Chloroplast Development In Euglena Research Articles

In-vitro transport of chloroplast proteins in a homologousEuglena system with particular reference to plastid leucyl-tRNA synthetase

In-vitro translations of total or polyadenylated RNA from chemoorganotrophic and photoautotrophicEuglena gracilis showed no substantial differences in the polypeptide patterns of the two cell types. By contrast, the corresponding patterns of in-vivo labelling indicated that posttranscriptional control of abundant cellular proteins occurred in illuminated cells. This type of control was confirmed forEuglena chloroplast proteins of cytoplasmic origin. The posttranslational transport of in-vitro-formed polypeptides into homologous chloroplasts allowed the plastid-targeted proteins to be recognized. Estimations of the amounts of in-vitro-translated polypeptides showed that the mRNA levels for nuclearencoded chloroplast proteins were almost constant throughout chloroplast development inEuglena. The import of the in-vitro-translation products into the chloroplasts was demonstrated (i) by kinetic determination of radioactivity increasing inside and decreasing outside the organelles, (ii) by autoradiographic analysis of the transported translation products among two-dimensionally separated chloroplast proteins, and (iii) by autoradiographic estimation of the decrease in labelled polypeptides in the incubation medium. After 60 min of incubation in the presence of chloroplasts, about 20% of the in-vitro-labelled translation products, corresponding to 30 protein spots, were found to be redistributed into the organelles. Low-abundance chloroplast leucyl-tRNA synthetase (LeuRS) was detected immunologically among the in-vitro translation products as a precursor protein (pre-LeuRS) of 112 kilodaltons (kDa), which was transported into chloroplasts where it was present in the form of the 105-kDa mature enzyme. Processing of pre-LeuRS also seemed to occur using wheat-germ translation system; however, the mature enzyme was not sequestered intoEuglena chloroplasts. Our results indicate that a nuclear-encoded chloroplastic aminoacyl-tRNA synthetase, the product of a low-abundance mRNA, is transported into and processed in chloroplasts in vitro.

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

Control of gene expression in Euglena was examined during light-induced chloroplast development. Greening was achieved under standard conditions which allowed the synthesis of all plastid proteins in both cytoplasmic and chloroplastic compartments, or under experimentally modified conditions inducing the preferential synthesis of the photosystem II (PSII) light-harvesting antenna or reaction centers. The relative composition of total mRNAs in cellular, cytoplasmic or chloroplastic fractions, as analyzed by their in-vitro translation products in cell-free systems did not significantly change during the in-vivo protein-synthesis processes which are specific to each greening system. By contrast, cytoplasmic polysomal mRNAs extracted during the selective recovery phase of PSII light-harvesting antennae provided a major in-vitro synthesis product of 28 kDa which could correspond to a precursor of the main 26-kDa apoprotein of the light-harvesting chlorophyll a/b protein complex. Similarly, the in-vivo selective synthesis of the 41-kDa and 51-kDa polypeptides of PSII reaction centers was concomitant with an enrichment of plastid polysomes in mRNA species coding for polypeptides of the same molecular weight. These observations confirm that protein synthesis during chloroplast development in Euglena is weakly regulated at the transcription level and they demonstrate that translational regulation occurs in both the cytoplasmic and the chloroplastic compartments.

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.

Sensitivity of Chloroplast Development in Euglena to Acridine Orange: Bleaching and Recovery from Damage

Treatment of light-grown Euglena cells with acridine orange (AO) at non-lethal concentrations resulted in the permanent loss of their ability to form chloroplasts. However, dark-grown cells were insensitive to AO. Starvation of the cells under light culture or the addition of chloramphenicol (CM) deformed the chloroplasts and made them less AO sensitive. Cells with mature chloroplasts seemed to show the most sensitivity to AO. Illumination with intense visible light of the AO-treated cells, whether they had been light- or dark-grown, made a greater proportion of them liable to be bleached or to be killed. The photosensitization was evident over the range of wavelengths of 450–500 nm, where the absorption maximum of AO is located. When light-grown cells, after AO treatment, were held in darkness in a nongrowth medium for 24 hr, the bleaching effect was reduced. However, if held under a light of 620–670 nm, where cells were not photosensitized but were able to form chloroplasts, the AO-damage was not reversed. The possibility of repair after AO-treatment is discussed.

Nutritional Regulation of Organelle Biogenesis in Euglena

Nitrogen deficiency and the presence of specific organic carbon sources prevent chloroplast development in Euglena. In exponentially growing cultures, chlorophyll levels were low and independent of the nitrogen content of the growth medium. Chlorophyll levels increased in stationary phase and the amount of chlorophyll formed was proportional to the initial nitrogen content of the growth medium; the greater the concentration of nitrogen, the greater the amount of chlorophyll synthesized during stationary phase. Washing experiments demonstrated that the major nutritional factor inhibiting chlorophyll synthesis in stationary phase cultures grown on medium containing a high carbon to nitrogen ratio was the absence of nitrogen rather than the presence of utilizable organic carbon.The light-induced synthesis of chlorophyll and of NADP-glyceraldehyde-3-phosphate dehydrogenase was inhibited when acetate or ethanol was added at the time of exposure of dark-grown resting cells to light. Malate addition, however, stimulated chlorophyll and enzyme synthesis. Both cell number and total cell protein increased after ethanol, acetate, or malate addition, indicating that the resting cells were not nitrogen-deficient. Ethanol and acetate specifically repress light-induced chlorophyll synthesis. NADP-glyceraldehyde-3-phosphate dehydrogenase synthesis was inhibited at a time, the first 24 hours of light exposure, when chlorophyll synthesis was unaffected by carbon addition.

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

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.

Studies on Chloroplast Development and Replication in Euglena

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.It is also shown that different bleached substrains exhibit substantial variation, both in total carotenoids and in Euglena alkaline DNase activity. These results are discussed in terms of the possibility that a cytoplasmic photoreceptor system is influencing the light-induced increase in Euglena alkaline DNase activity.

Insensitivity of development of photosynthetic competence to doses of ultraviolet light which block green colony formation in Euglena

The Do for inactivation of chloroplast inheritance in Euglena gracilis var. bacillaris is 5.6 ergs/mm2. In contrast, the Do for the inactivation of light-induced chloroplast development from the proplastid in non-dividing cells as measured by chlorophyll formation and appearance of photosynthetic carbon dioxide fixation is 200–300 ergs/mm2. This strongly suggests that DNA replication in Euglena chloroplasts is far more sensitive to ultraviolet inactivation than is DNA transcription. Carrying this model further, a useful comparison can be made between Euglena chloroplasts and certain strains of Escherichia coli. Since the Do for inactivation of viability in two strains of E. coli lacking dark repair is about the same as the Do for inactivation of chloroplast inheritance, it is suggested that the chloroplast lacks a dark repair system. Furthermore, since the bacterial sensitivity has been ascribed to a few inactivations in the DNA which prevent successful replication, this has been adopted to explain the blockage of plastid replication. Inhibition of transcription of the DNA into RNA in these bacterial strains, however, requires much higher doses since many cistrons must be inactivated and the dose required compares favorably with that required for inactivation of chloroplast development in Euglena. From the data presented in this paper, previous data on the Euglena system, and the model derived from a comparison with E. coli, the chloroplast can be viewed as a triploid procaryote lacking dark repair and possessing exceptional ultraviolet sensitivity of chloroplast inheritance compared with development which can be ascribed to the amounts of damage in DNA required to affect DNA replication or transcription.

Differential effects of actinomycin D on cell division and light-induced chloroplast development in Euglena

Light-induced chloroplast development in resting cells of Euglena gracilis var. bacillaris, as judged from measurements of the kinetics of chlorophyll formation, photosynthetic carbon dioxide fixation, and observations in the fluorescence microscope, is progressively inhibited by increasing concentrations of actinomycin D (AM D) with 80% inhibition occurring at about 45 μg/ml. The extent of inhibition finally achieved is also related to the length of time that the cells have been exposed to AM D. In contrast to the high concentration of AM D necessary to produce a significant inhibition of chloroplast development, cell division is extremely sensitive and is completely inhibited at concentrations of 7–10 μg/ml. At all concentrations of AM D employed, colony formation from treated cells was impaired to a large extent, but all colonies observed were green, indicating that AM D does not selectively block chloroplast replication. The effects of AM D, in general, were potentiated by carrying out chloroplast development and colony formation in red light, which presumably prevents photodestruction of the inhibitor. The extensive labeling of cellular RNA with 32PO 4 3− that normally accompanies light-light-induced chloroplast development is almost completely inhibited by 45 μg/ml of AM D, as is the minimal labeling of the RNA of the dark controls. The differential sensitivities of chloroplast development, chloroplast replication, and cell division to AM D is discussed with reference to its known effect as an inhibitor of DNA-dependent RNA synthesis.

A comparison of light-dependent RNA metabolism in wild-type Euglena with that of mutants impaired for chloroplast development

The possibility that (32)PO 4 (3-) ((32)Pi) labeling of both chloroplast and non-chloroplast RNAs during light-induced chloroplast development in Euglena is due, in part, to the break-down of existing RNAs and their resynthesis into labeled RNAs has been examined by comparing the RNA content of dark-grown, non-dividing cells after completion of light-induced chloroplast development with that of identical cells maintained in darkness for the same period of time. The involvement of the photo-conversion of protochlorophyll to chlorophyll and other photoreceptor systems in the labeling of RNA during chloroplast development has been considered by comparing the labeling pattern obtained with wild-type cells with the patterns obtained with mutants of Euglena which either lack detectable amounts of protochlorophyll and chlorophyll or form only rudimentary chloroplasts upon light induction.No significant difference in RNA content between dark-grown, non-dividing cells containing fully developed chloroplasts and the same cells maintained in darkness for the development period can be detected. This observation is interpreted to mean that in non-dividing cells precursors for chloroplast-associated RNAs are derived from pools and pre-existing RNAs, including non-chloroplast RNAs, and that the matebolic entrapment of (32)Pi involves a light-dependent turnover and DNA-directed RNA synthesis in wild-type cells.The RNA profiles on sucrose gradients of mutants of Euglena show no remarkable deviation from the profile established for wild-type cells. The labeling patterns obtained after 24 hours of incubation in light and in darkness differ from that obtained for wild-type cells in that all mutants show less of a light-minus-dark difference than wild-type and that mutants lacking plastid-associated DNA and detectable amounts of chlorophyll incorporate considerably more (32)Pi into RNA in darkness than wild-type. One such mutant shows no significant difference in its light-dark labeling pattern.These observations indicate that cells possessing normal proplastids capable of forming functional chloroplasts regulate metabolism of RNA in darkness in a different manner than with either rudimentary chloroplasts or containing no detectable plastids structures. The possible involvement of more than one photoreceptor system in metabolic control is discussed.

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.

Studies of Chloroplast Development in Euglena: XIV. Sequential Interactions of Ultraviolet Light and Photoreactivating Light in Green Colony Formation

Photoreactivation (PR) of green colony-forming ability in Euglena is pH-insensitive from pH 6.0 to 8.0 and temperature-sensitive with a maximum rate at 35 degrees C. There is no PR at 0 degrees C. The rate of PR varies with the growth stage of the cells; PR of exponential phase cells is slower than that of stationary phase cells. The reciprocity rule holds for PR over a 6-fold range of intensity. The shape of PR curves is a function of the UV dose; there appears to be a progressive increase in multiplicity until a limiting multiplicity is reached as indicated by the fact that curves for high doses are superposable. Dark-grown and light-grown cells give the same PR response for comparable UV doses. UV inactivation of cells which have been treated with UV and then with PR light shows that, if the PR dose is sufficiently large, the same UV-inactivation curve is obtained as for nonpretreated control cells. Doses of PR lower than the saturating dose produce UV-inactivation curves, the ultimate slopes of which are parallel to the slope of the control curve, but which show reduced multiplicity. The multiplicity of these curves increases with increasing PR dose. The UV inactivation of green colony-forming ability in Euglena is completely photoreactivable at the doses studied, in contrast with the UV inactivation of colony-forming ability, which occurs at considerably higher UV doses and behaves like most other photoreactivable systems, showing a photoreactivable sector of 0.32.

Studies of chloroplast development in Euglena. 9. Chloroplast antigens and their appearance during chloroplast development.

SYNOPSIS. Antibodies to purified chloroplast preparations from Euglena gracilis var. bacillaris were prepared in rabbits. Success was achieved only after removal of lipids by forming acetone powders af chloroplasts. Ouchterlony agar‐diffusion procedures showed that light‐grown cells (containing chloroplasts) have at least 3 antigens reacting with these antibodies and not detectable in the dark‐grown cells (which contained proplastids but no chloroplasts), The antigens uniquely associated with the chloroplast appear sequentially during the development of the proplastid into the chloroplast on light induction of the dark‐grown cells. The first of these antigens appears at about 10 hr of development. Mutants of the parent strain showed varying numbers of antigens which were cor‐related with their capacities to form plastid structures. Cells developing abnormal chloroplasts at low light intensities did not reveal any antigenic differences from cells developing normal chloroplasts. Various antigenic bands were also found which were common to all cell types studied. These can be ascribed either to non‐chloroplast contaminants in the original preparation used to immunize the rabbits or to antigens which, although not in the chloroplasts, are cross‐reacting with chloroplast materials. In the case of the dark‐grown cells, it is likely that portions of the proplastids are cross‐reacting with chloroplast materials.

Studies of chloroplast development in Euglena: XII. Two types of satellite DNA

Three species of DNA from wild-type cells of Euglena gracilis var. bacillaris are described. Main band DNA (density = 1·707 g/cm3) most probably is assignable to the nucleus and is found in all strains examined. The satellite region (density = 1·688 g/cm3) previously described ( Leff, Mandel, Epstein & Schiff, 1963 ) has now been resolved into two DNA satellites, Sc (density = 1·686 g/cm3) and Sx (density = 1·691 g/cm3), both of which are present in wild-type cells, Sc is found in enriched quantities in the chloroplast fraction of green cells and is found in light-grown and dark-grown wild-type cells and in all mutants capable of forming at least a partial chloroplast, but is absent from all mutants incapable of plastid formation. Conversion of strains capable of chloroplast formation to strains incapable of this by means of ultraviolet treatment brings about a loss of Sc. This is consistent with ScDNA being a constituent of the chloroplasts. Sx is found in wild-type cells and in all mutants examined, whether capable or incapable of forming chloroplasts. On cell fractionation, Sx is absent from the chloroplast fraction and is found in the small-particle fraction of the cells along with mitochondrial cytochromes. From density calculations, main band DNA contains 48% guanine+cytosine : 52% adenine+thymine; Sx contains 31% guanine+cytosine: 69% adenine+thymine; and Sc contains 26% guanine+cytosine : 74% adenine+thymine. All three DNA species appear to be double-stranded.

Studies of Chloroplast Development in Euglena. VI. Light Intensity as a Controlling Factor in Development

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Studies of Chloroplast Development in Euglena. V. Pigment Biosynthesis, Photosynthetic Oxygen Evolution and Carbon Dioxide Fixation during Chloroplast Development

Our studies with Euglena (2, 6, 14, 19, 20) have suggested that dark-grown cells contain approximately 30 proplastids which develop into about 10 chloroplasts when the cells are exposed to light. The existence of these proplastids, originally inferred from studies on ultraviolet inactivation and photoreactivation of chloroplast formation (14,19), was confirmed by fluorescence and electron microscopy (2, 6). Other workers have studied some of the initial steps involved when dark-grown cells are exposed to light, such as the conversion of protochlorophyll to chlorophyll (17). Other studies also exist of the related problem of the physiology of chloroplast development in higher plants (3, 7, 23, 27). Our earlier work (20) also showed that chloroplast development in Euglena could be separated experimentally into 2 phases: replication of the system which manufactures the chloroplast and development of the proplastid into the mature chlor6plast. In this study we correlate the onset and kinetics of pigment formation, 02 evolution and CO2 fixation with the development of the proplastid into the mature chloroplast. Materials and Methods

Studies of Chloroplast Development in Euglena. 4. Electron and Fluorescence Microscopy of the Proplastid and its Development into a Mature Chloroplast.*

Electron and fluorescence microscopy reveal a proplastid about 1 μ in diameter in dark‐grown Euglena gracilis quite like those in higher plants. After the cells are placed in the light these structures lengthen, probably in part by coalescence of several proplastids. Full size is reached in some cells after about one day in light. Lamellae, growing as blebs off the inner proplastid membrane, develop in a roughly linear fashion with time, art average of one new lamella appearing after each 6 hours in the light. The maturation of the proplastid thus appears to differ appreciably from that in higher plants since the latter presumably create several lamellae simultaneously, and these appear able to replicate themselves in a geometrically parallel fashion. The question is raised whether the apparent differences in development may not be due to lesser discrimination in observing the sequence in the higher plants, for there are no observations necessarily inconsistent with the sequence found in Euglena.

Studies of chloroplast development in Euglena I. Inactivation of green colony formation by U.V. light

Irradiation of Euglena gracilis with non-lethal doses of u.v. light prevents the formation of chloroplasts in the progeny. This inactivation is photoreactivable with efficiencies approaching 100%. The albino cells produced through irradiation form colorless clones which never revert to green, indicating that this is a heritable effect. The action spectrum for u.v. inactivation of chloroplast formation shows peaks at 260 and 280 mμ.implicating nucleoprotein as the probable site of u.v. action. Evidence obtained from the inactivation kinetics of dark-grown and light-grown cells, the presence of sectored colonies after u.v., the lack of lethality, and the multiplicity of inactivation sites suggest to us that the u.v. sensitive site is cytoplasmic. The evidence leads us, then, to the hypothesis that there are self-reproducing cytoplasmic entities in Euglena which contain nucleoprotein and which are responsible for chloroplast formation.

Studies of chloroplast development in Euglena II. Photoreversal of the U.V. inhibition of green colony formation

The inhibition of chloroplast formation in u.v. irradiated progeny of Euglena gracilis can be reversed by visible light with efficiencies approaching 100%. If the cells are permitted to divide after u.v. treatment, photoreactivability is rapidly lost. The kinetics of this loss are fairly consistent with the hypothesis that entities responsible for chloroplast formation are prevented from being replicated by u.v. light and are diluted out among the progeny. There is no decay of photoreactivability in non-dividing cells. The action spectrum for photoreactivation of chloroplast formation shows a single broad peak in the near-u.v. region of the spectrum clearly separating this phenomenon from the light induction of chloroplast formation which has effectiveness peaks in the blue and red regions of the spectrum. The evidence presented is consistent with the hypothesis of cytoplasmic entities which are replicated at cell division and which control chloroplast formation.