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Contributions from the Plastid and the Nucleocytoplasmic Compartments to the Events Associated with Heat-bleaching in Euglena gracilis
J.PlantPhysiol. Vol. 139.pp. 543-548{1992}
Contributions from the Plastid and the Nucleocytoplasmic Compartments to the Events Associated with Heat-bleaching in
Euglena gracilis WILLIAM ORTIZ Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, Oklahoma 73019-0245, USA Received June 4, 1991 . Accepted August 17, 1991
Summary Growing photo heterotrophic cultures of Euglena gracilis Z show a time-dependent loss of chlorophyll per cell that results in the production of bleached cultures of the alga on prolonged incubation at 33°C. In terms of chlorophyll accumulation, the bleaching process involves an initial rise in chlorophyll accumulation per cell, peaking at about 15 h, followed by a second phase in which chlorophyll levels per cell actually decline. The primary objective of this work was to establish whether loss of chloroplast function at 33°C is associated with a loss of translational activity on chloroplast ribosomes or a loss in the synthesis of chloroplast proteins of nucleocytoplasmic origin, or both. To this effect, pulse-labeling experiments were carried out in vivo on cultures of the alga in the second phase of the temperature response. The results show that synthesis and import of proteins from the nucleocytoplasmic compartment remain high in bleaching plastids during the first 41 h of the temperature treatment. By contrast, protein synthesis inside plastids is gradually lost at the elevated temperature, and only low levels can be detected in 41-h bleached cultures. Consequently, the great majority of the proteins accumulating in 41-h bleached plastids are imported from the nucleocytoplasmic compartment. The loss of translational activity inside the bleaching plastid could explain why Euglena is unable to maintain photosynthetically competent plastids at the elevated temperature.
Key words: Euglena gracilis Z; heat-bleaching; protein synthesis. Abbreviations: ChI = chlorophyll; CHX = cycloheximide; LHCn = light-harvesting chlorophyllbinding protein of Photosystem II; LSU and SSU = large and small subunit; PM SF = phenylmethylsulfonyl fluoride; QB = quinone-binding protein; RUBIS CO = ribulosebisphosphate carboxylase; TCA = trichloracetic acid. Introduction The temperature-induced loss of total ChI in growing photoheterotrophic cultures of Euglena gracilis is commonly known as heat-bleaching (Pringsheim and Pringsheim, 1952). The phenomenon is of interest to us because the alga is unable to maintain functional chloroplasts at the moderately elevated temperature of 33 to 34°C. The effect of temperature on ChI accumulation takes place in two phases (Ortiz and Wilson, 1988). Chlorophyll accu© 1992 by Gustav Fischer Verlag. Stuttgart
mulation per cell initially rises and peaks at about 15 h after the onset of the temperature treatment. The rise in ChI is followed by a second phase characterized by a steady decline in ChI levels per cell. In this context, we have documented changes in the profiles of stainable polypeptides in cultures maintained at the bleaching temperature for nearly 60 h (Ortiz and Wilson, 1988). These changes include a significant accumulation of presumably novel polypeptides of 45 and 60 kDa in the thylakoids and of 63 kDa in the stroma. Other changes involve the loss of one of two polypeptides asso-
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WILLIAM ORTIZ
ciated with the LHcn and the loss of the LSU and the SSU of RUBIS CO. Loss of the LSU and the SSU of RUBISCO, in particular, prompted the following question: Is loss of chloroplast function associated with a decline in the synthesis of chloroplast proteins on cytoplasmic ribosomes or on chloroplast ribosomes, or both? The impact of temperature on the synthesis of plastid proteins of chloroplast origin and of nucleocytoplasmic origin was studied during the second phase of the temperature response. I report that, in general, synthesis and import of chloroplast polypeptides of nucleocytoplasmic origin remain high in cultures of Euglena maintained at the bleaching temperature for up to 41 h. By contrast, protein synthesis in plastids is lost affecting all of the major polypeptides synthesized by the organelle.
Materials and Methods Cell Culture Photoheterotrophic cultures of Euglena gracilis (Klebs}z strain (Pringsheim) were grown at room temperature (unbleached) or at 33°C for 24, 33, and 41 h in modified Hutner's medium (Ortiz et al., 1980) containing 50 ng/L vitamin B12 • Cultures were grown under cool-white fluorescent light at a fluence rate of 5.3 W .m- 2 with continuous shaking (Ortiz and Wilson, 1988).
Table 1: Synthesis of plastid proteins in photoheterotrophic Euglena grown at room temperature or bleached at 33°C. In vivo pulselabeling was carried out with [lsS]sodium sulfate (4 j.1Ci per mL culture) using room temperature (unbleached) and partially bleached cultures of the alga. Plastids were isolated in isoosmotic gradients of Percoll and fractionated in sucrose step-gradients. 3sS-incorporation was determined by the disc method. Culture
3SS-incorporation Stromaa Thylakoidsa
Unbleached 30.3 24 h-bleached 69.5 33 h-bleached 62.5 41 h-bleached 56.3 " - incpm x 10- 3 · mgProtein.
11.6 49.1 17.8 12.0
The gel was prepared for fluorography according to the Burckhardt et al. (1979) modification on the method of Laskey and Mills (1975).
Other Methods Protein determinations were carried out according to Larson et al. (1986). Cell counts were determined with a hemacytometer. An average was calculated from at least 10 independent cell counts.
Results Pulse-labeling in Vivo Unbleached and partially bleached cultures of the alga at the exponential phase of growth were harvested by centrifugation and resuspended in fresh photoheterotrophic low-sulfate medium (Monroy et al., 1987) where MgS04 was replaced by MgCh. Normal concentrations of FeS04, MnS04, and ZnS04 are still present in the medium. Pulse-labeling in vivo was initiated by the addition of j.1Ci of [3sSJsodium sulfate (43 Ci/mgS; ICN Biochemicals) per milliliter of cell culture. Protein synthesis in plastids was studied in vivo in the presence of 50 mg/L CHX to inhibit protein synthesis on cytoplasmic ribosomes. Pulse-labeling in the presence of CHX was carried out for relatively short periods of time (3 h). Incubations in the presence of CHX contained 8 !lCi of [3sS]sodium sulfate per milliliter of cell culture. In all cases, pulse-labeling in vivo was carried out at the original growth temperature in the light with continuous shaking.
Isolation and Subfractionation of Plastids At the end of the 3-h radiolabeling period, plastids were isolated on isoosmotic gradients of Percoll (Ortiz et aI., 1980; Ortiz and Wilson, 1988), lysed, and subfractionated on sucrose step-gradients according to Douce and Joyard (1982). 3sS-incorporation into hot TCA-precipitable material of the stroma and the thylakoids was determined by the disc method of Bollum (1966).
Gel Electrophoresis and Fluorography Polypeptides of the stroma and the thylakoids were separated by SDS-polyacrylamide gel electrophoresis on 10 to 15 % linear gradients (Chua, 1980) containing 2M urea in both the stacking and resolving gels. Samples (100 j.1g protein) were solubilized in sample buffer (Chua, 1980) containing 2M urea and 1 mM PMSF. Electrophoresis was carried out overnight at a constant current of 6 rnA.
Synthesis 0/ Plastid Proteins in Bleaching Euglena Pulse-labeling experiments were carried out to study the synthesis of plastid proteins of nucleocytoplasmic and plastid origin in cultures that have been bleached for up to 41 h. In terms of Chi accumulation per cell, cultures treated at 33°C for 24 h have essentially experienced no net loss of Chi compared to an unbleached culture. Prolonged incubations for 36, 48 and 60 h at the elevated temperature bring about a loss of Chi per cell of about 25, 50 and 70 %, respectively. Table 1 shows that synthesis of plastid proteins on cytoplasmic and chloroplast ribosomes remains high in cultures that have been kept at 33°C for up to 41 h. Synthesis of thylakoid proteins is 2.3-fold higher in cultures bleached for 24 h than in an unbleached culture. Although synthesis of
Table 2: Protein synthesis in plastids from photoheterotrophic Eu· glena grown at room temperature and from partially bleached cells. Cultures of the alga grown at room temperature (unbleached) or at the bleaching temperature were pulse-labeled in vivo (8 j.1Ci [35S]Sodium sulfate per mL culture) in the presence of CHX. Plastids were isolated on isoosmotic gradients of Percoll and fractionated on sucrose step-gradients. 35S-incorporation was measured by the disc method. Culture
3sS-incorporation Thylakoids·
Unbleached 58.2 24 h-bleached 33.3 26.7 33 h-bleached 11.5 41 h-bleached a _ in cpm x 10- 3 . mg Protein.
Stroma" 68.6 12.4 16.0 6.3
Synthesis of plastid proteins in bleaching Euglena thylakoid proteins decreases slightly in the 33- and 41-h cultures, synthesis is still 1.85-fold higher after 41 h than in the unbleached culture. Likewise, synthesis of stromal proteins on cytoplasmic and chloroplast ribosomes is higher in the 24 h-bleached culture than in the unbleached culture. Although synthesis of stromal proteins in the 33- and 41-h cultures falls below the levels measured on the 24-h culture, synthesis at 33 and 41 h still compares favorably with the levels of the unbleached culture. Table 2 shows that synthesis of thylakoid proteins on plastid ribosomes declines steadily in cultures bleached for increasing periods of time. Synthesis is about 80 % lower in the 41-h culture than in the unbleached culture. Synthesis of stromal proteins on plastid ribosomes decreases even more precipitously with values ranging from 75 to 90 % lower than the unbleached culture.
o
24
33 41
o
545
24 33 41
-
64
-
54
41
-
28
20
-
o o f:J.
45-=
o o
Fig.!: Synthesis of thylakoid proteins in bleaching Euglena. Growing cultures of photoheterotrophic Euglena were incubated at the bleaching temperature for the times indicated (h) on the figure and pulse-labeled in vivo with [35S]sodium sulfate at the original growth temperature. Plastids were isolated on isoosmotic gradients of PercolI, lysed, and subfractionated on step-gradients of sucrose. Thylakoid proteins were separated by gel electrophoresis followed by fluorography for the detection of the radiolabeled polypeptides. Protein standards (bovine serum albumin, ovalbumin, and trypsinogen) are identified with small arrowheads. Numbers at the top of the figure indicate the incubation time at the bleaching temperature in hours while numbers on the side refer to the Mr of the polypeptide in thousands.
Fig. 2: Thylakoid polypeptides synthesized in bleaching plastids. Growing cultures of photoheterotrophic Euglena were incubated at the bleaching temperature for various time intervals (h) and pulse-labeled in vivo in the presence of CHX to inhibit protein synthesis on cytoplasmic ribosomes. Plastids were isolated on isoosmotic gradients of Percoll, lysed, and subfractionated on step-gradients of sucrose. Polypeptides of the thylakoid were separated by gel electrophoresis followed by fluorography. Small arrowheads indicate the position of the molecular weight standards: bovine serum albumin, ovalbumin, trypsinogen, and lysozyme. Numbers at the top of the figure indicate the time of incubation at the bleaching temperature in hours. Numbers on the side refer to the Mr of the polypeptide in thousands.
Synthesis of Thylakoid Proteins Thylakoids from radiolabeled Euglena were analyzed by gel electrophoresis followed by fluorography for the detection of the radiolabeled products. Fig. 1 shows a profile of the polypeptides synthesized during the pulse-labeling period. Since no-antibiotic was present in the radiolabeling mix, the radiolabeled polypeptides are products synthesized on cytoplasmic and plastid ribosomes. The profile of radiolabeled polypeptides from partially bleached thylakoids is strikingly different from those of an unbleached culture suggesting a qualitative transformation in the make-up of the thylakoid at this stage of the bleaching process. Although in a previous study a number of novel polypeptides could be identified in thylakoids from 15-h bleached cells (Ortiz, 1990), the changes have become more evident in cultures bleached for longer periods of time. A more careful inspection of Fig. 1 also permits the identification of polypeptides whose synthesis is only transient at 33 °C {examples of these
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WILLIAM ORTIZ
are indicated with a triangle) and of polypeptides whose synthesis appears to increase significantly as bleaching progresses (examples of these are indicated with a circle). In the latter group one finds the presumably novel polypeptides of 45 and 60 kDa whose synthesis appears to remain high throughout the bleaching period studied here. Synthesis of thylakoid proteins on plastid ribosomes was studied in vivo in the presence of CHX to block the contribution by cytoplasmic ribosomes (Ortiz, 1990). The profile of radiolabeled thylakoid polypeptides synthesized in plastids of unbleached and partially bleached cells appears in Fig. 2. As expected, synthesis of thylakoid proteins on plastid ribosomes decreases with prolonged treatment at the elevated temperature based on the information presented in Table 2. The inhibitory effect of temperature is widespread affecting all of the major thylakoid polypeptides synthesized on plastid ribosomes such as the Chl-a-binding polypeptides of Photosystems I and II (64, 54, and 41 kDa) (Cunningham
o
24
33
41
-
63~
ssu~
Fig. 3: Synthesis of stromal proteins in bleaching cells. Growing photo heterotrophic cultures of Euglena incubated at the bleaching temperature for the times indicated (h) were pulse-labeled in vivo at the original growth temperature. Plastids were isolated on isoosmotic gradients of Percoll, lysed and subfractionated on sucrose step-gradients. Stromal proteins were separated by gel electrophoresis followed by fluorography for the detection of the radiolabeled species. The small arrowheads indicate the position of the molecular weight standards: bovine serum albumin, ovalbumin, trypsinogen, and lysozyme. The numbers at the top of the figure represent the time of incubation at the elevated temperature in hours. Numbers on the side represent the Mr of the polypeptide in thousands.
and Schiff, 1986) and QB (28 kDa) (Reardon and Price, 1984). Except for the limited synthesis of a 54-kDa polypeptide, protein synthesis in plastids is almost completely lost after 41 h of treatment at the bleaching temperature. This observation is in agreement with Brandt (1988) who reports a decrease in translation and insertion of thylakoid proteins at 34.5 °C particularly of polypeptides associated with Photosystems I and II. With regards to the temperature-induced synthesis of polypeptides of plastid origin, a presumably novel but minor polypeptide of 20 kDa is evident in cultures treated for 24 h and 33 h at 33°C but not in the unbleached culture. The appearance of this polypeptide in thylakoids is transitory, however, since its presence is not evident at 41 h or in the profile of 15-h plastids reported in earlier work (Ortiz, 1990). Synthesis of Stromal Proteins
The strategy employed to study the synthesis of thylakoid proteins was extended to the synthesis of stromal proteins. Fig. 3 presents a profile of radiolabeled polypeptides of the stroma following pulse-labeling in vivo of unbleached and partially bleached cultures of the alga. The profile shows translation products synthesized on cytoplasmic and chloroplast ribosomes since pulse-labeling was carried out in the absence of CHX. In general, synthesis of stromal proteins increases significantly in cultures treated at 33°C for 24 h compared with an unbleached culture. For the most part, the increase involves polypeptides that are already present at low levels in the unbleached culture but whose synthesis increases significantly at the elevated temperature. At later times (33 and 41 h), synthesis of stromal proteins decreases from the 24 h levels with the exception of a previously reported novel polypeptide of 63 kDa (Ortiz, 1990) whose synthesis remains high after 41 h at 33°C. In vivo pulse-labeling in the presence of CHX was also employed to study the effect of temperature on the synthesis of stromal proteins of plastid origin. Fig. 4 presents a profile of stromal proteins synthesized on plastid ribosomes from unbleached and from partially bleached cells. Synthesis of the LSU of RUBIS CO decreased significantly in partially bleached plastids. The inhibitory effect of temperature is most strongly felt by 41 h when synthesis of the LSU is barely detectable. Nevertheless, despite a strong effect on the synthesis of the LSU, synthesis of a 10.5-kDa polypeptide is still evident even at 41 h. Synthesis of the SSU remains high (Fig. 3) despite a sharp decrease in the synthesis of the LSU (Fig. 4). Even in the 41-h bleached culture, where levels have decreased with respect to the 24-h culture, synthesis of the SSU still compares favorably with the unbleached culture. Conclusive evidence regarding changes in the synthesis of SSU during bleaching requires a more specific assay such as immunoprecipitation with anti-SSU antibodies. Nevertheless, continued synthesis of the SSU despite low levels of LSU synthesis suggests a loose coordination in the activities of the protein synthesis machineries of the cytoplasm and the plastid with regards to the synthesis of the subunits of RUBISCO. Brandt et al. (1989) have also observed a loose coordination in the accumulation of the SSU and LSU during part of the light cycle
Synthesis of plastid proteins in bleaching Euglena
o
24
33
41
LSU~ . .
10.5
Fig. 4: Polypeptides of the stroma synthesized in bleaching plastids. Growing cultures of photoheterotrophic Euglena were incubated at the bleaching temperature for the times indicated (h). Pulse-labeling in vivo was carried out in the presence of CHX to inhibit protein synthesis on cytoplasmic ribosomes followed by the isolation of plastids on gradients of Percoll, lysis and subfractionation on sucrose step-gradients. Polypeptides of the stroma were separated by gel electrophoresis followed by fluorography for the detection of the radiolabeled polypeptides. The position of the molecular weight standards is indicated with small arrowheads: bovine serum albumin, ovalbumin, trypsinogen, and lysozyme. Numbers at the top of the figure represent times of incubation at the elevated temperature in hours while numbers on the side indicate the Mr of the polypeptide in thousands.
in synchronized cultures of Euglena. Profiles of stainable polypeptides of the stroma show no accumulation of the SSU in 33- and 41-h bleached cells (Ortiz and Wilson, 1988). Thus, in view of the sustained levels of synthesis and import of the SSU and the virtual absence of SSU accumulation, it is possible to suggest that the levels of the SSU in bleaching plastids are regulated at a post-translational level through a rapid degradation of the unassembled polypeptide. A similar mechanism has been shown to occur in chloroplasts of Chlamydomonas resulting in the rapid and selective degradation of the SSU in the absence of LSU accumulation (Schmidt and Mishkind, 1983) and of plastocyanin, a copper protein, in the absence of Cu(II) (Merchant and Bogorad, 1986).
547
Discussion The transformation of chloroplasts into non-photosynthetic entities at the bleaching temperature in Euglena is associated with a gradual loss of plastid protein synthesis. In this report I show that loss of protein synthesis in plastids affects most of the major polypeptides synthesized on plastid ribosomes. With the exception of a 10.5-kDa polypeptide of the stroma and of trace amounts of a 54-kDa polypeptide associated with Photosystem II, synthesis of all major polypeptides of plastid origin is totally lost after 41 h of incubation at the elevated temperature. Inhibition, however, is not uniform throughout the bleaching process since synthesis of some polypeptides appears to be more sensitive to the temperature treatment than others. For example, synthesis of LSU and the Chl-a-binding polypeptides of 64 and 41 kDa appears to be most sensitive to temperature since inhibition was already evident in 15-h bleached cultures (Ortiz, 1990). By contrast, inhibition of QB was apparent later in 24-h cultures while a clear inhibition in the synthesis of the 54-kDa polypeptide became evident at 33 h. Synthesis of a 10.5-kOa polypeptide of the stroma, however, appeared to be the least sensitive to treatment since this polypeptide could still be observed in the 41 h-bleached culture at levels comparable to the unbleached culture. Although inhibition could result from a general impairment in the operation of the chloroplast protein synthesis machinery, this mechanism appears to be unlikely since inhibition is manifested in a hierarchical manner and does not affect the synthesis of all polypeptides equally during the initial 41-h period. Since protein synthesis occurs to a limited extent even after 41 h of treatment, it is possible to conclude that the protein synthesis machinery is still functional in bleaching plastids. A considerable number of polypeptides of nucleocytoplasmic origin are imported and accumulated by bleaching plastids. In the particular case of 41-h bleached cells, imported proteins constitute the majority of the plastid proteins synthesized in bleaching cells since protein synthesis inside the bleaching plastid is almost totally lost. Continued synthesis and import of proteins (including some novel polypeptides) from the nucleocytoplasmic compartment during bleaching suggests that during the initial 33 to 41 h the chloroplast is gradually transformed into a structurally and functionally different entity. The fact that major qualitative changes in the synthesis of chloroplast proteins are already evident in 15-h cultures (Ortiz, 1990), when Chi accumulation is at its peak, suggests that the transformation process begins before Chi levels have started their steady decline. Contrary to what might have been expected, temperature does not bring about a total loss of those cellular activities responsible for the development and maintenance of the organelle. Instead, as plastid protein synthesis contributes fewer proteins for the maintenance of the organelle, control over the fate of the organelle appears to be transferred almost completely to the nucleocytoplasmic compartment. In this regard, loss of chloroplast function could be explained in part by an inhibition of transcription of chloroplast genes at the elevated temperature. Brandt and Weissner (1977) have reported a strong inhibition of chloroplast RNA polymerase activity in vitro at 34° and 35°C.
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WILUAM ORTIZ
In summary, the results presented in this report suggest that synthesis and import of proteins of nucleocytoplasmic origin into bleaching plastids remains remarkably strong in cultures treated at 33 °C, bringing about a fundamental change in the profile of proteins synthesized and accumulated in plastids. Synthesis of stromal and thylakoid proteins on plastid ribosomes, on the other hand, is strongly inhibited as bleaching progresses. Consequently, loss of translation by bleaching plastids could account for the inability of bleaching cultures of Euglena to maintain photosynthetically competent plastids at the elevated temperature. Acknowledgements The author wishes to thank Professor C. A. Price for helpful discussions. The author also thanks Ms. B. Richey for her help in typing the manuscript and the Office of Research Administration at the University of Oklahoma. This work was supported by a grant from the National Science Foundation DCB-8715422.
References ARNON, D. I.: Copper enzymes in isolated chloroplasts. Polyphenol oxidases in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). BOLLUM, F. J.: Filter paper disk technique for assaying radioactive macromolecules. In: CANTONI, G. L. and R. D. DAVIS (eds.): Procedures in Nucleic Acid Research, 296-300. Harper and Row, New York (1966). BRANDT, P.: Strain and stage-specific high temperature treatment of Euglena gracilis causes permanent apochlorosis. J. Plant Physiol. 133,281-287 (1988). BRANDT, P., E. BREIDENBACH, B. PRESTIN, and A. BOSCHETTI: Transcriptional, translational and assembly control of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase during the cell cycle of Euglena gracilis. J. Plant Physiol. 134, 420-426 (1989). BRANDT, P. and W. WIESSNER: Different temperature optima of the DNA-dependent RNA polymerase of Euglena gracilis strain Z and their role for the experimental bleaching by higher temperature. Z. Pflanzenphysiol. 85, 53-60 (1977).
BURCKHARDT, J., J. TELFORD, and M. L. BIRNSTIEL: Detection of labelled RNA species by contact hybridization. Nucleic Acid Res. 6,2963 -2971 (1979). CHUA, N. H.: Electrophoretic analysis of chloroplast proteins. Methods in Enzymol. 69, 434-446 (1980). CUNNINGHAM, F. X. and J. A. SCHIFF: Chlorophyll-protein complexes from Euglena gracilis and mutants deficient in chlorophyll b. II. Polypeptide composition. Plant Physiol. 80, 231-238 (1986). DOUCE, R. and J. JOYARD: Purification of the chloroplast envelope. In: EDELMAN, M., R. B. HALLlCK, and N. H. CHUA (eds.): Methods in Chloroplast Molecular Biology, 239 - 256. Academic Press, New York (1982). LARSON, E., B. HOWLETT, and A. JAGENDORF: Artificial reductant enhancement of the Lowry method for protein determination. Anal. Biochem. 155,243-248 (1986). LASKEY, R. A. and D. MILLS: Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341 (1975). MERCHANT, S. and L. BOGORAD: Rapid degradation of apoplastocyanin in Cu(II}-deficient cells of Chlamydomonas reinhardtii. J. BioI. Chern. 261, 15850-15853 (1986). MONROY, A. F., S. A. MCCARTHY, and S. D. SCHWARTZBACH: Evidence for translational regulation of chloroplast and mitochondrial biogenesis in Euglena. Plant Science 51, 61-76 (1987). ORTIZ, W.: Protein synthesis during the initial phase of the temperature-induced bleaching response in Euglena gracilis. Plant Physiol. 93, 141-147 (1990). ORTIZ, W., E. M. REARDON, and C. A. PRICE: Preparation of chloroplasts from Euglena highly active in protein synthesis. Plant Physiol. 66, 291-294 (1980). ORTIZ, W. and C. J. WILSON: Induced changes in chloroplast protein accumulation during heat bleaching in Euglena gracilis. Plant Physiol. 86, 554-561 (1988). PRINGSHEIM, E. G. and O. PRINGSHEIM: Experimental elimination of chromatophores and eye-spot in Euglena gracilis. New Phytol. 51,65-76 (1952). REARDON, E. M. and C. A. PRICE: Synthesis of the psbA gene product in Euglena. In organello and in vitro. Plant Physiol. 75, 246-248 (1984). SCHMIDT, G. W. and M. L. MISHKlND: Rapid degradation of unassembled ribulose-l,5-bisphosphate carboxylase small subunits in chloroplasts. Proc. Nat. Acad. Sci. USA 80, 2632-2636 (1983).
Contributions from the Plastid and the Nucleocytoplasmic Compartments to the Events Associated with Heat-bleaching in
Euglena gracilis WILLIAM ORTIZ Department of Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, Oklahoma 73019-0245, USA Received June 4, 1991 . Accepted August 17, 1991
Summary Growing photo heterotrophic cultures of Euglena gracilis Z show a time-dependent loss of chlorophyll per cell that results in the production of bleached cultures of the alga on prolonged incubation at 33°C. In terms of chlorophyll accumulation, the bleaching process involves an initial rise in chlorophyll accumulation per cell, peaking at about 15 h, followed by a second phase in which chlorophyll levels per cell actually decline. The primary objective of this work was to establish whether loss of chloroplast function at 33°C is associated with a loss of translational activity on chloroplast ribosomes or a loss in the synthesis of chloroplast proteins of nucleocytoplasmic origin, or both. To this effect, pulse-labeling experiments were carried out in vivo on cultures of the alga in the second phase of the temperature response. The results show that synthesis and import of proteins from the nucleocytoplasmic compartment remain high in bleaching plastids during the first 41 h of the temperature treatment. By contrast, protein synthesis inside plastids is gradually lost at the elevated temperature, and only low levels can be detected in 41-h bleached cultures. Consequently, the great majority of the proteins accumulating in 41-h bleached plastids are imported from the nucleocytoplasmic compartment. The loss of translational activity inside the bleaching plastid could explain why Euglena is unable to maintain photosynthetically competent plastids at the elevated temperature.
Key words: Euglena gracilis Z; heat-bleaching; protein synthesis. Abbreviations: ChI = chlorophyll; CHX = cycloheximide; LHCn = light-harvesting chlorophyllbinding protein of Photosystem II; LSU and SSU = large and small subunit; PM SF = phenylmethylsulfonyl fluoride; QB = quinone-binding protein; RUBIS CO = ribulosebisphosphate carboxylase; TCA = trichloracetic acid. Introduction The temperature-induced loss of total ChI in growing photoheterotrophic cultures of Euglena gracilis is commonly known as heat-bleaching (Pringsheim and Pringsheim, 1952). The phenomenon is of interest to us because the alga is unable to maintain functional chloroplasts at the moderately elevated temperature of 33 to 34°C. The effect of temperature on ChI accumulation takes place in two phases (Ortiz and Wilson, 1988). Chlorophyll accu© 1992 by Gustav Fischer Verlag. Stuttgart
mulation per cell initially rises and peaks at about 15 h after the onset of the temperature treatment. The rise in ChI is followed by a second phase characterized by a steady decline in ChI levels per cell. In this context, we have documented changes in the profiles of stainable polypeptides in cultures maintained at the bleaching temperature for nearly 60 h (Ortiz and Wilson, 1988). These changes include a significant accumulation of presumably novel polypeptides of 45 and 60 kDa in the thylakoids and of 63 kDa in the stroma. Other changes involve the loss of one of two polypeptides asso-
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WILLIAM ORTIZ
ciated with the LHcn and the loss of the LSU and the SSU of RUBIS CO. Loss of the LSU and the SSU of RUBISCO, in particular, prompted the following question: Is loss of chloroplast function associated with a decline in the synthesis of chloroplast proteins on cytoplasmic ribosomes or on chloroplast ribosomes, or both? The impact of temperature on the synthesis of plastid proteins of chloroplast origin and of nucleocytoplasmic origin was studied during the second phase of the temperature response. I report that, in general, synthesis and import of chloroplast polypeptides of nucleocytoplasmic origin remain high in cultures of Euglena maintained at the bleaching temperature for up to 41 h. By contrast, protein synthesis in plastids is lost affecting all of the major polypeptides synthesized by the organelle.
Materials and Methods Cell Culture Photoheterotrophic cultures of Euglena gracilis (Klebs}z strain (Pringsheim) were grown at room temperature (unbleached) or at 33°C for 24, 33, and 41 h in modified Hutner's medium (Ortiz et al., 1980) containing 50 ng/L vitamin B12 • Cultures were grown under cool-white fluorescent light at a fluence rate of 5.3 W .m- 2 with continuous shaking (Ortiz and Wilson, 1988).
Table 1: Synthesis of plastid proteins in photoheterotrophic Euglena grown at room temperature or bleached at 33°C. In vivo pulselabeling was carried out with [lsS]sodium sulfate (4 j.1Ci per mL culture) using room temperature (unbleached) and partially bleached cultures of the alga. Plastids were isolated in isoosmotic gradients of Percoll and fractionated in sucrose step-gradients. 3sS-incorporation was determined by the disc method. Culture
3SS-incorporation Stromaa Thylakoidsa
Unbleached 30.3 24 h-bleached 69.5 33 h-bleached 62.5 41 h-bleached 56.3 " - incpm x 10- 3 · mgProtein.
11.6 49.1 17.8 12.0
The gel was prepared for fluorography according to the Burckhardt et al. (1979) modification on the method of Laskey and Mills (1975).
Other Methods Protein determinations were carried out according to Larson et al. (1986). Cell counts were determined with a hemacytometer. An average was calculated from at least 10 independent cell counts.
Results Pulse-labeling in Vivo Unbleached and partially bleached cultures of the alga at the exponential phase of growth were harvested by centrifugation and resuspended in fresh photoheterotrophic low-sulfate medium (Monroy et al., 1987) where MgS04 was replaced by MgCh. Normal concentrations of FeS04, MnS04, and ZnS04 are still present in the medium. Pulse-labeling in vivo was initiated by the addition of j.1Ci of [3sSJsodium sulfate (43 Ci/mgS; ICN Biochemicals) per milliliter of cell culture. Protein synthesis in plastids was studied in vivo in the presence of 50 mg/L CHX to inhibit protein synthesis on cytoplasmic ribosomes. Pulse-labeling in the presence of CHX was carried out for relatively short periods of time (3 h). Incubations in the presence of CHX contained 8 !lCi of [3sS]sodium sulfate per milliliter of cell culture. In all cases, pulse-labeling in vivo was carried out at the original growth temperature in the light with continuous shaking.
Isolation and Subfractionation of Plastids At the end of the 3-h radiolabeling period, plastids were isolated on isoosmotic gradients of Percoll (Ortiz et aI., 1980; Ortiz and Wilson, 1988), lysed, and subfractionated on sucrose step-gradients according to Douce and Joyard (1982). 3sS-incorporation into hot TCA-precipitable material of the stroma and the thylakoids was determined by the disc method of Bollum (1966).
Gel Electrophoresis and Fluorography Polypeptides of the stroma and the thylakoids were separated by SDS-polyacrylamide gel electrophoresis on 10 to 15 % linear gradients (Chua, 1980) containing 2M urea in both the stacking and resolving gels. Samples (100 j.1g protein) were solubilized in sample buffer (Chua, 1980) containing 2M urea and 1 mM PMSF. Electrophoresis was carried out overnight at a constant current of 6 rnA.
Synthesis 0/ Plastid Proteins in Bleaching Euglena Pulse-labeling experiments were carried out to study the synthesis of plastid proteins of nucleocytoplasmic and plastid origin in cultures that have been bleached for up to 41 h. In terms of Chi accumulation per cell, cultures treated at 33°C for 24 h have essentially experienced no net loss of Chi compared to an unbleached culture. Prolonged incubations for 36, 48 and 60 h at the elevated temperature bring about a loss of Chi per cell of about 25, 50 and 70 %, respectively. Table 1 shows that synthesis of plastid proteins on cytoplasmic and chloroplast ribosomes remains high in cultures that have been kept at 33°C for up to 41 h. Synthesis of thylakoid proteins is 2.3-fold higher in cultures bleached for 24 h than in an unbleached culture. Although synthesis of
Table 2: Protein synthesis in plastids from photoheterotrophic Eu· glena grown at room temperature and from partially bleached cells. Cultures of the alga grown at room temperature (unbleached) or at the bleaching temperature were pulse-labeled in vivo (8 j.1Ci [35S]Sodium sulfate per mL culture) in the presence of CHX. Plastids were isolated on isoosmotic gradients of Percoll and fractionated on sucrose step-gradients. 35S-incorporation was measured by the disc method. Culture
3sS-incorporation Thylakoids·
Unbleached 58.2 24 h-bleached 33.3 26.7 33 h-bleached 11.5 41 h-bleached a _ in cpm x 10- 3 . mg Protein.
Stroma" 68.6 12.4 16.0 6.3
Synthesis of plastid proteins in bleaching Euglena thylakoid proteins decreases slightly in the 33- and 41-h cultures, synthesis is still 1.85-fold higher after 41 h than in the unbleached culture. Likewise, synthesis of stromal proteins on cytoplasmic and chloroplast ribosomes is higher in the 24 h-bleached culture than in the unbleached culture. Although synthesis of stromal proteins in the 33- and 41-h cultures falls below the levels measured on the 24-h culture, synthesis at 33 and 41 h still compares favorably with the levels of the unbleached culture. Table 2 shows that synthesis of thylakoid proteins on plastid ribosomes declines steadily in cultures bleached for increasing periods of time. Synthesis is about 80 % lower in the 41-h culture than in the unbleached culture. Synthesis of stromal proteins on plastid ribosomes decreases even more precipitously with values ranging from 75 to 90 % lower than the unbleached culture.
o
24
33 41
o
545
24 33 41
-
64
-
54
41
-
28
20
-
o o f:J.
45-=
o o
Fig.!: Synthesis of thylakoid proteins in bleaching Euglena. Growing cultures of photoheterotrophic Euglena were incubated at the bleaching temperature for the times indicated (h) on the figure and pulse-labeled in vivo with [35S]sodium sulfate at the original growth temperature. Plastids were isolated on isoosmotic gradients of PercolI, lysed, and subfractionated on step-gradients of sucrose. Thylakoid proteins were separated by gel electrophoresis followed by fluorography for the detection of the radiolabeled polypeptides. Protein standards (bovine serum albumin, ovalbumin, and trypsinogen) are identified with small arrowheads. Numbers at the top of the figure indicate the incubation time at the bleaching temperature in hours while numbers on the side refer to the Mr of the polypeptide in thousands.
Fig. 2: Thylakoid polypeptides synthesized in bleaching plastids. Growing cultures of photoheterotrophic Euglena were incubated at the bleaching temperature for various time intervals (h) and pulse-labeled in vivo in the presence of CHX to inhibit protein synthesis on cytoplasmic ribosomes. Plastids were isolated on isoosmotic gradients of Percoll, lysed, and subfractionated on step-gradients of sucrose. Polypeptides of the thylakoid were separated by gel electrophoresis followed by fluorography. Small arrowheads indicate the position of the molecular weight standards: bovine serum albumin, ovalbumin, trypsinogen, and lysozyme. Numbers at the top of the figure indicate the time of incubation at the bleaching temperature in hours. Numbers on the side refer to the Mr of the polypeptide in thousands.
Synthesis of Thylakoid Proteins Thylakoids from radiolabeled Euglena were analyzed by gel electrophoresis followed by fluorography for the detection of the radiolabeled products. Fig. 1 shows a profile of the polypeptides synthesized during the pulse-labeling period. Since no-antibiotic was present in the radiolabeling mix, the radiolabeled polypeptides are products synthesized on cytoplasmic and plastid ribosomes. The profile of radiolabeled polypeptides from partially bleached thylakoids is strikingly different from those of an unbleached culture suggesting a qualitative transformation in the make-up of the thylakoid at this stage of the bleaching process. Although in a previous study a number of novel polypeptides could be identified in thylakoids from 15-h bleached cells (Ortiz, 1990), the changes have become more evident in cultures bleached for longer periods of time. A more careful inspection of Fig. 1 also permits the identification of polypeptides whose synthesis is only transient at 33 °C {examples of these
546
WILLIAM ORTIZ
are indicated with a triangle) and of polypeptides whose synthesis appears to increase significantly as bleaching progresses (examples of these are indicated with a circle). In the latter group one finds the presumably novel polypeptides of 45 and 60 kDa whose synthesis appears to remain high throughout the bleaching period studied here. Synthesis of thylakoid proteins on plastid ribosomes was studied in vivo in the presence of CHX to block the contribution by cytoplasmic ribosomes (Ortiz, 1990). The profile of radiolabeled thylakoid polypeptides synthesized in plastids of unbleached and partially bleached cells appears in Fig. 2. As expected, synthesis of thylakoid proteins on plastid ribosomes decreases with prolonged treatment at the elevated temperature based on the information presented in Table 2. The inhibitory effect of temperature is widespread affecting all of the major thylakoid polypeptides synthesized on plastid ribosomes such as the Chl-a-binding polypeptides of Photosystems I and II (64, 54, and 41 kDa) (Cunningham
o
24
33
41
-
63~
ssu~
Fig. 3: Synthesis of stromal proteins in bleaching cells. Growing photo heterotrophic cultures of Euglena incubated at the bleaching temperature for the times indicated (h) were pulse-labeled in vivo at the original growth temperature. Plastids were isolated on isoosmotic gradients of Percoll, lysed and subfractionated on sucrose step-gradients. Stromal proteins were separated by gel electrophoresis followed by fluorography for the detection of the radiolabeled species. The small arrowheads indicate the position of the molecular weight standards: bovine serum albumin, ovalbumin, trypsinogen, and lysozyme. The numbers at the top of the figure represent the time of incubation at the elevated temperature in hours. Numbers on the side represent the Mr of the polypeptide in thousands.
and Schiff, 1986) and QB (28 kDa) (Reardon and Price, 1984). Except for the limited synthesis of a 54-kDa polypeptide, protein synthesis in plastids is almost completely lost after 41 h of treatment at the bleaching temperature. This observation is in agreement with Brandt (1988) who reports a decrease in translation and insertion of thylakoid proteins at 34.5 °C particularly of polypeptides associated with Photosystems I and II. With regards to the temperature-induced synthesis of polypeptides of plastid origin, a presumably novel but minor polypeptide of 20 kDa is evident in cultures treated for 24 h and 33 h at 33°C but not in the unbleached culture. The appearance of this polypeptide in thylakoids is transitory, however, since its presence is not evident at 41 h or in the profile of 15-h plastids reported in earlier work (Ortiz, 1990). Synthesis of Stromal Proteins
The strategy employed to study the synthesis of thylakoid proteins was extended to the synthesis of stromal proteins. Fig. 3 presents a profile of radiolabeled polypeptides of the stroma following pulse-labeling in vivo of unbleached and partially bleached cultures of the alga. The profile shows translation products synthesized on cytoplasmic and chloroplast ribosomes since pulse-labeling was carried out in the absence of CHX. In general, synthesis of stromal proteins increases significantly in cultures treated at 33°C for 24 h compared with an unbleached culture. For the most part, the increase involves polypeptides that are already present at low levels in the unbleached culture but whose synthesis increases significantly at the elevated temperature. At later times (33 and 41 h), synthesis of stromal proteins decreases from the 24 h levels with the exception of a previously reported novel polypeptide of 63 kDa (Ortiz, 1990) whose synthesis remains high after 41 h at 33°C. In vivo pulse-labeling in the presence of CHX was also employed to study the effect of temperature on the synthesis of stromal proteins of plastid origin. Fig. 4 presents a profile of stromal proteins synthesized on plastid ribosomes from unbleached and from partially bleached cells. Synthesis of the LSU of RUBIS CO decreased significantly in partially bleached plastids. The inhibitory effect of temperature is most strongly felt by 41 h when synthesis of the LSU is barely detectable. Nevertheless, despite a strong effect on the synthesis of the LSU, synthesis of a 10.5-kDa polypeptide is still evident even at 41 h. Synthesis of the SSU remains high (Fig. 3) despite a sharp decrease in the synthesis of the LSU (Fig. 4). Even in the 41-h bleached culture, where levels have decreased with respect to the 24-h culture, synthesis of the SSU still compares favorably with the unbleached culture. Conclusive evidence regarding changes in the synthesis of SSU during bleaching requires a more specific assay such as immunoprecipitation with anti-SSU antibodies. Nevertheless, continued synthesis of the SSU despite low levels of LSU synthesis suggests a loose coordination in the activities of the protein synthesis machineries of the cytoplasm and the plastid with regards to the synthesis of the subunits of RUBISCO. Brandt et al. (1989) have also observed a loose coordination in the accumulation of the SSU and LSU during part of the light cycle
Synthesis of plastid proteins in bleaching Euglena
o
24
33
41
LSU~ . .
10.5
Fig. 4: Polypeptides of the stroma synthesized in bleaching plastids. Growing cultures of photoheterotrophic Euglena were incubated at the bleaching temperature for the times indicated (h). Pulse-labeling in vivo was carried out in the presence of CHX to inhibit protein synthesis on cytoplasmic ribosomes followed by the isolation of plastids on gradients of Percoll, lysis and subfractionation on sucrose step-gradients. Polypeptides of the stroma were separated by gel electrophoresis followed by fluorography for the detection of the radiolabeled polypeptides. The position of the molecular weight standards is indicated with small arrowheads: bovine serum albumin, ovalbumin, trypsinogen, and lysozyme. Numbers at the top of the figure represent times of incubation at the elevated temperature in hours while numbers on the side indicate the Mr of the polypeptide in thousands.
in synchronized cultures of Euglena. Profiles of stainable polypeptides of the stroma show no accumulation of the SSU in 33- and 41-h bleached cells (Ortiz and Wilson, 1988). Thus, in view of the sustained levels of synthesis and import of the SSU and the virtual absence of SSU accumulation, it is possible to suggest that the levels of the SSU in bleaching plastids are regulated at a post-translational level through a rapid degradation of the unassembled polypeptide. A similar mechanism has been shown to occur in chloroplasts of Chlamydomonas resulting in the rapid and selective degradation of the SSU in the absence of LSU accumulation (Schmidt and Mishkind, 1983) and of plastocyanin, a copper protein, in the absence of Cu(II) (Merchant and Bogorad, 1986).
547
Discussion The transformation of chloroplasts into non-photosynthetic entities at the bleaching temperature in Euglena is associated with a gradual loss of plastid protein synthesis. In this report I show that loss of protein synthesis in plastids affects most of the major polypeptides synthesized on plastid ribosomes. With the exception of a 10.5-kDa polypeptide of the stroma and of trace amounts of a 54-kDa polypeptide associated with Photosystem II, synthesis of all major polypeptides of plastid origin is totally lost after 41 h of incubation at the elevated temperature. Inhibition, however, is not uniform throughout the bleaching process since synthesis of some polypeptides appears to be more sensitive to the temperature treatment than others. For example, synthesis of LSU and the Chl-a-binding polypeptides of 64 and 41 kDa appears to be most sensitive to temperature since inhibition was already evident in 15-h bleached cultures (Ortiz, 1990). By contrast, inhibition of QB was apparent later in 24-h cultures while a clear inhibition in the synthesis of the 54-kDa polypeptide became evident at 33 h. Synthesis of a 10.5-kOa polypeptide of the stroma, however, appeared to be the least sensitive to treatment since this polypeptide could still be observed in the 41 h-bleached culture at levels comparable to the unbleached culture. Although inhibition could result from a general impairment in the operation of the chloroplast protein synthesis machinery, this mechanism appears to be unlikely since inhibition is manifested in a hierarchical manner and does not affect the synthesis of all polypeptides equally during the initial 41-h period. Since protein synthesis occurs to a limited extent even after 41 h of treatment, it is possible to conclude that the protein synthesis machinery is still functional in bleaching plastids. A considerable number of polypeptides of nucleocytoplasmic origin are imported and accumulated by bleaching plastids. In the particular case of 41-h bleached cells, imported proteins constitute the majority of the plastid proteins synthesized in bleaching cells since protein synthesis inside the bleaching plastid is almost totally lost. Continued synthesis and import of proteins (including some novel polypeptides) from the nucleocytoplasmic compartment during bleaching suggests that during the initial 33 to 41 h the chloroplast is gradually transformed into a structurally and functionally different entity. The fact that major qualitative changes in the synthesis of chloroplast proteins are already evident in 15-h cultures (Ortiz, 1990), when Chi accumulation is at its peak, suggests that the transformation process begins before Chi levels have started their steady decline. Contrary to what might have been expected, temperature does not bring about a total loss of those cellular activities responsible for the development and maintenance of the organelle. Instead, as plastid protein synthesis contributes fewer proteins for the maintenance of the organelle, control over the fate of the organelle appears to be transferred almost completely to the nucleocytoplasmic compartment. In this regard, loss of chloroplast function could be explained in part by an inhibition of transcription of chloroplast genes at the elevated temperature. Brandt and Weissner (1977) have reported a strong inhibition of chloroplast RNA polymerase activity in vitro at 34° and 35°C.
548
WILUAM ORTIZ
In summary, the results presented in this report suggest that synthesis and import of proteins of nucleocytoplasmic origin into bleaching plastids remains remarkably strong in cultures treated at 33 °C, bringing about a fundamental change in the profile of proteins synthesized and accumulated in plastids. Synthesis of stromal and thylakoid proteins on plastid ribosomes, on the other hand, is strongly inhibited as bleaching progresses. Consequently, loss of translation by bleaching plastids could account for the inability of bleaching cultures of Euglena to maintain photosynthetically competent plastids at the elevated temperature. Acknowledgements The author wishes to thank Professor C. A. Price for helpful discussions. The author also thanks Ms. B. Richey for her help in typing the manuscript and the Office of Research Administration at the University of Oklahoma. This work was supported by a grant from the National Science Foundation DCB-8715422.
References ARNON, D. I.: Copper enzymes in isolated chloroplasts. Polyphenol oxidases in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). BOLLUM, F. J.: Filter paper disk technique for assaying radioactive macromolecules. In: CANTONI, G. L. and R. D. DAVIS (eds.): Procedures in Nucleic Acid Research, 296-300. Harper and Row, New York (1966). BRANDT, P.: Strain and stage-specific high temperature treatment of Euglena gracilis causes permanent apochlorosis. J. Plant Physiol. 133,281-287 (1988). BRANDT, P., E. BREIDENBACH, B. PRESTIN, and A. BOSCHETTI: Transcriptional, translational and assembly control of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase during the cell cycle of Euglena gracilis. J. Plant Physiol. 134, 420-426 (1989). BRANDT, P. and W. WIESSNER: Different temperature optima of the DNA-dependent RNA polymerase of Euglena gracilis strain Z and their role for the experimental bleaching by higher temperature. Z. Pflanzenphysiol. 85, 53-60 (1977).
BURCKHARDT, J., J. TELFORD, and M. L. BIRNSTIEL: Detection of labelled RNA species by contact hybridization. Nucleic Acid Res. 6,2963 -2971 (1979). CHUA, N. H.: Electrophoretic analysis of chloroplast proteins. Methods in Enzymol. 69, 434-446 (1980). CUNNINGHAM, F. X. and J. A. SCHIFF: Chlorophyll-protein complexes from Euglena gracilis and mutants deficient in chlorophyll b. II. Polypeptide composition. Plant Physiol. 80, 231-238 (1986). DOUCE, R. and J. JOYARD: Purification of the chloroplast envelope. In: EDELMAN, M., R. B. HALLlCK, and N. H. CHUA (eds.): Methods in Chloroplast Molecular Biology, 239 - 256. Academic Press, New York (1982). LARSON, E., B. HOWLETT, and A. JAGENDORF: Artificial reductant enhancement of the Lowry method for protein determination. Anal. Biochem. 155,243-248 (1986). LASKEY, R. A. and D. MILLS: Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341 (1975). MERCHANT, S. and L. BOGORAD: Rapid degradation of apoplastocyanin in Cu(II}-deficient cells of Chlamydomonas reinhardtii. J. BioI. Chern. 261, 15850-15853 (1986). MONROY, A. F., S. A. MCCARTHY, and S. D. SCHWARTZBACH: Evidence for translational regulation of chloroplast and mitochondrial biogenesis in Euglena. Plant Science 51, 61-76 (1987). ORTIZ, W.: Protein synthesis during the initial phase of the temperature-induced bleaching response in Euglena gracilis. Plant Physiol. 93, 141-147 (1990). ORTIZ, W., E. M. REARDON, and C. A. PRICE: Preparation of chloroplasts from Euglena highly active in protein synthesis. Plant Physiol. 66, 291-294 (1980). ORTIZ, W. and C. J. WILSON: Induced changes in chloroplast protein accumulation during heat bleaching in Euglena gracilis. Plant Physiol. 86, 554-561 (1988). PRINGSHEIM, E. G. and O. PRINGSHEIM: Experimental elimination of chromatophores and eye-spot in Euglena gracilis. New Phytol. 51,65-76 (1952). REARDON, E. M. and C. A. PRICE: Synthesis of the psbA gene product in Euglena. In organello and in vitro. Plant Physiol. 75, 246-248 (1984). SCHMIDT, G. W. and M. L. MISHKlND: Rapid degradation of unassembled ribulose-l,5-bisphosphate carboxylase small subunits in chloroplasts. Proc. Nat. Acad. Sci. USA 80, 2632-2636 (1983).