Sensitivity of light-grown and dark-grown Euglena cells to gamma-irradiation.

Light-grown cells which contain fully developed chloroplasts were found to be more resistant to gamma-irradiation than dark-grown cells which are devoid of chloroplasts. The radio-resistance of dark-grown cells progressively increased during light-induced development of chloroplasts and, conversely, radio-resistance of light-grown cells decreased progressively with chloroplast de-development during growth in the dark. The presence of chloroplasts seemed to play a major role in the capacity of cells to recover from radiation damage, the efficiency of cellular recovery being correlatable with the degree of chloroplast development.     

Gamma radiation-induced single strand breaks in DNA and their repair in spheroplasts and nuclei of light-grown and dark-grown Euglena cells

Exposure of light-grown and dark-grown Euglena cells to gamma radiation causes single strand breaks in nuclear DNA as assessed by sedimentation analysis in alkaline sucrose density gradients. The number of radiation-induced single strand breaks in nuclear DNA of light-grown cells is found to be less than that in dark-grown cells. Post-irradiation incubation of both types of cells in 0 . 1 M phosphate buffer, pH 7 . 0 at 25 degrees C for 1 hour results in restitution of the strand breaks in DNA. Light-grown cells (cells with chloroplasts) are able to rejoin all the single strand breaks in DNA produced by gamma irradiation at D50 and D5 doses. On the other hand, dark-grown cells (cells devoid of chloroplasts) are unable to rejoin all the strand breaks caused by irradiation at either of the doses. The rate of DNA repair in dark-grown cells is also much slower than that in light-grown cells. Radiation-induced single strand breaks in DNA and their repair in nuclei from both types of cells is found to be similar to that observed in the spheroplasts. It is suggested that some factor(s) elaborated by chloroplasts may contribute towards the efficiency of nuclear DNA repair in Euglena cells.     

Glycollate-pathway enzymes in mitochondria from phototrophic,organotrophic and mixotrophic cells of Euglena

Glycollate dehydrogenase and NADFH-glyoxylate reductase are constitutive enzymes in Percoll-purified mitochondria from phototrophic, mixotrophic and organotrophic cells of Euglena gracilis Klebs strain z Pringsheim. Glycollate oxidation by isolated mitochondria is stimulated four-fold by the addition of glutamate but rates of glycine oxidation are low in mitochondria from all cell types, the ratio of malate to glycine oxidation always being greater than 4:1. Measurement of the rate of NADPH oxidation in intact mitochondria and mitoplasts showed that the outer mitochondrial membrane is impermeable to NADPH and in the absence of NADPH-dehydrogenase activity the oxidation of NADPH by mitoplasts is dependent on the presence of glyoxylate for NADPH-glyoxylate-reductase activity. It is concluded that glycollate oxidation in the mitochondrion provides glyoxylate which, in the presence of a suitable amino-donor, can be converted to glycine by glutamate-glyoxylate amino-transferase so providing essential intermediates for biosynthesis. Glycollate oxidation outside the mitochondrion is concerned with photorespiratory metabolism and the inability of mitochondria to oxidise exogenous glycine at appreciable rates means that the separation of photorespiratory metabolism from the biosynthesis of essential intermediates is effected.     

Presence of glyoxylate cycle enzymes in the mitochondria of Euglena gracilis

Isocitrate lyase and malate synthase are specific enzymes of the glyoxylate cycle, used here as glyoxysomal markers. Both enzymes were found in the mitochondrial fraction after organelle fractionation by isopycnic centrifugation. Electron microscopy of this fraction indicated that mitochondria were the only recognizable organelles. Using an immunogold labeling method with anti-(malate synthase) antiserum, the only organelles stained in cells were the mitochondria. These results show that the glyoxylate cycle is present in mitochondria in Euglena.     

Effect of benzyladenine on some enzymes of mitochondria and microbodies in excised sunflower cotyledons

Benzyladenine (BA) increases the rate of expansion of dark-grown sunflower (Helianthus annuus L.) cotyledons. The hormone slightly enhances the development of the two glyoxysomal enzymes, isocitrate lyase and malate synthetase, during the first 3 days of germination and greatly accelerates their decay in the 2 following days. The levels of the peroxisomal enzymes, glycolate oxidase and glyoxylate reductase, are enhanced by BA more than those of the two glyoxysomal enzymes. These effects of BA on microbody enzymes are very similar to those of white light. Mitochondrial enzyme activities are increased to a varying extent by BA: the increase is minimal for fumarase, and maximal for cytochrome oxidase. The level of cytochrome oxidase is enhanced 346% at the 5th day of germination. Also, the rate of O₂ consumption is increased by BA, but the time course of this increased O₂ consumption does not match with that of cytochrome oxidase. Fusicoccin, a fungal toxin, mimics the effect of BA on cotyledon expansion, but fails to duplicate its action on microbody enzymes. This suggests that the effect of BA on microbody enzymes is not closely linked with the mechanism of growth promotion.     

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.     

Isolation and characterization of an acyl-CoA thioesterase from dark-grown Euglena gracilis

An acyl-CoA hydrolase from dark-grown Euglena gracilis Z was purified 700-fold by subjecting the 105,000g supernatant of the cell-free extract to (NH4)2SO4 precipitation, acid precipitation, calcium phosphate gel treatment, gel filtration on Sephadex G-100, and chromatography on QAE-Sephadex, hydroxylapatite, and CM-Sephadex. Polyacrylamide disc gel electrophoresis of the purified enzyme showed a major protein band (greater than 80%) which contained thioesterase activity and a minor protein band with no thioesterase activity. Molecular weight estimated by gel filtration was 37,000 and sodium dodecyl sulfate-electrophoresis showed one major band (greater than 80%) corresponding to a molecular weight of 37,000 and a minor band of molecular weight 32,000, suggesting that the enzyme was monomeric. The pH optimum of the purified enzyme progressively increased with the chain length of the substrate, with hexanoyl-CoA showing a pH optimum at 4.5 and stearoyl-CoA at 7.0. The rate of hydrolysis of acyl-CoA showed a nonlinear dependence on protein concentration, and bovine serum albumin overcame this effect as well as stimulated the rate. The extent of stimulation by albumin increased with chain length of the substrate up to lauroyl-CoA and then decreased as chain length increased; albumin inhibited the hydrolysis of stearoyl-CoA. This enzyme hydrolyzed CoA esters of C6 to C18 fatty acids with a maximal rate of 17 mumol min-1 mg protein-1 for C14. Typical substrate saturation patterns were obtained with all substrates except that high concentrations were inhibitory. Studies on the effect of pH on the apparent Km and Vmax values for octanoyl-CoA, lauroyl-CoA, and palmitoyl-CoA showed that in all cases Vmax was greatest and Km was lowest at the respective pH optima. Active-serine-directed reagents severely inhibited the thioesterase activity, suggesting the participation of an active serine residue in catalysis; thiol-directed reagents were not effective inhibitors. Diethylpyrocarbonate also inhibited the enzyme and hydroxylamine reversed this inhibition, suggesting the involvement of a histidine residue in catalysis as expected for enzymes containing active serine. This thioesterase did not affect the chain length distribution of the products generated by the Euglena fatty acid synthase I.     

The regulation of synthesis of mitochondrial enzymes in regreening and division-synchronized Euglena cultures

The effect of light and carbon nutrition on the synthesis of citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) in dark-grown resting (carbon deficient) and in phototrophic division-synchronized cultures of Euglena gracilis Klebs strain z were investigated. Exposure of dark-grown Euglena to white or red light produced a transient increase in the specific activities of citrate synthase and malate dehydrogenase but blue light (of equal energy) was ineffective. Citrate-synthase activity increased at the end of the light phase and in early dark phase in phototrophic cultures division-synchronized by a regime of 14 h light-10 h dark. The addition of ethanol or malate produced a twofold increase in citrate-synthase activity compared with phototrophic cultures. White and blue light, but not red light, produced a transient repression of the metabolite-induced increase in citrate-synthase activity in division-synchronized cultures. Since only red light could effect a transient increase in the specific activity of mitochondrial enzymes, and the blue-red plastid receptor should respond to both blue and red light, the synthesis of mitochondrial enzymes in regreening cultures may be under the control of a new photoreceptor responding only to red light. In division-synchronized phototrophic cells the primary effector of synthesis of mitochondrial enzymes is not light but carbon nutrition.     

Treatment of dark-grown Arabidopsis thaliana with a brassinosteroid-biosynthesis inhibitor, brassinazole, induces some characteristics of light-grown plants

When a brassinosteroid biosynthesis inhibitor, brassinazole (Brz), was applied at concentrations ranging from 0.1 to 2 μM, Arabidopsis thaliana (L.) Heynh seedlings grown in the dark exhibited morphological features of light-grown plants, i.e. short hypocotyls, expanded cotyledons, and true leaves, in a dose-dependent manner. Control (non Brz-treated) seedlings grown in the dark for 40 d did not develop leaf primordia. However, treatment with the lowest concentration of Brz induced the development of leaf buds, although it hardly induced any short hypocotyls, and treatment with the highest concentration of Brz induced both short hypocotyls and leaves. Labeling experiments with the thymidine analogue 5-bromo-2′-deoxyuridine revealed that amplification of cell nuclei and organellar nucleoids is activated in the shoot apical meristems of dark-grown Brz-treated seedlings. These results suggest that Brz-treatment induces development of true leaves. Furthermore, condensation and scattering of plastid nucleoids, which is known to occur during the differentiation of etioplasts into chloroplasts, was observed in the plastids of dark-grown Brz-treated cotyledons. In addition, high levels of ribulose-1,5-bisphosphate carboxylase-oxygenase proteins accumulated in the plastids of the cotyledons. Electron microscopy showed that the plastids were etioplasts with a prolamellar body and few thylakoid membranes. These results suggest that Brz treatment in the dark induces the initial steps of plastid differentiation, which occur prior to the development of thylakoid membranes. This is a novel presumed function of brassinosteroids. These cytological changes seen in Brz-treated Arabidopsis were exactly the same as those seen in a brassinosteroid-biosynthesis-deficient mutant, det2, supporting the hypothesis that Brz has no side-effects except inhibiting brassinosteroid biosynthesis, and should prove a useful tool in clarifying the role of brassinosteroids. Received: 10 February 2000 / Accepted: 11 April 2000     

Chlorophyll organization in dark-grown and light-grown pine (Pinus brutia) and barley (Hordeum vulgare)

A study was conducted comparing the organization of chlorophyll during development of the photosynthetic apparatus in dark-grown and light-grown pine and barley. The rationale was that gymnosperms, but not angiosperms, have a capacity to synthesize chlorophyll in darkness. Seedlings of Pinus brutia were germinated and grown in darkness or under photoperiodic (day/night) conditions. The low-temperature (77 K) fluorescence spectra of newly-emerging dark-grown seedlings exhibited a single fluorescence band peaking at 678–679 nm, which decayed primarily with a ∼5.5 ns lifetime. Over the first few days of growth, the emission shifted to longer wavelengths and a subnanosecond lifetime component became prevalent. After several days of dark growth the emission spectrum and lifetime profile of the low temperature fluorescence came to resemble those of light-grown pine and barley. At room temperature, dark-grown pine showed little variable fluorescence, though addition of DCMU caused a substantial fluorescence rise. Illumination with moderate light for a few hours was sufficient to 'photoinduce' the appearance of normal variable fluorescence. At 77 K, DCMU-treated samples clearly showed a very long-lived (∼40 ns) fluorescence lifetime component in light-grown pine and barley. This component was undetectable in dark-grown pine. If, however, dark-grown samples were illuminated either before or after DCMU addition and then frozen to 77 K, the ∼40 ns lifetime component appeared at a fluorescence intensity similar to that in light-grown samples. These results are explained primarily in terms of photoactivation of the photosystem II (PSII) donor side. The temporary maintenance of PSII in an inactive, highly-quenched state is suggested to provide an available, yet protected precursor for active PSII.     

Phosphorylation of thylakoid proteins during chloroplast biogenesis in greening etiolated and light-grown wheat leaves

Phosphorylation of polypeptides in isolated thylakoids was examined during chloroplast biogenesis in greening etiolated wheat leaves and 4 day-old wheat leaves grown under a diurnal light regime. At early stages of plastid development standard thylakoid preparations were heavily contaminated with nuclear proteins, which distorted the polypeptide phosphorylation profiles. Removal of contamination from membranes by sucrose density centrifugation demonstrated that the major membrane phosphoprotein in etioplasts was at 35 kDa. During etioplast greening a number of phosphoproteins appeared, of which the 25–27 kDa apoproteins of the light-harvesting chlorophylla/b protein complex associated with photosystem II (LHCII) became the most dominant. At the early stages of thylakoid development found at the base of the 4-day-old light grown leaf the LHCII apoproteins were evident as phosphoproteins; however the major phosphoprotein was polypeptide atca. 9kDA. Phosphorylation of both the LHCII apoproteins and the 9 kDa polypeptide in these thylakoids was not light-dependent. In the older thylakoids isolated from the leaf tip the LHCII apoproteins were the major phosphoproteins and their phosphorylation had become light-regulated; however phosphorylation of the 9 kDa polypeptide remained insensitive to light.