Light-independent and dependent phases of proplastid development in Euglena gracilis W3BUL

Cells of Euglena gracilis Klebs var. bacillaris Cori mutant W3BUL grown in darkness on Hutner's pH 3.5 medium without agitation accumulate wax ester. These cells have undeveloped proplastid remnants characteristic of this mutant. If these cells are transferred to an inorganic medium and bubbled with 2-3% CO2 in air, the wax disappears and the proplastid expands and develops in darkness to form prolamellar bodies and membrane vessicles within 96 h. No further development takes place in darkness, but if these cultures are illuminated at 96 h formation of prothylakoids is observed. Thus the wax ester accumulated during growth can be used subsequently to support proplastid development up to the prolamellar body stage, but the formation of prothylakoids is strictly light-dependent. Development in this system takes place at a slower rate than in cells grown with shaking and lacking wax which are transferred to resting medium. As previously shown, all of proplastid development requires light under these conditions. It is suggested that the oxygen-requiring utilization of wax in darkness can provide energy and metabolites for a part of proplastid development but the later steps in these cells, or the entire development in cells lacking wax is supported by paramylum degradation which is strictly light-dependent. However, a specific light reaction required for prothylakoid organization is not ruled out.     

Regulation of Protochlorophyll(ide) Levels in Dark-Grown Non-Dividing Euglena 3. Proplastid Structure during Loss and Regeneration of Protochlorophyll(ide)

Cells of Euglena gracilis Klebs var. bacillaris Cori growingin darkness on a complete medium have small undifferentiatedproplastids. On transfer to an incomplete (resting) medium indarkness, the cells cease division within 72 h. During thistime the proplastid expands and several prothylakoids and prolamellarbodies develop even though phototransformable protochlorophyll(ide)[PT-Pchl(ide)] is decreasing. As PT-Pchl(ide) decreases furtherand reaches a stable plateau after 4–5 more days in darkness,the proplastid structure becomes highly reduced. Forty minutesof light plus a one h dark period, or addition of glutamateor malate for 7 h does not change the proplastid structure significantlyeven though PT-Pchl(ide) returns to the level found in growingcells. Upon prolonged incubation in darkness after light treatment(72 h) an expanded proplastid containing prothylakoids, prolamellarbodies and membrane whorls with mitochondria in close associationis seen; most of the cellular paramylum is lost during thisperiod leaving cavities in the cytoplasm. Without light, prolongedincubation in darkness (72 h) with malate leads to accumulationof cellular paramylum but no change in proplastid structurewhile prolonged treatment with glutamate (72 h) allows the formationof a few prothylakoids but no prolamellar bodies. ¹Supported by Grants GM 14595 from the National Institutes ofHealth. ²Permanent address: Department of Microbiology, Tokyo MedicalCollege, 6-1-1 Shinjuku, Tokyo 160, Japan. ³Abraham and Etta Goodman Professor of Biology. (Received July 23, 1983; Accepted September 22, 1983)     

Kinetics of Accumulation of Ribulose-1,5-bisphosphate Carboxylase during Greening in Euglena gracilis: Nutritional Regulation

The accumulation of ribulose-1,5-bisphosphate carboxylase (RuBPCase) in resting Euglena gracilis strain Z during greening is photo-regulated (Freyssinet, Eichholz, Buetow 1984 Plant Physiol 75: 850-857. Greening resting cells are not photosynthetically competent for about the first 24 hours in the light. Therefore, substrates for a net synthesis of the enzyme must come from endogenous constituents. During this time, degradation of endogenous paramylum (carbohydrate) reserves provides the main source of substrates. By about 24 hours of greening, resting cells are photosynthetically competent and RuBPCase accumulation becomes highly sensitive to 3-(3,4 dichlorophenyl)-1,1-dimethylurea. Therefore, from about 24 hours of greening onward, substrates (and/or energy) for RuBPCase synthesis are provided by photosynthesis. Ethanol, a nutritional substrate ordinarily used constitutively by Euglena for growth, inhibits RuBPCase accumulation when added to the resting medium in the light. The alcohol exerts this negative regulatory effect by limiting the availability of substrates needed for a net synthesis of the enzyme.     

Behavioral mutants of Euglena gracilis: functional and spectroscopic characterization

Three mutant strains of the phytoflagellate Euglena gracilis Z have been characterized in order to analyze the signal perception and signal transduction pathways involved in photo- and gravitaxis. Using the fluorescence of the chromophoric groups believed to be involved in photoperception (flavins and pterins) a method was developed for an in situ and in vivo detection of the paraxonemal body, the proposed location of the photoreceptor molecules. Two of the mutant strains, 1224-5/9f and 1224-5/1f, do not possess a stigma and also lack a paraxonemal body, as indicated by fluorescence measurements. The third strain, FB, has a small stigma, but only some cells contain a paraxonemal body. In contrast to the present hypothesis on photoorientation of Euglena, all strains were able to orient with respect to the light direction. However, the mutant strains did not show any orientation at low irradiances. At medium and high irradiances the strains 1224-5/9f and 1224-5/1f oriented perpendicular to the light direction (diaphototaxis) while cells of strains of FB showed partly negative phototaxis and partly diaphototaxis. Diaphototaxis was never observed in the wild type strain. Strains 1224-5/9f and 1224-5/1f showed normal graviresponses compared with the wild type. Astasia longa, a nonphtototactic relative of E. gracilis, as well as strain FB were both negative and positive gravitactic at all culture ages tested. This result confirmed the hypothesis that the paraxonemal body is not directly involved in graviperception.     

EFFECTS OF DARKNESS AND OF STREPTOMYCIN ON THE FINE STRUCTURE OF EUGLENA GRACILIS

Siegesmund , Kenneth A., Walter G. Rosen , and Stanley R. Gawlik . (Marquette (J., Milwaukee, Wis.) Effects of darkness and of streptomycin on the fine structure of Euglena gracilis. Amer. Jour. Bot. 49 (2) : 137–145. Illus. 1962.—Dark-grown Euglena gracilis cells, transferred from streptomycin (SM)-containing medium to SM-free medium 5 days before transfer to light, turn green normally, indicating that proplastids are unaffected by SM. SM-bleached cells, grown in light, contain numerous bodies composed of concentric lamellae (CL bodies). These differ from chloroplasts in that their lamellae lack electron-dense dots, are not coalesced, and they lack a 3-layered limiting membrane and pyrenoids. CL bodies are absent from dark-grown normal and dark-grown SM-bleached cells, as well as from light-grown normal cells. It is suggested that CL bodies result from a derangement of chloroplast synthesis caused by SM blockage of chlorophyll synthesis.     

Light-Induced Synthesis of Anthocyanin in Carrot Cells in Suspension: I. THE FACTORS AFFECTING ANTHOCYANIN PRODUCTION

Takeda, J. 1988. Light-induced synthesis of anthocyanin in carrotcells in suspension. I. The factors affecting anthocyanin production.—J.exp. Bot. 39: 1065–1077. A light-triggered anthocyanin-synthesizing system was establishedfor carrot cells in suspension. A few days after transfer ofthe cells to a 2,4-dichlorophenoxyacetic acid (2,4-D)-free mediumin the dark, light irradiation triggered anthocyanin synthesisand concomitantly stopped expansion growth. Over 90% of thecells synthesized anthocyanin without cell division. By loweringthe concentration of phosphate or both nitrogen and phosphateand delaying the time of onset of irradiation, the productionof anthocyanin per cell increased to a maximum level of 0–8µmol anthocyanin per 10⁶ cells. A change in the physiologicalstate of cells (light-insensitive to light-sensitive state)induced by the transfer to 2,4-D-free medium is suggested tobe a prerequisite for the light-triggered synthesis of anthocyanin. Key words: Anthocyanin production, cultured cells, Daucus carota, light-triggered, 2,4-D     

Synthetic abilities of Euglena chloroplasts in darkness

Protein synthesis, normally a light-dependent process in isolated mature chloroplasts of Euglena gracilis var. bacillaris will take place in darkness if ATP and Mg2+ (ATP/Mg) are supplied. Either 5 or 10 mM ATP plus 15 mM MgCl2 are optimal and rates equal to those in the light can be obtained. Since ATP and Mg2+ are not stoichiometrically related, and since the optimal Mg2+ concentration is similar to that which stabilizes chloroplast ribosomes in vitro, it is suggested that the chloroplast is freely permeable to Mg2+ under these conditions. Protein synthesis under these conditions is not inhibited appreciably by DCMU, FCCP, cycloheximide, or by the addition of ribonuclease, but is highly sensitive to chloramphenicol. Carbon dioxide fixation is also a light-dependent process in isolated mature chloroplasts from Euglena, but addition of ATP (5 mM) and fructose bisphosphate (5 mM) plus aldolase (1.0 unit/ml) (fructose-1,6-bisphosphate/aldolase) yields CO2 fixation rates in darkness that are 43% of those normally obtained in the light. Mg2+ higher than 1.0 mM (e.g., 16 mM) is somewhat inhibitory. Chlorophyll synthesis from 5-aminolevulinate in 36 h developing chloroplasts from Euglena is also light-dependent, but addition of ATP/Mg and fructose-1,6-bis-phosphate/aldolase in darkness brings about the accumulation of a compound having the same RF on chromatography as protochlorophyllide from Barley; a subsequent brief illumination of the chloroplasts converts this compound to a compound with the RF of chlorophyll. Thus Euglena chloroplasts supplied with appropriate additions can carry out protein synthesis, carbon dioxide fixation and most of chlorophyll synthesis in darkness. This versatility is appropriate in photosynthetic organelles isolated from photo-organotrophic cells.     

Euglena gracilis: Phenotypic resistance to 2,4-dinitrophenol

Euglena gracilis, when grown on a medium containing 10^?5m 2,4-dinitrophenol, will initially bleach, cease to divide, and about one-half will die off. After a prolonged lag period of 6–8 days, the remaining cells green and begin to multiply. The resultant cells are resistant to dinitrophenol and will grow in its presence at rates close to those in normal medium. The resistant cells do not differ greatly from the nonresistant ones, except that they show no sign of respiratory control, their photosynthetic activity is somewhat reduced, and their size is larger. The resistant cells appear less motile, their flagellar movement is slower, and their motility appears disturbed (they tend to swim in circles). The resistance is lost when the cells are grown for 2–3 generations on medium lacking dinitrophenol.     

Cytological Characterization of a Giant Strain of Euglena gracilis Obtained from Dark-starved Cultures

Euglena gracilis green cells were dark-starved for four months. After this period almost the entire population died, while a few giant, viable cells appeared in the culture. The giantism was maintained after repeated subcultures in growth medium in light or dark conditions. However, the phenomenon was not permanent, and the morphological characteristics of the wild-type Euglena were gradually restored. In giant cells nuclei enlarged greatly, DNA content increased and the Golgi apparatus greatly proliferated. Chloroplasts and mitochondria increased in number and size and often presented structural modifications when compared with normal Euglena. Importantly, in the giant cells that were maintained in darkness in resting or growth conditions chloroplasts persisted as structured organelles which appeared red-fluorescent under UV illumination. Whether giantism is a phenotypic or a genotypic change is still debated. In our case, the evolution of this phenomenon, chiefly the enhanced DNA content, suggests that teratism is a multiploid mutation with the possibility of a return to the normoploid condition. Constitutive chloroplasts are present in most algae, except for a few species, among which is Euglena gracilis. The persistence of differentiated plastids in darkness in giant Euglena is considered to be a return to an ancestral condition and may, therefore, be phylogenetically important.     

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; )     

Cotton embryogenesis: The tissues of the stigma and style and their relation to the pollen tube

Summary The stigma of cotton (Gossypium hirsutum) is covered by unicellular hairs. The cytoplasm of these hairs degenerates before the stigma becomes receptive. The vacuole remains intact, but the hair cytoplasm becomes a mass of dark, amorphous material with only a few organelles still being visible. The rest of the stigma consists of thin-walled parenchyma cells with large vacuoles and large amounts of starch. The cells of the style are differentiated into a uniseriate epidermis, vascular tissue, a cortex of thin-walled, vacuolate parenchyma cells, and the transmitting tissue. This latter tissue occupies the center of the style and consists of thick-walled cells with few vacuoles. The cells are rich in starch, ribosomes, endoplasmic reticulum and dictyosomes. They also contain deposits of calcium salts in the form of druses. The pollen germinates on the stigmatic hairs, grows down the outside of the hair and between the cells of the stigma to the transmitting tissue of the style. There the tubes grow between the walls of the cells but do not enter the cells themselves. Some transmitting cells adjacent to the pollen tube degenerate after the tip of the pollen tube has grown past them. However, not all degenerate, and those that do show no fixed spatial relationship to one another. The cells which do degenerate follow a characteristic pattern of breakdown. No ultrastructural evidence was found for the secretion of hydrolytic enzymes by the pollen tube.     

Cooperation of cytoplasmic and plastidial translation in formation of the photosynthetic apparatus and its stage-specific efficiency

In synchronized Euglena gracilis, strain Z, the synthesis of the apoproteins for the chlorophyll-protein-complexes CPI, CPa, and LHCP is light-dependent and takes place in the light period in a characteristic consecutive fashion: CPI at 1 to 2 hours, CPa at 7 to 12 hours, and LHCP at 8 to 12 hours. The syntheses sequence of the chlorophyll-protein-complexes coincides with the efficiency alterations of the photosynthetic apparatus of E. gracilis during the light period of the cell cycle. In particular, the synthesis onset of the photosystem II-related polypeptides CPa and LHCP aligns with the decrease of oxygen evolution at 6 hours of the light period and is discussed as reorganization process in the thylakoids.     

Action of Myxin on the Chloroplast System of Euglena gracilis

SYNOPSIS. Myxin (1-hydroxy-6-methoxy-phenazine-5,10-dioxide), a wide spectrum antibiotic, inhibits chloroplast replication in Euglena gracilis strain Z at concentrations which have no effect upon growth or survival. Myxin also inhibits the synthesis of chlorophyll when etiolated Euglena are illuminated in resting medium. By analogy with its action on bacteria, it is suggested that myxin may cause selective inhibition of chloroplast nucleic acid synthesis.     

Effect of Light on Glucose Utilization by Euglena gracilis

The effect of light on glucose consumption by wild-type Euglena gracilis Z. and mutant cells has been studied. When dark- or light-grown wild-type cells are transferred from a medium containing sodium butyrate as the only carbon source to a glucose-containing medium, glucose consumption is blocked for 6 to 7 days when cultures are incubated under a light intensity of at least 600 lux. During this time cells multiply at the same rate as controls kept on media devoid of any utilizable organic carbon source. This light-induced inhibition of glucose consumption and of growth on glucose-containing medium is not related to photosynthesis since: (a) glucose consumption is inhibited by light intensities much lower than those required for high phototrophic growth; (b) the inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea does not overcome the inhibition of glucose consumption; and (c) nonphototrophic-growing mutants also show light-induced inhibition of glucose consumption and of growth on glucose-containing medium. This inhibition of growth by light might be explained by modification in the permeability of the cellular membrane.     

Influence of Cell Division on the Degree of Streptomycin Bleaching of Euglena gracilis

SYNOPSIS. Streptomycin (SM) inhibition of greening of Euglena gracilis , strain z, was studied. The antibiotic was most effective if present during cell division in the absence of light. The next most effective condition was that which allowed cell division in the light, and the least effective conditions were those that allowed only minimal cell division in the dark or light (i.e., under "resting" conditions). In the dark, 20–200 times higher concentrations of SM were required for the same degree of inhibition under resting conditions as under growing conditions. The observation of Kirk ( Biochim. Biophys. Acta , 1962, 56 , 139–51)-that the pH of the resting medium influenced the degree of inhibition–was confirmed.     

Developmentally Regulated Secreted Factors Control Expression of Muscarinic Receptor Subtypes in Embryonic Chick Retina

Abstract: Two molecular mass subtypes of muscarinic receptor are expressed by the chick retina (72 and 86 kDa). During development, the ratio of subtypes changes, with the 72-kDa form becoming predominant. We have found that subtype switch can occur in retina cell culture, and have investigated factors that influence this in vitro increase in the 72-kDa receptor. Increases similar to those in vivo occurred when cells were cultured at 10⁵ cells/cm², but not at 10-fold lower density. High-density cultures, maintained on coverslips, showed no receptor development when transferred to large volumes of fresh medium, indicating that cell-cell contact alone was not responsible for induction. However, replacement of fresh medium with conditioned medium (from high-density cultures) resulted in normal induction. There were no morphological differences between cultures with high and low levels of the 72-kDa receptor. Conditioned medium also induced 72-kDa receptors in low-density cultures, consistent with a minimal role for cell-cell contact. Efficacy of conditioned medium was markedly dependent on age. Media from cells cultured 1–4 days had no effect, but media from cells cultured 5–8 and 1–8 days elicited 1.6-fold and fourfold increases in the 72-kDa subtype, respectively. The data indicate that maturing retina cells secrete developmentally regulated factors that are necessary for abundant expression of the 72-kDa muscarinic receptor subtype.     

Studies of Chloroplast Development in Euglena: XIII. Variation of Ultraviolet Sensitivity with Extent of Chloroplast Development

Ultraviolet (UV) inactivation of green colony-forming ability of several different types of Euglena gracilis var. bacillaris was studied. The observed target numbers are not widely different, while the doses required to produce a single inactivation event (D(o)) vary with the type of cell used. In dark-grown cells adapting to the light in resting medium and in an X-ray-induced mutant, D(o) is proportional to the chlorophyll content of the cells. However, in hyperdeveloped cells which contain abnormally high amounts of chlorophyll, the correlation does not hold, suggesting that it is not chlorophyll per se which is responsible for the differences observed. D(o)'s of colony-forming ability (viability) of light-grown and dark-grown cells are found to differ by the same factor as those of green colony-forming ability. Stationary phase and exponential phase cells show a small difference in D(o) with no obvious difference in target multiplicity. The multiplicity of the various target curves has been re-evaluated by computer and found to be between 30 and 40.     

Accumulation of N-acetylneuraminic acid (sialic acid) in human fibroblasts cultured in the presence of N-acetylmannosamine

Human skin fibroblasts incubated for 72 h in medium containing 10 mM N-acetyl-D-mannosamine accumulate material that yields a chromophore in the presence of thiobarbituric acid. This material was tentatively identified as free (unbound) sialic acid due to its reactivity with thiobarbituric acid prior to acid hydrolysis, its solubility in 10% trichloroacetic acid, its chromatographic properties on an anion-exchange column and its enzymatic susceptibility to acylneuraminate pyruvate-lyase. Mass spectrometry analysis established that the accumulated material was, in fact, N-acetylneuraminic acid. Loading studies demonstrated a linear relationship between the amount of N-acetylmannosamine in the medium and the level of sialic acid accumulating within the cells. Cells grown in the absence of N-acetylmannosamine contained an average of 5 nmol free sialic acid/mg protein, while cells cultured for 72 h in 20 mM amounts of this material contained an average of 156.3 nmol free sialic acid/mg protein. When the cells were removed from the N-acetylmannosamine-enriched medium and incubated in regular medium, more than 80% of the accumulated, intracellular sialic acid disappeared within the first 96 h. It was concluded from these data that normal fibroblasts cultured in medium enriched with N-acetylmannosamine store large amounts of N-acetylneuraminic acid and can thus serve as an excellent model for the study of both normal and abnormal sialic acid metabolism, transport, storage and/or metabolic (feedback) regulation in human tissue.