3-6 /i, which compares favourably with figures of 0-6-4-0/* obtained from randomly sectioned chloroplasts. In shadowed thylakoids (Fig. 8) there is some evidence of the presence of large subunits, or quanta- somes, similar to those which have been described from the thylakoids of angiosperms. The chloroplast matrix or stroma, as is normal in all chloroplasts, contains granular material with much variation in the size of the granules. The larger particles (compare Fig. 4) are probably ribosomes as they stain densely with uranyl salts and are 140-200 A in diameter. Occasionally one finds large areas of stroma (Fig. 3) not crossed by any lamellae. These may be regions which will become extruded from the chloroplast as pyrenoids or they may simply be areas of chloroplast where the lamellae are still forming. It was found that when cells were grown in higher light intensity than normal (500 ft-c instead of 100) the lamellae were spaced further apart than usual. As with most chloroplasts, those of Aureodinium frequently contain lipid droplets (Fig. 3) and occasionally fibrillar areas are found which, as they can be removed by treatment with DNase, consist of DNA. DNA is not nearly so common as in the chloroplasts of Woloszynskia (Leadbeater & Dodge, 1966).

Pyrenoids It would appear that in the class Dinophyceae as a whole several types of pyrenoid are found. The present account will be confined to the simple stalked pyrenoids which have so far been found in Aureodinium pigmentosum and in Glenodinium (Florida isolate). These pyrenoids are situated on the inner side of the chloroplasts (Fig. 2), to which they are connected by a short stalk. Sometimes the stalk is quite narrow (Fig. 12), but in what appear to be developing pyrenoids (Figs. 10, 11) it is almost non- existent. The body of the pyrenoid is surrounded by a continuation of the chloroplast envelope and it contains uniformly granular material (Figs. 10-13) which contrasts with the irregular granularity of the chloroplast stroma. It would appear to lack ribosomes and probably consists of protein. Occasional pyrenoids are found to contain one or more pieces of chloroplast lamella, usually consisting of not more than two thylakoids (Fig. 13). Surrounding what appear to be mature pyrenoids (that is, those with stalks) is found a wide halo of electron-transparent material (Figs. 12, 13) which sometimes contains bands of rather electron-opaque material. The pyrenoid and halo appeared to be much larger in material grown under high light intensity. The halo almost certainly consists of polysaccharide and when tested with iodine and examined by light micro- scopy the pyrenoids became stained, but not apparently the very deep blue colour given by starch. No membrane surrounds the pyrenoid halo and it abuts on normal cell . Dinoflagellate chloroplasts andpyrenoids 45

DISCUSSION The fine structure of algal chloroplasts has been found to have a distinctive form in many of the algal classes. This structure shows what may be a developmental series from the primitive arrangement in the blue-, to the simple structure in the and ultimately to the complex arrangement of the lamellae in many of the green algae. One point which this paper tries to establish is the position of the Dino- phyceae in this emerging pattern. Clearly the dinoflagellate chloroplasts are more complex than those of the Rhodo- phyceae where the lamellae consist of single thylakoids (Bouck, 1962; Gibbs, 1962 c; Gantt & Conti, 1965; Nichols, Ridgeway & Bold, 1966) and they differ from those of the Phaeophyceae where the adjacent thylakoids are not fused and where girdle lamellae and endoplasmic reticulum outer envelopes are found (Gibbs, 1962 c; Bouck, 1965; Evans, 1966). The dinoflagellates also differ from certain (Drum & Pancratz, 1964; Manton & von Stosch, 1966), the Chrysophyceae (Gibbs, 1962 a; Manton & Harris, 1966) and the Xanthophyceae (Greenwood, 1959) which all possess girdle lamellae. The , reputedly closely allied to the Dinophyceae, differ from them in regularly having two-thylakoid lamellae and also possessing an outer endoplasmic reticulum envelope (Gibbs, 1962c; Greenwood, 1967). Apart from the , which generally have much more complex chloroplasts, we are left with the Haptophyceae and the Euglenophyceae. In the former class both Chryso- chromulina chiton (Manton, 1966) and Prymnesium parvum (Manton, 1964) have chloroplasts which could be mistaken for those of a dinoflagellate but for the fact that they have a double endoplasmic reticulum envelope outside the double chloroplast envelope. The Euglenophyceae (Gibbs, i960; Leedale, Pringsheim & Meuse, 1965) have chloroplasts which appear very similar to those of the Dinophyceae, even to the triple-layered envelope. Gibbs stated that the envelope was double but did say that on occasion it looked more complex and Leedale et al. (1965) described the membrane as ' compound'. It has now been found (G. F. Leedale, personal communication) that it is in fact composed of three layers and has connexions with the nuclear envelope by way of tubular endoplasmic reticulum. In the Xanthophyceae a triple chloroplast envelope has been found in Vaucheria, Botrydium (Greenwood, 1964) and various other genera (G. F. Leedale, D. Hibberd & A. Massalski, personal communication). Here again the envelope has distinct endoplasmic reticulum connexions. Thus the only structural feature which distinguishes the chloroplast of the Dinophyceae from other chloroplasts is the absence of endoplasmic reticulum connexions with the three-layered envelope. It is not easy to account for the triple chloroplast envelope, for most biological structures which employ membranes (as mitochondria, Golgi bodies, nuclear envelope, endoplasmic reticulum) normally use only two. A possible explanation is suggested by the work on the Euglenophyceae and Xanthophyceae cited above. Here the outer of the three membranes is continuous with the endoplasmic reticulum. As most organisms with endoplasmic reticulum attached to the chloroplast (Chrysophyceae, Phaeophyceae, Haptophyceae) have a quadruple envelope, in which the outer two membranes are part of the endoplasmic reticulum, it would seem possible that the 46 J. D. Dodge triple condition has derived from this by fusion of two of the membranes. In the present work it has been noticed that the central membrane sometimes appears slightly thicker than the outer two, although this difference has not yet been adequately measured. One point against the endoplasmic reticulum hypothesis is that in the Dinophyceae no ribosomes have been found adhering to the chloroplast envelope. If the outer membrane is part of the reticulum ribosomes would have been expected and are present, for example, on the outer of the four membranes surrounding the chloroplasts of the Haptophyceae (Manton, 1964, 1966). The simple stalked pyrenoids described in the present paper are similar, apart from the apparent absence of an outer endoplasmic reticulum sheath, to stalked pyrenoids which have been found in one member of the Haptophyceae, ChrysochromuUna chiton (Manton, 1966), several members of the Phaeophyceae (Bouck, 1965; Evans, 1966) and some members of the Euglenophyceae (G. F. Leedale, personal communication). This similar pyrenoid structure in organisms which are reasonably closely related might be thought to have some phylogenetic significance were it not for the fact that other dinoflagellates are known which have at least three other differing types of pyrenoid (Gibbs, 19626; Leadbeater & Dodge, 1966, and unpublished observations). Drum & Pancratz (1964) found a similar situation in the Bacillariophyceae. It is clear that pyrenoids will never provide the distinctive character for dinoflagellates such as is already provided by the nuclei (Dodge, 1966), the flagella (Leadbeater & Dodge, 1967), and to some extent by the chloroplasts. However, the different pyrenoid types may be of some significance at the generic level, and this is currently being investigated.

Acknowledgments are due to the Science Research Council, to those named above who supplied cultures and to G. Lawes, J. Bhola and V. Morris for technical assistance.

REFERENCES BOUCK, G. B. (1962). Chromatophore development, pits, and other fine structure in the red alga Lomentaria bcdleyana (Harv.) Farlow. J. Cell Biol. 12, 553-569. BOUCK, G. B. (1965). Fine structure and organelle associations in . J. Cell Biol. 26, 523-537- BOUCK, G. B. & SWEENEY, B. M. (1966). The fine structure and ontogeny of trichocysts in marine dinoflagellates. Protoplasma 61, 205-233. DODGE, J. D. (1966). The Dinophyceae. In The Chromosomes of the Algae (ed. M. B. E. Godward). London: Arnold. DODGE, J. D. (1967). Fine structure of the dinoflagellate Aureodinium pigmentosum gen. et sp. nov. Br. phycol. Bull. 3, 327-336. DRUM, R. W. & PANCRATZ, H. S. (1964). Pyrenoids, raphes, and other fine structure in diatoms. Am.J. Bot. 51, 405-418. EVANS, L. V. (1966). Distribution of pyrenoids among some brown algae. J'. Cell Set. 1, 449-454. GANTT, E. & CONTI, S. E. (1965). The ultxastructure of Porphyridium cruentttm. J. Cell Biol. 36, 365-381. GIBBS, S. P. (i960). The fine structure of gracilis with special reference to the chloro- plasts and pyrenoids. J. Ultrastruct. Res. 4, 127-148. GIBBS, S. P. (1962a). Nuclear envelope-chloroplast relationships in algae. J. Cell Biol. 14, 433-444- GIBBS, S. P. (19626). The ultrastructure of the pyrenoids of algae, exclusive of the green algae. J. Ultrastruct. Res. 7, 247-261. Dinoflagellate chloroplasts and pyrenoids 47 GIBBS, S. P. (1962c). The ultrastructure of the chloroplasts of algae, J. Ultrastruct. Res. 7, 418-435- GREENWOOD, A. D. (1959). Observations on the structure of the zoospores of Vaucheria II. J. exp. Bot. 10, 55-68. GREENWOOD, A. D. (1964). The structure of chloroplasts in lower plants. Abstr. Xth Int. Congr. Bot. pp. 212-213. Edinburgh. GREENWOOD, A. D. (1967). Quoted in J. T. O. Kirk & R. A. E. Tilney-Bassett, The . London: Freeman. GRELL, K. G. & WOHLFARTH-BOTTERMAN, K. E. (1957). Licht- und elektronenmikroskopische Untereuchungen an dem Dinoflagellaten Amphidimum elegans n.sp. Z. Zellforsch. mikrosk. Anat. 47, 7-17. LEADBEATER, B. & DODGE, J. D. (1966). The fine structure of Woloszynskia micra sp. nov., a new marine dinoflagellate. Br. phycol. Bull. 3, 1—17. LEADBEATER, B. & DODGE, J. D. (1967). An electron microscope study of dinoflagellate flagella. J. gen. Microbiol. 46, 305-314. LEEDALE, G. F., PRINGSHEIM, E. G. & MEUSE, B. J. D. (1965). Structure and physiology of Euglena spirogyra. II. Cytology and fine structure. Arch. Mikrobiol. 50, 70-102. MANTON, I. (1964). Observations with the electron microscope on the division cycle in the Prymnesium parvum Carter. Jl R. microsc. Soc. 83, 317-325. MANTON, I. (1966). Further observations on the fine structure of Ckrysochromtdina chiton, with special reference to the pyrenoid. J. Cell Set. 1, 187-192. MANTON, I. & HARRIS, K. (1966). Observations on the micro-anatomy of the brown flagellate Sphaleromantis tetragona Skuja with special reference to the flagellar apparatus and scales. J. Linn. Soc. (Bot.) 59, 395~4°3- MANTON, I. & STOSCH, H. A. VON. (1966). Observations on the fine structure of the male gamete of the marine centric Lithodesmium undulatum.jfl R. microsc. Soc. 85, 119-134. NICHOLS, H. W., RTDGEWAY, J. E. & BOLD, H. C. (1966). A preliminary ultrastructural study of the freshwater red alga Compsopogon. Ann. Mo. bot. Gdn 53, 17-27. UEDA, K. (1961). Structure of plant cells with special reference to lower plants. VI. Structure of chloroplasts of algae. Cytologia 26, 344-358. {Received 30 June 1967) J. D. Dodge

Except where stated all figures are of Aureodinium pigmentosum and are of material fixed in glutaraldehyde/osmium and stained with uranyl acetate and lead citrate. Journal of Cell Science, Vol. 3, No. 1

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Fig. 2. A longitudinal section of a cell to show the peripheral arrangement of chloro- plasts (c), parts of several pyrenoids (p), the nucleus («) and other organelles. x 20 600. J. D. DODGE (Facing p. 48) Journal of Cell Science, Vol. 3, No.

Fig. 3. A median section through a chloroplast to show the large number of more or less parallel lamellae composed of 2-4 thylakoids, the granular stroma with dark lipid droplets and the thick chloroplast envelope. Stained with lead citrate, x 70000. J. D. DODGE Journal of Cell Science, Vol. 3, No. 1

Fig. 4. Section of a chloroplast situated immediately beneath the . Note the variable number of thylakoids per lamella, x 80000. Fig. 5. A chloroplast with few lamellae, most of which consist of at least 10 thylakoids. x 70000. J. D. DODGE Journal of Cell Science, Vol. 3, No. 1

Figs. 6, 7. Parts of two chloroplasts highly magnified in order to show the three- membrane structure of the chloroplast envelope (e). x 104500.

J. D. DODGE

Figs. 8, 9. Thylakoids of disrupted chloroplasts of Woloszynskia micra. Fig. 8. Shadowed preparation showing some evidence of granular structure in the surface of the thylakoid membrane, x 35000. Fig. 9. Negatively stained preparation (PTA), showing a concentric arrangement of thylakoid discs of decreasing diameter, x 50000. Journal of Cell Science, Vol. 3, No. 1

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J. D. DODGE Figs. 10-13. Pyrenoids. All x 50000. Fig. 10. A small, possibly developing, pyrenoid attached to the inner surface of a chloroplast. Fig. 11. A larger pyrenoid clearly surrounded by a complex membrane but lacking any polysaccharide halo. Fig. 12. A well-developed stalked pyrenoid of Glenodinium grown in strong-light conditions. Note the broad, electron-transparent halo. Fig. 13. An unusually shaped pyrenoid showing the complex membrane around the granular body but the absence of any membrane around the clear halo. Note the presence of 2-thylakoid lamellae within the pyrenoid. Journal of Cell Science, Vol. 3, No. 1

J. D. DODGE