The effects of 24-h exposure to spectinomycin (100 μg/ml) and ethidium bromide (1 μg/ml) on the accumulation of chloroplast and mitochondrial rRNAs and on organelle ultra-structure were studied in greening cells of Ochromonas danica. Cells treated with ethidium bromide for 24 h divide at the same rate as controls but contain less than one third the normal amount of mitochondrial rRNA. Ultrastructural observations showed that these cells contain only 10% the number of mitochondrial ribosomes found in controls as well as fewer mitochondrial cristae. Ethidium bromide has no effect on chloroplast ultrastructure in Ochromonas.

Greening cells treated with spectinomycin grow at close to control rates but contain 30–40% less chloroplast rRNA than do controls. Electron microscopy showed that spectino-mycin disrupts the organization of chloroplast membranes and reduces the number of chloroplast ribosomes by 30%. Under these conditions, spectinomycin has no effect on mitochondrial rRNA or ultrastructure. Since spectinomycin is a specific inhibitor of translation on 70s ribosomes, these results are consistent with the possibility that at least some chloroplast ribosomal proteins are synthesized in the chloroplast of Ochromonas.

Although chloroplasts contain all the components necessary for the synthesis of proteins, only some of their constituent proteins are synthesized on chloroplast ribosomes; the rest are synthesized on cytoplasmic ribosomes and subsequently transported into the chloroplast. Numerous studies have been directed toward the question of where chloroplast ribosomal proteins are synthesized but their results considered as a whole are conflicting. In Chlor ella (Galling, Salzmann & Speiss, 1973), Ochromonas (Smith-Johannsen & Gibbs, 1972), Marchantía and Pylaiella (Loiseaux, 1976), pea (Ellis & Hartley, 1971) and lupin (Mesquita, 1971), inhibitors of chloroplast protein synthesis inhibit the synthesis of plastid ribosomes. Similarly, in pea (Ellis & Hartley, 1971), spinach (Detchon & Possingham, 1975) and radish (Ingle, 1968), chloroplast rRNA is markedly reduced in plants treated with inhibitors of chloroplast protein synthesis. These studies suggest that in these species, at least some of the proteins of the chloroplast ribosomes are synthesized in the plastid. However, in Chlamydomonas where there is evidence that some chloroplast ribosomal proteins are coded in the chloroplast and others in the nucleus (Ohta, Sager & Inouye, 1975; Mets & Bogorad, 1972; Davidson, Hanson & Bogorad, 1974), inhibitors of protein synthesis on chloroplast ribosomes have no effect on the number of plastid ribosomes (Goodenough, 1971). Moreover, inhibitor studies on Acetabularia indicate that chloroplast ribosomal proteins are not synthesized in the plastid of this species (Kloppstech & Schweiger, 1974).

In view of the importance of this problem and its controversial nature, we decided to re-examine the question of the site of synthesis of chloroplast ribosomal proteins in Ochromonas. In our previous study (Smith-Johannsen & Gibbs, 1972), chloramphenicol was used to block translation on plastid ribosomes. However, since chloram-phenicol in high concentrations may also inhibit mitochondrial respiration (Firkin & Linnane, 1968; Freeman & Haidar, 1968) spectinomycin was employed in the present investigation to inhibit protein synthesis on plastid ribosomes (Ellis, 1970). In some experiments, ethidium bromide which intercalates with mitochondrial DNA and blocks its replication (Goldring et al. 1970) and transcription (Zylber, Vesco & Penman, 1969) was used to inhibit mitochondrial rRNA synthesis. The present communication reports the effects of spectinomycin on the synthesis of chloroplast rRNA and chloroplast ribosomes in greening cells of Ochromonas. The effects of spectinomycin and ethidium bromide on chloroplast and mitochondrial ultrastructure are also described.

Biological material and growth conditions

Stocks of Ochromonas danica Pringsheim were obtained from the Culture Collection of Algae which is now located at the University of Texas in Austin (Culture no. 1298). Cells were grown at 29 °C in 250-ml Erlenmeyer flasks containing 150 ml medium as described previously (Smith-Johannsen & Gibbs, 1972). The light intensity at the culture surface, was 600 ft-c. (6·5 × 103 lux).

The density of cultures at the beginning of experiments was o·2–1·5 × 106 cells/ml. At this concentration dark-grown cells divide exponentially. Spectinomycin was added to cultures just prior to illumination. Ethidium bromide was added from freshly prepared stock solutions (0·15 mg/ml in distilled water). Cell counts were made with a Model ZB1 Coulter Counter using a 100μm aperture.

Biochemical procedures

The extraction and electrophoresis of total RNA from Ochromonas was carried out at o-4°C as described elsewhere (Smith-Johannsen, Gibbs & Fromson, 1979). Briefly, cells were lysed in a 1:1 mixture of buffer (0 05 M Tris-acetate pH 7 ·6, 0·1 M NaCl, 0 ·01 M disodium EDTA) containing 0 5% sodium dodecylsarcosinate and phenol-chloroform. After shaking for 15 min, the preparation was centrifuged at 4000 g for 15 min and the resulting aqueous phase was re-extracted with phenol-chloroform until no material remained at the interface. The RNA was precipitated by adding 01 vol. 2 M potassium acetate (pH 5·5) and 2·5 vol. absolute ethanol to the final supernatant and allowing the solution to stand for a minimum of 18 h at—20 °C. Gels containing 0 ·5% agarose (Peacock & Dingham, 1968) and 2-4% acrylamide were prepared in glass tubes (o·8 × 10 cm) according to a procedure adapted from Loening (1969). The gels were pre-run at 5 mA/gel for 40 min at 4 °C in electrophoresis buffer (0-36 M Tris, pH 7 8, 0·3 M NaH2PO4, 1 mM disodium EDTA). Samples of 10-12 μg RNA in 50 μl electrophoresis buffer containing 20% glycerol were then loaded on each gel. Electrophoresis (5 mA/gel) was for 3 h at 4 °C. The gels were removed from the tubes, soaked in distilled water for 30 min at 4 °C and scanned at 260 nm in a Gilford Model 240 spectrophotometer equipped with a linear transport attachment.

Analysis of electropherograms

Relative proportions of RNA species represented by peaks in the electrophoretic profiles were determined by measuring the areas bounded by the curves. Tracings of 3-12 replicate gels of each sample from one or more separate RNA extractions were photocopied and the peaks extrapolated to the base line using a straight edge. Some variability in the background absorbance of gels persisted within and between experiments. Therefore the base line was usually determined by connecting the troughs between the major peaks in the tracing. Individual peaks were then cut out and weighed. Since the cytoplasmic heavy rRNA peak often ran off-scale, values for amounts of organelle rRNA were normalized with respect to the cytoplasmic light rRNA species. Thus, amounts of organelle rRNA are expressed as a ratio of the organelle rRNA peak to the amount of cytoplasmic light rRNA.

The ratios obtained by analysis of the electropherograms of RNA from control, spectinomycin- and ethidium bromide-treated cells were transformed to arcsin values (Sokal & Rohlf, 1969). The data were then subjected to single classification analysis of variants after confirmation of homogeneity of variance by Bartlett’s test (Snedecor, 1956). Dunnett’s procedure (Steele & Torrie, 1960) was employed to determine levels of confidence between values for control and treated cells.

Electron microscopy

Greening cultures were exposed to 100 μg/ml spectinomycin and/or 1 μg/ml ethidium bromide for 24 h. Cells were collected using a clinical centrifuge at 2200 g for 3 min and each sample was processed for electron microscopy at room temperature by a standard fixation (cells fixed in glutaraldehyde and then in osmium tetroxide) and also by a co-fixation (cells fixed in a mixture of glutaraldehyde and osmium tetroxide followed by osmium tetroxide alone) as described previously (Smith-Johannsen & Gibbs, 1972).

Samples were dehydrated through an ethanol series and embedded in low viscosity epoxy resin (Spurr, 1969). Sections were cut on a Porter-Blum Mt-2 ultramicrotome, stained for 10 min with lead citrate (Reynolds, 1963), and examined in a Philips EM 200 electron microscope.

Analysis of electron micrographs

Chloroplast and mitochondrial ribosomes were counted on electron micrographs of co-fixed cells printed at a total magnification of 73 000 times. A window representing a square of 0·25μm side was placed at random over the organelle and the number of electron-dense particles of ribosome size counted. Since membranes usually occupied some fraction of the counting field, the values obtained by this method express ribosomes/total chloroplast area rather than ribosomes/area chloroplast matrix. Each value for ribosomes/unit organelle area given in Tables 2 and 4 is an average of approximately 130 counts from a minimum of 25 chloroplasts or approximately 100 counts from at least 75 mitochondria.

The per cent volume of the cell occupied by the chloroplast under control and experimental conditions was determined from the fractional area occupied by the chloroplast in approximately 100 sections through as many cells selected at random from each sample. Chloroplast and cell areas were measured with a compensating polar planimeter (Geotec 349–1838) on micrographs printed at a final magnification of 25900 times.

The per cent chloroplast volume was then converted to absolute chloroplast volume by multiplying by the mean cell volume of the population. Cell volumes were measured with a Model ZB1 Coulter Counter, calibrated with paper mulberry pollen. The absolute chloroplast volumes thus determined were used to compute total ribosomes per chloroplast.

The data was subjected to statistical analysis and levels of confidence were determined by a Student’s t-test.

Chemicals and equipment

Ethidium bromide was obtained from Sigma Chemical Co., St. Louis, Mo. and spectinomycin was purchased from the Upjohn Co., Kalamazoo, Mich.

Sodium dodecyl sarcosinate (Sarkosyl NL 97) was a gift from Geigy Limited, Toronto, Ont. Agarose was obtained from Seakem, Rockland, Me. Acrylamide bisacrylamide, ammonium persulphate and NNNN′ tetramethylethylenediamine were purchased from Biorad Laboratories, Richmond, Cal. The electrophoresis apparatus was from the E-C Apparatus Corporation, St. Petersburg, Fla..

Glutaraldehyde was obtained from Ladd Research Industries, Burlington, Vt, and osmium tetroxide was purchased from BDH Chemicals, Toronto, Ont.

Effect of spectinomycin and ethidium bromide on cell division and chlorophyll synthesis

Fig. 1 shows the effect of spectinomycin (100 μg/ml) and ethidium bromide (1 μg/ml) on the growth rate of greening cells. The inhibitors were added in the dark to exponentially growing cultures just prior to illumination. Cells treated with spectinomycin continued to grow exponentially for about 48 h at a rate 85–90% that of controls. Cells treated with ethidium bromide grew at the same rate as the control cells for 24 h, but then the growth rate declined. Cells exposed to both ethidium bromide and spectinomycin grew at a rate similar to that of the spectinomycin-treated cells for 24 h and then declined to a rate comparable to that of ethidium bromide-treated cells.

The amount of chlorophyll a per cell was determined after 24 h of greening in the absence and presence of inhibitors as an index of chloroplast development. As shown in Table 1, spectinomycin-treated cells had 44% less chlorophyll a than the control cells, whereas the chlorophyll content of ethidium bromide-treated cells was not significantly reduced. Cells treated with both inhibitors had 36% less chlorophyll than control cells.

Effect of spectinomycin on the light-induced synthesis of chloroplast rRNA

Fig. 2 A shows a representative electrophoretic profile of total RNA from cells of Ochromonas which have been allowed to green in the light for 24 h. Five peaks of high molecular weight RNA, designated a, b, c, d and e, as well as another peak containing low molecular weight RNAs are resolved. We have shown elsewhere (Smith-Johannsen et al. 1979) that peaks a and c contain the cytoplasmic heavy (1·18× 106 Daltons) and light (0·66× 106 Daltons) rRNAs respectively. Peak b is a composite peak containing both the chloroplast and mitochondrial heavy rRNAs (0·94 ×106 Daltons). Peak d contains the mitochondrial light rRNA (0·55 × 106 Daltons) and peak e consists of the chloroplast light rRNA (0·50 × 106 Daltons).

The electrophoretic profiles of RNA extracted from cells which had been allowed to green in the absence and presence of spectinomycin for 24 h are presented in Fig. 2 A, B, respectively. The amounts of rRNA in peaks b and e are selectively reduced with respect to peaks a and c in the extract from spectinomycin-treated cells, whereas the relative amount of rRNA in peak d remains approximately the same. Since peaks b and e contain chloroplast heavy and light rRNA these results indicate that spectinomycin inhibits the normal light-induced increase of chloroplast rRNA during greening. Since peak d is the mitochondrial light rRNA, these observations also suggest that spectinomycin does not affect the synthesis of mitochondrial rRNA.

In Fig. 3 the electrophoretic profile of RNA extracted from greening cells treated with ethidium bromide is compared with that of greening cells exposed to both spectinomycin and ethidium bromide for 24 h. Since ethidium bromide treatment eliminates a large part of the mitochondrial rRNA present in the cells (see Fig. 3 A and Table 2), treatment of the cells with spectinomycin in conjunction with ethidium bromide allows one to assess more readily the effect of spectinomycin on chloroplast rRNA. It can be seen that spectinomycin caused a decrease in the amount of chloroplast rRNA compared to the amount of cytoplasmic rRNA.

To quantify the effect of spectinomycin on the accumulation of chloroplast rRNA in greening cells, 9 to 12 electropherograms each of RNA from controls and from cells treated with spectinomycin, ethidium bromide and ethidium bromide plus spectinomycin were analysed. Cytoplasmic light rRNA was used as an internal standard and the amount of organelle rRNA expressed as a ratio with respect to this rRNA species. The results are presented in Table 2 which shows that in cells exposed to spectinomycin, the amount of chloroplast rRNA relative to cytoplasmic rRNA is reduced by approximately 30%. Mitochondrial rRNA is unaffected.

Effect of spectinomycin and ethidium bromide on organelle ribosomes

It was assumed that a reduction in the amount of chloroplast rRNA would be accompanied by a corresponding decrease in the number of chloroplast ribosomes. To check this assumption, control and spectinomycin-treated greening cells were fixed simultaneously in glutaraldehyde and osmium tetroxide and processed for electron microscopy. Counts were made on the number of chloroplast ribosomes per unit area and these counts converted to the absolute number of ribosomes per plastid from measurements of the section thickness, cell volume, and the per cent of the cell occupied by the chloroplast.

Fig. 4 shows a section of a plastid from a control cell which had been exposed to light for 24 h. Numerous chloroplast ribosomes are present in the chloroplast matrix. Most appear to lie free in the chloroplast matrix with only a few appearing to make contact with a thylakoid membrane. Fig. 5 is a section of a chloroplast from a cell which had been allowed to green in the presence of spectinomycin for 24 h. Ribosomelike particles are present in the chloroplast matrix, but they are fewer than in the control cells.

Table 3 shows that treatment with spectinomycin reduces the total number of plastid ribosomes in 24-h greening cells by approximately 30%. Since the volume of the chloroplast in the spectinomycin-treated cells is the same as in the control cells, the reduction in ribosome number observed in the treated cells is due exclusively to the lower concentration of ribosomes in the chloroplast of these cells.

Ethidium bromide, as expected, had no effect on the number of ribosomes per plastid (Table 3), but it did have a marked effect on the number of mitochondrial ribosomes. Figs. 6-8 illustrate mitochondria from control, ethidium bromide-treated and spectinomycin-treated cells. Ribosomes are abundant in the mitochondria from control and spectinomycin-treated cells, but are sparse in the ethidium bromide-treated cells. Table 4 gives the number of mitochondrial ribosomes per unit area. It can be seen that ethidium bromide treatment reduces the concentration of ribosomes in mitochondria by 90%. Mitochondrial volume was not directly determined in this study; instead the length of the mitochondria in sections was measured. The average length of mitochondria in ethidium bromide-treated cells was found to be 30% less than in the control cells and there was no apparent increase in the number of mito-chondrial profiles per cell. Thus ethidium bromide appears to cause a decrease in mitochondrial volume, so the reduction in the number of mitochondrial ribosomes caused by the dye is even greater than that indicated in Table 4.

Effect of spectinomycin and ethidium bromide on other aspects of organelle ultrastructure

The chloroplast of cells which have been illuminated for 24 h, although still only half the size of the fully developed plastid, already has the characteristic ultrastructure of the mature chloroplast. The thylakoids are arranged in bands of 3; those which encircle the rim of the chloroplast are designated girdle bands (Fig. 4) (Smith-Johannsen & Gibbs, 1972). Treatment with spectinomycin caused marked changes in chloroplast ultrastructure. Counts of the number of thylakoids per plastid section showed that the total amount of thylakoid membrane is considerably reduced in spectinomycin-treated cells (data not given). In addition, the arrangement of the thylakoids is disturbed. Thylakoids are often single or are arranged in stacks of more than 3. Stacks consisting of 4 to 11 thylakoids are common. These changes are illustrated in Figs. 5 and 9. The cell in Fig. 9 has been exposed to both spectinomycin. and ethidium bromide for 24 h. Since ethidium bromide alone was shown to have no effect on chloroplast ultrastructure, the abnormal chloroplast ultrastructure observed is due to spectinomycin alone.

Another feature of chloroplast ultrastructure which is affected by spectinomycin is the location of the chloroplast DNA. Characteristically the plastid DNA molecules are arranged in a ring-shaped nucleoid which lies at the rim of the chloroplast just inside the girdle bands (Fig. 4) (Gibbs, Cheng & Slankis, 1974). In spectinomycin-treated cells, the girdle bands are frequently abnormal (Fig. 5) or absent (Fig. 9). In such plastids, the chloroplast DNA no longer has its peripheral location, but appears as electron-translucent areas located between the central thylakoids (Figs-5, 9)

The chloroplast of Ochromonas is surrounded by a sac of endoplasmic reticulum which is continuous with the nuclear envelope (Fig. 9). Characteristically, a single layer of tubules and vesicles, the periplastidal reticulum, is present between the nucleus and chloroplast. In cells treated with spectinomycin this membrane system is hypertrophied (Fig. 9). A similar proliferation of the periplastidal reticulum was observed in chloramphenicol-treated cells (Smith-Johannsen & Gibbs, 1972).

The ultrastructural changes caused by treatment with ethidium bromide were limited to the mitochondria. In addition to virtually eliminating mitochondrial ribosomes, ethidium bromide caused a marked reduction in the number of mito-chondrial cristae (Figs. 7, 9).

Ethidium bromide selectively affects mitochondria

The biological activity of ethidium bromide is correlated with its ability to intercalate between DNA base pairs (Waring, 1968) and, in particular, its selective affinity for closed, circular DNA at low dye concentrations (Bauer & Vinograd, 1968). In eukaryotic cells, the target of ethidium bromide action is the mitochondrion. Mitochondria from nearly all lower (Borst, 1970) and higher plants (Kolodner & Tewari, 1972), as well as from all animals except some protozoa (Borst, 1970), contain covalently closed, circular DNA molecules. Both the replication (Goldring et al. 1970) and transcription (Zylber et al. 1969) of mitochondrial DNA in yeast and mammalian cells have been shown to be selectively inhibited by the dye. Presumably, binding of ethidium bromide to mitochondrial DNA in vivo precludes its normal functioning by distorting the tertiary structure of the supercoiled DNA molecules (Smith, Jordan & Vinograd, 1971; Nass, 1972).

Although the growth of Ochromonas cells cultured in medium containing 1 μg/ml ethidium bromide is normal for approximately 24 h, the morphology of the mitochondria in cells exposed to the dye during this interval is dramatically altered; the number of mitochondrial ribosomes is reduced by more than 90% and the number of cristae is also markedly reduced compared to control cells. Quantification of the various species of organelle rRNA from control and ethidium bromide-treated cells is in agreement with ultrastructural observations and shows that the transcription of mitochondrial rRNAs is selectively inhibited by the dye.

The abrupt decline in the rate of cell division of ethidium bromide-treated cells after 24 h is most likely a consequence of a depressed respiration rate due to the loss of the mitochondrial cristae. The effect of ethidium bromide on mitochondrial membrane is probably not direct but secondary due to the decrease in the number of mitochondrial ribosomes on which some cristae proteins are synthesized (e.g. Rubin & Tzagoloff, 1973). A similar loss of mitochondrial cristae in Ochromonas was observed when translation of mitochondrial ribosomes was inhibited by chloramphenicol (Smith-Johannsen & Gibbs, 1972). Ethidium bromide (1·5 μg/ml) caused aberrations in the arrangement of mitochondrial cristae as well as a reduction in their number in bleached cells of Euglena (Nass & Ben Shaul, 1973), while 25 μg/ml ethidium bromide induces dilation of the mitochondrial cristae in Acetabularia (Heilporn & Limbosch, 1971)

In contrast to its effect on mitochondria, ethidium bromide does not perceptibly alter the ultrastructure of the chloroplast in Ochromonas. Ethidium bromide-treated cells also contain normal amounts of chloroplast rRNA. These observations are in accordance with the lack of effect of ethidium bromide on the ultrastructure of chloroplasts in Euglena (Nass & Ben-Shaul, 1973). Although the chloroplasts in Acetabularia become swollen with storage products in the presence of ethidium bromide, their ultrastructure, as well as photosynthetic capacity, is otherwise normal (Heilporn & Limbosch, 1971). Chloroplasts from Euglena (Manning et al. 1971) and higher plants (Manning, Wolstenholme & Richards, 1972) have been shown to contain supercoiled DNA. If one assumes that the closed, circular nature of chloroplast DNA is universal, then the apparent insensitivity of the chloroplasts in Ochromonas, Acetabularia and Euglena to ethidium bromide seems to contradict the known mode of action of this dye. The discrepancy between the predicted and observed effects of ethidium bromide on chloroplasts might be explained if chloroplast DNA does not bind the dye. Chloroplast DNA isolated from Antirrhinum, Beta and Spinachia, however, has been shown to bind ethidium bromide (Herrmann, Bohnert, Kowallik & Schmitt, 1975). Another possible explanation for the lack of effect of ethidium bromide on the chloroplast is that this organelle is impermeable to the dye. This is unlikely since ethidium bromide was shown to inhibit all synthesis of cytoplasmic DNA in Acetabularia (Heilporn & Limbosch, 1971). Moreover, Flechtner & Sager (1973) reported that 10μg/ml ethidium bromide induces the loss of about 85% of the chloroplast DNA in Chlamydomonas. These authors proposed that one or more ‘master’ copies of chloroplast DNA in Chlamydomonas are sequestered in a region inaccessible to the dye.

In a recent study of SV40-transformed hamster cells resistant to ethidium bromide, preliminary evidence was obtained suggesting that an altered enzyme such as DNA polymerase may explain the resistance of these cells (Wolf, Tejmar, Borell & Kliet-mann, 1978). Perhaps the native properties of chloroplast DNA polymerase allow it to function in the presence of ethidium bromide. Further studies are required to sub-stantiate these theories or to explain in some other way the differential response of chloroplast and mitochondrial circular DNAs to ethidium bromide.

Spectinomycin affects chloroplast rRNA synthesis and ultrastructure, but not mitochondria

Spectinomycin is an antibiotic which inhibits translation on bacterial ribosomes by interfering with translocation (Pestka, 1971). It has also been shown to be a potent inhibitor of protein synthesis on chloroplast ribosomes from algae (Schlanger & Sager, 1974) and higher plants (Ellis, 1970), while not affecting protein synthesis on cytoplasmic ribosomes (Ellis, 1970).

Theoretically, one would expect the spectinomycin would inhibit protein synthesis on mitochondrial ribosomes. In cells of Chlamydomonas subjected to prolonged exposure to 90 μg/ml spectinomycin, cyanide-sensitive respiration is abolished (Boynton, Burton, Gillham & Harris, 1973). This observation suggests that protein synthesis on mitochondrial ribosomes in Chlamydomonas is sensitive to spectinomycin. However, this antibiotic does not substantially slow the heterotrophic growth of Chlorella (Galling et al. 1973) nor, as we observed, that of Ochromonas. Moreover, spectinomycin has no effect on the mitochondrial cristae in Ochromonas, in contrast to the result of chloramphenicol treatment, which markedly reduces the number of mitochondrial cristae (Smith-Johannsen & Gibbs, 1972). This suggests that in these algae, the mitochondrial ribosomes are insensitive to the concentrations of spectino-mycin used or that spectinomycin does not penetrate the mitochondria of these cells.

In greening cells of Ochromonas spectinomycin has a dramatic effect on the arrangement of membranes associated with the chloroplast. All of these alterations were also seen in cells treated with chloramphenicol (Smith-Johannsen & Gibbs, 1972). The formation of abnormally large stacks of thylakoids as well as single unassociated thylakoids in the presence of these inhibitors suggests that a component required for the organization of these photosynthetic membranes is synthesized on plastid ribo-somes. The abnormal proliferation of the periplastidal reticulum in response to chloramphenicol and spectinomycin treatment has led Gibbs (1979) to suggest that this membrane network normally serves as a transportation mechanism to deliver plastid proteins synthesized on cytoplasmic ribosomes into the chloroplast.

The results of this study have shown that, although cells of Ochromonas exposed to spectinomycin during the first 24 h of greening divide at a rate almost 90% that of controls, the amount of chloroplast rRNA relative to cytoplasmic rRNA is reduced by approximately 30%. Similarly, the total number of plastid ribosomes as measured by electron microscopy is reduced by 30%. One possible explanation of these observations is that spectinomycin inhibits chloroplast rRNA synthesis by blocking the synthesis of chloroplast rRNA polymerase. However, Ellis & Hartley (1971) have shown that lincomycin, another specific inhibitor of translation on plastid ribosomes, did not prevent the light-induced synthesis of plastid RNA polymerase. This finding suggests that chloroplast RNA polymerase is synthesized on cytoplasmic ribosomes. We did not assay RNA polymerase activity in this study, but assuming the biogenesis of this enzyme in Ochromonas is similar to that in pea, our observations cannot be accounted for by an inhibition of chloroplast RNA polymerase synthesis. An alternative interpretation of the effects of spectinomycin in Ochromonas is that the inhibitor blocks the synthesis of an enzyme(s) involved in the scission or methylation of chloroplast rRNA during processing or in the post-translational modification of a chloroplast ribosomal protein required for complete assembly. Our data do not permit the elimination of this possibility. Similarly, we cannot exclude the possibility that spectinomycin may inhibit the synthesis of a protein(s) required for the entry or utilization of a cytoplasmically synthesized ribosomal protein (s).

Although the above hypotheses merit consideration, the most direct explanation for the inhibition of chloroplast rRNA and ribosome synthesis in the presence of spectinomycin is that this antibiotic prevents the synthesis of ribosomal proteins required for the assembly of chloroplast ribosomes. Newly transcribed rRNA is associated with proteins (Hamkalo & Miller, 1973) and, presumably, is rapidly degraded if not incorporated into a ribonucleoprotein complex. Therefore, if chloro-plast ribosomal proteins are not available, chloroplast rRNA cannot accumulate. This is consistent with the results of Ellis & Hartley (1971) and Heizmann (1974), who observed that the incorporation of precursors into pea and Euglena chloroplast rRNAs continued in the presence of lincomycin, while the accumulation of these rRNA species was inhibited.

In conclusion, we have presented evidence which suggests that at least some chloroplast ribosomal proteins are synthesized in the chloroplast of Ochromonas. Recently, Freyssinet (1978) reported that out of 39 Euglena chloroplast ribosomal proteins analysed, 9 are synthesized in the chloroplast, while the remainder are either synthesized or regulated by the cytoplasm. This observation indicates that chloroplast ribosomal proteins are the products of 2 cell compartments, rather than derived exclusively from either the chloroplast or nucleus/cytoplasm. Indeed, evidence based on analysis of antibiotic-resistant mutants of Chlamydomonas suggests that some chloroplast ribosomal proteins are coded by nuclear genes and others by chloroplast genes (Davidson et al. 1974; Ohta et al. 1975; Freyssinet, 1977). Further studies on the biogenesis of chloroplast ribosomes should lead to a greater understanding of the complex regulatory mechanisms which exist between a cell and its chloroplast(s).

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