Planta (Berl.) 111, 73--84 (1973) 9 by Springer-Verlag 1973
Transcription and Translation for Carotenoid Synthesis in Chlamydomonas reinhardtii Reidun Sirevs Botanical Laboratory, University of Oslo, Oslo, Norway R o b e r t Paul Levine The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138, U.S.A. Received December 4, 1972 Summary. The sites of transcription and translation of carotenoid pigments were studied in synchronously grown cells of Chlamydomonas reinhardtii Dang. 1Rifampiein, cycloheximide and spectinomyein were used to distinguish between the nuclear-cytoplasmic genetic system and the genetic system of the chloroplast. Since rifampicin is without effect, chloroplast DNA appears not to possess information required for the synthesis of carotenoids. Carotenoid synthesis parallels chlorophyll synthesis in these cells. Carotenoid synthesis is dependent on de hove protein S~Ulthesis both on cytoplasmic and chloroplast ribosomes, for both cyeloheximide and spectinomycin are effective inhibitors. However, the ceils are able to form about 40% of the expected increase in earotenoids when cytoplasmic and chloroplast ribosomes are simultaneously inhibited. Of the major carotenoids in C. reinhardtii, Iutein appears the least dependent on de nero protein synthesis. The synthesis of fi.carotene and trollein appears to be completely dependent on the function of cytoplasmic ribosomes.
Introduction The chloroplasts of the unicellular green alga, Chlamydomonas reinhardtii Dang., contain D N A and possess the necessary equipment for protein synthesis (Chun et al., 1963 ; Sager and Ishida, 1963 ; Kirk, 1966 ; Hoober and Blobel, 1969; Hoober, 1970, 1972). However, it has become increasingly evident (Surzyeki et al., 1970 ; Armstrong et al., 1971 ; Sirevs and Levine, 1972) t h a t the synthesis of the components of this organelle as well as its development are under control of both the nuclear-cytoplasmic genetic system and the genetic system of the chloroplast. The carotenoids of eukaryotic photosynthetic organisms are integral components of their chloroplast membranes, and it is therefore of interest to determine the degree to which the nuclear-cy%oplasmic genetic system and the chloroplast genetic system function in the synthesis of proteins u p o n which carotenoid biosynthesis m a y depend. This paper represents the results obtained from ~ s t u d y of the effects t h a t inhibitors of genetic transcription and translation have on carotenoid synthesis in synchronous cultures of C. reinhardtii.
74
R. Sirev's and 1~. P. Levine:
Antibiotics t h a t selectively inhibit transcription and translation in either the nuclear-cytoplasmic system or in the chloroplast can be import a n t tools in studying the formation of the chloroplast. R i / a m p i c i n has been shown to inhibit R N A synthesis in the chloroplasts of C. reinhardtii (Surzycki, 1969) and Chlorella pyrenoidosa (Galling, 1971). Thus, this inhibitor apparently prevents the genes of the chloroplast from being transcribed. Spectinolnycin has been shown to bind specifically to 70S chloroplast ribosomes in C. reinhardtii (Burton, 1970, 1972), while cycloheximide is k n o w n to inhibit protein synthesis on 80S cytoplasmic ribosomes (Siegal and Sisler, 1964; Hoober et al., 1969). Accordingly, comparative studies using speetinomycin and cyclohexilnide can show whether chloroplast or cytoplasmic ribosomes are the sites of translation of various proteins. I n a previous paper from this laboratory (Armstrong et al., 1971), a system for synchronous growth of C. reinhardtii was described in which rifampicin, spectinolnycin, and cyclohexilnide were used in an a t t e m p t to determine the sites of genetic transcription and translation of certain i m p o r t a n t chloroplast components. I t was pointed out t h a t synchronously grown cells are especially suited for studies with antibiotics, since all the cells in the culture are in the same physiological state at a given tilne. Furthermore, an antibiotic can be added to the culture and its effects observed during a time period in which there is little R N A synthesis and the cells are non-dividing. During this time period, however, chloroplast and other cell components are being synthesized in preparation for the subsequent cellular division. Also, the time of exposure of the cells to the antibiotic is relatively short, thus reducing b u t not eliminating the chances of secondary effects unrelated to protein synthesis.
Materials and Methods Organism and Culture Conditions. C. reinhardtii, a substrain of wild-type strain 137c of mating type + , was used. This strain is capable of synchronous growth in the minimal medium of Sueoka (1960) as modified by Armstrong et al. (1971). The cells were synchronized by using a 12-h light/12-h dark cycle according to the method of Kates and Jones (1964). Light and temperature conditions were as described by Armstrong et al. (1971). In experiments employing rifampicin, it was necessary to increase the light intensity from 5000 lux to 8000 ]ux since the inhibitor itself is light absorbing. This was achieved by substituting tungsten reflector flood lamps for the white and daylight4ype fluorescent lamps used for the synchronous cultures. All experiments were carried out during the third light period after inoculation and at cell concentrations ill the synchronous cultures of 0.5-1.0 • 106 cell/ml. The time at which the 3rd light period began is designated zero time. Sterile conditions were maintained throughout. Inhibitors. l~ifampicin (Mann lZesearch Labs., Orangeburg, New York, U.S.A.) was dissolved in sterile, unbufferd 0.01 M KHsPOa to give a concentration of ]5 mg/ ml. The solution was stirred overnight at room temperature and added to the cul-
Transcription a n d Translation for Carotenoid Synthesis in Chlamydomonas
75
ture 50 rain before the onset of the light period to give a final concentration of 250 ,ag/ml. Speetinomyein (a gift from Upjohn Co., KMamazoo, Michigan, U.S.A.) was dissolved in distilled water to give a concentration of 5 mg/ml and sterilized b y passing through a sterile Millipore membrane. The inhibitor was added to the culture 50 min before the onset of the 3rd light period to give a final concentration of 3 ~g/ml. Cycloheximide (Sigma Chemical Co., St. Louis, Missouri, U.S.A.)was dissolved in distilled water to give a concentration of 1.5 mg/ml, sterilized b y passing through a sterile Millipore membrane, a n d added to the culture 3 h after the onset of the 3rd light period. The final concentration of eyeloheximide in the cultures was 1 #g/ml unless otherwise stated.
Cell Number. 5-ml samples were withdrawn from the cultures a t the beginning and a t the end of the 3rd light period. Cell n u m b e r was determined with the aid of a hemacy~ometer. Chlorophyll. Chlorophyll was determined by a modification (Arnon, 1949) of the method of Mackinney (1941). Samples of cells from the synchronous cultures were treated as described by Armstrong et al. (1971). Carotenoids. Samples of 150 ml were taken as indicated from the synchronous cultures for analysis of total carotenoids. The cells were harvested by cengrifugation at 17000 X g for 5 rain, washed once in I mM phosphate buffer (pI-I 7.0), and resuspended in same to i ml. The carotenoid content of the cells was analyzed directly or after storage of the cells for no nmre than 48 h at --20 ~ C. The analysis used was a slight modification of the procedure of Krinsky and Levine (1964). After addition of 5 ml 6 % (w/v) KOI-I in methanol and saponification in the dark at 40 ~ C for 5 min, the suspension was rapidly cooled and centrifuged at 3 000 • g for 5 rain. The supern a t a n t was transferred to a separatory funnel and the cell residue re-extracted repeatedly with 5 ml methanol until colorless. The methanol extracts werer.,combined , and the carotenoids were then extracted into diethyl ether (peroxide free). Addition of water facilitated separation of the two layers. The methanol extracts were reextracted with petroleum ether (b.p. 37-46.2 ~ C). To remove alkali the combined extracts were washed 3 times with water until phenolphthalin no longer t u r n e d pink; after this the extracts were filtered through NazSO 4 (anhydrous) a n d made up to volume with petroleum ether. For estimation of total earotenoids, the absorbancy a t 450 n m was read, a n d an E l 1%cm vame of 2500 was used. The results are expressed as #g earogenoids formed per ml of synchronous culture. For analysis of individual earotenoids the extract was taken to dryness in vaeuo a t 38 ~ C. The dry pigments were redissolved in diethyl ether when used for thinlayer chromatography. Two-dimensional t h i n layer chromatography was carried out in the dark on commercially prepared sheets (ITLC, Type S G, Gelman I n s t r u m e n t Co., A n n Arbor, Michigan, U.S.A.). 10 Ezl (approximately 10 ~zg) of extract were applied, and separation was achieved using a solvent system of petroleum e t h e r - - a c e t o n e (7:3 v/v) for the first dimension, and petroleum e t h e r - - e t h a n o l (95:5 v/v) for the second dimension. The individual spots were rapidly cut out and eluted in diethyl ether, petroleum ether, or ethanol. The eluates were t a k e n to dryness in a stream of nitrogen, and made up to a known volume in petroleum ether or ethanol. The absorption spectra of the individual carotenoids were determined in a model 14 Cary recording spectrophotometer.
76
1~. Sirevhg and R. P. Levine:
To estimate the quantities of the individual carotenoids, the maximum absorbaneies of the individual pigments were added, the sum taken as 100%, and the percentage contributed by each of the carotenoids calculated on this basis.
Results 1. Synthesis o/Total Carotenoids Synthesis of total carotenoids parallels chlorophyll synthesis in synchronous cultures of C. reinhardtii, the major increase taking place at the eighth hour of the light period as is shown in Fig. 1. I n contrast to chlorophyll synthesis, which levels off after the 10th hour, carotenoid synthesis continues to the end of the light period but stops once the cells have entered the following dark period.
2. E/]ect o/Inhibitors on Total Carotenoid Synthesis Rifampicin had no effect on the expected increase in carotenoid formation during the 3rd light period of synchronous growth of C. reinhardtii (Fig. 2). The high levels of carotenoids formed in the control culture in this case are probably due to the high light intensity used in the experiments with rifampicin. Synthesis of total carotenoids was affected both b y cycloheximide and spectinomycin, the extent of inhibition by each drug varying from ca. 45% to 65 %. Fig. 3 shows the results from one such experiment. Since the concentration of cycloheximide used in these experiments was low compared to what has been used by other workers (Bishop and Smillie, 1970; Kirk and Allen, 1963), the effect of increasing concentrations of cycloheximide on the synthesis of total carotenoids was tested. The data in Table 1 show that the percentage of inhibition remains the same even when the concentration is increased 5-fold. Chlorophyll synthesis, on the other hand, is much more affected b y cycloheximide; as Table 1 shows, 5 ~g/ml cycloheximidc completely inhibited the synthesis o~ this pigment. To test whether carotenoid synthesis was completely dependent on protein synthesis occurring simultaneously in the cytoplasm and the chloroplast, cycloheximide and spectinomycin were added to the same cell culture. A viable cell count assured that the cells were not killed by this treatment. Fig. 4 shows t h a t carotenoid synthesis still proceeds, although at a greatly reduced rate, in the presence of both drugs. 3. E//ect o/Inhibitors on the Synthesis o/Individual Carotenoids When carotenoid extracts from synchronously grown C. reinhardtii were chromatographed, five major spots appeared. On the basis of their spectral properties, their position on the chromatogram, and color reac-
Transcription and Translation for Carotenoid Synthesis in Chlamydomonas
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06
6.0
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0.4
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0.3
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2.0
0.I
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0
2
4
6
(3
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77
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Hours of s y n c h r o n o u s
growth in the light
Fig. 1. The increase in total earotenoids and chlorophyll during the third light period of synchronous growth of C. reinhardtii. Synchronous cultures were obtained by using a 12-h dark/12-h light cycle. The light intensity was 5000 lux, the temperature 21 ~ C. Samples were taken in the 3rd light period at times indicated and analyzed for pigments as described in Materials and Methods. Tile amount of pigment is expressed as ~g formed per ml of synchronous culture. Cell concentration in the synchronous culture was 0.8 • 106 eells/ml, o earotenoids; 9 chlorophyll
t i o n w i t h c o n c e n t r a t e d hydrochloric acid, t h e spots were identified as/~carotene, lutein, v i o l a x a n t h i n , trollein, a n d n e o x a n t h i n . T h e epoxides, v i o l a x a n t h i n a n d n e o x a n t h i n , were f u r t h e r identified b y their characteristic h y p o s c h r o m i c spectral shift in t h e presence of dilute acid. These are t h e same m a j o r carotenoids as were described b y K r i n s k y a n d Levine (1964) for C. reinhardtii. W h e n t h e spots were e l a t e d from t h e c h r o m a t o g r a m s a n d t h e a m o u n t of each c a r o t e n o i d c a l c u l a t e d from t h e i r i n d i v i d u a l a b s o r p t i o n spectra, i t was f o u n d t h a t t h e r a t i o of fl-earotene to lutein v a r i e d s o m e w h a t from one e x p e r i m e n t to another. I n m o s t cases t h e level of lutein was a b o v e t h a t of fl-carotene in t h e s y n c h r o n o u s l y grown cultures. W h e n t h e cells were t r e a t e d with r i f a m p i c i n a n d a n a l y z e d for individ u a l carotenoids, t h e r e was a l m o s t no effect on t h e r a t i o of one carotenoid to a n o t h e r c o m p a r e d to t h e control w i t h o u t rifampicin. This is shown in T a b l e 2. The high levels of fl-carotene in t h e control m a y be t h e consequence of t h e high light i n t e n s i t y used in t h e e x p e r i m e n t s w i t h rifampicin.
78
1~. Sirevs and R. P. Levine:
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0.4
0,3
0. 2
it" 0
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I 4
I 6
I 8
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Hours of synchronous growth in the light Fig. 2. Effect of rifampicin on the increase in total carotenoids during the3rd light
period of synchronous growth of C. rdnhardtii. Experimental conditions as described in Materials and Methods and Fig. 1. The light intensity was 8000 lu:(. o control; o 250 ~g/ml of rifampicin. Rifampicin was added 50 rain before zero time
Table 3 shows the results from an experiment in which the cells were treated with cyeloheximide of spectinomycin. I n this experiment cycloheximide inhibits total carotenoid synthesis by 67 %, but it can be seen that the individual carotenoids are affected differently. At the time when eycloheximide is added to the cells (3 h in the light), the level of each carotenoid has increased somewhat in the cell. Addition of eycloheximide completely stops synthesis of fi-earotene and tro]lein, whereas synthesis of violaxanthin appears to be unaffected, and that of lutein is inhibited 45%. The individual carotenoids are also affected differently by spectinomycin. Speetinomyein inhibited total carotenoid synthesis 54 % in this experiment, the synthesis of fi-carotene being inhibited the most. Violaxanthin was inhibited 66 %, while trollein synthesis was only inhibited 12 %.
Transcription and Translation for Carotenoid Synthesis in Chlamydomonas
c5
79
0.5
0.4
0
2
4
6
8
I0
12
Hours of synchronous growth in the light Fig. 3. Effects of eycloheximide and speetinomycin on the increase in total carotenoids during the 3rd light period of synchronous growth of C. reinhardtii. Experimental conditions as described in Materials and Methods and Fig. 1. o control; 1 ag/ml of eycloheximide; 9 3 txg/ml of spectinomycin. Speetinomycin was added 50 rain before zero time; eycloheximide was added 3 h after zero time
Table 1. Effect of different concentrations of cyeloheximide on formation of total carotenoids and chlorophyll in synchronous cultures of Chlamydomonas reinhardtii Culture conditions were as described for Fig. 1. Cycloheximide was added 3 h after the onset of the third light period. Samples were taken 12 h in the light and analyzed for pigment as described in Materials and Methods. Cyeloheximide /zg/ml
Carotenoids % inhibition
Chlorophyll % inhibition
1 2 5
62.5 62.5 62.5
73.5 94.5 100.0
Discussion A l t h o u g h chloroplasts c o n t a i n DNA, this D N A does n o t appear to c o n t a i n all of the i n f o r m a t i o n necessary for the f o r m a t i o n of the organelle itself (Kirk, 1966 ; A r m s t r o n g et al., 1971). E v e n such a n i m p o r t a n t chloroplast c o m p o n e n t as chlorophyll appears to be completely u n d e r the con-
80
R. Sirev~g and 1~. P. Levine: 0.6
0.5
i
0.4
0.5
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0.2
0.1
~L 2
4
6
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Hours of synchronous growth in the light Fig. 4. Effects of cycloheximide and cycloheximide combined with spectinomycin on the increase in total carotcnoids during the 3rd light period of synchronous growth of C. reinhardtii. Experimental conditions as described in Materials and Methods, and Figs. 1 and 2. o control; ~ 1 ~g/ml of cycloheximide; 9 1 ~g/ml of cycloheximide + 3 y.g/ml of spectinomycin Table 2. Effect of rifampicin on the formation of the major carotenoids in synchronous cultures of Chlamydomonas reinhardtii. Culture conditions were as described for Figs. 1 and 2. Samples were taken after 12 h in the light and analyzed for individual carotenoids as described in Materials and Methods Carotenoid
fl-Carotene Lutein Violaxanthin Trollein Neoxanthin
Percentage of total carotenoids Control
Rifampicin
41 25 14 13 7
38 22 13 19 8
trol of nuclear genes in C. reinhardtii (Armstrong et al., 1971). The results reported here from the experiments with rifampicin show t h a t , as i n the case of chlorophyll, the biosynthesis of the carotenoids i n C. reinhardtii is unaffected b y this antibiotic. Although negative d a t a from experiments with rifampicin are inconclusive, since the lifetime of chloroplast messen-
Transcription and Translation for Carotenoid Synthesis in Ghlamydomonas
81
Table 3. Effect of cyeloheximide and spectinomycin on the formation of the individual major earotenoids as seen in the llth hour of synchronous culture of C. reinhardtii Culture conditions were as described for Figs. 1 and 3. Samples were taken after 11 h in the light and analyzed for individual carotenoids as described in Materials and Methods. Carotenoid
fl-Carotene Lutein Violaxanthin Trollein Neoxanthin
Control Cycloheximide (~g/ml formed) ~g/ml % formed inhibition
~g/ml formed
% inhibition
0.i13 0.154 0.059 0.043 0.022
0.027 0.080 0.020 0.038 0.015
76 48 66 12 32
0.000 0.084 0.058 0.000 0.015
100 45 0 100 32
Spectinomycin
ger RNAs is not known, it appears that the synthesis of carotenoids is independent of information in chloroplast DNA and by inference dependent upon information enclosed in nuclear DNA. This is in agreement with the conclusion of Kirk (1966) based on studies of carotenoid-deficient mutant strains of higher plants that show Mendelian inheritance and are thus presumed to be the consequence of mutation of nuclear genes. The carotenoid pigments of eukaryotie photosynthetic organisms are found within the chloroplast membranes (Goodwin, 1959; Weier and Benson, 1966). They function as accessory pigments b y transferring absorbed energy to chlorophyll (Duysens, 1956). Further, they are thought to play a role in protecting chlorophyll against photooxidation (Griffiths et al., 1955; Sistrom et al., 1956; Krinsky, 1966). I t is therefore not surprising that the synthesis of these pigments closely parallels chlorophyll synthesis in synchronously grown C. reinhardtii. The major carotenoid pigments in C. reinhardtii are fl-carotene, lutein, violaxanthin, trollein, and neoxanthin (Krinsky and Levine, 1964). They are believed to arise from common precursors, but their biosynthesis and regulation are complex, involving light and many different enzymatic steps, most of which are poorly understood (Porter and Anderson, 1967). fl-carotene is an important compound in this respect because it is an intermediate in the formation of violaxanthin, trollein, and neoxanthin. Lutein, on the other hand, is thought to arise from ~-carotene (Porter and Anderson, 1967). Although the site of carotenoid formation is thought to be the chloroplast (Goodwin, 1969), our results with eycloheximide and spectinomycin show that their formation is dependent on de-novo protein synthesis on both cytoplasmic and chloroplast ribosomes. The fact that the levels of 6
P l a n t a (Berl.), Bd. 111
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1~. Sirevs and R. P. Levine:
carotenoids in the cell increased to a large extent even when translation on the cytoplasmic and chloroplast ribosomes was inhibited simultaneously, indicates that the enzymes required for carotenoid biosynthesis are present in the cells at the onset of the light period and in amount sufficient to synthesize at least 39 % (Fig. 4) of the carotenoids found in the control cultures. From the data obtained by the analysis of the individual carotenoids, it appears that these enzymes might be mainly concerned with lutein synthesis, since the synthesis of this pigment was much less affected by the drugs than was that of the other individual carotenoids. The extensive inhibition of/~-carotene formation by either eycloheximide or spectinomycin suggests that the synthesis of this pigment is completely dependent on translational steps both on cytoplasmic and chloroplast ribosomes. There still exists the possibility, however, that some flcarotene is formed in the presence of the drugs and is then immediately converted to other carotenoids. Thus, the finding that violaxanthin formarion is unaffected by cycloheximide indicates that its precursor, /?carotene, may be formed to some extent in the presence of this inhibitor. In addition, the inhibition of violaxanthin formation by speetinomycin demonstrates that protein synthesis on chloroplast ribosomes is involved in the conversion of fl-carotene to violaxanthin. On the other hand, it appears that the formation of trollein from fl-carotene requires de novo protein synthesis only on the cytoplasmic ribosomes since the formation of this pigment is completely inhibited by cycloheximide and only slightly affected by spectinomycin. I t appears from this study that the formation of the five major carotenoids of C. reinhardtii depends on proteins (most likely enzymes of carotenoid biosynthesis) that are synthesized on both chloroplast and cytoplasmic ribosomes. I t is likely that both types of ribosomes are involved, for the synthesis of the five pigments undoubtedly entails several different biochemical reactions. The membrane-bound nature of the pigments must also be kept in mind, for it is possible that the antibiotics are inhibiting the synthesis of proteins that are essential for the association of the carotenoids with the membranes. A sharing of responsibility between chloroplast and cytoplasmic ribosomes may not be rare, for the synthesis of cytochrome 563 and ribulose 1,5-diphosphate carboxylase in synchronous cultures of C. reinhardtii appears to depend on both types of ribosomes (Armstrong et al., 1971). A dependence on both classes of ribosomes has also been reported for the synthesis of ribulose 1,5-diphosphate earboxylase in barley (Criddle et al., 1970). The authors wish to thank Professor Norman Krinsky, Tufts Medical School, Boston, Mass. for his helpful suggestions during the course of the work reported here. This work has been supported by the National Science Foundation, grant GB 18666, to the second author.
Transcription and Translation for Ca,rotenoid Synthesis in Chlamydomonas 83 Relerences Armstrong, J. J., Surzycki, S. J., Moll, B., Levine, R. P. : Genetic transcription and translation specifying chloroplast components in Chlamydomo~as reinhardi. Biochemistry lO, 692-701 (1971). Arnon, D. I. : Copper enzymes in isolated chloroplasts: polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). Bishop, D. G., Smillie, R. M. : The effect of ehloramphenicol and cyclohexamide on lipid synthesis during chloroplast development in Euglena gracilis. Arch. Biochem. Biophys. 137, 179-189 (1970). Burton, W. G. : The binding of spectinomycinto chloroplast ribosomes of Chlamydomonas reinhardi. J. Cell Biol. 47, 28a (1970). Burton, W. G. : Dihydrospectinomycin binding to chloroplast ribosomes from antibiotic-sensitive and -resistant strains of Chlamydomonas reinhardi. Biochim. biophys. Aeta (Amst.) 272, 305-311 (1972). Chun, E. It., Vaughan, M. It., Rich, A. : The isolation and characterization of DNA associated with chloroplast preparations. J. molec. Biol. 7, 130-141 (1963). Criddle, R. S., Dau, B., Kleinkopf, G. E., ttuffaker, R. C. : Differential synthesis of ribulosediphosphate carboxylase subunits. Biochim. biophys. Res. Commun. 41, 621-627 (1970). Duysens, L. N. M. : Energy transformations in photosynthesis. Ann. Rev. Plant Physiol. 7, 25-50 (1956). Galling, G. : Der EinfluB yon Rifampicin, Chloramphenicol und Cycloheximid auf den Uridin-Einbanin chloroplastid~re Ribosomenvorstufen yon Chlorella. Planta (Berl.) 98, 50-62 (1971). Goodwin, T. W. : Biosynthesis and function of carotenoid pigments. In: Advances in enzymology, vol. XXI, p. 296-368, Nord, F . F . , ed. New York-London: Interseience 1959. Goodwin, T. W.: Carotenoid biosynthesis in chloroplasts. In: Progress in photosynthesis research, vol. II, p. 669-674, Metzner, H., ed. Tfibingen 1969. Griffiths, M., Sistrom, W. R., Cohen-Baz]re, G., Stanier, R. Y. : Function of carotenoids in photosynthesis. Nature (Lond.) 176, 1211-1215 (1955). ttoober, J. K. : Sites of synthesis of chloroplast membrane polypeptides in Chlamydomonas relnhardi. J. biol. Chem. 245, 4327-4334 (1970). Hoober, J. K. : A major polypeptide of chloroplast membranes of Chlamydomonas reinhardi: Evidence for synthesis in the cytoplasm as a soluble component. J. Cell Biol. 52, 84-96 (1972). Hoober, J. K., Blobel, G. : Characterization of the ehloroplastic and cytoplasmic ribosomes in Chlamydomonas reinhardi. J. molee. Biol. 41, 121-138 (1969). Hoober, J. K., Siekevitz, P., Palade, G. E. : Formation of chloroplast membranes in Chlamydomonas reinhardi y-1. J. biol. Chem. 244, 2621-2631 (1969). Kates, J. R., Jones, R. F. : The control of gametic differentiation in liquid cultures of Chlamydomonas. J. cell. eomp. Physiol. 63, 157-164 (1964). Kirk, J. T. O. : Nature and function of chloroplast DNA. In: Biochemistry of chloroplasts, vol. I, p. 319-340, Goodwin, T. W., ed. London-New York: Acad. Press 1966. Kirk, J. T. 0., Allen, R. L. : Dependence of chloroplast pigment synthesis on prorein synthesis: Effect of aetidione. Biochem. biophys. Res. Commun. 21,523-530 (1963). Krinsky, N. I, : The role of carotenoid pigments as protective agents against photosensitized oxidation in chloroplasts. In: Biochemistry of chloroplasts, vol. I. p. 432-430, Goodwin, T. W., ed. London-New York: Aead. Press 1966. 6*
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Krinsky, N. I., Levine, R. P. : Carotenoids of wild-type and mutant strains of the green alga, Chlamydomonas reinhardi. Plant Physiol. 39, 680-687 (1964). Mackinney, G. : Absorption of light by chlorophyll solutions. J. biol. Chem. 140, 315-322 (1941). Porter, J., Anderson, D.: Biosynthesis of carotenes. Ann. Rev. Plant Physiol. 18, 197-228 (1967). Sager, R., Ishida, M. R. : Chloroplast DNA in Chlamydomonas. Prec. nat. Acad. Sci. (Wash.) 50, 725-730 (1963). Siegal, ~ . , Sisler, H. D. : Site of action of cycloheximide in cells of Saccharomyces pastorianus. II. The nature of inhibition of protein synthesis in a cell-free system. Biochim. biophys. Aeta (Amst.) 87, 83-89 (1964). Sirev~g, R., Levine, R. P. : F a t t y acid synthetase from Chlamydomonas relnhardi: Sites of transcription and translation. J. biol. Chem. 247, 2586-2591 (1972) Sistrom, W. R., Griffiths, M., Stanier, R. Y. : The biology of a photosynthetic bacterium which lacks colored carotenoids. J. cell. comp. Physiol. 48, 473-515 (1956). Sueoka, N. : l~Iitotie replication of deoxyribonucleicacid in Chlamydomonasreinhardi. Prec. nat. Acad. Sci. (Wash.) 46, 86-91 (1960). Surzyeki, S. J. : Genetic functions of the chloroplast of Chlamydomonas reinhardi: Effect of rifampiein on chloroplast DNA-dependent RNA polymerase. Prec. nat. Acad. Sci. (Wash.) 63, 1327-1334 (1969). Surzycki, S. J., Goodenough, U. W., Levine, R. P., Armstrong, J. 5.: Nuclear and chloroplast control of chloroplast structure and function in ChIamydomonas reinhardl. Syrup. Soc. exp. Biol. 24 13-37 (1970). Weier, T. E., Benson, A. A. : The molecular nature of chloroplast membranes. In: Biochemistry of chloroplasts, vol. I, p. 91-113, Goodwin, T. W., ed., LondonNew York: Acad. Press 1966.
Transcription and Translation for Carotenoid Synthesis in Chlamydomonas reinhardtii Reidun Sirevs Botanical Laboratory, University of Oslo, Oslo, Norway R o b e r t Paul Levine The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138, U.S.A. Received December 4, 1972 Summary. The sites of transcription and translation of carotenoid pigments were studied in synchronously grown cells of Chlamydomonas reinhardtii Dang. 1Rifampiein, cycloheximide and spectinomyein were used to distinguish between the nuclear-cytoplasmic genetic system and the genetic system of the chloroplast. Since rifampicin is without effect, chloroplast DNA appears not to possess information required for the synthesis of carotenoids. Carotenoid synthesis parallels chlorophyll synthesis in these cells. Carotenoid synthesis is dependent on de hove protein S~Ulthesis both on cytoplasmic and chloroplast ribosomes, for both cyeloheximide and spectinomycin are effective inhibitors. However, the ceils are able to form about 40% of the expected increase in earotenoids when cytoplasmic and chloroplast ribosomes are simultaneously inhibited. Of the major carotenoids in C. reinhardtii, Iutein appears the least dependent on de nero protein synthesis. The synthesis of fi.carotene and trollein appears to be completely dependent on the function of cytoplasmic ribosomes.
Introduction The chloroplasts of the unicellular green alga, Chlamydomonas reinhardtii Dang., contain D N A and possess the necessary equipment for protein synthesis (Chun et al., 1963 ; Sager and Ishida, 1963 ; Kirk, 1966 ; Hoober and Blobel, 1969; Hoober, 1970, 1972). However, it has become increasingly evident (Surzyeki et al., 1970 ; Armstrong et al., 1971 ; Sirevs and Levine, 1972) t h a t the synthesis of the components of this organelle as well as its development are under control of both the nuclear-cytoplasmic genetic system and the genetic system of the chloroplast. The carotenoids of eukaryotic photosynthetic organisms are integral components of their chloroplast membranes, and it is therefore of interest to determine the degree to which the nuclear-cy%oplasmic genetic system and the chloroplast genetic system function in the synthesis of proteins u p o n which carotenoid biosynthesis m a y depend. This paper represents the results obtained from ~ s t u d y of the effects t h a t inhibitors of genetic transcription and translation have on carotenoid synthesis in synchronous cultures of C. reinhardtii.
74
R. Sirev's and 1~. P. Levine:
Antibiotics t h a t selectively inhibit transcription and translation in either the nuclear-cytoplasmic system or in the chloroplast can be import a n t tools in studying the formation of the chloroplast. R i / a m p i c i n has been shown to inhibit R N A synthesis in the chloroplasts of C. reinhardtii (Surzycki, 1969) and Chlorella pyrenoidosa (Galling, 1971). Thus, this inhibitor apparently prevents the genes of the chloroplast from being transcribed. Spectinolnycin has been shown to bind specifically to 70S chloroplast ribosomes in C. reinhardtii (Burton, 1970, 1972), while cycloheximide is k n o w n to inhibit protein synthesis on 80S cytoplasmic ribosomes (Siegal and Sisler, 1964; Hoober et al., 1969). Accordingly, comparative studies using speetinomycin and cyclohexilnide can show whether chloroplast or cytoplasmic ribosomes are the sites of translation of various proteins. I n a previous paper from this laboratory (Armstrong et al., 1971), a system for synchronous growth of C. reinhardtii was described in which rifampicin, spectinolnycin, and cyclohexilnide were used in an a t t e m p t to determine the sites of genetic transcription and translation of certain i m p o r t a n t chloroplast components. I t was pointed out t h a t synchronously grown cells are especially suited for studies with antibiotics, since all the cells in the culture are in the same physiological state at a given tilne. Furthermore, an antibiotic can be added to the culture and its effects observed during a time period in which there is little R N A synthesis and the cells are non-dividing. During this time period, however, chloroplast and other cell components are being synthesized in preparation for the subsequent cellular division. Also, the time of exposure of the cells to the antibiotic is relatively short, thus reducing b u t not eliminating the chances of secondary effects unrelated to protein synthesis.
Materials and Methods Organism and Culture Conditions. C. reinhardtii, a substrain of wild-type strain 137c of mating type + , was used. This strain is capable of synchronous growth in the minimal medium of Sueoka (1960) as modified by Armstrong et al. (1971). The cells were synchronized by using a 12-h light/12-h dark cycle according to the method of Kates and Jones (1964). Light and temperature conditions were as described by Armstrong et al. (1971). In experiments employing rifampicin, it was necessary to increase the light intensity from 5000 lux to 8000 ]ux since the inhibitor itself is light absorbing. This was achieved by substituting tungsten reflector flood lamps for the white and daylight4ype fluorescent lamps used for the synchronous cultures. All experiments were carried out during the third light period after inoculation and at cell concentrations ill the synchronous cultures of 0.5-1.0 • 106 cell/ml. The time at which the 3rd light period began is designated zero time. Sterile conditions were maintained throughout. Inhibitors. l~ifampicin (Mann lZesearch Labs., Orangeburg, New York, U.S.A.) was dissolved in sterile, unbufferd 0.01 M KHsPOa to give a concentration of ]5 mg/ ml. The solution was stirred overnight at room temperature and added to the cul-
Transcription a n d Translation for Carotenoid Synthesis in Chlamydomonas
75
ture 50 rain before the onset of the light period to give a final concentration of 250 ,ag/ml. Speetinomyein (a gift from Upjohn Co., KMamazoo, Michigan, U.S.A.) was dissolved in distilled water to give a concentration of 5 mg/ml and sterilized b y passing through a sterile Millipore membrane. The inhibitor was added to the culture 50 min before the onset of the 3rd light period to give a final concentration of 3 ~g/ml. Cycloheximide (Sigma Chemical Co., St. Louis, Missouri, U.S.A.)was dissolved in distilled water to give a concentration of 1.5 mg/ml, sterilized b y passing through a sterile Millipore membrane, a n d added to the culture 3 h after the onset of the 3rd light period. The final concentration of eyeloheximide in the cultures was 1 #g/ml unless otherwise stated.
Cell Number. 5-ml samples were withdrawn from the cultures a t the beginning and a t the end of the 3rd light period. Cell n u m b e r was determined with the aid of a hemacy~ometer. Chlorophyll. Chlorophyll was determined by a modification (Arnon, 1949) of the method of Mackinney (1941). Samples of cells from the synchronous cultures were treated as described by Armstrong et al. (1971). Carotenoids. Samples of 150 ml were taken as indicated from the synchronous cultures for analysis of total carotenoids. The cells were harvested by cengrifugation at 17000 X g for 5 rain, washed once in I mM phosphate buffer (pI-I 7.0), and resuspended in same to i ml. The carotenoid content of the cells was analyzed directly or after storage of the cells for no nmre than 48 h at --20 ~ C. The analysis used was a slight modification of the procedure of Krinsky and Levine (1964). After addition of 5 ml 6 % (w/v) KOI-I in methanol and saponification in the dark at 40 ~ C for 5 min, the suspension was rapidly cooled and centrifuged at 3 000 • g for 5 rain. The supern a t a n t was transferred to a separatory funnel and the cell residue re-extracted repeatedly with 5 ml methanol until colorless. The methanol extracts werer.,combined , and the carotenoids were then extracted into diethyl ether (peroxide free). Addition of water facilitated separation of the two layers. The methanol extracts were reextracted with petroleum ether (b.p. 37-46.2 ~ C). To remove alkali the combined extracts were washed 3 times with water until phenolphthalin no longer t u r n e d pink; after this the extracts were filtered through NazSO 4 (anhydrous) a n d made up to volume with petroleum ether. For estimation of total earotenoids, the absorbancy a t 450 n m was read, a n d an E l 1%cm vame of 2500 was used. The results are expressed as #g earogenoids formed per ml of synchronous culture. For analysis of individual earotenoids the extract was taken to dryness in vaeuo a t 38 ~ C. The dry pigments were redissolved in diethyl ether when used for thinlayer chromatography. Two-dimensional t h i n layer chromatography was carried out in the dark on commercially prepared sheets (ITLC, Type S G, Gelman I n s t r u m e n t Co., A n n Arbor, Michigan, U.S.A.). 10 Ezl (approximately 10 ~zg) of extract were applied, and separation was achieved using a solvent system of petroleum e t h e r - - a c e t o n e (7:3 v/v) for the first dimension, and petroleum e t h e r - - e t h a n o l (95:5 v/v) for the second dimension. The individual spots were rapidly cut out and eluted in diethyl ether, petroleum ether, or ethanol. The eluates were t a k e n to dryness in a stream of nitrogen, and made up to a known volume in petroleum ether or ethanol. The absorption spectra of the individual carotenoids were determined in a model 14 Cary recording spectrophotometer.
76
1~. Sirevhg and R. P. Levine:
To estimate the quantities of the individual carotenoids, the maximum absorbaneies of the individual pigments were added, the sum taken as 100%, and the percentage contributed by each of the carotenoids calculated on this basis.
Results 1. Synthesis o/Total Carotenoids Synthesis of total carotenoids parallels chlorophyll synthesis in synchronous cultures of C. reinhardtii, the major increase taking place at the eighth hour of the light period as is shown in Fig. 1. I n contrast to chlorophyll synthesis, which levels off after the 10th hour, carotenoid synthesis continues to the end of the light period but stops once the cells have entered the following dark period.
2. E/]ect o/Inhibitors on Total Carotenoid Synthesis Rifampicin had no effect on the expected increase in carotenoid formation during the 3rd light period of synchronous growth of C. reinhardtii (Fig. 2). The high levels of carotenoids formed in the control culture in this case are probably due to the high light intensity used in the experiments with rifampicin. Synthesis of total carotenoids was affected both b y cycloheximide and spectinomycin, the extent of inhibition by each drug varying from ca. 45% to 65 %. Fig. 3 shows the results from one such experiment. Since the concentration of cycloheximide used in these experiments was low compared to what has been used by other workers (Bishop and Smillie, 1970; Kirk and Allen, 1963), the effect of increasing concentrations of cycloheximide on the synthesis of total carotenoids was tested. The data in Table 1 show that the percentage of inhibition remains the same even when the concentration is increased 5-fold. Chlorophyll synthesis, on the other hand, is much more affected b y cycloheximide; as Table 1 shows, 5 ~g/ml cycloheximidc completely inhibited the synthesis o~ this pigment. To test whether carotenoid synthesis was completely dependent on protein synthesis occurring simultaneously in the cytoplasm and the chloroplast, cycloheximide and spectinomycin were added to the same cell culture. A viable cell count assured that the cells were not killed by this treatment. Fig. 4 shows t h a t carotenoid synthesis still proceeds, although at a greatly reduced rate, in the presence of both drugs. 3. E//ect o/Inhibitors on the Synthesis o/Individual Carotenoids When carotenoid extracts from synchronously grown C. reinhardtii were chromatographed, five major spots appeared. On the basis of their spectral properties, their position on the chromatogram, and color reac-
Transcription and Translation for Carotenoid Synthesis in Chlamydomonas
~,
06
6.0
0.5i
5.0
0.4
~.0
0.3
3.0
0.2
2.0
0.I
l.O
0
2
4
6
(3
I0
77
12
Hours of s y n c h r o n o u s
growth in the light
Fig. 1. The increase in total earotenoids and chlorophyll during the third light period of synchronous growth of C. reinhardtii. Synchronous cultures were obtained by using a 12-h dark/12-h light cycle. The light intensity was 5000 lux, the temperature 21 ~ C. Samples were taken in the 3rd light period at times indicated and analyzed for pigments as described in Materials and Methods. Tile amount of pigment is expressed as ~g formed per ml of synchronous culture. Cell concentration in the synchronous culture was 0.8 • 106 eells/ml, o earotenoids; 9 chlorophyll
t i o n w i t h c o n c e n t r a t e d hydrochloric acid, t h e spots were identified as/~carotene, lutein, v i o l a x a n t h i n , trollein, a n d n e o x a n t h i n . T h e epoxides, v i o l a x a n t h i n a n d n e o x a n t h i n , were f u r t h e r identified b y their characteristic h y p o s c h r o m i c spectral shift in t h e presence of dilute acid. These are t h e same m a j o r carotenoids as were described b y K r i n s k y a n d Levine (1964) for C. reinhardtii. W h e n t h e spots were e l a t e d from t h e c h r o m a t o g r a m s a n d t h e a m o u n t of each c a r o t e n o i d c a l c u l a t e d from t h e i r i n d i v i d u a l a b s o r p t i o n spectra, i t was f o u n d t h a t t h e r a t i o of fl-earotene to lutein v a r i e d s o m e w h a t from one e x p e r i m e n t to another. I n m o s t cases t h e level of lutein was a b o v e t h a t of fl-carotene in t h e s y n c h r o n o u s l y grown cultures. W h e n t h e cells were t r e a t e d with r i f a m p i c i n a n d a n a l y z e d for individ u a l carotenoids, t h e r e was a l m o s t no effect on t h e r a t i o of one carotenoid to a n o t h e r c o m p a r e d to t h e control w i t h o u t rifampicin. This is shown in T a b l e 2. The high levels of fl-carotene in t h e control m a y be t h e consequence of t h e high light i n t e n s i t y used in t h e e x p e r i m e n t s w i t h rifampicin.
78
1~. Sirevs and R. P. Levine:
I
(9,/
0.5
0.4
0,3
0. 2
it" 0
I 2
I 4
I 6
I 8
I
12
Hours of synchronous growth in the light Fig. 2. Effect of rifampicin on the increase in total carotenoids during the3rd light
period of synchronous growth of C. rdnhardtii. Experimental conditions as described in Materials and Methods and Fig. 1. The light intensity was 8000 lu:(. o control; o 250 ~g/ml of rifampicin. Rifampicin was added 50 rain before zero time
Table 3 shows the results from an experiment in which the cells were treated with cyeloheximide of spectinomycin. I n this experiment cycloheximide inhibits total carotenoid synthesis by 67 %, but it can be seen that the individual carotenoids are affected differently. At the time when eycloheximide is added to the cells (3 h in the light), the level of each carotenoid has increased somewhat in the cell. Addition of eycloheximide completely stops synthesis of fi-earotene and tro]lein, whereas synthesis of violaxanthin appears to be unaffected, and that of lutein is inhibited 45%. The individual carotenoids are also affected differently by spectinomycin. Speetinomyein inhibited total carotenoid synthesis 54 % in this experiment, the synthesis of fi-carotene being inhibited the most. Violaxanthin was inhibited 66 %, while trollein synthesis was only inhibited 12 %.
Transcription and Translation for Carotenoid Synthesis in Chlamydomonas
c5
79
0.5
0.4
0
2
4
6
8
I0
12
Hours of synchronous growth in the light Fig. 3. Effects of eycloheximide and speetinomycin on the increase in total carotenoids during the 3rd light period of synchronous growth of C. reinhardtii. Experimental conditions as described in Materials and Methods and Fig. 1. o control; 1 ag/ml of eycloheximide; 9 3 txg/ml of spectinomycin. Speetinomycin was added 50 rain before zero time; eycloheximide was added 3 h after zero time
Table 1. Effect of different concentrations of cyeloheximide on formation of total carotenoids and chlorophyll in synchronous cultures of Chlamydomonas reinhardtii Culture conditions were as described for Fig. 1. Cycloheximide was added 3 h after the onset of the third light period. Samples were taken 12 h in the light and analyzed for pigment as described in Materials and Methods. Cyeloheximide /zg/ml
Carotenoids % inhibition
Chlorophyll % inhibition
1 2 5
62.5 62.5 62.5
73.5 94.5 100.0
Discussion A l t h o u g h chloroplasts c o n t a i n DNA, this D N A does n o t appear to c o n t a i n all of the i n f o r m a t i o n necessary for the f o r m a t i o n of the organelle itself (Kirk, 1966 ; A r m s t r o n g et al., 1971). E v e n such a n i m p o r t a n t chloroplast c o m p o n e n t as chlorophyll appears to be completely u n d e r the con-
80
R. Sirev~g and 1~. P. Levine: 0.6
0.5
i
0.4
0.5
,,~
0.2
0.1
~L 2
4
6
8
I0
12
Hours of synchronous growth in the light Fig. 4. Effects of cycloheximide and cycloheximide combined with spectinomycin on the increase in total carotcnoids during the 3rd light period of synchronous growth of C. reinhardtii. Experimental conditions as described in Materials and Methods, and Figs. 1 and 2. o control; ~ 1 ~g/ml of cycloheximide; 9 1 ~g/ml of cycloheximide + 3 y.g/ml of spectinomycin Table 2. Effect of rifampicin on the formation of the major carotenoids in synchronous cultures of Chlamydomonas reinhardtii. Culture conditions were as described for Figs. 1 and 2. Samples were taken after 12 h in the light and analyzed for individual carotenoids as described in Materials and Methods Carotenoid
fl-Carotene Lutein Violaxanthin Trollein Neoxanthin
Percentage of total carotenoids Control
Rifampicin
41 25 14 13 7
38 22 13 19 8
trol of nuclear genes in C. reinhardtii (Armstrong et al., 1971). The results reported here from the experiments with rifampicin show t h a t , as i n the case of chlorophyll, the biosynthesis of the carotenoids i n C. reinhardtii is unaffected b y this antibiotic. Although negative d a t a from experiments with rifampicin are inconclusive, since the lifetime of chloroplast messen-
Transcription and Translation for Carotenoid Synthesis in Ghlamydomonas
81
Table 3. Effect of cyeloheximide and spectinomycin on the formation of the individual major earotenoids as seen in the llth hour of synchronous culture of C. reinhardtii Culture conditions were as described for Figs. 1 and 3. Samples were taken after 11 h in the light and analyzed for individual carotenoids as described in Materials and Methods. Carotenoid
fl-Carotene Lutein Violaxanthin Trollein Neoxanthin
Control Cycloheximide (~g/ml formed) ~g/ml % formed inhibition
~g/ml formed
% inhibition
0.i13 0.154 0.059 0.043 0.022
0.027 0.080 0.020 0.038 0.015
76 48 66 12 32
0.000 0.084 0.058 0.000 0.015
100 45 0 100 32
Spectinomycin
ger RNAs is not known, it appears that the synthesis of carotenoids is independent of information in chloroplast DNA and by inference dependent upon information enclosed in nuclear DNA. This is in agreement with the conclusion of Kirk (1966) based on studies of carotenoid-deficient mutant strains of higher plants that show Mendelian inheritance and are thus presumed to be the consequence of mutation of nuclear genes. The carotenoid pigments of eukaryotie photosynthetic organisms are found within the chloroplast membranes (Goodwin, 1959; Weier and Benson, 1966). They function as accessory pigments b y transferring absorbed energy to chlorophyll (Duysens, 1956). Further, they are thought to play a role in protecting chlorophyll against photooxidation (Griffiths et al., 1955; Sistrom et al., 1956; Krinsky, 1966). I t is therefore not surprising that the synthesis of these pigments closely parallels chlorophyll synthesis in synchronously grown C. reinhardtii. The major carotenoid pigments in C. reinhardtii are fl-carotene, lutein, violaxanthin, trollein, and neoxanthin (Krinsky and Levine, 1964). They are believed to arise from common precursors, but their biosynthesis and regulation are complex, involving light and many different enzymatic steps, most of which are poorly understood (Porter and Anderson, 1967). fl-carotene is an important compound in this respect because it is an intermediate in the formation of violaxanthin, trollein, and neoxanthin. Lutein, on the other hand, is thought to arise from ~-carotene (Porter and Anderson, 1967). Although the site of carotenoid formation is thought to be the chloroplast (Goodwin, 1969), our results with eycloheximide and spectinomycin show that their formation is dependent on de-novo protein synthesis on both cytoplasmic and chloroplast ribosomes. The fact that the levels of 6
P l a n t a (Berl.), Bd. 111
82
1~. Sirevs and R. P. Levine:
carotenoids in the cell increased to a large extent even when translation on the cytoplasmic and chloroplast ribosomes was inhibited simultaneously, indicates that the enzymes required for carotenoid biosynthesis are present in the cells at the onset of the light period and in amount sufficient to synthesize at least 39 % (Fig. 4) of the carotenoids found in the control cultures. From the data obtained by the analysis of the individual carotenoids, it appears that these enzymes might be mainly concerned with lutein synthesis, since the synthesis of this pigment was much less affected by the drugs than was that of the other individual carotenoids. The extensive inhibition of/~-carotene formation by either eycloheximide or spectinomycin suggests that the synthesis of this pigment is completely dependent on translational steps both on cytoplasmic and chloroplast ribosomes. There still exists the possibility, however, that some flcarotene is formed in the presence of the drugs and is then immediately converted to other carotenoids. Thus, the finding that violaxanthin formarion is unaffected by cycloheximide indicates that its precursor, /?carotene, may be formed to some extent in the presence of this inhibitor. In addition, the inhibition of violaxanthin formation by speetinomycin demonstrates that protein synthesis on chloroplast ribosomes is involved in the conversion of fl-carotene to violaxanthin. On the other hand, it appears that the formation of trollein from fl-carotene requires de novo protein synthesis only on the cytoplasmic ribosomes since the formation of this pigment is completely inhibited by cycloheximide and only slightly affected by spectinomycin. I t appears from this study that the formation of the five major carotenoids of C. reinhardtii depends on proteins (most likely enzymes of carotenoid biosynthesis) that are synthesized on both chloroplast and cytoplasmic ribosomes. I t is likely that both types of ribosomes are involved, for the synthesis of the five pigments undoubtedly entails several different biochemical reactions. The membrane-bound nature of the pigments must also be kept in mind, for it is possible that the antibiotics are inhibiting the synthesis of proteins that are essential for the association of the carotenoids with the membranes. A sharing of responsibility between chloroplast and cytoplasmic ribosomes may not be rare, for the synthesis of cytochrome 563 and ribulose 1,5-diphosphate carboxylase in synchronous cultures of C. reinhardtii appears to depend on both types of ribosomes (Armstrong et al., 1971). A dependence on both classes of ribosomes has also been reported for the synthesis of ribulose 1,5-diphosphate earboxylase in barley (Criddle et al., 1970). The authors wish to thank Professor Norman Krinsky, Tufts Medical School, Boston, Mass. for his helpful suggestions during the course of the work reported here. This work has been supported by the National Science Foundation, grant GB 18666, to the second author.
Transcription and Translation for Ca,rotenoid Synthesis in Chlamydomonas 83 Relerences Armstrong, J. J., Surzycki, S. J., Moll, B., Levine, R. P. : Genetic transcription and translation specifying chloroplast components in Chlamydomo~as reinhardi. Biochemistry lO, 692-701 (1971). Arnon, D. I. : Copper enzymes in isolated chloroplasts: polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). Bishop, D. G., Smillie, R. M. : The effect of ehloramphenicol and cyclohexamide on lipid synthesis during chloroplast development in Euglena gracilis. Arch. Biochem. Biophys. 137, 179-189 (1970). Burton, W. G. : The binding of spectinomycinto chloroplast ribosomes of Chlamydomonas reinhardi. J. Cell Biol. 47, 28a (1970). Burton, W. G. : Dihydrospectinomycin binding to chloroplast ribosomes from antibiotic-sensitive and -resistant strains of Chlamydomonas reinhardi. Biochim. biophys. Aeta (Amst.) 272, 305-311 (1972). Chun, E. It., Vaughan, M. It., Rich, A. : The isolation and characterization of DNA associated with chloroplast preparations. J. molec. Biol. 7, 130-141 (1963). Criddle, R. S., Dau, B., Kleinkopf, G. E., ttuffaker, R. C. : Differential synthesis of ribulosediphosphate carboxylase subunits. Biochim. biophys. Res. Commun. 41, 621-627 (1970). Duysens, L. N. M. : Energy transformations in photosynthesis. Ann. Rev. Plant Physiol. 7, 25-50 (1956). Galling, G. : Der EinfluB yon Rifampicin, Chloramphenicol und Cycloheximid auf den Uridin-Einbanin chloroplastid~re Ribosomenvorstufen yon Chlorella. Planta (Berl.) 98, 50-62 (1971). Goodwin, T. W. : Biosynthesis and function of carotenoid pigments. In: Advances in enzymology, vol. XXI, p. 296-368, Nord, F . F . , ed. New York-London: Interseience 1959. Goodwin, T. W.: Carotenoid biosynthesis in chloroplasts. In: Progress in photosynthesis research, vol. II, p. 669-674, Metzner, H., ed. Tfibingen 1969. Griffiths, M., Sistrom, W. R., Cohen-Baz]re, G., Stanier, R. Y. : Function of carotenoids in photosynthesis. Nature (Lond.) 176, 1211-1215 (1955). ttoober, J. K. : Sites of synthesis of chloroplast membrane polypeptides in Chlamydomonas relnhardi. J. biol. Chem. 245, 4327-4334 (1970). Hoober, J. K. : A major polypeptide of chloroplast membranes of Chlamydomonas reinhardi: Evidence for synthesis in the cytoplasm as a soluble component. J. Cell Biol. 52, 84-96 (1972). Hoober, J. K., Blobel, G. : Characterization of the ehloroplastic and cytoplasmic ribosomes in Chlamydomonas reinhardi. J. molee. Biol. 41, 121-138 (1969). Hoober, J. K., Siekevitz, P., Palade, G. E. : Formation of chloroplast membranes in Chlamydomonas reinhardi y-1. J. biol. Chem. 244, 2621-2631 (1969). Kates, J. R., Jones, R. F. : The control of gametic differentiation in liquid cultures of Chlamydomonas. J. cell. eomp. Physiol. 63, 157-164 (1964). Kirk, J. T. O. : Nature and function of chloroplast DNA. In: Biochemistry of chloroplasts, vol. I, p. 319-340, Goodwin, T. W., ed. London-New York: Acad. Press 1966. Kirk, J. T. 0., Allen, R. L. : Dependence of chloroplast pigment synthesis on prorein synthesis: Effect of aetidione. Biochem. biophys. Res. Commun. 21,523-530 (1963). Krinsky, N. I, : The role of carotenoid pigments as protective agents against photosensitized oxidation in chloroplasts. In: Biochemistry of chloroplasts, vol. I. p. 432-430, Goodwin, T. W., ed. London-New York: Aead. Press 1966. 6*
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