Photo and Nutritional Regulation of the Light-Harvesting Chlorophyll a/b-Binding Protein of Photosystem II mRNA Levels in Euglena

In Euglena gracillis var bacillaris, light exposure increases the level of mRNA encoding the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) approximately twofold. LHCPII mRNA levels increased in the dark upon either malate or ethanol addition. LHCPII mRNA is present but LHCPII is not synthesized in the bleached mutants W₃BUL and W₁₀BSmL, which lack protochlorophyll(ide) and most if not all of the chloroplast genome. Light exposure increased LHCPII mRNA levels in W₃BUL but not in W₁₀BSmL. Carbon availability and light acting through a nonchloroplast photoreceptor appear to regulate LHCPII mRNA levels. A chloroplast photoreceptor and/or a product produced by the chloroplast appear to regulate LHCPII mRNA translation.     

Nutritional regulation of organelle biogenesis inEuglena: Photo- and metabolite induction of mitochondria

Exposure of dark-grown restingEuglena gracilis Klebs var.bacillaris Cori to light, ethanol, or malate produced an increase in the specific activity of fumarase (EC. 4.2.1.2) and succinate dehydrogenase (EC. 1.3.99.1) during the first 8–12 h of exposure to inducer, followed by a decrease in the specific activity of both mitochondrial enzymes between 12 and 72 h. The increased specific activity represented a net increase in the level of active enzyme, and it was dependent upon cytoplasmic protein synthesis. The photoinduction of fumarase required continuous illumination while the subsequent decrease in fumarase specific activity was independent of light. Light had little effect on the ethanol and malate induction of fumarase and succinate dehydrogenase. In the mutant W₃BUL, which has no detectable protochlorophyll(ide) and chloroplast DNA, light induced both mitochondrial enzymes and the kinetics of enzyme induction were similar to the induction kinetics in wild-type cells. The induction of mitochondrial enzymes appears to be controlled by a non-chloroplast photoreceptor. Dark-grown resting cells of the plastidless mutant W₁₀SmL have lost the ability to regulate fumarase levels. In this mutant, the specific activity of fumarase fluctuated and light had little effect on these fluctuations, indicating that fumarase synthesis was uncoupled from the nonchloroplast photoreceptor. Ethanol addition produced transient changes in fumarase specific activity in W₁₀SmL indicating that in this mutant, mitochondrial enzymes are still inductible by metabolites. Fumarase synthesis in wild-type cells was not induced in the dark by levulinic acid, a chemical inducer of the breakdown ofEuglena storage carbohydrates. Taken together, our results indicate that the photoinduction of mitochondrial enzyme synthesis is not a result of the photoinduction of carbohydrate breakdown. The mechanisms by which light and organic carbon induce the synthesis ofEuglena mitochondria may differ.     

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.     

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.     

A consideration of Euglena gracilis W3BUL as a cytoplasmic control for the wild-type phenylalanyl-tRNA synthetase system

The studies described indicate that the UV bleached mutant, Euglena gracilis W₃BUL does not serve as a suitable cytoplasmic control for the phenylalanyl-tRNA synthetase system. Chromatography of wild-type E. gracilis on Sephadex G100 revealed three peaks of activity identified as the chloroplastic, cytoplasmic and mitochondrial enzymes. The chloroplastic activity was greater in log than in stationary phase cells and was the only activity recovered from purified chloroplasts. Cell-free extracts of the achloroplastic mutant, E. gracilis W₃BUL, contained wild-type levels of the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases. However, no chloroplastic synthetase was detected in the mutant extracts. Anomalies in the aminoacylation behavior of the W₃BUL system were observed which suggest the possibility of a mutation affecting non-chloroplastic tRNAs in this UV-induced mutant. These anomalies significantly reduce the ability of the E. gracilis W₃BUL mutant to serve as a cytoplasmic control in the phenylalanyl-tRNA synthetase system.     

Photoreactivating enzyme from Euglena and the control of its intracellular level

Photoreactivating (PR) enzyme activity has already been demonstrated by us in cell-free extracts of Euglena gracilis var. bacillaris Pringsheim using the Hemophilus transformation assay. This activity can also be detected in extracts using a direct non-biological assay for the photorepair of thymine dimers in DNA. PR enzyme is found in extracts of both wild-type cells and cells of an aplastidic mutant, W₃BUL, lacking detectable chloroplast DNA, indicating that the PR enzyme is neither coded nor translated exclusively in the chloroplast, but is probably coded in the nucleus and translated in the cytoplasm. Growing cultures of wild-type cells manifest a large increase in PR enzyme activity in vitro upon entering stationary phase. This correlates with the increased photoreactivability of chloroplast inheritance in vivo in stationary phase cells, previously found for Euglena, and suggests that a substantial part of the newly synthesized PR enzyme is available to repair plastid DNA. When dark-grown nondividing wild-type cells are exposed to light, there is a large increase in the specific activity of PR enzyme measured in vitro. This increase is prevented by cycloheximide but not by chloramphenicol or streptomycin, indicating that the enzyme is synthesized on 87s cytoplasmic ribosomes rather than 68s chloroplast ribosomes. Wavelengths of light effective for PR of chloroplast DNA in vivo are also effective for the light induction of PR enzyme. A brief illumination (45 min) of dark-grown nondividing wild-type cells triggers the synthesis of PR enzyme which continues in the absence of light. Growing cultures of W₃BUL also exhibit a preferential synthesis of PR enzyme in the staionary phase of growth, but the specific activity in vitro is consistently ten times higher than that of wild-type. Dark-grown non-dividing cultures of W₃BUL also show a cycloheximide-sensitive light induction of PR enzyme synthesis which, however, is dependent on the continued presence of light. The light induction of PR enzyme synthesis can be regarded as the induction of an enzyme by one of its substrates.     

Events surrounding the early development of euglena chloroplasts: v. Control of paramylum degradation

The degradation of the storage carbohydrate, paramylum, is induced by light in wild-type Euglena gracilis Klebs var. bacillaris Pringsheim and in a mutant, W₃BUL, which lacks detectable plastid DNA. Treatment of wild type with cycloheximide in the dark produces 60% as much paramylum breakdown as light, whereas treatment with levulinic acid in the dark yields a slightly greater response than light. Both cycloheximide and levulinic acid produce a greater paramylum breakdown in the light than they do in the dark. Treatment of W₃BUL with levulinic acid in darkness produces a larger paramylum degradation than light, with values similar to wild type in the light. Treatment of W₃BUL with cycloheximide induces paramylum degradation in darkness, and as with wild type, light is slightly stimulatory in the presence of both cycloheximide or levulinic acid. Streptomycin brings about only a very small amount of paramylum breakdown in the dark and only slightly inhibits breakdown in the light. Thus paramylum breakdown induced by light does not require the synthesis of proteins on cytoplasmic or plastid ribosomes. A model which explains these results postulates the existence of a protein which inhibits paramylum breakdown. When the synthesis of this protein is prevented either by light, cycloheximide, or by levulinic acid acting as a regulatory analog of delta amino levulinic acid, paramylum breakdown takes place. Because levulinic acid is a better inducer than light in W₃BUL, W₃BUL may not be able to form as much delta amino levulinic acid in light as wild type. The small amount of induction by streptomycin is viewed as a secondary regulatory effect attributable to interference with plastid protein synthesis which affects regulatory signals from the plastid to the rest of the cell.     

Nutritional Regulation of Organelle Biogenesis in Euglena: INDUCTION OF MICROBODIES

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 W₃BUL, enzyme levels were always higher in the dark than in the light.     

Studies of sulfate utilization of algae: 15. Enzymes of assimilatory sulfate reduction in euglena and their cellular localization

Crude extracts of wild-type Euglena grown in the light (WTL) or in the dark (WTD) and a mutant lacking detectable plastid DNA (W₃BUL) contain adenosine 5′-phosphosulfate (APS) sulfotransferase. Isotope dilution experiments indicate that adenosine 3′-phosphate 5′-phosphosulfate (PAPS) sulfotransferase is absent.     

Photocontrol of chloroplast and mitochondrial polypeptide levels in euglena

Two dimensional polyacrylamide gel electrophoresis resolved protein from intact chloroplasts of wild type Euglena gracilis Klebs var. bacillaris Cori into 185 polypeptides of which 55 were localized on the whole cell polypeptide map. Of these chloroplast polypeptides, the relative amounts of 49 increased, the relative amounts of two decreased, and the relative amounts of four polypeptides were unaltered by exposure of dark grown resting cells to light for 72 hours. Proteins from intact purified mitochondria obtained from a bleached mutant (W₁₀BSmL) lacking plastids were resolved into 193 polypeptides of which 44 were localized on the whole cell polypeptide map from wild type cells. Of these mitochondrial polypeptides, the relative amount of one increased, the relative amounts of 12 were unaltered, and the relative amounts of 31 decreased after exposure of the dark grown resting cells to light. Since it is known that the development of the chloroplast in Euglena occurs without a net increase in total cellular protein and without a change in the size of the cellular amino acid pools, the degradation of mitochondrial polypeptides represents a major source of amino acids for the synthesis of chloroplast polypeptides.     

Effects of 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea on the Cell Cycle in Euglena gracilis

The cell cycle of the photosynthetic unicellular alga Euglena gracilis growing in phototrophic medium is regulated by light. To investigate the relationship of this cell cycle response to light stimulated photosynthesis, we have tested the effect of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on Euglena cell cycle transit. While DCMU does not block light stimulated cells from entering the S phase of the cell cycle, it does inhibit the transit through G₂/M. The specificity of this response and its relationship to photosynthesis was studied by looking at the effect of DCMU on dark grown wild-type cells, and on two bleached variants of Euglena (W₃BUL and W₁₀BSmL) that lack chloroplasts. The drug does block G₂/M in these cells, but not entrance into the cell cycle. Our studies show that entrance of cells into the cell cycle from a quiescent state does not require active photosynthesis, and that DCMU has effects on G₂/M transit that are independent of the photosynthetic capacity of the cells.     

A chloroplast-associated fatty acid synthetase system in Euglena

Fatty acid synthetase activity in etiolated Euglena gracilis strain Z is independent of added ACP and associated with a high-molecular-weight complex of the type found in yeast. Cells grown in the dark and then greened by illumination in a resting medium develop a second enzyme system which is dependent on added ACP and generally resembles the corresponding E. coli and plant enzymes. Cycloheximide has no effect on the appearance of the ACP-dependent fatty acid synthetase in greening cells whereas chloramphenicol causes complete inhibition at concentrations which decrease chlorophyll synthesis by 66%. An induction of the ACP-dependent fatty acid synthetase in the absence of chloroplast development occurs on exposure of dark-grown cells to doses of ultraviolet light which selectively affect proplastid nucleoprotein. This enzyme induction by ultraviolet light is inhibited by chloramphenicol. The protein synthesis machinery of the chloroplast appears to be responsible, either directly or indirectly, for the appearance of the ACP-dependent fatty acid synthetase of Euglena.     

RNA metabolism during light-induced chloroplast development in euglena

Methods are described which provide good recoveries of non-degraded chloroplast and non-chloroplast RNAs from Euglena gracilis var. bacillaris. These have been characterized by comparing the RNA from W₃BUL (an aplastidic mutant of Euglena), with that of wild-type cells which have been resolved into chloroplast and non-chloroplast fractions. Using E. coli RNA as a standard, the RNAs from W₃BUL and from the non-chloroplast fraction of green cells exhibit optical density peaks, upon sucrose gradient centrifugation, at 4S, 10S, and 19S. The chloroplast fraction exhibits optical density peaks at 19S and 14S with the 19S component predominating. Application of various techniques for the separation of RNAs to the problem of separating the chloroplast and non-chloroplast RNAs, without prior separation of the organelle, have not proven successful.

³²P_i is readily incorporated into RNA by cells undergoing light-induced chloroplast development, and fractionation at the end of development reveals that although chloroplast RNAs have a higher specific activity, the other RNAs of the cells are significantly labeled as well. The succession of labeling patterns of total cellular RNA as light-induced chloroplast development proceeds are displayed and reveal that all RNA species mentioned above eventually become labeled. In contrast, cells kept in darkness during this period incorporate little ³²P_i into any RNA fraction. In addition, a heavy RNA component, designated as 28S, while representing a negligible fraction of the total RNA, becomes significantly labeled during the first 24 hours of illumination. While there is light stimulated uptake of ³²P_i into the cells, this uptake is never limiting in the light or dark, for RNA labeling.

On the basis of these findings, we suggest that extensive activation of non-chloroplast RNA labeling during chloroplast development is the result of the activation of the cellular synthetic machinery external to the chloroplast necessary to provide metabolic precursors for plastid development. Thus the plastid is viewed as an auxotrophic resident within the cell during development. Other possibilities for interaction at this and other levels are also discussed.

     

Cytosolic and plastid forms of 5-enolpyruvylshikimate-3-phosphate synthase in Euglena gracilis are differentially expressed during light-induced chloroplast development

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), the target of the herbicide glyphosate [N-(phosphonomethyl)glycine], exists in two molecular forms in Euglena gracilis. One form has previously been characterized as a monofunctional 59 kDa protein. The other form constitutes a single domain of the multifunctional 165 kDa arom protein. The two enzyme forms are inversely regulated at the protein and mRNA levels during light-induced chloroplast development, as demonstrated by the determination of their enzyme activities after non-denaturing polyacrylamide gel electrophoresis and Northern hybridization analysis with a Saccharomyces cerevisiae ARO1 gene probe. The arom protein and its mRNA predominate in dark-grown cells, and the levels of both decline upon illumination. In contrast, the monofunctional EPSP synthase and its mRNA are induced by light, the increase in mRNA abundance preceding accumulation of the protein. The two enzymes are localized in different subcellular compartments, as demonstrated by comparing total protein patterns with those of isolated organelles. Glyphosate-adapted wild-type cells and glyphosate-tolerant cells of a plastid-free mutant of E. gracilis, W₁₀BSmL, were used for organelle isolation and protein extraction, as these cell lines overproduce EPSP synthase and the arom protein, respectively. Evidence was obtained for the cytosolic localization of the arom protein and the plastid compartmentalization of the monofunctional EPSP synthase. These conclusions are further supported by the observation that EPSP synthase precursor, produced by in vitro translation of the hybrid-selected mRNA, was efficiently taken up and processed to mature size by isolated chloroplasts from photoautotrophic wild-type E. gracilis cells, while the in vitro-synthesized arom protein was not sequestered by isolated Euglena plastids.Dedicated to Prof. Dr. A. Trebst on the occasion of his 65th birthday     

Events Surrounding the Early Development of Euglena Chloroplasts: 14. Biosynthesis of Cytochrome c-552 in Wild Type and Mutant Cells

Lack of a suitable assay has thwarted attempts to measure cytochrome c-552 in dark-grown wild type cells of Euglena gracilis var. bacillaris in mutants and in other situations where the concentrations are low. Purification methods are described based on electrofocusing which provide a cytochrome c-552 preparation homogeneous enough to elicit a single reactive antibody in rabbits; this antibody is then used as a specific and sensitive assay for cytochrome c-552. Dark-grown cells of wild type and of mutants O₁BS, O₂BX, G₁BU and P₁BXL (which make normal sized chloroplasts with abnormal internal structure in the light) have 0.02 to 0.1 × 10⁻¹¹ micromoles of cytochrome c-552 per cell, 10 to 150 times less than light-grown cells. Light-grown cells of these mutants and of wild type show a ratio of chlorophyll to cytochrome of about 300 (mole to mole). Cytochrome c-552 is undetectable in dark-grown Y₁BXD, Y₃BUD, and W₃₄ZUD which cannot carry plastid development beyond the proplastid in light; the light-grown cells of these mutants have levels of cytochrome similar to or lower than dark-grown wild type cells. Cytochrome c-552 is undetectable in light- and dark-grown mutants in which plastid DNA is undetectable (such as Y₂BUL, W₃BUL, W₈BHL, and W₁₀BSmL) consistent with the view, but not proving, that this molecule may be coded, at least in part, in plastid DNA. During light-induced chloroplast development in resting cells, cytochrome c-552 formation behaves in all respects like chlorophyll except that the dark-grown cells contain low amounts of the cytochrome c-552 but lack chlorophyll. Thus, both cytochrome c-552 and chlorophyll show the same lag period even when the length is changed by nutritional manipulation; preillumination largely eliminates the lag in the formation of both molecules, cycloheximide and streptomycin both inhibit the biosynthesis of chlorophyll and cytochrome c-552 in the same manner, and the formation of both during chloroplast development is strictly light-dependent. It is shown that chloroplasts isolated from Euglena by methods thought to give intact organelles, lack 95% of the cytochrome c-552; this and the loss of similar molecules may explain why these isolated chloroplasts are not photosynthetically active.     

Chlorophyll biosynthesis from glutamate or 5-aminolevulinate in intact Euglena chloroplasts

Chloroplasts observed, by electron microscopy, to be intact and uncontaminated, with high rates of light-dependent protein synthesis and CO₂ fixation were isolated from cells grown on low-vitamin-B₁₂ medium in the light or from cells grown in the same medium in the dark and then exposed to light for 36 h. Both types of chloroplasts were active but less variability was encountered with developing chloroplasts from 36-h cells. The 36-h chloroplasts showed good light-dependent incorporation of 5-amino-levulinic acid (ALA) or l-glutamic acid into chlorophyll (Chl) a which was linear for approx. 1 h. The specific activity of the Chl a remained the same after conversion to pheophytin a, methylpheophorbide a or pyromethylpheophorbide a and rechromatography, indicating that the label was in the tetrapyrrole. Incorporation of ALA was inhibited by levulinic acid, and by chloramphenicol and other inhibitors of translation of 70S-type chloroplast ribosomes at concentrations which did not appreciably inhibit photosynthesis but which blocked plastid protein synthesis nearly completely. Cycloheximide, an inhibitor of translation on 87S cytoplasmic ribosomes of Euglena, was without effect. The 70S inhibitors did not block uptake of labeled ALA. Although labeled glycine was taken up by the plastids, no incorporation into Chl a was observed. Thus the developing chloroplasts appear to contain all of the enzymatic machinery necessary to convert glutamic acid to Chl via the C5 pathway of ALA formation but the Shemin pathway from succinyl coenzyme A and glycine to ALA appears to be absent. The requirement for plastid protein synthesis concomitant with Chl synthesis indicates a regulatory interaction and also indicates that at least one protein influencing Chl synthesis is synthesized on 70S-type plastid ribosomes and is subject to metabolic turnover.Abbreviations ALA 5-aminolevulinic acid - Chl chlorophyll     

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.     

Alteration of cyanobacterial glutamine synthetase activity in vivo in response to light and NH 4 +

Extractable glutamine synthetase activity of the cyanobacterium Anabaena cylindrica was reduced by approximately 50% when N₂-fixing cultures were treated with 10 mM NH ₄ ⁺ or were placed in darkness. The deactivated enzyme could be rapidly reactivated (within 5 min) by adding 40 mM 2-mercaptoethanol to the biosynthetic reaction mixture. The enzyme could also be reactivated in vivo by replacing the culture in light or by removing NH ₄ ⁺ . When the enzyme was deactivated by simultaneously adding NH ₄ ⁺ and placing the culture in darkness, reactivation occurred on reillumination and removal of NH ₄ ⁺ . The removal of NH ₄ ⁺ in darkness did not result in reactivation. On in vitro reactivation of glutamine synthetase from dark or NH ₄ ⁺ -treated cultures the maximum glutamine synthetase activity observed frequently exceeded that of glutamine synthetase extracted from untreated cultures. Anacystis nidulans showed a similar type of reversible dark deactivation to A. cylindrica but Plectonema boryanum and a Nostoc did not. With A. cylindrica, a direct positive correlation between the size of the intracellular pool of glutamate and biosynthetic glutamine synthetase activity occurred during light/dark shifts, and on treatment with NH ₄ ⁺ . The changes in activity of glutamine synthetase in A. cylindrica in response to light resemble in some respects the light modulation of enzymes of the oxidative and reductive pentose phosphate pathways noted in cyanobacteria by others.     

Biosynthesis of ribulose-1.5-bisphosphate carboxylase in greening cells of Euglena gracilis

Light induction of chloroplast development in Euglena leads to quantitative changes in the protein composition of the soluble cell part. One major part of these is the observed accumulation of ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBPCase) enzyme (EC 4.1.1.39). As measured by immunoelectrophoresis, a small amount of RuBPCase (about 10^-6 pmol) is present in a dark-grown cell, whereas a greening cell (72h) contains 10–20 pmol enzyme. Both the cytoplasmic and chloroplastic translation inhibitors, cycloheximide and spectinomycin, have a strong inhibitory effect on the synthesis of the enzyme throughout the greening process of Euglena cells. Electrophoretic and immunological analyses of the soluble phase prepared from etiolated or greening cells do not show the presence of free subunits of the enzyme. For each antibiotic-treated greening cell, the syntheses of both subunits are blocked. Our data indicate that tight reciprocal control between the syntheses of the two classes of subunits occurs in Euglena. In particular, the RuBPCase small subunit synthesis in greening Euglena seems more dependent on the protein synthesis activity of the chloroplast than the syntheses of other stromal proteins from cytoplasmic origin.Abbreviations LSU large subunit of ribulose-1.5-bisphosphate carboxylase - RuBP ribulose-1.5-bisphosphate - RuBP-Case ribulose-1.5-bisphosphate carboxylase - SSU small subunit of ribulose-1.5-bisphosphate carboxylase