J. Cell Set. 49, 401-409 (1981)
Printed in Great Britain © Company of Biologists Limited 1081
STRUCTURE OF THE CHLOROPLAST AND
ITS DNA IN CHLOROMONADOPHYCEAN
ALGAE
ANNETTE W. COLEMAN AND PETER HEYWOOD
Division of Biology and Medicine, Brown University, Providence,
Rhode Island 02912, U.S.A.
SUMMARY
The arrangement and ultrastructure of chloroplasts is described for the Chloromonadophycean
algae Gonyostomum semen Diesing and Vacuolaria virescens Cienkowsky. The chloroplasts are
present in large numbers and are discoid structures approximately 3-4 /im in length by 2-3 /i-m
in width. In Gonyostomum semen the chloroplasts form a single layer immediately interior to the
cell membrane; frequently their longitudinal axis parallels the longitudinal axis of the cell.
The chloroplasts in Vacuolaria virescens are more than 1 layer deep and do not appear to be
preferentially oriented. In both organisms, chloroplast bands usually consist of 3 apposed
thylakoids, although fusion and interconnections between adjacent bands frequently occur.
External to the girdle band (the outermost thylakoids) is the chloroplast envelope. This is
bounded by endoplasmic reticulum but there is no immediately apparent continuity between
this endoplasmic reticulum and the nuclear envelope. Electron-dense spheres in the chloroplast
stroma are thought to be lipid food reserve. Ring-shaped electron-translucent regions in the
chloroplast contain chloroplast DNA. The DNA is distributed along this ring in an uneven
fashion and, when stained, resembles a string of beads. Each plastid has 1 ring, and the ring
is unbroken in the intact plastid.
INTRODUCTION
Some features of chloroplast ultrastructure in the Chloromonadophyceae have
been described in previously published accounts of these algae (Heywood, 1977,
1980; Koch & Schnepf, 1967; Mignot, 1967, 1976). The purpose of this paper is to
describe the chloroplasts of Gonyostomum semen and Vacuolaria virescens in greater
detail and to correlate these findings with observations on light-microscope preparations in which the chloroplast DNA has been rendered fluorescent with 4',6-diamidino
-2-phenylindole (DAPI).
MATERIALS AND METHODS
Unialgal cultures of Gonyostomum semen and Vacuolaria virescens were grown at 18 ± 1 deg. C
on defined medium (Heywood, 1973), which was aerated with a gas mixture of 4 % C0 2 in air,
or in soil/water tubes lacking CaCO3 supplementation. Illumination (2000 lx) was supplied
by Ecko daylight fluorescent tubes either continuously or intermittently (16 h light alternating
with 8 h dark). Olisthodiscus luteus was grown in f/2 medium (Guillard, 1975) at 24 °C in
1000 lx fluorescent light.
402
A. W. Coleman and P. Heywood
Chloromonadophycean chloroplasts
403
For electron microscopy, cells harvested by gentle centrifugation were fixed for 1 h in a 2 %
solution of glutaraldehyde buffered with 0-07 M-phosphate buffer at pH6'S. After several
washes in buffer they were post-fixed in buffered 1 % osmium tetroxide, dehydrated in a
graded ethanol series, and embedded in Epon. Light microscopy of cells prepared in this
manner utilized io-/tm thick sections, which were examined with Nomarski differential
interference optics (e.g. Fig. 2). Ultrathin sections were cut with a diamond knife, stained with
aqueous uranyl acetate and lead citrate, and examined using a Philips 201S electron microscope
(e.g. Figs. 1, 3-18). Ribbons of serial sections were collected on single-hole copper grids coated
with Formvar and carbon.
Observations of chloroplast DNA were made in situ using fluorescence microscopy of cells
stained with the DNA-binding fluorochrome, DAPI, with or without prior enzyme treatment
as described by Coleman (1979). Chloroplasts isolated into the H medium of Shepard (1970)
were vitally stained with 0-5 /ig/ml DAPI and observed directly with an epifluorescence
microscope. Similar staining (0-5-0-1 /tg/ml DAPI in Mcllvaine's buffer, pH 4-0) was applied
to cells or isolated plastids, variously manipulated, that had been pressed beneath a cover slip
onto gelatin-coated slides, frozen in liquid nitrogen, and fixed in 3 :1 (ethanol: acetic acid) or
in 70 % ethanol.
OBSERVATIONS
Gonyostomum semen and Vacuolaria virescens are biflagellate unicells that frequently
reach 60-80 /tm in length. The cells contain 200-500 chloroplasts, which are approximately 3-4 /tm in length by 2-3 /tm in width and are disk-shaped or plano-convex
(Fig. 1). In Gonyostomum semen the plano-convex chloroplasts usually have their
flattened surface adjacent to the cell membrane (Heywood, 1968; Hovasse, 1945);
in this organism the chloroplasts form a single layer immediately interior to the cell
membrane (Fig. 2) and frequently their longitudinal axis parallels the longitudinal
axis of the cell (Heywood, 1968; Hovasse, 1945), but this is not always the case
(Mignot, 1976). In Vacuolaria virescens the chloroplasts are several layers deep and
do not appear to be oriented in any particular direction (Fig. 1).
Chloroplast bands are arranged approximately parallel to the longitudinal axis of
the chloroplast; frequent interconnections exist between adjacent bands (Fig. 3).
Each band consists of 3 thylakoids. The girdle band, which forms the periphery of
the chloroplast, has the same structure as the other bands and is often in continuity
Fig. 1. Section through a cell of Vacuolaria virescens. Chloroplasts occur between the
cell membrane and the layer of cytoplasm surrounding the nucleus (n). Also present
is an extensive Golgi region (g), which is adjacent to the contractile vacuole (cv).
x 4000.
Fig. 2. Two cells of Gonyostomum semen photographed using Nomarski differential
interference microscopy. The cell on the left is in oblique longitudinal section while
the cell on the right is in transverse section. The chloroplasts (c) occur as a single row
immediately interior to the cell membrane and a nucleus (n) is also present, x 1000.
Fig. 3. A chloroplast of Gonyostomum.semen. Note the lipid droplet (/) and the
interconnections between bands (1) and between bands and the girdle band (arrowheads). External to the girdle band (g) are the chloroplast envelope and endoplasmic
reticulum (ce/er). x 25 000. An electron-translucent region of the chloroplast occurs
immediately adjacent to the girdle band; this region (solid arrow) is shown at higher
magnification in the inset. It contains fine fibrillar structures (open arrow) which are
presumptive chloroplast DNA. The second inset (from another chloroplast) also
shows this same type offibrillarstructure. Inset magnification is x 80000.
A. W. Coleman and P. Heywood
8
Chloromonadophycean chloroplasts
405
with them. The chloroplast stroma contains large numbers of small electron-opaque
structures, which are assumed to be chloroplast ribosomes. Also present in the stroma
are osmiophilic spheres (Fig. 3), which are thought to be a lipid food reserve. Pyrenoids
occur in Chattonella subsalsa (Mignot, 1976) but they appear to be absent in Gonyostomum semen and Vacuolaria virescens. Eyespots have not been observed but the cells
are positively phototactic towards light of moderate intensity (Chapman & Haxo,
1966; Poisson & Hollande, 1943) and a diurnal movement has been reported in
natural populations of Gonyostomum semen (Cowles & Brambel, 1936).
External to the girdle band is the chloroplast envelope, which is bounded by
endoplasmic reticulum (Figs. 3-15). Presumably the latter helps to maintain the
chloroplast in position and also ensures some degree of membrane contact between
the chloroplast and other organelles, but a direct continuity between the nuclear
envelope and this endoplasmic reticulum is not apparent.
Longitudinal sections through chloroplasts frequently reveal the presence of an
electron-translucent region immediately interior to the girdle band (Figs. 3-18).
This region lacks chloroplast bands and resembles the ring-shaped chloroplast
nucleoids in the Bacillariophyceae, Chrysophyceae, Phaeophyceae and Xanthophyceae
(Bisalputra, 1974; Gibbs, 1970; Taylor, 1976). Serial sections (Figs. 4-15, Figs. 16-18)
confirm that this electron-translucent region is a continuous structure, and indicate
that its width is variable (compare Figs. 6, 7 with Fig. 8 and Figs. 11—13 with Figs. 14,
15). The electron-translucent region contains irregularly arranged fibrils, which
resemble DNA in morphology and dimensions (Fig. 3).
Confirmation of the presence of chloroplast DNA is provided by examining
Vacuolaria (Fig. 19) and Gonyostomum (Fig. 21) cells stained with DAPI, a fluorochrome with high affinity for double-stranded DNA. Both in isolated, unfixed
chloroplasts and in fixed material there is 1 ring of DNA per plastid (Fig. 22). The
normal, nodulated appearance of the ring is shown clearly in Figs. 19-21. The
physical continuity of this complex nucleoid is apparent when a ring isolated from a
plastid is stretched before fixation (Fig. 23). Fixed preparations treated with DNase
and then stained fail to reveal any such rings, while pretreatment with RNase or
trypsin leaves the rings intact, as detected by DAPI staining. Similar brightlystaining rings can be observed in Olisthodiscus (Fig. 24).
Figs. 4-15. Serial sections through one end of a chloroplast of Vacuolaria virescens.
Fig. 4 is a section close to one side of the chloroplast while Fig. 15 is an approximately
median longitudinal section. The asterisk in Fig. 4 has been placed in the electrontranslucent structure adjacent to the girdle band. Successive sections indicate that the
electron-translucent region is a continuouus structure whose width is variable.
Chloroplast DNA is not distinguishable in these micrographs. The chloroplast
envelope and its surrounding layer of endoplasmic reticulum (ce/er) occur exterior
to the girdle band (g) and are usually in close apposition, but these layers can be
distinguished in Fig. 14. x 23 300.
Figs. 16-18. Serial sections through one side of a chloroplast of Vacuolaria virescens.
The asterisks in Fig. 16 have been placed in the electron-translucent structure
adjacent to the girdle band. A few chloroplast bands are present in Figs. 16, 17 but
these are absent in Fig. 18, which is a glancing section through the girdle band and
the electron-translucent region immediately interior to it. x 18000.
A. W. Coletnan and P. Heywood
406
19
Fig. 19. Montage of squashed Vacuolaria virescens cell, fixed and treated with RNase
prior to staining with DAPI. Fluorescence photomicrograph reveals a large, pawshaped structure, the broken nucleus, and many chloroplast DNA rings spread about,
some slightly stretched. Bar, io/(m. x 700.
Fig. zo. Fluorescent chloroplast DNA rings from isolated Vacuolaria plastids,
pretreated with RNase and then stained with DAPI. Beaded aspect is characteristic,
x 1700.
Fig. 21. DAPI fluorescence photomicrograph of squashed, stained cell of Gonyostomum semen, illustrating bright centrally-located nucleus and surrounding plastid
DNA rings. Bar, 10 fim. x 1400.
Fig. 32. A (fluorescence) and B (phase contrast) photomicrographs of a field of isolated
Gonyostomum chloroplasts, fixed in 70 % ethanol to retain the characteristic plastid
phase density, and stained with DAPI. Each plastid contains one DNA ring, seen
here either in face or edge view, x 2000.
Fig. 23. Fluorescence photomicrograph of stretched DNA rings from isolated
Gonyostomum plastids, fixed and stained with DAPI. Longest ring visible is 55 fim
in circumference, x 1000.
Fig. 24. Whole cell of OHsthodiscus, slightly squashed, fixed and stained with DAPI.
x 1200.
Chloromonadophycean chloroplasts
408
A. W. Coletnan and P. Heywood
DISCUSSION
Chloromonadophycean chloroplasts are often a distinctive bright green colour
(Drouet & Cohen, 1935; Poisson & Hollande, 1943). The presence of chlorophylls a
and c has been demonstrated by Guillard & Lorenzen (1972), and features of their
structure are shared by 4 of the 5 other major algal groups that possess these chlorophylls, the Bacillariophyceae, Chrysophyceae, Phaeophyceae and Xanthophyceae.
In particular, the Chloromonadophyceae resemble these algae in the possession of a
ring-shaped electron-transparent region immediately inside the girdle band. Fibrils
that resemble DNA in morphology and dimensions (Fig. 3) occur in this region. The
presence of 1 ring of DNA per chloroplast has been demonstrated in both Gonyostomum semen (Figs. 21, 22) and Vacuolaria virescens (Fig. 19). Similarly, there is one
ring of DNA per chloroplast in Olisthodiscus (Fig. 24), an organism which may be a
member of the Chloromonadophyceae (for a discussion of this point see Gibbs, Chu
& Magnussen, 1980, and Loeblich & Fine, 1977). Fluorochrome staining has revealed
similar rings in diatom and brown algal cells (Coleman, 1979).
Biochemical evidence suggests that the chloroplast nucleoid in Olisthodiscus is
multiploid (Cattolicco, 1978; Aldrich & Cattolicco, 1979), and preliminary microspectrophotometric evaluation of the Gonyostomum plastid DNA ring suggests a
similar situation (Coleman, unpublished observations). This multi-genome state of
the plastid in organisms with ring-shaped DNA nucleoids is analogous to the situation
in green algae and higher plants, where the plastid DNA is also multiploid (reviewed
by Herrman & Possingham, 1980). In this latter situation, DNA aggregates can be
seen irregularly dispersed among the thylakoids stacks; the possible continuity of the
various DNA centres within a single plastid has not yet been established.
We are grateful to Dr R. R. L. Guillard for the gift of an Olisthodiscus luteus culture, and to
Mr Mark Maguire for his dedicated assistance. This investigation was supported by a Biomedical Sciences Support Grant from Brown University and by NSF Grants DEB-76-82919,
PCM 78-15783 and PCM-79-23054.
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(Received 13 October 1980)