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. DODGEDepartment of Botany, Birkbeck College, London, W.C. 1

SUMMARYThe 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 arearranged, in a more or less parallel manner, many lamellae normally composed of three apposedthylakoids, 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 shapewith a diameter of 0*15-3-6 /* and a mean thickness of 240 A. Ribosomes, lipid droplets andDNA areas are present in the chloroplast stroma. No connexions were seen between thechloroplasts and any other organelles, nor did the chloroplasts contain girdle lamellae. Stalkedpyrenoids, which are found in some dinoflagellates, are shown to arise from the inner face of thechloroplasts, to contain a finely granular material and to be frequently surrounded by anelectron-transparent area. These findings are discussed in relation to the fine structure of thechloroplasts and pyrenoids of other algal classes.

INTRODUCTION

Although the chloroplasts and pyrenoids of many algae have been extensivelystudied by electron microscopy over the past few years, those of dinoflagellates havereceived little attention. The first published electron micrograph of a sectioneddinoflagellate was of Amphidinium elegans (Grell & Wohlfarth-Botterman, 1957). Thisshowed the chloroplast to be lamellate with each lamella consisting of a number ofparallel membranes. Ueda (1961) reported that the chloroplasts of Ceratium andDinophysis were four-lamellate. In the present terminology this meant that thelamellae each consisted of three thylakoids. Later, Gibbs (1962 b, c), in a survey of thechloroplasts and pyrenoids of several algal classes, found that in Amphidinium carterithe lamellae (or bands) consisted of three or four apposed thylakoids (or discs).Occasional lamellae split into two, and the lamellae were normally so close togetherthat there was little space for chloroplast matrix. This result may have been caused bythe particular osmium tetroxide fixative used. Gibbs also found that A. carteri hadone single, central, starch-sheathed pyrenoid with a number of chloroplast lamellaepenetrating its dense ground substance. Bouck & Sweeney (1966), in a study ofdinoflagellate trichocysts, incidentally showed sections of the radially orientatedchloroplasts of Gonyaulax polyedra. These appeared to contain numerous two or threethylakoid lamellae arranged parallel to the long axis of the chloroplast. A somewhatsimilar 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 oflamellae was observed. In this organism elongated or flattened pyrenoids were foundbetween the lamellae of the chloroplast.

In the present paper the detailed structure of the chloroplasts of some small marinedinoflagellates will be described in detail for the first time and compared with thestructure of chloroplasts in other algae. The single-stalked pyrenoids found in anumber 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 atpH 7-0 with sucrose added to give a molarity of 0-2 M. This fixative was used eithercold for 1-2 h or at 20 °C for 5 min to i\ h and followed, after several washings inbuffer, 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 orEpon, sectioned with an LKB microtome and examined in a Zeiss EM 9 electronmicroscope.

Whole mounts of broken chloroplasts were prepared by the following method. Adense suspension of cells was transferred to o-8 M sucrose in TRIS buffer at pH 7-8and 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 containedmostly 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 asAureodinium pigmentosum they are probably saucer-shaped and are peripheral inposition (Fig. 2). In larger organisms such as Gonyaulax tamarensis they are frequentlylens-shaped and radial in position. A peripheral reticulate arrangement is seen inExuviaella and Prorocentrum. The number of chloroplasts seems to be variable evenwithin a species.

The chloroplasts are surrounded by a distinct bounding membrane. With certainfixations 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 verywrinkled, 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 thanthe width of a single thylakoid in the same micrograph, suggesting that whateverswelling 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 theouter surface of the envelope.

I 230 A

240 A

Fig. 1. Diagrammatic representation of part of a dinoflagellate chloroplast. A. Thestructure of the chloroplast envelope with its three membranes. B. Part of a lamellaconsisting of three apposed thylakoids.

The chloroplasts contain numerous lamellae which are oriented parallel to thelonger 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, wherebranching has been found, there are normally no interconnexions between lamellae.Girdle lamellae have not been seen in any of the dinoflagellates examined. Eachnormal lamella consists of a number of apposed thylakoids (or 'discs' in the olderterminology) giving in cross-section the appearance: thin dark line, clear space, thickdark line, and so on, where the thick dark lines correspond to two apposed thylakoidmembranes. Most frequently each lamella consists of three thylakoids, but four andtwo (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 theyhave generally been found in cells from old cultures.

The thickness of the thylakoids, as seen in section, shows a considerable variationwhich 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 only190 A in width, but in the separate pairs of thylakoids of Fig. 6 (both Figs. 5 and 6 arefrom the same fixation) the width averages 380 A. The mean value for all measurementsmade is 240 A (Fig. 1), of which the outer membranes account for about 60 A eachand the central space about 120 A.

44 J- D. Dodge

From sectioned cells it is difficult to ascertain the shape of individual lamellae andthylakoids. However, by breaking cells and separating the chloroplast fraction it waspossible to examine the form of the thylakoids. They are seen (Figs. 8, 9) almostalways to be circular discs which exhibit a considerable variation in diameter. InWoloszynskia the size ranged from 0-15 to 3-6 /i, which compares favourably withfigures of 0-6-4-0/* obtained from randomly sectioned chloroplasts. In shadowedthylakoids (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 granularmaterial with much variation in the size of the granules. The larger particles (compareFig. 4) are probably ribosomes as they stain densely with uranyl salts and are 140-200 Ain diameter. Occasionally one finds large areas of stroma (Fig. 3) not crossed by anylamellae. These may be regions which will become extruded from the chloroplast aspyrenoids or they may simply be areas of chloroplast where the lamellae are stillforming. 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 bytreatment with DNase, consist of DNA. DNA is not nearly so common as in thechloroplasts of Woloszynskia (Leadbeater & Dodge, 1966).

Pyrenoids

It would appear that in the class Dinophyceae as a whole several types of pyrenoidare found. The present account will be confined to the simple stalked pyrenoids whichhave so far been found in Aureodinium pigmentosum and in Glenodinium (Floridaisolate). These pyrenoids are situated on the inner side of the chloroplasts (Fig. 2), towhich 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 chloroplastenvelope and it contains uniformly granular material (Figs. 10-13) which contrastswith the irregular granularity of the chloroplast stroma. It would appear to lackribosomes and probably consists of protein. Occasional pyrenoids are found to containone or more pieces of chloroplast lamella, usually consisting of not more than twothylakoids (Fig. 13).

Surrounding what appear to be mature pyrenoids (that is, those with stalks) isfound a wide halo of electron-transparent material (Figs. 12, 13) which sometimescontains bands of rather electron-opaque material. The pyrenoid and halo appeared tobe much larger in material grown under high light intensity. The halo almost certainlyconsists of polysaccharide and when tested with iodine and examined by light micro-scopy the pyrenoids became stained, but not apparently the very deep blue colourgiven by starch. No membrane surrounds the pyrenoid halo and it abuts on normalcell cytoplasm.

Dinoflagellate chloroplasts andpyrenoids 45

DISCUSSION

The fine structure of algal chloroplasts has been found to have a distinctive form inmany of the algal classes. This structure shows what may be a developmental seriesfrom the primitive arrangement in the blue-green algae, to the simple structure in thered algae and ultimately to the complex arrangement of the lamellae in many of thegreen 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 ofthe Phaeophyceae where the adjacent thylakoids are not fused and where girdlelamellae and endoplasmic reticulum outer envelopes are found (Gibbs, 1962 c; Bouck,1965; Evans, 1966). The dinoflagellates also differ from certain diatoms (Drum &Pancratz, 1964; Manton & von Stosch, 1966), the Chrysophyceae (Gibbs, 1962 a;Manton & Harris, 1966) and the Xanthophyceae (Greenwood, 1959) which all possessgirdle lamellae. The Cryptophyceae, reputedly closely allied to the Dinophyceae,differ from them in regularly having two-thylakoid lamellae and also possessing anouter endoplasmic reticulum envelope (Gibbs, 1962c; Greenwood, 1967). Apart fromthe Chlorophyceae, which generally have much more complex chloroplasts, we areleft with the Haptophyceae and the Euglenophyceae. In the former class both Chryso-chromulina chiton (Manton, 1966) and Prymnesium parvum (Manton, 1964) havechloroplasts which could be mistaken for those of a dinoflagellate but for the factthat they have a double endoplasmic reticulum envelope outside the double chloroplastenvelope. The Euglenophyceae (Gibbs, i960; Leedale, Pringsheim & Meuse, 1965)have chloroplasts which appear very similar to those of the Dinophyceae, even to thetriple-layered envelope. Gibbs stated that the envelope was double but did say that onoccasion 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 isin fact composed of three layers and has connexions with the nuclear envelope by wayof tubular endoplasmic reticulum. In the Xanthophyceae a triple chloroplast envelopehas been found in Vaucheria, Botrydium (Greenwood, 1964) and various other genera(G. F. Leedale, D. Hibberd & A. Massalski, personal communication). Here again theenvelope has distinct endoplasmic reticulum connexions. Thus the only structuralfeature which distinguishes the chloroplast of the Dinophyceae from other chloroplastsis the absence of endoplasmic reticulum connexions with the three-layered envelope.

It is not easy to account for the triple chloroplast envelope, for most biologicalstructures which employ membranes (as mitochondria, Golgi bodies, nuclear envelope,endoplasmic reticulum) normally use only two. A possible explanation is suggestedby the work on the Euglenophyceae and Xanthophyceae cited above. Here the outerof the three membranes is continuous with the endoplasmic reticulum. As mostorganisms with endoplasmic reticulum attached to the chloroplast (Chrysophyceae,Phaeophyceae, Haptophyceae) have a quadruple envelope, in which the outer twomembranes 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 thepresent work it has been noticed that the central membrane sometimes appearsslightly thicker than the outer two, although this difference has not yet been adequatelymeasured. One point against the endoplasmic reticulum hypothesis is that in theDinophyceae no ribosomes have been found adhering to the chloroplast envelope. Ifthe outer membrane is part of the reticulum ribosomes would have been expected andare present, for example, on the outer of the four membranes surrounding thechloroplasts of the Haptophyceae (Manton, 1964, 1966).

The simple stalked pyrenoids described in the present paper are similar, apart fromthe apparent absence of an outer endoplasmic reticulum sheath, to stalked pyrenoidswhich 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 relatedmight be thought to have some phylogenetic significance were it not for the fact thatother dinoflagellates are known which have at least three other differing types ofpyrenoid (Gibbs, 19626; Leadbeater & Dodge, 1966, and unpublished observations).Drum & Pancratz (1964) found a similar situation in the Bacillariophyceae. It is clearthat pyrenoids will never provide the distinctive character for dinoflagellates such as isalready provided by the nuclei (Dodge, 1966), the flagella (Leadbeater & Dodge, 1967),and to some extent by the chloroplasts. However, the different pyrenoid types may beof some significance at the generic level, and this is currently being investigated.

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

REFERENCESBOUCK, 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 brown algae. 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 Euglena 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 Plastids.London: Freeman.

GRELL, K. G. & WOHLFARTH-BOTTERMAN, K. E. (1957). Licht- und elektronenmikroskopischeUntereuchungen 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 newmarine 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 ofEuglena spirogyra. II. Cytology and fine structure. Arch. Mikrobiol. 50, 70-102.

MANTON, I. (1964). Observations with the electron microscope on the division cycle in theflagellate Prymnesium parvum Carter. Jl R. microsc. Soc. 83, 317-325.

MANTON, I. (1966). Further observations on the fine structure of Ckrysochromtdina chiton, withspecial reference to the pyrenoid. J. Cell Set. 1, 187-192.

MANTON, I. & HARRIS, K. (1966). Observations on the micro-anatomy of the brown flagellateSphaleromantis 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 malegamete of the marine centric diatom Lithodesmium undulatum.jfl R. microsc. Soc. 85, 119-134.

NICHOLS, H. W., RTDGEWAY, J. E. & BOLD, H. C. (1966). A preliminary ultrastructural studyof 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 ofchloroplasts 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 materialfixed in glutaraldehyde/osmium and stained with uranyl acetate and lead citrate.

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> 2Fig. 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)

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Fig. 3. A median section through a chloroplast to show the large number of more orless parallel lamellae composed of 2-4 thylakoids, the granular stroma with darklipid droplets and the thick chloroplast envelope. Stained with lead citrate, x 70000.

J. D. DODGE

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Fig. 4. Section of a chloroplast situated immediately beneath the cell membrane. Notethe 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

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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.

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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 thepresence of 2-thylakoid lamellae within the pyrenoid.

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