Electron microscope studies on the morphogenesis of plastids

Streptomycin sulphate (2 mg/ml) did not affect the formation of proplastids or the elaboration of prolamellar bodies. The plastids of the streptomycin (SM)-treated cotyledons contained both crystalline prolamellar bodies and ribosomes, and were undistinguishable from the plastids of the water-grown cotyledon. However, plastids from dark-grown SM-treated cotyledons were no longer able to differentiate to more advanced stages of development, even after exposure to light. The plastids of light and dark-grown SM-treated cotyledons often contained prolamellar bodies and abnormal giant grana. Variegation developed in the cotyledons germinated in Hoagland's solution plus SM. The plastids in pale green tissue contained stroma-lamellae and one or two giant grana, whereas in those of pale yellow tissue, many osmiophilic globules, large vacuoles and crystal bodies were observed. It is suggested that the formation of prolamellar bodies may depend on cytoplasmic protein synthesis whereas functional stroma- and grana-lamellae may depend on protein synthesis within the plastids. The inhibitory effects of SM on protein synthesis were used as a tool to test this hypothesis. This work was carried out in the Department of Botany, University of California, Davis, by Grant-GB-11906 from National Science Foundation of U.S.A.     

Functional and Structural Organization of Chlorophyll in the Developing Photosynthetic Membranes of Euglena gracilis Z: II. CHARACTERISTICS OF THE LATE FORMATION OF ACTIVE PHOTOSYSTEM II REACTION CENTERS DURING FIRST STAGES OF GREENING

During light-induced greening of dark-grown, nondividing Euglena gracilis Z, there is a delay of about 10 hours in the formation of active photosystem II (PSII) reaction centers compared to chlorophyll synthesis. Experiments with greening under different light intensities rule out the possibility that this delay results from a late induction of active PSII reaction center formation when a definite amount of chlorophyll is attained in the early greened cells. Experiments on greening after preillumination show that this delay does not originate in a long, light-induced formation of specific synthesizing machinery for reaction center components. Experiments with greening in the presence of streptomycin show that, when this inhibitor of protein synthesis by chloroplastic ribosomes is added to dark-grown, preilluminated cells or to cells already greened for 24 hours, the formation of active PSII reaction centers is inhibited after a time which depends on the light intensity used for greening. Under very low light intensity (150 lux), the addition of streptomycin to 24-hour greened cells does not prevent further development of functional chloroplasts. These observations lead to the conclusion that streptomycin-insensitive chloro-plast development occurs due to syntheses of cytoplasmic origin and of light-induced pools of components synthesized early by chloroplastic ribo-somes. Conformational changes requiring time may allow the insertion of components necessary for the reorganization of PSII reaction centers in the developing thylakoid after synthesis. This hypothesis accounts for the observed delay in PSII reaction center formation compared to chlorophyll synthesis.     

Comparaison, à la lumière et à l'obscurité, des synthèses spécifiques d'ARN chez Euglena gracilis Z en cultures synchrones sur milieu lactate et étude du Chondriome

Summary Synchronization of Euglena gracilis (Z) on lactate medium is shown to be independent of illumination. The existence of a mitochondrial cycle in lightgrown as well as in dark-grown Euglena is demonstrated. When RNA synthesis is studied by pulse labeling with tritiated uracil in synchronously growing cells, a discontinuous RNA synthesis is found. Two peaks of preferential RNA synthesis in dark-grown cells and three peaks in light-grown cells are seen; the significance of the third peak of RNA synthesis in light-grown Euglena is discussed.     

Light-triggered organization of the stigma in dark-grown nondividing cells of Euglena gracilis

Dark-grown cells of Euglena gracilis Klebs var. bacillaris Cori contain amorphous stigma material. When these cells are placed on resting medium for 3 days in darkness, the cells cease division; the organization of a normal stigma from the amorphous material requires continuous illumination for 72-96 hr. We have now found that if dark-grown cells are placed on resting medium for 8 days, a 40-min light pulse is sufficient to cause normal organization of the stigma in a subsequent 72-hr dark period. Thus stigma development is light-dependent at 3 days of resting but becomes light-triggered at 8 days. Other examples of light-triggered phenomena in Euglena are discussed and a model based on turnover of protein molecules repressing development that are ordinarily removed by exposure to light is presented; it is suggested that as the cells become more starved their ability to replace repressor molecules removed by light becomes limited and the system thereby becomes light-triggered rather than light-dependent.     

Antibiotics and apochlorosis

Hydroxylamine, an inhibitor of deoxyribonucleic acids (DNA), ribonucleic acids (RNA) and proteosynthesis interferes with the bleaching effect of streptomycin on growing cells ofEuglena gracilis. The addition of hydroxylamine to a green autotrophic culture ofEuglena gracilis inhibits, depigmentation of the culture by streptomycin. Otherwise, streptomycin alone, without, hydroxylamine, is a powerful bleaching agent and when added to a growing culture ofEuglena gracilis, transforms the green, autotrophic cells to permanently colourless, heterotrophic cells. Phenethyl alcohol, an inhibitor of RNA synthesis, and chloramphenicol, an inhibitor of proteosynthesis, do not block the bleaching effect of streptomycin. It can be concluded from these results that the bleaching effect of streptomycin is related to its interference in the plastid DNA.     

Light Regulation of delta-Aminolevulinic Acid Biosynthetic Enzymes and tRNA in Euglena gracilis

Chlorophyll synthesis in Euglena, as in higher plants, occurs only in the light. The key chlorophyll precursor, δ-aminolevulinic acid (ALA), is formed in Euglena, as in plants, from glutamate in a reaction sequence catalyzed by three enzymes and requiring tRNA^Glu. ALA formation from glutamate occurs in extracts of light-grown Euglena cells, but activity is very low in dark-grown cell extracts. Cells grown in either red (650-700 nanometers) or blue (400-480 nanometers) light yielded in vitro activity, but neither red nor blue light alone induced activity as high as that induced by white light or red and blue light together, at equal total fluence rates. Levels of the individual enzymes and the required tRNA were measured in cell extracts of light- and dark-grown cells. tRNA capable of being charged with glutamate was approximately equally abundant in extracts of light- and dark-grown cells. tRNA capable of supporting ALA synthesis was approximately three times more abundant in extracts of light-grown cells than in dark-grown cell extracts. Total glutamyl-tRNA synthetase activity was nearly twice as high in extracts of light-grown cells as in dark-grown cell extracts. However, extracts of both light- and dark-grown cells were able to charge tRNA^Glu isolated from light-grown cells to form glutamyl-tRNA that could function as substrate for ALA synthesis. Glutamyl-tRNA reductase, which catalyzes pyridine nucleotide-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde (GSA), was approximately fourfold greater in extracts of light-grown cells than in dark-grown cell extracts. GSA aminotransferase activity was detectable only in extracts of light-grown cells. These results indicate that both the tRNA and enzymes required for ALA synthesis from glutamate are regulated by light in Euglena. The results further suggest that ALA formation from glutamate in dark-grown Euglena cells may be limited by the absence of GSA aminotransferase activity.     

Monogalactosyl and digalactosyl diglycerides from heterotrophic, hetero-autotrophic, and photobiotic Euglena gracilis

The lipid of Euglena gracilis, dark-grown in a complete medium, contained 2% galactose. The lipid of Euglena gracilis, light-grown in either a complete or an inorganic medium, contained 13-14% galactose. Pure monogalactosyl and digalactosyl diglyceride fractions, isolated by column plus thin-layer chromatography, contained 50% of the lipid-bound galactose of dark-grown cells, and 80% of that of light-grown cells. Molar ratios of monogalactosyl to digalactosyl compounds ranged from 2 to 3. The results show that galactosyl diglycerides, stored in large amount in light-grown cells, persist in small amount in the dark-grown cells. Fatty acids in both the monogalactosyl and the digalactosyl diglycerides were mainly of the 16- and 18-carbon varieties, with high proportions of trienes. The monogalactosyl diglycerides were rich in hexadecatetraenoic acid. Strictly photobiotic cells had twice as much hexadecadienoic and hexadecatetraenoic acids in their monogalactosyl diglycerides, and three times as much hexadecadienoic and octadecadienoic acids in their digalactosyl diglycerides as did illuminated cells grown in a complete medium. Dark-grown (obligate) heterotrophs contained galactosyl diglycerides with high percentages of monoenes. Great compositional variations in the galactosyl diglycerides are thus induced by light and also by nonlipid exogenous metabolites.     

Effects of streptomycin on plastids in dividing Euglena

Summary The plastids of dividing Euglena cells growing in the light in the presence of streptomycin decreased in length after a lag period of seven generations. The typical structure of the chloroplast was lost after a similar lag period. This loss of structure did not follow a regular pattern. After 11 generations the plastids resembled normal proplastids of dark-grown cells. Initial chlorophyll loss of treated cells was slow, but after 3 generations the rate of loss was about 0.5/generation, indicating a cessation of synthesis and a dilution among the progeny.     

Biosynthesis of Phosphatidylcholine in Euglena gracilis*†

SYNOPSIS. Extracts of Euglena gracilis carry out a very rapid but limited synthesis of phosphatidylcholine when S-adenosylmethionine or ATP and methionine are supplied. Cytidinediphosphocholine apparently is not utilized. Qualitatively the same results are obtained whether the cells are light- or dark-grown.     

A POSSIBLE MECHANISM FOR THE MORPHOGENESIS OF LAMELLAR SYSTEMS IN PLANT CELLS

A mechanism for the formation of lamellar systems in the plant cell has been proposed as a result of electron microscope observations of young and mature cells of Nitella cristata and the plastids of Zea mays in normal plants, developing plants, and certain mutant types. The results are compatible with the concept that lamellar structures arise by the fusion or coalescence of small vesicular elements, giving rise initially to closed double membrane Structures (cisternae). In the chloroplasts of Zea, the cisternae subsequently undergo structural transformations to give rise to a compound layer structure already described for the individual chloroplast lamellae. During normal development, the minute vesicles in the young chloroplast are aggregated into one or more dense granular bodies (prolamellar bodies) which often appear crystalline. Lamellae grow out from these bodies. In fully etiolated leaves lamellae are absent and the prolamellar bodies become quite large, presumably because of inhibition of the fusion step which appears to require chlorophyll. Lamellae develop rapidly on exposure of the plant to light, and subsequent development closely parallels that seen under normal conditions. The plastids of white and very pale green mutants of Zea similarly lack lamellae and contain only vesicular elements. A specialized peripheral zone immediately below the double limiting membrane in Zea chloroplasts appears to be responsible for the production of vesicles. These may be immediately converted to lamellae under normal conditions, but accumulate to form a prolamellar body if lamellar formation is prevented, as in the case of etiolation and chlorophyll-deficient mutation, or when the rate of lamellar formation is slower than that of the production of precursor material (as appears to be the case in the early stages of normal development).     

An investigation of the mechanism of activation of tryptophan by tryptophanyl-tRNA synthetase from beef pancreas

Exposure of dark-grown Euglena to white or red light, but not blue light, produced a twofold increase in the specific activity of citrate synthase. A 400-fold purification of mitochondrial citrate synthase (subunit Mr = 44000) was achieved from cells of Euglena gracilis by affinity chromatography on ATP-activated agarose. Antisera, raised against the homogeneously pure enzyme, were used to demonstrate that the increase in citrate synthase activity on exposure of dark-grown cells to light resulted from an increase in citrate synthase protein. Anti-(citrate synthase) was used to detect precursor citrate synthase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. Citrate synthase mRNA was found to be present in cells at all stages of regreening. However, extraction and translation of polyadenylated RNA from free polysomes isolated from darkgrown and regreening cells demonstrated that appreciable translation of citrate synthase mRNA was only occurring in regreening cells.     

Carotenoid Production by Streptomycin-Bleached Euglena

SYNOPSIS. Streptomycin-bleached Euglena gracilis , strain Z, was cultivated under conditions which yielded good growth rates and high cell densities. Dividing cells produced only small amounts of carotenoid. After the cessation of cell division the carotenoid content of the cells increased rapidly. During the major period of carotenoid synthesis the cell number remained unchanged but the packed cell volume decreased. Some similar observations on carotenoid production by normal dark-grown Euglena are noted.     

Events surrounding the early development of Euglena chloroplasts 13. Photocontrol of protein synthesis

Protein synthesis measured as leucine incorporation was followedduring the early hours of light exposure of dark-grown cellsof wild type cells of Euglena gratilis var. bacillaris and ofbleached mutants W₃BUL and W₁₀SmL which lack detectable plastidDNA. In all strains, linear rates of leucine incorporation wereobserved in dark-grown resting cells and on exposure to light,this rate increased. After about 3 hr light exposure in wildtype cells and somewhat later in the mutants, the rate of proteinsynthesis sharply declined below that of the dark-grown anddark-incubated cells. Experiments in wild type cells showedthat leucine uptake was not rate limiting for protein synthesisalthough light exposure decreased the rate of uptake. The changesin rate found during continuous labeling of wild type cellswere verified by pulse-labeling experiments in continuous light.Exposure of dark-grown wild type cells to a two hour pulse oflight produced a transient increase in the rate of leucine incorporationwhich subsequently returned in darkness to the level of thedark-grown cells which received no light; thus the changes inrate of leucine incorporation are light-dependent. Since theeffects of light on leucine incorporation can be reproducedin mutants lacking detectable plastid DNA, the photoreceptormachinery involved cannot be coded in plastid DNA, and probablyoriginates in nuclear DNA. The role of light in programmingprotein synthesis and turnover in early chloroplast developmentis discussed. ¹Supported by Grant Number GM-14595 from the National Institutesof Health. ²Microbiology trainee of the National Institutes of Health,Grant Number GM1586. Portions of the material in this paperwere taken from a dissertation submitted by S. D. S. to theGraduate Faculty of Brandeis University in partial fulfillmentof the requirements for the Ph.D. degree. Present address: Schoolof Life Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska68588, U. S. A. ³Abraham and Etta Goodman Professor of Biology and Director,Institute for Photobiology of Cells and Organdies, BrandeisUniversity, Waltham, MA, U. S. A. 02154, to whom reprint requestsshould be sent. (Received February 8, 1979; )     

ACID PHOSPHATASE ACTIVITY AND ULTRASTRUCTURE OF AGED CELLS OF EUGLENA GRANULATA1,2

Ultrastructure and acid phosphatase activity of aged calls of Euglena granulata are reported. Cells are spherical, quiescent, and nonflagellated. The most conspicuous attribute of aged cells is the accumulation of cyloplasmic vacuoles and lysosome-like structures containing heavily stained, pigmented bodies and membrane fragments. In chloroplasts, portions of whorled lamellae arc abscised and subsequently incorporated into lysosome-like structures; osmiophilic granules increase in number. Membranes surrounding eyespot granules disappear and the granules themselves become diffuse; the usual association with microtubules is not seen in aged cells. Acid phosphatase precipitation accumulates largely at the maturing face of dictyosomes and associated vesicles; there is also activity in multivesicular and lysosome-like vacuoles.     

LIGHT AND ELECTRON MICROSCOPIC CHARACTERIZATION OF TWO TYPES OF PYRENOIDS IN GONIUM (GONIACEAE,CHLOROPHYTA)1

The single, basal pyrenoids of Gonium quadratum Pringsheim ex Nozaki and G. pectorale Müller (Goniaceae, Chlorophyta) differed in appearance when vegetative colonies were cultured photoheterotrophically in medium containing sodium acetate. Chloroplasts of G. quadratum had distinct pyrenoids when grown in medium without major carbon compounds. However, the pyrenoids degenerated and were markedly reduced in size when such cells were inoculated into a medium containing 400 mg·L^?1 of sodium acetate. No pyrenoids were visible under the light microscope; however, with electron microscopy small pyrenoids and electron-dense bodies were visible within the degenerating chloroplasts, which had only single layers of thylakoid lamellae at the periphery. The chloroplasts subsequently developed distinct pyrenoids and several layers of thylakoid lamellae as the culture aged. In contrast, vegetative cells of G. pectorale always showed distinct pyrenoids when cells were inoculated into medium containing sodium acetate, sodium pyruvic acid, sodium lactate, and/or yeast extract. Therefore, we propose two terms, “unstable pyrenoids” and “stable pyrenoids,” for pyrenoids of G. quadratum and G. pectorale, respectively. Chloroplasts of the colonial green flagellates should thus be examined under various culture conditions in order to determine whether their pyrenoids are unstable or stable when pyrenoids are used as taxonomic indicators. Immunogold electron microscopy showed that the ratios of gold particle density of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) between pyrenoid matrix and chloroplast stroma in G. quadratum grown in medium with or without sodium acetate were lower than those of G. pectorale. Heavy labeling by anti-RuBisCO was observed in both the electron-dense bodies and pyrenoid matrix of G. quadratum. This is the first electron microscopic demonstration of degeneration and development of both pyrenoids and thylakoid lamellae in the chloroplast as a function of culture condition in green algae.     

Amminoacidi Liberi in Euglena

Abstract

Free amino acids in EUGLENA. — The free amino acids present in Euglena gracilis strain Z, cultivated in the light and the dark, as well as in the strain bleached by streptomycin, have been identified by the conventional paper chromatographic method.

Variations have been observed when the photosynthetic apparatus is lost. Generally in the photosynthetic Euglena cells the pool of aminoacids reminds that of green algae, whereas bleached cells are similar to protozoa. It is possible that these variations are linked to the metabolic patways of green and bleached forms.     

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.     

THE EFFECT OF 3-AMINO-1,2,4-TRIAZOLE ON THE ULTRASTRUCTURE OF PLASTIDS OF TRITICUM VULGARE SEEDLINGS

The application of sublethal doses of 3-amino-1,2,4-triazole (AT) to germinating, light-grown wheat grains causes chlorosis of the resulting leaves. An ultrastructural examination of the leaf tissue reveals that the plastids lack normal grana-fret membrane systems and chloroplast ribosomes. A few disorganized membranes are always present in these chloroplasts. However, AT-treated, dark-grown seedlings contain proplastids with non-crystalline prolamellar bodies and ribosomes. When these etiolated, treated plants are exposed to 600 ft-c light for various periods of time, the proplastids fail to develop into normal, grana-containing chloroplasts.     

Formation of tyrosine O-sulfate by mitochondria and chloroplasts of Euglena

Mitochondria that have been purified from cells of light-grown wild-type Euglena gracilis Klebs var. bacillaris Cori or dark-grown mutant W10BSmL and incubated with 35SO4(2-) and ATP accumulate a labeled compound in the surrounding medium. This compound is also labeled when mitochondria are incubated with [14C]tyrosine and nonradioactive sulfate under the same conditions. This compound shows exact coelectrophoresis with synthetic tyrosine O-sulfate at pH 2.0, 5.8, and 8.0, and yields sulfate and tyrosine on acid hydrolysis. Treatment with aryl sulfatase from Aerobacter aerogenes yields sulfate and tyrosine but no tyrosine methyl ester; no hydrolysis of tyrosine methyl ester to tyrosine is observed under identical conditions, ruling out methyl esterase activity in the aryl sulfatase preparation. Thus the compound is identified as tyrosine O-sulfate. No tyrosine O-sulfate is found outside purified developing chloroplasts of Euglena incubated with 35SO4(2-) and ATP, but both chloroplasts and mitochondria accumulate labeled tyrosine-O-sulfate externally when incubated with adenosine 3'-phosphate 5'-phospho[35S]-sulfate (PAP35S). Since tyrosine does not need to be added, it must be provided from endogenous sources. Labeled tyrosine O-sulfate is found in the free pools of light-grown Euglena cells grown on 35SO4(2-) or in dark-grown cells incubated with 35SO4(2-) in light, but none is found in the medium after cell growth. No labeled tyrosine O-sulfate is found in Euglena proteins (including those in extracellular mucus) after growth or incubation of cells with 35SO4(2-) or after incubation of organelles with 35SO4(2-) and ATP or PAP35S, ruling out sulfation of the tyrosine in protein or incorporation of free-pool tyrosine O-sulfate into protein. The system forming tyrosine O-sulfate is membrane-bound and may be involved in transporting tyrosine out of the organelles.     

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