J. Cell Set. is, 481-494 (1974)
Printed m Great Britain
VARIATION IN EYESPOT ULTRASTRUCTURE
IN CHLAMYDOMONAS REINHARDI (ac-31)
HELEN E. GRUBER
Department of General Science, Oregon State University,
Corvallis, Oregon 97331, U.S.A.
AND BENJAMIN ROSARIO
Biology Dept., Battelle Pacific Northwest Laboratories,
Richland, Washington 99352, U.S.A.
SUMMARY
Several morphological variations in eyespot complex fine structure were exhibited in some
cells of the pale-green mutant strain ac-31 of Chlamydomonas reinhardi. The cells were grown
in minimal medium supplemented with 0-2 % sodium acetate and were harvested by centrifugation and prepared for electron-microscopic examination.
Microtubules were seen near the flagella, confirming previous observations. Microtubules
were also seen near the eyespot complex. Although a direct connexion between the microtubules
of the flagellar and eyespot region was never observed, this does not exclude the possibility
that it exists and thus provides a structural and functional connexion between the twoorganelles.
Occasionally irregular curved bodies intimately associated with the eyespot complex of some
cells appeared to displace the chloroplast and cell membranes. These bodies often appeared to
be nearly covered by a limiting membrane and were found near empty ' cavities' in the eyespot
plate. Crystalline arrays of dense bodies were observed in some sections. The significance of
these bodies is discussed in relation to the functional state of the carotenoid pigments making
up the eyespot granules. An hypothesis for the formation of the rod-like structures is presented,
based upon the observation of granules which had fused together to form a helix.
INTRODUCTION
The eyespot of algae has remained an enigma for more than a century. Ehrenberg
(1838) studied and observed it in freshwater dinoflagellates, and hypothesized that it
functioned as an eye. Others envisioned that this pigmented area of the cell was a
highly organized series of lenses and pigment cups which focused light, causing a
flagellar response (Mast, 1927). Many of the early hypotheses have been disproved by
recent experimental techniques, but as pointed out by Dodge (1969) knowledge of
the function of the eyespot is still only speculative. How it functions and whether or
not it is light-sensitive are still unanswered questions. Current thinking on eyespot
function has focused upon 2 possibilities (Arnott & Brown, 1967): that the eyespot is
truly a primary photoreceptor, sensing light and transferring this information to the
motor apparatus of the cell; or that the eyespot acts as a shade over some other photosensitive area of the cell. Dodge (1969) stated that algal eyespots, which vary widely
in structure and complexity, have only 2 common features: they occupy a specific
location in the cell, and they are composed of carotenoid pigments localized in lipid
or osmiophilic granules.
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The work presented here describes the ultrastructure of the eyespot of a pale-green
mutant strain of Chlamydomonas reinhardi. Originally isolated and characterized
ultrastructurally by Goodenough, Armstrong & Levine (1969), this acetate-requiring
strain shows no stacking of its chloroplast thylakoids - the disks formed by the
chloroplast membranes are not as closely and uniformly associated as they are in the
wild-type cell. Since this variation in chloroplast morphology was already known to
occur in this mutant, the present study was undertaken to investigate the structure of
the chloroplast-associated organelle, the eyespot.
MATERIALS AND METHODS
Cultures of Chlamydomonas reinliardi, mutant strain ac-31, mating-type minus, kindly provided by Dr R. P. Levine (Goodenough et al. 1969) were grown in a Hotpack Programmed
Refrigerated Incubator at 20-22 °C under a light-dark synchronization regime of 12 h light of
approximately 3230 lx followed by 12 h of darkness. Cultures were grown in minimal media
supplemented with 0-2 % sodium acetate in 30-ml tissue culture flasks (Falcon Plastics) with
loosely fitting caps.
Cells were harvested by centrifugation midway in the light period, fixed 1 h in Karnovsky's
fixative (Karnovsky, 1965) at about 4 °C, rinsed several times with 02 M cacodylate buffer containing 05 % CaCl2, and rinsed overnight in buffer. The cells were then fixed with 2 % OsO«
containing 1 mg/ml CaCl2, dehydrated and embedded in Araldite 502 epoxy resin (Luft,
1961). Sections were examined in a Philips EM 300.
RESULTS
The eyespot is sometimes considered to be a highly differentiated portion of the
chloroplast (Sager & Palade, 1957), and, in Chlamydomonas reinhardi, is found midway
between the anterior and posterior regions of the cell (Fig. 1 A). AS in all of the green
algae, the eyespot is not associated with the flagella (Fig. 1 B). In wild-type Chlamydomonas and some ac-31 cells the eyespot can be seen to consist of two or three layers
or plates of granules (Fig. 1 c). The granules or globules have been reported to be
100-140/im in diameter, with approximately 150 granules per plate (Sager & Palade,
1957). In Fig. i c the granules do not appear to be membrane bound, but lie in an
orderly arrangement' sandwiched' between the chloroplast lamellae. Others have also
reported the lack of a limiting membrane around the granules themselves in other
Chlamydomonas species, Carteria and Tetracystis (Arnott & Brown, 1967; Sager &
Palade, 1957; Lembi & Lang, 1965). When sectioned tangentially, Chlamydomonas'
eyespot plates can be seen to consist of granules which exhibit hexagonal close-packing
(Fig. ID).
In several favourable sections in the present study, microtubules were seen in close
proximity to the eyespot region (arrows in Figs. 2, 3 A).
In the majority of the acetate-requiring cells studied in section, various additional
structures were seen in the area of the eyespot. Representative illustrations are presented in Figs. 2-8. Some cells were seen to possess irregularly shaped, curving,
electron-dense rod-shaped structures which extended various distances across the
eyespot complex. (The appearance of these bodies did not differ significantly when
they were examined in unstained sections.) Three such rod-shaped structures are
Eyespot ultrastructure in Chlamydomonas
483
visible in Fig. 2; the central region of the eyespot, where the bodies occur, seems
somewhat distended. In this section they lie well within the chloroplast and cell
membranes. In Fig. 3 A, however, the central area seems greatly distorted, and the
rod-shaped mass seems to have displaced both the chloroplast and cell membranes.
In Fig. 3B the eyespot complex seems even more distorted and swollen and the membranes appear distended. A possible association between the longer bodies and the
cell wall is indicated at the arrow in Fig. 3B.
The rod-like structures assumed various twisted shapes (Figs. 4, 5B). In Fig. 4 the
denser regions appear at the right-hand edge of the eyespot complex (arrow) and
continue outward, again distending the cell membrane and possibly progressing
through it. In this section the rod-shaped body appears to be nearly covered by a
limiting membrane.
Figs. 5 A, B illustrate yet another property of eyespot ultrastructure that was
observed. Often the dense, irregular bodies were found adjacent to empty 'cavities'
in the eyespot plate. It appears that these spaces were previously occupied by the
eyespot granules. Fig. 5 B also illustrates the presence of a membrane in the central
area of one of the dense rods (arrow). These have been observed to be present over
considerable lengths of the rods.
An 'empty space' was usually present between the granules and the distended
chloroplast and cell membranes. Occasionally, as illustrated in Fig. 6, the rods and
eyespot plate were separated by a narrow isthmus of ribosome-free cytoplasm.
A different type of configuration can be seen in Fig. 7. In addition to the presence
of the dense rods and empty 'cavities', 3 regularly shaped crystalline objects can be
seen on the periphery of the eyespot area (Fig. 7, arrows).
One of the most thought-provoking conformations observed is pictured in the
stereo-pair of micrographs in Fig. 8. On first glance this view appeared to be a section
through 2 plates of granules, each consisting of 2 rows of granules, with 1 row slightly
above and to the side of the other. Upon closer stereo examination, however, it can
be seen that the granules are fused together to form a helix.
DISCUSSION
The presence of microtubules in the eyespot region of Chlamydomonas merits
further consideration. Investigators have long believed that the eyespot and flagella
could work together in the coordination of cellular orientation to light. In Euglena
this is a reasonable assumption, for the flagella insert into a reservoir in the region of
the eyespot (Walne & Arnott, 1967). This concept appears less likely for the green
algae, because the eyespot and flagella are separated by a fairly broad expanse of cytoplasm (Fig. IB). Walne & Arnott (1967) proposed that microtubules could provide a
method of communication between eyespot and flagella, but a direct association
between the 2 organelleshad not been shown. In Chlamydomonas Ringo (1967) demonstrated bands of microtubules passing beneath the cell surface from the region of the
base of theflagella.Figs. 2 and 3 A show the presence of microtubules near the eyespot,
providing some support for such a proposed coordination between the 2 organelles.
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Admittedly, these figures do not reveal the origin or termination of the tubules. There
is good agreement on the close association of such tubules with the flagella in the
anterior region of the cell, but it has not been determined what organelle, if any, is
associated with the other end of these tubules.
The eyespot has been reported to replicate prior to cytokinesis in Chlamydomonas
and Euglena (Ettl, 1971; Kivic & Vesk, 1972). De novo formation in Chlamydomonas
has been proposed, claiming that the eyespot granules could derive from osmiophilic
granules commonly found in the chloroplast, such as those shown in Fig. 3 A. Vegetative cells in forms such as Tetracystis do not have eyespots, so it has been assumed
that they arise de novo. Two pathways for eyespot development have been proposed for
Tetracystis (Arnott & Brown, 1967): the granules could grow by acquiring new substances, or the granules might migrate to the eyespot region after being formed elsewhere in the chloroplast.
Considerably more is known about the physical and chemical nature of the granules.
The action spectra peaks for phototaxis coincide with the absorption maximum for
eyespot granules (Arnott & Brown, 1967; Sager & Palade, 1957; Lembi & Lang, 1965;
Batra & TolJin, 1964) and chemically they are comprised of lipid-soluble carotenoids
(thus differing from the lipoidal osmiophilic granules found in the chloroplast). Four
pigments were isolated from Euglena eyespot granules: lutein and cryptoxanthin
(together forming 83 % of the total carotenoids), /^-carotene, and an unidentified
component (Batra & Tollin, 1964). In the euglenoid eyespot, pigment formation is
inhibited by chloramphenicol, but not by cycloheximide (Kivic & Vesk, 1972). One
of the first cellular responses to a 6-h exposure of Chlamydomonas to colchicine is the
dissociation of the eyespot and its appearance in the posterior region of the cell
(Walne, 1967).
In tangential section the individual granules show hexagonal close-packing, an
arrangement to be expected when spherical bodies of equal size are packed together
(Arnott & Brown, 1967). Compression of granules could occur either from increasing
granule size or number, or growth of the chloroplast, with concomitant encroachment
upon the space occupied by the eyespot granules.
The granules are not enclosed in a membrane, but chloroform extraction of Chlamydomonas removed the central portion of the granules, coincident with the appearance
of 'individual delimiting membranes' (Walne & Arnott, 1967). The authors believed
that the membrane probably represented the 'interface' between the granule and the
surrounding portion of the chloroplast.
Two instances have been reported in the literature in which irregularly shaped
bodies were found in the eyespot region. In Euglena they were present in etiolated
(bleached) cells, where the globules often fused and were less electron-dense than the
globules of light-grown cells (Kivic & Vesk, 1972). In Chlamydomonas eugametos,
irregular curved bodies have been observed (Walne & Arnott, 1967), but these did
not protrude from the cell surface. It was proposed that the curved bodies might be
due to a change in oxidation state of the carotenoid pigments, such as a- to /9-carotene,
or to lycopene. Walne & Arnott (1967) also proposed that this could result in a change
from a lipid-soluble state to a crystalline state. This has special significance with
Eyespot ultrastructure in Chlamydomonas
485
respect to the structures shown in Fig. 7. A change in functional state or cellular ageing
have also been suggested as causes for the structures observed in Chlamydomonas
eugametos (Walne & Arnott, 1967). Senescence seems a less likely explanation in view
of the formation of similar bodies in Euglena after bleaching. A change in function is
more in agreement with their presence in the acetate species of Chlamydomonas used
in this study, since it requires the acetate supplement in addition to normal photosynthetic activity.
The conformation observed in Fig. 8 suggests that fusion of granules from adjacent
or the same row of granules could account for the formation of the twisting irregular
bodies. Fusion of rows could explain the membranes observed in Figs. 4 and 5B, and
the 'cavities' observed in some plates. A change in functional or chemical state, such
as that proposed by Walne & Arnott (1967) might be one of the initiators of this
morphological change. This could be common to algal strains which do not rely
entirely upon photosynthesis for their energy source, but which in part utilize alternate pathways.
The authors would like to express sincere thanks to Dr James Hampton for his guidance
and encouragement in this work and his comments on the manuscript, and Roy Adee and Victor
Faubert for their helpful discussions and suggestions.
This research was supported by the USAEC Contracts AT(4S-i)-2O42 and A T ( 4 5 - I ) - I 8 3 O
while H. Gruber was on a Northwest College and University Association for Science Appointment.
REFERENCES
ARNOTT, H. J. & BROWN,R., JR. (1967). Ultrastructure of the eyespot and its possible significance
in phototaxis of Tetracystis excentrica. J. Protozool. 14, 529-539.
BATRA, P. P. & TOLLIN, G. (1964). Phototaxis in Euglena. I. Isolation of the eye-spot granules
and identification of the eye-spot pigments. Biochim. biophys. Ada 79, 371-378.
DODGE, J. D. (1969). A review of the fine structure of algal eyespots. Br. Phycol.J. 4, 199-210.
EHRENBERC, C. G. (1838). DieInfusionsthierchenah vollkommene Organismen. Leipzig: Cited in:
Mast, S. O. (1927). Structure and function of the eye-spot in unicellular and colonial organisms. Arch. Protistenk. 60, 197-220.
ETTL, H. (1971). The first protoplast division in the course of asexual reproduction in Chlamydomonas (On the knowledge o(Chlamydomonas). Ost bot. Z. 119, 521-530. (Biol. Abstr. (1973)
55, no. 6933.)
GOODENOUGH, U. W., ARMSTRONG, J. J. & LEVINE, R. P. (1969). Photosynthetic properties of
ac-31, a mutant strain of Chlamydomonas reinhardi devoid of chloroplast membrane stacking.
Plant Physiol., Lancaster 44, 1001-1012.
GRAY, E. G. & WILLIS, R. A. (1968). Problems of electron stereoscopy of biological tissue.
J. Cell Set. 3, 309-326.
KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use in
electron microscopy. .7. Cell Biol. 27, 137A.
KIVIC, P. A. & VESK, M. (1972). Structure and function in the euglenoid eyespot apparatus:
the fine structure, and response to environmental changes. Planta 105, 1-14.
LEMBI, C. A. & LANG, N. J. (1965). Electron microscopy of Carteria and Chlamydomonas.
Am. J. Bot. 52, 464-477.
LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. biophys. biochem.
Cytol. 9, 409-414.
MAST, S. O. (1927). Structure and function of the eye-spot in unicellular and colonial organisms. Arch. Protistenk. 60, 197-220.
RINGO, D. L. (1967). Flagellar motion and fine structure of the flagellar apparatus in Chlamydomonas. J. Cell Biol. 33, 543-571.
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R. & PALADE, G. E. (1957). Structure and development of the chloroplast in Chlamydomonas. I. The normal green cell. J. biophys. biochem. Cytol. 3, 463-488.
WALNE, P. L. (1967). The effects of colchicine on cellular organization in Chlamydomonas. II.
Infrastructure. Am. J. Bot. 54, 564-577.
WALNE, P. L. & ARNOTT, H. J. (1967). The comparative ultrastructure and possible function
of eyespots: Euglena granulata and Chlamydomonas eugametos. Planta 77, 325-353.
SAGER,
(Received 22 January 1974)
Fig. 1. Electron micrographs showing the position and appearance of the eyespot in
cells sectioned in several different planes.
Fig. 1 A: in longitudinal section the eyespot (es) is located beneath the cell wall,
midway between the ends of the cell. The nucleus (n) and pyrenoid(p) are also shown,
x 20350.
Fig. IB: shows the position of the eyespot in relation to one of the cell'sflagella(/);
the eyespot is at the lower left, cv, contractile vacuole; n, nucleus, x 16650.
Fig. 1 c: at higher magnification, the arrangement of eyespot granules in 2 rows or
plates can be seen in association with the chloroplast. cw, cell wall, x 57200.
Fig. I D : eyespot granules in horizontal section exhibit hexagonal close-packing,
x 57200.
Eyespot ultrastructure in Chlamydomonas
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H. E. Gruber and B. Rosario
Fig. 2. Irregular rod-shaped bodies are evident in the eyespot complex. Microtubules
(arrows) are nearby, x 57200.
Fig. 3. Rod-shaped bodies, longer and more tortuous than those illustrated in Fig. 2,
distort the chloroplast (cp) and plasma membranes (pvi).
Fig. 3 A: microtubules (arrow) and osmiophilic granules (06) are shown, x 57200.
Fig. 3B: close relationship between the rod-shaped body and the cell wall is shown
at the arrow, x 57 200.
Eyespot ultrastructure in Chlamydomonas
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H. E. Gruber and B. Rosario
Fig. 4. Dense material is apparent at the edge of the eyespot complex (arrow). The
rod-shaped body is covered by a limiting membrane, x 57200.
Fig. 5 A. Clear vacuoles in the eyespot plate were often observed near the irregular
bodies, x 67200.
Fig. 5 B. A membrane is present in the central area of the dense rods (arrow, and shown
in inset), x 52800. Inset, x 99000.
Eyespot ultrastructure in Chlamydomonas
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H. E. Gruber and B. Rosario
Fig. 6. The rod-shaped bodies appear to be attached to the cell by a pedicle of
agranular cytoplasm, x 70400.
Fig. 7. Three crystalline bodies (arrows), similar in density to the eyespot granules
and rod-shaped bodies, are present at the periphery of the eyespot complex, x 59000.
Eyespot ultrastructure in Chlamydomonas
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H. E. Gruber and B. Rosario
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Fig. 8. Stereo-pair micrographs, taken at tilts of 6° left and right, show that the granules
within plates fuse to form helices. For instructions in obtaining a stereo image see
Gray & Willis (1968). x 54000.