Ultrastructural Changes in Euglena After Ultraviolet Irradiation

J. Cell Set. 13, 799-809 (1973) 799 Printed in Great Britain

ULTRASTRUCTURAL CHANGES IN EUGLENA AFTER ULTRAVIOLET IRRADIATION

A. MICHAELS* AND A. GIBOR Biology Department, University of California, Santa Barbara, California 93106, U.S.A.

SUMMARY The structural changes associated with the ultraviolet-induced bleaching of light-grown cells of Euglena gracilis were investigated. Our light- and electron-microscopic observations of the bleaching process indicate that there is a continuity of plastid structure in cells 5 generations after receiving a bleaching dose of ultraviolet light. There seems to be a continuous dilution of the plastid thylakoids and a decrease in plastid size in the bleaching cells. There also seems to be a change in the position of the plastids in relation to the mitochondria in the bleaching cells. The plastids and possibly the mitochondria are the only organelles which are affected by the ultraviolet irradiation. The continuity of plastids in bleaching cells of Euglena is discussed in relation to the proposed effect of the ultraviolet light.

INTRODUCTION Euglena gracilis strain z is a unicellular alga which has distinct advantages for the investigation of cytoplasmic organelles. The alga can be cultured in the light, autotrophically, or in the dark, heterotrophically. The chloroplast of light-grown Euglena is bounded by a double membrane and is comprised of 2-4 pairs of membranous thylakoids (Gibbs, i960). If green cells are cultured in the dark, the chloroplasts lose their chlorophyll and their characteristic internal structure in subsequent generations. The plastids decrease in size and return eventually to the proplastid state. The proplastid appears in the electron microscope to be a sack about 4 /im long (Schiff, 1971), delimited by a double membrane. It contains very little internal structure but develops into a normal chloroplast on exposure to light (Ben-Shaul, Schiff & Epstein, 1964). Changes in the plastid system of Euglena can be induced by a number of mutagens. Nitrosoguanidine (McCalla, 1965, 1967), streptomycin (Provasoli, Hunter & Shatz, 1968; Erbringer, Nemec, Santova & Folitnova, 1970) and naladixic acid (Erbringer, 1970) to note only a few, are all very effective in causing plastid mutations in Euglena. It was discovered that a sublethal dose of ultraviolet (u.v.) irradiation will subsequently cause all the cells to lose their potential to form chloroplasts (Pringsheim, 1958). This loss of ability to form green colonies is called ' bleaching'. It is a permanent, inheritable modification. It has been shown that the induction of bleaching by u.v. can be reversed by visible light, though red light (above 660 nm) is not effective in reversing the bleaching process (Lyman, Epstein & Schiff, 1959; Schiff, Lyman & Epstein, 1961). • Present address: Smithsonian Institution, Radiation Biology Laboratory, 12441 Parklawn Drive, Rockville, Maryland 20852, U.S.A.

51-2 8oo A. Michaels and A. Gibor In cells of bleached colonies one fails to find characteristic chloroplast structures. It is not easily possible to identify structures found in such cells as representing the modified progeny of the original plastids since they do not possess identifiable lamellae or prolamellar bodies. In this report we describe our observations on some of the intermediate stages in the formation of bleached cells. The ultrastructural changes in the chloroplasts of light- grown cells of Euglena were used as indicators of the u.v. effect on the plastids.

MATERIALS AND METHODS Irradiation and culture conditions The ethanol medium described by Buetow & Padilla (1963) was used at all times. Light- grown cells were irradiated with a G.E. germicidal lamp as described by Gibor (1969). Liquid cultures were then incubated under 50 J m""1 s"1 of red light (light transmission above 660 run). Efficiency of bleaching was checked by plating an aliquot of cells on solid medium. The plates were incubated under white light. Counts revealed less than 1 % green colonies. Cell division in liquid cultures was monitored with a haemocytometer or an automatic cell counter.

Electron microscopy Samples of cells at 3, 4 and 5 divisions after u.v. irradiation were fixed with 4 % glutaraldehyde for 20 min in an ice bath. The cells were washed and then postfixed with 1 % OsO* for 30 nun in the cold. They were then dehydrated in a graded series of aqueous acetone, transferred to propylene oxide and embedded in Epon. Thin sections were cut on a Porter-Blum ultra- microtome with a diamond knife, then stained on grids with uranyl acetate and lead citrate and viewed with a Siemens Elmiskop I.

Light microscopy Live cells were examined with a Reichert light microscope, equipped for fluorescence studies. Cells were photographed by the use of phase-contrast and fluorescence microscopy.

RESULTS Green cells were irradiated and then transferred to culture medium and grown under red light. Cell counts were done daily. Three divisions after u.v. irradiation under red light, cells irradiated with a bleaching dose of u.v. are green and contain chloroplasts. These organelles have a structure similar to that of chloroplasts of light- grown cells (Fig. 5). The majority of the thylakoids are fused into grana (Gibbs, 1970). Only one area indicates some irregularity (arrow). This seems to be a break in a few long thylakoids. At this stage the chloroplasts may have swollen thylakoids (Fig. 6). Mitochondria seem to increase in size (Fig. 6) and number, and are quite distinct from chloroplasts, especially in the density of their matrix. After 4 divisions, plastids are still discernible in the cells and many seem to contain fewer lamellae (Fig. 9). Consequently, one can see more of the ground substance of the plastid. Pyrenoids are absent altogether or are difficult to distinguish. Figs. 3 and 4, which are light and fluorescence micrographs of u.v. irradiated cells after 5 divisions under red light, reveal many red fluorescing bodies. These fluorescing centres are similar to those normally associated with chloroplasts of non-u.v.-treated Ultraviolet irradiation of Euglena 801 green cells grown under red light (Figs. 1, 2). The photographs of live cells indicate that the plastids are not all at the cell periphery as in unirradiated cells, but are posi- tioned around the nucleus. In sections viewed through the electron microscope this location of the plastids of u.v.-treated cells is not as striking (Fig. 10). The plastids of the fifth generation after u.v. irradiation contain relatively few thylakoids (Fig. 7). The decrease in the number of thylakoids in successive generations after u.v. irradiation is presented in Table 1.

Table 1. Number of thylakoids in bleaching Euglena

No. of divisions after u.v. No. of thylakoids Standard deviation 3 12-3 ±3"3(39)t 4 7-4 + 2-3 (40) 5 4'4 ±1-4(25) • Standard deviation ±-J[(E d*/N)]. f Number of individual chloroplasts measured.

The free thylakoids seem to vesiculate during their degeneration (Figs. 8, 10, 11). Figs. 10 and 11 show 2 successive sections in series through a fifth-generation cell. They show that thylakoid vesiculation occurs at one end of the plastid which seems normal at the other end. Some areas in the plastid contain no thylakoids, or if a few are present they are pressed close to the outer membranes so that an area containing primarily plastid 'ground substance' is created (Fig. 8). This type of structural organization is similar to that found by Schiff (1971) in the most recent published pictures of the proplastid of dark-grown Euglena. Third generation plastids have many ribosomes (Fig. 6), while after 5 divisions there are very few (Fig. 8, arrows). Figs. 10 and 11 show the extent of undulation of the mitochondria. These 2 micrographs indicate that there may be only one continuous mitochondrion in this cell. Fifth generation mitochondria are characteristically enlarged and may contain a series of stacked cristae (Fig. 7). All other structures of the u.v.-treated cells appear to be normal. The only apparent changes are the diminishing plastids and the proliferating mitochondria.

DISCUSSION Ultraviolet irradiation changes the cells of Euglena so that they lose the potential to develop green colonies (Lyman et al. 1959). The action spectrum of this effect indicates that a nucleoprotein is the absorbing chromophore (Lyman, Epstein & Schiff, 1961). Gibor & Granick (1962 a) have demonstrated that bleaching can occur in Euglena when the nucleus is shielded from the u.v.; thus the u.v. primarily effects a cytoplasmic target. Two phases of plastid biology are readily distinguishable in the Euglena system; chloroplast development from proplastids, and the perpetuation of the plastids either as chloroplastids or proplastids in the cell line (Schiff et al. 1961; Lyman et al. 1959, 802 A. Michaels and A. Gibor 1961). The u.v. treatment does not inhibit the ability of the proplastids to develop into photosynthetically competent chloroplasts in non-dividing cultures (Diamond & Schiff, 1970). On the other hand Edelman, Schiff & Epstein (1965) have shown that chloroplast DNA is not detectable in u.v.-bleached strains of Euglena. They have concluded from this study that the u.v. prevents chloroplast replication. However, there has been some opposition to the hypothesis that chloroplasts cease to divide after u.v. irradiation. Gibor & Granick (19626) have shown that u.v.-irradiated microcultures of Euglena display red fluorescing bodies characteristic of chloroplasts after several generations under non-photoreactivating conditions. These results indicate plastid continuity in the u.v.-treated cells. Moriber, Hershneov, Aronson & Bensky (1963) have examined u.v.-bleached strains of Euglena in the electron microscope. They found bodies containing crista-like structures which they conclude to be chloroplast remnants. The effect of u.v. irradiation in our experiments is to prevent the maintenance of a developed chloroplast in growing Euglena cultures. From light- and electron- microscopic studies we conclude that there is no apparent inhibition of plastid division after u.v. treatment. The structural changes which occur in the Euglena plastid appear to be similar to those described for the degeneration of senescing higher plant chloroplasts (Kirk & Tilney-Bassett, 1967) and plastids of the ac-20 mutant of Chlamydo- monas (Goodenough & Levine, 1970). The chloroplast of autotrophically grown Chlamydomonas ac-20 contains normal levels of 70s ribosomes. When the cells are transferred to a mixotrophic growth medium, the mutant cells lose their plastid ribosomes. The plastids of u.v.-irradiated Euglena also seem to have fewer chloroplast ribosomes. Changes in the structure of Euglena mitochondria result after treatment with mutagens (Calvayrac, VanLente & Buetow, 1971). The change in the mitochondria reported here after u.v. treatment is probably a secondary phenomenon associated with the loss of functional chloroplasts. The proliferation of the mitochondria is correlated with the de-development of the plastids. The mitochondria may contain stacked cristae, a feature not found in normal mitochondria. Such mitochondria may have been mistaken for plastid remnants in bleached strains (Moriber et al. 1963). We have chosen to investigate the u.v. effect with light-grown cells of Euglena to facilitate the identification of the plastids. Since the light-grown cells have chloroplasts with normal complements of lamellae, we have used the lamellae as indicators of plastids and plastid remnants. A striking change occurs after u.v. treatment in the relative position of plastids and mitochondria within the cell. In a normal green cell the chloroplasts occupy the peripheral region, while the mitochondria lie internally. After irradiation, with the proliferation of the mitochondria and degeneration of the chloroplast, the relative positions of the organelles are reversed. Mitochondria now occupy the position peripheral to the plastids. SchifF and his collaborators (Schiff & Epstein, 1965, 1968; Schiff & Zeldin, 1968) have also studied cells of bleached clones of Euglena with the electron microscope. In these cases, many generations after u.v. irradiation, no plastid-like structures were observed. The authors, however, did note some structures resembling proplastids Ultraviolet irradiation of Euglena 803 which they marked with a question mark. We believe that these unidentified structures were the remaining perpetuating plastids because in our material, similar proplastid- like bodies arise from the degenerating chloroplasts. Our results are similar to those obtained by others (Ophir & Ben-Shaul, 1968; Ben-Shaul, Silman & Ophir, 1972) in an investigation of the ultrastructural changes in Euglena plastids during streptomycin- induced bleaching. Thus, the process of degeneration of the plastid after u.v. irradiation is similar to the return of the chloroplast to the proplastid state when cells are placed in the dark (Ben-Shaul, Epstein & Schiff, 1965). The rate of lamellar loss in this study of normal green cells grown in the dark was 0-3 lamellae per generation. In our study it is about 0-4. These 2 rates are lower than the expected loss of 0-5 per generation, according to a dilution hypothesis. There is considerable error in this type of measurement and differences from a theoretical rate are not surprising. It is known that many components of the chloroplast are under nuclear control (Kirk, 1971; Woodcock & Bogorad, 1971). Since the u.v. affects a cytoplasmic nucleoprotein one can speculate that the structures that are perpetuated (e.g. plastid envelope) are controlled by nuclear genes (if indeed all the chloroplast DNA is missing in the u.v.-bleached clones). It is possible that synthesis of some structural protein of plastid thylakoids is controlled by cytoplasmic genes (i.e. chloroplast DNA) because u.v. treatment causes the loss of plastid DNA and then the ability to synthesize the thylakoids.

REFERENCES BEN-SHAUL, Y., EPSTEIN, H. T. & SCHIFF, J. A. (1965). Studies of chloroplast development in Euglena. X. The return of the chloroplast to the proplastid condition. Can.J.Bot.43, 129-136. BEN-SHAUL, Y., SCHIFF, J. A. & EPSTEIN, H. T. (1964). Studies on chloroplast development in Euglena. VII. Fine structure of the developing plastid. Plant Physiol. 39, 231-239. BEN-SHAUL, Y., SILMAN, R. & OPHIR, I. (1972). Effects of streptomycin on the ultrastructure of plastids in Euglena. Physiol. vig. 10, 255-268. BUETOW, D. E. & PADILLA, G. M. (1963). Growth of Astasia longa on ethanol. J. Protozool. 10, 121-123. CALVAYRAC, R., VANLENTE, F. & BUETOW, R. A. (1971). Euglena gracilis: formation of giant mitochondria. Science, N. Y. 173, 252-254. DIAMOND, I. & SCHIFF, J. A. (1970). Insensitivity of development of photosynthetic competence to doses of ultraviolet light which block green colony formation in Euglena. Can. J. Bot. 48, 1277-1283. EDELMAN, M., SCHIFF, J. A. & EPSTEIN, H. T. (1965). Studies of chloroplast development in Euglena. XII. Two types of satellite DNA. J. molec. Biol. 11, 760-774. ERBRINGER, L. (1970). Action of naladixic acid on Euglena plastids. J. gen. Microbiol. 61, I4I-I44- ERBRINGER, L., NEMEC, P., SANTOVA, H. & FOLITNOVA, P. (1970). Changes of the plastid system of Euglena gracilis induced with streptomycin and dihydrostreptomycin. Arch. Mikrobiol. 73, 268-280. GIBBS, S. P. (i960). The fine structure of Euglena gracilis with special reference to the chloroplasts and pyrenoids. J. Ultrastruct. Res. 4, 127-148. GIBBS, S. P. (1970). Comparative ultrastructure of the algal chloroplast. Ann. N.Y. Acad. Sci. I7S. 454-473- GIBOR, A. (1969). Effect of ultraviolet irradiation on DNA metabolism of Euglena gracilis. J. Protozool. 16, 190—193. 804 A. Michaels and A. Gibor GIBOR, A. & GRANICK, S. (1962a). Ultraviolet sensitive factors in the cytoplasm that affect the differentiation of Euglena plastids. J. Cell Biol. 15, 599-603. GIBOR, A. & GRANICK, S. (19626). The plastid system of normal and bleached Euglena gracilis. J. Protozool. 9, 327-334- GOODENOUGH, U. & LEVINE, R. P. (1970). Chloroplast structure and function in ac-20, a mutant strain of Chlamydomonas reinhardi. III. Chloroplast ribosome and membrane organization. J. Cell Biol. 44, 547-562. KIRK, J. T. O. (1971). Chloroplast structure and biogenesis. A. Rev. Biochem. 40, 161-196. KIRK, J. T. O. & TILNEY-BASSETT, R. A. E. (1967). In The Plastids, p. 608. London and San Francisco: Freeman. LYMAN, H., EPSTEIN, H. T. & SCHIFF, J. A. (1959). Inactivation and photoreactivation of chloroplast development in Euglena without cell death. J. Protozool. 6, 264-265. LYMAN, H., EPSTEIN, H. T. & SCHIFF, J. A. (1961). Studies of chloroplast development in Euglena. I. Inactivation of green colony formation by u.v. light. Biochim. biophys. Ada 50, 301-309. MORIBER, L. B., HERSHNEOV, B., ARONSON, S. A. & BENSKY, B. (1963). Teratological chloroplast structures in Euglena gracilis permanently bleached by exogenous physical and chemical agents. J. Protozool. 10, 80-86. MCCALLA, D. R. (1965). Effect of nitrofurans on the chloroplast system of Euglena gracilis. J. Protozool. 12, 34-41. MCCALLA, D. R. (1967). Mutation of the Euglena chloroplast system: The mechanism of bleaching by nitrosoguanidine and related compounds. J. Protozool. 14, 480-482. OPHIR, I. & BEN-SHAUL, Y. (1968). Kinetics of fine structure changes in Euglena cells bleached by streptomycin. In Proc. 4th Europ. reg. Conf. Electron Microsc, vol. 2 (ed. D. S. Bocciarelli), pp. 419—420. Rome: Tipografia Poliglotta Vaticana. PRINGSHEIM, E. (1958). Die Apoplastididie bei Euglena gracilis. Revue algol. 4, 41-56. PROVASOLI, L., HUNTER, S. H. & SHATZ, A. (1968). Streptomycin-induced chlorophyll-less races of Euglena. Proc. Soc. exp. Biol. Med. 69, 279-282. SCHIFF, J. A. (1971). Developmental interactions among cellular compartments in Euglena. In Autonomy and Biogenesis of Mitochondria and Chloroplasts (ed. N. K. Boardman, A. W. Linnane & R. M. Smillie), pp. 98-118. New York: Elsevier. SCHIFF, J. A., & EPSTEIN, H. T. (1965). The continuity of the chloroplast in Euglena. In Reproduction: Molecular, Subcellular and Cellular (ed. M. Locke), pp. 131-189. New York and London: Academic Press. SCHIFF, J. A. & EPSTEIN, H. T. (1968). The continuity of the chloroplast in Euglena. In The Biology of Euglena, vol. 2 (ed. D. Buetow), pp. 285-334. New York and London: Academic Press. SCHIFF, J. A., LYMAN, H. & EPSTEIN, H. T. (1961). Studies of chloroplast development in Euglena. II. Photoreversal of the ultraviolet inhibition of green colony formation. Biochim. biophys. Acta 50, 310-338. SCHIFF, J. A. & ZELDIN, M. (1968). The developmental aspect of chloroplast continuity in Euglena. J. cell. Physiol. (suppl.) 72, 103-128. WOODCOCK, C. L. F. & BOGORAD, L. (1971). Nucleic acids and information processing in chloroplasts. In Structure and Function of Chloroplasts (ed. M. Gibbs), pp. 89-128. New York and Berlin: Springer-Verlag. (Received 20 December 1972) Ultraviolet irradiation of Euglena 805

..r 4

Fig. 1. Phase-contrast micrograph of unirradiated Euglena cells cultured under red light, x 1040. Fig. 2. Fluorescence micrograph of the cells in Fig. 1. The red fluorescence of chlorophyll appears dark in this micrograph, x 1040. Fig. 3. Light micrograph of u.v.-treated cells cultured for 5 divisions under red light, x 1040. Fig. 4. Fluorescence micrograph of the cells in Fig. 3. The cell in the middle of the field contains red fluorescing plastids which appear dark and have irregular contours, x 1040. 8o6 A. Michaels and A. Gibor

Fig. 5. An example of a plasrid (p) from a cell after u.v. treatment and 3 divisions under red light. Arrow indicates the only area of irregularity, m, mitochondrion, x 30000. Fig. 6. A cell 3 divisions after u.v. treatment cultured under red light. The plasrid (p) seems to have swollen lamellae (/). Other than having this one characteristic, the organ- elle seems to be normal with normal amounts of ribosomes (r). Note the enlarged mitochondrion (m). The crystals (cr) present in the cytoplasm are of unknown origin, x 30000. Ultraviolet irradiation of Euglena 807

Fig. 7. Section of a cell 5 generations after u.v. treatment. The plastids (p) shown contain lamellae (/) in the process of degeneration. The lamellae vesiculate (v) until there are very few left (e.g. the plastid on the right edge of the figure). A cytolysosome (c/)isin theprocessof digesting what appears to be a mitochondrion. The mitochondria (m) in the cytoplasm are enlarged. One contains stacked cristae (sc). Paramylon (pa) and endoplasmic reticulum can also be seen, og, osmiophillic globuli. x 24000. 8o8 A. Michaels and A. Gibor rSJ3!3PT r

Fig. 8. Enlargement of one of the plastids in Fig. 7. This micrograph shows the details of the vesiculation of the lamellae (/). Vesicles (v) seem to arise from the thylakoids. There are few ribosomes (r) present in these plastids compared to 3-division plastids (Fig. 6). Cytoplasmic ribosomes are easily noted (r). x 40500. Fig. 9. Section of a cell 4 divisions after u.v. irradiation. The plastid shown contains swollen lamellae. The pyrenoid (py) in the plastid seems to be degenerating. The mitochondrion (rn) directly under the plastid shows the beginnings of stacking of cristae. x 26000. Ultraviolet irradiation of Euglena 809

Fig. 10. A cell 5 divisions after receiving a bleaching dose of u.v. The plastids (p) are shown in various stages of degeneration. Three highly lobed mitochondria («,, m2, m,) are also noted, v, vesicle, x 20000. Fig. 11. Another section in series of the cell shown in Fig. 10. Only the locomotory flagellum is present (J{). The 3 mitochondria seen in Fig. 10 seem to be interconnected in this section, an indication that there is one giant mitochondrion (m) in this cell, p, plastid; v, vesicle, x 24000.