Planta

Planta (1986) 167:51~-520

9 Springer-Verlag 1986

Translational regulation of protein synthesis during light-induced chloroplast development in Euglena C. Bouet 1 *, R. Schantz 2, G. Dubertret 1, B. Pineau 1 and G. Ledoigt I ** 1 Laboratoire de Cytophysiologie de la Photosynth6se, C.N.R.S., F-91190 Gif sur Yvette, France, and 2 Institut de Botanique, Laboratoire de Physiologie V6g6tale, 28, Rue Goethe, F-67083 Strasbourg, France

Abstract. Control of gene expression in Euglena was examined during light-induced chloroplast development. Greening was achieved under standard conditions which allowed the synthesis of all plastid proteins in both cytoplasmic and chloroplastic compartments, or under experimentally modified conditions inducing the preferential synthesis of the photosystem II (PSII) light-harvesting antenna or reaction centers. The relative composition of total mRNAs in cellular, cytoplasmic or chloroplastic fractions, as analyzed by their in-vitro translation products in cell-free systems did not significantly change during the in-vivo protein-synthesis processes which are specific to each greening system. By contrast, cytoplasmic polysomal mRNAs extracted during the selective recovery phase of PSII light-harvesting antennae provided a major in-vitro synthesis product of 28 kDa which could correspond to a precursor of the main 26-kDa apoprotein of the light-harvesting chlorophyll a/b protein complex. Similarly, the in-vivo selective synthesis of the 4J-kDa and 51-kDa polypeptides of PSII reaction centers was concomitant with an enrichment of plastid polysomes in m R N A species coding for polypeptides of the same molecular weight. These observations confirm that protein synthesis during chloroplast development in Euglena is weakly regulated at the transcription level and they demonstrate that translational regulation occurs in both the cytoplasmic and the chloroplastic compartments. * Present address." Laboratoire de Physiologie V6g6tale Mol6-

culaire Universit6 Paris-Sud, F-91405 Orsay, France ** Present address: Laboratoire de Biologie et Physiologie V6-

g6tale, Universit6 Clermont II, 4 Rue Ledrn, F-63038 Clermont Ferrand, France Abbreviations: LHCP = light-harvesting chlorophyll a/b protein complex; PS I, II = photosystem I, II; RuBPcase = ribulose-] ,5bisphosphate carboxylase

Key words: Chloroplast development - Euglena m R N A - Photosystem II - Polyribosome - Protein synthesis (regulation).

Introduction

The unicellular alga Euglena gracilis is a eukaryotic organism in which the relationship between the expression of plastid and nuclear genomes can easily be studied during chloroplast development. In heterotrophic growth in darkness, Euglena are devoid of functional chloroplasts and it has been shown that the chloroplast genome is transcribed both in dark-grown cells containing undifferentiated proplasts, and in light-grown cells containing fully developed chloroplasts (Nigon and Heizmann 1978). When dark-grown Euglena are illuminated, a sequence of macromolecular events occurs which leads to the formation of mature chloroplasts (Ledoigt 1976; Schiff 1973; Heizmann et al. 1978; Dubertret 1981; Timko et al. 1982). During light-induced plastid development, changes in transcription products are mainly quantitative (Nigon and Heizmann 1978). Among the major transcription products are the ribosomal (r) RNAs and transfer (t) RNAs, but less is known about the nature and the differential expression of mRNAs encoding specific chloroplast proteins. Hybridization studies have shown only small differences in the transcription of plastid DNA during greening of Euglena cells (Chelm and Hallick 1976; Rawson and Boerma 1976; Rawson and Boerma 1979; Chelm et al. 1979; Rawson et al. 1981). Recently, several transcripts were shown to be developmentally expressed (Dix and Rawson 1983; Price et al. 1983). Thus, the control of gene expression during plastid development is currently a topic of intense interest.

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Our approach to this problem was to compare in-vitro translational products directed by total mRNA or by polysomal mRNA extracted from greening cells at different stages of chloroplast development. For the sake of simplification, we have extended such analyses to two greening systems experimentally modified in order to trigger specifically the synthesis of a limited number of polypeptide species, including those involved in PSII formation. Development of photosynthetic membranes devoid either of PSII light-harvesting antennae or of PSII reaction centers was first induced by exposing greening cells to intermittent light (Dubertret and Lefort-Tran 1981) or to specific inhibitors of plastid translation such as clindamycin (Dubertret and Pineau 1984). The restoration of normal conditions, obtained by exposing the greening cells to permanent light or by washing off the inhibitor, thus allows the preferential synthesis of the missing light-harvesting antenna or reaction-center polypeptides in the cytoplasmic or chloroplastic compartments. The results obtained demonstrate that during chloroplast development in Euglena, chloroplastic and cytoplasmic syntheses are mainly post-transcriptionally regulated.

Material and methods Culture and greening conditions. Etiolated Euglena gracilis Z (Cambridge n ~ 1224-5D) were first grown in the dark in lactatecontaining medium (Calvayrac 1970). All greening experiments were performed with non-dividing, stirred cells obtained by transferring the cultures into a resting medium according to Stern et al. (1964). For greening under permanent light, the cultures were exposed to 1200-1x white light provided by banks of daylight, fluorescent tubes. For intermittent-light experiments, cells were placed 30 cm from two banks of 100-W incandescent lamps providing ~5 s light for every 15 rain of darkness (Dubertret and Lefort-Tran 1981). For greening in the presence of an inhibitor of plastid synthesis, cells were first greened for 24 h. Clindamycin (Upjohn; Paris-la Defense, France) was then aseptically added to a final concentration of 10 -a M, and the culture was exposed to a higher light intensity (3000 Ix) for the subsequent 48 h in order to photodestroy PSII reaction centers. For clindamycin removal, cells were centrifuged, washed, resuspended in fresh resting medium and exposed to low light intensity (300 lx) in order to induce PSII reactioncenter formation without appreciable chlorophyll synthesis. For in-vivo radioactive-labelling experiments, a mixture of 15 I~Camino acids 17" 105 Bq per atom of C, CEA, Gif-sur-Yvette, France) was added to concentrated cultures to give a final concentration of 3.77-104 Bq-ml - ~. Measurement of chlorophyll concentration and PSH functional parameters. Chlorophyll concentration was measured in acetone extracts according to MeKenney (1941). The size of the PSII light-harvesting antennae and the concentration of the PSII reaction center were estimated from the half-rise time and

from the area above the fluorescence induction curve in the presence of 10-3 M 3-(3', 4'-dichlorophenyl)-l,l-dimethylurea (DCMU), as previously described (Dubertret and Lefort-Tran 1981).

Preparation of thylakoids. Ceils were broken with a French Press and thylakoids were purified by differential and gradient centrifugations as previously described (Pineau et al. 1985). Preparation ofmRNA. (a) Cytoplasmic mRNA. Cells were harvested, resuspended in sterile buffer A (2-amino-2-(hydroxymethyl)-l,3-propanediol) (Tris-HC1) 50 mM, pH 7.5, sucrose 0.25 M, KC1 25 raM, MgClz 5 mM) and broken with a French Press at 1.5.10 7 Pa. Chloroplasts, mitochondria and large cell debris were eliminated in the pellet of a 15 000-g, 20-rain centrifugation. Total cytoplasmic m R N A was extracted from a sample of the supernatant with an equal volume of a mixture of watersaturated phenol and chloroform (1:1, v/v), and nucleic acids were precipitated in the aqueous phase with 2.5 vol. of cold ethanol overnight at - 20 ~ C. For polysomal m R N A isolation, cytoplasmic polysomes were purified according to Avadhani and Buetow (1972); the 15000-g supernatant was solubilized with desoxycholate (1% final concentration) and 6 ml were layered on 3 ml of a 2-M sucrose cushion prepared in sterile buffer A. The polyribosome pellet obtained after a centrifugation at 200000 g for 3 h was suspended in 500 gl of buffer B and R N A was extracted using the chloroform-phenol method. (b) Chloroplastic mRNA. Cells were harvested, suspended in sterile buffer B (Tris-HC1 50 mM pH 8.5, fl mercaptoethanol 7 raM, KC1 40 raM, Mg-acetate 20 mM) plus sucrose 0.25 M and disrupted with a French Press at 1.5.107 Pa. A chloroplastenriched pellet was obtained by two sets of differential centrifugations at 120 g for 1 min and 2000 g for 5 rain. For total chloroplastic m R N A extraction, the chloroform-phenol method was applied to this chloroplast-enriched pellet. For extraction of m R N A from chloroplastic polysomes, chloroplasts were solubilized in buffer B containing 2% Triton X-100. Debris and residual membrane structures were eliminated in the pellet of a 12000-g, 15-rain centrifugation and the supernatant (7 ml) was layered on a discontinuous sucrose gradient (0.7 and 1.7 M sucrose in Buffer B, 2.5 ml each) and centrifuged at 200000 g for 3 h. The chloroform-phenol method was used to extract RNAs from the polysomal pellet, Cell-free synthesis. The different m R N A fractions were collected by centrifugation, washed three times with cold 2 M LiC1 and with cold ethanol. The precipitates were used for in-vitro protein synthesis in a rabbit reticulocyte (Amersham Buchler, Braunschweig, FRG) or in a wheat-germ (Bethesda Res. Lab. Gaethersburg, Md., USA) translation system using asS-labelled methione (3.77.104 B q - g l - t CEA). Reactions were stopped by incubation with 1 vol. sohibilizing buffer containing TrisHC1 0.06 M pH 8.2, glycerol 20%, ethylenediaminetetraacetic acid (EDTA) 0.3 mM, fl-mercaptoethanol 2%, sodium dodecyl sulfate (SDS) 4%, and the mixtures were heated at 90~ for 10 min before electrophoresis. Electrophoresis and fluorography. Membrane proteins and invitro translation products were analyzed by electrophoresis on slab gels according to Laemmli (1970). Details concerning thylakoid solubilization and two-dimensional electrophoreses are given elsewhere (Pineau et al. 1985). Gels were stained with Coomassie blue and treated with 2,5-diphenyloxazole (Bonner and Laskey 1974) or sodium salicylate (Chamberlain 1979) as enhancers for fluorography using Royal RP Xomat (Kodak) films.

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Results

Greening under permanent light Cell growth and division is inhibited in the restingmedium system used. Changes in protein composition and RNA contents observed during greening are thus mainly related to biosynthetic events involved in the light-induced differentiation of proplastids into functional chloroplasts.

Synthesis of thylakoid polypeptides. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved about 30 polypeptides from thylakoids purified at different stages of greening (Fig. 1). However, the polypeptide pattern drastically changed after 6 h of light exposure, concomitant with the onset of active chlorophyll synthesis. While some bands decreased or disappeared, others, involved in the formation of chlorophyll-protein complexes, became predominant. After 24 h of greening, the main membrane component appeared to be a 26-kDa polypeptide related to the major apoprotein of the LHCP (Pineau et al. 1985). The other main bands apparently synthesized during the greening process corresponded to the second 29-kDa apoprotein of the LHCP and to polypeptides of 68, 51 and 41 kDa; these are involved in chlorophyll-protein complex I (CPI) and PSII reaction-center formation, respectively (Pineau et al. 1985).

Fig. 1. Coomassie-blue stained 9-15% acrylamide gel of Euglena plastid membranes developed for 0, 3, 6, 12 and 24 h under permanent light. Thylakoids were purified on Percoll gradients according to Price and Reardon (1982). The arrows indicate the peptides involved in PSI (68 kDa) and PSII (51 and 41 kDa) reaction centers and in the light-harvesting antenna (29 and 26 kDa)

mRNA content. Translation products directed by the corresponding total mRNA fractions, in wheat-germ or rabbit reticulocyte systems are shown in Figs. 2A, B. The polypeptide patterns obtained were different, indicating different selections of the mRNA species effectively translated in each of these systems. Anyhow, none of these systems showed marked qualitative differences in

Fig. 2A, B. Autoradiograms of the electrophoretic pattern of polypeptides synthesized in vitro in the presence of total m R N A fractions extracted from cells greened for 0, 3, 6, 12 and 24 h. A Wheat-germ translation system; B Reticulocyte-lysate translation system. Cell-free syntheses were carried out in the presence of [35S]-methionine (3.77-104 Bq-gl- 1); polypeptides synthesized in each system without added mRNA are shown in tracks c

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Fig. 3. Coomassie-blue stained 8-18% acrylamide gel of Euglena thylakoids developed under different light conditions. IL 96 h intermittent light (15 s light/15 rain dark); PL, 96 h intermittent light plus 15 h permanent light; C, control exposed for 96 h to permanent light. The arrows indicate the position of the two apoproteins (29 and 26 kDa) of Euglena LHCP. Equal amounts of chlorophyll (30 p,g) were loaded on each lane

the pattern of in-vitro translation products during greening. This result shows that, by contrast to protein composition, chloroplast development proceeds without important changes in the relative levels of translatable mRNAs. Light exposure of etiolated cells, therefore, increases the relative rate of synthesis of several main in-vivo translation products in the absence of a corresponding increase in the relative levels of some m R N A species.

Greening under intermittent light Greening of etiolated cells for 4 d under intermittent light induces the formation of functional chloroplasts devoid of the PSII light-harvesting antennae (Dubertret and Lefort-Tran 1981). The subsequent exposure of such cells to permanent light allows the cytoplasmic biosynthetic events associated with the recovery of the missing light-harvesting antennae to be studied.

Fig. 4. Autoradiogram of the electrophoretic patterns of polypeptides synthesized in the reticulocyte system in the presence of cytoplasmic mRNA fractions extracted from cells greened for 96 h under intermittent light (IL), then exposed for 15 h to permanent light (PL) and from control cells (C) greened for 96 h under permanent light. In-vitro syntheses were carried out using reticulocyte lysate in the presence of [35S]methionine (3.77.104 Bq. gl- 1), Approximately equal amounts of radioactivity were loaded on each lane, Controls without added m R N A provided the same result as in Fig. 2B

Thylakoid polypeptides. Stained gels of polypeptides extracted from cells greened under intermittent light (Fig. 3, IL) revealed the presence of all polypeptides observed in the control sample (Fig. 3, C) except those involved in LHCP formation, i.e. the major 26-kDa apoprotein which was absent and the minor 29-kDa which was markedly reduced. The subsequent exposure of cells to permanent light for 15 h resulted in the synthesis andor the integration of principally both the 29- and 26-kDa apoproteins of the LHCP (Fig. 3, PL) and of a 17.5-kDa polypeptide which is associated with CPI (Pineau et at. 1985). mRNA. Total cytoplasmic mRNAs extracted from control cells (Fig. 4, C) and from cells first greened under intermittent light (Fig. 4, IL), then exposed for 15 h to permanent light (Fig. 4, PL) were translated in the reticulocyte system. The polypeptide profiles revealed by fluorography (Fig. 4) were identical indicating that intermittent light and the

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of total cytoplasmic m R N A (Fig. 4) and of polysomal m R N A (Fig. 5). Transfer to permanent light, therefore, increased the proportion of cytoplasmic polyribosomes containing the corresponding m R N A species. The increase in-vitro synthesis of this 28-kDa polypeptide was concomitant with the appearance of the 26-kDa apoprotein of the LHCP in the thylakoids; this 28-kDa polypeptide could, therefore, correspond to a precursor form of the 26-kDa apoprotein of the LHCP.

Greening in the presence of clindamycin

Fig. 5. Autoradiogram of the electrophoretic pattern of polypeptides synthesized in vitro (reticulocyte system) in the presence of cytoplasmic polysomal mRNA extracted from cells greened for 96 h under intermittent light (IL) then exposed for 15 h to permanent light (PL). Same experimental conditions as in Fig. 4

subsequent permanent light did not exert any appreciable effect on translatable mRNA. By contrast, mRNAs extracted from polyribosomes of cells greened under intermittent light and transferred to permanent light, directed the synthesis of a major 28-kDa polypeptide which appeared only as a faint band in the translation products

The addition of clindamycin to greening cultures, 24 h after the onset of light does not change the overall structural development of plastids, as compared with control cells. Under these conditions, the major effect of this inhibitor of protein synthesis on plastid ribosomes is to prevent the renewal of PSII reaction centers which are destroyed by high light intensity. Greening in the presence of clindamycin thus leads to the formation of chloroplasts largely devoid of PSII reaction centers (Dubertret and Pineau 1984). The subsequent elimination of the inhibitor allows the chloroplastic biosynthetic events associated with the recovery of PSII reaction centers to be analysed.

Thylakoid polypeptides. Control, clindamycintreated and washed cells were labelled for 24 h with a mixture of ~4C-amino acids and two-dimensional electrophoresis of thylakoid polypeptides was performed in order to resolve the 51-kDa apoprotein of chlorophyll-protein complex a (CPa) from a comigrating 50-kDa polypeptide (Pineau et al. 1985). The same amount of radioactivity was loaded in each track. Fluorography of the seconddimension electrophoresis (Fig. 6) showed that, in

Fig. 6. Autoradiogram of the two-dimensional electrophoretic pattern of thylakoid polypeptides of control cells (C), of clindamycintreated cells (+cI) and ctindamycin-washed cells (-c/). Clindamycin-treated cells were washed and the three parallel cultures were simultaneously labelled for 24 h with a mixture of 14C-amino acids 3.77-104 Bq-gl-1). Chlorophyll-protein complexes were first separated in a 7% acrylamide gel and polypeptides were resolved by a second-dimension electrophoresis in 8-18% acrylamide gel. Equal amounts of radioactivity were loaded in each track, arrows indicate the apoproteins of chlorophyll-protein complex I (68 kDa) and those involved in PSII reaction centers (5t and 41 kDa)

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Fig. 7. Autoradiogram of the electrophoretic pattern of polypeptides synthesized in vitro (reticulocyte system) in the presence of plastid-enriched mRNA extracted from control cells (C), clindamycin-treated cells ( + c o and clindamycin-washed cells (-cO. Same experimental conditions as in Fig. 4

control thylakoids (C), labelling was distributed among numerous polypeptides including 68-, 53 to 51-, 31-, 26-kDa and low-molecular-weight species. Cells kept in the presence of clindamycin (+cl), in which plastid syntheses were inhibited, incorporated radioactivity principally in low-molecular-weight thylakoid polypeptides. By contrast, most of the radioactivity incorporated by clindamycin-washed cells ( - c l ) was located in the 51and 41-kDa and to a lesser extent, in the 68-kDa polypeptides, which were previously assigned respectively to PSII and PSI reaction centers. Clindamycin removal thus resulted in the rapid synthesis on plastid ribosomes of a limited number of polypeptides including those of 51 and 41 kDa which are constituents of PSII reaction centers, and to a lesser extent the 68-kDa polypeptide involved in PSI activity. Plastid mRNA. Fractions enriched in plastid total m R N A were extracted from cells grown in the three culture conditions. They provided similar polypeptide patterns on fluorographs when translated in vitro in the reticulocyte lysate (Fig. 7). The population of translatable m R N A was thus unal-

Fig. 8. Autoradiogram of the electrophoretic pattern of polypeptide syntheses in vitro in the presence of plastid polysomal mRNA extracted from control cells (C), clindamycin-treated cells ( + c o and clindamycin-washed cells (-cO. The polypeptide pattern obtained from - c l cells of another experiment is presented on the right. Same experimental conditions as in Fig. 4

tered by clindamycin treatment and the in-vivo active synthesis of PSII reaction-center polypeptides in the chloroplasts was therefore unrelated to any appreciable increase in some of the plastid m R N A species. By contrast, m R N A isolated from plastid polyribosomes of control, clindamycin-treated and washed cells provided qualitative differences in translation products obtained with the same invitro synthesis system (Fig. 8). Although stimulation of [35S]methionine incorporation by the same amounts of polysomal m R N A was much lower when extracted from clindamycin-treated cells than from control cells, both samples displayed qualitatively similar polypeptides patterns. This indicates that weak amounts of most of the plastid m R N A species remained bound to plastid polyribosomes in clindamycin-treated cells. However, polysomal m R N A of washed cells promoted the in-vitro synthesis of a few major polypeptides of 55, 51 and 41 kDa. Depending on the experiment, the relative amount of the 41-kDa polypeptide appeared to be variable (see Fig. 8); it increased most when the proportion of PSII reaction centers still active before clindamycin removal was low. This limited

C. Bouet et al. : Translational regulation of chloroplast protein synthesis in Euglena

number of in-vitro-synthesized polypeptides indicates that removal of the drug resulted in the selective translation of a few mRNA species. The 55-kDa polypeptide can tentatively be attributed to the large subunit of ribulose bisphosphate carboxylase (RuBPCase) and/or to the subunits of ATP synthetase. The 51- and 41-kDa polypeptides synthesized in vitro obviously correspond to the 51- and 41-kDa polypeptides of PSII reaction centers with which their in-vivo synthesis is concomitant. Discussion

Exposure to light of etiolated, resting Euglena cells induces the differentiation of proplastids into functional chloroplasts without concomitant cell divisions. The structural and functional development of chloroplasts is then associated with a sequence of specific biosynthetic events resulting in the increase or the de-novo synthesis of plastid components. Even if analyzed at the simple level of thylakoid polypeptide biosynthesis, this developmental process appears to be quite complex since numerous polypeptides, principally involved in the formation of PSII and PSI chlorophyll-protein complexes accumulated while some species disappeared (Fig. 1). Simplification of this biosynthesis process should therefore provide a favourable tool for the analysis of protein-synthesis regulation. Pretreatment of greening cells with clindamycin and high light intensity and the subsequent transfer to normal conditions allowed the synthesis on plastid ribosomes of large amounts of a few polypeptides including the 41- and 5]-kDa species involved in PSII reaction centers (Fig. 6). By contrast, the transfer to permanent light of cells first greened under intermittent light resulted in the preferential synthesis on cytoplasmic ribosomes of the 29- and principally of the 26-kDa apoproteins of PSII light-harvesting antenna (Fig. 3). The evolution of mRNA relative composition in each of these greening systems was determined by analysing the relative composition of translation products in cell-free systems. Since the E. coli protein-synthesizing system appeared to be inefficient in translating Euglena mRNA (results not shown and Edelman et al. 1977), we tried the wheat germ and the reticulocyte-lysate systems. The different polypeptide patterns obtained from the same cellular mRNA fractions with these heterologous systems (Fig. 2 A, B) indicate that artifacts could arise from messenger recognition by ribosomes and-or from premature termination of polypeptide elongation. Translations in such cell-free

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systems are less reliable than in homologous systems and results therefore only provide indications of mRNA levels and composition. Nevertheless, taking into account these limitations, the results show that no major changes can be observed in the translation products of mRNA isolated from cells undergoing greening in the different experimental conditions described (Figs. 2, 4, 7). Quantitatively, the small increase in the stimulation of 35S incorporation, when observed, shows that the large amount of in-vivo protein synthesis during greening under permanent light or under modified conditions is not associated with a proportional increase in mRNA content. Moreover, the qualitatively similar polypeptide patterns obtained in each type of experiment indicate that the in-vivo synthesis of polypeptides involved in thylakoid development is not correlated with the selective synthesis of the corresponding mRNA species. The absence of a relationship between mRNA composition and the selective synthesis of some polypeptides during chloroplast development therefore confirms that protein synthesis in Euglena is largely post-transcriptionally regulated. Limited changes in plastid mRNA level (Miller et al. 1983) and in cellular mRNA composition (McCarthy and Schwartzbach 1984) during Euglena chloroplast development had already been observed using cell-free translation systems and were interpreted as indicating a weak transcriptional regulation of protein synthesis in Euglena. Measurement of transcription during chloroplast development by hybridization of plastid restriction fragments with total cellular RNA (Rawson and Boerma 1979; Dix and Rawson 1983) showed that the great majority of the transcripts are present in dark-grown Euglena cells and, therefore, are constitutively expressed. However, several transcripts appeared to be developmentally expressed. In similar experiments with cloned restriction fragments containing identified chloroplast protein genes, Hallick et al. (1983) showed that transcription of the large subunit of RuBPCase, of the flsubunit of the coupling factor and of the 32-kDa polypeptide loci is developmentally regulated while the level of mRNA detected with the probe for the transfer, unstable (Tu) protein synthesis elongation factor, remains constant through the first 60 h of chloroplast development. More recently, Schantz (1985) demonstrated, using the hybridization method, that transcription of genes of LHCP and CPa apoproteins does not markedly increase during Euglena chloroplast development. Results obtained by several authors with different methods thus agree in demonstrating a weak

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regulation of transcription and, therefore, lead to the conclusion that protein synthesis during Euglena chloroplast development may be largely regulated at the translational and-or at a post-translational level. According to this hypothesis, the translation process on polysomes should involve a limited number of m R N A species among the mRNAs present. The modified greening systems we used here, allow the selective enhancement of the synthesis and-or the integration of a limited number of thylakoid polypeptides, including those involved in the formation of PSII light-harvesting antenna or reaction centers, and therefore facilitate the analysis of concomitant events at the polysomal level. The polypeptide patterns obtained by in-vitro translation of total cytoplasmic or chloroplastic mRNAs are different from those displayed by the corresponding polysomal m R N A fractions. Obviously, only some m R N A species enter into the translation process during each of the analyzed recovery systems. The preferential in-vivo synthesis of the 41- and 51-kDa PSII reaction-center polypeptides subsequent to clindamycin removal is concomitant with the formation of plastid polyribosomes containing mRNAs translated in vitro principally as the 41- and 51-kDa polypeptides (Fig. 8). Similarly, cytoplasmic polyribosomes are enriched in an m R N A species translated in vitro as a 28-kDa polypeptide (Fig. 5) when the 26- and, to a lesser extent, the - 29-kDa apoproteins of LHCP are synthesized in vivo in the intermittentlight system. Immunological identification of these translation products of polysomal mRNAs would allow us to ascertain whether or not they are effectively constituents of the LHCP and CPa. Unfortunately, antibodies directed against the LHCP and CPa apoproteins do not recognized peptides of the expected size among the in-vitro translation products of Euglena m R N A (Devic and Schantz 1984). This result is peculiar to Euglena since the same antibodies with the same procedure provide the usual immunoprecipitated peptides when translations are directed by RNAs extracted from Chlamydomonas and from several higher plants. Immunological identification of Euglena polypeptides synthesized in vitro thus cannot be reliable as long as these unusual results are not clearly understood. Anyhow, although the identity of these polypeptides may be questionable, our results undoubtedly demonstrate that during Euglena development the synthesis of some thylakoid polypeptides, probably involved in LHCP and PSII reaction centers, is regulated at the translational level.

Moreover, if, as is probable, the major in-vitro translation products of polysomal mRNAs extracted from clindamycin-washed cells correspond to PSII reaction-center polypeptides, the similarity of their molecular weight (51 and 41 kDa) would indicate that they are not markedly modified by post-translational events. By contrast, the major in-vitro translation product of molecular weight 28-kDa obtained with the intermittent-light system seems to correspond to a precursor form of the 26-kDa apoprotein of LHCP which is actively synthesized in vivo. The post-translational processing of the native polypeptide by elimination of a "transit sequence" would be consistent with similar post-translational maturation of LHCP apoproteins observed in higher plants and algae (Apel and Kloppstech 1978; Tobin 1981; Shepherd et al. 1983). Post-transcriptional events have been reported in stressed cells of higher plants (Baszczynski et al. 1983), and many regions of mustard plastid DNA have recently been found to be constitutively expressed during chloroplast development (Link 1984). A weak transcriptional regulation of the large subunit of RuBPCase has also been described in Chlamydomonas (Howell 1978; Mishkind and Schmidt 1983). However, the fact that the regulation of protein synthesis occurs mainly at the translational or post-translational levels actually seems to be a characteristic of Euglena. This raises the question of the mechanisms of translational regulation. Leu et al. (1984) and Minami and Watanabe (1984) recently showed that in Chlamydomonas and spinach chloroplasts, stromal and thylakoid-bound polysomes contain the same m R N A species while synthesizing different proteins. This observation seems to rule out the hypothesis of a selective recognition of messengers by ribosomes, depending on their localization within the chloroplast, and favours the idea that thylakoids could exert a regulatory role on the elongation process after polysome formation. However, such a mechanism does not account for the changes in m R N A composition of plastid and cytoplasmic polysomes observed here. The important translational regulation of protein synthesis during the light-induced chloroplast development of Euglena, therefore, arises either from the ribosomal recognition of translatable messengers or from the instability of polyribosomes containing m R N A species which are not actually translated. We thank Mrs. C. G6rard-Hirne for experimental assistance. This work was supported by two grants of the Centre National de la Recherche Scientifique (ATP Conversion de l'&nergie dans

C. Bouet et al. : Translational regulation of chloroplast protein synthesis in Euglena les membranes biologiques et ATP Organisation et expression du g~nome dans les cellules eucaryotes) and a grant MRT "Mission des Biotechnologies" n o 82 V 12 66.

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Translational regulation of protein synthesis during light-induced chloroplast development in Euglena.

Control of gene expression in Euglena was examined during light-induced chloroplast development. Greening was achieved under standard conditions which...
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