J. Cell Sri. 3, 41-48 (1968)
Printed in Great Britain
41
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 consisting 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 conditions. 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 phosphate 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 mitochondria 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 endoplasmic reticulum or any other organelle, nor haveribosomesbeen 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 chloroplast 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 encountered (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 quantasomes, 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 nonexistent.
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 microscopy 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 cytoplasm.
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-green algae, to the simple structure in the
red algae 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 Dinophyceae in this emerging pattern.
Clearly the dinoflagellate chloroplasts are more complex than those of the Rhodophyceae 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 diatoms (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 Cryptophyceae, 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 Chlorophyceae, which generally have much more complex chloroplasts, we are
left with the Haptophyceae and the Euglenophyceae. In the former class both Chrysochromulina 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), theflagella(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.
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S. P. (1962c). The ultrastructure of the chloroplasts of algae, J. Ultrastruct. Res. 7,
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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.
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LEEDALE, G. F., PRINGSHEIM, E. G. & MEUSE, B. J. D. (1965). Structure and physiology of
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{Received 30 June 1967)
GIBBS,
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
> 2
Fig. 2. A longitudinal section of a cell to show the peripheral arrangement of chloroplasts (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 cell membrane. 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 threemembrane 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