variation in eyespot ultrastructure in …jcs.biologists.org/content/joces/15/3/481.full.pdf ·...

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 somecells of the pale-green mutant strain ac-31 of Chlamydomonas reinhardi. The cells were grownin minimal medium supplemented with 0-2 % sodium acetate and were harvested by centrifuga-tion and prepared for electron-microscopic examination.

Microtubules were seen near the flagella, confirming previous observations. Microtubuleswere also seen near the eyespot complex. Although a direct connexion between the microtubulesof the flagellar and eyespot region was never observed, this does not exclude the possibilitythat it exists and thus provides a structural and functional connexion between the twoorganelles.

Occasionally irregular curved bodies intimately associated with the eyespot complex of somecells appeared to displace the chloroplast and cell membranes. These bodies often appeared tobe nearly covered by a limiting membrane and were found near empty ' cavities' in the eyespotplate. Crystalline arrays of dense bodies were observed in some sections. The significance ofthese bodies is discussed in relation to the functional state of the carotenoid pigments makingup 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 itfunctioned as an eye. Others envisioned that this pigmented area of the cell was ahighly organized series of lenses and pigment cups which focused light, causing aflagellar response (Mast, 1927). Many of the early hypotheses have been disproved byrecent experimental techniques, but as pointed out by Dodge (1969) knowledge ofthe function of the eyespot is still only speculative. How it functions and whether ornot it is light-sensitive are still unanswered questions. Current thinking on eyespotfunction has focused upon 2 possibilities (Arnott & Brown, 1967): that the eyespot istruly a primary photoreceptor, sensing light and transferring this information to themotor apparatus of the cell; or that the eyespot acts as a shade over some other photo-sensitive area of the cell. Dodge (1969) stated that algal eyespots, which vary widelyin structure and complexity, have only 2 common features: they occupy a specificlocation in the cell, and they are composed of carotenoid pigments localized in lipidor osmiophilic granules.

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482 H. E. Gruber and B. Rosario

The work presented here describes the ultrastructure of the eyespot of a pale-greenmutant strain of Chlamydomonas reinhardi. Originally isolated and characterizedultrastructurally by Goodenough, Armstrong & Levine (1969), this acetate-requiringstrain shows no stacking of its chloroplast thylakoids - the disks formed by thechloroplast membranes are not as closely and uniformly associated as they are in thewild-type cell. Since this variation in chloroplast morphology was already known tooccur in this mutant, the present study was undertaken to investigate the structure ofthe chloroplast-associated organelle, the eyespot.

MATERIALS AND METHODS

Cultures of Chlamydomonas reinliardi, mutant strain ac-31, mating-type minus, kindly pro-vided by Dr R. P. Levine (Goodenough et al. 1969) were grown in a Hotpack ProgrammedRefrigerated Incubator at 20-22 °C under a light-dark synchronization regime of 12 h light ofapproximately 3230 lx followed by 12 h of darkness. Cultures were grown in minimal mediasupplemented with 0-2 % sodium acetate in 30-ml tissue culture flasks (Falcon Plastics) withloosely fitting caps.

Cells were harvested by centrifugation midway in the light period, fixed 1 h in Karnovsky'sfixative (Karnovsky, 1965) at about 4 °C, rinsed several times with 02 M cacodylate buffer contain-ing 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 thechloroplast (Sager & Palade, 1957), and, in Chlamydomonas reinhardi, is found midwaybetween the anterior and posterior regions of the cell (Fig. 1 A). AS in all of the greenalgae, the eyespot is not associated with the flagella (Fig. 1 B). In wild-type Chlamy-domonas and some ac-31 cells the eyespot can be seen to consist of two or three layersor plates of granules (Fig. 1 c). The granules or globules have been reported to be100-140/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 anorderly arrangement' sandwiched' between the chloroplast lamellae. Others have alsoreported the lack of a limiting membrane around the granules themselves in otherChlamydomonas 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 closeproximity to the eyespot region (arrows in Figs. 2, 3 A).

In the majority of the acetate-requiring cells studied in section, various additionalstructures were seen in the area of the eyespot. Representative illustrations are pre-sented in Figs. 2-8. Some cells were seen to possess irregularly shaped, curving,electron-dense rod-shaped structures which extended various distances across theeyespot complex. (The appearance of these bodies did not differ significantly whenthey 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, seemssomewhat distended. In this section they lie well within the chloroplast and cellmembranes. In Fig. 3 A, however, the central area seems greatly distorted, and therod-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 mem-branes appear distended. A possible association between the longer bodies and thecell wall is indicated at the arrow in Fig. 3B.

The rod-like structures assumed various twisted shapes (Figs. 4, 5B). In Fig. 4 thedenser regions appear at the right-hand edge of the eyespot complex (arrow) andcontinue outward, again distending the cell membrane and possibly progressingthrough it. In this section the rod-shaped body appears to be nearly covered by alimiting membrane.

Figs. 5 A, B illustrate yet another property of eyespot ultrastructure that wasobserved. Often the dense, irregular bodies were found adjacent to empty 'cavities'in the eyespot plate. It appears that these spaces were previously occupied by theeyespot granules. Fig. 5 B also illustrates the presence of a membrane in the centralarea of one of the dense rods (arrow). These have been observed to be present overconsiderable lengths of the rods.

An 'empty space' was usually present between the granules and the distendedchloroplast and cell membranes. Occasionally, as illustrated in Fig. 6, the rods andeyespot 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 presenceof the dense rods and empty 'cavities', 3 regularly shaped crystalline objects can beseen on the periphery of the eyespot area (Fig. 7, arrows).

One of the most thought-provoking conformations observed is pictured in thestereo-pair of micrographs in Fig. 8. On first glance this view appeared to be a sectionthrough 2 plates of granules, each consisting of 2 rows of granules, with 1 row slightlyabove and to the side of the other. Upon closer stereo examination, however, it canbe seen that the granules are fused together to form a helix.

DISCUSSION

The presence of microtubules in the eyespot region of Chlamydomonas meritsfurther consideration. Investigators have long believed that the eyespot and flagellacould work together in the coordination of cellular orientation to light. In Euglenathis is a reasonable assumption, for the flagella insert into a reservoir in the region ofthe eyespot (Walne & Arnott, 1967). This concept appears less likely for the greenalgae, because the eyespot and flagella are separated by a fairly broad expanse of cyto-plasm (Fig. IB). Walne & Arnott (1967) proposed that microtubules could provide amethod of communication between eyespot and flagella, but a direct associationbetween the 2 organelleshad not been shown. In Chlamydomonas Ringo (1967) demon-strated bands of microtubules passing beneath the cell surface from the region of thebase of the flagella. 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.

31-2


Admittedly, these figures do not reveal the origin or termination of the tubules. Thereis good agreement on the close association of such tubules with the flagella in theanterior region of the cell, but it has not been determined what organelle, if any, isassociated with the other end of these tubules.

The eyespot has been reported to replicate prior to cytokinesis in Chlamydomonasand Euglena (Ettl, 1971; Kivic & Vesk, 1972). De novo formation in Chlamydomonashas been proposed, claiming that the eyespot granules could derive from osmiophilicgranules commonly found in the chloroplast, such as those shown in Fig. 3 A. Vege-tative cells in forms such as Tetracystis do not have eyespots, so it has been assumedthat they arise de novo. Two pathways for eyespot development have been proposed forTetracystis (Arnott & Brown, 1967): the granules could grow by acquiring new sub-stances, or the granules might migrate to the eyespot region after being formed else-where 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 foreyespot 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). Fourpigments were isolated from Euglena eyespot granules: lutein and cryptoxanthin(together forming 83 % of the total carotenoids), /^-carotene, and an unidentifiedcomponent (Batra & Tollin, 1964). In the euglenoid eyespot, pigment formation isinhibited by chloramphenicol, but not by cycloheximide (Kivic & Vesk, 1972). Oneof the first cellular responses to a 6-h exposure of Chlamydomonas to colchicine is thedissociation 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, anarrangement to be expected when spherical bodies of equal size are packed together(Arnott & Brown, 1967). Compression of granules could occur either from increasinggranule size or number, or growth of the chloroplast, with concomitant encroachmentupon the space occupied by the eyespot granules.

The granules are not enclosed in a membrane, but chloroform extraction of Chlamy-domonas removed the central portion of the granules, coincident with the appearanceof 'individual delimiting membranes' (Walne & Arnott, 1967). The authors believedthat the membrane probably represented the 'interface' between the granule and thesurrounding portion of the chloroplast.

Two instances have been reported in the literature in which irregularly shapedbodies 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 theglobules of light-grown cells (Kivic & Vesk, 1972). In Chlamydomonas eugametos,irregular curved bodies have been observed (Walne & Arnott, 1967), but these didnot protrude from the cell surface. It was proposed that the curved bodies might bedue 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 changefrom a lipid-soluble state to a crystalline state. This has special significance with


respect to the structures shown in Fig. 7. A change in functional state or cellular ageinghave also been suggested as causes for the structures observed in Chlamydomonaseugametos (Walne & Arnott, 1967). Senescence seems a less likely explanation in viewof the formation of similar bodies in Euglena after bleaching. A change in function ismore in agreement with their presence in the acetate species of Chlamydomonas usedin this study, since it requires the acetate supplement in addition to normal photo-synthetic activity.

The conformation observed in Fig. 8 suggests that fusion of granules from adjacentor the same row of granules could account for the formation of the twisting irregularbodies. Fusion of rows could explain the membranes observed in Figs. 4 and 5B, andthe 'cavities' observed in some plates. A change in functional or chemical state, suchas that proposed by Walne & Arnott (1967) might be one of the initiators of thismorphological change. This could be common to algal strains which do not relyentirely upon photosynthesis for their energy source, but which in part utilize alter-nate pathways.

The authors would like to express sincere thanks to Dr James Hampton for his guidanceand encouragement in this work and his comments on the manuscript, and Roy Adee and VictorFaubert 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 Owhile H. Gruber was on a Northwest College and University Association for Science Appoint-ment.

REFERENCES

ARNOTT, H. J. & BROWN,R., JR. (1967). Ultrastructure of the eyespot and its possible significancein phototaxis of Tetracystis excentrica. J. Protozool. 14, 529-539.

BATRA, P. P. & TOLLIN, G. (1964). Phototaxis in Euglena. I. Isolation of the eye-spot granulesand 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 organ-isms. Arch. Protistenk. 60, 197-220.

ETTL, H. (1971). The first protoplast division in the course of asexual reproduction in Chlamy-domonas (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 ofac-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 inelectron 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 organ-isms. Arch. Protistenk. 60, 197-220.

RINGO, D. L. (1967). Flagellar motion and fine structure of the flagellar apparatus in Chlamy-domonas. J. Cell Biol. 33, 543-571.


SAGER, R. & PALADE, G. E. (1957). Structure and development of the chloroplast in Chlamy-domonas. 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 functionof eyespots: Euglena granulata and Chlamydomonas eugametos. Planta 77, 325-353.

(Received 22 January 1974)

Fig. 1. Electron micrographs showing the position and appearance of the eyespot incells 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. I B : shows the position of the eyespot in relation to one of the cell's flagella (/);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 orplates 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

«< es


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.


Fig. 4. Dense material is apparent at the edge of the eyespot complex (arrow). Therod-shaped body is covered by a limiting membrane, x 57200.Fig. 5 A. Clear vacuoles in the eyespot plate were often observed near the irregularbodies, x 67200.Fig. 5 B. A membrane is present in the central area of the dense rods (arrow, and shownin inset), x 52800. Inset, x 99000.


5B


Fig. 6. The rod-shaped bodies appear to be attached to the cell by a pedicle ofagranular cytoplasm, x 70400.Fig. 7. Three crystalline bodies (arrows), similar in density to the eyespot granulesand rod-shaped bodies, are present at the periphery of the eyespot complex, x 59000.


\

i -if- •aFig. 8. Stereo-pair micrographs, taken at tilts of 6° left and right, show that the granuleswithin plates fuse to form helices. For instructions in obtaining a stereo image seeGray & Willis (1968). x 54000.

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