Chloroplast Protein Synthesis Research Articles

Sites of synthesis of chloroplast ribosomal proteins in Chlamydomonas.

Cells of Chlamydomonas reinhardtii were pulse-labeled in vivo in the presence of inhibitors of cytoplasmic (anisomycin) or chloroplast (lincomycin) protein synthesis to ascertain the sites of synthesis of chloroplast ribosomal proteins. Fluorographs of the labeled proteins, resolved on two-dimensional (2-D) charge/SDS and one-dimensional (1-D) SDS-urea gradient gels, demonstrated that five to six of the large subunit proteins are products of chloroplast protein synthesis while 26 to 27 of the large subunit proteins are synthesized on cytoplasmic ribosomes. Similarly, 14 of 31 small subunit proteins are products of chloroplast protein synthesis, while the remainder are synthesized in the cytoplasm. The 20 ribosomal proteins shown to be made in the chloroplast of Chlamydomonas more than double the number of proteins known to be synthesized in the chloroplast of this alga.

Translational sites of thylakoid membrane proteins. Studies based on selective inhibition of cytoplasmic and chloroplast ribosomes by cycloheximide and chloramphenicol

To inhibit chloroplast and cytoplasmic protein synthesis, respectively, excised shoots of 19-day-old Vicia faba plants and 10-day-old Hordeum vulgare plants were incubated with d-threochloramphenicol or cycloheximide in combination with l-[ 35S]methionine. Incubation was performed for 22 h under light subsequent to a 30 min inhibitor pre-incubation without methionine. In order to analyse the translational sites of thylakoid membrane polypeptides, isolated thylakoids were dispersed by sodium dodecyl sulfate and separated by means of two electrophoretic systems. Based on results obtained with electrophoretic system III, 19 of the Vicia faba thylakoid membrane polypeptides sufficiently labeled with l-[ 35S]methionine were inhibited by chloramphenicol and 17 were inhibited by cycloheximide. Nine polypeptides were found to be sufficiently stained but very weakly labeled, indicating low metabolic activity or low methionine content. These polypeptides of low specific radioactivity did not allow analysis of inhibitor effects. Chloramphenicol inhibition included three chlorophyll a-apoproteins. In contrast, the synthesis of three chlorophyll a b- apoproteins , besides some unidentified polypeptides found in different light-harvesting complex preparations of various composition, was severely inhibited by cycloheximide. Comparison of V. faba and H. vulgare thylakoids revealed significant differences in the polypeptide patterns. Both plant species, however, are characterized by three rapidly labeled polypeptides located closely together on the polyacrylamide gels. The identity of these rapidly labeled polypeptide(s) with a herbicide binding protein is discussed.

Ertyhromycin as a tool for discriminating in vivo between mitochondrial and chloroplastic protein synthesis in Nicotiana sylvestris

In plant cells it has not been possible to discriminate between mitochondrial (MPS) and chloroplastic protein synthesis (ClPS) in vivo by means of compartment specific inhibitors. In this paper data are reported about the specificity of erythromycin (ERY) which inhibited ClPS but not MPS in tissue cultures of Nicotiana sylvestris. The data supporting this conclusion are: (a) ERY produced bleaching without affecting the earlier phases of growth; (b) protein synthesis in isolated chloroplasts was inhibited by the drug, whereas protein synthesis in isolated mitochondria was almost unaffected; (c) the synthesis of cytochrome oxidase (a mitochondrial-dependent process) was unaffected by ERY; (d) electron micrographs of the ultra-thin sections of plantlets and tissue cultures showed that the structure of mitochondria was unaffected by the drug, but the structure of chloroplasts was altered completely.

Protein synthetic patterns during the asexual life cycle of Volvox carteri

The polypeptide labeling patterns of somatic cells, gonidia (asexual reproductive cells), embryos, and juvenile spheroids of Volvox carteri cultures synchronized by a light/dark cycle were studied as a function of developmental stage and incubation condition. Specimens were exposed to 35SO = 4 for 1-hr periods at selected intervals throughout the asexual life cycle; proteins were then extracted and analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. Although sulfation accounts for more than half the 35S incorporated, the conditions of extraction and electrophoresis employed resulted in exclusion of most sulfated products and inclusion of nearly all products bearing incorporated sulfur amino acids in the electrophoretic analysis. Hence SDS-PAGE profiles reflect relative rates of synthesis of major polypeptides. The first phase of these studies involved examination of stage-specific differences in protein synthetic patterns. Because a single developmental stage exhibits different protein synthetic patterns in light and darkness, detailed developmental comparisons were made only on organisms or cells exposed to label in the light. They yielded the following results: Shortly after the completion of embryogenesis (while all cells are still linked by numerous cytoplasmic bridges) presumptive somatic cells and gonidia exhibit a nearly identical pattern of labeling of the major polypeptides. In just a few hours, however, as cytoplasmic bridges begin to break down, the synthetic patterns of the two cell types begin to diverge; with passing time this divergence becomes progressively greater. By the time gonidia are mature, the patterns of labeling of major polypeptides by somatic cells and gonidia exhibit far more differences than similarities. Embryos derived from these mature gonidia then exhibit numerous, reproducible, stage-specific changes in polypeptide labeling throughout embryogenesis. However, two glycoproteins that previous authors implicated in the control of the differentiative cleavage division are here shown to be labeled in the parental somatic cells, not in the embryos as was previously supposed; hence a central role for them in embryonic development seems highly unlikely. In the second phase of this study the effects of light on protein synthetic patterns of organisms at selected developmental stages were analyzed. At all stages marked, rapid, reversible changes in the pattern of labeling of major polypeptides occur when cultures are transferred from light to dark or vice versa, but these changes are most marked in juvenile spheroids at the end of the dark period during which they had completed their embryogenesis. Some, but by no means all, of the changes induced by light can be attributed to stimulated synthesis of chloroplast proteins, on both chloroplast and cytosol ribosomes. The proteins made at the beginning of one light period are not identical to those made at the end of the preceding light period. We postulate that it is the selective activation, by light, of protein synthesis on transcripts accumulated during the dark period that accounts for the fact that the life cycle can be synchronized by light.

Protein synthesis in Petunia hybrida chloroplasts isolated from leaves and cell cultures

Isolation and incubation conditions were established for Petunia hybrida chloroplasts capable of performing in vitro protein and RNA synthesis. Under these conditions, chloroplasts from leaves as well as from the non-photoautotrophic mutant green cell culture AK-2401 are able to incorporate labeled amino acids into polypeptides. Intact chloroplasts can use light as an energy source; photosynthetically-inactive chloroplasts require the addition for ATP for this protein synthesis. Sodium dodecylsulphate polyacrylamide slab gel electrophoresis shows that in isolated leaf chloroplasts at least twenty-five radioactive polypeptide species are synthesized. The three major products synthesized have molecular weights of 52,000, 32,000 and 17,000. Coomassie brilliant-bluestained polypeptide patterns from plastids isolated from the mutant green cell culture AK-2401 differ considerably from those obtained from leaf chloroplasts. The pattern of radioactive polypeptides synthesized in these isolated cell culture plastids also shows differences. These results indicate that the difference in developmental stage observed between plastids from the cell culture AK-2401 and leaves is reflected in an altered expression of the chloroplast DNA.

Nutritional Regulation of Organelle Biogenesis in Euglena

Exposure of dark grown resting Euglena to ethanol produced a transient increase in the specific activity of the glyoxysomal enzyme malate synthase. Enzyme specific activity increased during the first 24 hours of ethanol treatment and then declined. Light exposure or malate addition failed to increase enzyme specific activity. The increase and decrease in enzyme specific activity represented changes in the amount of active enzyme. In both wild type cells and the plastidless mutant W(3)BUL, enzyme levels were always higher in the dark than in the light.The specific activity of the peroxisomal enzyme glycolate dehydrogenase began to increase 24 hours after dark grown resting Euglena were exposed to light. Ethanol, but not malate, prevented the increase and promoted a decrease in glycolate dehydrogenase levels. Cycloheximide produced a decline in enzyme levels similar to the decline produced by ethanol addition. Glycolate dehydrogenase was present in the plastidless mutant W(3)BUL indicating that it is coded in the nucleus and synthesized on cytoplasmic ribosomes. Streptomycin, a specific inhibitor of chloroplast protein synthesis and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthetic CO(2) fixation, inhibited the photoinduction of glycolate dehydrogenase while having no effect on the photoinduction of NADP dependent glyceraldehyde-3-phosphate dehydrogenase, another light induced, nuclear coded, cytoplasmically synthesized enzyme. Taken together, these results suggest that microbodies are continually synthesized in resting Euglena and their enzyme complement is determined through substrate induction of glyoxysomal and peroxisomal enzymes.

A uniparental mutant of Chlamydomonas reinhardtii resistant to chloramphenicol

A uniparental mutant of Chlamydomonas resistant to chloramphenicol was selected following treatment of wild-type cells with 5-fluorodeoxyuridine. Under heterotrophic conditions, growth and chloroplast protein synthesis of this mutant (CAP1) are resistant to chloramphenicol. Under phototrophic conditions, CAP1 is sensitive to chloramphenicol. In addition CAP1 displays thermosensitivity when grown phototrophically in the absence of antibiotics: at the restrictive temperature, a specific reduction of those thylakoid membrane polypeptides which are synthesized inside the chloroplast is observed. Alternative explanations for the pleiotropic phenotype of CAP1 are discussed.

Synthesis of Chloroplast Proteins during Germination and Early Development of Cucumber

Cell-free protein synthesizing systems have been used to study the developmental changes in the synthesis of chloroplast proteins in the cotyledons of cucumber seedlings grown in the light or in the dark. Escherichia coli and wheat germ in vitro protein synthesizing systems have been used to assay the changes in the levels of the mRNA's coding for ribulose 1,5-bisphosphate carboxylase (RuBPCase). The large subunit of cucumber RuBPCase has been identified among the translation products of the E. coli system. The wheat germ system translates the cucumber mRNA coding for the small subunit of RuBPCase to produce a 25,000 molecular weight precursor polypeptide. Plastids isolated from light-grown cotyledons were used to study developmental changes in their capacity to synthesize protein. The data obtained indicate that in the light there is an initial 48-hour period of accumulation of the mRNA's coding for the large and small subunits of RuBPCase, coupled with an increase in the capacity of the isolated plastids to synthesize protein. This is followed by a decline. This decline is not reflected in the accumulation of RuBPCase in the cotyledons which remains constant over the period of study.

Identification of chloroplast NADP-linked malate dehydrogenase as a product of chloroplast protein synthesis

Identification of chloroplast NADP-linked malate dehydrogenase as a product of chloroplast protein synthesis

Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates.

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light-dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development.

Purification of Euglena gracilis chloroplast elongation factor G and comparison with other prokaryotic and eukaryotic translocases.

Euglena gracilis chloroplast protein synthesis elongation factor G (EF-Gchl) has been purified to about 80% homogeneity by a two-step procedure which removes all traces of the cytoplasmic and mitochondrial translocases (EF-2 and EF-Gmt). The purification scheme generally results in approximately 130-fold purification with 20% recovery of the total EF-G activity present in whole cell extracts. The Euglena EF-Gchl is a monomeric protein with a molecular weight of approximately 85,000. As observed for all translocases to date, the activity of Euglena EF-Gchl is inhibited by treatment with low concentrations of N-ethylmaleimide, suggesting that a free sulfhydryl group is required for catalytic activity. Treatment with 3 microM fusidic acid results in a 50% inhibition of Euglena EF-Gchl activity and of the EF-G activity present in Chlamydomonas reinhardtii. About 10-fold higher concentrations of this antibiotic are required to inhibit the mitochondrial EF-G of Euglena and Escherichia coli EF-G to the same extent. Yeast mitochondrial EF-G is clearly distinguishable from the other organellar translocases tested, requiring 1 mM fusidic acid for 50% inhibition. Fusidic acid also inhibits the cytoplasmic translocases from yeast, wheat germ, and Euglena, although a wide range of sensitivities is observed. When antiserum raised against highly purified Euglena EF-Gchl is used to inhibit enzymatic translocation, a low degree of cross-reaction of the antiserum with Chlamydomonas EF-G and with E. coli EF-G is observed. The EF-G activity present in spinach is very slightly inhibited by the antiserum, whereas that of yeast is not affected. The mitochondrial and cytoplasmic translocases of Euglena are also unaffected by the antiserum against EF-Gchl. The evolutionary implications of these observations are discussed.

Photosynthetic recovery of resurrection spikemosses from different hydration regimes.

Patterns and mechanisms of recovery of photosynthetic activity of two resurrection spikemoss species from different hydration environments in the Chihuahuan desert of southwest Texas, USA were compared in the laboratory. Selaginella lepidophylla Hook. and Grev. is the dominant poikilohydric plant found in hot, arid low-elevation desert scrub communities and S. pilifera A. Br. is one of many poikilohydric species found in more mesic, high-elevation woodland communities in Big Bend National Park, Texas. Infrared gas analysis of CO2 exchange during hydration of desiccated plants indicated that photosynthetic competence was achieved significantly more rapidly in S. lepidophylla tha S. pilifera. Chloramphenicol, a chloroplast protein synthesis inhibitor at 100 μg·ml-1 in the hydration medium, significantly inhibited the resumption of CO2 uptake in both species, but the level of inhibition was significantly greater in S. pilifera. The level of conserved ribulose 1,5 bis phosphate carboxylase specific activity in desiccated plants was significantly greater in S. lepidophylla. These results support the hypothesis that poikilohydric plants from increasingly xeric environments are capable of more rapid photosynthetic recovery, due to increased conservation of the photosynthetic system during desiccation and a reduced requirement for photosynthetic system repair during hydration, than those from more mesic environments.

Studies on frost hardiness in Chlorella ellipsoidea. V. The role of glucose and related compounds

Chlorella ellipsoidea cells at an intermediate stage in the ripening phase of the life cycle were hardened at 3°C. d-Glucose added during hardening in the dark induced a tolerance to freezing. Sucrose, d-galactose and d-fructose were also capable of increasing hardiness, but pyruvate, succinate and citrate were not. These results suggest the involvement of the pentose-phosphate cycle in the hardening process. The amounts of monosaccharide and dextrin in Chlorella cells did not change during hardening. Sucrose concentration increased during hardening in both the light and the dark in the presence of glucose. Starch concentration remained unchanged in the dark in the presence of glucose, but increased remarkably in the light. These results suggest that the hardening process involves an accumulation of sucrose, and indicate that there is no direct correlation between starch content and frost hardiness. Glucose 6-phosphate dehydrogenase activity increased greatly during the early phase of hardening; 6-phosphogluconate dehydrogenase activity also increased with hardiness. These results suggest that pentose-phosphate cycle activity occurs at a high level during hardening. Cycloheximide and oligomycin completely inhibited the hardiness increase in the dark in the presence of glucose; chloramphenicol and DCMU had no effect. These results suggest that protein synthesis on cytoplasmic ribosomes and ATP synthesis in mitochondria are involved in the process of hardening in the dark in the presence of glucose, but protein and ATP synthesis in chloroplasts are not. The inhibition of the hardiness increase by sodium dodecyl sulfate was greater in the light than in the dark in the presence of glucose. Triton X-100 inhibited completely the hardiness increase in both the light and the dark in the presence of glucose. It seems likely, therefore, that membrane alterations during hardening in the dark in the presence of glucose may occur; these alterations are different from those which occur in the light.

Initiation of protein synthesis in isolated mitochondria and chloroplasts

N 5-Formyltetrahydrofolate, a competitive inhibitor of the formylation of the initiator Met-tRNA fMet in an in vitro assay, is a powerful inhibitor of amino acid incorporation in isolated Saccharomyces cerevisiae mitochondria and in Euglena gracilis chloroplasts. Thus, a large part of the incorporation is dependent upon new initiation acts. On the contrary, the rate of incorporation can be largely increased by addition of the specific formyl group donor, N 10-formyltetrahydrofolate. Experiments are also reported strongly suggesting that the formylation of Met-tRNA fMet is an absolute requirement in order to initiate protein synthesis in chloroplasts, as has been shown in mitochondria.

Protein synthesis in chloroplasts IX. Assembly of newly-synthesized large subunits into ribulose bishopshate carboxylase in isolated intact pea chloroplasts

Isolated pea ( Pisum sativum) chloroplasts incorporate [ 35S]methionine into the large subunit of the chloroplast enzyme ribulose bisphosphate carboxylase. When chloroplasts are incubated in a medium containing KCl as osmoticum, newly-synthesised large subunits are not incorporated into the holoenzyme but can be separated from pre-existing enzyme by gel electrophoresis under non-denaturing conditions. Furthermore, newly-synthesised large subunits are not precipitated by antibodies which precipitate pre-existing holoenzyme and large subunit prepared from holoenzyme. When chloroplasts are incubated in a medium containing sorbitol as osmoticum, some of the newly-synthesised large subunits comigrate with holoenzyme on both 3% and 5% polyacrylamide non-denaturing gels. Such comigrating large subunits are precipitated by antibodies raised against the holoenzyme. These results indicate assembly of large subunits into ribulose bisphosphate carboxylase in the sorbitol medium. Time course experiments indicate that there is a time-lag of several minutes between the onset of synthesis of large subunits and the onset of assembly. Newly-synthesised large subunits which do not comigrate with holoenzyme on both 3% and 5% polyacrylamide non-denaturing gels are associated with a protein of subunit molecular weight 60 000. This protein may be specifically combined with newly-synthesised large subunits, and the resulting aggregate be involved in the assembly of complete molecules of ribulose bisphosphate carboxylase.

Comparison of the Polypeptide Compositions of the Internal Membranes of Chloroplasts, Etioplasts and Ribosome-Deficient Heat-Bleached Plastids from Rye Leaves

When rye seedlings ( Secale cereale L.) are grown at a non-permissive temperature of 32°C in light 70S ribosome-deficient and chlorotic leaves are formed, due to a previously described preferential high-temperature-sensitivity of the plastidic ribosome formation that allows the selective elimination of the chloroplastic protein synthesis. From bleached ribosome-deficient plastids obtained from 32°-grown rye leaves the internal membranes were isolated. Their polypeptide composition was analysed by sodium dodecylsulfate polyacrylamide gel electrophoresis and compared to that of the internal membranes of chloroplasts and etioplasts from leaves grown at a permissive temperature of 22°C. For about 50% of the major polypeptides of normal chloroplast thylakoids corresponding polypeptide bands of equal electrophoretic mobility occurred in the internal membranes of the ribosome-deficient plastids, thereby indicating that they were of cytoplasmic origin. The bleached plastids contained also numerous additional membrane polypeptides which did not occur in chloroplast thylakoids. Several of those were characteristic for etioplast membranes from 22°-grown leaves. The etioplast internal membranes contained most of the chloroplast thylakoid polypeptides and a number of specific additional polypeptides. The α- and β-subunits of the coupling factor CF 1 were present in chloroplasts and etioplasts from 22°-grown leaves but missing in the bleached plastids of 32°-grown leaves. Both the etioplasts and the bleached ribosome-deficient plastids contained a polypeptide corresponding in its electrophoretic mobility to that of the main apoprotein (relative molecular mass 69,100) of the chlorophyllprotein complex I. The apoprotein of the chlorophyll-protein complex II (relative molecular mass 24,600) was missing in etioplasts but present, though in relatively small amount, in the ribosome-deficient plastids.

Protein synthesis in chloroplasts VIII. Differential synthesis of chloroplast proteins during spinach leaf development

Excised primary leaves of spinach ( Spinacia oleracea) incorporate [ 35S]-methionine into a number of chloroplast polypeptides. The ratio of incorporation of isotope into the large subunit of ribulose bisphosphate carboxylase relative to a thylakoid polypeptide (peak D) decreases during leaf development in whole leaves; this changing pattern of incorporation is also observed in isolated chloroplasts where these two polypeptides are the major products of protein synthesis. Chloroplast RNA prepared from developing leaves was translated in a reticulocyte lysate extract to yield full-length carboxylase large subunit and peak D polypeptides. The fidelity of translation of these two polypeptides was checked by partial protease digestion. Changes in the synthesis of the large subunit of the carboxylase and peak D in developing leaves are reflected in changes in the amount of translatable mRNA for these two polypeptides.

Identification of P700—Chlorophyll a—Protein complex as a product of chloroplast protein synthesis

Identification of P700—Chlorophyll a—Protein complex as a product of chloroplast protein synthesis

Inhibition of Protein Synthesis in Chloroplasts from Plant Cells by Virginiamycin

The light-driven incorporation of amino acids by isolated spinach chloroplasts is inhibited by the M component (VM) and not by the S component (VS) of virginiamycin. This inhibitory effect is partially reversible. In chloroplast extracts, poly(U)-directed polyphenylalanine formation is strongly inhibited by VM and not by VS. The in vivo synergistic effect of VM and VS observed in bacteria and algae, does not occur in isolated chloroplasts and chloroplast extracts.

Effect of Manganese Deficiency on Chloroplasts of Lemon Leaves

AbstractManganese deficiency in chloroplasts of Eureka lemon leaves resulted in 23% and 40% increase of chloroplast nitrogen and protein, respectively, on a chlorophyll unit basis. Acrylamide gel electrophoresis carried out on extracts of these chloroplasts disclosed also qualitative differences between the normal and deficient leaves. Calculated on chloroplast N basis there is an increase of 17% in the chloroplast protein under Mn deficiency. This increase apparently indicates a more intense protein synthesis in the Mn deficient chloroplasts. Hill activity of the –Mn leaves was about one‐third of the analog control leaves. Manganese infiltration into detached but intact leaves restored the activity in the –Mn leaves up to 70% of the control. Lemon leaves affected by other macro‐ and micro‐ nutrient deficiencies did not respond to the manganese infiltration; therefore, the use of this infiltration method is suggested for the evaluation of the manganese nutrition status of citrus and probably other higher plants.