Chlorophyll Formation and Photosynthetic Competence in Euglena During Light-Induced Chloroplast Development in the Presence of 3, (3,4-dichlorophenyl) 1,1-Dimethyl Urea (DCMU)

The possibility that photosynthetic competence is gratuitous for light-induced chloroplast development in Euglena gracilis var. bacillaris was examined by incubating dark-grown resting cells in the light with DCMU, an inhibitor of photosynthesis. Under these conditions photosynthetic carbon dioxide fixation was inhibited essentially completely at all times during chloroplast development, but about 70% of the chlorophyll was formed with essentially the same pattern of accumulation found for cells incubated in the absence of the inhibitor. Electron microscopy of cells incubated with DCMU in the light revealed the formation of morphologically recognizable chloroplasts having comparable overall dimensions and structural elements to those found in normally developed chloroplasts, but frequently lacking a readily detectable pyrenoid with paramylum sheaths, and often containing increased numbers of discs per lamella. Such abnormalities are considered minor since upon removal of DCMU by centrifugation, the cells usually regained almost full photosynthetic competence on a chlorophyll basis.

It is concluded that photosynthetic competence is not necessary for chloroplast development in Euglena and supports the hypothesis, already suggested from other evidence, that light induction results in activation of synthetic machinery external to the developing chloroplast.

     

CpLEPA Is Critical for Chloroplast Protein Synthesis Under Suboptimal Conditions in Arabidopsis thaliana

LEPA is one of the most conserved translation factors and is found from bacteria to higher plants. However, the physiological function of the chloroplast LEPA homolog in higher plants remains unknown. Herein, we demonstrate the physiological role of cpLEPA in enabling efficient photosynthesis in higher plants. The cplepa-1 mutant displays slightly high chlorophyll fluorescence and pale green phenotypes under normal growth conditions. The growth of the cplepa-1 mutant is reduced when grown on soil, and greater reduction is observed under intense light illumination. Photosynthetic activity is impaired in the cplepa-1 mutants, which is reflected in the decreased steady-state levels of chloroplast proteins. In vivo protein labeling experiments explained the decrease in the steady-state levels of chloroplast proteins. An abnormal association of the chloroplast-encoded mRNAs with ribosomes suggests that the protein synthesis deficiencies in cplepa-1 are due to defects in translation initiation in the chloroplasts. The cpLEPA protein appears to be an essential translation factor that promotes the efficiency of chloroplast protein synthesis.     

EFFECTS OF CHLORAMPHENICOL ON CHLOROPLAST AND MITOCHONDRIAL ULTRASTRUCTURE IN OCHROMONAS DANICA

The effect of chloramphenicol (CAP) on cell division and organelle ultrastructure was studied during light-induced chloroplast development in the Chrysophyte alga, Ochromonas danica. Since the growth rate of the CAP-treated cells is the same as that of the control cells for the first 12 hr in the light, CAP is presumed to be acting during that interval solely by inhibiting protein synthesis on chloroplast and mitochondrial ribosomes. CAP markedly inhibits chloroplast growth and differentiation. During the first 12 hr in the light, chlorophyll synthesis is inhibited by 93%, the formation of new thylakoid membranes is reduced by 91%, and the synthesis of chloroplast ribosomes is inhibited by 81%. Other chloroplast-associated abnormalities which occur during the first 12 hr and become more pronounced with extended CAP treatment are the presence of prolamellar bodies and of abnormal stacks of thylakoids, the proliferation of the perinuclear reticulum, and the accumulation of dense granular material between the chloroplast envelope and the chloroplast endoplasmic reticulum. CAP also causes a progressive loss of the mitochondrial cristae, which is paralleled by a decline in the growth rate of the cells, but it has no effect on the synthesis of mitochondrial ribosomes. We postulate that one or more chloroplast ribosomal proteins are synthesized on chloroplast ribosomes, whereas mitochondrial ribosomal proteins are synthesized on cytoplasmic ribosomes.     

Chloroplast elongation factor Tu: Evidence that it is the product of a chloroplast gene in Euglena

The chloroplast protein synthesizing factor responsible for the binding of aminoacyl-tRNA to ribosomes (EF-Tu_chl) has been identified in extracts of Euglena gracilis. This factor is present in low levels when Euglena is grown in the dark and can be induced more than 10-fold when the organism is exposed to light. The induction of the chloroplast EF-Tu by light is inhibited by streptomycin, an inhibitor of protein synthesis on chloroplast ribosomes, indicating that protein synthesis within the chloroplast itself is required for the induction of this factor. The induction of the chloroplast EF-Tu by light is also inhibited by cycloheximide, a specific inhibitor of protein synthesis on cytoplasmic ribosomes. The effect of cycloheximide probably results from the inhibition of chloroplast ribosome synthesis which requires the synthesis of many proteins by the cytoplasmic translational system. Chloroplast EF-Tu cannot be induced by light in an aplastidic mutant (strain W₃BUL) of Euglena which has neither significant plastid structure nor detectable chloroplast DNA. These data strongly suggest that the genetic information for chloroplast EF-Tu resides in the chloroplast genome and that this protein is synthesized within the organelle itself.     

Effect of High Temperature on Chloroplast Ribosomes and Biosynthesis of Chloroplast Proteins in Wheat

The chloroplasts of wheat have chanced greatly at high temperature condition(34℃). When wheat grown at 34℃ for 10 days, its chlorophyll content was 6 times less than that under the normal condition(22℃). The ribosomes were isolated from the leaves by sucrose density gradient centrifugation. It is found that only 80 S ribosomes existed in wheat leaves grown at the high temperature and the formation of 70 S ribosomes is specifically prevented. Since the absence of 70 S ribosomes in chloroplast, proteins synthesis can no longer proceed. Analysis of SDS-polyacrylamide gel electrophoresis indicates that the bands of chloroplast proteins from the leaves of wheat at the high temperature are less than those under normal condition. One of the poly- peptides the large subunit(MW=57000 daltons) of ribulose bisphosphate carboxylase, which is coded for by chloroplast genome and synthesized on 70 S ribosome in chloroplast, was lost. The photosynthetic intensity is decreased due to the blocking synthesis in chloroplast of some polypeptides which play the important role in photosynthesis.     

Resistance and Sensitivity of Chloroplast Ribosomes to Streptomycin in Mutants of Chlamydomonas reinhardi

• 1 In a mendelian (sr₃) and an uniparental (sr₃₅) streptomycin resistant mutant of Chlamydomonas reinhardi the influence of streptomycin on protein synthesis on the chloroplast and cytoplasmic ribosomes was investigated in vitro. Hetero-, mixo- and phototrophic agar cultures and heterotrophic liquid cultures were used.
• 2 Protein synthesis on the cytoplasmic ribosomes, measured by the activity of glyceraldehyde-3-phosphate: NADP dehydrogenase (EC 1.2.1.9), was not inhibited, but rather stimulated by streptomycin.
• 3 Protein synthesis on the chloroplast ribosomes of sr₃, measured by the activity of ribulose-1,5-diphosphate carboxylase (EC 4.1.1.39), was greatly inhibited by streptomycin, especially in hetero- and mixotrophic cultures. In sr₃₅ the chloroplast ribosomes were resistant to streptomycin.
• 4 Heterotrophically grown cultures of sr₃ and of a streptomycin-sensitive strain are yellow in the presence of streptomycin and form no or only reduced thylakoids on solid media. But 70-S organelle-ribosomes are present in a normal amount.
• 5 The relationship between chloroplast protein synthesis and thylakoid formation is discussed.

     

Evidence for the nuclear location of the gene for chloroplast elongation factor G.

The chloroplast protein synthesis factor responsible for the translocation step of polypeptide synthesis on chloroplast ribosomes (chloroplast elongation factor G [EF-G]) has been detected in whole cell extracts and in isolated chloroplasts from Euglena gracilis. This factor can be detected by its ability to catalyze translocation on 70 S prokaryotic ribosomes such as those from E. coli. Chloroplast EF-G is present in low levels when Euglena is grown in the dark and can be induced more than 20-fold when the organism is grown in the light. The induction of this factor by light is inhibited by cycloheximide, a specific inhibitor of protein synthesis on cytoplasmic ribosomes. However, inhibitors of chloroplast protein synthesis such as streptomycin or spectinomycin have no effect on the induction of this factor by light. Furthermore, chloroplast EF-G can be partially induced by light in an aplastidic mutant (strain W₃BUL) which has neither significant plastid structure nor detectable chloroplast DNA. These data strongly suggest that the genetic information for chloroplast EF-G resides in the nuclear genome, and that this protein is synthesized on cytoplasmic ribosomes prior to compartmentalization within the chloroplasts.     

Analysis and characterization of DCMU-resistant Euglena gracilis

Dark-grown, DCMU-adapted Euglena gracilis Z (ZR) are able to undergo light-induced chloroplast development in the presence or absence of DCMU. The differentiated chloroplasts are photosynthetically active and are resistant not only to DCMU, but also to an analog, o-phenanthrolene. When DCMU overdoses are added to ZR cells or to chloroplasts isolated from these cells, photosynthesis is partially inhibited. A brief period of darkness removes this inhibition. This recovery phenomenon is related to DCMU resistance, since it is not exhibited by non-resistant control cells. The chloroplast protein synthesis apparatus is not involved in DCMU resistance. Rather, this phenomenon is apparently related to new characteristics of thylakoids. It is shown that photosynthetic recovery by ZR cells depends on the accessibility and fluid properties of membranes. The analysis of fluorescence induction kinetics shows that changes in the environmental conformation of photosystem II units occur during recovery.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ZR DCMU-adapted Euglena gracilis Z I and II=Calvayrac et al., in press (a, b)     

Studies on Chloroplast Development and Replication in Euglena: III. A Study of the Site of Synthesis of Alkaline Deoxyribonuclease Induced during Chloroplast Development in Euglena gracilis

During chloroplast development in Euglena, the activity of a specific DNase, Euglena alkaline DNase, increases in a manner similar to that of chlorophyll synthesis, but without the lag customarily associated with the early hours of chlorophyll synthesis. The increase in Euglena alkaline DNase activity is not inhibited by chloramphenicol or by streptomycin, but is inhibited by cycloheximide. Euglena alkaline DNase activity is present in a group of aplastidic substrains which contain carotenoids. These results are interpreted to mean that this chloroplast-related DNase is synthesized in the cytoplasm, and that the genetic information for this enzyme is probably nuclear.     

Polypeptides of chloroplastic and cytoplastic origin required for development of Photosystem II activity,and chlorophyll-protein complexes,in Euglena gracilis Z chloroplast membranes

The development of photosynthetic activity and synthesis of chloroplast membrane polypeptides was studied during greening of Euglena gracilis Z in alternate light-dark-light cycles. The results show: (a) The development of both Photosystem II and Photosystem I can be dissociated from chlorophyll synthesis. (b) Most of the polypeptides required for development of Photosystem I are already synthesized during the initial light period (10–12 h); the further rise in Photosystem I activity in the dark is not inhibited by cycloheximide nor by chloramphenicol. (c) The development of Photosystem II requires continuous de novo synthesis of polypeptides and is inhibited by chloramphenicol. The water-splitting activity already present at the end of the first light period decays in the presence of chloramphenicol while that of 1,5-diphenylcarbazide oxidation is only partially retained. The activity can be repaired in the absence of chlorophyll synthesis and is correlated with the de novo synthesis of polypeptides of 50 000–60 000 daltons. The synthesis of these polypeptides and associated repair of Photosystem II activity is not inhibited by cycloheximide. (d) The chloroplast membranes can be resolved into about 40 distinct polypeptides, among them several in the molecular weight range 50 000–60 000, 20 000–35 000 and 10 000–15 000, which are major membrane constitutents. (e) The synthesis of two major polypeptides (M_r = 20 000–30 000) required for the formation of chlorophyll-protein complex(es) containing chlorophyll a and traces of chlorophyll b (CPII?) is light-dependent and cycloheximide-inhibited. It is concluded that the synthesis and addition to the growing membrane of chlorophyll and polypeptides required for the formation of Photosystem II and Photosystem I complexes can be dissociated in time. The H₂O-splitting enzyme(s) and possibly other components of Photosystem II complex are of chloroplastic origin and turn over in the dark while at least some of the chlorophyll binding polypeptides are of cytoplastic origin and their synthesis is light-controlled.     

Periodic variations in the ratio of free to thylakoid-bound chloroplast ribosomes during the cell cycle of Chlamydomonas reinhardtii

The ratio of free to thylakoid-bound chloroplast ribosomes in Chlamydomonas reinhardtii undergoes periodic changes during the synchronous light-dark cycle. In the light, when there is an increase in the chlorophyll content and synthesis of thylakoid membrane proteins, about 20-30% of the chloroplast ribosomes are bound to the thylakoid membranes. On the other hand, only a few or no bound ribosomes are present in the dark when there is no increase in the chlorophyll content. The ribosome-membrane interaction depends not only on the developmental stage of the cell but also on light. Thus, bound ribosomes were converted to the free variety after cultures at 4 h in the light had been transferred to the dark for 10 min. Conversely, a larger number of chloroplast ribosomes became attached to the membranes after cultures at 4 h in the dark had been illuminated for 10 min. Under normal conditions, when there was slow cooling of the cultures during cell harvesting, chloroplast polysomal runoff occurred in vivo leading to low levels of thylakoid-bound ribosomes. This polysomal runoff could be arrested by either rapid cooling of the cells or the addition of chloramphenicol or erythromycin. Each of these treatments prevented polypeptide chain elongation on chloroplast ribosomes and thus allowed the polyosomes to remain bound to the thylakoids. Addition of lincomycin, an inhibitor of chain initiation on 70S ribosomes, inhibited the assembly of polysome-thylakoid membrane complex in the light. These results support a model in which initiation of mRNA translation begins in the chloroplast stroma, and the polysome subsequently becomes attached to the thylakoid membrane. Upon natural chain termination, the chloroplast ribosomes are released from the membrane into the stroma.     

Events surrounding the early development of Euglena chloroplasts. 16. Plastid thylakoid polypeptides during greening

Using sulfolipid to locate plastid thylakoid membranes in gradients from dark-grown resting cells it has been possible to study the plastid thylakoid membrane polypeptides of Euglena gracilis var. bacillaris undergoing light-induced chloroplast development. All plastid thylakoid bands seen in dark-growing wild-type cells and in mutant W₃BUL in which plastid DNA is undetectable, are observed to increase in amount during plastid development. Others, which are undetectable in dark-grown wild-type and W₃BUL increase greatly during plastid development and appear to be those associated with pigment-protein complexes. The data obtained from experiments where the polypeptides were labeled with ³⁵S during development, either continuously or in pulses, were consistent with these findings. Cycloheximide strongly inhibited the increases in amount in all bands and chloramphenicol or streptomycin produced a lower level of inhibition in all bands indicating tight control of the formation of each plastid membrane constituent by the others. The formation of a polypeptide band of 25 000 molecular weight, thought to be a part of a pigment-protein complex of the thylakoid, and chlorophyll synthesis were inhibited identically by these antibiotics.     

Inhibition of protein synthesis by aminoglycoside antibiotics in polyamine-requiring bacteria

The effect of streptomycin and other aminoglycosides on protein synthesis has been studied using various streptomycin-sensitive strains unable to synthesize polyamines. We have confirmed and extended our previous results showing that the strong inhibition of translation caused by the antibiotic in polyamine-supplemented bacteria was markedly reduced in polyamine-starved cells. The analysis of polypeptides synthesized in the absence and presence of streptomycin in bacteria grown with and without putrescine has shown that the antibiotic provoked the accumulation of low molecular weight peptides partially bound to ribosomes in polyamine-unstarved cells. On the contrary, the drug did not induce major alterations in the patterns of proteins obtained from polyamine-depleted bacteria. The addition of the antibiotic did not evoke any change of proteolytic activity.     

Chloroplast genes in Chlamydomonas affecting organelle ribosomes. Genetic and biochemical analysis of analysis of antibiotic-resistant mutants at several gene loci.

Six chloroplast gene mutants of Chlamydomonas reinhardtii resistant to spectinomycin, erythromycin, or streptomycin have been assessed for antibiotic resistance of their chloroplast ribosomes. Four of these mutations clearly confer high levels of antibiotic resistance on the chloroplast ribosomes both in vivo. Although one mutant resistant to streptomycin and one resistant to spectinomycin have chloroplast ribosomes as sensitive to antibiotics as those of wild type in vivo, these mutations can be shown to alter the wildtype sensitivity of chloroplast ribosomes in polynucleotide-directed amino acid incorporation in vitro. Genetic analysis of these six chloroplast mutants and three similar mutants (Sager, 1972), two of which have been shown to affect chloroplast ribosomes (Mets and Bogorad, 1972; Schlanger and Sager, 1974), indicates that in Chlamydomonas at least three chloroplast gene loci can affect streptomycin resistance of chloroplast ribosomes and that two can affect erythromycin resistance. The three spectinomycin-resistant mutants examined appear to be alleles at a single chloroplast gene locus, but may represent mutations at two different sites within the same gene. Unlike wild type, the streptomycin and spectinomycin resistant mutants which have chloroplast ribosomes sensitive to antibiotics in vivo, grow well in the presence of antibiotic by respiring exogenously supplied acetate as a carbon source, and have normal levels of cytochrome oxidase activity and cyanide-sensitive respiration. We conclude that mitochondrial protein synthesis in these mutants is resistant to these antibiotics, whereas in wild type it is sensitive. To explain the behavior of these two chloroplast gene mutants as well as other one-step mutants which are resistant both photosynthetically and when respiring acetate in the dark, we have postulated that a mutation in a single chloroplast gene may result in alteration of both chloroplast and mitochondrial ribosomes. Mitochondrial resistance would appear to be the minimal necessary condition for survival of all such mutants, and antibiotic-resistant chloroplast ribosomes would be necessary for survival only under photosynthetic conditions.     

Inhibition of chloroplast development in Euglena by streptomycin: Differential inhibition of the appearance of photosynthesis in the presence of the continued synthesis of chlorophyll

Summary Streptomycin effectively inhibits chloroplast development in dark-grown non-dividing Euglena gracilis when added at the onset of greening but apparently exerts a diminished effect on chlorophyll synthesis when added at later stages. We have further investigated this phenomenon and show that streptomycin added to a Euglena culture at 24 h of development has a differential inhibitory action on the extent of synthesis of several chloroplast-associated parameters. Chlorophyll, carotenoids, cytochrome 552 and photosystem I Hill activity are all slightly, if at all, inhibited and to approximately the same extent between 24 and 72 h of development. We find a very strong inhibition of both ribulose diphosphate carboxylase synthesis and photosystem II Hill activity.Abbreviations A absorbance - chl chlorophyll - Cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPIP 2,6-dichlorophenolindophenol - DPC diphenylcarbazide - PS photosystem - Rib-5-P ribose-5-phosphate - RuDP ribulose-1,5-diphosphate - Sm Streptomycin Present Adress: Laboratoire de Photosynthèse C.N.R.S. 91190 Gif-sur-Yvette, France. After 1 June 1976: 50 rue Pasteur. 78460 Chevreuse, France     

A method for complementation analysis of nuclear and chloroplast mutants of photosynthesis in chlamydomonas

The photosynthetic properties of young zygotes of Chlamydomonas reinhardi were analyzed. In heterozygotes for two nuclear or two chloroplast mutations affecting photosynthesis, recovery of photosynthetic activity was observed that is most likely the result of intergenic complementation.——We observed that chloramphenicol inhibited the recovery of activity in double heterozygotes for mutants lacking at least one thylakoid polypeptide of chloroplast origin, while it had not effect on wild-type homozygotes. This indicates that the recovery of activity in double heterozygotes could result from the repair of existing thylakoid membranes by de novo synthesis of the missing polypeptides.     

Events surrounding the early development of Euglena chloroplasts. 16. Plastid thylakoid polypeptides during greening.

Using sulfolipid to locate plastid thylakoid membranes in gradients from dark-grown resting cells it has been possible to study the plastid thylakoid membrane polypeptides of Euglena gracilis var. bacillaris undergoing light-induced chloroplast development. All plastid thylakoid bands seen in dark-growing wild-type cells and in mutant W3BUL in which plastid DNA is undetectable, are observed to increase in amount during plastid development. Others, which are undetectable in dark-grown wild-type and W3BUL increase greatly during plastid development and appear to be those associated with pigment-protein complexes. The data obtained from experiments where the polypeptides were labeled with 35S during development, either continuously or in pulses, were consistent with these findings. Cycloheximide strongly inhibited the increases in amount in all bands and chloramphenicol or streptomycin produced a lower level of inhibition in all bands indicating tight control of theformation of each plastid membrane constituent by the others. The formation of a polypeptide band of 25 000 molecular weight, thought to be a part of a pigment-protein complex of the thylakoid, and chlorophyll synthesis were inhibited identically by these antibiotics.     

In vitro synthesis and assembly of photosystem II proteins of spinach chloroplasts

The synthesis and assembly of photosystem II (PS II) proteins of spinach chloroplasts were investigated in three different in vitro systems, i.e., protein synthesis in isolated chloroplasts (in organello translation), read-out translation of thylakoid-bound ribosomes, and transport of translation products from spinach leaf polyadenylated RNA into isolated chloroplasts. Polyacrylamide gel electrophoresis of labeled thylakoid polypeptides in the presence of sodium dodecyl sulfate revealed that the first two systems were capable of synthesizing the reaction center proteins of PS II (47 and 43 kDa), the herbicide-binding protein, and cytochrome b559. The reaction center proteins synthesized in organello were shown to bind chlorophyll and to assemble properly into the PS II core complex. One of the reaction center proteins translated by the thylakoid-bound ribosomes (47 kDa) was also found to be integrated in situ into the complex but was lacking bound chlorophyll. Incorporation of radioactivity into the three extrinsic proteins of the oxygen-evolution system (33, 24, and 18 kDa) was detected only when intact chloroplasts were incubated with the translation products from polyadenylated RNA, showing that these proteins are coded for by nuclear DNA. The occurrence of a precursor polypeptide 6 kDa larger than the 33-kDa protein was immunochemically detected in the translation products.     

G-418, an elongation inhibitor of 80 S ribosomes

The mode of action of the aminoglycoside G-418 was studied in wheat-germ, cell-free translation systems programmed with rat-liver polyadenylated RNA. Incorporation of amino acids into protein was effectively inhibited by G-418 in the microM concentration range. The inhibition pattern obtained was not uniform. The synthesis of polypeptides with higher molecular weights was more inhibited than that of smaller polypeptides. An identical inhibition pattern within a similar range of concentrations was obtained with cycloheximide, a known elongation inhibitor. Translation activity was abolished when the wheat-germ 80 S ribosomes were removed and could be partially reconstructed upon addition of the ribosomes. Incubation with G-418 prior to isolation yielded ribosomes defective in their reconstruction ability. The inhibition pattern was not uniform and exhibited again the same relationship between the size of a polypeptide and the extent of inhibition of its synthesis. Therefore, we suggest that in wheat-germ, cell-free translation systems G-418 affects the 80 S ribosomes and inhibits the elongation cycle.