J. Cell Sri. 3, 41-48 (1968) 41 Printed in Great Britain THE FINE STRUCTURE OF CHLOROPLASTS AND PYRENOIDS IN SOME MARINE DINOFLAGELLATES 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 chloroplast are arranged, in a more or less parallel manner, many lamellae normally composed of three apposed thylakoids, 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 algae 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 dinoflagellate 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 Dinophysis 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 trichocysts, incidentally showed sections of the radially orientated chloroplasts of Gonyaulax polyedra. These appeared to contain numerous two or three thylakoid 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.