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Sample records for chlamydomonas reinhardtii influences

  1. Selenoprotein-Transgenic Chlamydomonas reinhardtii

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    Jiazuan Ni

    2013-02-01

    Full Text Available Selenium (Se deficiency is associated with the occurrence of many diseases. However, excessive Se supplementation, especially with inorganic Se, can result in toxicity. Selenoproteins are the major forms of Se in vivo to exert its biological function. Expression of those selenoproteins, especially with the application of a newly developed system, is thus very important for studying the mechanism of Se in nutrition. The use of Chlamydomonas reinhardtii (C. reinhardtii as a biological vector to express an heterogeneous protein is still at the initial stages of development. In order to investigate the possibility of using this system to express selenoproteins, human 15-KDa selenoprotein (Sep15, a small but widely distributed selenoprotein in mammals, was chosen for the expression platform test. Apart from the wild-type human Sep15 gene fragment, two Sep15 recombinants were constructed containing Sep15 open reading frame (ORF and the selenocysteine insertion sequence (SECIS element from either human Sep15 or C. reinhardtii selenoprotein W1, a highly expressed selenoprotein in this alga. Those Sep15-containing plasmids were transformed into C. reinhardtii CC-849 cells. Results showed that Sep15 fragments were successfully inserted into the nuclear genome and expressed Sep15 protein in the cells. The transgenic and wild-type algae demonstrated similar growth curves in low Se culture medium. To our knowledge, this is the first report on expressing human selenoprotein in green alga.

  2. Iron economy in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Glaesener, Anne G; Merchant, Sabeeha S; Blaby-Haas, Crysten E

    2013-09-02

    While research on iron nutrition in plants has largely focused on iron-uptake pathways, photosynthetic microbes such as the unicellular green alga Chlamydomonas reinhardtii provide excellent experimental systems for understanding iron metabolism at the subcellular level. Several paradigms in iron homeostasis have been established in this alga, including photosystem remodeling in the chloroplast and preferential retention of some pathways and key iron-dependent proteins in response to suboptimal iron supply. This review presents our current understanding of iron homeostasis in Chlamydomonas, with specific attention on characterized responses to changes in iron supply, like iron-deficiency. An overview of frequently used methods for the investigation of iron-responsive gene expression, physiology and metabolism is also provided, including preparation of media, the effect of cell size, cell density and strain choice on quantitative measurements and methods for the determination of metal content and assessing the effect of iron supply on photosynthetic performance.

  3. Iron economy in Chlamydomonas reinhardtii

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    Anne G. Glaesener

    2013-09-01

    Full Text Available While research on iron nutrition in plants has largely focused on iron-uptake pathways, photosynthetic microbes such as the unicellular green alga Chlamydomonas reinhardtii provide excellent experimental systems for understanding iron metabolism at the subcellular level. Several paradigms in iron homeostasis have been established in this alga, including photosystem remodeling in the chloroplast and preferential retention of some pathways and key iron-dependent proteins in response to suboptimal iron supply. This review presents our current understanding of iron homeostasis in Chlamydomonas, with specific attention on characterized responses to changes in iron supply, like iron-deficiency. An overview of frequently used methods for the investigation of iron-responsive gene expression, physiology and metabolism is also provided, including preparation of media, the effect of cell size, cell density and strain choice on quantitative measurements and methods for the determination of metal content and assessing the effect of iron supply on photosynthetic performance.

  4. Trophic status of Chlamydomonas reinhardtii influences the impact of iron deficiency on photosynthesis

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    Terauchi, Aimee M.; Peers, Graham; Kobayashi, Marilyn C.; Krishna K Niyogi; Merchant, Sabeeha S.

    2010-01-01

    To investigate the impact of iron deficiency on bioenergetic pathways in Chlamydomonas, we compared growth rates, iron content, and photosynthetic parameters systematically in acetate versus CO2-grown cells. Acetate-grown cells have, predictably (2-fold) greater abundance of respiration components but also, counter-intuitively, more chlorophyll on a per cell basis. We found that phototrophic cells are less impacted by iron deficiency and this correlates with their higher iron content on a per...

  5. Elucidation of Basal Body and Centriole Functions in Chlamydomonas reinhardtii

    National Research Council Canada - National Science Library

    Dutcher, Susan K

    2003-01-01

    .... They are needed for the assembly of flagella or cilia as well as for cell division. Chlamydomonas reinhardtii provides an excellent model organism for the study of the basal body and centrioles...

  6. Trophic status of Chlamydomonas reinhardtii influences the impact of iron deficiency on photosynthesis.

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    Terauchi, Aimee M; Peers, Graham; Kobayashi, Marilyn C; Niyogi, Krishna K; Merchant, Sabeeha S

    2010-07-01

    To investigate the impact of iron deficiency on bioenergetic pathways in Chlamydomonas, we compared growth rates, iron content, and photosynthetic parameters systematically in acetate versus CO(2)-grown cells. Acetate-grown cells have, predictably (2-fold) greater abundance of respiration components but also, counter-intuitively, more chlorophyll on a per cell basis. We found that phototrophic cells are less impacted by iron deficiency and this correlates with their higher iron content on a per cell basis, suggesting a greater capacity/ability for iron assimilation in this metabolic state. Phototrophic cells maintain both photosynthetic and respiratory function and their associated Fe-containing proteins in conditions where heterotrophic cells lose photosynthetic capacity and have reduced oxygen evolution activity. Maintenance of NPQ capacity might contribute to protection of the photosynthetic apparatus in iron-limited phototrophic cells. Acetate-grown iron-limited cells maintain high growth rates by suppressing photosynthesis but increasing instead respiration. These cells are also able to maintain a reduced plastoquinone pool.

  7. Developing molecular tools for Chlamydomonas reinhardtii

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    Noor-Mohammadi, Samaneh

    Microalgae have garnered increasing interest over the years for their ability to produce compounds ranging from biofuels to neutraceuticals. A main focus of researchers has been to use microalgae as a natural bioreactor for the production of valuable and complex compounds. Recombinant protein expression in the chloroplasts of green algae has recently become more routine; however, the heterologous expression of multiple proteins or complete biosynthetic pathways remains a significant challenge. To take full advantage of these organisms' natural abilities, sophisticated molecular tools are needed to be able to introduce and functionally express multiple gene biosynthetic pathways in its genome. To achieve the above objective, we have sought to establish a method to construct, integrate and express multigene operons in the chloroplast and nuclear genome of the model microalgae Chlamydomonas reinhardtii. Here we show that a modified DNA Assembler approach can be used to rapidly assemble multiple-gene biosynthetic pathways in yeast and then integrate these assembled pathways at a site-specific location in the chloroplast, or by random integration in the nuclear genome of C. reinhardtii. As a proof of concept, this method was used to successfully integrate and functionally express up to three reporter proteins (AphA6, AadA, and GFP) in the chloroplast of C. reinhardtii and up to three reporter proteins (Ble, AphVIII, and GFP) in its nuclear genome. An analysis of the relative gene expression of the engineered strains showed significant differences in the mRNA expression levels of the reporter genes and thus highlights the importance of proper promoter/untranslated-region selection when constructing a target pathway. In addition, this work focuses on expressing the cofactor regeneration enzyme phosphite dehydrogenase (PTDH) in the chloroplast and nuclear genomes of C. reinhardtii. The PTDH enzyme converts phosphite into phosphate and NAD(P)+ into NAD(P)H. The reduced

  8. Systemic Cold Stress Adaptation of Chlamydomonas reinhardtii*

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    Valledor, Luis; Furuhashi, Takeshi; Hanak, Anne-Mette; Weckwerth, Wolfram

    2013-01-01

    Chlamydomonas reinhardtii is one of the most important model organisms nowadays phylogenetically situated between higher plants and animals (Merchant et al. 2007). Stress adaptation of this unicellular model algae is in the focus because of its relevance to biomass and biofuel production. Here, we have studied cold stress adaptation of C. reinhardtii hitherto not described for this algae whereas intensively studied in higher plants. Toward this goal, high throughput mass spectrometry was employed to integrate proteome, metabolome, physiological and cell-morphological changes during a time-course from 0 to 120 h. These data were complemented with RT-qPCR for target genes involved in central metabolism, signaling, and lipid biosynthesis. Using this approach dynamics in central metabolism were linked to cold-stress dependent sugar and autophagy pathways as well as novel genes in C. reinhardtii such as CKIN1, CKIN2 and a hitherto functionally not annotated protein named CKIN3. Cold stress affected extensively the physiology and the organization of the cell. Gluconeogenesis and starch biosynthesis pathways are activated leading to a pronounced starch and sugar accumulation. Quantitative lipid profiles indicate a sharp decrease in the lipophilic fraction and an increase in polyunsaturated fatty acids suggesting this as a mechanism of maintaining membrane fluidity. The proteome is completely remodeled during cold stress: specific candidates of the ribosome and the spliceosome indicate altered biosynthesis and degradation of proteins important for adaptation to low temperatures. Specific proteasome degradation may be mediated by the observed cold-specific changes in the ubiquitinylation system. Sparse partial least squares regression analysis was applied for protein correlation network analysis using proteins as predictors and Fv/Fm, FW, total lipids, and starch as responses. We applied also Granger causality analysis and revealed correlations between proteins and

  9. Systemic cold stress adaptation of Chlamydomonas reinhardtii.

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    Valledor, Luis; Furuhashi, Takeshi; Hanak, Anne-Mette; Weckwerth, Wolfram

    2013-08-01

    Chlamydomonas reinhardtii is one of the most important model organisms nowadays phylogenetically situated between higher plants and animals (Merchant et al. 2007). Stress adaptation of this unicellular model algae is in the focus because of its relevance to biomass and biofuel production. Here, we have studied cold stress adaptation of C. reinhardtii hitherto not described for this algae whereas intensively studied in higher plants. Toward this goal, high throughput mass spectrometry was employed to integrate proteome, metabolome, physiological and cell-morphological changes during a time-course from 0 to 120 h. These data were complemented with RT-qPCR for target genes involved in central metabolism, signaling, and lipid biosynthesis. Using this approach dynamics in central metabolism were linked to cold-stress dependent sugar and autophagy pathways as well as novel genes in C. reinhardtii such as CKIN1, CKIN2 and a hitherto functionally not annotated protein named CKIN3. Cold stress affected extensively the physiology and the organization of the cell. Gluconeogenesis and starch biosynthesis pathways are activated leading to a pronounced starch and sugar accumulation. Quantitative lipid profiles indicate a sharp decrease in the lipophilic fraction and an increase in polyunsaturated fatty acids suggesting this as a mechanism of maintaining membrane fluidity. The proteome is completely remodeled during cold stress: specific candidates of the ribosome and the spliceosome indicate altered biosynthesis and degradation of proteins important for adaptation to low temperatures. Specific proteasome degradation may be mediated by the observed cold-specific changes in the ubiquitinylation system. Sparse partial least squares regression analysis was applied for protein correlation network analysis using proteins as predictors and Fv/Fm, FW, total lipids, and starch as responses. We applied also Granger causality analysis and revealed correlations between proteins and

  10. Studies on flagellar shortening in Chlamydomonas reinhardtii

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    Cherniack, J.

    1985-01-01

    Flagellar shortening of Chlamydomonas reinhardtii was promoted by sodium chloride, pyrophosphate (sodium, potassium and ammonium salts), EDTA and EGTA, succinate, citrate and oxalate (sodium salts), caffeine and aminophylline. Removal of calcium from the medium potentiated the effects of these agents in inducing shortening. Investigations of the release of phosphorylated compounds to the medium during pyrophosphate-induced flagellar shortening of cells pre-labelled with /sup 32/P, revealed an as yet unidentified /sup 32/P-labelled compound with distinct chromatographic properties. Chromatography and electrophoresis indicates that it is a small, highly polar molecule with a high charge to mass ratio, containing thermo- and acid-labile phosphate linkages. Investigations showed of the release of /sup 35/S-labelled protein to the medium from cells pre-labelled with /sup 35/S-sulfate showed that flagellated cells released two prominent polypeptides which comigrated with ..cap alpha..- and ..beta..-flagellar tubulin on SDS polyacrylamide gel electrophoresis, while deflagellated cells did not.

  11. Influence of agglomeration of cerium oxide nanoparticles and speciation of cerium(III) on short term effects to the green algae Chlamydomonas reinhardtii

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    Röhder, Lena A. [Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, Dübendorf 8600 (Switzerland); ETH-Zurich, Institute of Biogeochemistry and Pollutant Dynamics, Zürich 8092 (Switzerland); Brandt, Tanja [Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, Dübendorf 8600 (Switzerland); Sigg, Laura [Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, Dübendorf 8600 (Switzerland); ETH-Zurich, Institute of Biogeochemistry and Pollutant Dynamics, Zürich 8092 (Switzerland); Behra, Renata, E-mail: Renata.behra@eawag.ch [Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, Dübendorf 8600 (Switzerland)

    2014-07-01

    Highlights: • Phosphate-dispersed CeO₂ NP did not affect photosynthetic yield in C. reinhardtii. • Agglomerated CeO₂ NP slightly decreased photosynthetic yield. • Cerium(III) was shown to affect photosynthetic yield and intracellular ROS level. • Slight effects of CeO₂ NP were caused by dissolved Ce³⁺ ions present in suspensions. • Wild type and cell wall free mutant of C. reinhardtii showed the same sensitivity. - Abstract: Cerium oxide nanoparticles (CeO₂ NP) are increasingly used in industrial applications and may be released to the aquatic environment. The fate of CeO₂ NP and effects on algae are largely unknown. In this study, the short term effects of CeO₂ NP in two different agglomeration states on the green algae Chlamydomonas reinhardtii were examined. The role of dissolved cerium(III) on toxicity, its speciation and the dissolution of CeO₂ NP were considered. The role of cell wall of C. reinhardtii as a barrier and its influence on the sensitivity to CeO₂ NP and cerium(III) was evaluated by testing both, the wild type and the cell wall free mutant of C. reinhardtii. Characterization showed that CeO₂ NP had a surface charge of ~0 mV at physiological pH and agglomerated in exposure media. Phosphate stabilized CeO₂ NP at pH 7.5 over 24 h. This effect was exploited to test CeO₂ NP dispersed in phosphate with a mean size of 140 nm and agglomerated in absence of phosphate with a mean size of 2000 nm. The level of dissolved cerium(III) in CeO₂ NP suspensions was very low and between 0.1 and 27 nM in all tested media. Exposure of C. reinhardtii to Ce(NO₃)₃ decreased the photosynthetic yield in a concentration dependent manner with EC₅₀ of 7.5 ± 0.84 μM for wild type and EC₅₀ of 6.3 ± 0.53 μM for the cell wall free mutant. The intracellular level of reactive oxygen species (ROS) increased upon exposure to Ce(NO₃)₃ with effective concentrations similar to those inhibiting photosynthesis. The agglomerated Ce

  12. Alteration of proteins and pigments influence the function of photosystem I under iron deficiency from Chlamydomonas reinhardtii.

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    Venkateswarlu Yadavalli

    Full Text Available BACKGROUND: Iron is an essential micronutrient for all organisms because it is a component of enzyme cofactors that catalyze redox reactions in fundamental metabolic processes. Even though iron is abundant on earth, it is often present in the insoluble ferric [Fe (III] state, leaving many surface environments Fe-limited. The haploid green alga Chlamydomonas reinhardtii is used as a model organism for studying eukaryotic photosynthesis. This study explores structural and functional changes in PSI-LHCI supercomplexes under Fe deficiency as the eukaryotic photosynthetic apparatus adapts to Fe deficiency. RESULTS: 77K emission spectra and sucrose density gradient data show that PSI and LHCI subunits are affected under iron deficiency conditions. The visible circular dichroism (CD spectra associated with strongly-coupled chlorophyll dimers increases in intensity. The change in CD signals of pigments originates from the modification of interactions between pigment molecules. Evidence from sucrose gradients and non-denaturing (green gels indicates that PSI-LHCI levels were reduced after cells were grown for 72 h in Fe-deficient medium. Ultrafast fluorescence spectroscopy suggests that red-shifted pigments in the PSI-LHCI antenna were lost during Fe stress. Further, denaturing gel electrophoresis and immunoblot analysis reveals that levels of the PSI subunits PsaC and PsaD decreased, while PsaE was completely absent after Fe stress. The light harvesting complexes were also susceptible to iron deficiency, with Lhca1 and Lhca9 showing the most dramatic decreases. These changes in the number and composition of PSI-LHCI supercomplexes may be caused by reactive oxygen species, which increase under Fe deficiency conditions. CONCLUSIONS: Fe deficiency induces rapid reduction of the levels of photosynthetic pigments due to a decrease in chlorophyll synthesis. Chlorophyll is important not only as a light-harvesting pigment, but also has a structural role

  13. Monitoring Autophagy in the Model Green Microalga Chlamydomonas reinhardtii

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    María Esther Pérez-Pérez

    2017-10-01

    Full Text Available Autophagy is an intracellular catabolic system that delivers cytoplasmic constituents and organelles in the vacuole. This degradative process is mediated by a group of proteins coded by autophagy-related (ATG genes that are widely conserved from yeasts to plants and mammals. Homologs of ATG genes have been also identified in algal genomes including the unicellular model green alga Chlamydomonas reinhardtii. The development of specific tools to monitor autophagy in Chlamydomonas has expanded our current knowledge about the regulation and function of this process in algae. Recent findings indicated that autophagy is regulated by redox signals and the TOR network in Chlamydomonas and revealed that this process may play in important role in the control of lipid metabolism and ribosomal protein turnover in this alga. Here, we will describe the different techniques and approaches that have been reported to study autophagy and autophagic flux in Chlamydomonas.

  14. Adaptation prevents the extinction of Chlamydomonas reinhardtii under toxic beryllium

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    Beatriz Baselga-Cervera

    2016-03-01

    Full Text Available The current biodiversity crisis represents a historic challenge for natural communities: the environmental rate of change exceeds the population’s adaptation capability. Integrating both ecological and evolutionary responses is necessary to make reliable predictions regarding the loss of biodiversity. The race against extinction from an eco-evolutionary perspective is gaining importance in ecological risk assessment. Here, we performed a classical study of population dynamics—a fluctuation analysis—and evaluated the results from an adaption perspective. Fluctuation analysis, widely used with microorganisms, is an effective empirical procedure to study adaptation under strong selective pressure because it incorporates the factors that influence demographic, genetic and environmental changes. The adaptation of phytoplankton to beryllium (Be is of interest because human activities are increasing the concentration of Be in freshwater reserves; therefore, predicting the effects of human-induced pollutants is necessary for proper risk assessment. The fluctuation analysis was performed with phytoplankton, specifically, the freshwater microalgae Chlamydomonas reinhardtii, under acute Be exposure. High doses of Be led to massive microalgae death; however, by conducting a fluctuation analysis experiment, we found that C. reinhardtii was able to adapt to 33 mg/l of Be due to pre-existing genetic variability. The rescuing adapting genotype presented a mutation rate of 9.61 × 10−6 and a frequency of 10.42 resistant cells per million wild-type cells. The genetic adaptation pathway that was experimentally obtained agreed with the theoretical models of evolutionary rescue (ER. Furthermore, the rescuing genotype presented phenotypic and physiologic differences from the wild-type genotype, was 25% smaller than the Be-resistant genotype and presented a lower fitness and quantum yield performance. The abrupt distinctions between the wild-type and the Be

  15. Identification of an NADP/thioredoxin system in Chlamydomonas reinhardtii

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    Huppe, H. C.; Picaud, A.; Buchanan, B. B.; Miginiac-Maslow, M.

    1991-01-01

    The protein components of the NADP/thioredoxin system, NADP-thioredoxin reductase (NTR) and thioredoxin h, have been purified and characterized from the green alga, Chlamydomonas reinhardtii. The analysis of this system confirms that photoautotrophic Chlamydomonas cells resemble leaves in having both an NADP- and ferrodoxin-linked thioredoxin redox system. Chlamydomonas thioredoxin h, which is smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than thioredoxin m from the same source, cross-reacted with antisera to thioredoxin h from spinach (Spinacia oleracea L.) and wheat germ (Triticum vulgaris L.) but not with antisera to m or f thioredoxins. In these properties, the thioredoxin h resembled a thioredoxin from Chlamydomonas, designated Ch1, whose sequence was reported recently (P. Decottignies et al., 1991, Eur. J. Biochem. 198, 505-512). The differential reactivity of thioredoxin h with antisera was used to demonstrate that thioredoxin h is enriched outside the chloroplast. The NTR was purified from Chlamydomonas using thioredoxin h from the same source. Similar to its counterpart from other organisms, Chlamydomonas NTR had a subunit size of approx. 36 kDa and was specific for NADPH. Chlamydomonas NTR effectively reduced thioredoxin h from the same source but showed little activity with the other thioredoxins tested, including spinach thioredoxin h and Escherichia coli thioredoxin. Comparison of the reduction of Chlamydomonas thioredoxins m and h by each of the endogenous thioredoxin reductases, NTR and ferredoxin-thioredoxin reductase, revealed a differential specificity of each enzyme for thioredoxin. Thus, NTR showed increased activity with thioredoxin h and ferredoxin-thioredoxin reductase with thioredoxins m and f.

  16. Establishing Chlamydomonas reinhardtii as an industrial biotechnology host.

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    Scaife, Mark A; Nguyen, Ginnie T D T; Rico, Juan; Lambert, Devinn; Helliwell, Katherine E; Smith, Alison G

    2015-05-01

    Microalgae constitute a diverse group of eukaryotic unicellular organisms that are of interest for pure and applied research. Owing to their natural synthesis of value-added natural products microalgae are emerging as a source of sustainable chemical compounds, proteins and metabolites, including but not limited to those that could replace compounds currently made from fossil fuels. For the model microalga, Chlamydomonas reinhardtii, this has prompted a period of rapid development so that this organism is poised for exploitation as an industrial biotechnology platform. The question now is how best to achieve this? Highly advanced industrial biotechnology systems using bacteria and yeasts were established in a classical metabolic engineering manner over several decades. However, the advent of advanced molecular tools and the rise of synthetic biology provide an opportunity to expedite the development of C. reinhardtii as an industrial biotechnology platform, avoiding the process of incremental improvement. In this review we describe the current status of genetic manipulation of C. reinhardtii for metabolic engineering. We then introduce several concepts that underpin synthetic biology, and show how generic parts are identified and used in a standard manner to achieve predictable outputs. Based on this we suggest that the development of C. reinhardtii as an industrial biotechnology platform can be achieved more efficiently through adoption of a synthetic biology approach. © 2015 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

  17. Estudio del metabolismo del glicolato en celulas de Chlamydomonas reinhardtii

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    Borrero Rubio, José Antonio

    1994-01-01

    En este trabajo pretendemos contribuir a los actuales conocimientos sobre el metabolismo del glicolato enChlamydomonas reinhardtii desde tres aproximaciones:1. Por una parte , analizar las actividades enzimáticas implicadas en la biosíntesis y oxidación de glicolato desde la perspectiva de su evolución en el ciclo celular y desde el contexto de fotorrespiración en que fisiológicamente participan, para lo cual se desarrolla un estudio comparado de estas enzimas en alto y bajo CO2.2. Por otra p...

  18. Lipidomic Analysis of Chlamydomonas reinhardtii under Nitrogen and Sulfur Deprivation.

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    Dawei Yang

    Full Text Available Chlamydomonas reinhardtii accumulates lipids under complete nutrient starvation conditions while overall growth in biomass stops. In order to better understand biochemical changes under nutrient deprivation that maintain production of algal biomass, we used a lipidomic assay for analyzing the temporal regulation of the composition of complex lipids in C. reinhardtii in response to nitrogen and sulfur deprivation. Using a chip-based nanoelectrospray direct infusion into an ion trap mass spectrometer, we measured a diversity of lipid species reported for C. reinhardtii, including PG phosphatidylglycerols, PI Phosphatidylinositols, MGDG monogalactosyldiacylglycerols, DGDG digalactosyldiacylglycerols, SQDG sulfoquinovosyldiacylglycerols, DGTS homoserine ether lipids and TAG triacylglycerols. Individual lipid species were annotated by matching mass precursors and MS/MS fragmentations to the in-house LipidBlast mass spectral database and MS2Analyzer. Multivariate statistics showed a clear impact on overall lipidomic phenotypes on both the temporal and the nutrition stress level. Homoserine-lipids were found up-regulated at late growth time points and higher cell density, while triacyclglycerols showed opposite regulation of unsaturated and saturated fatty acyl chains under nutritional deprivation.

  19. Coupling of Carbon Dioxide Fixation to the Oxyhydrogen Reaction in the Isolated Chloroplast of Chlamydomonas reinhardtii

    National Research Council Canada - National Science Library

    Changguo Chen; Martin Gibbs

    1992-01-01

    The oxyhydrogen reaction (the reduction of O to water by H ) in the presence of CO was studied in the isolated Chlamydomonas reinhardtii chloroplast by monitoring the rate of CO incorporation into acid-stable products in the dark...

  20. Cd2+ Toxicity to a green alga Chlamydomonas reinhardtii as influenced by its adsorption on TiO2 engineered nanoparticles.

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    Wei-Wan Yang

    Full Text Available In the present study, Cd(2+ adsorption on polyacrylate-coated TiO(2 engineered nanoparticles (TiO(2-ENs and its effect on the bioavailability as well as toxicity of Cd(2+ to a green alga Chlamydomonas reinhardtii were investigated. TiO(2-ENs could be well dispersed in the experimental medium and their pH(pzc is approximately 2. There was a quick adsorption of Cd(2+ on TiO(2-ENs and a steady state was reached within 30 min. A pseudo-first order kinetics was found for the time-related changes in the amount of Cd(2+ complexed with TiO(2-ENs. At equilibrium, Cd(2+ adsorption followed the Langmuir isotherm with the maximum binding capacity 31.9, 177.1, and 242.2 mg/g when the TiO(2-EN concentration was 1, 10, and 100 mg/l, respectively. On the other hand, Cd(2+ toxicity was alleviated in the presence of TiO(2-ENs. Algal growth was less suppressed in treatments with comparable total Cd(2+ concentration but more TiO(2-ENs. However, such toxicity difference disappeared and all the data points could be fitted to a single Logistic dose-response curve when cell growth inhibition was plotted against the free Cd(2+ concentration. No detectable amount of TiO(2-ENs was found to be associated with the algal cells. Therefore, TiO(2-ENs could reduce the free Cd(2+ concentration in the toxicity media, which further lowered its bioavailability and toxicity to C. reinhardtii.

  1. Metabolic responses to ethanol and butanol in Chlamydomonas reinhardtii.

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    Jiang, Yongguang; Xiao, Peng; Shao, Qing; Qin, Huan; Hu, Zhangli; Lei, Anping; Wang, Jiangxin

    2017-01-01

    Microalgae have been demonstrated to be among the most promising phototrophic species for producing renewable biofuels and chemicals. Ethanol and butanol are clean energy sources with good chemical and physical properties as alternatives to gasoline. However, biosynthesis of these two biofuels has not been achieved due to low tolerance of algal cells to ethanol or butanol. With an eye to circumventing these problems in the future and engineering the robust alcohol-producing microalgal hosts, we investigated the metabolic responses of the model green alga Chlamydomonas reinhardtii to ethanol and butanol. Using a quantitative proteomics approach with iTRAQ-LC-MS/MS technologies, we detected the levels of 3077 proteins; 827 and 730 of which were differentially regulated by ethanol and butanol, respectively, at three time points. In particular, 41 and 59 proteins were consistently regulated during at least two sampling times. Multiple metabolic processes were affected by ethanol or butanol, and various stress-related proteins, transporters, cytoskeletal proteins, and regulators were induced as the major protection mechanisms against toxicity of the organic solvents. The most highly upregulated butanol response protein was Cre.770 peroxidase. The study is the first comprehensive view of the metabolic mechanisms employed by C. reinhardtii to defend against ethanol or butanol toxicity. Moreover, the proteomic analysis provides a resource for investigating potential gene targets for engineering microalgae to achieve efficient biofuel production.

  2. Influence of carbon dioxide, temperature, medium kind and light intensity on the growth of algae Chlamydomonas reinhardtii and Scenedesmus obliquus

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    Olejnik Przemysław Piotr

    2016-01-01

    Full Text Available Microalgae attracts the attention of scientists because of the possibility of using in the energy industry as one of the substrates for the production of renewable energy. So far, the greatest emphasis was put on attempts to obtain strains, and technologies of their culturing, in order to efficiently acquire fat from cells and its further conversion to biodiesel using transesterification reaction. Increasingly, algae are considered also as an efficient biomass producer, which can be used as a substrate for methane production in biogas plants. In this study the influence of different physical and chemical conditions, on the growth of two algae species: Chlamydomonasreinhardtii and Scenedesmus obliquus was investigated. Based on the literature and the data obtained for the algae growth on the standard medium and the digestate remaining after fermentation, one may suggest further investigations on the use of other liquid waste from agriculture and industry for algae breeding, including chemical. analysis and supplementation of these mediums so as to provide the best conditions for their growth.

  3. Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii1

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    Chen, Changguo; Gibbs, Martin

    1991-01-01

    Chloroplastic respiration was monitored by measuring 14CO2 from 14C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of 14CO2 evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The Km for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of 14CO2 was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO2 evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO2 evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH4Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO2 and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism. PMID:16667985

  4. Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii.

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    Chen, C; Gibbs, M

    1991-01-01

    Chloroplastic respiration was monitored by measuring (14)CO(2) from (14)C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of (14)CO(2) evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K(m) for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of (14)CO(2) was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO(2) evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO(2) evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH(4)Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO(2) and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.

  5. Effect of cadmium accumulation on green algae Chlamydomonas reinhardtii and acid-tolerant Chlamydomonas CPCC 121.

    Science.gov (United States)

    Samadani, Mahshid; Perreault, François; Oukarroum, Abdallah; Dewez, David

    2018-01-01

    Cadmium is one of the most dangerous metals found in wastewater since exposure to low concentrations are highly toxic for cellular functions. In this study, the effect of cadmium accumulation on Chlamydomonas reinhardtii and acid-tolerant strain CPCC 121 was investigated during 48 h under 100-600 μM of Cd and two pH conditions (4 and 7). The toxicity of accumulated Cd was determined by the change of cellular and photosynthetic parameters. Obtained results showed that the maximum capacity of Cd accumulation in algal biomass was reached for both strains at 24 h of exposure to 600 μM of Cd. Under this condition, C. reinhardtii showed a higher uptake of Cd compared to the strain CPCC 121, inducing a stronger cellular toxic impact. Chlamydomonas CPCC 121 showed a tolerance for Cd due to the exclusion of Cd at the cell wall surface, which was higher at pH 4 than pH 7. TEM images and EDX spectrum of Cd distribution within the cell confirmed the role of the cell wall as a barrier for Cd uptake. Although Cd 2+ concentration was the highest in the medium, CPCC 121 was the most tolerant at pH 4, but was not enough efficient to be considered for the phycoremediation of Cd. At neutral pH, the efficiency of C. reinhardtii for the removal of Cd was limited by its toxicity, which was dependent to the concentration of Cd in the medium and the time of exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Thomas Julie

    2010-02-01

    Full Text Available Abstract Background Genome-wide computational analysis of alternative splicing (AS in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs. Results Our analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy. Conclusions The extent of AS in Chlamydomonas that we observed is much

  7. Oxidative stress in the algae Chlamydomonas reinhardtii exposed to biocides.

    Science.gov (United States)

    Almeida, Ana Catarina; Gomes, Tânia; Langford, Katherine; Thomas, Kevin V; Tollefsen, Knut Erik

    2017-08-01

    The toxicity of biocides can be associated with the formation of reactive oxygen species (ROS) and subsequent oxidative damage, interfering with the normal function of photosynthetic organisms. This study investigated the formation and effects of ROS in the unicellular green algae Chlamydomonas reinhardtii exposed to three environmentally relevant biocides, aclonifen, dichlofluanid and triclosan. After a first screening to identify which biocides induced ROS, a 24h multi-endpoint analysis was used to verify the possible consequences. A battery of high-throughput methods was applied in algae for measuring ROS formation, reduced glutathione (GSH), lipid peroxidation (LPO), photosystem (PS) II performance and pigments (chlorophylls a, b and carotenoids). Results show that only aclonifen induced ROS after the first 6h exposure, with the other two biocides not showing any ROS formation. Aclonifen, a Protox and carotenoid inhibitor, induced a concentration-dependent ROS formation, LPO and interfered with algae pigments content, while no alterations were detected in GSH content. A significant effect was also seen in the photosynthetic process, especially a reduction in the maximum and effective quantum yields, accompanied by alterations in energy dissipation in PSII reaction centers and the impairment of the electron transport rate. This study demonstrated the successful use of a battery of high-throughput methods for quickly screening biocides capacity to induce the formation of ROS and the subsequent effects in C. reinhardtii, thus revealing their mode of action (MoA) at concentrations before an impact on growth can become effective. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Glucose respiration in the intact chloroplast of Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Changguo Chen; Gibbs, M. (Brandeis Univ., Waltham, MA (United States))

    1991-01-01

    Chloroplastic respiration was monitored by measuring {sup 14}CO{sub 2} from {sup 14}C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast, The patterns of {sup 14}CO{sub 2} evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolypyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K{sub m} for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of {sup 14}CO{sub 2} was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO{sub 2} evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO{sub 2} evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH{sub 4}Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolypyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to Co{sub 2} and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.

  9. Photosynthetic efficiency of Chlamydomonas reinhardtii in flashing light.

    Science.gov (United States)

    Vejrazka, Carsten; Janssen, Marcel; Streefland, Mathieu; Wijffels, René H

    2011-12-01

    Efficient light to biomass conversion in photobioreactors is crucial for economically feasible microalgae production processes. It has been suggested that photosynthesis is enhanced in short light path photobioreactors by mixing-induced flashing light regimes. In this study, photosynthetic efficiency and growth of the green microalga Chlamydomonas reinhardtii were measured using LED light to simulate light/dark cycles ranging from 5 to 100 Hz at a light-dark ratio of 0.1 and a flash intensity of 1000 µmol m(-2) s(-1). Light flashing at 100 Hz yielded the same photosynthetic efficiency and specific growth rate as cultivation under continuous illumination with the same time-averaged light intensity (i.e., 100 µmol m(-2) s(-1)). The efficiency and growth rate decreased with decreasing flash frequency. Even at 5 Hz flashing, the rate of linear electron transport during the flash was still 2.5 times higher than during maximal growth under continuous light, suggesting storage of reducing equivalents during the flash which are available during the dark period. In this way the dark reaction of photosynthesis can continue during the dark time of a light/dark cycle. Understanding photosynthetic growth in dynamic light regimes is crucial for model development to predict microalgal photobioreactor productivities. Copyright © 2011 Wiley Periodicals, Inc.

  10. Paralytic shellfish toxins inhibit copper uptake in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Cusick, Kathleen D; Wetzel, Randall K; Minkin, Steven C; Dodani, Sheel C; Wilhelm, Steven W; Sayler, Gary S

    2013-06-01

    Paralytic shellfish toxins are secondary metabolites produced by several species of dinoflagellates and cyanobacteria. Known targets of these toxins, which typically occur at detrimental concentrations during harmful algal blooms, include voltage-gated ion channels in humans and other mammals. However, the effects of the toxins on the co-occurring phytoplankton community remain unknown. The present study examined the molecular mechanisms of the model photosynthetic alga Chlamydomonas reinhardtii in response to saxitoxin exposure as a means of gaining insight into the phytoplankton community response to a bloom. Previous work with yeast indicated that saxitoxin inhibited copper uptake, so experiments were designed to examine whether saxitoxin exhibited a similar mode of action in algae. Expression profiling following exposure to saxitoxin or a copper chelator produced similar profiles in copper homeostasis genes, notably induction of the cytochrome c6 (CYC6) and copper transporter (COPT1, CTR1) genes. Cytochrome c6 is used as an alternative to plastocyanin under conditions of copper deficiency, and immunofluorescence data showed this protein to be present in a significantly greater proportion of saxitoxin-exposed cells compared to controls. Live-cell imaging with a copper-sensor probe for intracellular labile Cu(I) confirmed that saxitoxin blocked copper uptake. Extrapolations of these data to phytoplankton metabolic processes along with the copper transporter as a molecular target of saxitoxin based on existing structural models are discussed. Copyright © 2013 SETAC.

  11. Metabolic acclimation to excess light intensity in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Davis, Maria C; Fiehn, Oliver; Durnford, Dion G

    2013-07-01

    There are several well-described acclimation responses to excess light in green algae but the effect on metabolism has not been thoroughly investigated. This study examines the metabolic changes during photoacclimation to high-light (HL) stress in Chlamydomonas reinhardtii using nuclear magnetic resonance and mass spectrometry. Using principal component analysis, a clear metabolic response to HL intensity was observed on global metabolite pools, with major changes in the levels of amino acids and related nitrogen metabolites. Amino acid pools increased during short-term photoacclimation, but were especially prominent in HL-acclimated cultures. Unexpectedly, we observed an increase in mitochondrial metabolism through downstream photorespiratory pathways. The expression of two genes encoding key enzymes in the photorespiratory pathway, glycolate dehydrogenase and malate synthase, were highly responsive to the HL stress. We propose that this pathway contributes to metabolite pools involved in nitrogen assimilation and may play a direct role in photoacclimation. Our results suggest that primary and secondary metabolism is highly pliable and plays a critical role in coping with the energetic imbalance during HL exposure and a necessary adjustment to support an increased growth rate that is an effective energy sink for the excess reducing power generated during HL stress. © 2013 John Wiley & Sons Ltd.

  12. Phosphorylation of nuclear and flagellar basal apparatus proteins during flagellar regeneration in Chlamydomonas reinhardtii

    OpenAIRE

    1993-01-01

    The antiphosphoprotein monoclonal antibody MPM-2 was used to investigate protein phosphorylation during flagellar regeneration in Chlamydomonas reinhardtii. MPM-2 recognizes a phosphorylated epitope and detects several Chlamydomonas proteins by Western immunoblot analysis. Two MPM-2 reactive proteins (34 and 90 kD) increase in Western immunoblot intensity after flagellar excision and decrease in intensity during flagellar regeneration. Immunofluorescence and immunogold labeling revealed MPM-2...

  13. Ultraviolet modification of Chlamydomonas reinhardtii for carbon capture

    Directory of Open Access Journals (Sweden)

    Gopal NS

    2016-04-01

    Full Text Available Nikhil S Gopal,1 K Sudhakar2 1The Lawrenceville School, Lawrenceville, NJ, USA; 2Bioenergy Laboratory, Malauna Azad National Institute of Technology, Bhopal, India Purpose: Carbon dioxide (CO2 levels have been rising rapidly. Algae are single-cell organisms with highly efficient CO2 uptake mechanisms. Algae yield two to ten times more biomass versus terrestrial plants and can grow nearly anywhere. Large scale CO2 sequestration is not yet sustainable due to high amounts of nitrogen (N and phosphate (P needed to grow algae in media. Methods: Mutant strains of Chlamydomonas reinhardtii were created using ultraviolet light (2.2–3 K J/m2 and natural selection using media with 20%–80% lower N and P compared to standard Sueoka's high salt medium. Strains were selected based upon growth in media concentrations varying from 20% to 80% less N/P compared to control. Biomass was compared to wild-type control (CC-125 using direct counts, optical density dry weight, and mean doubling time. Results: Mean doubling time was 20 and 25 hours in the low N and N/P strains, respectively (vs 66 hours in control. Using direct counts, growth rates of mutant strains of low N and N/P cultures were not statistically different from control (P=0.37 and 0.70, respectively. Conclusion: Two new strains of algae, as well as wild-type control, were able to grow while using 20%–40% less N and P. Ultraviolet light-based modification of algae is an inexpensive and alternative option to genetic engineering techniques. This technique might make larger scale biosequestration possible. Keywords: biosequestration, ultraviolet, carbon sequestration, carbon capture, algae

  14. Light-harvesting complex II (LHCII) and its supramolecular organization in Chlamydomonas reinhardtii

    NARCIS (Netherlands)

    Drop, B.A.; Webber-Birungi, M.; Yadav, S.K.N.; Filipowicz-Szymanska, A.; Fusetti, F.; Boekema, E.J.; Croce, R.

    2014-01-01

    LHCII is the most abundant membrane protein on earth. It participates in the first steps of photosynthesis by harvesting sunlight and transferring excitation energy to the core complex. Here we have analyzed the LHCII complex of the green alga Chlamydomonas reinhardtii and its association with the

  15. ATP Production in Chlamydomonas reinhardtii Flagella by Glycolytic Enzymes

    DEFF Research Database (Denmark)

    Mitchell, Beth F; Pedersen, Lotte B; Feely, Michael

    2005-01-01

    Eukaryotic cilia and flagella are long, thin organelles, and diffusion from the cytoplasm may not be able to support the high ATP concentrations needed for dynein motor activity. We discovered enzyme activities in the Chlamydomonas reinhardtii flagellum that catalyze three steps of the lower half...

  16. Triclosan-induced transcriptional and biochemical alterations in the freshwater green algae Chlamydomonas reinhardtii

    NARCIS (Netherlands)

    Pan, Chang Gui; Peng, Feng-Jiao; Shi, Wen Jun; Hu, Li Xin; Wei, Xiao Dong; Ying, Guang Guo

    2018-01-01

    Triclosan (TCS) is an antibacterial and antifungal agent widely used in personal care products (PCPs). We investigated the effects of TCS (20 μg/L, 100 μg/L and 500 μg/L) on Chlamydomonas reinhardtii by measuring the algal growth, chlorophyll content, lipid peroxidation, and transcription of the

  17. Light-harvesting complex II (LHCII) and its supramolecular organization in Chlamydomonas reinhardtii

    NARCIS (Netherlands)

    Drop, Bartlomiej; Webber-Birungi, Mariam; Yadav, Sathish K. N.; Filipowicz-Szymanska, Alicja; Fusetti, Fabrizia; Boekema, Egbert J.; Croce, Roberta

    LHCII is the most abundant membrane protein on earth. It participates in the first steps of photosynthesis by harvesting sunlight and transferring excitation energy to the core complex. Here we have analyzed the LHCII complex of the green alga Chlamydomonas reinhardtii and its association with the

  18. Evolutionarily conserved Δ25(27)-olefin ergosterol biosynthesis pathway in the alga Chlamydomonas reinhardtii

    OpenAIRE

    Miller, Matthew B.; Haubrich, Brad A; Wang, Qian; Snell, William J.; Nes, W. David

    2012-01-01

    Ergosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ24(28)-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphat...

  19. Effects of benzophenone-3 on the green alga Chlamydomonas reinhardtii and the cyanobacterium Microcystis aeruginosa.

    Science.gov (United States)

    Mao, Feijian; He, Yiliang; Kushmaro, Ariel; Gin, Karina Yew-Hoong

    2017-12-01

    Effects of benzophenone-3 (BP-3) on the green alga, Chlamydomonas reinhardtii, and the cyanobacterium, Microcystis aeruginosa, were investigated. The tested organisms were exposed to environmental levels of BP-3 for 10 days, at nominal concentrations from 0.01 to 5000μgL-1. Specific growth rate and photosynthetic pigments were employed to evaluate the toxic responses. The two tested algae had distinct toxic responses towards BP-3 stress, with the green alga C. reinhardtii being more sensitive than the cyanobacterium M. aeriginosa, based on EC20 and EC50 values. Uptake of BP-3 from the medium occurred in both species, with M. aeruginosa showing greater overall uptake (27.2-77.4%) compared to C. reinhardtii (1.1-58.4%). The effects of BP-3 on C. reinhardtii were variable at concentrations lower than 100μgL-1. At higher concentrations, the specific growth rate of C. reinhardtii decreased following a reduction in chlorophyll a (chl-a) content. Further experiments showed that BP-3 regulated the growth of C. reinhardtii by affecting the production of chl-a, chlorophyll b and carotenoids. In M. aeruginosa, specific growth rate was only moderately affected by BP-3. Additionally, the production of chl-a was significantly inhibited over the different exposure concentrations, while the production of carotenoids was stimulated. These results indicate a potential detrimental effect on prokaryotes and eukaryotes and that the mechanism of action varies with species. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Investigating the link between fermentative metabolism and hydrogen production in the unicellular green alga Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Burgess, S.J.; Nixon, P.J. [Imperial College London (United Kingdom)

    2010-07-01

    In the model green alga Chlamydomonas reinhardtii, the electrons required for hydrogen production can come from both the biophotolysis of water and from the fermentation of carbohydrate reserves. Anoxia leads to the activation of several fermentative pathways, which produce a number of end products including formic, malic and acetic acid along with ethanol, carbon dioxide and hydrogen. It has been proposed that by switching off competing fermentative pathways hydrogen production can be increased. Therefore the aim of this study was to devise an experimental strategy to down-regulate the expression of enzymes thought to control C. reinhardtii's fermentative metabolism. We demonstrate here that it is possible to use artificial microRNA (amiRNA) technology to generate knock-down mutants with reduced expression of pyruvate formate lyase (PFL1), a key fermentative enzyme in C. reinhardtii. This work opens up new possibilities to improve hydrogen yields through metabolic engineering. (orig.)

  1. Acclimation to NaCl and light stress of heterotrophic Chlamydomonas reinhardtii for lipid accumulation.

    Science.gov (United States)

    Fan, Jianhua; Zheng, Lvhong

    2017-09-01

    Salt stress has been proven very effective in enhancing the lipid content among many photoautotrophically grown microalgae species including marine and freshwater algae. Nevertheless, its effect on heterotrophic grown cells and lipid accumulation is scarcely known. This study sought to demonstrate a new train of thought for cost-effective biofuels production by heterotrophic culture of Chlamydomonas reinhardtii coupling with subsequent salt and light stress. NaCl treatments (25-200 mM) gradually suppressed the cell growth. After one day's acclimation, the cells restored slow growth with light supplement (200 μmol/m2/s) in low salt concentration (0-50 mM). However, high concentration of NaCl (200 mM) dose caused permanent damage, with over 47% cells death after 3 days treatment. The highest lipid content of 35.8% and lipid productivity of 28.6 mg/L/d were achieved by 50 mM NaCl stress and light treatment upon heterotrophic grown cells. Cells lost their green pigmentation and became yellowish under 100-200 mM NaCl conditions, whereas cells grown in 0-50 mM NaCl retained their dark-green pigmentation. Variable-to-maximum fluorescence ratio (Fv/Fm) and non-photochemical quenching (NPQ) value were markedly influenced under salt and light stress, indicating that severe inhibition of photosynthetic ability was occurred. Moreover, we further demonstrated the dynamic changes of cell growth and lipid accumulation would potentially be caused by the increase of intracellular redox state. To our knowledge, this study is the first instance in which C. reinhardtii was applied to oil accumulation by using combination of heterotrophic culture and multiple stress, and opened up a new territory for the further development of microalgae-based biofuels production. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Rapid Induction of Lipid Droplets in Chlamydomonas reinhardtii and Chlorella vulgaris by Brefeldin A

    Science.gov (United States)

    Ko, Donghwi; Yamaoka, Yasuyo; Otsuru, Masumi; Kawai-Yamada, Maki; Ishikawa, Toshiki; Oh, Hee-Mock; Nishida, Ikuo; Li-Beisson, Yonghua; Lee, Youngsook

    2013-01-01

    Algal lipids are the focus of intensive research because they are potential sources of biodiesel. However, most algae produce neutral lipids only under stress conditions. Here, we report that treatment with Brefeldin A (BFA), a chemical inducer of ER stress, rapidly triggers lipid droplet (LD) formation in two different microalgal species, Chlamydomonas reinhardtii and Chlorella vulgaris. LD staining using Nile red revealed that BFA-treated algal cells exhibited many more fluorescent bodies than control cells. Lipid analyses based on thin layer chromatography and gas chromatography revealed that the additional lipids formed upon BFA treatment were mainly triacylglycerols (TAGs). The increase in TAG accumulation was accompanied by a decrease in the betaine lipid diacylglyceryl N,N,N-trimethylhomoserine (DGTS), a major component of the extraplastidic membrane lipids in Chlamydomonas, suggesting that at least some of the TAGs were assembled from the degradation products of membrane lipids. Interestingly, BFA induced TAG accumulation in the Chlamydomonas cells regardless of the presence or absence of an acetate or nitrogen source in the medium. This effect of BFA in Chlamydomonas cells seems to be due to BFA-induced ER stress, as supported by the induction of three homologs of ER stress marker genes by the drug. Together, these results suggest that ER stress rapidly triggers TAG accumulation in two green microalgae, C. reinhardtii and C. vulgaris. A further investigation of the link between ER stress and TAG synthesis may yield an efficient means of producing biofuel from algae. PMID:24349166

  3. Rapid induction of lipid droplets in Chlamydomonas reinhardtii and Chlorella vulgaris by Brefeldin A.

    Directory of Open Access Journals (Sweden)

    Sangwoo Kim

    Full Text Available Algal lipids are the focus of intensive research because they are potential sources of biodiesel. However, most algae produce neutral lipids only under stress conditions. Here, we report that treatment with Brefeldin A (BFA, a chemical inducer of ER stress, rapidly triggers lipid droplet (LD formation in two different microalgal species, Chlamydomonas reinhardtii and Chlorella vulgaris. LD staining using Nile red revealed that BFA-treated algal cells exhibited many more fluorescent bodies than control cells. Lipid analyses based on thin layer chromatography and gas chromatography revealed that the additional lipids formed upon BFA treatment were mainly triacylglycerols (TAGs. The increase in TAG accumulation was accompanied by a decrease in the betaine lipid diacylglyceryl N,N,N-trimethylhomoserine (DGTS, a major component of the extraplastidic membrane lipids in Chlamydomonas, suggesting that at least some of the TAGs were assembled from the degradation products of membrane lipids. Interestingly, BFA induced TAG accumulation in the Chlamydomonas cells regardless of the presence or absence of an acetate or nitrogen source in the medium. This effect of BFA in Chlamydomonas cells seems to be due to BFA-induced ER stress, as supported by the induction of three homologs of ER stress marker genes by the drug. Together, these results suggest that ER stress rapidly triggers TAG accumulation in two green microalgae, C. reinhardtii and C. vulgaris. A further investigation of the link between ER stress and TAG synthesis may yield an efficient means of producing biofuel from algae.

  4. Production and characterization of algae extract from Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Weston Kightlinger

    2014-01-01

    Conclusions: This study showed that algae extract derived from C. reinhardtii is similar, if not superior, to commercially available yeast extract in nutrient content and effects on the growth and metabolism of E. coli and S. cerevisiae. Bacto™ yeast extract is valued at USD $0.15–0.35 per gram, if algae extract was sold at similar prices, it would serve as a high-value co-product in algae-based fuel processes.

  5. Metabolic flux analysis of heterotrophic growth in Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Nanette R Boyle

    Full Text Available Despite the wealth of knowledge available for C. reinhardtii, the central metabolic fluxes of growth on acetate have not yet been determined. In this study, 13C-metabolic flux analysis (13C-MFA was used to determine and quantify the metabolic pathways of primary metabolism in C. reinhardtii cells grown under heterotrophic conditions with acetate as the sole carbon source. Isotopic labeling patterns of compartment specific biomass derived metabolites were used to calculate the fluxes. It was found that acetate is ligated with coenzyme A in the three subcellular compartments (cytosol, mitochondria and plastid included in the model. Two citrate synthases were found to potentially be involved in acetyl-coA metabolism; one localized in the mitochondria and the other acting outside the mitochondria. Labeling patterns demonstrate that Acetyl-coA synthesized in the plastid is directly incorporated in synthesis of fatty acids. Despite having a complete TCA cycle in the mitochondria, it was also found that a majority of the malate flux is shuttled to the cytosol and plastid where it is converted to oxaloacetate providing reducing equivalents to these compartments. When compared to predictions by flux balance analysis, fluxes measured with 13C-MFA were found to be suboptimal with respect to biomass yield; C. reinhardtii sacrifices biomass yield to produce ATP and reducing equivalents.

  6. Measurement of Lipid Droplet Accumulation Kinetics in Chlamydomonas reinhardtii Using Seoul-Fluor

    Directory of Open Access Journals (Sweden)

    Noo Li Jeon

    2013-10-01

    Full Text Available Alternative energy resources have become an important issue due to the limited stocks of petroleum-based fuel. Microalgae, a source of renewable biodiesel, use solar light to convert CO2 into lipid droplets (LDs. Quantification of LDs in microalgae is required for developing and optimizing algal bioprocess engineering. However, conventional quantification methods are both time and labor-intensive and difficult to apply in high-throughput screening systems. LDs in plant and mammalian cells can be visualized by staining with various fluorescence probes such as the Nile Red, BODIPY, and Seoul-Fluor (SF series. This report describes the optimization of LD staining in Chlamydomonas reinhardtii with SF probes via systematic variations of dye concentration, staining time, temperature, and pH. A protocol for quantitative measurement of accumulation kinetics of LDs in C. reinhardtii was developed using a spectrofluorimeter and the accuracy of LD size measurement was confirmed by transmission electron microscopy (TEM. Our results indicate that our spectrofluorimeter-based measurement approach can monitor kinetics of intracellular LDs (in control and nitrogen-source-starved Chlamydomonas reinhardtii accumulation that has not been possible in the case of conventional imaging-based methods. Our results presented here confirmed that an SF44 can be a powerful tool for in situ monitoring and tracking of intracellular LDs formation.

  7. Triclosan-induced transcriptional and biochemical alterations in the freshwater green algae Chlamydomonas reinhardtii.

    Science.gov (United States)

    Pan, Chang-Gui; Peng, Feng-Jiao; Shi, Wen-Jun; Hu, Li-Xin; Wei, Xiao-Dong; Ying, Guang-Guo

    2018-02-01

    Triclosan (TCS) is an antibacterial and antifungal agent widely used in personal care products (PCPs). We investigated the effects of TCS (20μg/L, 100μg/L and 500μg/L) on Chlamydomonas reinhardtii by measuring the algal growth, chlorophyll content, lipid peroxidation, and transcription of the antioxidant-related genes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), glutathione S-transferase (GST), plastid terminal oxidase 2 (PTOX) and thioredoxin (TRX)) as well as biochemical alterations. The results showed significant dose-related effects of TCS on the algal species in terms of growth and chlorophyll content. Malondialdehyde (MDA) increased with increasing TCS concentrations and showed significant difference between the treatment of 405.3μg/L TCS and control group. Transcription analysis revealed that the expression of SOD mRNA was most sensitive to TCS among the selected genes. In addition, Fourier-transform infrared spectroscopy showed time- and concentration-specific biochemical responses in C. reinhardtii when exposed to TCS. The biochemical alterations associated with different doses of TCS were mainly attributed to structural changes associated with lipid, protein, nucleic acid and carbohydrate. The findings from this study reveal that TCS in the aquatic environment may affect algal growth, chlorophyll synthesis, oxidative stress responses and cause biochemical alterations. This study provided important information to achieve a better understanding of the toxic mechanism of triclosan on algae Chlamydomonas reinhardtii. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. An efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Pratheesh, P T; Vineetha, M; Kurup, G Muraleedhara

    2014-06-01

    Algal-based recombinant protein production has gained immense interest in recent years. The development of algal expression system was earlier hindered due to the lack of efficient and cost-effective transformation techniques capable of heterologous gene integration and expression. The recent development of Agrobacterium-mediated genetic transformation method is expected to be the ideal solution for these problems. We have developed an efficient protocol for the Agrobacterium-mediated genetic transformation of microalga Chlamydomonas reinhardtii. Pre-treatment of Agrobacterium in TAP induction medium (pH 5.2) containing 100 μM acetosyringone and 1 mM glycine betaine and infection of Chlamydomonas with the induced Agrobacterium greatly improved transformation frequency. This protocol was found to double the number of transgenic events on selection media compared to that of previous reports. PCR was used successfully to amplify fragments of the hpt and GUS genes from transformed cells, while Southern blot confirmed the integration of GUS gene into the genome of C. reinhardtii. RT-PCR, Northern blot and GUS histochemical analyses confirm GUS gene expression in the transgenic cell lines of Chlamydomonas. This protocol provides a quick, efficient, economical and high-frequency transformation method for microalgae.

  9. Metabolism of xenobiotics by Chlamydomonas reinhardtii: Phenol degradation under conditions affecting photosynthesis.

    Science.gov (United States)

    Nazos, Theocharis T; Kokarakis, Emmanouel J; Ghanotakis, Demetrios F

    2017-01-01

    In the present work, the biodegradation of phenol by axenic cultures of the unicellular microalga Chlamydomonas reinhardtii was investigated. Biodegradation proved to be a dynamic bioenergetic process, affected by changes in the culture conditions. Microalgae biodegraded defined amounts of phenol, as a result of the induced stress caused at high concentrations, despite the fact that this process proved to be energy demanding and thus affected growth of the culture. High levels of biodegradation were observed both in the absence of an alternative carbon source and in the presence of acetate as a carbon source. Biodegradation of phenol by Chlamydomonas proved to be an aerobic, photoregulated process. This is the first time that Chlamydomonas reinhardtii has been used for bioremediation purposes. This study has demonstrated that the most important factor in the biodegradation of phenol is the selection of the appropriate culture conditions (presence or absence of alternative carbon source, light intensity, and oxygen availability) that provide the best bioenergetic balance among growth, induced stress, and biodegradation of phenol.

  10. Synthesizing and Salvaging NAD+: Lessons Learned from Chlamydomonas reinhardtii

    Science.gov (United States)

    Lin, Huawen; Kwan, Alan L.; Dutcher, Susan K.

    2010-01-01

    The essential coenzyme nicotinamide adenine dinucleotide (NAD+) plays important roles in metabolic reactions and cell regulation in all organisms. Bacteria, fungi, plants, and animals use different pathways to synthesize NAD+. Our molecular and genetic data demonstrate that in the unicellular green alga Chlamydomonas NAD+ is synthesized from aspartate (de novo synthesis), as in plants, or nicotinamide, as in mammals (salvage synthesis). The de novo pathway requires five different enzymes: L-aspartate oxidase (ASO), quinolinate synthetase (QS), quinolate phosphoribosyltransferase (QPT), nicotinate/nicotinamide mononucleotide adenylyltransferase (NMNAT), and NAD+ synthetase (NS). Sequence similarity searches, gene isolation and sequencing of mutant loci indicate that mutations in each enzyme result in a nicotinamide-requiring mutant phenotype in the previously isolated nic mutants. We rescued the mutant phenotype by the introduction of BAC DNA (nic2-1 and nic13-1) or plasmids with cloned genes (nic1-1 and nic15-1) into the mutants. NMNAT, which is also in the de novo pathway, and nicotinamide phosphoribosyltransferase (NAMPT) constitute the nicotinamide-dependent salvage pathway. A mutation in NAMPT (npt1-1) has no obvious growth defect and is not nicotinamide-dependent. However, double mutant strains with the npt1-1 mutation and any of the nic mutations are inviable. When the de novo pathway is inactive, the salvage pathway is essential to Chlamydomonas for the synthesis of NAD+. A homolog of the human SIRT6-like gene, SRT2, is upregulated in the NS mutant, which shows a longer vegetative life span than wild-type cells. Our results suggest that Chlamydomonas is an excellent model system to study NAD+ metabolism and cell longevity. PMID:20838591

  11. Katanin localization requires triplet microtubules in Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Jessica M Esparza

    Full Text Available Centrioles and basal bodies are essential for a variety of cellular processes that include the recruitment of proteins to these structures for both centrosomal and ciliary function. This recruitment is compromised when centriole/basal body assembly is defective. Mutations that cause basal body assembly defects confer supersensitivity to Taxol. These include bld2, bld10, bld12, uni3, vfl1, vfl2, and vfl3. Flagellar motility mutants do not confer sensitivity with the exception of mutations in the p60 (pf19 and p80 (pf15 subunits of the microtubule severing protein katanin. We have identified additional pf15 and bld2 (ε-tubulin alleles in screens for Taxol sensitivity. Null pf15 and bld2 alleles are viable and are not essential genes in Chlamydomonas. Analysis of double mutant strains with the pf15-3 and bld2-6 null alleles suggests that basal bodies in Chlamydomonas may recruit additional proteins beyond katanin that affect spindle microtubule stability. The bld2-5 allele is a hypomorphic allele and its phenotype is modulated by nutritional cues. Basal bodies in bld2-5 cells are missing proximal ends. The basal body mutants show aberrant localization of an epitope-tagged p80 subunit of katanin. Unlike IFT proteins, katanin p80 does not localize to the transition fibers of the basal bodies based on an analysis of the uni1 mutant as well as the lack of colocalization of katanin p80 with IFT74. We suggest that the triplet microtubules are likely to play a key role in katanin p80 recruitment to the basal body of Chlamydomonas rather than the transition fibers that are needed for IFT localization.

  12. Phosphopantetheinylation in the green microalgae Chlamydomonas reinhardtii

    DEFF Research Database (Denmark)

    Sonnenschein, Eva; Pu, Yuan; Beld, Joris

    2016-01-01

    is responsible for holding the product. Fatty acid biosynthesis is initiated through posttranslational modification of the ACP by the phosphopantetheinyl transferase (PPTase). We identified two PPTases, PptC1 and PptC2, in the model alga Chlamydomonas reinhardtii by genome analysis and phylogenetic...... and structural comparison. Both PPTases are of Sfp-type, the archetypical PPTase type for non-ribosomal peptide and polyketide biosynthetic pathways in bacteria and cyanobacteria. In vitro analysis revealed that PptC2 has a broader substrate range than PptC1. Both PPTases were able to activate the cognate ACP...... of the type II FAS, while PptC2 also recognized ACP of Escherichia coli type II FAS and actinorhodin type II polyketide synthase. Besides FAS as PPTase target, the C. reinhardtii genome encodes a single type I PKS, and we hypothesize that PptC2 is responsible for its activation. Screening of the currently...

  13. Trophic transfer of gold nanoparticles from Euglena gracilis or Chlamydomonas reinhardtii to Daphnia magna.

    Science.gov (United States)

    Lee, Woo-Mi; Yoon, Sung-Ji; Shin, Yu-Jin; An, Youn-Joo

    2015-06-01

    Understanding the trophic transfer of nanoparticles (NPs) is important because NPs are small enough to easily penetrate into organisms. In this study, we evaluated the trophic transfer of gold NPs (AuNPs) within the aquatic food chain. We observed AuNPs transfer from 2 species of primary producers (Chlamydomonas reinhardtii or Euglena gracilis) to the primary consumer (Daphnia magna). Also, bioaccumulation of AuNPs in E. gracilis was higher than that in C. reinhardtii. The reasons for the difference in Au accumulation may be the physical structure of these organisms, and the surface area that is available for interaction with NPs. C. reinhardtii has a cell wall that may act as a barrier to the penetration of NPs. The size of E. gracilis is larger than that of C. reinhardtii. This study demonstrates the trophic transfer of AuNPs from a general producer to a consumer in an aquatic environment. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Phototaxis beyond turning: persistent accumulation and response acclimation of the microalga Chlamydomonas reinhardtii

    CERN Document Server

    Arrieta, Jorge; Chioccioli, Maurizio; Polin, Marco; Tuval, Idan

    2016-01-01

    Phototaxis is an important reaction to light displayed by a wide range of motile microorganisms. Flagellated eukaryotic microalgae in particular, like the model organism Chlamydomonas reinhardtii, steer either towards or away from light by a rapid and precisely timed modulation of their flagellar activity. Cell steering, however, is only the beginning of a much longer process which ultimately allows cells to determine their light exposure history. This process is not well understood. Here we present a first quantitative study of the long timescale phototactic motility of Chlamydomonas at both single cell and population levels. Our results reveal that the phototactic strategy adopted by these microorganisms leads to an efficient exposure to light, and that the phototactic response is modulated over typical timescales of tens of seconds. The adaptation dynamics for phototaxis and chlorophyll fluorescence show a striking quantitative agreement, suggesting that photosynthesis controls quantitatively how cells nav...

  15. Protocol: methodology for chromatin immunoprecipitation (ChIP in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Strenkert Daniela

    2011-11-01

    Full Text Available Abstract We report on a detailed chromatin immunoprecipitation (ChIP protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR.

  16. CrMAPK3 regulates the expression of iron-deficiency-responsive genes in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Fei, Xiaowen; Yu, Junmei; Li, Yajun; Deng, Xiaodong

    2017-05-16

    Under iron-deficient conditions, Chlamydomonas exhibits high affinity for iron absorption. Nevertheless, the response, transmission, and regulation of downstream gene expression in algae cells have not to be investigated. Considering that the MAPK pathway is essential for abiotic stress responses, we determined whether this pathway is involved in iron deficiency signal transduction in Chlamydomonas. Arabidopsis MAPK gene sequences were used as entry data to search for homologous genes in Chlamydomonas reinhardtii genome database to investigate the functions of mitogen-activated protein kinase (MAPK) gene family in C. reinhardtii under iron-free conditions. Results revealed 16 C. reinhardtii MAPK genes labeled CrMAPK2-CrMAPK17 with TXY conserved domains and low homology to MAPK in yeast, Arabidopsis, and humans. The expression levels of these genes were then analyzed through qRT-PCR and exposure to high salt (150 mM NaCl), low nitrogen, or iron-free conditions. The expression levels of these genes were also subjected to adverse stress conditions. The mRNA levels of CrMAPK2, CrMAPK3, CrMAPK4, CrMAPK5, CrMAPK6, CrMAPK8, CrMAPK9, and CrMAPK11 were remarkably upregulated under iron-deficient stress. The increase in CrMAPK3 expression was 43-fold greater than that in the control. An RNA interference vector was constructed and transformed into C. reinhardtii 2A38, an algal strain with an exogenous FOX1:ARS chimeric gene, to silence CrMAPK3. After this gene was silenced, the mRNA levels and ARS activities of FOX1:ARS chimeric gene and endogenous CrFOX1 were decreased. The mRNA levels of iron-responsive genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were also remarkably reduced. CrMAPK3 regulates the expression of iron-deficiency-responsive genes in C. reinhardtii.

  17. Stimulation of growth and photosynthetic carbon metabolism in Chlamydomonas reinhardtii with triacontanol

    Energy Technology Data Exchange (ETDEWEB)

    Houtz, R.L.

    1985-01-01

    Treatment of Chlamydomonas reinhardtii Dangeard cells (-, strain N. 90), cultured at 5% CO/sub 2/, with 1 to 1000 ..mu..g/L triacontanol (TRIA) resulted in a 21% to 35% increase in cell density, 7% to 31% increase in total chlorophyll, and 20% to 100% increase in photosynthetic CO/sub 2/ assimilation. Chlamydomonas cells responded to a broad range of TRIA concentrations that were at least 10-fold above the optimum concentration for higher plants. Octacosanol inhibited the effect of TRIA on photosynthetic CO/sub 2/ assimilation. TRIA did not alter glycolate excretion, the CO/sub 2/ compensation point or sensitivity of photosynthetic CO/sub 2/ assimilation to O/sub 2/ in Chlamydomonas. Kinetic analysis of TRIA-treated cells showed that the increase in photosynthetic CO/sub 2/ assimilation was a result of an increase in the whole-cell apparent Vmax. The activity of RuBP carboxylase/oxygenase was significantly higher in cell lysates from TRIA-treated cells than those from control cells. However, quantification of RuBP carboxylase/oxygenase levels by /sup 14/CABP binding did not show increased enzyme levels in TRIA-treated cells. Therefore, there was an increase in the specific activity of RuBP carboxylase/oxygenase extracted from Chlamydomonas cells treated with TRIA. TRIA alone had no effect in vitro on the activity of RuBPcarboxylase/oxygenase purified from spinach (Spinacia oleracea) leaves or from cell lysates of Chlamydomonas. RuBP levels were significantly higher in TRIA-treated cells at high and low CO/sub 2/. Increased RuBP levels in TRIA-treated Chlamydomonas cells were also observed in the absence of CO/sub 2/ with atmospheres of N/sub 2/ and 21% O/sub 2/.

  18. Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii

    Science.gov (United States)

    2013-01-01

    Background Oils produced by microalgae are precursors to biodiesel. To achieve a profitable production of biodiesel from microalgae, identification of factors governing oil synthesis and turnover is desirable. The green microalga Chlamydomonas reinhardtii is amenable to genetic analyses and has recently emerged as a model to study oil metabolism. However, a detailed method to isolate various types of oil mutants that is adapted to Chlamydomonas has not been reported. Results We describe here a forward genetic approach to isolate mutants altered in oil synthesis and turnover from C. reinhardtii. It consists of a three-step screening procedure: a primary screen by flow cytometry of Nile red stained transformants grown in 96-deep-well plates under three sequential conditions (presence of nitrogen, then absence of nitrogen, followed by oil remobilization); a confirmation step using Nile red stained biological triplicates; and a validation step consisting of the quantification by thin layer chromatography of oil content of selected strains. Thirty-one mutants were isolated by screening 1,800 transformants generated by random insertional mutagenesis (1.7%). Five showed increased oil accumulation under the nitrogen-replete condition and 13 had altered oil content under nitrogen-depletion. All mutants were affected in oil remobilization. Conclusion This study demonstrates that various types of oil mutants can be isolated in Chlamydomonas based on the method set-up here, including mutants accumulating oil under optimal biomass growth. The strategy conceived and the protocol set-up should be applicable to other microalgal species such as Nannochloropsis and Chlorella, thus serving as a useful tool in Chlamydomonas oil research and algal biotechnology. PMID:24295516

  19. Cross-reconstitution of the extrinsic proteins and photosystem II complexes from Chlamydomonas reinhardtii and Spinacia oleracea.

    Science.gov (United States)

    Suzuki, T; Ohta, H; Enami, I

    2005-06-01

    Cross-reconstitution of the extrinsic proteins and Photosystem II (PS II) from a green alga, Chlamydomonas reinhardtii, and a higher plant,Spinacia oleracea, was performed to clarify the differences of binding properties of the extrinsic proteins between these two species of organisms. (1) Chlamydomonas PsbP and PsbQ directly bound to Chlamydomonas PS II independent of the other extrinsic proteins but not to spinach PS II. (2) Chlamydomonas PsbP and PsbQ directly bound to the functional sites of Chlamydomonas PS II independent of the origins of PsbO, while spinach PsbP and PsbQ only bound to non-functional sites on Chlamydomonas PS II. (3) Both Chlamydomonas PsbP and spinach PsbP functionally bound to spinach PS II in the presence of spinach PsbO. (4) While Chlamydomonas PsbP functionally bound to spinach PS II in the presence of Chlamydomonas PsbO, spinach PsbP bound loosely to spinach PS II in the presence of Chlamydomonas PsbO with no concomitant restoration of oxygen evolution. (5) Chlamydomonas PsbQ bound to spinach PS II in the presence of Chlamydomonas PsbP and PsbO or spinach PsbO but not to spinach PS II in the presence of spinach PsbP and Chlamydomonas PsbO or spinach PsbO. (6) Spinach PsbQ did not bind to spinach PS II in the presence of Chlamydomonas PsbO and PsbP. On the basis of these results, we showed a simplified scheme for binding patterns of the green algal and higher plant extrinsic proteins with respective PS II.

  20. Photosynthetic efficiency and oxygen evolution of Chlamydomonas reinhardtii under continuous and flashing light.

    Science.gov (United States)

    Vejrazka, Carsten; Janssen, Marcel; Benvenuti, Giulia; Streefland, Mathieu; Wijffels, René H

    2013-02-01

    As a result of mixing and light attenuation in a photobioreactor (PBR), microalgae experience light/dark (L/D) cycles that can enhance PBR efficiency. One parameter which characterizes L/D cycles is the duty cycle; it determines the time fraction algae spend in the light. The objective of this study was to determine the influence of different duty cycles on oxygen yield on absorbed light energy and photosynthetic oxygen evolution. Net oxygen evolution of Chlamydomonas reinhardtii was measured for four duty cycles (0.05, 0.1, 0.2, and 0.5) in a biological oxygen monitor (BOM). Oversaturating light flashes were applied in a square-wave fashion with four flash frequencies (5, 10, 50, and 100 Hz). Algae were precultivated in a turbidostat and acclimated to a low photon flux density (PFD). A photosynthesis-irradiance (PI) curve was measured under continuous illumination and used to calculate the net oxygen yield, which was maximal between a PFD of 100 and 200 μmol m⁻² s⁻¹. Net oxygen yield under flashing light was duty cycle-dependent: the highest yield was observed at a duty cycle of 0.1 (i.e., time-averaged PFD of 115 μmol m⁻² s⁻¹). At lower duty cycles, maintenance respiration reduced net oxygen yield. At higher duty cycles, photon absorption rate exceeded the maximal photon utilization rate, and, as a result, surplus light energy was dissipated which led to a reduction in net oxygen yield. This behavior was identical with the observation under continuous light. Based on these data, the optimal balance between oxygen yield and production rate can be determined to maximize PBR productivity.

  1. Characterization of Hydrocortisone Biometabolites and 18S rRNA Gene in Chlamydomonas reinhardtii Cultures

    Directory of Open Access Journals (Sweden)

    Seyed Bagher Mosavi-Azam

    2008-10-01

    Full Text Available A unicellular microalga, Chlamydomonas reinhardtii, was isolated from rice paddy-field soil and water samples and used in the biotransformation of hydrocortisone (1. This strain has not been previously tested for steroid bioconversion. Fermentation was carried out in BG-11 medium supplemented with 0.05% substrate at 25ºC for 14 days of incubation. The products obtained were chromatographically purified and characterized using spectroscopic methods. 11b,17b-Dihydroxyandrost-4-en-3-one (2, 11b-hydroxyandrost-4-en-3,17-dione (3, 11b,17a,20b,21-tetrahydroxypregn-4-en-3-one (4 and prednisolone (5 were the main products of the bioconversion. The observed bioreaction features were the side chain degradation of the substrate to give compounds 2 and 3 and the 20-ketone reduction and 1,2-dehydrogenation affording compounds 4 and 5, respectively. A time course study showed the accumulation of product 2 from the second day of the fermentation and of compounds 3, 4 and 5 from the third day. All the metabolites reached their maximum concentration in seven days. Microalgal 18S rRNA gene was also amplified by PCR. PCR products were sequenced to confirm their authenticity as 18S rRNA gene of microalgae. The result of PCR blasted with other sequenced microalgae in NCBI showed 100% homology to the 18S small subunit rRNA of two Chlamydomonas reinhardtii spp.

  2. Chlamydomonas reinhardtii LFO1 Is an IsdG Family Heme Oxygenase.

    Science.gov (United States)

    Lojek, Lisa J; Farrand, Allison J; Wisecaver, Jennifer H; Blaby-Haas, Crysten E; Michel, Brian W; Merchant, Sabeeha S; Rokas, Antonis; Skaar, Eric P

    2017-01-01

    Heme is essential for respiration across all domains of life. However, heme accumulation can lead to toxicity if cells are unable to either degrade or export heme or its toxic by-products. Under aerobic conditions, heme degradation is performed by heme oxygenases, enzymes which utilize oxygen to cleave the tetrapyrrole ring of heme. The HO-1 family of heme oxygenases has been identified in both bacterial and eukaryotic cells, whereas the IsdG family has thus far been described only in bacteria. We identified a hypothetical protein in the eukaryotic green alga Chlamydomonas reinhardtii, which encodes a protein containing an antibiotic biosynthesis monooxygenase (ABM) domain consistent with those associated with IsdG family members. This protein, which we have named LFO1, degrades heme, contains similarities in predicted secondary structures to IsdG family members, and retains the functionally conserved catalytic residues found in all IsdG family heme oxygenases. These data establish LFO1 as an IsdG family member and extend our knowledge of the distribution of IsdG family members beyond bacteria. To gain further insight into the distribution of the IsdG family, we used the LFO1 sequence to identify 866 IsdG family members, including representatives from all domains of life. These results indicate that the distribution of IsdG family heme oxygenases is more expansive than previously appreciated, underscoring the broad relevance of this enzyme family. IMPORTANCE This work establishes a protein in the freshwater alga Chlamydomonas reinhardtii as an IsdG family heme oxygenase. This protein, LFO1, exhibits predicted secondary structure and catalytic residues conserved in IsdG family members, in addition to a chloroplast localization sequence. Additionally, the catabolite that results from the degradation of heme by LFO1 is distinct from that of other heme degradation products. Using LFO1 as a seed, we performed phylogenetic analysis, revealing that the IsdG family is

  3. Towards elucidation of the toxic mechanism of copper on the model green alga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Jiang, Yongguang; Zhu, Yanli; Hu, Zhangli; Lei, Anping; Wang, Jiangxin

    2016-09-01

    Toxic effects of copper on aquatic organisms in polluted water bodies have garnered particular attention in recent years. Microalgae play an important role in aquatic ecosystems, and they are sensitive to heavy metal pollution. Thus, it is important to clarify the mechanism of copper toxicity first for ecotoxicology studies. In this study, the physiological, biochemical and gene expression characteristics of a model green microalga, Chlamydomonas reinhardtii, with 0, 50, 150 and 250 μM copper treatments were investigated. The response of C. reinhardtii to copper stress was significantly shown at a dose dependent manner. Inhibition of cell growth and variation of total chlorophyll content were observed with copper treatments. The maximum photochemical efficiency of PSII, actual photochemical efficiency of PSII and photochemical quenching value decreased in the 250 μM copper treatment with minimum values equal to 28, 24 and 60 % of the control values respectively. The content of lipid peroxidation biomarker malondialdehyde with copper treatments increased with a maximum value sevenfold higher than the control value. Inhibition of cell growth and photosynthesis was ascribed to peroxidation of membrane lipids. The glutathione content and activities of antioxidant enzymes, glutathione S-transferase, glutathione peroxidase, superoxide dismutase and peroxidase were induced by copper. Interestingly, the expression of antioxidant genes and the photosynthetic gene decreased in most copper treatments. In conclusion, oxidative stress caused by production of excess reactive oxidative species might be the major mechanism of copper toxicity on C. reinhardtii.

  4. Light Intensity is Important for Hydrogen Production in NaHSO3-Treated Chlamydomonas reinhardtii.

    Science.gov (United States)

    Wei, Lanzhen; Yi, Jing; Wang, Lianjun; Huang, Tingting; Gao, Fudan; Wang, Quanxi; Ma, Weimin

    2017-03-01

    Chlamydomonas reinhardtii is a unicellular green alga that can use light energy to produce H2 from H2O in the background of NaHSO3 treatment. However, the role of light intensity in such H2 production remains elusive. Here, light intensity significantly affected the yield of H2 production in NaHSO3-treated C. reinhardtii, which was consistent with its effects on the content of O2 and the expression and activity of hydrogenase. Further, NaHSO3 was found to be able to remove O2 via a reaction of bisulfite with superoxide anion produced at the acceptor side of PSI, and light intensity affected the reaction rate significantly. Accordingly, high light and strong light but not low light can create an anaerobic environment, which is important to activate hydrogenase and produce H2. Based on the above results, we conclude that light intensity plays an important role in removing O2 and consequently activating hydrogenase and producing H2 in NaHSO3-treated C. reinhardtii. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. Binding of recombinant protein disulfide isomerase I (PDII) from Chlamydomonas reinhardtii to the 5' untranslated region of Chlamydomonas rbcL transcripts

    OpenAIRE

    Warsame, Ahmed Mohamed Ali

    2011-01-01

    Chlamydomonas reinhardtii is a widely used model organism to study chloroplast gene expression and photosynthetic processes. In this project we worked to identify a novel trans-acting factor that is thought to contribute to the stability of rbcL mRNA. These types of factors are thought to bind to specific cis-sequence elements and protect the transcript from nuclease degradation. Experimental studies have proven that the 5‟untranslated region of Chlamydomonas rbcL mRNA plays a key role in ...

  6. Gene regulation of iron-deficiency responses is associated with carbon monoxide and heme oxydase 1 in Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Zhang Liping

    Full Text Available Carbon monoxide (CO as an endogenous gaseous molecule regulates a variety of biological processes in animals. However, CO regulating nutrient stress responses in green alga is largely unknown. On the other hand, heme oxydase (HO1 as a rate-limiting enzyme of the first step for heme degration and to catalyze heme into biliverdin (BV, which is concomitant with releasing of CO and ferrous ions, probably participates in the process of CO-regulating response to nutrient stress in green alga. In this paper, we described an observation that CO could regulate iron-homeostasis in iron-starving Chlamydomonas reinhardtii. Exogenous CO at 8 µM was able to prevent the iron deficient-inducing chlorosis and improve chlorophyll accumulation. Expression pattern of FOX1, FTR1 and ferredoxin was up-regulated by CO exposure in iron-deficient mediam. treatment with external CO increasing iron accumulation in iron-deficient C. reinhardtii. Moreover, to get insights into the regulatory role of HO1, we constructed a transgenic alga overexpressing HO1 and HO1 knock-out mutants. The results show that there was no significant influence on chlorosis with HO1 overexpression of C. reinhardtii under iron-deficiency and the chlorophyll accumulation, and gene expression associated with iron deficiency of mutant were greatly improved. Otherwise, those results from HO1 knock-out mutants were opposite to HO1 overexpression mutants. Finally, CO exposure induced NO accumulation in cells. However, such an action could be blocked by NO scavenger cPTIO. These results indicate that CO/HO1 may play an important role in improving green algae adaptation to iron deficiency or cross-talking with NO under the iron deficiency.

  7. Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles.

    Science.gov (United States)

    Barwal, Indu; Ranjan, Peeyush; Kateriya, Suneel; Yadav, Subhash Chandra

    2011-12-07

    Elucidation of molecular mechanism of silver nanoparticles (SNPs) biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution) of SNPs. The unicellular algae Chlamydomonas reinhardtii was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis. The C. reinhardtii cell free extract (in vitro) and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro) SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP⁺ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro. Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver nanoparticles using C. reinhardtii as a model system.

  8. Cellular oxido-reductive proteins of Chlamydomonas reinhardtii control the biosynthesis of silver nanoparticles

    Directory of Open Access Journals (Sweden)

    Barwal Indu

    2011-12-01

    Full Text Available Abstract Background Elucidation of molecular mechanism of silver nanoparticles (SNPs biosynthesis is important to control its size, shape and monodispersity. The evaluation of molecular mechanism of biosynthesis of SNPs is of prime importance for the commercialization and methodology development for controlling the shape and size (uniform distribution of SNPs. The unicellular algae Chlamydomonas reinhardtii was exploited as a model system to elucidate the role of cellular proteins in SNPs biosynthesis. Results The C. reinhardtii cell free extract (in vitro and in vivo cells mediated synthesis of silver nanoparticles reveals SNPs of size range 5 ± 1 to 15 ± 2 nm and 5 ± 1 to 35 ± 5 nm respectively. In vivo biosynthesized SNPs were localized in the peripheral cytoplasm and at one side of flagella root, the site of pathway of ATP transport and its synthesis related enzymes. This provides an evidence for the involvement of oxidoreductive proteins in biosynthesis and stabilization of SNPs. Alteration in size distribution and decrease of synthesis rate of SNPs in protein-depleted fractions confirmed the involvement of cellular proteins in SNPs biosynthesis. Spectroscopic and SDS-PAGE analysis indicate the association of various proteins on C. reinhardtii mediated in vivo and in vitro biosynthesized SNPs. We have identified various cellular proteins associated with biosynthesized (in vivo and in vitro SNPs by using MALDI-MS-MS, like ATP synthase, superoxide dismutase, carbonic anhydrase, ferredoxin-NADP+ reductase, histone etc. However, these proteins were not associated on the incubation of pre-synthesized silver nanoparticles in vitro. Conclusion Present study provides the indication of involvement of molecular machinery and various cellular proteins in the biosynthesis of silver nanoparticles. In this report, the study is mainly focused towards understanding the role of diverse cellular protein in the synthesis and capping of silver

  9. A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Fan, J.; Xu, C.; Andre, C.

    2011-06-23

    Neutral lipid metabolism has been extensively studied in yeast, plants and mammals. In contrast, little information is available regarding the biochemical pathway, enzymes and regulatory factors involved in the biosynthesis of triacylglycerol (TAG) in microalgae. In the conventional TAG biosynthetic pathway widely accepted for yeast, plants and mammals, TAG is assembled in the endoplasmic reticulum (ER) from its immediate precursor diacylglycerol (DAG) made by ER-specific acyltransferases, and is deposited exclusively in lipid droplets in the cytosol. Here, we demonstrated that the unicellular microalga Chlamydomonas reinhardtii employs a distinct pathway that uses DAG derived almost exclusively from the chloroplast to produce TAG. This unique TAG biosynthesis pathway is largely dependent on de novo fatty acid synthesis, and the TAG formed in this pathway is stored in lipid droplets in both the chloroplast and the cytosol. These findings have wide implications for understanding TAG biosynthesis and storage and other areas of lipid metabolism in microalgae and other organisms.

  10. Chlamydomonas reinhardtii: a protein expression system for pharmaceutical and biotechnological proteins.

    Science.gov (United States)

    Griesbeck, Christoph; Kobl, Iris; Heitzer, Markus

    2006-10-01

    Recombinant proteins have become more and more important for the pharmaceutical and chemical industry. Although various systems for protein expression have been developed, there is an increasing demand for inexpensive methods of large-scale production. Eukaryotic algae could serve as a novel option for the manufacturing of recombinant proteins, as they can be cultivated in a cheap and easy manner and grown to high cell densities. Being a model organism, the unicellular green alga Chlamydomonas reinhardtii has been studied intensively over the last decades and offers now a complete toolset for genetic manipulation. Recently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its ability for biotechnological applications.

  11. Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii (I) aerobic conditions.

    Science.gov (United States)

    Alric, Jean; Lavergne, Jérôme; Rappaport, Fabrice

    2010-01-01

    Assimilation of atmospheric CO2 by photosynthetic organisms such as plants, cyanobacteria and green algae, requires the production of ATP and NADPH in a ratio of 3:2. The oxygenic photosynthetic chain can function following two different modes: the linear electron flow which produces reducing power and ATP, and the cyclic electron flow which only produces ATP. Some regulation between the linear and cyclic flows is required for adjusting the stoichiometric production of high-energy bonds and reducing power. Here we explore, in the green alga Chlamydomonas reinhardtii, the onset of the cyclic electron flow during a continuous illumination under aerobic conditions. In mutants devoid of Rubisco or ATPase, where the reducing power cannot be used for carbon fixation, we observed a stimulation of the cyclic electron flow. The present data show that the cyclic electron flow can operate under aerobic conditions and support a simple competition model where the excess reducing power is recycled to match the demand for ATP.

  12. Real-time monitoring of genetically modified Chlamydomonas reinhardtii during the Foton M3 space mission

    Science.gov (United States)

    Lambreva, M.; Rea, G.; Antonacci, A.; Serafini, A.; Damasso, M.; Pastorelli, S.; Margonelli, A.; Johanningmeier, U.; Bertalan, I.; Pezzotti, G.; Giardi, M. T.

    2008-09-01

    Long-term space exploration, colonization or habitation requires biological life support systems capable to cope with the deleterious space environment. The use of oxygenic photosynthetic microrganisms is an intriguing possibility mainly for food, O2 and nutraceutical compounds production. The critical points of utilizing plants- or algae-based life support systems are the microgravity and the ionizing radiation, which can influence the performance of these organisms. The aim of the present study was to assess the effects of space environment on the photosynthetic activity of various microrganisms and to select space stresstolerant strains. Photosystem II D1 protein sitedirected and random mutants of the unicellular green alga Chlamydomonas reinhardtii [1] were used as a model system to test and select the amino acid substitutions capable to account for space stress tolerance. We focussed our studies also on the accumulation of the Photosystem II photoprotective carotenoids (the xantophylls violaxanthin, anteraxanthin and zeaxanthin), powerful antioxidants that epidemiological studies demonstrated to be human vision protectors. For this purpose some mutants modified at the level of enzymes involved in the biosynthesis of xanthophylls were included in the study [2]. To identify the consequences of the space environment on the photosynthetic apparatus the changes in the Photosystem II efficiency were monitored in real time during the ESA-Russian Foton- M3 mission in September 2007. For the space flight a high-tech, multicell fluorescence detector, Photo-II, was designed and built by the Centre for Advanced Research in Space Optics in collaboration with Kayser-Italy, Biosensor and DAS. Photo-II is an automatic device developed to measure the chlorophyll fluorescence and to provide a living conditions for several different algae strains (Fig.1). Twelve different C. reinhardti strains were analytically selected and two replications for each strain were brought to space

  13. Characterization of Chlamydomonas reinhardtii phosphatidylglycerophosphate synthase in Synechocystis sp. PCC 6803

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    Chun-Hsien eHung

    2015-08-01

    Full Text Available Phosphatidylglycerol (PG is an indispensable phospholipid class with photosynthetic function in plants and cyanobacteria. However, its biosynthesis in eukaryotic green microalgae is poorly studied. Here, we report the isolation and characterization of two homologs (CrPGP1 and CrPGP2 of phosphatidylglycerophosphate synthase (PGPS, the rate-limiting enzyme in PG biosynthesis, in Chlamydomonas reinhardtii. Heterologous complementation of Synechocystis sp. PCC 6803 pgsA mutant by CrPGP1 and CrPGP2 rescued the PG-dependent growth phenotype, but the PG level and its fatty acid composition were not fully rescued in the complemented strains. As well, oxygen evolution activity was not fully recovered, although electron transport activity of photosystem II was restored to the wild-type level. Gene expression study of CrPGP1 and CrPGP2 in nutrient-starved C. reinhardtii showed differential response to phosphorus and nitrogen deficiency. Taken together, these results highlight the distinct and overlapping function of PGPS in cyanobacteria and eukaryotic algae.

  14. Transcriptome Analysis of Manganese-deficient Chlamydomonas reinhardtii Provides Insight on the Chlorophyll Biosynthesis Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Lockhart, Ainsley; Zvenigorodsky, Natasha; Pedraza, Mary Ann; Lindquist, Erika

    2011-08-11

    The biosynthesis of chlorophyll and other tetrapyrroles is a vital but poorly understood process. Recent genomic advances with the unicellular green algae Chlamydomonas reinhardtii have created opportunity to more closely examine the mechanisms of the chlorophyll biosynthesis pathway via transcriptome analysis. Manganese is a nutrient of interest for complex reactions because of its multiple stable oxidation states and role in molecular oxygen coordination. C. reinhardtii was cultured in Manganese-deplete Tris-acetate-phosphate (TAP) media for 24 hours and used to create cDNA libraries for sequencing using Illumina TruSeq technology. Transcriptome analysis provided intriguing insight on possible regulatory mechanisms in the pathway. Evidence supports similarities of GTR (Glutamyl-tRNA synthase) to its Chlorella vulgaris homolog in terms of Mn requirements. Data was also suggestive of Mn-related compensatory up-regulation for pathway proteins CHLH1 (Manganese Chelatase), GUN4 (Magnesium chelatase activating protein), and POR1 (Light-dependent protochlorophyllide reductase). Intriguingly, data suggests possible reciprocal expression of oxygen dependent CPX1 (coproporphyrinogen III oxidase) and oxygen independent CPX2. Further analysis using RT-PCR could provide compelling evidence for several novel regulatory mechanisms in the chlorophyll biosynthesis pathway.

  15. Integration of carbon assimilation modes with photosynthetic light capture in the green alga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Berger, Hanna; Blifernez-Klassen, Olga; Ballottari, Matteo; Bassi, Roberto; Wobbe, Lutz; Kruse, Olaf

    2014-10-01

    The unicellular green alga Chlamydomonas reinhardtii is capable of using organic and inorganic carbon sources simultaneously, which requires the adjustment of photosynthetic activity to the prevailing mode of carbon assimilation. We obtained novel insights into the regulation of light-harvesting at photosystem II (PSII) following altered carbon source availability. In C. reinhardtii, synthesis of PSII-associated light-harvesting proteins (LHCBMs) is controlled by the cytosolic RNA-binding protein NAB1, which represses translation of particular LHCBM isoform transcripts. This mechanism is fine-tuned via regulation of the nuclear NAB1 promoter, which is activated when linear photosynthetic electron flow is restricted by CO(2)-limitation in a photoheterotrophic context. In the wild-type, accumulation of NAB1 reduces the functional PSII antenna size, thus preventing a harmful overexcited state of PSII, as observed in a NAB1-less mutant. We further demonstrate that translation control as a newly identified long-term response to prolonged CO(2)-limitation replaces LHCII state transitions as a fast response to PSII over-excitation. Intriguingly, activation of the long-term response is perturbed in state transition mutant stt7, suggesting a regulatory link between the long- and short-term response. We depict a regulatory circuit operating on distinct timescales and in different cellular compartments to fine-tune light-harvesting in photoheterotrophic eukaryotes. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.

  16. Methanol-Promoted Lipid Remodelling during Cooling Sustains Cryopreservation Survival of Chlamydomonas reinhardtii.

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    Duanpeng Yang

    Full Text Available Cryogenic treatments and cryoprotective agents (CPAs determine the survival rate of organisms that undergo cryopreservation, but their mechanisms of operation have not yet been characterised adequately. In particular, the way in which membrane lipids respond to cryogenic treatments and CPAs is unknown. We developed comparative profiles of the changes in membrane lipids among cryogenic treatments and between the CPAs dimethyl sulfoxide (DMSO and methanol (MeOH for the green alga Chlamydomonas reinhardtii. We found that freezing in liquid nitrogen led to a dramatic degradation of lipids, and that thawing at warm temperature (35°C induced lipid remodelling. DMSO did not protect membranes, but MeOH significantly attenuated lipid degradation. The presence of MeOH during cooling (from 25°C to -55°C at a rate of 1°C/min sustained the lipid composition to the extent that membrane integrity was maintained; this phenomenon accounts for successful cryopreservation. An increase in monogalactosyldiacylglycerol and a decrease in diacylglycerol were the major changes in lipid composition associated with survival rate, but there was no transformation between these lipid classes. Phospholipase D-mediated phosphatidic acid was not involved in freezing-induced lipid metabolism in C. reinhardtii. Lipid unsaturation changed, and the patterns of change depended on the cryogenic treatment. Our results provide new insights into the cryopreservation of, and the lipid metabolism in, algae.

  17. Valorization of Spent Escherichia coli Media Using Green Microalgae Chlamydomonas reinhardtii and Feedstock Production

    Directory of Open Access Journals (Sweden)

    Jian-Guo Zhang

    2017-06-01

    Full Text Available The coupling of Chlamydomonas reinhardtii biomass production for nutrients removal of Escherichia coli anaerobic broth (EAB is thought to be an economically feasible option for the cultivation of microalgae. The feasibility of growing microalgae in using EAB high in nutrients for the production of more biomass was examined. EAB comprised of nutrient-abundant effluents, which can be used to produce microalgae biomass and remove environment pollutant simultaneously. In this study, C. reinhardtii 21gr (cc1690 was cultivated in different diluted E. coli anaerobic broth supplemented with trace elements under mixotrophic and heterotrophic conditions. The results showed that C. reinhardtii grown in 1×, 1/2×, 1/5× and 1/10×E. coli anaerobic broth under mixotrophic conditions exhibited specific growth rates of 2.71, 2.68, 1.45, and 1.13 day-1, and biomass production of 201.9, 184.2, 175.5, and 163.8 mg L-1, respectively. Under heterotrophic conditions, the specific growth rates were 1.80, 1.86, 1.75, and 1.02 day-1, and biomass production were 45.6, 29.4, 15.8, and 12.1 mg L-1, respectively. The removal efficiency of chemical oxygen demand, total-nitrogen and total-phosphorus from 1×E. coli anaerobic broth was 21.51, 22.41, and 15.53%. Moreover, the dry biomass had relatively high carbohydrate (44.3% and lipid content (18.7%. Therefore, this study provides an environmentally sustainable as well economical method for biomass production in promising model microalgae and subsequently paves the way for industrial use.

  18. Spontaneous mutations in the ammonium transport gene AMT4 of Chlamydomonas reinhardtii.

    Science.gov (United States)

    Kim, Kwang-Seo; Feild, Eithne; King, Natalie; Yaoi, Takuro; Kustu, Sydney; Inwood, William

    2005-06-01

    Evidence in several microorganisms indicates that Amt proteins are gas channels for NH(3) and CH(3)NH(2), and this has been confirmed structurally. Chlamydomonas reinhardtii has at least four AMT genes, the most reported for a microorganism. Under nitrogen-limiting conditions all AMT genes are transcribed and Chlamydomonas is sensitive to methylammonium toxicity. All 16 spontaneous methylammonium-resistant mutants that we analyzed had defects in accumulation of [(14)C]methylammonium. Genetic crosses indicated that 12 had lesions in a single locus, whereas two each had lesions in other loci. Lesions in different loci were correlated with different degrees of defect in [(14)C]methylammonium uptake. One mutant in the largest class had an insert in the AMT4 gene, and the insert cosegregated with methylammonium resistance in genetic crosses. The other 11 strains in this class also had amt4 lesions, which we characterized at the molecular level. Properties of the amt4 mutants were clearly different from those of rh1 RNAi lines. They indicated that the physiological substrates for Amt and Rh proteins, the only two members of their protein superfamily, are NH(3) and CO(2), respectively.

  19. The contractile vacuole as a key regulator of cellular water flow in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Komsic-Buchmann, Karin; Wöstehoff, Luisa; Becker, Burkhard

    2014-11-01

    Most freshwater flagellates use contractile vacuoles (CVs) to expel excess water. We have used Chlamydomonas reinhardtii as a green model system to investigate CV function during adaptation to osmotic changes in culture medium. We show that the contractile vacuole in Chlamydomonas is regulated in two different ways. The size of the contractile vacuoles increases during cell growth, with the contraction interval strongly depending on the osmotic strength of the medium. In contrast, there are only small fluctuations in cytosolic osmolarity and plasma membrane permeability. Modeling of the CV membrane permeability indicates that only a small osmotic gradient is necessary for water flux into the CV, which most likely is facilitated by the aquaporin major intrinsic protein 1 (MIP1). We show that MIP1 is localized to the contractile vacuole, and that the expression rate and protein level of MIP1 exhibit only minor fluctuations under different osmotic conditions. In contrast, SEC6, a protein of the exocyst complex that is required for the water expulsion step, and a dynamin-like protein are upregulated under strong hypotonic conditions. The overexpression of a CreMIP1-GFP construct did not change the physiology of the CV. The functional implications of these results are discussed. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  20. The small molecule fenpropimorph rapidly converts chloroplast membrane lipids to triacylglycerols in Chlamydomonas reinhardtii

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    Hanul eKim

    2015-02-01

    Full Text Available Concern about global warming has prompted an intense interest in developing economical methods of producing biofuels. Microalgae provide a promising platform for biofuel production, because they accumulate high levels of lipids, and do not compete with food or feed sources. However, current methods of producing algal oil involve subjecting the microalgae to stress conditions, such as nitrogen deprivation, and are prohibitively expensive. Here, we report that the fungicide fenpropimorph rapidly causes high levels of neutral lipids to accumulate in Chlamydomonas reinhardtii cells. When treated with fenpropimorph (10 μg mL–1 for 1 h, Chlamydomonas cells accumulated at least four-fold the amount of triacylglycerols (TAGs present in the untreated control cells. Furthermore, the quantity of TAGs present after 1 h of fenpropimorph treatment was over two-fold higher than that formed after 9 days of nitrogen starvation in medium with no acetate supplement. Biochemical analysis of lipids revealed that the accumulated TAGs were derived mainly from chloroplast polar membrane lipids. Such a conversion of chloroplast polar lipids to TAGs is desirable for biodiesel production, because polar lipids are usually removed during the biodiesel production process. Thus, our data exemplified that a cost and time effective method of producing TAGs is possible using fenpropimorph or similar drugs.

  1. Introducing Dunaliella LIP promoter containing light-inducible motifs improves transgenic expression in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Baek, Kwangryul; Lee, Yew; Nam, Onyou; Park, Seunghye; Sim, Sang Jun; Jin, EonSeon

    2016-03-01

    Promoter of the light-inducible protein gene (LIP) of Dunaliella was recently isolated in our laboratory. The aim of this work is to find the light-inducible motif in the Dunaliella LIP promoter and verify its regulatory motif with a Gaussia luciferase reporter gene transformed in Chlamydomonas reinhardtii. 400 bp upstream to the translational start site of the Dunaliella LIP gene was gradually truncated and analyzed for the luciferase expression. Furthermore, this promoter comprising duplicated or triplicated light-responsive motifs was tested for its augmentation of light response. Two putative light-responsive motifs, GT-1 binding motif and sequences over-represented in light-repressed promoters (SORLIP) located in the 200 bp LIP promoter fragment were analyzed for their light responsibility. It is turned out that SORLIP was responsible for the light-inducible activity. With the copy number of SORLIP up to three showed stronger high light response compared with the native LIP promoter fragment. Therefore, we found a light-responsive DNA motif operating in Chlamydomonas and confirm a synthetic promoter including this motif displayed light inducibility in heterologously transformed green algae for the first time. This light-inducible expression system will be applied to various area of algal research including algal biotechnology. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Deletion of CGLD1 Impairs PSII and Increases Singlet Oxygen Tolerance of Green Alga Chlamydomonas reinhardtii

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    Jiale Xing

    2017-12-01

    Full Text Available The green alga Chlamydomonas reinhardtii is a key model organism for studying photosynthesis and oxidative stress in unicellular eukaryotes. Using a forward genetics approach, we have identified and characterized a mutant x32, which lacks a predicted protein named CGLD1 (Conserved in Green Lineage and Diatom 1 in GreenCut2, under normal and stress conditions. We show that loss of CGLD1 resulted in minimal photoautotrophic growth and PSII activity in the organism. We observed reduced amount of PSII complex and core subunits in the x32 mutant based on blue-native (BN/PAGE and immunoblot analysis. Moreover, x32 exhibited increased sensitivity to high-light stress and altered tolerance to different reactive oxygenic species (ROS stress treatments, i.e., decreased resistance to H2O2/or tert-Butyl hydroperoxide (t-BOOH and increased tolerance to neutral red (NR and rose bengal (RB that induce the formation of singlet oxygen, respectively. Further analysis via quantitative real-time PCR (qRT-PCR indicated that the increased singlet-oxygen tolerance of x32 was largely correlated with up-regulated gene expression of glutathione-S-transferases (GST. The phenotypical and physiological implications revealed from our experiments highlight the important roles of CGLD1 in maintaining structure and function of PSII as well as in protection of Chlamydomonas under photo-oxidative stress conditions.

  3. Characterization of DNA repair deficient strains of Chlamydomonas reinhardtii generated by insertional mutagenesis.

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    Andrea Plecenikova

    Full Text Available While the mechanisms governing DNA damage response and repair are fundamentally conserved, cross-kingdom comparisons indicate that they differ in many aspects due to differences in life-styles and developmental strategies. In photosynthetic organisms these differences have not been fully explored because gene-discovery approaches are mainly based on homology searches with known DDR/DNA repair proteins. Here we performed a forward genetic screen in the green algae Chlamydomonas reinhardtii to identify genes deficient in DDR/DNA repair. We isolated five insertional mutants that were sensitive to various genotoxic insults and two of them exhibited altered efficiency of transgene integration. To identify genomic regions disrupted in these mutants, we established a novel adaptor-ligation strategy for the efficient recovery of the insertion flanking sites. Four mutants harbored deletions that involved known DNA repair factors, DNA Pol zeta, DNA Pol theta, SAE2/COM1, and two neighbouring genes encoding ERCC1 and RAD17. Deletion in the last mutant spanned two Chlamydomonas-specific genes with unknown function, demonstrating the utility of this approach for discovering novel factors involved in genome maintenance.

  4. Novel shuttle markers for nuclear transformation of the green alga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Meslet-Cladière, Laurence; Vallon, Olivier

    2011-12-01

    The green alga Chlamydomonas reinhardtii today is a premier model organism for the study of green algae and plants. Yet the efficient engineering of its nuclear genome requires development of new antibiotic resistance markers. We have recoded, based on codon usage in the nuclear genome, the AadA marker that has been used previously for chloroplast transformation. The recoded AadA gene, placed under the control of the HSP70A-RBCS2 hybrid promoter and preceded by the RbcS2 chloroplast-targeting peptide, can be integrated into the nuclear genome by electroporation, conferring resistance to spectinomycin and streptomycin. Transformation efficiency is markedly increased when vector sequences are completely eliminated from the transforming DNA. Antibiotic resistance is stable for several months in the absence of selection pressure. Shuttle markers allowing selection in both Chlamydomonas and Escherichia coli would also be a useful asset. By placing an artificial bacterial promoter and Shine-Dalgarno sequence in frame within the AadA coding sequence, we generated such a shuttle marker. To our surprise, we found that the classical AphVIII construct already functions as a shuttle marker. Finally, we developed a method to introduce the AadA and AphVIII markers into the vector part of the bacterial artificial chromosomes (BACs) of the Chlamydomonas genomic DNA library. Our aim was to facilitate complementation studies whenever the test gene cannot be selected for directly. After transformation of a petC mutant with a modified BAC carrying the AphVIII marker along with the PETC gene in the insert, almost half of the paromomycin-resistant transformants obtained showed restoration of phototrophy, indicating successful integration of the unselected test gene. With AadA, cotransformation was also observed, but with a lower efficiency.

  5. Nucleotide diversity of the Chlamydomonas reinhardtii plastid genome: addressing the mutational-hazard hypothesis.

    Science.gov (United States)

    Smith, David Roy; Lee, Robert W

    2009-05-27

    The mutational-hazard hypothesis argues that the noncoding-DNA content of a genome is a consequence of the mutation rate (mu) and the effective number of genes per locus in the population (N(g)). The hypothesis predicts that genomes with a high N(g)mu will be more compact than those with a small N(g)mu. Approximations of N(g)mu can be gained by measuring the nucleotide diversity at silent sites (pi(silent)). We addressed the mutation-hazard hypothesis apropos plastid-genome evolution by measuring pi(silent) of the Chlamydomonas reinhardtii plastid DNA (ptDNA), the most noncoding-DNA-dense plastid genome observed to date. The data presented here in conjunction with previously published values of pi(silent) for the C. reinhardtii mitochondrial and nuclear genomes, which are respectively compact and bloated, allow for a complete analysis of nucleotide diversity and genome compactness in all three genetic compartments of this model organism. In C. reinhardtii, the mean estimate of pi(silent) for the ptDNA (14.5 x 10(-3)) is less than that of the nuclear DNA (32 x 10(-3)) and greater than that of the mitochondrial DNA (8.5 x 10(-3)). On average, C. reinhardtii has approximately 4 times more silent-site ptDNA diversity than the mean value reported for land plants, which have more compact plastid genomes. The silent-site nucleotide diversity of the different ptDNA loci that were studied varied significantly: from 0 to 71 x 10(-3) for synonymous sites and from 0 to 42 x 10(-3) for intergenic regions. Our findings on silent-site ptDNA diversity are inconsistent with what would be expected under the mutational-hazard hypothesis and go against the documented trend in other systems of pi(silent) positively correlating with genome compactness. Overall, we highlight the lack of reliable nucleotide-diversity measurements for ptDNA and hope that the values presented here will act as sound data for future research concerning the mutational-hazard hypothesis and plastid evolution in

  6. Pilot-scale cultivation of wall-deficient transgenic Chlamydomonas reinhardtii strains expressing recombinant proteins in the chloroplast

    DEFF Research Database (Denmark)

    Zedler, Julie A.Z.; Gangl, Doris; Guerra, Tiago

    2016-01-01

    Microalgae have emerged as potentially powerful platforms for the production of recombinant proteins and high-value products. Chlamydomonas reinhardtii is a potentially important host species due to the range of genetic tools that have been developed for this unicellular green alga. Transformation...... growth data for C. reinhardtii grown at pilot scale, and the growth of cell wall-deficient strains has not been reported at all. Here, we report the first pilot-scale growth study for transgenic, cell wall-deficient C. reinhardtii strains. Strains expressing a cytochrome P450 (CYP79A1) or bifunctional...... diterpene synthase (cis-abienol synthase, TPS4) were grown for 7 days under mixotrophic conditions in a Tris-acetate-phosphate medium. The strains reached dry cell weights of 0.3 g/L within 3–4 days with stable expression levels of the recombinant proteins during the whole upscaling process. The strains...

  7. Intact anthracene inhibits photosynthesis in algal cells: a fluorescence induction study on Chlamydomonas reinhardtii cw92 strain.

    Science.gov (United States)

    Aksmann, Anna; Tukaj, Zbigniew

    2008-12-01

    Short-term (24h) experiments were performed to examine the effect of anthracene (ANT) on Chlamydomonas reinhardtii cw92 grown in a batch culture system aerated with 2.5% CO(2). At concentrations ranging from 0.7 to 5.6 microM, ANT inhibited the growth of population in a concentration-dependent manner and EC(50) calculated amounted to 1.6 microM. At concentrations from 0.7 to 4.2 microM ANT stimulated respiration and inhibited the intensity of photosynthesis but did not affect chlorophyll content in the cells. ANT influenced chlorophyll a fluorescence parameters, measured by OJIP test (O, J, I and P are the different steps of fluorescence induction curve). ANT diminished the performance index (PI), the yield of primary photochemistry (phi(Po)), the yield of electron transport (phi(Epsilonomicron), the efficiency of moving the electron beyond Qa(-) (Psi(0)) and the fraction of active oxygen evolving complexes (OEC). The fraction of active PS II reaction centres in the treated samples dramatically dropped. The most pronounced changes in ANT-treated cells were observed in the stimulation of energy dissipation parameter (DI(0)/RC). The only OJIP parameter that was not influenced by ANT was energy absorption by photosynthetic antennae (ABS). The results lead to a conclusion that the inhibition of photosynthesis may be a consequence of unspecific ANT-membrane interaction, resulting from hydrophobic character of this hydrocarbon.

  8. Evolutionarily conserved Delta(25(27))-olefin ergosterol biosynthesis pathway in the alga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Miller, Matthew B; Haubrich, Brad A; Wang, Qian; Snell, William J; Nes, W David

    2012-08-01

    Ergosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ(24(28))-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphate not present in fungi. Here, we report that C. reinhardtii synthesizes the protosterol cycloartenol and converts it to ergosterol (C24β-methyl) and 7-dehydroporiferasterol (C24β-ethyl) through a highly conserved sterol C24- methylation-C25-reduction (Δ(25(27))-olefin) pathway that is distinct from the well-described acetate-mevalonate pathway to fungal lanosterol and its conversion to ergosterol by the Δ(24(28))-olefin pathway. We isolated and characterized 23 sterols by a combination of GC-MS and proton nuclear magnetic resonance spectroscopy analysis from a set of mutant, wild-type, and 25-thialanosterol-treated cells. The structure and stereochemistry of the final C24-alkyl sterol side chains possessed different combinations of 24β-methyl/ethyl groups and Δ(22(23))E and Δ(25(27))-double bond constructions. When incubated with [methyl-(2)H(3)]methionine, cells incorporated three (into ergosterol) or five (into 7-dehydroporiferasterol) deuterium atoms into the newly biosynthesized 24β-alkyl sterols, consistent only with a Δ(25(27))-olefin pathway. Thus, our findings demonstrate that two separate isoprenoid-24-alkyl sterol pathways evolved in fungi and green algae, both of which converge to yield a common membrane insert ergosterol.

  9. The biosynthesis of nitrous oxide in the green alga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Plouviez, Maxence; Wheeler, David; Shilton, Andy; Packer, Michael A; McLenachan, Patricia A; Sanz-Luque, Emanuel; Ocaña-Calahorro, Francisco; Fernández, Emilio; Guieysse, Benoit

    2017-07-01

    Over the last decades, several studies have reported emissions of nitrous oxide (N2 O) from microalgal cultures and aquatic ecosystems characterized by a high level of algal activity (e.g. eutrophic lakes). As N2 O is a potent greenhouse gas and an ozone-depleting pollutant, these findings suggest that large-scale cultivation of microalgae (and possibly, natural eutrophic ecosystems) could have a significant environmental impact. Using the model unicellular microalga Chlamydomonas reinhardtii, this study was conducted to investigate the molecular basis of microalgal N2 O synthesis. We report that C. reinhardtii supplied with nitrite (NO2- ) under aerobic conditions can reduce NO2- into nitric oxide (NO) using either a mitochondrial cytochrome c oxidase (COX) or a dual enzymatic system of nitrate reductase (NR) and amidoxime-reducing component, and that NO is subsequently reduced into N2 O by the enzyme NO reductase (NOR). Based on experimental evidence and published literature, we hypothesize that when nitrate (NO3- ) is the main Nitrogen source and the intracellular concentration of NO2- is low (i.e. under physiological conditions), microalgal N2 O synthesis involves the reduction of NO3- to NO2- by NR followed by the reduction of NO2- to NO by the dual system involving NR. This microalgal N2 O pathway has broad implications for environmental science and algal biology because the pathway of NO3- assimilation is conserved among microalgae, and because its regulation may involve NO. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  10. High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Ramos-Martinez, Erick Miguel; Fimognari, Lorenzo; Sakuragi, Yumiko

    2017-09-01

    Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n , wherein n = 10 or 20]. The yields of the (SP)n -fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP)n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  11. Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii.

    Science.gov (United States)

    Cakmak, Turgay; Angun, Pinar; Demiray, Yunus Emre; Ozkan, Alper Devrim; Elibol, Zeynep; Tekinay, Turgay

    2012-08-01

    Biodiesel production from microalgae is a promising approach for energy production; however, high cost of its process limits the use of microalgal biodiesel. Increasing the levels of triacylglycerol (TAG) levels, which is used as a biodiesel feedstock, in microalgae has been achieved mainly by nitrogen starvation. In this study, we compared effects of sulfur (S) and nitrogen (N) starvation on TAG accumulation and related parameters in wild-type Chlamydomonas reinhardtii CC-124 mt(-) and CC-125 mt(+) strains. Cell division was interrupted, protein and chlorophyll levels rapidly declined while cell volume, total neutral lipid, carotenoid, and carbohydrate content increased in response to nutrient starvation. Cytosolic lipid droplets in microalgae under nutrient starvation were monitored by three-dimensional confocal laser imaging of live cells. Infrared spectroscopy results showed that relative TAG, oligosaccharide and polysaccharide levels increased rapidly in response to nutrient starvation, especially S starvation. Both strains exhibited similar levels of regulation responses under mineral deficiency, however, the degree of their responses were significantly different, which emphasizes the importance of mating type on the physiological response of algae. Neutral lipid, TAG, and carbohydrate levels reached their peak values following 4 days of N or S starvation. Therefore, 4 days of N or S starvation provides an excellent way of increasing TAG content. Although increase in these parameters was followed by a subsequent decline in N-starved strains after 4 days, this decline was not observed in S-starved ones, which shows that S starvation is a better way of increasing TAG production of C. reinhardtii than N starvation. Copyright © 2012 Wiley Periodicals, Inc.

  12. Sensitivity of the green algae Chlamydomonas reinhardtii to gamma radiation: Photosynthetic performance and ROS formation

    Energy Technology Data Exchange (ETDEWEB)

    Gomes, Tânia, E-mail: tania.gomes@niva.no [Norwegian Institute for Water Research (NIVA), Section of Ecotoxicology and Risk Assessment, Gaustadalléen 21, N-0349, Oslo (Norway); Centre for Environmental Radioactivity, Norwegian University of Life Sciences (NMBU), Post Box 5003, N-1432 Ås (Norway); Xie, Li [Norwegian Institute for Water Research (NIVA), Section of Ecotoxicology and Risk Assessment, Gaustadalléen 21, N-0349, Oslo (Norway); Centre for Environmental Radioactivity, Norwegian University of Life Sciences (NMBU), Post Box 5003, N-1432 Ås (Norway); Brede, Dag; Lind, Ole-Christian [Centre for Environmental Radioactivity, Norwegian University of Life Sciences (NMBU), Post Box 5003, N-1432 Ås (Norway); Department for Environmental Sciences, Faculty of Environmental Science & Technology, Norwegian University of Life Sciences (NMBU), Post Box 5003, N-1432, Ås (Norway); Solhaug, Knut Asbjørn [Centre for Environmental Radioactivity, Norwegian University of Life Sciences (NMBU), Post Box 5003, N-1432 Ås (Norway); Department of Ecology and Natural Resource Management, Norwegian University of Life Sciences (NMBU), Postbox 5003, N-1432, Ås (Norway); Salbu, Brit [Centre for Environmental Radioactivity, Norwegian University of Life Sciences (NMBU), Post Box 5003, N-1432 Ås (Norway); Department for Environmental Sciences, Faculty of Environmental Science & Technology, Norwegian University of Life Sciences (NMBU), Post Box 5003, N-1432, Ås (Norway); and others

    2017-02-15

    Highlights: • Chlorophyll fluorescence parameters affected at higher dose rates. • Changes in PSII associated with electron transport and energy dissipation pathways. • Dose-dependent ROS production in algae exposed to gamma radiation. • Decrease in photosynthetic efficiency connected to ROS formation. - Abstract: The aquatic environment is continuously exposed to ionizing radiation from both natural and anthropogenic sources, making the characterization of ecological and health risks associated with radiation of large importance. Microalgae represent the main source of biomass production in the aquatic ecosystem, thus becoming a highly relevant biological model to assess the impacts of gamma radiation. However, little information is available on the effects of gamma radiation on microalgal species, making environmental radioprotection of this group of species challenging. In this context, the present study aimed to improve the understanding of the effects and toxic mechanisms of gamma radiation in the unicellular green algae Chlamydomonas reinhardtii focusing on the activity of the photosynthetic apparatus and ROS formation. Algal cells were exposed to gamma radiation (0.49–1677 mGy/h) for 6 h and chlorophyll fluorescence parameters obtained by PAM fluorometry, while two fluorescent probes carboxy-H{sub 2}DFFDA and DHR 123 were used for the quantification of ROS. The alterations seen in functional parameters of C. reinhardtii PSII after 6 h of exposure to gamma radiation showed modifications of PSII energy transfer associated with electron transport and energy dissipation pathways, especially at the higher dose rates used. Results also showed that gamma radiation induced ROS in a dose-dependent manner under both light and dark conditions. The observed decrease in photosynthetic efficiency seems to be connected to the formation of ROS and can potentially lead to oxidative stress and cellular damage in chloroplasts. To our knowledge, this is the first

  13. The phosphoproteome of a Chlamydomonas reinhardtii eyespot fraction includes key proteins of the light signaling pathway.

    Science.gov (United States)

    Wagner, Volker; Ullmann, Katharina; Mollwo, Anne; Kaminski, Marc; Mittag, Maria; Kreimer, Georg

    2008-02-01

    Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca(2+)-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii.

  14. SINGLET OXYGEN RESISTANT 1 links reactive electrophile signaling to singlet oxygen acclimation in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Fischer, Beat B; Ledford, Heidi K; Wakao, Setsuko; Huang, ShihYau Grace; Casero, David; Pellegrini, Matteo; Merchant, Sabeeha S; Koller, Andreas; Eggen, Rik I L; Niyogi, Krishna K

    2012-05-15

    Acclimation of Chlamydomonas reinhardtii cells to low levels of singlet oxygen, produced either by photoreactive chemicals or high light treatment, induces a specific genetic response that strongly increases the tolerance of the algae to subsequent exposure to normally lethal singlet oxygen-producing conditions. The genetic response includes the increased expression of various oxidative stress response and detoxification genes, like the glutathione peroxidase homologous gene GPXH/GPX5 and the σ-class glutathione-S-transferase gene GSTS1. To identify components involved in the signal transduction and activation of the singlet oxygen-mediated response, a mutant selection was performed. This selection led to the isolation of the singlet oxygen resistant 1 (sor1) mutant, which is more tolerant to singlet oxygen-producing chemicals and shows a constitutively higher expression of GPXH and GSTS1. Map-based cloning revealed that the SOR1 gene encodes a basic leucine zipper transcription factor, which controls its own expression and the expression of a large number of oxidative stress response and detoxification genes. In the promoter region of many of these genes, a highly conserved 8-bp palindromic sequence element was found to be enriched. This element was essential for GSTS1 induction by increased levels of lipophilic reactive electrophile species (RES), suggesting that it functions as an electrophile response element (ERE). Furthermore, GSTS1 overexpression in sor1 requires the ERE, although it is unknown whether it occurs through direct binding of SOR1 to the ERE. RES can be formed after singlet oxygen-induced lipid peroxidation, indicating that RES-stimulated and SOR1-mediated responses of detoxification genes are part of the singlet oxygen-induced acclimation process in C. reinhardtii.

  15. A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Kropat, Janette; Hong-Hermesdorf, Anne; Casero, David; Ent, Petr; Castruita, Madeli; Pellegrini, Matteo; Merchant, Sabeeha S; Malasarn, Davin

    2011-06-01

    Interest in exploiting algae as a biofuel source and the role of inorganic nutrient deficiency in inducing triacylglyceride (TAG) accumulation in cells necessitates a strategy to efficiently formulate species-specific culture media that can easily be manipulated. Using the reference organism Chlamydomonas reinhardtii, we tested the hypothesis that modeling trace element supplements after the cellular ionome would result in optimized cell growth. We determined the trace metal content of several commonly used Chlamydomonas strains in various culture conditions and developed a revised trace element solution to parallel these measurements. Comparison of cells growing in the revised supplement versus a traditional trace element solution revealed faster growth rates and higher maximum cell densities with the revised recipe. RNA-seq analysis of cultures growing in the traditional versus revised medium suggest that the variation in transcriptomes was smaller than that found between different wild-type strains grown in traditional Hutner's supplement. Visual observation did not reveal defects in cell motility or mating efficiency in the new supplement. Ni²⁺-inducible expression from the CYC6 promoter remained a useful tool, albeit with an increased requirement for Ni²⁺ because of the introduction of an EDTA buffer system in the revised medium. Other advantages include more facile preparation of trace element stock solutions, a reduction in total chemical use, a more consistent batch-to-batch formulation and long-term stability (tested up to 5 years). Under the new growth regime, we analyzed cells growing under different macro- and micronutrient deficiencies. TAG accumulation in N deficiency is comparable in the new medium. Fe and Zn deficiency also induced TAG accumulation, as suggested by Nile Red staining. This approach can be used to efficiently optimize culture conditions for other algal species to improve growth and to assay cell physiology. © 2011 The Authors

  16. Flocculation of Chlamydomonas reinhardtii with Different Phenotypic Traits by Metal Cations and High pH

    Directory of Open Access Journals (Sweden)

    Jianhua Fan

    2017-11-01

    Full Text Available Concentrating algal cells by flocculation as a prelude to centrifugation could significantly reduce the energy and cost of harvesting the algae. However, how variation in phenotypic traits such as cell surface features, cell size and motility alter the efficiency of metal cation and pH-induced flocculation is not well understood. Our results demonstrate that both wild-type and cell wall-deficient strains of the green unicellular alga Chlamydomonas reinhardtii efficiently flocculate (>90% at an elevated pH of the medium (pH 11 upon the addition of divalent cations such as calcium and magnesium (>5 mM. The trivalent ferric cation (at 10 mM proved to be essential for promoting flocculation under weak alkaline conditions (pH ∼8.5, with a maximum efficiency that exceeded 95 and 85% for wild-type CC1690 and the cell wall-deficient sta6 mutant, respectively. Near complete flocculation could be achieved using a combination of 5 mM calcium and a pH >11, while the medium recovered following cell removal could be re-cycled without affecting algal growth rates. Moreover, the absence of starch in the cell had little overall impact on flocculation efficiency. These findings contribute to our understanding of flocculation in different Chlamydomonas strains and have implications with respect to inexpensive methods for harvesting algae with different phenotypic traits. Additional research on the conditions (e.g., pH and metal ions used for efficient flocculation of diverse algal groups with diverse characteristics, at both small and large scale, will help establish inexpensive procedures for harvesting cell biomass.

  17. Manipulation of oil synthesis in Nannochloropsis strain NIES-2145 with a phosphorus starvation-inducible promoter from Chlamydomonas reinhardtii.

    Science.gov (United States)

    Iwai, Masako; Hori, Koichi; Sasaki-Sekimoto, Yuko; Shimojima, Mie; Ohta, Hiroyuki

    2015-01-01

    Microalgae accumulate triacylglycerols (TAGs) under conditions of nutrient stress. Phosphorus (P) starvation induces the accumulation of TAGs, and the cells under P starvation maintain growth through photosynthesis. We recently reported that P starvation-dependent overexpression of type-2 diacylglycerol acyl-CoA acyltransferase (CrDGTT4) from Chlamydomonas reinhardtii using a sulfoquinovosyldiacylglycerol synthase 2 (SQD2) promoter, which has increased activity during P starvation, enhances TAG accumulation in C. reinhardtii cells. As a result, the content of C18:1 fatty acid, a preferred substrate of CrDGTT4, is increased in TAGs. Here we isolated genes encoding SQD2 from strain NIES-2145 of the eustigmatophyte Nannochloropsis and showed that their expression, like that in C. reinhardtii, was up-regulated during P starvation. To enhance oil accumulation under P starvation, we transformed pCrSQD2-CrDGTT4 into Nannochloropsis strain NIES-2145. The transformants had a fatty acid composition that was more similar to that of C. reinhardtii, which resulted in enhanced TAG accumulation and higher 18:1(9) content. The results indicated that the P starvation-inducible promoter of C. reinhardtii was able to drive expression of the CrDGTT4 gene in Nannochloropsis strain NIES-2145 under P starvation. We conclude that the heterologous CrSQD2 promoter is effective in manipulating TAG synthesis in Nannochloropsis during P starvation.

  18. Manipulation of oil synthesis in Nannochloropsis strain NIES-2145 with a phosphorus starvation–inducible promoter from Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Masako eIwai

    2015-09-01

    Full Text Available Microalgae accumulate triacylglycerols (TAGs under conditions of nutrient stress. Phosphorus (P starvation induces the accumulation of TAGs, and the cells under P starvation maintain growth through photosynthesis. We recently reported that P starvation–dependent overexpression of type-2 diacylglycerol acyl-CoA acyltransferase (CrDGTT4 from Chlamydomonas reinhardtii using a sulfoquinovosyldiacylglycerol synthase 2 (SQD2 promoter, which has increased activity during P starvation, enhances TAG accumulation in C. reinhardtii cells. As a result, the content of C18:1 fatty acid, a preferred substrate of CrDGTT4, is increased in TAGs. Here we isolated genes encoding SQD2 from strain NIES-2145 of the eustigmatophyte Nannochloropsis and showed that their expression, like that in C. reinhardtii, was up-regulated during P starvation. To enhance oil accumulation under P starvation, we transformed pCrSQD2-CrDGTT4 into Nannochloropsis strain NIES-2145. The transformants had a fatty acid composition that was more similar to that of C. reinhardtii, which resulted in enhanced TAG accumulation and higher 18:1(9 content. The results indicated that the P starvation–inducible promoter of C. reinhardtii was able to drive expression of the CrDGTT4 gene in Nannochloropsis strain NIES-2145 under P starvation. We conclude that the heterologous CrSQD2 promoter is effective in manipulating TAG synthesis in Nannochloropsis during P starvation.

  19. Manipulation of oil synthesis in Nannochloropsis strain NIES-2145 with a phosphorus starvation–inducible promoter from Chlamydomonas reinhardtii

    Science.gov (United States)

    Iwai, Masako; Hori, Koichi; Sasaki-Sekimoto, Yuko; Shimojima, Mie; Ohta, Hiroyuki

    2015-01-01

    Microalgae accumulate triacylglycerols (TAGs) under conditions of nutrient stress. Phosphorus (P) starvation induces the accumulation of TAGs, and the cells under P starvation maintain growth through photosynthesis. We recently reported that P starvation–dependent overexpression of type-2 diacylglycerol acyl-CoA acyltransferase (CrDGTT4) from Chlamydomonas reinhardtii using a sulfoquinovosyldiacylglycerol synthase 2 (SQD2) promoter, which has increased activity during P starvation, enhances TAG accumulation in C. reinhardtii cells. As a result, the content of C18:1 fatty acid, a preferred substrate of CrDGTT4, is increased in TAGs. Here we isolated genes encoding SQD2 from strain NIES-2145 of the eustigmatophyte Nannochloropsis and showed that their expression, like that in C. reinhardtii, was up-regulated during P starvation. To enhance oil accumulation under P starvation, we transformed pCrSQD2-CrDGTT4 into Nannochloropsis strain NIES-2145. The transformants had a fatty acid composition that was more similar to that of C. reinhardtii, which resulted in enhanced TAG accumulation and higher 18:1(9) content. The results indicated that the P starvation–inducible promoter of C. reinhardtii was able to drive expression of the CrDGTT4 gene in Nannochloropsis strain NIES-2145 under P starvation. We conclude that the heterologous CrSQD2 promoter is effective in manipulating TAG synthesis in Nannochloropsis during P starvation. PMID:26441858

  20. Shewanella oneidensis: a new and efficient System for Expression and Maturation of heterologous [Fe-Fe] Hydrogenase from Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Sybirna Kateryna

    2008-09-01

    Full Text Available Abstract Background The eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium Shewanella oneidensis as a new and efficient system for expression and maturation of HydA1 from Chlamydomonas reinhardtii. Results Based on codon usage bias and hydrogenase maturation ability, the bacterium S. oneidensis, which possesses putative [Fe-Fe] and [Ni-Fe] hydrogenase operons, was selected as the best potential host for C. reinhardtii [Fe-Fe] hydrogenase expression. Hydrogen formation by S. oneidensis strain AS52 (ΔhydAΔhyaB transformed with a plasmid bearing CrHydA1 and grown in the presence of six different substrates for anaerobic respiration was determined. A significant increase in hydrogen evolution was observed for cells grown in the presence of trimethylamine oxide, dimethylsulfoxide and disodium thiosulfate, showing that the system of S. oneidensis is efficient for heterologous expression of algal [Fe-Fe] hydrogenase. Conclusion In the present work a new efficient system for heterologous expression and maturation of C. reinhardtii hydrogenase has been developed. HydA1 of C. reinhardtii was purified and shown to contain 6 Fe atoms/molecule of protein, as expected. Using DMSO, TMAO or thiosulfate as substrates for anaerobic respiration during the cell growth, 0.4 – 0.5 mg l-1(OD600 = 1 of catalytically active HydA1 was obtained with hydrogen evolution rate of ~700 μmol H2 mg-1 min-1.

  1. Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

    Science.gov (United States)

    Park, Jae Woo; Na, Sang Cheol; Nguyen, Thanh Qua; Paik, Sang-Min; Kang, Myeongwoo; Hong, Daewha; Choi, Insung S; Lee, Jae-Hyeok; Jeon, Noo Li

    2015-03-01

    This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the "biological glue." We take advantage of hydroxyproline (Hyp)-based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm(2) . Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable. © 2014 Wiley Periodicals, Inc.

  2. An omics based assessment of cadmium toxicity in the green alga Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Jamers, An; Blust, Ronny; De Coen, Wim [Laboratory for Ecophysiology, Biochemistry and Toxicology, Department of Biology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp (Belgium); Griffin, Julian L. [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 2QA (United Kingdom); Jones, Oliver A.H., E-mail: oliver.jones@rmit.edu.au [School of Applied Sciences, RMIT University, GPO Box 2476, Melbourne, VIC 3001 (Australia)

    2013-01-15

    The effects of cadmium were assessed in the freshwater alga Chlamydomonas reinhardtii. Algae were exposed to concentrations of 0, 8.1 or 114.8 {mu}M of cadmium and growth rates, gene transcription and metabolite profiles were examined after 48 and 72 h of exposure. In algae exposed to 8.1 {mu}M Cd, several genes were differentially transcribed after 48 h but no adverse growth related effects were detected. A transient effect on both gene transcription patterns and metabolite profiles could be discerned after 48 h of exposure but the majority of these changes disappeared after 72 h. In contrast, all effects were more pronounced at the 114.8 {mu}M cadmium exposure. Here growth was clearly reduced and transcription of a large number of genes involved in oxidative stress defense mechanisms was differentially increased. Metabolites involved in the glutathione synthesis pathway (an important antioxidant defense) were also affected but the effects of cadmium were found to be more pronounced at the transcript level than in the metabolome, suggesting that the former exhibits greater sensitivity toward cadmium exposure.

  3. Repeated production of hydrogen by sulfate re-addition in sulfur deprived culture of Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jun Pyo; Kim, Kyoung-Rok; Choi, Seung Phill; Sim, Sang Jun [Department of Chemical Engineering, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Han, Se Jong [Polar BioCenter, Korea Polar Research Institute, KORDI, Incheon 406-840 (Korea, Republic of); Kim, Mi Sun [Biomass Research Team, Korea Institute of Energy Research, Daejeon 305-343 (Korea, Republic of)

    2010-12-15

    Biological hydrogen production by the green alga, Chlamydomonas reinhardtii can be induced in conditions of sulfur deprivation. In this study, we investigated the repeated and enhanced hydrogen production afforded by the re-addition of sulfate with monitoring of pH and concentration of chlorophyll and sulfate. Without adjustment of the pH, the optimal concentration of re-added sulfate was 30 {mu}M for the hydrogen production. By the re-addition of 30 {mu}M of sulfate and the adjustment of the pH during 4 cycles of repeated production, we obtained the maximum amount of 789 ml H{sub 2} l{sup -1} culture, which is 3.4 times higher than that of one batch production without adjustment of pH, 236 ml H{sub 2} l{sup -1} culture. This means that the enhancement of the hydrogen production can be achieved by the careful control of the sulfate re-addition and pH adjustment in the sulfur deprived culture. (author)

  4. The diurnal logic of the expression of the chloroplast genome in Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Adam D Idoine

    Full Text Available Chloroplasts are derived from cyanobacteria and have retained a bacterial-type genome and gene expression machinery. The chloroplast genome encodes many of the core components of the photosynthetic apparatus in the thylakoid membranes. To avoid photooxidative damage and production of harmful reactive oxygen species (ROS by incompletely assembled thylakoid protein complexes, chloroplast gene expression must be tightly regulated and co-ordinated with gene expression in the nucleus. Little is known about the control of chloroplast gene expression at the genome-wide level in response to internal rhythms and external cues. To obtain a comprehensive picture of organelle transcript levels in the unicellular model alga Chlamydomonas reinhardtii in diurnal conditions, a qRT-PCR platform was developed and used to quantify 68 chloroplast, 21 mitochondrial as well as 71 nuclear transcripts in cells grown in highly controlled 12 h light/12 h dark cycles. Interestingly, in anticipation of dusk, chloroplast transcripts from genes involved in transcription reached peak levels first, followed by transcripts from genes involved in translation, and finally photosynthesis gene transcripts. This pattern matches perfectly the theoretical demands of a cell "waking up" from the night. A similar trend was observed in the nuclear transcripts. These results suggest a striking internal logic in the expression of the chloroplast genome and a previously unappreciated complexity in the regulation of chloroplast genes.

  5. Using natural selection to explore the adaptive potential of Chlamydomonas reinhardtii.

    Science.gov (United States)

    Perrineau, Marie-Mathilde; Gross, Jeferson; Zelzion, Ehud; Price, Dana C; Levitan, Orly; Boyd, Jeffrey; Bhattacharya, Debashish

    2014-01-01

    Improving feedstock is critical to facilitate the commercial utilization of algae, in particular in open pond systems where, due to the presence of competitors and pests, high algal growth rates and stress tolerance are beneficial. Here we raised laboratory cultures of the model alga Chlamydomonas reinhardtii under serial dilution to explore the potential of crop improvement using natural selection. The alga was evolved for 1,880 generations in liquid medium under continuous light (EL population). At the end of the experiment, EL cells had a growth rate that was 35% greater than the progenitor population (PL). The removal of acetate from the medium demonstrated that EL growth enhancement largely relied on efficient usage of this organic carbon source. Genome re-sequencing uncovered 1,937 polymorphic DNA regions in the EL population with 149 single nucleotide polymorphisms resulting in amino acid substitutions. Transcriptome analysis showed, in the EL population, significant up regulation of genes involved in protein synthesis, the cell cycle and cellular respiration, whereas the DNA repair pathway and photosynthesis were down regulated. Like other algae, EL cells accumulated neutral lipids under nitrogen depletion. Our work demonstrates transcriptome and genome-wide impacts of natural selection on algal cells and points to a useful strategy for strain improvement.

  6. Using natural selection to explore the adaptive potential of Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Marie-Mathilde Perrineau

    Full Text Available Improving feedstock is critical to facilitate the commercial utilization of algae, in particular in open pond systems where, due to the presence of competitors and pests, high algal growth rates and stress tolerance are beneficial. Here we raised laboratory cultures of the model alga Chlamydomonas reinhardtii under serial dilution to explore the potential of crop improvement using natural selection. The alga was evolved for 1,880 generations in liquid medium under continuous light (EL population. At the end of the experiment, EL cells had a growth rate that was 35% greater than the progenitor population (PL. The removal of acetate from the medium demonstrated that EL growth enhancement largely relied on efficient usage of this organic carbon source. Genome re-sequencing uncovered 1,937 polymorphic DNA regions in the EL population with 149 single nucleotide polymorphisms resulting in amino acid substitutions. Transcriptome analysis showed, in the EL population, significant up regulation of genes involved in protein synthesis, the cell cycle and cellular respiration, whereas the DNA repair pathway and photosynthesis were down regulated. Like other algae, EL cells accumulated neutral lipids under nitrogen depletion. Our work demonstrates transcriptome and genome-wide impacts of natural selection on algal cells and points to a useful strategy for strain improvement.

  7. Hydrogen Production by a Chlamydomonas reinhardtii Strain with Inducible Expression of Photosystem II

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    Khorcheska Batyrova

    2017-03-01

    Full Text Available Chlamydomonas reinhardtii cy6Nac2.49 is a genetically modified algal strain that activates photosynthesis in a cyclical manner, so that photosynthesis is not active constitutively in the presence of oxygen, but is turned on only in response to a metabolic trigger (anaerobiosis. Here, we further investigated hydrogen production by this strain comparing it with the parental wild-type strain under photoheterotrophic conditions in regular tris-acetate-phosphate (TAP medium with a 10-h:14-h light/dark regime. Unlike the wild-type, whose level of H2 production remained low during illumination, H2 production in the mutant strain increased gradually with each subsequent light period, and by the final light period was significantly higher than the wild-type. The relatively low Photosystem II (PSII activity of the mutant culture was shown by low fluorescence yield both in the dark (Fv/Fm and in the light (δF/Fm’ periods. Measurement of oxygen evolution confirmed the low photosynthetic activity of the mutant cells, which gradually accumulated O2 to a lesser extent than the wild-type, thus allowing the mutant strain to maintain hydrogenase activity over a longer time period and to gradually accumulate H2 during periods of illumination. Therefore, controllable expression of PSII can be used to increase hydrogen production under nutrient replete conditions, thus avoiding many of the limitations associated with nutrient deprivation approaches sometimes used to promote hydrogen production.

  8. Cellular toxicity pathways of inorganic and methyl mercury in the green microalga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Beauvais-Flück, Rebecca; Slaveykova, Vera I; Cosio, Claudia

    2017-08-14

    Contamination by mercury (Hg) is a worldwide concern because of Hg toxicity and biomagnification in aquatic food webs. Nevertheless, bioavailability and cellular toxicity pathways of inorganic (IHg) and methyl-Hg (MeHg) remain poorly understood. We analyzed the uptake, transcriptomic, and physiological responses in the microalga Chlamydomonas reinhardtii exposed to IHg or MeHg. Bioavailability of MeHg was up to 27× higher than for IHg. Genes involved in cell processes, energy metabolism and transport were dysregulated by both Hg species. Physiological analysis revealed an impact on photosynthesis and reduction-oxidation reaction metabolism. Nevertheless, MeHg dysregulated a larger number of genes and with a stronger fold-change than IHg at equivalent intracellular concentration. Analysis of the perturbations of the cell's functions helped to derive a detailed mechanistic understanding of differences in cellular handling of IHg and MeHg resulting in MeHg having a stronger impact. This knowledge is central for the prediction of impact of toxicants on organisms.

  9. Phytotoxicity of 15 common pharmaceuticals on the germination of Lactuca sativa and photosynthesis of Chlamydomonas reinhardtii.

    Science.gov (United States)

    Pino, Ma Rosa; Muñiz, Selene; Val, Jonatan; Navarro, Enrique

    2016-11-01

    Pharmaceuticals reach terrestrial environments through the application of treated wastewaters and biosolids to agricultural soils. We have investigated the toxicity of 15 common pharmaceuticals, classified as nonsteroidal anti-inflammatory drugs (NSAIDs), blood lipid-lowering agents, β-blockers and antibiotics, in two photosynthetic organisms. Twelve pharmaceuticals caused inhibitory effects on the radicle and hypocotyl elongation of Lactuca sativa seeds. The EC 50 values obtained were in the range of 170-5656 mg L -1 in the case of the radicle and 188-4558 mg L -1 for the hypocotyl. Propranolol was the most toxic drug for both root and hypocotyl elongation, followed by the NSAIDs, then gemfibrozil and tetracycline. Other effects, such as root necrosis, inhibition of root growth and curly hairs, were detected. However, even at the highest concentrations tested (3000 mg L -1 ), seed germination was not affected. NSAIDs decreased the photosynthetic yield of Chlamydomonas reinhardtii, but only salicylic acid showed EC 50 values below 1000 mg L -1 . The first effects detected at low concentrations, together with the concentrations found in environmental samples, indicate that the use of biosolids and wastewaters containing pharmaceuticals should be regulated and their compositions assessed in order to prevent medium- and long-term impacts on agricultural soils and crops.

  10. Coupling of Carbon Dioxide Fixation to the Oxyhydrogen Reaction in the Isolated Chloroplast of Chlamydomonas reinhardtii.

    Science.gov (United States)

    Chen, C; Gibbs, M

    1992-11-01

    The oxyhydrogen reaction (the reduction of O(2) to water by H(2)) in the presence of CO(2) was studied in the isolated Chlamydomonas reinhardtii chloroplast by monitoring the rate of (14)CO(2) incorporation into acid-stable products in the dark. The endogenous rate of CO(2) uptake (50-125 nmol/mg chlorophyll per h) was increased about 3- to 4-fold by ATP and additionally when combined with glucose, ribose-5-phosphate, and glycerate-3-phosphate. The rate was diminished 50 to 75%, respectively, when H(2) was replaced by N(2) or by air. Decrease in CO(2) uptake by dl-glyceraldehyde was taken to indicate that the regenerative phase and complete Calvin cycle turnover were involved. Diminution of CO(2) incorporation by rotenone, antimycin A, and 2,5-dibromo-3-methyl-6-isopropanol-p-benzoquinone was attributed to an inhibition of the oxyhydrogen reaction, resulting in an elevated NADPH/NADP ratio. If so, then the diminished CO(2) uptake could have been by "product inhibition" of the carbon metabolic network. Our data are consistent with the proposal (H. Gaffron [1942] J Gen Physiol 26: 241-267) that CO(2) fixation coupled to the oxyhydrogen reaction is dependent to some extent on exchloroplastic metabolism. This support is primarily ATP provided by mitochondrial respiration.

  11. Flow Cytometric Methods for Indirect Analysis and Quantification of Gametogenesis in Chlamydomonas reinhardtii (Chlorophyceae)

    Science.gov (United States)

    Tomkins, Joseph L.

    2016-01-01

    Induction of sexual reproduction in the facultatively sexual Chlamydomonas reinhardtii is cued by depletion of nitrogen. We explore the capacity for indirect monitoring of population variation in the gametogenic process using flow cytometry. We describe a high-throughput method capable of identifying fluorescence, ploidy and scatter profiles that track vegetative cells entering and undergoing gametogenesis. We demonstrate for the first time, that very early and late growth phases reduce the capacity to distinguish putative gametes from vegetative cells based on scatter and fluorescence profiles, and that early/mid-logarithmic cultures show the optimal distinction between vegetative cells and gamete scatter profiles. We argue that early/mid logarithmic cultures are valuable in such high throughput comparative approaches when investigating optimisation or quantification of gametogenesis based on scatter and fluorescence profiles. This approach provides new insights into the impact of culture conditions on gametogenesis, while documenting novel scatter and fluorescence profile shifts which typify the process. This method has potential applications to; enabling quick high-throughput monitoring, uses in increasing efficiency in the quantification of gametogenesis, as a method of comparing the switch between vegetative and gametic states across treatments, and as criteria for enrichment of gametic phenotypes in cell sorting assays. PMID:27676075

  12. Advances in the biotechnology of hydrogen production with the microalga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Torzillo, Giuseppe; Scoma, Alberto; Faraloni, Cecilia; Giannelli, Luca

    2015-01-01

    Biological hydrogen production is being evaluated for use as a fuel, since it is a promising substitute for carbonaceous fuels owing to its high conversion efficiency and high specific energy content. The basic advantages of biological hydrogen production over other "green" energy sources are that it does not compete for agricultural land use, and it does not pollute, as water is the only by-product of the combustion. These characteristics make hydrogen a suitable fuel for the future. Among several biotechnological approaches, photobiological hydrogen production carried out by green microalgae has been intensively investigated in recent years. A select group of photosynthetic organisms has evolved the ability to harness light energy to drive hydrogen gas production from water. Of these, the microalga Chlamydomonas reinhardtii is considered one of the most promising eukaryotic H2 producers. In this model microorganism, light energy, H2O and H2 are linked by two excellent catalysts, the photosystem 2 (PSII) and the [FeFe]-hydrogenase, in a pathway usually referred to as direct biophotolysis. This review summarizes the main advances made over the past decade as an outcome of the discovery of the sulfur-deprivation process. Both the scientific and technical barriers that need to be overcome before H2 photoproduction can be scaled up to an industrial level are examined. Actual and theoretical limits of the efficiency of the process are also discussed. Particular emphasis is placed on algal biohydrogen production outdoors, and guidelines for an optimal photobioreactor design are suggested.

  13. A simple and non-invasive method for nuclear transformation of intact-walled Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Sora Kim

    Full Text Available Genetic engineering in microalgae is gaining attraction but nuclear transformation methods available so far are either inefficient or require special equipment. In this study, we employ positively charged nanoparticles, 3-aminopropyl-functionalized magnesium phyllosilicate (aminoclay, approximate unit cell composition of [H2N(CH23]8Si8Mg6O12(OH4, for nuclear transformation into eukaryotic microalgae. TEM and EDX analysis of the process of transformation reveals that aminoclay coats negatively-charged DNA biomolecules and forms a self-assembled hybrid nanostructure. Subsequently, when this nanostructure is mixed with microalgal cells and plated onto selective agar plates with high friction force, cell wall is disrupted facilitating delivery of plasmid DNA into the cell and ultimately to the nucleus. This method is not only simple, inexpensive, and non-toxic to cells but also provides efficient transformation (5.03×10(2 transformants/µg DNA, second only to electroporation which needs advanced instrumentation. We present optimized parameters for efficient transformation including pre-treatment, friction force, concentration of foreign DNA/aminoclay, and plasticity of agar plates. It is also confirmed the successful integration and stable expression of foreign gene in Chlamydomonas reinhardtii through molecular methods.

  14. 3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process.

    Science.gov (United States)

    Stern, D B; Kindle, K L

    1993-01-01

    Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms. Images PMID:8455609

  15. Photobiological hydrogen production with the unicellular green alga Chlamydomonas reinhardtii under process engineering aspects; Photobiologische Wasserstoffproduktion mit der einzelligen Gruenalge Chlamydomonas reinhardtii unter verfahrenstechnischen Aspekten

    Energy Technology Data Exchange (ETDEWEB)

    Geier, Stephanie

    2011-07-01

    Hydrogen is of high interest as a clean and environmentally friendly energy source as its combustion only emits water and energy. However, currently hydrogen is produced in energy demanding processes by the consumption of fossil fuels. An alternative way of sustainable and non-polluting hydrogen production could be provided by use of photosynthetic active microalgae. Within this work, the photobiological hydrogen production with the unicellular green algae Chlamydomonas reinhardtii is investigated under the aspects of bioprocess-engineering and economics. Objectives are, besides the increase of the photochemical efficiency, the cultivation of the algae and subsequent hydrogen production under cost-free sunlight. It could be demonstrated that outdoor cultivation of C. reinhardtii is possible in Central Europe throughout the year by using e.g. waste heat. Similar cell numbers in the range from 1,2.10{sup 7} cells ml{sup -1} to 1,7.10{sup 7} cells ml{sup -1} could be achieved in closed photobioreactors of the type Photobioreactor Screening Module under controlled laboratory conditions and both continuous illumination (200 {mu}mol.m{sup -2}.s{sup -1}) and simulated outdoor conditions according to the light intensity of idealized summer day as well as in outdoor experiments (up to 2000 {mu}mol.m{sup -2}.s{sup -1}).The use of 10 % CO{sub 2} corresponding to the CO{sub 2} content in flue gas led to a doubling of cell numbers under continuous illumination to 4,2.10{sup 7} cells ml{sup -1}, compared to the reference culture bubbled with 3 % CO{sub 2}. A significant increase of cell numbers under the light profiles of an idealized summer day could not be achieved. The cultivation under the light profile of a winter day at 25 C reduced cell growth to 54 %, compared to the summer simulation. In open 30 L outdoor ponds, only 0,26.10{sup 7} cells ml{sup -1} could be achieved under photoheterotrophic conditions during the summer months, which corresponds to 20 % of the cell

  16. Refactoring the six-gene photosystem II core in the chloroplast of the green algae Chlamydomonas reinhardtii

    DEFF Research Database (Denmark)

    Gimpel, Javier A.; Nour-Eldin, Hussam Hassan; Scranton, Melissa A.

    2016-01-01

    production, particularly under specific environmental conditions. PSII is a complex multisubunit enzyme with strong interdependence among its components. In this work, we have deleted the six core genes of PSII in the eukaryotic alga Chlamydomonas reinhardtii and refactored them in a single DNA construct....... Complementation of the knockout strain with the core PSII synthetic module from three different green algae resulted in reconstitution of photosynthetic activity to 85, 55, and 53% of that of the wild-type, demonstrating that the PSII core can be exchanged between algae species and retain function. The strains...

  17. Acclimation of Chlamydomonas reinhardtii to ultraviolet radiation and its impact on chemical toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Korkaric, Muris; Xiao, Mao [Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, 8600 Duebendorf (Switzerland); ETH Zürich, Institute of Biogeochemistry and Pollutant Dynamics, 8092 Zürich (Switzerland); Behra, Renata [Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, 8600 Duebendorf (Switzerland); Eggen, Rik I.L., E-mail: rik.eggen@eawag.ch [Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, 8600 Duebendorf (Switzerland); ETH Zürich, Institute of Biogeochemistry and Pollutant Dynamics, 8092 Zürich (Switzerland)

    2015-10-15

    Highlights: • Systematic study of UVR acclimation and its impact on chemical toxicity in C. reinhardtii. • UVR acclimation is mediated through fast and reversible physiological defense mechanisms. • Pigment analysis suggests a role of lutein in UVR acclimation. • Co-tolerance to rose bengal suggests a role of singlet oxygen defense in UVR acclimation. • Knowledge on the toxic mechanism of chemicals needed to predict co-tolerance. - Abstract: The toxicity of chemical pollutants can be modulated under stressful environmental conditions, such as increased temperature, salinity or ultraviolet radiation (UVR), due to the interaction of effects during simultaneous stressor exposure. However, organisms may acclimate to such conditions by activation of physiological and biochemical defence mechanisms. In sequential exposures, organisms acclimated to environmental stressors may display an increased sensitivity or co-tolerance towards chemical pollutants. It has been suggested that co-tolerance might be expected for similarly acting stressors due to common defence mechanisms. To test this for combinations of UVR and chemical stressors, we first acclimatized the model green alga Chlamydomonas reinhardtii to UVR and subsequently compared the sensitivity of UVR pre-exposed and control algae towards chemicals. Selected chemicals all act on photosynthesis and thus share a common physiological target, but display distinct toxicity mechanisms. Results showed that UVR pre-exposure for four days partially inhibited algal growth and photosynthesis, but also increased algal tolerance to higher UVR levels, confirming UVR acclimation. HPLC analysis of algal pigments indicated that UVR acclimation might in part be explained by the protective function of lutein while the contribution of UVR absorbing compounds was less clear. Challenge exposure to chemicals in the absence of UVR showed that acclimated algae were co-tolerant to the photosensitizer rose bengal, but not to the

  18. Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9.

    Science.gov (United States)

    Greiner, Andre; Kelterborn, Simon; Evers, Heide; Kreimer, Georg; Sizova, Irina; Hegemann, Peter

    2017-10-01

    The fast-growing biflagellated single-celled chlorophyte Chlamydomonas reinhardtii is the most widely used alga in basic research. The physiological functions of the 18 sensory photoreceptors are of particular interest with respect to Chlamydomonas development and behavior. Despite the demonstration of gene editing in Chlamydomonas in 1995, the isolation of mutants lacking easily ascertained newly acquired phenotypes remains problematic due to low DNA recombination efficiency. We optimized gene-editing protocols for several Chlamydomonas strains (including wild-type CC-125) using zinc-finger nucleases (ZFNs), genetically encoded CRISPR/associated protein 9 (Cas9) from Staphylococcus aureus and Streptococcus pyogenes, and recombinant Cas9 and developed protocols for rapidly isolating nonselectable gene mutants. Using this technique, we disrupted the photoreceptor genes COP1/2, COP3 (encoding channelrhodopsin 1 [ChR1]), COP4 (encoding ChR2), COP5, PHOT, UVR8, VGCC, MAT3, and aCRY and created the chr1 chr2 and uvr8 phot double mutants. Characterization of the chr1, chr2, and mat3 mutants confirmed the value of photoreceptor mutants for physiological studies. Genes of interest were disrupted in 5 to 15% of preselected clones (∼1 out of 4000 initial cells). Using ZFNs, genes were edited in a reliable, predictable manner via homologous recombination, whereas Cas9 primarily caused gene disruption via the insertion of cotransformed DNA. These methods should be widely applicable to research involving green algae. © 2017 American Society of Plant Biologists. All rights reserved.

  19. Experimental Definition and Validation of Protein Coding Transcripts in Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Kourosh Salehi-Ashtiani; Jason A. Papin

    2012-01-13

    Algal fuel sources promise unsurpassed yields in a carbon neutral manner that minimizes resource competition between agriculture and fuel crops. Many challenges must be addressed before algal biofuels can be accepted as a component of the fossil fuel replacement strategy. One significant challenge is that the cost of algal fuel production must become competitive with existing fuel alternatives. Algal biofuel production presents the opportunity to fine-tune microbial metabolic machinery for an optimal blend of biomass constituents and desired fuel molecules. Genome-scale model-driven algal metabolic design promises to facilitate both goals by directing the utilization of metabolites in the complex, interconnected metabolic networks to optimize production of the compounds of interest. Using Chlamydomonas reinhardtii as a model, we developed a systems-level methodology bridging metabolic network reconstruction with annotation and experimental verification of enzyme encoding open reading frames. We reconstructed a genome-scale metabolic network for this alga and devised a novel light-modeling approach that enables quantitative growth prediction for a given light source, resolving wavelength and photon flux. We experimentally verified transcripts accounted for in the network and physiologically validated model function through simulation and generation of new experimental growth data, providing high confidence in network contents and predictive applications. The network offers insight into algal metabolism and potential for genetic engineering and efficient light source design, a pioneering resource for studying light-driven metabolism and quantitative systems biology. Our approach to generate a predictive metabolic model integrated with cloned open reading frames, provides a cost-effective platform to generate metabolic engineering resources. While the generated resources are specific to algal systems, the approach that we have developed is not specific to algae and

  20. Structural Analysis of the Rubisco-Assembly Chaperone RbcX-II from Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Andreas Bracher

    Full Text Available The most prevalent form of the Rubisco enzyme is a complex of eight catalytic large subunits (RbcL and eight regulatory small subunits (RbcS. Rubisco biogenesis depends on the assistance by specific molecular chaperones. The assembly chaperone RbcX stabilizes the RbcL subunits after folding by chaperonin and mediates their assembly to the RbcL8 core complex, from which RbcX is displaced by RbcS to form active holoenzyme. Two isoforms of RbcX are found in eukaryotes, RbcX-I, which is more closely related to cyanobacterial RbcX, and the more distant RbcX-II. The green algae Chlamydomonas reinhardtii contains only RbcX-II isoforms, CrRbcX-IIa and CrRbcX-IIb. Here we solved the crystal structure of CrRbcX-IIa and show that it forms an arc-shaped dimer with a central hydrophobic cleft for binding the C-terminal sequence of RbcL. Like other RbcX proteins, CrRbcX-IIa supports the assembly of cyanobacterial Rubisco in vitro, albeit with reduced activity relative to cyanobacterial RbcX-I. Structural analysis of a fusion protein of CrRbcX-IIa and the C-terminal peptide of RbcL suggests that the peptide binding mode of RbcX-II may differ from that of cyanobacterial RbcX. RbcX homologs appear to have adapted to their cognate Rubisco clients as a result of co-evolution.

  1. Inorganic polyphosphate occurs in the cell wall of Chlamydomonas reinhardtii and accumulates during cytokinesis

    Directory of Open Access Journals (Sweden)

    Freimoser Florian M

    2007-09-01

    Full Text Available Abstract Background Inorganic polyphosphate (poly P, linear chains of phosphate residues linked by energy rich phosphoanhydride bonds, is found in every cell and organelle and is abundant in algae. Depending on its localization and concentration, poly P is involved in various biological functions. It serves, for example, as a phosphate store and buffer against alkali, is involved in energy metabolism and regulates the activity of enzymes. Bacteria defective in poly P synthesis are impaired in biofilm development, motility and pathogenicity. PolyP has also been found in fungal cell walls and bacterial envelopes, but has so far not been measured directly or stained specifically in the cell wall of any plant or alga. Results Here, we demonstrate the presence of poly P in the cell wall of Chlamydomonas reinhardtii by staining with specific poly P binding proteins. The specificity of the poly P signal was verified by various competition experiments, by staining with different poly P binding proteins and by correlation with biochemical quantification. Microscopical investigation at different time-points during growth revealed fluctuations of the poly P signal synchronous with the cell cycle: The poly P staining peaked during late cytokinesis and was independent of the high intracellular poly P content, which fluctuated only slightly during the cell cycle. Conclusion The presented staining method provides a specific and sensitive tool for the study of poly P in the extracellular matrices of algae and could be used to describe the dynamic behaviour of cell wall poly P during the cell cycle. We assume that cell wall poly P and intracellular poly P are regulated by distinct mechanisms and it is suggested that cell wall bound poly P might have important protective functions against toxic compounds or pathogens during cytokinesis, when cells are more vulnerable.

  2. L,L-diaminopimelate aminotransferase from Chlamydomonas reinhardtii: a target for algaecide development.

    Directory of Open Access Journals (Sweden)

    Renwick C J Dobson

    Full Text Available In some bacterial species and photosynthetic cohorts, including algae, the enzyme L,L-diaminopimelate aminotransferase (DapL (E.C. 2.6.1.83 is involved in the anabolism of the essential amino acid L-lysine. DapL catalyzes the conversion of tetrahydrodipicolinate (THDPA to L,L-diaminopimelate (L,L-DAP, in one step bypassing the DapD, DapC and DapE enzymatic reactions present in the acyl DAP pathways. Here we present an in vivo and in vitro characterization of the DapL ortholog from the alga Chlamydomonas reinhardtii (Cr-DapL. The in vivo analysis illustrated that the enzyme is able to functionally complement the E. coli dap auxotrophs and was essential for plant development in Arabidopsis. In vitro, the enzyme was able to inter-convert THDPA and L,L-DAP, showing strong substrate specificity. Cr-DapL was dimeric in both solution and when crystallized. The structure of Cr-DapL was solved in its apo form, showing an overall architecture of a α/β protein with each monomer in the dimer adopting a pyridoxal phosphate-dependent transferase-like fold in a V-shaped conformation. The active site comprises residues from both monomers in the dimer and shows some rearrangement when compared to the apo-DapL structure from Arabidopsis. Since animals do not possess the enzymatic machinery necessary for the de novo synthesis of the amino acid L-lysine, enzymes involved in this pathway are attractive targets for the development of antibiotics, herbicides and algaecides.

  3. Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii.

    Science.gov (United States)

    Young, Rosanna E B; Purton, Saul

    2016-05-01

    There is a growing interest in the use of microalgae as low-cost hosts for the synthesis of recombinant products such as therapeutic proteins and bioactive metabolites. In particular, the chloroplast, with its small, genetically tractable genome (plastome) and elaborate metabolism, represents an attractive platform for genetic engineering. In Chlamydomonas reinhardtii, none of the 69 protein-coding genes in the plastome uses the stop codon UGA, therefore this spare codon can be exploited as a useful synthetic biology tool. Here, we report the assignment of the codon to one for tryptophan and show that this can be used as an effective strategy for addressing a key problem in chloroplast engineering: namely, the assembly of expression cassettes in Escherichia coli when the gene product is toxic to the bacterium. This problem arises because the prokaryotic nature of chloroplast promoters and ribosome-binding sites used in such cassettes often results in transgene expression in E. coli, and is a potential issue when cloning genes for metabolic enzymes, antibacterial proteins and integral membrane proteins. We show that replacement of tryptophan codons with the spare codon (UGG→UGA) within a transgene prevents functional expression in E. coli and in the chloroplast, and that co-introduction of a plastidial trnW gene carrying a modified anticodon restores function only in the latter by allowing UGA readthrough. We demonstrate the utility of this system by expressing two genes known to be highly toxic to E. coli and discuss its value in providing an enhanced level of biocontainment for transplastomic microalgae. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  4. Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: Screening cytotoxicity and genotoxicity endpoints

    Energy Technology Data Exchange (ETDEWEB)

    Esperanza, Marta; Cid, Ángeles; Herrero, Concepción; Rioboo, Carmen, E-mail: carmen.rioboo@udc.es

    2015-08-15

    Highlights: • Mitochondrial membrane potential constituted the most sensitive parameter assayed. • Several genotoxicity methods were applied for first time in ecotoxicological studies. • Oxidative DNA base damage (8-OHdG) was induced by paraquat exposure. • Cells with DNA strand breakage and subG1-nuclei increased in treated cultures. • Typical apoptosis hallmarks were observed in microalgal cells exposed to paraquat. - Abstract: Since recent evidence has demonstrated that many types of chemicals exhibit oxidative and/or genotoxic potential on living organisms, reactive oxygen species (ROS) formation and DNA damage are currently the best accepted paradigms to assess the potential hazardous biological effects of a wide range of contaminants. The goal of this study was to evaluate the sensitivity of different cytotoxicity and genotoxicity responses on the model microalga Chlamydomonas reinhardtii exposed to the prooxidant herbicide paraquat. In addition to the growth endpoint, cell viability, mitochondrial membrane potential and presence of reactive oxygen species (ROS) were assayed as potential markers of cytotoxicity using flow cytometry (FCM). To study the effects of paraquat on C. reinhardtii DNA, several genotoxicity approaches were implemented for the first time in an ecotoxicological study on microalgae. Oxidative DNA base damage was analysed by measuring the oxidative DNA lesion 8-OHdG by FCM. DNA fragmentation was analysed by different methods: comet assay, and cell cycle analysis by FCM, with a particular focus on the presence of subG1-nuclei. Finally, effects on morphology of nuclei were monitored through DAPI staining. The evaluation of these endpoints showed that several physiological and biochemical parameters reacted to oxidative stress disturbances with greater sensitivity than integrative parameters such as growth rates or cell viability. The experiments revealed concentration-dependent cytotoxicity (ROS formation, depolarization of

  5. The Mechanism of Starch Over-Accumulation in Chlamydomonas reinhardtii High-Starch Mutants Identified by Comparative Transcriptome Analysis

    Directory of Open Access Journals (Sweden)

    Kwang M. Koo

    2017-05-01

    Full Text Available The focus of this study was the mechanism of starch accumulation in Chlamydomonas reinhardtii high-starch mutants. Three C. reinhardtii mutants showing high-starch content were generated using gamma irradiation. When grown under nitrogen-deficient conditions, these mutants had more than twice as much starch than a wild-type control. The mechanism of starch over-accumulation in these mutants was studied with comparative transcriptome analysis. In all mutants, induction of phosphoglucomutase 1 (PGM1 expression was detected; PGM1 catalyzes the inter-conversion of glucose 1-phosphate and glucose 6-phosphate in both starch biosynthetic and glycolytic pathway. Interestingly, transcript levels of phosphoglucose isomerase 1 (PGI1, fructose 1,6-bisphosphate aldolase 1 and 2 (FBA1 and FBA2 were down-regulated in all mutants; PGI1, FBA1, and FBA2 act on downstream of glucose 6-phosphate conversion in glycolytic pathway. Therefore, down-regulations of PGI1, FBA1, and FBA2 may lead to accumulation of upstream metabolites, notably glucose 6-phosphate, resulting in induction of PGM1 expression through feed-forward regulation and that PGM1 overexpression caused starch over-accumulation in mutants. These results suggest that PGI1, FBA1, FBA2, and PGM1 correlate with each other in terms of coordinated transcriptional regulation and play central roles for starch over-accumulation in C. reinhardtii.

  6. Interactive effects of copper oxide nanoparticles and light to green alga Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Cheloni, Giulia; Marti, Elodie; Slaveykova, Vera I., E-mail: vera.slaveykova@unige.ch

    2016-01-15

    Highlights: • Comparable stability of CuO-NP suspensions under different light conditions. • UVR* inhibits growth, bleaches chlorophyll fluorescence and damages membrane. • Below 1 mg L{sup −1} CuO-NPs do not attenuate light in algal suspension. • SNL enhances significantly the effect of 0.8 mg L{sup −1} CuO-NPs on microalgae. • Synergistic interactions between UVR* and CuO-NPs. - Abstract: The present study explores the effect of light with different spectral composition on the stability of CuO-nanoparticle (CuO-NP) dispersions and their effects to green alga Chlamydomonas reinhardtii. The results showed that simulated natural light (SNL) and light with enhanced UVB radiation (UVR*) do not affect the dissolution of CuO-NPs as compared to light irradiation conditions typically used in laboratory incubator (INC). Comparable values of ζ-potential and hydrodynamic size during 24 h were found under all studied conditions. Concentrations of CuO-NPs below 1 mg L{sup −1} do not attenuate the light penetration in the algal suspensions in comparison with NP-free system. Exposure to a combination of 8 μg L{sup −1} or 0.8 mg L{sup −1} CuO-NPs and INC or SNL has no significant effect on the algal growth inhibition, algal fluorescence and membrane integrity under short-term exposure. However, an enhancement of the percentage of cells experiencing oxidative stress was observed upon exposure to 0.8 mg L{sup −1} CuO-NPs and SNL for 4 and 8 h. Combination of UVR* and 0.8 mg L{sup −1} CuO-NPs resulted in synergistic effects for all biological endpoints. Despite the photocatalytic properties of CuO-NPs no significant increase in abiotic reactive oxygen species (ROS) production under simulated solar radiation was observed suggesting that the synergistic effect observed might be correlated to other factors than CuO-NP-mediated ROS photoproduction. Tests performed with CuSO{sub 4} confirmed the important role of dissolution as toxicity driving force for lower

  7. Nucleotide diversity in the mitochondrial and nuclear compartments of Chlamydomonas reinhardtii: investigating the origins of genome architecture

    Directory of Open Access Journals (Sweden)

    Lee Robert W

    2008-05-01

    Full Text Available Abstract Background The magnitude of intronic and intergenic DNA can vary substantially both within and among evolutionary lineages; however, the forces responsible for this disparity in genome compactness are conjectural. One explanation, termed the mutational-burden hypothesis, posits that genome compactness is primarily driven by two nonadaptive processes: mutation and random genetic drift – the effects of which can be discerned by measuring the nucleotide diversity at silent sites (πsilent, defined as noncoding sites and the synonymous sites of protein-coding regions. The mutational-burden hypothesis holds that πsilent is negatively correlated to genome compactness. We used the model organism Chlamydomonas reinhardtii, which has a streamlined, coding-dense mitochondrial genome and an noncompact, intron-rich nuclear genome, to investigate the mutational-burden hypothesis. For measuring πsilent we sequenced the complete mitochondrial genome and portions of 7 nuclear genes from 7 geographical isolates of C. reinhardtii. Results We found significantly more nucleotide diversity in the nuclear compartment of C. reinhardtii than in the mitochondrial compartment: net values of πsilent for the nuclear and mitochondrial genomes were 32 × 10-3 and 8.5 × 10-3, respectively; and when insertions and deletions (indels are factored in, these values become 49 × 10-3 for the nuclear DNA and 11 × 10-3 for the mitochondrial DNA (mtDNA. Furthermore, our investigations of C. reinhardtii revealed 4 previously undiscovered mitochondrial introns, one of which contains a fragment of the large-subunit (LSU rRNA gene and another of which is found in a region of the LSU-rRNA gene not previously reported (for any taxon to contain introns. Conclusion At first glance our results are in opposition to the mutational-burden hypothesis: πsilent was approximately 4 times greater in the nuclear compartment of C. reinhardtii relative to the mitochondrial compartment

  8. Localization of the enzymes involved in the photoevolution of H sub 2 from acetate in Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Willeford, K.O.; Gibbs, M. (Brandeis Univ., Waltham, MA (USA))

    1989-07-01

    The localization of a series of enzymes involved in the anaerobic photodissimilation of acetate in Chlamydomonas reinhardtii F-60 adapted to a hydrogen metabolism was determined through the enzymatic analyses of the chloroplastic, cytoplasmic, and mitochondrial fractions obtained with a cellular fractionation procedure that incorporated cell wall removal by treatment with autolysine, digestion of the plasmalemma with the detergent digitonin, and fractionation by differential centrifugation on a Percoll step gradient. The sequence of events leading to the photoevolution of H{sub 2} from acetate includes the conversion of acetate into succinate via the extraplastidic glyoxylate cycle, the oxidation of succinate to fumarate by chloroplastic succinate dehydrogenase, and the oxidation of malate to oxaloacetate in the chloroplast by NAD dependent malate dehydrogenase. The level of potential activity for the enzymes assayed were sufficient to accommodate the observed rate of the photoanaerobic dissimilation of acetate and the photoevolution of H{sub 2}.

  9. The Effect of DNA and Sodium Cholate Dispersed Single-Walled Carbon Nanotubes on the Green Algae Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Ryan M. Williams

    2014-01-01

    Full Text Available Increasing use of single-walled carbon nanotubes (SWCNTs will lead to their increased release into the environment. Previous work has shown negative effects of SWCNT on growth and survival of model organisms. The aim of the current study was to determine the effect of SWCNT well-dispersed by either DNA or sodium cholate (SC on the unicellular green algae Chlamydomonas reinhardtii in stagnant water conditions. Growth measurements were taken up to ten days for algae treated with varied levels of DNA:SWCNT or SC:SWCNT or controls, and chlorophyll content after 10 days was determined. Results show no effect on either growth or chlorophyll content of algae at any concentration or duration. This is in contradiction to prior work showing toxicity of SWCNT to environmental model organisms.

  10. Artificial miRNA inhibition of phosphoenolpyruvate carboxylase increases fatty acid production in a green microalga Chlamydomonas reinhardtii.

    Science.gov (United States)

    Wang, Chaogang; Chen, Xi; Li, Hui; Wang, Jiangxin; Hu, Zhangli

    2017-01-01

    Nutrient limitation, such as nitrogen depletion, is the most widely used method for improving microalgae fatty acid production; however, these harsh conditions also inhibit algal growth significantly and even kill cells at all. To avoid these problems, we used artificial microRNA (amiRNA) technology as a useful tool to manipulate metabolic pathways to increase fatty acid contents effectively in the green microalga Chlamydomonas reinhardtii. We down-regulated the expression of phosphoenolpyruvate carboxylase (PEPC), which catalyzes the formation of oxaloacetate from phosphoenolpyruvate and regulates carbon flux. amiRNAs against two CrPEPC genes were designed and transformed into Chlamydomonas cells and amiRNAs were induced by heat shock treatment. The transcription levels of amiRNAs increased 16-28 times, resulting in the remarkable decreases of the expression of CrPEPCs. In the end, inhibiting the expression of the CrPEPC genes dramatically increased the total fatty acid content in the transgenic algae by 28.7-48.6%, which mostly increased the content of C16-C22 fatty acids. Furthermore, the highest content was that of C18:3n3 with an average increase of 35.75%, while C20-C22 fatty acid content significantly increased by 85-160%. Overall our results suggest that heat shock treatment induced the expression of amiRNAs, which can effectively down-regulate the expression of CrPEPCs in C. reinhardtii, resulting in an increase of fatty acid synthesis with the most significant increase occurring for C16 to C22 fatty acids.

  11. The energy balance of the biomass generation of Chlamydomonas acidophila under acidic and neutral conditions and Chlamydomonas reinhardtii; Die Energiebilanz der Biomasseneubildung von Chlamydomonas acidophila unter sauren und neutralen Bedingungen und von Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Langner, Uwe

    2009-01-16

    In this study the influence of pH < 3 as an extreme environment has been investigated for the eukaryotic green alga Chlamydomonas (C.) acidophila. The limited number of trophic levels, consisting of bacteria, phytoplankton, zooplankton and macrophytes, is a special characteristic of extreme acidic water bodies. C. acidophila was isolated from an extreme acidic mining lake (RL 111) (Bissinger et al. 2000). A special feature of the examined algal species is its wide tolerance range of external pH values from 2 to 7 (Cassin 1874, Gerloff-Elias et al. 2005a). C. acidophila is a dominant species in the acidic mining lakes, it can grow up to chlorophyll maxima of 500 {mu}g L{sup -1} during the summer time (Nixdorf et al. 1998, 2003). The alga can be found elsewhere in extreme acidic water bodies around the world. The hydrochemistry of the acidic mining lakes in the central regions of Germany and Lusatia show clear differences compared to neutral water bodies. Some of the characteristics of acidic mining lakes are high metal and heavy metal (aluminum) concentrations, high ion concentrations, which lead to high conductivity, as well as low phosphate concentrations, ammonium as the only nitrogen source and CO{sub 2} as the only inorganic carbon source. Many eukaryotic microalgae in acidic water bodies including C. acidophila show a neutral cytosolic pH. This is provided by special adaptations of the organisms. Thus, organisms in extreme acidic environments have a positive cell surface charge, a very efficient H{sup +}-ATPase and high internal buffer capacities. These mechanisms work contrary to the proton influx and the acidification of the cytosol and are therefore proof for the physiological impact of osmoregulation by microalgae in extreme acidic environments (Sekler et al.1991, Weiss et al. 1999). Hence, these mechanisms also complicate the access to nutrients for the algal cell. The passive influx of positively charged ions such as potassium or ammonium is reduced by

  12. State transitions in Chlamydomonas reinhardtii. The role of the Mehler reaction in state 2-to-state 1 transition.

    Science.gov (United States)

    Forti, Giorgio; Caldiroli, Giovanni

    2005-02-01

    The light intensity-dependent transition to state 1 of dark-adapted anaerobic state 2 Chlamydomonas reinhardtii cells is stimulated by oxygen and by other electron acceptors for photosystem I, such as oxaloacetate and methylviologen. This suggests that the transition to state 1 requires the oxidation of the intersystem chain by photosystem I photochemistry. On the other hand, the mere oxidation in the dark of the chain-by addition of O2-leads only to a slow and incomplete transition. The light-driven stimulation by O2 of the state 1 transition is saturated at an O2 concentration of 15 to 20 microm, definitely higher than that of respiration. We suggest that this may represent the affinity for oxygen of the Mehler reaction, a conclusion that is confirmed by the observations that mitochondrial respiration is apparently not involved in modulating state 2-to-state 1 transition. The catalysis of the state 2-to-state 1 transition upon illumination of anaerobically adapted algae might represent, therefore, a relevant physiological role of this process in C. reinhardtii.

  13. mRNA imaging in the chloroplast of Chlamydomonas reinhardtii using the light-up aptamer Spinach.

    Science.gov (United States)

    Guzmán-Zapata, Daniel; Domínguez-Anaya, Yael; Macedo-Osorio, Karla S; Tovar-Aguilar, Andrea; Castrejón-Flores, José L; Durán-Figueroa, Noé V; Badillo-Corona, Jesús A

    2017-06-10

    Light-up aptamers are practical tools to image RNA localization in vivo. A now classical light-up aptamer system is the combination of the 3,5-difluoro-4-hydroxybenzylidene (DFHBI) fluorogen and the RNA aptamer Spinach, which has been successfully used in bacterial and mammalian cells. However, light-up aptamers have not been used in algae. Here, we show that a simple vector, carrying Spinach, transcriptionally fused to the aphA-6 gene, can be effectively used to generate a functional light-up aptamer in the chloroplast of Chlamydomonas reinhardtii. After incubation with DFHBI, lines expressing the aphA-6/Spinach mRNA were observed with laser confocal microscopy to evaluate the functionality of the light-up aptamer in the chloroplast of C. reinhardtii. Clear and strong fluorescence was localized to the chloroplast, in the form of discrete spots. There was no background fluorescence in the strain lacking Spinach. Light-up aptamers could be further engineered to image RNA or to develop genetically encoded biosensors in algae. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. The oxidative stress in allelopathy: Participation of prenyllipid antioxidants in the response to juglone in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Nowicka, Beatrycze; Żądło, Andrzej; Pluciński, Bartosz; Kruk, Jerzy; Kuczyńska, Paulina

    2017-09-21

    Allelopathy is a phenomenon, where one species releases compounds able to inhibit the growth of other species. Juglone, 5-hydroxy-1,4-naphtoquinone, is an allelochemical produced by walnut trees. The main mode of juglone toxicity is the formation of semiquinone radicals, able to reduce O2 to superoxide. Prenyllipid antioxidants such as tocopherol and plastoquinone are important for antioxidant defense in photosynthetic organisms. Here we assess their participation in the response to juglone. The impact of 20 μM juglone on the content of photosynthetic pigments and prenyllipid antioxidants in green microalga Chlamydomonas reinhardtii was measured over an incubation period of 7.5 h in low light and over 40 min under high light or in darkness. The decrease in pigment and prenyllipid content, accompanied by an increase in lipid hydroperoxides was observed over a longer incubation period with juglone. Simultaneous exposure to high light and juglone led to a pronounced decrease in carotenoids and prenyllipids, while there was no decrease in high light alone and no decrease or only a slight decrease in the series with juglone alone. The fact that semiquinone radicals are generated in juglone-exposed cells was confirmed using EPR spectroscopy. This article also shows that C. reinhardtii may be a suitable model for studies on some modes of phytotoxic action of allelochemicals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Jeong, Jooyeon; Baek, Kwangryul; Kirst, Henning; Melis, Anastasios; Jin, EonSeon

    2017-01-01

    The Chlamydomonas reinhardtii truncated light-harvesting antenna 4 (tla4) DNA transposon mutant has a pale green phenotype, a lower chlorophyll (Chl) per cell and a higher Chl a/b ratio in comparison with the wild type. It required a higher light intensity for the saturation of photosynthesis and displayed a greater per chlorophyll light-saturated rate of oxygen evolution than the wild type. The Chl antenna size of the photosystems in the tla4 mutant was only about 65% of that measured in the wild type. Molecular genetic analysis revealed that a single plasmid DNA insertion disrupted two genes on chromosome 11 of the mutant. A complementation study identified the "chloroplast signal recognition particle 54" gene (CpSRP54), as the lesion causing the tla4 phenotype. Disruption of this gene resulted in partial failure to assemble and, therefore, lower levels of light-harvesting Chl-binding proteins in the C. reinhardtii thylakoids. A comparative in silico 3-D structure-modeling analysis revealed that the M-domain of the CpSRP54 of C. reinhardtii possesses a more extended finger loop structure, due to different amino acid composition, as compared to that of the Arabidopsis CpSRP54. The work demonstrated that CpSRP54 deletion in microalgae can serve to generate tla mutants with a markedly smaller photosystem Chl antenna size, improved solar energy conversion efficiency, and photosynthetic productivity in high-density cultures under bright sunlight conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Using single cell cultivation system for on-chip monitoring of the interdivision timer in Chlamydomonas reinhardtii cell cycle

    Directory of Open Access Journals (Sweden)

    Soloviev Mikhail

    2010-09-01

    Full Text Available Abstract Regulation of cell cycle progression in changing environments is vital for cell survival and maintenance, and different regulation mechanisms based on cell size and cell cycle time have been proposed. To determine the mechanism of cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii, we developed an on-chip single-cell cultivation system that allows for the strict control of the extracellular environment. We divided the Chlamydomonas cell cycle into interdivision and division phases on the basis of changes in cell size and found that, regardless of the amount of photosynthetically active radiation (PAR and the extent of illumination, the length of the interdivision phase was inversely proportional to the rate of increase of cell volume. Their product remains constant indicating the existence of an 'interdivision timer'. The length of the division phase, in contrast, remained nearly constant. Cells cultivated under light-dark-light conditions did not divide unless they had grown to twice their initial volume during the first light period. This indicates the existence of a 'commitment sizer'. The ratio of the cell volume at the beginning of the division phase to the initial cell volume determined the number of daughter cells, indicating the existence of a 'mitotic sizer'.

  17. Photoregulation of fructose and glucose respiration in the intact chloroplasts of Chlamydomonas reinhardtii F-60 and spinach

    Energy Technology Data Exchange (ETDEWEB)

    Singh, K.K.; Changguo Chen; Gibbs, M. (Brandeis Univ., Waltham, MA (United States))

    1993-04-01

    The photoregulation of chloroplastic respiration was studied by monitoring in darkness and in light the release of [sup 14]CO[sub 2] from whole chloroplasts of Chlamydomonas reinhardtii F-60 and spinach (Spinacia oleracea L.) supplied externally with [[sup 14]C]glucose and [[sup 14]C]fructose, respectively. CO[sub 2] release was inhibited more than 90% in both chloroplasts by a light intensity of 4 W m[sup [minus]2]. Oxidants, oxaloacetate in Chlamydomonas, nitrite in spinach, and phenazine methosulfate in both chloroplasts, reversed the inhibition. The onset of the photoinhibitory effect on CO[sub 2] release was relatively rapid compared to the restoration of CO[sub 2] release following illumination. In both darkened chloroplasts, dithiothreitol inhibited release. Of the four enzymes (fructokinase, phosphoglucose isomerase, glucose-6-P dehydrogenase, and gluconate-6-P dehydrogenase) in the pathway catalyzing the release of CO[sub 2] from fructose, only glucose-6-P dehydrogenase was deactivated by light and by dithiothreitol. 33 refs., 3 figs., 4 tabs.

  18. Evidence for chloroplastic succinate dehydrogenase participating in the chloroplastic respiratory and photosynthetic electron transport chains of Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Willeford, K.O.; Gombos, Z.; Gibbs, M. (Brandeis Univ., Waltham, MA (USA))

    1989-07-01

    A method for isolating intact chloroplasts from Chlamydomonas reinhardtii F-60 was developed from the Klein, Chen, Gibbs, Platt-Aloia procedure. Protoplasts, generated by treatment with autolysine, were lysed with a solution of digitonin and fractionated on Percoll step gradients. The chloroplasts were assessed to be 90% intact (ferricyanide assay) and free from cytoplasmic contamination (NADP isocitrate dehydrogenase activity) and to range from 2 to 5% in mitochondrial contamination (cytochrome c oxidase activity). About 25% of the cellular succinate dehydrogenase activity (21.6 micromoles per milligram chlorophyll per hour, as determined enzymically) was placed within the chloroplast. Chloroplastic succinate dehydrogenase had a K{sub m} for succinate of 0.55 millimolar and was associated with the thylakoidal material derived from the intact chloroplasts. This same thylakoidal material, with an enzymic assay of 21.6 micromoles per milligram chlorophyll per hour was able to initiate a light-dependent uptake of oxygen at a rate of 16.4 micromoles per milligram chlorophyll per hour when supplied with succinate and methyl viologen. Malonate was an apparent competitive inhibitor of this reaction. The succinate dehydrogenase activity present in the chloroplast was sufficient to account for the photoanaerobic rate of acetate dissimilation in H{sub 2} adapted Chlamydomonas.

  19. Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes

    Science.gov (United States)

    Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Marshall, Wallace F.

    2005-01-01

    The important role that cilia and flagella play in human disease creates an urgent need to identify genes involved in ciliary assembly and function. The strong and specific induction of flagellar-coding genes during flagellar regeneration in Chlamydomonas reinhardtii suggests that transcriptional profiling of such cells would reveal new flagella-related genes. We have conducted a genome-wide analysis of RNA transcript levels during flagellar regeneration in Chlamydomonas by using maskless photolithography method-produced DNA oligonucleotide microarrays with unique probe sequences for all exons of the 19,803 predicted genes. This analysis represents previously uncharacterized whole-genome transcriptional activity profiling study in this important model organism. Analysis of strongly induced genes reveals a large set of known flagellar components and also identifies a number of important disease-related proteins as being involved with cilia and flagella, including the zebrafish polycystic kidney genes Qilin, Reptin, and Pontin, as well as the testis-expressed tubby-like protein TULP2.

  20. Modeling the dependence of respiration and photosynthesis upon light, acetate, carbon dioxide, nitrate and ammonium in Chlamydomonas reinhardtii using design of experiments and multiple regression.

    Science.gov (United States)

    Gérin, Stéphanie; Mathy, Grégory; Franck, Fabrice

    2014-08-16

    In photosynthetic organisms, the influence of light, carbon and inorganic nitrogen sources on the cellular bioenergetics has extensively been studied independently, but little information is available on the cumulative effects of these factors. Here, sequential statistical analyses based on design of experiments (DOE) coupled to standard least squares multiple regression have been undertaken to model the dependence of respiratory and photosynthetic responses (assessed by oxymetric and chlorophyll fluorescence measurements) upon the concomitant modulation of light intensity as well as acetate, CO₂, nitrate and ammonium concentrations in the culture medium of Chlamydomonas reinhardtii. The main goals of these analyses were to explain response variability (i.e. bioenergetic plasticity) and to characterize quantitatively the influence of the major explanatory factor(s). For each response, 2 successive rounds of multiple regression coupled to one-way ANOVA F-tests have been undertaken to select the major explanatory factor(s) (1st-round) and mathematically simulate their influence (2nd-round). These analyses reveal that a maximal number of 3 environmental factors over 5 is sufficient to explain most of the response variability, and interestingly highlight quadratic effects and second-order interactions in some cases. In parallel, the predictive ability of the 2nd-round models has also been investigated by k-fold cross-validation and experimental validation tests on new random combinations of factors. These validation procedures tend to indicate that the 2nd-round models can also be used to predict the responses with an inherent deviation quantified by the analytical error of the models. Altogether, the results of the 2 rounds of modeling provide an overview of the bioenergetic adaptations of C. reinhardtii to changing environmental conditions and point out promising tracks for future in-depth investigations of the molecular mechanisms underlying the present

  1. A cost-effective approach to produce15N-labelled amino acids employing Chlamydomonas reinhardtii CC503.

    Science.gov (United States)

    Nicolás Carcelén, Jesús; Marchante-Gayón, Juan Manuel; González, Pablo Rodríguez; Valledor, Luis; Cañal, María Jesús; Alonso, José Ignacio García

    2017-08-18

    The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described. The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, 15 N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15 NH 4 Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15 N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC-MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL. Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only 13

  2. Filling Knowledge Gaps in Biological Networks: integrating global approaches to understand H2 metabolism in Chlamydomonas reinhardtii - Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Posewitz, Matthew C

    2011-06-30

    The green alga Chlamydomonas reinhardtii (Chlamydomonas) has numerous genes encoding enzymes that function in fermentative pathways. Among these genes, are the [FeFe]-hydrogenases, pyruvate formate lyase, pyruvate ferredoxin oxidoreductase, acetate kinase, and phosphotransacetylase. We have systematically undertaken a series of targeted mutagenesis approaches to disrupt each of these key genes and omics techniques to characterize alterations in metabolic flux. Funds from DE-FG02-07ER64423 were specifically leveraged to generate mutants with disruptions in the genes encoding the [FeFe]-hydrogenases HYDA1 and HYDA2, pyruvate formate lyase (PFL1), and in bifunctional alcohol/aldehyde alcohol dehydrogenase (ADH1). Additionally funds were used to conduct global transcript profiling experiments of wildtype Chlamydomonas cells, as well as of the hydEF-1 mutant, which is unable to make H2 due to a lesion in the [FeFe]-hydrogenase biosynthetic pathway. In the wildtype cells, formate, acetate and ethanol are the dominant fermentation products with traces of CO2 and H2 also being produced. In the hydEF-1 mutant, succinate production is increased to offset the loss of protons as a terminal electron acceptor. In the pfl-1 mutant, lactate offsets the loss of formate production, and in the adh1-1 mutant glycerol is made instead of ethanol. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars, and a decline in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant performs a complete rerouting of the glycolytic carbon to lactate and glycerol. Lastly, transcriptome data have been analysed for both the wildtype and hydEF-1, that correlate with our

  3. The involvement of carbohydrate reserves in hydrogen photoproduction by the green alga Chlamydomonas reinhardtii; L'implication des reserves carbonees dans la photoproduction d'hydrogene chez l'algue verte Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Chochois, V.

    2009-09-15

    The unicellular green alga Chlamydomonas reinhardtii is able to produce hydrogen, using water as an electron donor, and sunlight as an energy source. Although this property offers interesting biotechnological perspectives, a major limitation is related to the sensitivity of hydrogenase to oxygen which is produced by photosynthesis. It had been previously shown that in conditions of sulfur deprivation, C. reinhardtii is able to produce hydrogen during several days (Melis et an. 2000). During this process, two pathways, one direct depending on photosystem II (PSII) activity and the other involving only the PSI, are involved, starch reserves being supposed to play a role in both of these pathways. The purpose of this phD thesis was to elucidate the mechanisms linking starch catabolism to the hydrogen photoproduction process. Firstly, the analysis of mutants affected in starch biosynthesis (sta6 and sta7) showed that if starch reserves are essential to the functioning of the indirect pathway, they are not involved in the direct one. Secondly, in order to identify metabolic steps and regulatory processes involved in starch breakdown, we developed a genetic approach based on the search of mutants affected in starch reserves mobilization. Eight mutant (std1 to std8) diversely affected in their ability to degrade starch after an accumulation phase have been isolated from an insertional mutant library of 15,000 clones. One of these mutants, std1, is affected in a kinase related to the DYRK family (dual-specificity tyrosine regulated serine threonine kinase). Although the targets of this putative kinase remain to be identified, the analysis of the granule bound proteome displayed profound alterations in the expression profile of starch phosphorylases, potentially involved in starch breakdown. STD1 represents the first starch catabolism regulator identified to date in plants. (author)

  4. Characterization of singlet oxygen-accumulating mutants isolated in a screen for altered oxidative stress response in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Eggen Rik IL

    2010-12-01

    Full Text Available Abstract Background When photosynthetic organisms are exposed to harsh environmental conditions such as high light intensities or cold stress, the production of reactive oxygen species like singlet oxygen is stimulated in the chloroplast. In Chlamydomonas reinhardtii singlet oxygen was shown to act as a specific signal inducing the expression of the nuclear glutathione peroxidase gene GPXH/GPX5 during high light stress, but little is known about the cellular mechanisms involved in this response. To investigate components affecting singlet oxygen signaling in C. reinhardtii, a mutant screen was performed. Results Mutants with altered GPXH response were isolated from UV-mutagenized cells containing a GPXH-arylsulfatase reporter gene construct. Out of 5500 clones tested, no mutant deficient in GPXH induction was isolated, whereas several clones showed constitutive high GPXH expression under normal light conditions. Many of these GPXH overexpressor (gox mutants exhibited higher resistance to oxidative stress conditions whereas others were sensitive to high light intensities. Interestingly, most gox mutants produced increased singlet oxygen levels correlating with high GPXH expression. Furthermore, different patterns of altered photoprotective parameters like non-photochemical quenching, carotenoid contents and α-tocopherol levels were detected in the various gox mutants. Conclusions Screening for mutants with altered GPXH expression resulted in the isolation of many gox mutants with increased singlet oxygen production, showing the relevance of controlling the production of this ROS in photosynthetic organisms. Phenotypic characterization of these gox mutants indicated that the mutations might lead to either stimulated triplet chlorophyll and singlet oxygen formation or reduced detoxification of singlet oxygen in the chloroplast. Furthermore, changes in multiple protection mechanisms might be responsible for high singlet oxygen formation and GPXH

  5. Multiple stressor effects in Chlamydomonas reinhardtii – Toward understanding mechanisms of interaction between effects of ultraviolet radiation and chemical pollutants

    Energy Technology Data Exchange (ETDEWEB)

    Korkaric, Muris [Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, 8600, Duebendorf (Switzerland); ETH Zürich, Institute of Biogeochemistry and Pollutant Dynamics, 8092 Zürich (Switzerland); Behra, Renata; Fischer, Beat B. [Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, 8600, Duebendorf (Switzerland); Junghans, Marion [Swiss Center for Applied Ecotoxicology Eawag-EPFL, 8600, Duebendorf (Switzerland); Eggen, Rik I.L., E-mail: rik.eggen@eawag.ch [Eawag, Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, 8600, Duebendorf (Switzerland); ETH Zürich, Institute of Biogeochemistry and Pollutant Dynamics, 8092 Zürich (Switzerland)

    2015-05-15

    Highlights: • Systematic study of multiple stressor effects of UVR and chemicals in C. reinhardtii. • UVR and chemicals did not act independently on algal photosynthesis and reproduction. • Multiple stressor effects of UVR and chemicals depended on chemical MOA. • Synergistic effect interactions not limited to oxidative stress inducing chemicals. • Multiple MOAs of UVR may limit applicability of current prediction models. - Abstract: The effects of chemical pollutants and environmental stressors, such as ultraviolet radiation (UVR), can interact when organisms are simultaneously exposed, resulting in higher (synergistic) or lower (antagonistic) multiple stressor effects than expected based on the effects of single stressors. Current understanding of interactive effects is limited due to a lack of mechanism-based multiple stressor studies. It has been hypothesized that effect interactions may generally occur if chemical and non-chemical stressors cause similar physiological effects in the organism. To test this hypothesis, we exposed the model green alga Chlamydomonas reinhardtii to combinations of UVR and single chemicals displaying modes of action (MOA) similar or dissimilar to the impact of UVR on photosynthesis. Stressor interactions were analyzed based on the independent action model. Effect interactions were found to depend on the MOA of the chemicals, and also on their concentrations, the exposure time and the measured endpoint. Indeed, only chemicals assumed to cause effects on photosynthesis similar to UVR showed interactions with UVR on photosynthetic yield: synergistic in case of Cd(II) and paraquat and antagonistic in case of diuron. No interaction on photosynthesis was observed for S-metolachlor, which acts dissimilarly to UVR. However, combined effects of S-metolachlor and UVR on algal reproduction were synergistic, highlighting the importance of considering additional MOA of UVR. Possible mechanisms of stressor effect interactions are

  6. The Phosphoproteome of a Chlamydomonas reinhardtii Eyespot Fraction Includes Key Proteins of the Light Signaling Pathway1[W

    Science.gov (United States)

    Wagner, Volker; Ullmann, Katharina; Mollwo, Anne; Kaminski, Marc; Mittag, Maria; Kreimer, Georg

    2008-01-01

    Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca2+-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii. PMID:18065559

  7. Site Energies of Active and Inactive Pheophytins in the Reaction Center of Photosystem II from Chlamydomonas Reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Acharya, K.; Neupane, B.; Zazubovich, V.; Sayre, R. T.; Picorel, R.; Seibert, M.; Jankowiak, R.

    2012-03-29

    It is widely accepted that the primary electron acceptor in various Photosystem II (PSII) reaction center (RC) preparations is pheophytin {alpha} (Pheo {alpha}) within the D1 protein (Pheo{sub D1}), while Pheo{sub D2} (within the D2 protein) is photochemically inactive. The Pheo site energies, however, have remained elusive, due to inherent spectral congestion. While most researchers over the past two decades placed the Q{sub y}-states of Pheo{sub D1} and Pheo{sub D2} bands near 678-684 and 668-672 nm, respectively, recent modeling [Raszewski et al. Biophys. J. 2005, 88, 986-998; Cox et al. J. Phys. Chem. B 2009, 113, 12364-12374] of the electronic structure of the PSII RC reversed the assignment of the active and inactive Pheos, suggesting that the mean site energy of Pheo{sub D1} is near 672 nm, whereas Pheo{sub D2} ({approx}677.5 nm) and Chl{sub D1} ({approx}680 nm) have the lowest energies (i.e., the Pheo{sub D2}-dominated exciton is the lowest excited state). In contrast, chemical pigment exchange experiments on isolated RCs suggested that both pheophytins have their Q{sub y} absorption maxima at 676-680 nm [Germano et al. Biochemistry 2001, 40, 11472-11482; Germano et al. Biophys. J. 2004, 86, 1664-1672]. To provide more insight into the site energies of both Pheo{sub D1} and Pheo{sub D2} (including the corresponding Q{sub x} transitions, which are often claimed to be degenerate at 543 nm) and to attest that the above two assignments are most likely incorrect, we studied a large number of isolated RC preparations from spinach and wild-type Chlamydomonas reinhardtii (at different levels of intactness) as well as the Chlamydomonas reinhardtii mutant (D2-L209H), in which the active branch Pheo{sub D1} is genetically replaced with chlorophyll {alpha} (Chl {alpha}). We show that the Q{sub x}-/Q{sub y}-region site energies of Pheo{sub D1} and Pheo{sub D2} are {approx}545/680 nm and {approx}541.5/670 nm, respectively, in good agreement with our previous assignment

  8. A C2H2 zinc finger protein FEMU2 is required for fox1 expression in Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Xiaodong Deng

    Full Text Available Chlamydomonas reinhardtii fox1 gene encodes a ferroxidase that is involved in cellular Fe uptake and highly induced during Fe deficient conditions. In an effort to identify fox1 promoter regulatory elements, an insertional library was generated in a transgenic Chlamydomonas strain (2A38 harboring an arylsulfatase (ARS reporter gene driven by the fox1 promoter. Mutants with a defective response to low iron conditions were selected for further study. Among these, a strain containing a disrupted femu2 gene was identified. Activation of the fox1 promoter by the femu2 gene product was confirmed by silencing the femu2 gene using RNA interference. In three femu2 RNAi transgenic lines (IR3, IR6, and IR7, ARS reporter gene activities declined by 84.3%, 86.4%, and 88.8%, respectively under Fe deficient conditions. Furthermore, RT-PCR analysis of both the femu2 mutant and the RNAi transgenic lines showed significantly decreased transcript abundance of the endogenous fox1 gene under Fe deficient conditions. Amino acid sequence analysis of the femu2 gene product identified three potential C2H2 zinc finger (ZF motifs and a nuclear localization study suggests that FEMU2 is localized to the nucleus. In addition, a potential FEMU2 binding site ((G/TTTGG(G/T(G/TT was identified using PCR-mediated random binding site selection. Taken together, this evidence suggests that FEMU2 is involved in up-regulation of the fox1 gene in Fe deficient cells.

  9. High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii

    DEFF Research Database (Denmark)

    Ramos Martinez, Erick Miguel; Fimognari, Lorenzo; Sakuragi, Yumiko

    2017-01-01

    reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion...... yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n , wherein n = 10 or 20]. The yields of the (SP)n -fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum...... yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP)n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts...

  10. A Novel Negative Fe-Deficiency-Responsive Element and a TGGCA-Type-Like FeRE Control the Expression of FTR1 in Chlamydomonas reinhardtii

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    Xiaowen Fei

    2010-01-01

    Full Text Available We have reported three Fe-deficiency-responsive elements (FEREs, FOX1, ATX1, and FEA1, all of which are positive regulatory elements in response to iron deficiency in Chlamydomonas reinhardtii. Here we describe FTR1, another iron regulated gene and mutational analysis of its promoter. Our results reveal that the FeREs of FTR1 distinguish itself from other iron response elements by containing both negative and positive regulatory regions. In FTR1, the −291/−236 region from the transcriptional start site is necessary and sufficient for Fe-deficiency-inducible expression. This region contains two positive FeREs with a TGGCA-like core sequence: the FtrFeRE1 (ATGCAGGCT at −287/−279 and the FtrFeRE2 (AAGCGATTGCCAGAGCGC at −253/−236. Furthermore, we identified a novel FERE, FtrFeRE3 (AGTAACTGTTAAGCC localized at −319/−292, which negatively influences the expression of FTR1.

  11. Fe Sparing and Fe Recycling Contribute to Increased Superoxide Dismutase Capacity in Iron-Starved Chlamydomonas reinhardtii[W

    Science.gov (United States)

    Page, M. Dudley; Allen, Michael D.; Kropat, Janette; Urzica, Eugen I.; Karpowicz, Steven J.; Hsieh, Scott. I.; Loo, Joseph A.; Merchant, Sabeeha S.

    2012-01-01

    Fe deficiency is one of several abiotic stresses that impacts plant metabolism because of the loss of function of Fe-containing enzymes in chloroplasts and mitochondria, including cytochromes, FeS proteins, and Fe superoxide dismutase (FeSOD). Two pathways increase the capacity of the Chlamydomonas reinhardtii chloroplast to detoxify superoxide during Fe limitation stress. In one pathway, MSD3 is upregulated at the transcriptional level up to 103-fold in response to Fe limitation, leading to synthesis of a previously undiscovered plastid-specific MnSOD whose identity we validated immunochemically. In a second pathway, the plastid FeSOD is preferentially retained over other abundant Fe proteins, heme-containing cytochrome f, diiron magnesium protoporphyrin monomethyl ester cyclase, and Fe2S2-containing ferredoxin, demonstrating prioritized allocation of Fe within the chloroplast. Maintenance of FeSOD occurs, after an initial phase of degradation, by de novo resynthesis in the absence of extracellular Fe, suggesting the operation of salvage mechanisms for intracellular recycling and reallocation. PMID:22685165

  12. Down-regulation of catalase activity allows transient accumulation of a hydrogen peroxide signal in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Michelet, Laure; Roach, Thomas; Fischer, Beat B; Bedhomme, Mariette; Lemaire, Stéphane D; Krieger-Liszkay, Anja

    2013-06-01

    In photosynthetic organisms, excess light is a stress that induces production of reactive oxygen species inside the chloroplasts. As a response, the capacity of antioxidative defence mechanisms increases. However, when cells of Chlamydomonas reinhardtii were shifted from dark to high light, a reversible partial inactivation of catalase activity was observed, which correlated with a transient increase in the level of H2 O2 in the 10 μm range. This concentration range seems to be necessary to activate H2 O2 -dependent signalling pathways stimulating the expression of H2 O2 responsive genes, such as the heat shock protein HSP22C. Catalase knock-down mutants had lost the transient accumulation of H2 O2 , suggesting that a decrease in catalase activity was the key element for establishing a transient H2 O2 burst. Catalase was inactivated by a one-electron event consistent with the reduction of a single cysteine. We propose that under high light intensity, the redox state of the photosynthetic electron transport chain is sensed and transmitted to the cytosol to regulate the catalase activity. This allows a transient accumulation of H2 O2 , inducing a signalling event that is transmitted to the nucleus to modulate the expression of chloroplast-directed protection enzymes. © 2012 Blackwell Publishing Ltd.

  13. Gene elements that affect the longevity of rbcL sequence-containing transcripts in Chlamydomonas reinhardtii chloroplasts.

    Science.gov (United States)

    Singh, M; Boutanaev, A; Zucchi, P; Bogorad, L

    2001-02-27

    The chloroplast gene rbcL encodes the large subunit of the CO(2)-fixing enzyme ribulose-bisphosphate carboxylase. In previous work a target for photo-accelerated degradation of Chlamydomonas reinhardtii rbcL transcripts in vivo was found to lie within the first 63 nucleotides, and a sequence element required for increasing the longevity of transcripts of rbcL-reporter genes was found to occur between nucleotides 170 and 350. Photo-accelerated degradation of rbcL transcripts has been found to require nucleotides 21 to 41. Transcript nucleotides lying between 329 and 334 and between 14 and 27 are essential for stabilizing transcripts in vivo; mutations in either region reduce the longevity of transcripts. It is postulated that the effectiveness of photo-accelerated endonuclease attacks on the nucleotide 21 to 41 region is reduced by physical blockage or distortion of the target sequence by interacting proteins that associate with nucleotides in the 14 to 27 and 329 to 334 regions of the transcripts. Both the nucleotide +329 to +334 stabilizing sequence of rbcL and a transcription enhancing sequence that lies between +126 and +170 encode well conserved (cyanobacteria through angiosperms) amino acid sequences; the evolution of expression control elements within the protein coding sequence of rbcL is considered.

  14. CCS2, an Octatricopeptide-Repeat Protein, Is Required for Plastid Cytochrome c Assembly in the Green Alga Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Sara G. Cline

    2017-08-01

    Full Text Available In bacteria and energy generating organelles, c-type cytochromes are a class of universal electron carriers with a heme cofactor covalently linked via one or two thioether bonds to a heme binding site. The covalent attachment of heme to apocytochromes is a catalyzed process, taking place via three evolutionarily distinct assembly pathways (Systems I, II, III. System II was discovered in the green alga Chlamydomonas reinhardtii through the genetic analysis of the ccs mutants (cytochrome csynthesis, which display a block in the apo- to holo- form conversion of cytochrome f and c6, the thylakoid lumen resident c-type cytochromes functioning in photosynthesis. Here we show that the gene corresponding to the CCS2 locus encodes a 1,719 amino acid polypeptide and identify the molecular lesions in the ccs2-1 to ccs2-5 alleles. The CCS2 protein displays seven degenerate amino acid repeats, which are variations of the octatricopeptide-repeat motif (OPR recently recognized in several nuclear-encoded proteins controlling the maturation, stability, or translation of chloroplast transcripts. A plastid site of action for CCS2 is inferred from the finding that GFP fused to the first 100 amino acids of the algal protein localizes to chloroplasts in Nicotiana benthamiana. We discuss the possible functions of CCS2 in the heme attachment reaction.

  15. A mutation in the centriole-associated protein centrin causes genomic instability via increased chromosome loss in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Marshall Wallace F

    2005-05-01

    Full Text Available Abstract Background The role of centrioles in mitotic spindle function remains unclear. One approach to investigate mitotic centriole function is to ask whether mutation of centriole-associated proteins can cause genomic instability. Results We addressed the role of the centriole-associated EF-hand protein centrin in genomic stability using a Chlamydomonas reinhardtii centrin mutant that forms acentriolar bipolar spindles and lacks the centrin-based rhizoplast structures that join centrioles to the nucleus. Using a genetic assay for loss of heterozygosity, we found that this centrin mutant showed increased genomic instability compared to wild-type cells, and we determined that the increase in genomic instability was due to a 100-fold increase in chromosome loss rates compared to wild type. Live cell imaging reveals an increased rate in cell death during G1 in haploid cells that is consistent with an elevated rate of chromosome loss, and analysis of cell death versus centriole copy number argues against a role for multipolar spindles in this process. Conclusion The increased chromosome loss rates observed in a centrin mutant that forms acentriolar spindles suggests a role for centrin protein, and possibly centrioles, in mitotic fidelity.

  16. TEF30 Interacts with Photosystem II Monomers and Is Involved in the Repair of Photodamaged Photosystem II in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Muranaka, Ligia Segatto; Rütgers, Mark; Bujaldon, Sandrine; Heublein, Anja; Geimer, Stefan; Wollman, Francis-André; Schroda, Michael

    2016-02-01

    The remarkable capability of photosystem II (PSII) to oxidize water comes along with its vulnerability to oxidative damage. Accordingly, organisms harboring PSII have developed strategies to protect PSII from oxidative damage and to repair damaged PSII. Here, we report on the characterization of the THYLAKOID ENRICHED FRACTION30 (TEF30) protein in Chlamydomonas reinhardtii, which is conserved in the green lineage and induced by high light. Fractionation studies revealed that TEF30 is associated with the stromal side of thylakoid membranes. By using blue native/Deriphat-polyacrylamide gel electrophoresis, sucrose density gradients, and isolated PSII particles, we found TEF30 to quantitatively interact with monomeric PSII complexes. Electron microscopy images revealed significantly reduced thylakoid membrane stacking in TEF30-underexpressing cells when compared with control cells. Biophysical and immunological data point to an impaired PSII repair cycle in TEF30-underexpressing cells and a reduced ability to form PSII supercomplexes after high-light exposure. Taken together, our data suggest potential roles for TEF30 in facilitating the incorporation of a new D1 protein and/or the reintegration of CP43 into repaired PSII monomers, protecting repaired PSII monomers from undergoing repeated repair cycles or facilitating the migration of repaired PSII monomers back to stacked regions for supercomplex reassembly. © 2016 American Society of Plant Biologists. All Rights Reserved.

  17. Application of proton exchange membrane fuel cells for the monitoring and direct usage of biohydrogen produced by Chlamydomonas reinhardtii

    Science.gov (United States)

    Oncel, S.; Vardar-Sukan, F.

    Photo-biologically produced hydrogen by Chlamydomonas reinhardtii is integrated with a proton exchange (PEM) fuel cell for online electricity generation. To investigate the fuel cell efficiency, the effect of hydrogen production on the open circuit fuel cell voltage is monitored during 27 days of batch culture. Values of volumetric hydrogen production, monitored by the help of the calibrated water columns, are related with the open circuit voltage changes of the fuel cell. From the analysis of this relation a dead end configuration is selected to use the fuel cell in its best potential. After the open circuit experiments external loads are tested for their effects on the fuel cell voltage and current generation. According to the results two external loads are selected for the direct usage of the fuel cell incorporating with the photobioreactors (PBR). Experiments with the PEM fuel cell generate a current density of 1.81 mA cm -2 for about 50 h with 10 Ω load and 0.23 mA cm -2 for about 80 h with 100 Ω load.

  18. Comparison of tubular and panel type photobioreactors for biohydrogen production utilizing Chlamydomonas reinhardtii considering mixing time and light intensity.

    Science.gov (United States)

    Oncel, S; Kose, A

    2014-01-01

    Two different photobioreactor designs; tubular and panel, were investigated for the biohydrogen production utilizing a green microalgae Chlamydomonas reinhardtii strain CC124 following the two stage protocol. Mixing time and light intensity of the systems were adjusted to compare the productivity of both aerobic culture phase and the following anaerobic biohydrogen production phase. The results showed there was an effect on both phases related with the design. During the aerobic phase bigger illumination area serving more energy, tubular photobioreactor reached higher biomass productivity of 31.8±2.1 mg L(-1) h(-1) which was about 11% higher than the panel photobioreactor. On the other hand biohydrogen productivity in the panel photobioreactor reached a value of 1.3±0.05 mL L(-1) h(-1) based on the efficient removal of biohydrogen gas. According to the results it would be a good approach to utilize tubular design for aerobic phase and panel for biohydrogen production phase. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Coupling of Carbon Dioxide Fixation to the Oxyhydrogen Reaction in the Isolated Chloroplast of Chlamydomonas reinhardtii1

    Science.gov (United States)

    Chen, Changguo; Gibbs, Martin

    1992-01-01

    The oxyhydrogen reaction (the reduction of O2 to water by H2) in the presence of CO2 was studied in the isolated Chlamydomonas reinhardtii chloroplast by monitoring the rate of 14CO2 incorporation into acid-stable products in the dark. The endogenous rate of CO2 uptake (50-125 nmol/mg chlorophyll per h) was increased about 3- to 4-fold by ATP and additionally when combined with glucose, ribose-5-phosphate, and glycerate-3-phosphate. The rate was diminished 50 to 75%, respectively, when H2 was replaced by N2 or by air. Decrease in CO2 uptake by dl-glyceraldehyde was taken to indicate that the regenerative phase and complete Calvin cycle turnover were involved. Diminution of CO2 incorporation by rotenone, antimycin A, and 2,5-dibromo-3-methyl-6-isopropanol-p-benzoquinone was attributed to an inhibition of the oxyhydrogen reaction, resulting in an elevated NADPH/NADP ratio. If so, then the diminished CO2 uptake could have been by “product inhibition” of the carbon metabolic network. Our data are consistent with the proposal (H. Gaffron [1942] J Gen Physiol 26: 241-267) that CO2 fixation coupled to the oxyhydrogen reaction is dependent to some extent on exchloroplastic metabolism. This support is primarily ATP provided by mitochondrial respiration. PMID:16653129

  20. Gametogenesis in the Chlamydomonas reinhardtii minus Mating Type Is Controlled by Two Genes, MID and MTD1

    Science.gov (United States)

    Lin, Huawen; Goodenough, Ursula W.

    2007-01-01

    In the unicellular algae Chlamydomonas reinhardtii, the plus and minus mating types are controlled by a complex locus, MT, where the dominant MID gene in the MT− locus has been shown to be necessary for expression of minus-specific gamete-specific genes in response to nitrogen depletion. We report studies on MID expression patterns during gametogenesis and on a second gene unique to the MT− locus, MTD1. Vegetative cells express basal levels of MID. An early activation of MID transcription after nitrogen removal, and its sequence similarity to plant RWP-RK proteins involved in nitrogen-responsive processes, suggest that Mid conformation/activity may be nitrogen sensitive. A second stage of MID upregulation correlates with the acquisition of mating ability in minus gametes. Knockdown of MTD1 by RNAi in minus strains results in a failure to differentiate into gametes of either mating type after nitrogen deprivation. We propose that intermediate Mid levels are sufficient to activate MTD1 transcription and to repress plus gamete-specific genes and that MTD1 expression in turn allows the threshold-level MID expression needed to turn on minus gamete-specific genes. We further propose that an MTD1-equivalent system, utilizing at least one gene product encoded in the MT+ locus, is operant during plus gametogenesis. PMID:17435233

  1. Concerted action of the new Genomic Peptide Finder and AUGUSTUS allows for automated proteogenomic annotation of the Chlamydomonas reinhardtii genome.

    Science.gov (United States)

    Specht, Michael; Stanke, Mario; Terashima, Mia; Naumann-Busch, Bianca; Janssen, Ingrid; Höhner, Ricarda; Hom, Erik F Y; Liang, Chun; Hippler, Michael

    2011-05-01

    The use and development of post-genomic tools naturally depends on large-scale genome sequencing projects. The usefulness of post-genomic applications is dependent on the accuracy of genome annotations, for which the correct identification of intron-exon borders in complex genomes of eukaryotic organisms is often an error-prone task. Although automated algorithms for predicting intron-exon structures are available, supporting exon evidence is necessary to achieve comprehensive genome annotation. Besides cDNA and EST support, peptides identified via MS/MS can be used as extrinsic evidence in a proteogenomic approach. We describe an improved version of the Genomic Peptide Finder (GPF), which aligns de novo predicted amino acid sequences to the genomic DNA sequence of an organism while correcting for peptide sequencing errors and accounting for the possibility of splicing. We have coupled GPF and the gene finding program AUGUSTUS in a way that provides automatic structural annotations of the Chlamydomonas reinhardtii genome, using highly unbiased GPF evidence. A comparison of the AUGUSTUS gene set incorporating GPF evidence to the standard JGI FM4 (Filtered Models 4) gene set reveals 932 GPF peptides that are not contained in the Filtered Models 4 gene set. Furthermore, the GPF evidence improved the AUGUSTUS gene models by altering 65 gene models and adding three previously unidentified genes. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Determination of the Speciation and Bioavailability of Sm to Chlamydomonas reinhardtii in the Presence of Natural Organic Matter.

    Science.gov (United States)

    Rowell, Justine-Anne; Fillion, Marc-Alexandre; Smith, Scott; Wilkinson, Kevin J

    2018-02-03

    As technological interest and environmental emissions of the rare earth elements (REE) increase, it is becoming more important to assess their potential environmental impact. Samarium (Sm) is a lanthanide of intermediate molar mass that is used in numerous high technology applications including wind turbines, solar panels and electric vehicles. The present study relates the speciation of samarium (Sm) determined in the presence of natural organic matter (NOM) to its bioavailability to the unicellular green alga, Chlamydomonas reinhardtii. The free ion concentration was determined using a cation exchange resin (IET) in dynamic mode and compared to thermodynamic modelling. Short-term biouptake experiments were performed in the presence of 4 types of NOM: Suwannee River fulvic acids, Pahokee Peat fulvic acids, Suwannee River humic acids and a Luther Marsh dissolved organic matter isolate (90-95% humic acids). The results clearly showed that even a small amount of NOM (0.5 mg C L-1 ) resulted in a significant decrease (10x) in the Sm internalization fluxes. Furthermore, complexation with humic acids (and the corresponding reduction in Sm bioavailability) was stronger than for the fulvic acids. The results showed that the experimentally measured (free) Sm was a better predictor of Sm internalization than either the total concentrations or the free ion concentrations obtained using thermodynamic modelling. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  3. Acetate and bicarbonate assimilation and metabolite formation in Chlamydomonas reinhardtii: a 13C-NMR study.

    Science.gov (United States)

    Singh, Himanshu; Shukla, Manish R; Chary, Kandala V R; Rao, Basuthkar J

    2014-01-01

    Cellular metabolite analyses by (13)C-NMR showed that C. reinhardtii cells assimilate acetate at a faster rate in heterotrophy than in mixotrophy. While heterotrophic cells produced bicarbonate and CO2aq, mixotrophy cells produced bicarbonate alone as predominant metabolite. Experiments with singly (13)C-labelled acetate ((13)CH(3)-COOH or CH(3)-(13)COOH) supported that both the (13)C nuclei give rise to bicarbonate and CO2(aq). The observed metabolite(s) upon further incubation led to the production of starch and triacylglycerol (TAG) in mixotrophy, whereas in heterotrophy the TAG production was minimal with substantial accumulation of glycerol and starch. Prolonged incubation up to eight days, without the addition of fresh acetate, led to an increased TAG production at the expense of bicarbonate, akin to that of nitrogen-starvation. However, such TAG production was substantially high in mixotrophy as compared to that in heterotrophy. Addition of mitochondrial un-coupler blocked the formation of bicarbonate and CO2(aq) in heterotrophic cells, even though acetate uptake ensued. Addition of PSII-inhibitor to mixotrophic cells resulted in partial conversion of bicarbonate into CO2(aq), which were found to be in equilibrium. In an independent experiment, we have monitored assimilation of bicarbonate via photoautotrophy and found that the cells indeed produce starch and TAG at a much faster rate as compared to that in mixotrophy and heterotrophy. Further, we noticed that the accumulation of starch is relatively more as compared to TAG. Based on these observations, we suggest that acetate assimilation in C. reinhardtii does not directly lead to TAG formation but via bicarbonate/CO2(aq) pathways. Photoautotrophic mode is found to be the best growth condition for the production of starch and TAG and starch in C. reinhardtii.

  4. Acetate and bicarbonate assimilation and metabolite formation in Chlamydomonas reinhardtii: a 13C-NMR study.

    Directory of Open Access Journals (Sweden)

    Himanshu Singh

    Full Text Available Cellular metabolite analyses by (13C-NMR showed that C. reinhardtii cells assimilate acetate at a faster rate in heterotrophy than in mixotrophy. While heterotrophic cells produced bicarbonate and CO2aq, mixotrophy cells produced bicarbonate alone as predominant metabolite. Experiments with singly (13C-labelled acetate ((13CH(3-COOH or CH(3-(13COOH supported that both the (13C nuclei give rise to bicarbonate and CO2(aq. The observed metabolite(s upon further incubation led to the production of starch and triacylglycerol (TAG in mixotrophy, whereas in heterotrophy the TAG production was minimal with substantial accumulation of glycerol and starch. Prolonged incubation up to eight days, without the addition of fresh acetate, led to an increased TAG production at the expense of bicarbonate, akin to that of nitrogen-starvation. However, such TAG production was substantially high in mixotrophy as compared to that in heterotrophy. Addition of mitochondrial un-coupler blocked the formation of bicarbonate and CO2(aq in heterotrophic cells, even though acetate uptake ensued. Addition of PSII-inhibitor to mixotrophic cells resulted in partial conversion of bicarbonate into CO2(aq, which were found to be in equilibrium. In an independent experiment, we have monitored assimilation of bicarbonate via photoautotrophy and found that the cells indeed produce starch and TAG at a much faster rate as compared to that in mixotrophy and heterotrophy. Further, we noticed that the accumulation of starch is relatively more as compared to TAG. Based on these observations, we suggest that acetate assimilation in C. reinhardtii does not directly lead to TAG formation but via bicarbonate/CO2(aq pathways. Photoautotrophic mode is found to be the best growth condition for the production of starch and TAG and starch in C. reinhardtii.

  5. Bioavailability of wastewater derived dissolved organic nitrogen to green microalgae Selenastrum capricornutum, Chlamydomonas reinhardtii, and Chlorella vulgaris with/without presence of bacteria.

    Science.gov (United States)

    Sun, Jingyi; Simsek, Halis

    2017-07-01

    Effluent dissolved organic nitrogen (DON) is problematic in nutrient sensitive surface waters and needs to be reduced to meet demanding total dissolved nitrogen discharge limits. Bioavailable DON (ABDON) is a portion of DON utilized by algae or algae+bacteria, while biodegradable DON (BDON) is a portion of DON decomposable by bacteria. ABDON and BDON in a two-stage trickling filter (TF) wastewater treatment plant was evaluated using three different microalgal species, Selenastrum capricornutum, Chlamydomonas reinhardtii and Chlorella vulgaris and mixed cultured bacteria. Results showed that up to 80% of DON was bioavailable to algae or algae+bacteria inoculum while up to 60% of DON was biodegradable in all the samples. Results showed that C. reinhardtii and C. vulgaris can be used as a test species the same as S. capricornutum since there were no significant differences among these three algae species based on their ability to remove nitrogen species. Copyright © 2017. Published by Elsevier B.V.

  6. Growth of Chlamydomonas reinhardtii in acetate-free medium when co-cultured with alginate-encapsulated, acetate-producing strains of Synechococcus sp. PCC 7002.

    Science.gov (United States)

    Therien, Jesse B; Zadvornyy, Oleg A; Posewitz, Matthew C; Bryant, Donald A; Peters, John W

    2014-01-01

    The model alga Chlamydomonas reinhardtii requires acetate as a co-substrate for optimal production of lipids, and the addition of acetate to culture media has practical and economic implications for algal biofuel production. Here we demonstrate the growth of C. reinhardtii on acetate provided by mutant strains of the cyanobacterium Synechococcus sp. PCC 7002. Optimal growth conditions for co-cultivation of C. reinhardtii with wild-type and mutant strains of Synechococcus sp. 7002 were established. In co-culture, acetate produced by a glycogen synthase knockout mutant of Synechococcus sp. PCC 7002 was able to support the growth of a lipid-accumulating mutant strain of C. reinhardtii defective in starch production. Encapsulation of Synechococcus sp. PCC 7002 using an alginate matrix was successfully employed in co-cultures to limit growth and maintain the stability. The ability of immobilized strains of the cyanobacterium Synechococcus sp. PCC 7002 to produce acetate at a level adequate to support the growth of lipid-accumulating strains of C. reinhartdii offers a potentially practical, photosynthetic alternative to providing exogenous acetate into growth media.

  7. Study of metabolic pathways for hydrogen production in chlamydomonas reinhardtii and transposition on a torus photo bioreactor; Etude des voies metaboliques de production d'hydrogene chez la microalgue Chlamydomonas reinhardtii et transposition en photobioreacteur

    Energy Technology Data Exchange (ETDEWEB)

    Fouchard, S

    2006-04-15

    Considering the recent increase in energy consumption. aide associated environmental risks, new trails are followed today to develop the use of clean and renewable alternative energies. In this context hydrogen seems to be a serious solution and this study, based on micro-algae photosynthetic capacities exploitation, will allow to devise a process for hydrogen production from only water and solar energy without greenhouse gas release. The sulphur deprivation protocol on TAP medium, known to lead to hydrogen production in Chlamydomonas reinhardtii species was particularly studied. At the metabolic level, two important phenomena are induced under these conditions: an over-accumulation of the intracellular starch reserves and a simultaneous alteration of the PsII activity which leads to anoxia and Fe-hydrogenase induction, an enzyme with a strong specific activity responsible for the hydrogen production. The contribution of the two electron transfer pathways implied in the hydrogen production process (PsII-dependent and PSII-independent) as well as the importance of the previously accumulated starch were highlighted here. We also investigated the potential for designing autotrophic protocols for hydrogen photoproduction. Various protocols, considered to be relevant, were then transposed on a torus photo-bioreactor, specifically developed in this study and which allows the control of culture parameters as well as the precise measurement of gas release kinetics, in order to obtain first estimates of productivity of the system. Integration of the physical; aspects of the pilot and biological aspects of the process in a model, finally opens new prospects for subject development, in particular for a reasoned optimization of hydrogen production via this double physiology/process approach. (author)

  8. New features on the environmental regulation of metabolism revealed by modeling the cellular proteomic adaptations induced by light, carbon and inorganic nitrogen in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Stéphanie Gérin

    2016-08-01

    Full Text Available Microalgae are currently emerging to be very promising organisms for the production of biofuels and high-added value compounds. Understanding the influence of environmental alterations on their metabolism is a crucial issue. Light, carbon and nitrogen availability have been reported to induce important metabolic adaptations. So far, the influence of these variables has essentially been studied while varying only one or two environmental factors at the same time. The goal of the present work was to model the cellular proteomic adaptations of the green microalga Chlamydomonas reinhardtii upon the simultaneous changes of light intensity, carbon concentrations (CO2 and acetate and inorganic nitrogen concentrations (nitrate and ammonium in the culture medium. Statistical design of experiments (DOE enabled to define 32 culture conditions to be tested experimentally. Relative protein abundance was quantified by two dimensional differential in-gel electrophoresis (2D-DIGE. Additional assays for respiration, photosynthesis, and lipid and pigment concentrations were also carried out. A hierarchical clustering survey enabled to partition biological variables (proteins + assays into eight co-regulated clusters. In most cases, the biological variables partitioned in the same cluster had already been reported to participate to common biological functions (acetate assimilation, bioenergetic processes, light harvesting, Calvin cycle and protein metabolism. The environmental regulation within each cluster was further characterized by a series of multivariate methods including principal component analysis and multiple linear regressions. This metadata analysis enabled to highlight the existence of a clear regulatory pattern for every cluster and to mathematically simulate the effects of light, carbon and nitrogen. The influence of these environmental variables on cellular metabolism is described in details and thoroughly discussed. This work provides an overview

  9. New Features on the Environmental Regulation of Metabolism Revealed by Modeling the Cellular Proteomic Adaptations Induced by Light, Carbon, and Inorganic Nitrogen in Chlamydomonas reinhardtii.

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    Gérin, Stéphanie; Leprince, Pierre; Sluse, Francis E; Franck, Fabrice; Mathy, Grégory

    2016-01-01

    Microalgae are currently emerging to be very promising organisms for the production of biofuels and high-added value compounds. Understanding the influence of environmental alterations on their metabolism is a crucial issue. Light, carbon and nitrogen availability have been reported to induce important metabolic adaptations. So far, the influence of these variables has essentially been studied while varying only one or two environmental factors at the same time. The goal of the present work was to model the cellular proteomic adaptations of the green microalga Chlamydomonas reinhardtii upon the simultaneous changes of light intensity, carbon concentrations (CO2 and acetate), and inorganic nitrogen concentrations (nitrate and ammonium) in the culture medium. Statistical design of experiments (DOE) enabled to define 32 culture conditions to be tested experimentally. Relative protein abundance was quantified by two dimensional differential in-gel electrophoresis (2D-DIGE). Additional assays for respiration, photosynthesis, and lipid and pigment concentrations were also carried out. A hierarchical clustering survey enabled to partition biological variables (proteins + assays) into eight co-regulated clusters. In most cases, the biological variables partitioned in the same cluster had already been reported to participate to common biological functions (acetate assimilation, bioenergetic processes, light harvesting, Calvin cycle, and protein metabolism). The environmental regulation within each cluster was further characterized by a series of multivariate methods including principal component analysis and multiple linear regressions. This metadata analysis enabled to highlight the existence of a clear regulatory pattern for every cluster and to mathematically simulate the effects of light, carbon, and nitrogen. The influence of these environmental variables on cellular metabolism is described in details and thoroughly discussed. This work provides an overview of the

  10. Analysis of LhcSR3, a protein essential for feedback de-excitation in the green alga Chlamydomonas reinhardtii.

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    Giulia Bonente

    2011-01-01

    Full Text Available In photosynthetic organisms, feedback dissipation of excess absorbed light energy balances harvesting of light with metabolic energy consumption. This mechanism prevents photodamage caused by reactive oxygen species produced by the reaction of chlorophyll (Chl triplet states with O₂. Plants have been found to perform the heat dissipation in specific proteins, binding Chls and carotenoids (Cars, that belong to the Lhc family, while triggering of the process is performed by the PsbS subunit, needed for lumenal pH detection. PsbS is not found in algae, suggesting important differences in energy-dependent quenching (qE machinery. Consistent with this suggestion, a different Lhc-like gene product, called LhcSR3 (formerly known as LI818 has been found to be essential for qE in Chlamydomonas reinhardtii. In this work, we report the production of two recombinant LhcSR isoforms from C. reinhardtii and their biochemical and spectroscopic characterization. We found the following: (i LhcSR isoforms are Chl a/b- and xanthophyll-binding proteins, contrary to higher plant PsbS; (ii the LhcSR3 isoform, accumulating in high light, is a strong quencher of Chl excited states, exhibiting a very fast fluorescence decay, with lifetimes below 100 ps, capable of dissipating excitation energy from neighbor antenna proteins; (iii the LhcSR3 isoform is highly active in the transient formation of Car radical cation, a species proposed to act as a quencher in the heat dissipation process. Remarkably, the radical cation signal is detected at wavelengths corresponding to the Car lutein, rather than to zeaxanthin, implying that the latter, predominant in plants, is not essential; (iv LhcSR3 is responsive to low pH, the trigger of non-photochemical quenching, since it binds the non-photochemical quenching inhibitor dicyclohexylcarbodiimide, and increases its energy dissipation properties upon acidification. This is the first report of an isolated Lhc protein constitutively

  11. Fast forward genetics to identify mutations causing a high light tolerant phenotype in Chlamydomonas reinhardtii by whole-genome-sequencing.

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    Schierenbeck, Lisa; Ries, David; Rogge, Kristin; Grewe, Sabrina; Weisshaar, Bernd; Kruse, Olaf

    2015-02-06

    High light tolerance of microalgae is a desired phenotype for efficient cultivation in large scale production systems under fluctuating outdoor conditions. Outdoor cultivation requires the use of either wild-type or non-GMO derived mutant strains due to safety concerns. The identification and molecular characterization of such mutants derived from untagged forward genetics approaches was limited previously by the tedious and time-consuming methods involving techniques such as classical meiotic mapping. The combination of mapping with next generation sequencing technologies offers alternative strategies to identify genes involved in high light adaptation in untagged mutants. We used the model alga Chlamydomonas reinhardtii in a non-GMO mutation strategy without any preceding crossing step or pooled progeny to identify genes involved in the regulatory processes of high light adaptation. To generate high light tolerant mutants, wildtype cells were mutagenized only to a low extent, followed by a stringent selection. We performed whole-genome sequencing of two independent mutants hit1 and hit2 and the parental wildtype. The availability of a reference genome sequence and the removal of shared bakground variants between the wildtype strain and each mutant, enabled us to identify two single nucleotide polymorphisms within the same gene Cre02.g085050, hereafter called LRS1 (putative Light Response Signaling protein 1). These two independent single amino acid exchanges are both located in the putative WD40 propeller domain of the corresponding protein LRS1. Both mutants exhibited an increased rate of non-photochemical-quenching (NPQ) and an improved resistance against chemically induced reactive oxygen species. In silico analyses revealed homology of LRS1 to the photoregulatory protein COP1 in plants. In this work we identified the nuclear encoded gene LRS1 as an essential factor for high light adaptation in C. reinhardtii. The causative random mutation within this gene was

  12. Whole genome sequencing identifies a deletion in protein phosphatase 2A that affects its stability and localization in Chlamydomonas reinhardtii.

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    Huawen Lin

    Full Text Available Whole genome sequencing is a powerful tool in the discovery of single nucleotide polymorphisms (SNPs and small insertions/deletions (indels among mutant strains, which simplifies forward genetics approaches. However, identification of the causative mutation among a large number of non-causative SNPs in a mutant strain remains a big challenge. In the unicellular biflagellate green alga Chlamydomonas reinhardtii, we generated a SNP/indel library that contains over 2 million polymorphisms from four wild-type strains, one highly polymorphic strain that is frequently used in meiotic mapping, ten mutant strains that have flagellar assembly or motility defects, and one mutant strain, imp3, which has a mating defect. A comparison of polymorphisms in the imp3 strain and the other 15 strains allowed us to identify a deletion of the last three amino acids, Y313F314L315, in a protein phosphatase 2A catalytic subunit (PP2A3 in the imp3 strain. Introduction of a wild-type HA-tagged PP2A3 rescues the mutant phenotype, but mutant HA-PP2A3 at Y313 or L315 fail to rescue. Our immunoprecipitation results indicate that the Y313, L315, or YFLΔ mutations do not affect the binding of PP2A3 to the scaffold subunit, PP2A-2r. In contrast, the Y313, L315, or YFLΔ mutations affect both the stability and the localization of PP2A3. The PP2A3 protein is less abundant in these mutants and fails to accumulate in the basal body area as observed in transformants with either wild-type HA-PP2A3 or a HA-PP2A3 with a V310T change. The accumulation of HA-PP2A3 in the basal body region disappears in mated dikaryons, which suggests that the localization of PP2A3 may be essential to the mating process. Overall, our results demonstrate that the terminal YFL tail of PP2A3 is important in the regulation on Chlamydomonas mating.

  13. AlgaGEM--a genome-scale metabolic reconstruction of algae based on the Chlamydomonas reinhardtii genome.

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    Dal'Molin, Cristiana Gomes de Oliveira; Quek, Lake-Ee; Palfreyman, Robin W; Nielsen, Lars K

    2011-12-22

    Microalgae have the potential to deliver biofuels without the associated competition for land resources. In order to realise the rates and titres necessary for commercial production, however, system-level metabolic engineering will be required. Genome scale metabolic reconstructions have revolutionized microbial metabolic engineering and are used routinely for in silico analysis and design. While genome scale metabolic reconstructions have been developed for many prokaryotes and model eukaryotes, the application to less well characterized eukaryotes such as algae is challenging not at least due to a lack of compartmentalization data. We have developed a genome-scale metabolic network model (named AlgaGEM) covering the metabolism for a compartmentalized algae cell based on the Chlamydomonas reinhardtii genome. AlgaGEM is a comprehensive literature-based genome scale metabolic reconstruction that accounts for the functions of 866 unique ORFs, 1862 metabolites, 2249 gene-enzyme-reaction-association entries, and 1725 unique reactions. The reconstruction was compartmentalized into the cytoplasm, mitochondrion, plastid and microbody using available data for algae complemented with compartmentalisation data for Arabidopsis thaliana. AlgaGEM describes a functional primary metabolism of Chlamydomonas and significantly predicts distinct algal behaviours such as the catabolism or secretion rather than recycling of phosphoglycolate in photorespiration. AlgaGEM was validated through the simulation of growth and algae metabolic functions inferred from literature. Using efficient resource utilisation as the optimality criterion, AlgaGEM predicted observed metabolic effects under autotrophic, heterotrophic and mixotrophic conditions. AlgaGEM predicts increased hydrogen production when cyclic electron flow is disrupted as seen in a high producing mutant derived from mutational studies. The model also predicted the physiological pathway for H2 production and identified new targets

  14. Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis [v1; ref status: indexed, http://f1000r.es/17x

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    Phillip B Grovenstein

    2013-06-01

    Full Text Available The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel which inserts Mg2+ into protoporphyrin IX (PPIX, proto to form magnesium-protoporphyrin IX (MgPPIX, Mgproto, the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2 which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis and chloroplast to nucleus retrograde signaling in Chlamydomonas, which has never been studied before.

  15. Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis [v2; ref status: indexed, http://f1000r.es/1ic

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    Phillip B Grovenstein

    2013-07-01

    Full Text Available The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel which inserts Mg2+ into protoporphyrin IX (PPIX, proto to form magnesium-protoporphyrin IX (MgPPIX, Mgproto, the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2 which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 (chli1-1 mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1-1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1-1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis in Chlamydomonas, which has never been studied before.

  16. Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii

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    Atsushi eSato

    2014-09-01

    Full Text Available Triacylglycerol (TG synthesis is induced for energy and carbon storage in algal cells under nitrogen(N-starved conditions, and helps prevent reactive oxygen species production through fatty acid synthesis that consumes excessive reducing power. Here, the regulatory mechanism for the TG content in sulfur(S-starved cells of Chlamydomonas reinhardtii was examined, in comparison to that in N- or phosphorus(P-starved cells. S- and N-starved cells exhibited markedly increased TG contents with up-regulation of mRNA levels of diacylglycerol acyltransferase genes. S-Starvation also induced expression of the genes for phosphatidate synthesis. In contrast, P-starved cells exhibited little alteration of the TG content with almost no induction of these genes. The results implied deficient nutrient-specific regulation of the TG content. An arg9 disruptant defective in arginine synthesis, even without nutritional deficiencies, exhibited an increased TG content upon removal of supplemented arginine, which repressed protein synthesis. Repression of protein synthesis thus seemed crucial for TG accumulation in S- or N-starved cells. Meanwhile, the results of inhibitor experiments involving cells inferred that TG accumulation during S-starvation is supported by photosynthesis and de novo fatty acid synthesis. During S-starvation, sac1 and snrk2.2 disruptants, which are defective in the response to the ambient S-status, accumulated TG at lower and higher levels, respectively, than the wild type. The sac1 and snrk2.2 disruptants showed no or much greater up-regulation of diacylglycerol acyltransferase genes, respectively. In conclusion, TG synthesis would be activated in S-starved cells, through the diversion of metabolic carbon-flow from protein to TG synthesis, and simultaneously through up-regulation of the expression of a particular set of genes for TG synthesis at proper levels through the actions of SAC1 and SNRK2.2.

  17. Expression of type 2 diacylglycerol acyltransferse gene DGTT1 from Chlamydomonas reinhardtii enhances lipid production in Scenedesmus obliquus.

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    Chen, Chun-Yen; Kao, Ai-Ling; Tsai, Zheng-Chia; Chow, Te-Jin; Chang, Hsin-Yueh; Zhao, Xin-Qing; Chen, Po-Ting; Su, Hsiang-Yen; Chang, Jo-Shu

    2016-03-01

    Microalgal strains of Scenedesmus obliquus have the great potential for the production of biofuels, CO2 fixation, and bioremediation. However, metabolic engineering of S. obliquus to improve their useful phenotypes are still not fully developed. In this study, S. obliquus strain CPC2 was genetically engineered to promote the autotrophic growth and lipid productivity. The overexpression plasmid containing the type 2 diacylglycerol acyltransferse (DGAT) gene DGTT1 from Chlamydomonas reinhardtii was constructed and transformed into S. obliquus CPC2, and the positive transformants were obtained. The expression of DGTT1 gene was confirmed by reverse transcription PCR analysis. Enhanced lipid content of the transformant S. obliquus CPC2-G1 by nearly two-fold was observed. The biomass concentration of the recombinant strains was also 29% higher than that of the wild-type strain. Furthermore, the recombinant strain CPC2-G1 was successfully grown in 40 L tubular type photobioreactor and open pond system in an outdoor environment. The lipid content, biomass concentration, and biomass productivity obtained from 40 L tubular PBR were 127.8% 20.0%, and 232.6% higher than those obtained from the wild-type strain. The major aim of this work is to develop a tool to genetically engineer an isolated S. obliquus strain for the desired purpose. This is the first report that genetic engineering of S. obliquus has been successful employed to improve both the microalgal cell growth and the lipid production. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Biotic ligand model explains the effects of competition but not complexation for Sm biouptake by Chlamydomonas reinhardtii.

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    Tan, Qiao-Guo; Yang, Guang; Wilkinson, Kevin J

    2017-02-01

    The applicability of the biotic ligand model (BLM) was tested with respect to the biouptake of the lanthanide Sm by the freshwater green alga, Chlamydomonas reinhardtii. In the absence of organic ligands, Sm uptake was well described by the Michaelis-Menten equation, consistent with the BLM assumption of single transporter, with the maximum influx rate (Jmax) of 1.5 × 10-14 mol cm-2 s-1 and a binding constant (KSm) of 107.0 M-1. The addition of organic ligands (i.e., malic acid, diglycolic acid and citric acid) decreased Sm influx rates, however, the decreases were much less than that predicted by the BLM, possibly due to the direct contribution of the Sm complexes. Competition effects of two major cations (Ca2+ and Mg2+) and three lanthanide cations (La3+, Ce3+ and Eu3+) were successfully modeled by the BLM, with binding constants corresponding to KCa = 104.0 M-1, KMg = 102.7 M-1, KLa = 106.8 M-1, KCe = 106.9 M-1 and KEu = 107.0 M-1. The binding constants and Jmax were very similar among the four investigated lanthanides and varied progressively with atomic number; therefore, the results obtained in the present study can probably be extrapolated to other rare earth metals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Selenium bioaccumulation in Chlamydomonas reinhardtii and subsequent transfer to Corbicula fluminea: role of selenium speciation and bivalve ventilation.

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    Fournier, Elodie; Adam, Christelle; Massabuau, Jean-Charles; Garnier-Laplace, Jacqueline

    2006-10-01

    The uptake of Se by the freshwater alga Chlamydomonas reinhardtii and the subsequent transfer to the Asiatic clam Corbicula fluminea was investigated. The objective was to investigate the bioavailability of algal-bound Se for C. fluminea while taking into account Se speciation and bivalve ventilation. First, uptake rates of waterborne Se (selenite, selenate, and selenomethionine) in the algae during a 1-h exposure period were determined for a range of concentrations up to 2,000 microg/L. Fluxes for selenite uptake were constant in the range of concentrations tested, whereas fluxes for selenate and selenomethionine uptake decreased with increasing concentrations, suggesting a saturated transport system at high concentrations (approximately 1,000 microg/L for selenate and 100 microg/L for selenomethionine). These data were used to set the algal contamination for the study of trophic transfer to the clam. Three parameters were studied: The Se form, the algal density, and the Se burden in the algae. The results show that for a fixed algal density, an Se-contaminated algal diet does not modify ventilation. In this case, the driving factor for ventilation is the algal density, with ventilation being enhanced for low algal densities. On the basis of ventilatory flow rate measurements and Se burdens in algae, it was found that bioaccumulation of Se in C. fluminea was proportional to the total quantity of Se passing through the whole organism, but with a lesser extraction coefficient for selenomethionine than for the inorganic forms. These results underline the importance of both physiological factors and speciation in understanding the trophic transfer of Se.

  20. New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii.

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    Wannathong, Thanyanan; Waterhouse, Janet C; Young, Rosanna E B; Economou, Chloe K; Purton, Saul

    2016-06-01

    In recent years, there has been an increasing interest in the exploitation of microalgae in industrial biotechnology. Potentially, these phototrophic eukaryotes could be used for the low-cost synthesis of valuable recombinant products such as bioactive metabolites and therapeutic proteins. The algal chloroplast in particular represents an attractive target for such genetic engineering, both because it houses major metabolic pathways and because foreign genes can be targeted to specific loci within the chloroplast genome, resulting in high-level, stable expression. However, routine methods for chloroplast genetic engineering are currently available only for one species-Chlamydomonas reinhardtii-and even here, there are limitations to the existing technology, including the need for an expensive biolistic device for DNA delivery, the lack of robust expression vectors, and the undesirable use of antibiotic resistance markers. Here, we describe a new strain and vectors for targeted insertion of transgenes into a neutral chloroplast locus that (i) allow scar-less fusion of a transgenic coding sequence to the promoter/5'UTR element of the highly expressed endogenous genes psaA or atpA, (ii) employ the endogenous gene psbH as an effective but benign selectable marker, and (iii) ensure the successful integration of the transgene construct in all transformant lines. Transformation is achieved by a simple and cheap method of agitation of a DNA/cell suspension with glass beads, with selection based on the phototrophic rescue of a cell wall-deficient ΔpsbH strain. We demonstrate the utility of these tools in the creation of a transgenic line that produces high levels of functional human growth hormone.

  1. Direct and indirect effects of silver nanoparticles on freshwater and marine microalgae (Chlamydomonas reinhardtii and Phaeodactylum tricornutum).

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    Sendra, M; Yeste, M P; Gatica, J M; Moreno-Garrido, I; Blasco, J

    2017-07-01

    The last decade has seen a considerable increase in the use of silver nanoparticles (AgNPs), which are found in many every-day consumer products including textiles, plastics, cosmetics, household sprays and paints. The release of those AgNPs into aquatic environments could be causing ecological damage. In this study we assess the toxicity of AgNPs of different sizes to two species of microalgae, from freshwater and marine environment (Chlamydomonas reinhardtii and Phaeodactylum tricornutum respectively). Dissolution processes affect the form and concentration of AgNPs in both environments. Dissolution of Ag from AgNPs was around 25 times higher in marine water. Nevertheless, dissolution of AgNPs in both culture media seems to be related to the small size and higher surface area of NPs. In marine water, the main chemical species were AgCl 2 - (53.7%) and AgCl 3 -2 (45.2%). In contrast, for freshwater, the main chemical species were Ag + (26.7%) and AgCl - (4.3%). The assessment of toxicological responses, specifically growth, cell size, cell complexity, chlorophyll a, reactive oxygen species, cell membrane damage and effective quantum yield of PSII, corroborated the existence of different toxicity mechanisms for microalgae. Indirect effects, notably dissolved Ag ions, seem to control toxicity to freshwater microalgae, whereas direct effects, notably attachment onto the cell surface and the internalization of AgNPs inside cells, seem to determine toxicity to the marine species studied. This research contributes to knowledge on the role of intrinsic and extrinsic factors in determining the behavior of NPs in different aquatic environments and the interaction with microalgae. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Mutations of photosystem II D1 protein that empower efficient phenotypes of Chlamydomonas reinhardtii under extreme environment in space.

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    Maria Teresa Giardi

    Full Text Available Space missions have enabled testing how microorganisms, animals and plants respond to extra-terrestrial, complex and hazardous environment in space. Photosynthetic organisms are thought to be relatively more prone to microgravity, weak magnetic field and cosmic radiation because oxygenic photosynthesis is intimately associated with capture and conversion of light energy into chemical energy, a process that has adapted to relatively less complex and contained environment on Earth. To study the direct effect of the space environment on the fundamental process of photosynthesis, we sent into low Earth orbit space engineered and mutated strains of the unicellular green alga, Chlamydomonas reinhardtii, which has been widely used as a model of photosynthetic organisms. The algal mutants contained specific amino acid substitutions in the functionally important regions of the pivotal Photosystem II (PSII reaction centre D1 protein near the QB binding pocket and in the environment surrounding Tyr-161 (YZ electron acceptor of the oxygen-evolving complex. Using real-time measurements of PSII photochemistry, here we show that during the space flight while the control strain and two D1 mutants (A250L and V160A were inefficient in carrying out PSII activity, two other D1 mutants, I163N and A251C, performed efficient photosynthesis, and actively re-grew upon return to Earth. Mimicking the neutron irradiation component of cosmic rays on Earth yielded similar results. Experiments with I163N and A251C D1 mutants performed on ground showed that they are better able to modulate PSII excitation pressure and have higher capacity to reoxidize the QA (- state of the primary electron acceptor. These results highlight the contribution of D1 conformation in relation to photosynthesis and oxygen production in space.

  3. Mutations of photosystem II D1 protein that empower efficient phenotypes of Chlamydomonas reinhardtii under extreme environment in space.

    Science.gov (United States)

    Giardi, Maria Teresa; Rea, Giuseppina; Lambreva, Maya D; Antonacci, Amina; Pastorelli, Sandro; Bertalan, Ivo; Johanningmeier, Udo; Mattoo, Autar K

    2013-01-01

    Space missions have enabled testing how microorganisms, animals and plants respond to extra-terrestrial, complex and hazardous environment in space. Photosynthetic organisms are thought to be relatively more prone to microgravity, weak magnetic field and cosmic radiation because oxygenic photosynthesis is intimately associated with capture and conversion of light energy into chemical energy, a process that has adapted to relatively less complex and contained environment on Earth. To study the direct effect of the space environment on the fundamental process of photosynthesis, we sent into low Earth orbit space engineered and mutated strains of the unicellular green alga, Chlamydomonas reinhardtii, which has been widely used as a model of photosynthetic organisms. The algal mutants contained specific amino acid substitutions in the functionally important regions of the pivotal Photosystem II (PSII) reaction centre D1 protein near the QB binding pocket and in the environment surrounding Tyr-161 (YZ) electron acceptor of the oxygen-evolving complex. Using real-time measurements of PSII photochemistry, here we show that during the space flight while the control strain and two D1 mutants (A250L and V160A) were inefficient in carrying out PSII activity, two other D1 mutants, I163N and A251C, performed efficient photosynthesis, and actively re-grew upon return to Earth. Mimicking the neutron irradiation component of cosmic rays on Earth yielded similar results. Experiments with I163N and A251C D1 mutants performed on ground showed that they are better able to modulate PSII excitation pressure and have higher capacity to reoxidize the QA (-) state of the primary electron acceptor. These results highlight the contribution of D1 conformation in relation to photosynthesis and oxygen production in space.

  4. Genome-wide functional annotation and structural verification of metabolic ORFeome of Chlamydomonas reinhardtii

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    Fan Changyu

    2011-06-01

    Full Text Available Abstract Background Recent advances in the field of metabolic engineering have been expedited by the availability of genome sequences and metabolic modelling approaches. The complete sequencing of the C. reinhardtii genome has made this unicellular alga a good candidate for metabolic engineering studies; however, the annotation of the relevant genes has not been validated and the much-needed metabolic ORFeome is currently unavailable. We describe our efforts on the functional annotation of the ORF models released by the Joint Genome Institute (JGI, prediction of their subcellular localizations, and experimental verification of their structural annotation at the genome scale. Results We assigned enzymatic functions to the translated JGI ORF models of C. reinhardtii by reciprocal BLAST searches of the putative proteome against the UniProt and AraCyc enzyme databases. The best match for each translated ORF was identified and the EC numbers were transferred onto the ORF models. Enzymatic functional assignment was extended to the paralogs of the ORFs by clustering ORFs using BLASTCLUST. In total, we assigned 911 enzymatic functions, including 886 EC numbers, to 1,427 transcripts. We further annotated the enzymatic ORFs by prediction of their subcellular localization. The majority of the ORFs are predicted to be compartmentalized in the cytosol and chloroplast. We verified the structure of the metabolism-related ORF models by reverse transcription-PCR of the functionally annotated ORFs. Following amplification and cloning, we carried out 454FLX and Sanger sequencing of the ORFs. Based on alignment of the 454FLX reads to the ORF predicted sequences, we obtained more than 90% coverage for more than 80% of the ORFs. In total, 1,087 ORF models were verified by 454 and Sanger sequencing methods. We obtained expression evidence for 98% of the metabolic ORFs in the algal cells grown under constant light in the presence of acetate. Conclusions We functionally

  5. Disulphide bridges of phospholipase C of Chlamydomonas reinhardtii modulates lipid interaction and dimer stability.

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    Mayanka Awasthi

    Full Text Available BACKGROUND: Phospholipase C (PLC is an enzyme that plays pivotal role in a number of signaling cascades. These are active in the plasma membrane and triggers cellular responses by catalyzing the hydrolysis of membrane phospholipids and thereby generating the secondary messengers. Phosphatidylinositol-PLC (PI-PLC specifically interacts with phosphoinositide and/or phosphoinositol and catalyzes specific cleavage of sn-3- phosphodiester bond. Several isoforms of PLC are known to form and function as dimer but very little is known about the molecular basis of the dimerization and its importance in the lipid interaction. PRINCIPAL FINDINGS: We herein report that, the disruption of disulphide bond of a novel PI-specific PLC of C. reinhardtii (CrPLC can modulate its interaction affinity with a set of phospholipids and also the stability of its dimer. CrPLC was found to form a mixture of higher oligomeric states with monomer and dimer as major species. Dimer adduct of CrPLC disappeared in the presence of DTT, which suggested the involvement of disulphide bond(s in CrPLC oligomerization. Dimer-monomer equilibrium studies with the isolated fractions of CrPLC monomer and dimer supported the involvement of covalent forces in the dimerization of CrPLC. A disulphide bridge was found to be responsible for the dimerization and Cys7 seems to be involved in the formation of the disulphide bond. This crucial disulphide bond also modulated the lipid affinity of CrPLC. Oligomers of CrPLC were also captured in in vivo condition. CrPLC was mainly found to be localized in the plasma membrane of the cell. The cell surface localization of CrPLC may have significant implication in the downstream regulatory function of CrPLC. SIGNIFICANCE: This study helps in establishing the role of CrPLC (or similar proteins in the quaternary structure of the molecule its affinities during lipid interactions.

  6. Bioaccumulation and subcellular partitioning of Cr(III) and Cr(VI) in the freshwater green alga Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Aharchaou, Imad [Laboratoire Interdisciplinaire des Environnements Continentaux, UMR 7360, Université de Lorraine and CNRS, 8 rue du Général Delestraint, 57070 Metz (France); Rosabal, Maikel; Liu, Fengjie [Institut National de la Recherche Scientifique, Centre Eau Terre Environnement (INRS-ETE), 490 rue de la Couronne, Québec (Québec) G1K 9A9 (Canada); Battaglia, Eric; Vignati, Davide A.L. [Laboratoire Interdisciplinaire des Environnements Continentaux, UMR 7360, Université de Lorraine and CNRS, 8 rue du Général Delestraint, 57070 Metz (France); Fortin, Claude, E-mail: claude.fortin@ete.inrs.ca [Institut National de la Recherche Scientifique, Centre Eau Terre Environnement (INRS-ETE), 490 rue de la Couronne, Québec (Québec) G1K 9A9 (Canada)

    2017-01-15

    Highlights: • C. reinhardtii accumulated similar levels of Cr(III) and Cr(VI). • The subcellular partitioning of Cr(III) and Cr(VI) was similar. • Cr(III) and Cr(VI) associated mainly with organelles and heat-stable proteins. • Metallomic analysis showed two main Cr-binding biomolecules after 72 h of exposure. - Abstract: Chromium occurs in aquatic environments under two main redox forms, namely Cr(III) and Cr(VI), with different geochemical and biochemical properties. Cr(VI) readily crosses biological membranes of living organisms and once inside the cells it undergoes a rapid reduction to Cr(III). The route of entry for the latter form is, however, poorly known. Using the radioactive tracer {sup 51}Cr we compared the accumulation (absorption and adsorption) of the two Cr forms by the green unicellular alga Chlamydomonas reinhardii after 1 h and 72 h of exposure to 100 nM of either Cr(III) or Cr(VI) at pH 7. Both Cr forms had similar accumulation, with a major part in the extracellular (adsorbed) fraction after 1 h and a major part of total accumulated Cr in the intracellular (absorbed) fraction after 72 h. We also investigated the intracellular partitioning of Cr using an operational fractionation scheme and found that both Cr forms had similar distributions among fractions: Cr was mostly associated with organelles (23 ± 12% after 1 h and 37 ± 7% after 72 h) and cytosolic heat-stable proteins and peptides (39 ± 18% after 1 h and 35 ± 3% after 72 h) fractions. Further investigations using a metallomic approach (SEC-ICP-MS) were performed with the heat-stable proteins and peptides fraction to compare the distribution of the two Cr forms among various biomolecules of this fraction. One Cr-binding biomolecule (∼28 kDa) appeared after 1 h of exposure for both Cr species. After 72 h another biomolecule of lower molecular weight (∼0.7 kDa) was involved in binding Cr and higher signal intensities were observed for Cr(VI) than for Cr(III). We show, for the

  7. Submicron and nano formulations of titanium dioxide and zinc oxide stimulate unique cellular toxicological responses in the green microalga Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Gunawan, Cindy, E-mail: c.gunawan@unsw.edu.au [ARC Centre of Excellence for Functional Nanomaterials, School of Chemical Engineering, The University of New South Wales, Sydney, NSW (Australia); Sirimanoonphan, Aunchisa [ARC Centre of Excellence for Functional Nanomaterials, School of Chemical Engineering, The University of New South Wales, Sydney, NSW (Australia); Teoh, Wey Yang [Clean Energy and Nanotechnology (CLEAN) Laboratory, School of Energy and Environment, City University of Hong Kong, Kowloon, Hong Kong Special Administrative Region (Hong Kong); Marquis, Christopher P., E-mail: c.marquis@unsw.edu.au [School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW (Australia); Amal, Rose [ARC Centre of Excellence for Functional Nanomaterials, School of Chemical Engineering, The University of New South Wales, Sydney, NSW (Australia)

    2013-09-15

    Highlights: • Uptake of TiO{sub 2} solids by C. reinhardtii generates ROS as an early stress response. • Submicron and nanoTiO{sub 2} exhibit benign effect on cell proliferation. • Uptake of ZnO solids and leached zinc by C. reinhardtii inhibit the alga growth. • No cellular oxidative stress is detected with submicron and nano ZnO exposure. • The toxicity of particles is not necessarily mediated by cellular oxidative stress. -- Abstract: The work investigates the eco-cytoxicity of submicron and nano TiO{sub 2} and ZnO, arising from the unique interactions of freshwater microalga Chlamydomonas reinhardtii to soluble and undissolved components of the metal oxides. In a freshwater medium, submicron and nano TiO{sub 2} exist as suspended aggregates with no-observable leaching. Submicron and nano ZnO undergo comparable concentration-dependent fractional leaching, and exist as dissolved zinc and aggregates of undissolved ZnO. Cellular internalisation of solid TiO{sub 2} stimulates cellular ROS generation as an early stress response. The cellular redox imbalance was observed for both submicron and nano TiO{sub 2} exposure, despite exhibiting benign effects on the alga proliferation (8-day EC50 > 100 mg TiO{sub 2}/L). Parallel exposure of C. reinhardtii to submicron and nano ZnO saw cellular uptake of both the leached zinc and solid ZnO and resulting in inhibition of the alga growth (8-day EC50 ≥ 0.01 mg ZnO/L). Despite the sensitivity, no zinc-induced cellular ROS generation was detected, even at 100 mg ZnO/L exposure. Taken together, the observations confront the generally accepted paradigm of cellular oxidative stress-mediated cytotoxicity of particles. The knowledge of speciation of particles and the corresponding stimulation of unique cellular responses and cytotoxicity is vital for assessment of the environmental implications of these materials.

  8. Involvement of ethylene and nitric oxide in cell death in mastoparan-treated unicellular alga Chlamydomonas reinhardtii

    NARCIS (Netherlands)

    Yordanova, Z.P.; Iakimova, E.T.; Cristescu, S.M.; Harren, F.J.M.; Kapchina-Toteva, V.M.; Woltering, E.J.

    2010-01-01

    This work demonstrates a contribution of ethylene and NO in mastoparan (MP)-induced cell death in the green algae C. reinhardtii. Following MP treatment, C. reinhardtii showed massive cell death, expressing morphological features of programmed cell death (PCD). A pharmacological approach involving

  9. Respiration of sugars in spinach (Spinacia oleraces), maize (Zea mays), and Chlamydomonas reinhardtii F-60 chloroplasts with emphasis on the hexose kinases

    Energy Technology Data Exchange (ETDEWEB)

    Singh, K.K.; Chen, C.; Epstein, D.K.; Gibbs, M. (Brandeis Univ., Waltham, MA (United States))

    1993-06-01

    The role of hexokinase in carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of [sup 14]CO[sub 2] from darkened spinach (Spinacia oleracea), maize (Zea mays) mesophyll, and Chlamydomonas reinhardtii chloroplasts externally supplied with [sup 14]C-labeled fructose, glucose, mannose, galactose, maltose, and ribose. Glucose and ribose were the preferred substrates with the Chlamydomonas and maize chloroplasts, respectively. The rate of CO[sub 2] release from fructose was about twice that from glucose in the spinach chloroplast. externally supplied ATP stimulated the rate of CO[sub 2] release. The pH optimum for CO[sub 2] release was 7.5 with ribose and fructose and 8.5 with glucose as substrates. Probing the outer membrane polypeptides of the intact spinach chloroplast with two proteases, trypsin and thermolysin, decreased [sup 14]CO[sub 2] release from glucose about 50% but had little effect when fructose was the substrate. Tryptic digestion decreased CO[sub 2] release from glucose in the Chlamydomonas chloroplast about 70%. [sup 14]CO[sub 2] evolution from [1-[sup 14]C]-glucose-6-phosphate in both chloroplasts was unaffected by treatment with trypsin. Enzymic analysis of the supernatant (stroma) of the lysed spinach chloroplast indicated a hexokinase active primarily with fructose but with some affinity for glucose. The pellet (membranal fraction) contained a hexokinase utilizing both glucose and fructose but with considerably less total activity than the stormal enzyme. Treatment with trypsin and thermolysin eliminated more than 50% of the glucokinase activity but had little effect on fructokinase activity in the spinach chloroplast. Tryptic digestion of the Chlamydomonas chloroplast resulted in a loss of about 90% of glucokinase activity. 34 refs., 2 figs., 6 tabs.

  10. Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in Q(B)-site of Chlamydomonas reinhardtii.

    Science.gov (United States)

    Chen, Shiguo; Xu, Xiaoming; Dai, Xinbin; Yang, Chunlong; Qiang, Sheng

    2007-04-01

    Tenuazonic acid (TeA) is a natural phytotoxin produced by Alternaria alternata, the causal agent of brown leaf spot disease of Eupatorium adenophorum. Results from chlorophyll fluorescence revealed TeA can block electron flow from Q(A) to Q(B) at photosystem II acceptor side. Based on studies with D1-mutants of Chlamydomonas reinhardtii, the No. 256 amino acid plays a key role in TeA binding to the Q(B)-niche. The results of competitive replacement with [(14)C]atrazine combined with JIP-test and D1-mutant showed that TeA should be considered as a new type of photosystem II inhibitor because it has a different binding behavior within Q(B)-niche from other known photosystem II inhibitors. Bioassay of TeA and its analogues indicated 3-acyl-5-alkyltetramic and even tetramic acid compounds may represent a new structural framework for photosynthetic inhibitors.

  11. Relationships between PSII-independent hydrogen bioproduction and starch metabolism as evidenced from isolation of starch catabolism mutants in the green alga Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Chochois, Vincent; Constans, Laure; Beyly, Audrey; Soliveres, Melanie; Peltier, Gilles; Cournac, Laurent [CEA, DSV, IBEB, Laboratoire de Bioenergetique et Biotechnologie des Bacteries and Microalgues, Saint Paul Lez Durance, F-13108 (France); CNRS, UMR Biologie Vegetale and Microbiologie Environnementales, Saint Paul lez Durance, F-13108 (France); Aix-Marseille Universite, Saint Paul lez Durance, F-13108 (France); Dauvillee, David; Ball, Steven [Univ Lille Nord de France, F-59000 Lille (France); USTL, UGSF, F-59650 Villeneuve d' Ascq (France); CNRS, UMR 8576, F-59650 Villeneuve d' Ascq (France)

    2010-10-15

    Sulfur deprivation, which is considered as an efficient way to trigger long-term hydrogen photoproduction in unicellular green algae has two major effects: a decrease in PSII which allows anaerobiosis to be reached and carbohydrate (starch) storage. Starch metabolism has been proposed as one of the major factors of hydrogen production, particularly during the PSII-independent (or indirect) pathway. While starch biosynthesis has been characterized in the green alga Chlamydomonas reinhardtii, little remains known concerning starch degradation. In order to gain a better understanding of starch catabolism pathways and identify those steps likely to limit the starch-dependent hydrogen production, we have designed a genetic screening procedure aimed at isolating mutants of the green alga C. reinhardtii affected in starch mobilization. Using two different screening protocols, the first one based on aerobic starch degradation in the dark and the second one on anaerobic starch degradation in the light, eighteen mutants were isolated among a library of 15,000 insertion mutants, eight (std1-8) with the first screen and ten (sda1-10) with the second. Most of the mutant strains isolated in this study showed a reduction or a delay in the PSII-independent hydrogen production. Further characterization of these mutants should allow the identification of molecular determinants of starch-dependent hydrogen production and supply targets for future biotechnological improvements. (author)

  12. Engineering the chloroplast of Chlamydomonas reinhardtii to express the recombinant PfCelTOS-Il2 antigen-adjuvant fusion protein.

    Science.gov (United States)

    Shamriz, Shabnam; Ofoghi, Hamideh

    2018-01-20

    Malaria is an infectious disease having a large negative impact on economic growth. Vaccines are considered as a novel strategy to reduce the burden of malaria. Malaria parasite has a complex life cycle and attempts are being made to develop vaccines that target each stage of the life cycle. Oral vaccines seem to be more feasible to implement in poor countries, since they are relatively inexpensive, needle-free administrated, mostly stable at non-refrigerated conditions and painless. By using recombinant technology, suitable oral hosts could serve as antigen delivering vehicles in developing oral vaccines. Chlamydomonas reinhardtii offers beneficial attributes as oral recombinant protein expression platform. Moreover, C. reinhardtii chloroplast is an attractive platform for expressing malaria antigens because it is capable of folding complex proteins, including those requiring disulfide bond formation, while lacking the ability to glycosylate proteins; a valuable quality of any malaria protein expression system, since the Plasmodium parasite lacks N-linked glycosylation machinery. As a first step towards developing an oral vaccine candidate against malaria, here, we expressed a fusion protein consisting of PfCelTOS, a candidate for pre-erythrocytic and transmission-blocking vaccines, fused to human interleukin-2 (IL-2) as vaccine adjuvant in the chloroplast of C. reinhardtii. The effect of light and media on recombinant protein production and cell growth was then studied. Results demonstrated that expressed recombinant proteins accumulate as a soluble, properly folded and functional protein within algal chloroplasts. Moreover, results showed that the highest cell density can be achieved using mixotrophy mode. However, protein accumulation appears to be favored by cultivating in TAP medium in low light. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Effect of core-shell copper oxide nanoparticles on cell culture morphology and photosynthesis (photosystem II energy distribution) in the green alga, Chlamydomonas reinhardtii.

    Science.gov (United States)

    Saison, Cyril; Perreault, François; Daigle, Jean-Christophe; Fortin, Claude; Claverie, Jérôme; Morin, Mario; Popovic, Radovan

    2010-01-31

    The effect of core-shell copper oxide nanoparticles with sizes smaller than 100 nm on cellular systems is still not well understood. Documenting these effects is pressing since core-shell copper oxide nanoparticles are currently components of pigments used frequently as antifouling paint protecting boats from crustacean, weed and slime fouling. However, the use of such paints may induce strong deteriorative effects on different aquatic trophic levels that are not the intended targets. Here, the toxic effect of core-shell copper oxide nanoparticles on the green alga, Chlamydomonas reinhardtii was investigated with regards to the change of algal cellular population structure, primary photochemistry of photosystem II and reactive oxygen species formation. Algal cultures were exposed to 0.004, 0.01 and 0.02 g/l of core-shell copper oxide nanoparticles for 6h and a change in algal population structure was observed, while the formation of reactive oxygen species was determined using the 2',7'-dichlorodihydrofluorescein diacetate marker measured by flow cytometry. For the study of the photosystem II primary photochemistry we investigated the change in chlorophyll a rapid rise of fluorescence. We found that core-shell copper oxide nanoparticles induced cellular aggregation processes and had a deteriorative effect on chlorophyll by inducing the photoinhibition of photosystem II. The inhibition of photosynthetic electron transport induced a strong energy dissipation process via non-photochemical pathways. The deterioration of photosynthesis was interpreted as being caused by the formation of reactive oxygen species induced by core-shell copper oxide nanoparticles. However, no formation of reactive oxygen species was observed when C. reinhardtii was exposed to the core without the shell or to the shell only. Copyright (c) 2009 Elsevier B.V. All rights reserved.

  14. Acclimation to Very Low CO2: Contribution of Limiting CO2 Inducible Proteins, LCIB and LCIA, to Inorganic Carbon Uptake in Chlamydomonas reinhardtii1[OPEN

    Science.gov (United States)

    Spalding, Martin H.

    2014-01-01

    The limiting-CO2 inducible CO2-concentrating mechanism (CCM) of microalgae represents an effective strategy to capture CO2 when its availability is limited. At least two limiting-CO2 acclimation states, termed low CO2 and very low CO2, have been demonstrated in the model microalga Chlamydomonas reinhardtii, and many questions still remain unanswered regarding both the regulation of these acclimation states and the molecular mechanism underlying operation of the CCM in these two states. This study examines the role of two proteins, Limiting CO2 Inducible A (LCIA; also named NAR1.2) and LCIB, in the CCM of C. reinhardtii. The identification of an LCIA-LCIB double mutant based on its inability to survive in very low CO2 suggests that both LCIA and LCIB are critical for survival in very low CO2. The contrasting effects of individual mutations in LCIB and LCIA compared with the effects of LCIB-LCIA double mutations on growth and inorganic carbon-dependent photosynthetic O2 evolution reveal distinct roles of LCIA and LCIB in the CCM. Although both LCIA and LCIB are essential for very low CO2 acclimation, LCIB appears to function in a CO2 uptake system, whereas LCIA appears to be associated with a HCO3− transport system. The contrasting and complementary roles of LCIA and LCIB in acclimation to low CO2 and very low CO2 suggest a possible mechanism of differential regulation of the CCM based on the inhibition of HCO3− transporters by moderate to high levels of CO2. PMID:25336519

  15. Molecular mechanism of NADPH-glyceraldehyde-3-phosphate dehydrogenase regulation through the C-terminus of CP12 in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Erales, Jenny; Mekhalfi, Malika; Woudstra, Mireille; Gontero, Brigitte

    2011-04-12

    In Chlamydomonas reinhardtii, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) consists of four GapA subunits. This A4 GAPDH is not autonomously regulated, as the regulatory cysteine residues present on GapB subunits are missing in GapA subunits. The regulation of A4 GAPDH is provided by another protein, CP12. To determine the molecular mechanisms of regulation of A4 GAPDH, we mutated three residues (R82, R190, and S195) of GAPDH of C. reinhardtii. Kinetic studies of GAPDH mutants showed the importance of residue R82 in the specificity of GAPDH for NADPH, as previously shown for the spinach enzyme. The cofactor NADPH was not stabilized through the 2'-phosphate by the serine 195 residue of the algal GAPDH, unlike the case in spinach. The mutation of R190 also led to a structural change that was not observed in the spinach enzyme. This mutation led to a loss of activity for NADPH and NADH, indicating the crucial role of this residue in maintaining the algal GAPDH structure. Finally, the interaction between GAPDH mutants and wild-type and mutated CP12 was analyzed by immunoblotting experiments, surface plasmon resonance, and kinetic studies. The results obtained with these approaches highlight the involvement of the last residue of CP12, Asp80, in modulating the activity of GAPDH by preventing access of the cofactor NADPH to the active site. These results help us to bridge the gap between our knowledge of structure and our understanding of functional biology in GAPDH regulation.

  16. Truncated Photosystem Chlorophyll Antenna Size in the Green Microalga Chlamydomonas reinhardtii upon Deletion of the TLA3-CpSRP43 Gene1[C][W][OA

    Science.gov (United States)

    Kirst, Henning; Garcia-Cerdan, Jose Gines; Zurbriggen, Andreas; Ruehle, Thilo; Melis, Anastasios

    2012-01-01

    The truncated light-harvesting antenna size3 (tla3) DNA insertional transformant of Chlamydomonas reinhardtii is a chlorophyll-deficient mutant with a lighter green phenotype, a lower chlorophyll (Chl) per cell content, and higher Chl a/b ratio than corresponding wild-type strains. Functional analyses revealed a higher intensity for the saturation of photosynthesis and greater light-saturated photosynthetic activity in the tla3 mutant than in the wild type and a Chl antenna size of the photosystems that was only about 40% of that in the wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western-blot analyses showed that the tla3 strain was deficient in the Chl a/b light-harvesting complex. Molecular and genetic analyses revealed a single plasmid insertion in chromosome 4 of the tla3 nuclear genome, causing deletion of predicted gene g5047 and plasmid insertion within the fourth intron of downstream-predicted gene g5046. Complementation studies defined that gene g5047 alone was necessary and sufficient to rescue the tla3 mutation. Gene g5047 encodes a C. reinhardtii homolog of the chloroplast-localized SRP43 signal recognition particle, whose occurrence and function in green microalgae has not hitherto been investigated. Biochemical analysis showed that the nucleus-encoded and chloroplast-localized CrCpSRP43 protein specifically operates in the assembly of the peripheral components of the Chl a/b light-harvesting antenna. This work demonstrates that cpsrp43 deletion in green microalgae can be employed to generate tla mutants with a substantially diminished Chl antenna size. The latter exhibit improved solar energy conversion efficiency and photosynthetic productivity under mass culture and bright sunlight conditions. PMID:23043081

  17. Truncated photosystem chlorophyll antenna size in the green microalga Chlamydomonas reinhardtii upon deletion of the TLA3-CpSRP43 gene.

    Science.gov (United States)

    Kirst, Henning; Garcia-Cerdan, Jose Gines; Zurbriggen, Andreas; Ruehle, Thilo; Melis, Anastasios

    2012-12-01

    The truncated light-harvesting antenna size3 (tla3) DNA insertional transformant of Chlamydomonas reinhardtii is a chlorophyll-deficient mutant with a lighter green phenotype, a lower chlorophyll (Chl) per cell content, and higher Chl a/b ratio than corresponding wild-type strains. Functional analyses revealed a higher intensity for the saturation of photosynthesis and greater light-saturated photosynthetic activity in the tla3 mutant than in the wild type and a Chl antenna size of the photosystems that was only about 40% of that in the wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western-blot analyses showed that the tla3 strain was deficient in the Chl a/b light-harvesting complex. Molecular and genetic analyses revealed a single plasmid insertion in chromosome 4 of the tla3 nuclear genome, causing deletion of predicted gene g5047 and plasmid insertion within the fourth intron of downstream-predicted gene g5046. Complementation studies defined that gene g5047 alone was necessary and sufficient to rescue the tla3 mutation. Gene g5047 encodes a C. reinhardtii homolog of the chloroplast-localized SRP43 signal recognition particle, whose occurrence and function in green microalgae has not hitherto been investigated. Biochemical analysis showed that the nucleus-encoded and chloroplast-localized CrCpSRP43 protein specifically operates in the assembly of the peripheral components of the Chl a/b light-harvesting antenna. This work demonstrates that cpsrp43 deletion in green microalgae can be employed to generate tla mutants with a substantially diminished Chl antenna size. The latter exhibit improved solar energy conversion efficiency and photosynthetic productivity under mass culture and bright sunlight conditions.

  18. Bioaccumulation and biosorption of copper and lead by a unicellular algae Chlamydomonas reinhardtii in single and binary metal systems: a comparative study.

    Science.gov (United States)

    Flouty, Roula; Estephane, Georgette

    2012-11-30

    A comparative evaluation of bioaccumulation and biosorption of Cu (II) and Pb (II) ions by algal cells of Chlamydomonas reinhardtii was conducted in single and binary metal systems. Experiments were performed in solutions containing 5 × 10(-7) M of free metal at 30 °C and pH 6. Algal cells were used in the concentration of 0.2 g/L. Both processes tend to be more important as contact time between heavy metals and algal cells increases. Under studied conditions, dead cells showed higher removal efficiency than living cells for both metal ions. Removal efficiency of Pb increases from 8% to 40% when comparing the results obtained by living cells and dead cells. For Cu (II) ions, the removal efficiency of dead cells was about 2 times higher than living cells (55% vs. 28%). Living cells showed similar bioaccumulation capacity for both ions. Synergistic and antagonistic effects between copper and lead were observed in binary metal systems which imply that bioaccumulation process is much more dynamic than assumed in the equilibrium models. In contrast, dead algal cells showed a higher affinity for Pb (II) ions compared to Cu (II) ions and no competitive effect was observed in the biosorption of copper and lead by the inert cells in binary metal mixtures. Biosorption of Cu (II) and Pb (II) seems to occur at different binding sites on the surface of algal biomass. The obtained results showed that the mostly advantageous process of metal ions binding is biosorption and the biomass of C. reinhardtii is suitable for the development of an efficient and economic biosorbent for the removal of heavy metals from aqueous environments. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Genome-wide identification of regulatory elements and reconstruction of gene regulatory networks of the green alga Chlamydomonas reinhardtii under carbon deprivation.

    Directory of Open Access Journals (Sweden)

    Flavia Vischi Winck

    Full Text Available The unicellular green alga Chlamydomonas reinhardtii is a long-established model organism for studies on photosynthesis and carbon metabolism-related physiology. Under conditions of air-level carbon dioxide concentration [CO2], a carbon concentrating mechanism (CCM is induced to facilitate cellular carbon uptake. CCM increases the availability of carbon dioxide at the site of cellular carbon fixation. To improve our understanding of the transcriptional control of the CCM, we employed FAIRE-seq (formaldehyde-assisted Isolation of Regulatory Elements, followed by deep sequencing to determine nucleosome-depleted chromatin regions of algal cells subjected to carbon deprivation. Our FAIRE data recapitulated the positions of known regulatory elements in the promoter of the periplasmic carbonic anhydrase (Cah1 gene, which is upregulated during CCM induction, and revealed new candidate regulatory elements at a genome-wide scale. In addition, time series expression patterns of 130 transcription factor (TF and transcription regulator (TR genes were obtained for cells cultured under photoautotrophic condition and subjected to a shift from high to low [CO2]. Groups of co-expressed genes were identified and a putative directed gene-regulatory network underlying the CCM was reconstructed from the gene expression data using the recently developed IOTA (inner composition alignment method. Among the candidate regulatory genes, two members of the MYB-related TF family, Lcr1 (Low-CO 2 response regulator 1 and Lcr2 (Low-CO2 response regulator 2, may play an important role in down-regulating the expression of a particular set of TF and TR genes in response to low [CO2]. The results obtained provide new insights into the transcriptional control of the CCM and revealed more than 60 new candidate regulatory genes. Deep sequencing of nucleosome-depleted genomic regions indicated the presence of new, previously unknown regulatory elements in the C. reinhardtii genome

  20. Effect of chromium oxide (III) nanoparticles on the production of reactive oxygen species and photosystem II activity in the green alga Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Costa, Cristina Henning da [Department of Sanitary and Environmental Engineering, Federal University of Santa Catarina, Campus Universitário, CEP: 88040-970, Florianópolis, SC (Brazil); Perreault, François [School of Sustainable Engineering and the Built Environment, Arizona State University, Tempe, AZ 85287-3005 (United States); Oukarroum, Abdallah [Department of Chemistry, University of Quebec in Montréal, 2101, Jeanne Mance Street, Station Centre-Ville, Montréal, QC H2X 2J6 (Canada); Melegari, Sílvia Pedroso [Department of Sanitary and Environmental Engineering, Federal University of Santa Catarina, Campus Universitário, CEP: 88040-970, Florianópolis, SC (Brazil); Center of Marine Studies, Federal University of Parana, Beira-mar Avenue, 83255-976, Pontal do Parana, PR (Brazil); Popovic, Radovan [Department of Chemistry, University of Quebec in Montréal, 2101, Jeanne Mance Street, Station Centre-Ville, Montréal, QC H2X 2J6 (Canada); Matias, William Gerson, E-mail: william.g.matias@ufsc.br [Department of Sanitary and Environmental Engineering, Federal University of Santa Catarina, Campus Universitário, CEP: 88040-970, Florianópolis, SC (Brazil)

    2016-09-15

    With the growth of nanotechnology and widespread use of nanomaterials, there is an increasing risk of environmental contamination by nanomaterials. However, the potential implications of such environmental contamination are hard to evaluate since the toxicity of nanomaterials if often not well characterized. The objective of this study was to evaluate the toxicity of a chromium-based nanoparticle, Cr{sub 2}O{sub 3}-NP, used in a wide diversity of industrial processes and commercial products, on the unicellular green alga Chlamydomonas reinhardtii. The deleterious impacts of Cr{sub 2}O{sub 3}-NP were characterized using cell density measurements, production of reactive oxygen species (ROS), esterase enzymes activity, and photosystem II electron transport as indicators of toxicity. Cr{sub 2}O{sub 3}-NP exposure inhibited culture growth and significantly lowered cellular Chlorophyll a content. From cell density measurements, EC50 values of 2.05 ± 0.20 and 1.35 ± 0.06 g L{sup −1} Cr{sub 2}O{sub 3}-NP were obtained after 24 and 72 h of exposure, respectively. In addition, ROS levels were increased to 160.24 ± 2.47% and 59.91 ± 0.15% of the control value after 24 and 72 h of exposition to 10 g L{sup −1} Cr{sub 2}O{sub 3}-NP. At 24 h of exposure, the esterase activity increased to 160.24% of control value, revealing a modification of the short-term metabolic response of algae to Cr{sub 2}O{sub 3}-NP exposure. In conclusion, the metabolism of C. reinhardtii was the most sensitive to Cr{sub 2}O{sub 3}-NP after 24 h of treatment. - Highlights: • Cr{sub 2}O{sub 3} nanoparticles are unstable and form large aggregates in the medium. • EC50 for growth inhibition of C. reinhardtii is 1.35 g L{sup −1} at 72 h. • Cr{sub 2}O{sub 3} nanoparticles increase ROS levels at 10 g L{sup −1}. • Cr{sub 2}O{sub 3} nanoparticles affect photosynthetic electron transport.

  1. The microalga Chlamydomonas reinhardtii CW-15 as a solar cell for hydrogen peroxide photoproduction. Comparison between free and immobilized cells and thylakoids for energy conversion efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Scholz, W.; Galvan, F.; Rosa, F.F. de la [Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla y CSIC, Sevilla (Spain)

    1995-11-28

    Immobilized cells and thylakoid vesicles of the microalga Chlamydomonas reinhardtii CW-15 have been developed as a solar cell because of their capabilities of producing hydrogen peroxide. This compound is an efficient and clean fuel used for rocket propulsion, motors and for heating. Hydrogen peroxide is produced by the photosystem in a catalyst cycle in which a redox mediator (methyl viologen) is reduced by electrons obtained from water by the photosynthetic apparatus of the microalga and it is re-oxidized by the oxygen dissolved in the solution. The photoproduction has been investigated using a discontinuous system with whole cells, or thylakoid vesicles, free or immobilized on alginate. The stimulation by azide as an inhibitor of catalase has also been analyzed. Under determined optimum conditions, the photoproduction by Ca-alginate entrapped cells, with a rate of 33 {mu}mol H{sub 2}O{sub 2}/mg Chl.h, was maintained for several hours with an energy conversion efficiency of 0.25%

  2. CCS2, an Octatricopeptide-Repeat Protein, Is Required for Plastid CytochromecAssembly in the Green AlgaChlamydomonas reinhardtii.

    Science.gov (United States)

    Cline, Sara G; Laughbaum, Isaac A; Hamel, Patrice P

    2017-01-01

    In bacteria and energy generating organelles, c -type cytochromes are a class of universal electron carriers with a heme cofactor covalently linked via one or two thioether bonds to a heme binding site. The covalent attachment of heme to apocytochromes is a catalyzed process, taking place via three evolutionarily distinct assembly pathways (Systems I, II, III). System II was discovered in the green alga Chlamydomonas reinhardtii through the genetic analysis of the ccs mutants ( c ytochrome c s ynthesis), which display a block in the apo- to holo- form conversion of cytochrome f and c 6 , the thylakoid lumen resident c -type cytochromes functioning in photosynthesis. Here we show that the gene corresponding to the CCS2 locus encodes a 1,719 amino acid polypeptide and identify the molecular lesions in the ccs2-1 to ccs2-5 alleles. The CCS2 protein displays seven degenerate amino acid repeats, which are variations of the o ctatrico p eptide- r epeat motif (OPR) recently recognized in several nuclear-encoded proteins controlling the maturation, stability, or translation of chloroplast transcripts. A plastid site of action for CCS2 is inferred from the finding that GFP fused to the first 100 amino acids of the algal protein localizes to chloroplasts in Nicotiana benthamiana . We discuss the possible functions of CCS2 in the heme attachment reaction.

  3. Comparative Shotgun Proteomic Analysis of Wastewater-Cultured Microalgae: Nitrogen Sensing and Carbon Fixation for Growth and Nutrient Removal in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Patel, Anil K; Huang, Eric L; Low-Décarie, Etienne; Lefsrud, Mark G

    2015-08-07

    Chlamydomonas reinhardtii was batch-cultured for 12 days under continuous illumination to investigate nitrogen uptake and metabolic responses to wastewater processing. Our approach compared two conditions: (1) artificial wastewater containing nitrate and ammonia and (2) nutrient-sufficient control containing nitrate as sole form of nitrogen. Treatments did not differ in final biomass; however, comparison of group proteomes revealed significant differences. Label-free shotgun proteomic analysis identified 2358 proteins, of which 92 were significantly differentially abundant. Wastewater cells showed higher relative abundances of photosynthetic antenna proteins, enzymes related to carbon fixation, and biosynthesis of amino acids and secondary metabolites. Control cells showed higher abundances of enzymes and proteins related to nitrogen metabolism and assimilation, synthesis and utilization of starch, amino acid recycling, evidence of oxidative stress, and little lipid biosynthesis. This study of the eukaryotic microalgal proteome response to nitrogen source, availability, and switching highlights tightly controlled pathways essential to the maintenance of culture health and productivity in concert with light absorption and carbon assimilation. Enriched pathways in artificial wastewater, notably, photosynthetic carbon fixation and biosynthesis of plant hormones, and those in nitrate only control, most notably, nitrogen, amino acid, and starch metabolism, represent potential targets for genetic improvement requiring targeted elucidation.

  4. Combined Increases in Mitochondrial Cooperation and Oxygen Photoreduction Compensate for Deficiency in Cyclic Electron Flow in Chlamydomonas reinhardtii[W][OPEN

    Science.gov (United States)

    Dang, Kieu-Van; Plet, Julie; Tolleter, Dimitri; Jokel, Martina; Cuiné, Stéphan; Carrier, Patrick; Auroy, Pascaline; Richaud, Pierre; Johnson, Xenie; Alric, Jean; Allahverdiyeva, Yagut; Peltier, Gilles

    2014-01-01

    During oxygenic photosynthesis, metabolic reactions of CO2 fixation require more ATP than is supplied by the linear electron flow operating from photosystem II to photosystem I (PSI). Different mechanisms, such as cyclic electron flow (CEF) around PSI, have been proposed to participate in reequilibrating the ATP/NADPH balance. To determine the contribution of CEF to microalgal biomass productivity, here, we studied photosynthesis and growth performances of a knockout Chlamydomonas reinhardtii mutant (pgrl1) deficient in PROTON GRADIENT REGULATION LIKE1 (PGRL1)–mediated CEF. Steady state biomass productivity of the pgrl1 mutant, measured in photobioreactors operated as turbidostats, was similar to its wild-type progenitor under a wide range of illumination and CO2 concentrations. Several changes were observed in pgrl1, including higher sensitivity of photosynthesis to mitochondrial inhibitors, increased light-dependent O2 uptake, and increased amounts of flavodiiron (FLV) proteins. We conclude that a combination of mitochondrial cooperation and oxygen photoreduction downstream of PSI (Mehler reactions) supplies extra ATP for photosynthesis in the pgrl1 mutant, resulting in normal biomass productivity under steady state conditions. The lower biomass productivity observed in the pgrl1 mutant in fluctuating light is attributed to an inability of compensation mechanisms to respond to a rapid increase in ATP demand. PMID:24989042

  5. Immobilization of Chlamydomonas reinhardtii CLH1 on APTES-Coated Magnetic Iron Oxide Nanoparticles and Its Potential in the Production of Chlorophyll Derivatives.

    Science.gov (United States)

    Yen, Chih-Chung; Chuang, Yao-Chen; Ko, Chia-Yun; Chen, Long-Fang O; Chen, Sheau-Shyang; Lin, Chia-Jung; Chou, Yi-Li; Shaw, Jei-Fu

    2016-07-26

    Recombinant Chlamydomonas reinhardtii chlorophyllase 1 (CrCLH1) that could catalyze chlorophyll hydrolysis to chlorophyllide and phytol in vitro was successfully expressed in Escherichia coli. The recombinant CrCLH1 was immobilized through covalent binding with a cubic (3-aminopropyl) triethoxysilane (APTES) coating on magnetic iron oxide nanoparticles (MIONPs), which led to markedly improved enzyme performance and decreased biocatalyst costs for potential industrial application. The immobilized enzyme exhibited a high immobilization yield (98.99 ± 0.91 mg/g of gel) and a chlorophyllase assay confirmed that the immobilized recombinant CrCLH1 retained enzymatic activity (722.3 ± 50.3 U/g of gel). Biochemical analysis of the immobilized enzyme, compared with the free enzyme, showed higher optimal pH and pH stability for chlorophyll-a hydrolysis in an acidic environment (pH 3-5). In addition, compared with the free enzyme, the immobilized enzyme showed higher activity in chlorophyll-a hydrolysis in a high temperature environment (50-60 °C). Moreover, the immobilized enzyme retained a residual activity of more than 64% of its initial enzyme activity after 14 cycles in a repeated-batch operation. Therefore, APTES-coated MIONP-immobilized recombinant CrCLH1 can be repeatedly used to lower costs and is potentially useful for the industrial production of chlorophyll derivatives.

  6. Immobilization of Chlamydomonas reinhardtii CLH1 on APTES-Coated Magnetic Iron Oxide Nanoparticles and Its Potential in the Production of Chlorophyll Derivatives

    Directory of Open Access Journals (Sweden)

    Chih-Chung Yen

    2016-07-01

    Full Text Available Recombinant Chlamydomonas reinhardtii chlorophyllase 1 (CrCLH1 that could catalyze chlorophyll hydrolysis to chlorophyllide and phytol in vitro was successfully expressed in Escherichia coli. The recombinant CrCLH1 was immobilized through covalent binding with a cubic (3-aminopropyl triethoxysilane (APTES coating on magnetic iron oxide nanoparticles (MIONPs, which led to markedly improved enzyme performance and decreased biocatalyst costs for potential industrial application. The immobilized enzyme exhibited a high immobilization yield (98.99 ± 0.91 mg/g of gel and a chlorophyllase assay confirmed that the immobilized recombinant CrCLH1 retained enzymatic activity (722.3 ± 50.3 U/g of gel. Biochemical analysis of the immobilized enzyme, compared with the free enzyme, showed higher optimal pH and pH stability for chlorophyll-a hydrolysis in an acidic environment (pH 3–5. In addition, compared with the free enzyme, the immobilized enzyme showed higher activity in chlorophyll-a hydrolysis in a high temperature environment (50–60 °C. Moreover, the immobilized enzyme retained a residual activity of more than 64% of its initial enzyme activity after 14 cycles in a repeated-batch operation. Therefore, APTES-coated MIONP-immobilized recombinant CrCLH1 can be repeatedly used to lower costs and is potentially useful for the industrial production of chlorophyll derivatives.

  7. Assessing bio-available silver released from silver nanoparticles embedded in silica layers using the green algae Chlamydomonas reinhardtii as bio-sensors.

    Science.gov (United States)

    Pugliara, Alessandro; Makasheva, Kremena; Despax, Bernard; Bayle, Maxime; Carles, Robert; Benzo, Patrizio; BenAssayag, Gérard; Pécassou, Béatrice; Sancho, Maria Carmen; Navarro, Enrique; Echegoyen, Yolanda; Bonafos, Caroline

    2016-09-15

    Silver nanoparticles (AgNPs) because of their strong antibacterial activity are widely used in health-care sector and industrial applications. Their huge surface-volume ratio enhances the silver release compared to the bulk material, leading to an increased toxicity for microorganisms sensitive to this element. This work presents an assessment of the toxic effect on algal photosynthesis due to small (size optical properties of the nanostructures were studied by transmission electron microscopy and optical reflectance. The silver release from the nanostructures after 20h of immersion in buffered water was measured by inductively coupled plasma mass spectrometry and ranges between 0.02 and 0.49μM. The short-term toxicity of Ag to photosynthesis of Chlamydomonas reinhardtii was assessed by fluorometry. The obtained results show that embedding AgNPs reduces the interactions with the buffered water free media, protecting the AgNPs from fast oxidation. The release of bio-available silver (impacting on the algal photosynthesis) is controlled by the depth at which AgNPs are located for a given host matrix. This provides a procedure to tailor the toxicity of nanocomposites containing AgNPs. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Expression of a Low CO2–Inducible Protein, LCI1, Increases Inorganic Carbon Uptake in the Green Alga Chlamydomonas reinhardtii[W][OA

    Science.gov (United States)

    Ohnishi, Norikazu; Mukherjee, Bratati; Tsujikawa, Tomoki; Yanase, Mari; Nakano, Hirobumi; Moroney, James V.; Fukuzawa, Hideya

    2010-01-01

    Aquatic photosynthetic organisms can modulate their photosynthesis to acclimate to CO2-limiting stress by inducing a carbon-concentrating mechanism (CCM) that includes carbonic anhydrases and inorganic carbon (Ci) transporters. However, to date, Ci-specific transporters have not been well characterized in eukaryotic algae. Previously, a Chlamydomonas reinhardtii mutant (lcr1) was identified that was missing a Myb transcription factor. This mutant had reduced light-dependent CO2 gas exchange (LCE) activity when grown under CO2-limiting conditions and did not induce the CAH1 gene encoding a periplasmic carbonic anhydrase, as well as two as yet uncharacterized genes, LCI1 and LCI6. In this study, LCI1 was placed under the control of the nitrate reductase promoter, allowing for the induction of LCI1 expression by nitrate in the absence of other CCM components. When the expression of LCI1 was induced in the lcr1 mutant under CO2-enriched conditions, the cells showed an increase in LCE activity, internal Ci accumulation, and photosynthetic affinity for Ci. From experiments using indirect immunofluorescence, LCI1–green fluorescent protein fusions, and cell fractionation procedures, it appears that LCI1 is mainly localized to the plasma membrane. These results provide strong evidence that LCI1 may contribute to the CCM as a component of the Ci transport machinery in the plasma membrane. PMID:20870960

  9. X-ray dense cellular inclusions in the cells of the green alga Chlamydomonas reinhardtii as seen by soft-x-ray microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Stead, A.D.; Ford, T.W.; Page, A.M. [Univ. of London (United Kingdom); Brown, J.T.; Meyer-Ilse, W. [Ernest Orlando Lawrence Berkeley National Lab., CA (United States)

    1997-04-01

    Soft x-rays, having a greater ability to penetrate biological material than electrons, have the potential for producing images of intact, living cells. In addition, by using the so-called {open_quotes}water window{close_quotes} area of the soft x-ray spectrum, a degree of natural contrast is introduced into the image due to differential absorption of the wavelengths by compounds with a high carbon content compared to those with a greater oxygen content. The variation in carbon concentration throughout a cell therefore generates an image which is dependent upon the carbon density within the specimen. Using soft x-ray contact microscopy the authors have previously examined the green alga Chlamydomonas reinhardtii, and the most prominent feature of the cells are the numerous x-ray absorbing spheres, But they were not seen by conventional transmission electron microscopy. Similar structures have also been reported by the Goettingen group using their cryo transmission x-ray microscope at BESSY. Despite the fact that these spheres appear to occupy up to 20% or more of the cell volume when seen by x-ray microscopy, they are not visible by transmission electron microscopy. Given the difficulties and criticisms associated with soft x-ray contact microscopy, the present study was aimed at confirming the existence of these cellular inclusions and learning more of their possible chemical composition.

  10. Crystallization and preliminary X-ray crystallographic studies of CrArsM, an arsenic(III) S-adenosylmethionine methyltransferase from Chlamydomonas reinhardtii.

    Science.gov (United States)

    Packianathan, Charles; Pillai, Jitesh K; Riaz, Ahmed; Kandavelu, Palani; Sankaran, Banumathi; Rosen, Barry P

    2014-10-01

    Arsenic is one the most toxic environmental substances. Arsenic is ubiquitous in water, soil and food, and ranks first on the Environmental Protection Agency's Superfund Priority List of Hazardous Substances. Arsenic(III) S-adenosylmethionine methyltransferases (AS3MT in animals and ArsM in microbes) are key enzymes of arsenic biotransformation, catalyzing the methylation of inorganic arsenite to give methyl, dimethyl and trimethyl products. Arsenic methyltransferases are found in members of every kingdom from bacteria to humans (EC 2.1.1.137). In the human liver, hAS3MT converts inorganic arsenic into more toxic and carcinogenic forms. CrArsM, an ortholog of hAS3MT from the eukaryotic green alga Chlamydomonas reinhardtii, was purified by chemically synthesizing the gene and expressing it in Escherichia coli. Synthetic purified CrArsM was crystallized in an unliganded form. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to space group R3:H, with unit-cell parameters a = b = 157.8, c = 95.4 Å, γ = 120° and two molecules in the asymmetric unit. Complete data sets were collected and processed to a resolution of 2.40 Å.

  11. Circadian Clock Regulation of Starch Metabolism Establishes GBSSI as a Major Contributor to Amylopectin Synthesis in Chlamydomonas reinhardtii1[W][OA

    Science.gov (United States)

    Ral, Jean-Philippe; Colleoni, Christophe; Wattebled, Fabrice; Dauvillée, David; Nempont, Clément; Deschamps, Philippe; Li, Zhongyi; Morell, Matthew K.; Chibbar, Ravindra; Purton, Saul; d'Hulst, Christophe; Ball, Steven G.

    2006-01-01

    Chlamydomonas reinhardtii displays a diurnal rhythm of starch content that peaks in the middle of the night phase if the algae are provided with acetate and CO2 as a carbon source. We show that this rhythm is controlled by the circadian clock and is tightly correlated to ADP-glucose pyrophosphorylase activity. Persistence of this rhythm depends on the presence of either soluble starch synthase III or granule-bound starch synthase I (GBSSI). We show that both enzymes play a similar function in synthesizing the long glucan fraction that interconnects the amylopectin clusters. We demonstrate that in log phase-oscillating cultures, GBSSI is required to obtain maximal polysaccharide content and fully compensates for the loss of soluble starch synthase III. A point mutation in the GBSSI gene that prevents extension of amylopectin chains, but retains the enzyme's normal ability to extend maltooligosaccharides, abolishes the function of GBSSI both in amylopectin and amylose synthesis and leads to a decrease in starch content in oscillating cultures. We propose that GBSSI has evolved as a major enzyme of amylopectin synthesis and that amylose synthesis comes as a secondary consequence of prolonged synthesis by GBSSI in arrhythmic systems. Maintenance in higher plant leaves of circadian clock control of GBSSI transcription is discussed. PMID:16844835

  12. Light-harvesting complex gene expression is controlled by both transcriptional and post-transcriptional mechanisms during photoacclimation in Chlamydomonas reinhardtii

    CERN Document Server

    Durnford Dion, G; McKim, Sarah M; Sarchfield, Michelle L

    2003-01-01

    To compensate for increases in photon flux density (PFD), photosynthetic organisms possess mechanisms for reversibly modulating their photosynthetic apparatus to minimize photodamage. The photoacclimation response in Chlamydomonas reinhardtii was assessed following a 10-fold increase in PFD over 24h. In addition to a 50% reduction in the amount of chlorophyll and light-harvesting complexes (LHC) per cell, the expression of genes encoding polypeptides of the light-harvesting antenna were also affected. The abundance of Lhcb (a LHCH gene), Lhcb4 (a CP29-like gene), and Lhca (a LHCI gene) transcripts were reduced by 65 to 80%, within 1-2 h; however, the RNA levels of all three genes recovered to their low-light (LL) concentrations within 6-8 h. To determine the role of transcript turnover in this transient decline in abundance, the stability of all transcripts was measured. Although there was no change in the Lhcb or Lhca transcript turnover time, the Lhcb4 mRNA stability decreased 2.5-fold immediately following...

  13. The target of rapamycin kinase affects biomass accumulation and cell cycle progression by altering carbon/nitrogen balance in synchronized Chlamydomonas reinhardtii cells.

    Science.gov (United States)

    Jüppner, Jessica; Mubeen, Umarah; Leisse, Andrea; Caldana, Camila; Wiszniewski, Andrew; Steinhauser, Dirk; Giavalisco, Patrick

    2018-01-01

    Several metabolic processes tightly regulate growth and biomass accumulation. A highly conserved protein complex containing the target of rapamycin (TOR) kinase is known to integrate intra- and extracellular stimuli controlling nutrient allocation and hence cellular growth. Although several functions of TOR have been described in various heterotrophic eukaryotes, our understanding lags far behind in photosynthetic organisms. In the present investigation, we used the model alga Chlamydomonas reinhardtii to conduct a time-resolved analysis of molecular and physiological features throughout the diurnal cycle after TOR inhibition. Detailed examination of the cell cycle phases revealed that growth is not only repressed by 50%, but also that significant, non-linear delays in the progression can be observed. By using metabolomics analysis, we elucidated that the growth repression was mainly driven by differential carbon partitioning between anabolic and catabolic processes. Accordingly, the time-resolved analysis illustrated that metabolic processes including amino acid-, starch- and triacylglycerol synthesis, as well RNA degradation, were redirected within minutes of TOR inhibition. Here especially the high accumulation of nitrogen-containing compounds indicated that an active TOR kinase controls the carbon to nitrogen balance of the cell, which is responsible for biomass accumulation, growth and cell cycle progression. © 2017 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

  14. Preliminary investigation on the production of fuels and bio-char from Chlamydomonas reinhardtii biomass residue after bio-hydrogen production.

    Science.gov (United States)

    Torri, Cristian; Samorì, Chiara; Adamiano, Alessio; Fabbri, Daniele; Faraloni, Cecilia; Torzillo, Giuseppe

    2011-09-01

    The aim of this work was to investigate the potential conversion of Chlamydomonas reinhardtii biomass harvested after hydrogen production. The spent algal biomass was converted into nitrogen-rich bio-char, biodiesel and pyrolysis oil (bio-oil). The yield of lipids (algal oil), obtained by solvent extraction, was 15 ± 2% w/w(dry-biomass). This oil was converted into biodiesel with a 8.7 ± 1% w/w(dry-biomass) yield. The extraction residue was pyrolysed in a fixed bed reactor at 350 °C obtaining bio-char as the principal fraction (44 ± 1% w/w(dry-biomass)) and 28 ± 2% w/w(dry-biomass) of bio-oil. Pyrolysis fractions were characterized by elemental analysis, while the chemical composition of bio-oil was fully characterized by GC-MS, using various derivatization techniques. Energy outputs resulting from this approach were distributed in hydrogen (40%), biodiesel (12%) and pyrolysis fractions (48%), whereas bio-char was the largest fraction in terms of mass. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Stable expression of a bifunctional diterpene synthase in the chloroplast of Chlamydomonas reinhardtii

    DEFF Research Database (Denmark)

    Zedler, Julie Annemarie Zita; Gangl, Doris; Hamberger, Björn Robert

    2015-01-01

    is an alternative strategy, and we report here the stable expression of a large (91 kDa) protein in the chloroplast using a recently developed low-cost transformation protocol. Moreover, selection of transformants is based on restoration of prototrophy using an endogenous gene (psbH) as the marker, thereby allowing...... the generation of transgenic lines without the use of antibiotic-resistance genes. Here, we have expressed a bifunctional diterpene synthase in C. reinhardtii chloroplasts. Homoplasmic transformants were obtained with the expressed enzyme accounting for 3.7 % of total soluble protein. The enzyme was purified...

  16. Comprehensive comparison of iTRAQ and label-free LC-based quantitative proteomics approaches using two Chlamydomonas reinhardtii strains of interest for biofuels engineering.

    Science.gov (United States)

    Wang, Hongxia; Alvarez, Sophie; Hicks, Leslie M

    2012-01-01

    Comprehensive comparisons of quantitative proteomics techniques are rare in the literature, yet they are crucially important for optimal selection of approaches and methodologies that are ideal for a given proteomics initiative. In this study, two LC-based quantitative proteomics approaches--iTRAQ and label-free--were implemented using the LTQ-Orbitrap Velos platform. For this comparison, the model used was the total protein content from two Chlamydomonas reinhardtii strains in the context of alternative biofuels production. The strain comparison includes sta6 (a starch-less mutant of cw15) that produces twice as many lipid bodies (LB) containing triacylglycerols (TAGs) as its parental strain cw15 (a cell wall-deficient C. reinhardtii strain) under nitrogen starvation. Internal standard addition was used to rigorously assess the quantitation accuracy and precision of each method. Results from iTRAQ-4plex labeling using HCD (higher energy collision-induced dissociation) fragmentation were compared to those obtained using a label-free approach based on the peak area of intact peptides and collision-induced dissociation. The accuracy and precision, number of identified/quantified proteins and statistically significant protein differences detected, as well as efficiency of these two quantitative proteomics methods were evaluated and compared. Four technical and three biological replicates of each strain were performed to assess both the technical and biological variation of both approaches. A total of 896 and 639 proteins were identified with high confidence, and 329 and 124 proteins were quantified significantly with label-free and iTRAQ, respectively, using biological replicates. The results showed that both iTRAQ labeling and label-free methods provide high quality quantitative and qualitative data using nano-LC coupled with the LTQ-Orbitrap Velos mass spectrometer, but the selection of the optimal approach is dependent on experimental design and the biological

  17. A millifluidic study of cell-to-cell heterogeneity in growth-rate and cell-division capability in populations of isogenic cells of Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Shima P Damodaran

    Full Text Available To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers and a significant subpopulation of slowly dividing cells (slow-growers. These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

  18. Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

    Science.gov (United States)

    Chioccioli, Maurizio; Hankamer, Ben; Ross, Ian L.

    2014-01-01

    Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD750) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD750 measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth. PMID

  19. Flow cytometry pulse width data enables rapid and sensitive estimation of biomass dry weight in the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris.

    Directory of Open Access Journals (Sweden)

    Maurizio Chioccioli

    Full Text Available Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD750 for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD750 measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary

  20. Negative impact on growth and photosynthesis in the green alga Chlamydomonas reinhardtii in the presence of the estrogen 17α-ethynylestradiol.

    Directory of Open Access Journals (Sweden)

    Tessa Pocock

    Full Text Available It is well known that estrogenic compounds affect development of fertilized eggs of many species of birds, fish and amphibians through disrupted activity of carbonic anhydrase (CA. The most potent activity comes from the most commonly occurring synthetic sterol, 17α-Ethynylestradiol (EE2. Less is known about the responses of aquatic phytoplankton to these compounds. Here we show for the first time that, in comparision to the control, the addition of 7 µM EE2 reduced the growth rate of the green alga Chlamydomonas reinhardtii by 68% for cells grown at high CO2. When cells were grown in ambient air (low Ci with a fully activated carbon concentrating mechanism through the induction of CA activity, the growth rates were reduced by as much as 119%. A reduced growth rate could be observed at EE2 concentrations as low as 10 pM. This was accompanied by a reduced maximum capacity for electron transport in photosystem II as determined by a lower FV/FM for low Ci-grown cells, which indicates the involvement of CAH3, a CA specifically located in the thylakoid lumen involved in proton pumping across the thylakoid membranes. These results were in agreement with an observed reduction in the chloroplastic affinity for Ci as shown by a strong increase in the Michaelis-Menten K0.5 for HCO3-. In itself, a lowering of the growth rate of a green alga by addition of the sterol EE2 warrants further investigation into the potential environmental impact by the release of treated waste water.

  1. The Type II NADPH Dehydrogenase Facilitates Cyclic Electron Flow, Energy-Dependent Quenching, and Chlororespiratory Metabolism during Acclimation of Chlamydomonas reinhardtii to Nitrogen Deprivation1[OPEN

    Science.gov (United States)

    Grossman, Arthur R.

    2016-01-01

    When photosynthetic organisms are deprived of nitrogen (N), the capacity to grow and assimilate carbon becomes limited, causing a decrease in the productive use of absorbed light energy and likely a rise in the cellular reduction state. Although there is a scarcity of N in many terrestrial and aquatic environments, a mechanistic understanding of how photosynthesis adjusts to low-N conditions and the enzymes/activities integral to these adjustments have not been described. In this work, we use biochemical and biophysical analyses of photoautotrophically grown wild-type and mutant strains of Chlamydomonas reinhardtii to determine the integration of electron transport pathways critical for maintaining active photosynthetic complexes even after exposure of cells to N deprivation for 3 d. Key to acclimation is the type II NADPH dehydrogenase, NDA2, which drives cyclic electron flow (CEF), chlororespiration, and the generation of an H+ gradient across the thylakoid membranes. N deprivation elicited a doubling of the rate of NDA2-dependent CEF, with little contribution from PGR5/PGRL1-dependent CEF. The H+ gradient generated by CEF is essential to sustain nonphotochemical quenching, while an increase in the level of reduced plastoquinone would promote a state transition; both are necessary to down-regulate photosystem II activity. Moreover, stimulation of NDA2-dependent chlororespiration affords additional relief from the elevated reduction state associated with N deprivation through plastid terminal oxidase-dependent water synthesis. Overall, rerouting electrons through the NDA2 catalytic hub in response to photoautotrophic N deprivation sustains cell viability while promoting the dissipation of excess excitation energy through quenching and chlororespiratory processes. PMID:26858365

  2. RNAi knock-down of LHCBM1, 2 and 3 increases photosynthetic H2 production efficiency of the green alga Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Melanie Oey

    Full Text Available Single cell green algae (microalgae are rapidly emerging as a platform for the production of sustainable fuels. Solar-driven H2 production from H2O theoretically provides the highest-efficiency route to fuel production in microalgae. This is because the H2-producing hydrogenase (HYDA is directly coupled to the photosynthetic electron transport chain, thereby eliminating downstream energetic losses associated with the synthesis of carbohydrate and oils (feedstocks for methane, ethanol and oil-based fuels. Here we report the simultaneous knock-down of three light-harvesting complex proteins (LHCMB1, 2 and 3 in the high H2-producing Chlamydomonas reinhardtii mutant Stm6Glc4 using an RNAi triple knock-down strategy. The resultant Stm6Glc4L01 mutant exhibited a light green phenotype, reduced expression of LHCBM1 (20.6% ±0.27%, LHCBM2 (81.2% ±0.037% and LHCBM3 (41.4% ±0.05% compared to 100% control levels, and improved light to H2 (180% and biomass (165% conversion efficiencies. The improved H2 production efficiency was achieved at increased solar flux densities (450 instead of ∼100 µE m(-2 s(-1 and high cell densities which are best suited for microalgae production as light is ideally the limiting factor. Our data suggests that the overall improved photon-to-H2 conversion efficiency is due to: 1 reduced loss of absorbed energy by non-photochemical quenching (fluorescence and heat losses near the photobioreactor surface; 2 improved light distribution in the reactor; 3 reduced photoinhibition; 4 early onset of HYDA expression and 5 reduction of O2-induced inhibition of HYDA. The Stm6Glc4L01 phenotype therefore provides important insights for the development of high-efficiency photobiological H2 production systems.

  3. Linoleic Acid-Induced Ultra-Weak Photon Emission from Chlamydomonas reinhardtii as a Tool for Monitoring of Lipid Peroxidation in the Cell Membranes

    Science.gov (United States)

    Prasad, Ankush; Pospíšil, Pavel

    2011-01-01

    Reactive oxygen species formed as a response to various abiotic and biotic stresses cause an oxidative damage of cellular component such are lipids, proteins and nucleic acids. Lipid peroxidation is considered as one of the major processes responsible for the oxidative damage of the polyunsaturated fatty acid in the cell membranes. Various methods such as a loss of polyunsaturated fatty acids, amount of the primary and the secondary products are used to monitor the level of lipid peroxidation. To investigate the use of ultra-weak photon emission as a non-invasive tool for monitoring of lipid peroxidation, the involvement of lipid peroxidation in ultra-weak photon emission was studied in the unicellular green alga Chlamydomonas reinhardtii. Lipid peroxidation initiated by addition of exogenous linoleic acid to the cells was monitored by ultra-weak photon emission measured with the employment of highly sensitive charged couple device camera and photomultiplier tube. It was found that the addition of linoleic acid to the cells significantly increased the ultra-weak photon emission that correlates with the accumulation of lipid peroxidation product as measured using thiobarbituric acid assay. Scavenging of hydroxyl radical by mannitol, inhibition of intrinsic lipoxygenase by catechol and removal of molecular oxygen considerably suppressed ultra-weak photon emission measured after the addition of linoleic acid. The photon emission dominated at the red region of the spectrum with emission maximum at 680 nm. These observations reveal that the oxidation of linoleic acid by hydroxyl radical and intrinsic lipoxygenase results in the ultra-weak photon emission. Electronically excited species such as excited triplet carbonyls are the likely candidates for the primary excited species formed during the lipid peroxidation, whereas chlorophylls are the final emitters of photons. We propose here that the ultra-weak photon emission can be used as a non-invasive tool for the

  4. The Chlamydomonas reinhardtii LI818 gene represents a distant relative of the cabI/II genes that is regulated during the cell cycle and in response to illumination.

    Science.gov (United States)

    Savard, F; Richard, C; Guertin, M

    1996-11-01

    In the green unicellular alga Chlamydomonas reinhardtii, as in higher plants, the expression of the genes encoding the chlorophyll a/b-binding (CAB) polypeptides associated with photosystem I (PSI) and photosystem II (PSII) is regulated by endogenous (circadian clock) and exogenous signals (light and temperature). The circadian clock ensures that the oscillation in the levels of the different cab mRNAs is continuously kept in phase with light/dark (LD) cycles and is maximal by the middle of the day. On the other hand, light controls the amplitude of the oscillations. We report here the cloning and characterization of the C. reinhardtii LI818 gene, which identifies a CAB-related polypeptide and whose expression is regulated quite differently from the cab I/II genes. We show: (1) that in LD synchronized Chlamydomonas cells LI818 mRNA accumulation is subject to dual regulation that involves separable regulation by light and an endogenous oscillator; (2) that LI818 mRNA is fully expressed several hours before the cab I/II mRNAs and that the latter accumulate concomitantly; (3) that blocking the electron flow through PSII using DCMU prevents cells from accumulating cab I/II mRNAs but not LI818 mRNA and (4) that the accumulation of LI818 mRNA is abolished by blocking cytoplasmic protein synthesis, suggesting that these regulatory mechanisms are mediated by labile proteins.

  5. Assessing bio-available silver released from silver nanoparticles embedded in silica layers using the green algae Chlamydomonas reinhardtii as bio-sensors

    Energy Technology Data Exchange (ETDEWEB)

    Pugliara, Alessandro [nMat group-CEMES (Centre d' Elaboration de Matériaux et d' Etudes Structurales)-CNRS, Université de Toulouse, 29 rue Jeanne Marvig, BP 94347, F-31055 Toulouse Cedex 4 (France); LAPLACE (LAboratoire PLAsma et Conversion d' Energie), Université de Toulouse, CNRS, UPS, INPT, 118 route de Narbonne, F-31062 Toulouse (France); Makasheva, Kremena; Despax, Bernard [LAPLACE (LAboratoire PLAsma et Conversion d' Energie), Université de Toulouse, CNRS, UPS, INPT, 118 route de Narbonne, F-31062 Toulouse (France); Bayle, Maxime; Carles, Robert; Benzo, Patrizio; BenAssayag, Gérard; Pécassou, Béatrice [nMat group-CEMES (Centre d' Elaboration de Matériaux et d' Etudes Structurales)-CNRS, Université de Toulouse, 29 rue Jeanne Marvig, BP 94347, F-31055 Toulouse Cedex 4 (France); Sancho, Maria Carmen; Navarro, Enrique [IPE (Instituto Pirenaico de Ecología)-CSIC, Avda. Montañana 1005, Zaragoza 50059 (Spain); Echegoyen, Yolanda [I3A, Department of Analytical Chemistry, University of Zaragoza, C/ María de Luna 3, 50018, Zaragoza (Spain); Bonafos, Caroline, E-mail: bonafos@cemes.fr [nMat group-CEMES (Centre d' Elaboration de Matériaux et d' Etudes Structurales)-CNRS, Université de Toulouse, 29 rue Jeanne Marvig, BP 94347, F-31055 Toulouse Cedex 4 (France)

    2016-09-15

    Silver nanoparticles (AgNPs) because of their strong antibacterial activity are widely used in health-care sector and industrial applications. Their huge surface-volume ratio enhances the silver release compared to the bulk material, leading to an increased toxicity for microorganisms sensitive to this element. This work presents an assessment of the toxic effect on algal photosynthesis due to small (size < 20 nm) AgNPs embedded in silica layers. Two physical approaches were originally used to elaborate the nanocomposite structures: (i) low energy ion beam synthesis and (ii) combined silver sputtering and plasma polymerization. These techniques allow elaboration of a single layer of AgNPs embedded in silica films at defined nanometer distances (from 0 to 7 nm) beneath the free surface. The structural and optical properties of the nanostructures were studied by transmission electron microscopy and optical reflectance. The silver release from the nanostructures after 20 h of immersion in buffered water was measured by inductively coupled plasma mass spectrometry and ranges between 0.02 and 0.49 μM. The short-term toxicity of Ag to photosynthesis of Chlamydomonas reinhardtii was assessed by fluorometry. The obtained results show that embedding AgNPs reduces the interactions with the buffered water free media, protecting the AgNPs from fast oxidation. The release of bio-available silver (impacting on the algal photosynthesis) is controlled by the depth at which AgNPs are located for a given host matrix. This provides a procedure to tailor the toxicity of nanocomposites containing AgNPs. - Highlights: • Controlled synthesis of 2D arrays of silver nanoparticles embedded in silica. • Assessing bio-available silver release using the green algae as bio-sensors. • The Ag release can be controlled by the distance nanoparticles/dielectric surface. • All the Ag released in solution is in the form of Ag{sup +} ions. • Toxicity comparable to similar concentrations of

  6. Selenite -Se(4)- uptake mechanisms in the unicellular green alga Chlamydomonas reinhardtii: bioaccumulation and effects induced on growth and ultrastructure; Mecanismes de prise en charge du selenite - Se(4)-chez l'algue verte unicellulaire Chlamydomonas reinhardtii. Bioaccumulation et effets induits sur la croissance et l'ultrastructure

    Energy Technology Data Exchange (ETDEWEB)

    Morlon, H

    2005-03-15

    Selenium is an essential element, but becomes very toxic at higher concentrations. It occurs in the environment at concentrations ranging from nM to {mu}M and selenium pollution is a worldwide phenomenon. This works aims at improving the knowledge on the interactions between selenite - Se(IV) - and a freshwater phyto-planktonic organism: the unicellular green algae Chlamydomonas reinhardtii. The aim of the performed experiments were: i) to investigate selenite -Se(IV)- uptake mechanisms in C. reinhardtii, using Se{sup 75} as a tracer in short term exposures (<1 h); ii) to assess selenite toxicity as measured with growth impairment and ultrastructural damage (with EDAX-TEM analysis), using long term exposures (96 h) to stable selenite; iii) to evaluate the bioaccumulation capacity of selenite and its potential links with toxicity. Short-term experiments revealed a negligible adsorption and a time-dependent linear absorption with an estimated absorbed flux of about 0.2 nmol.m{sup -2}.nM{sup -1}.h{sup -1}. The uptake was proportional to ambient levels in a broad range of intermediate concentrations (from nM to {mu}M). However, fluxes were higher at very low concentrations (< nM), and decrease with increasing high concentrations ( > {mu}M), suggesting that a high affinity but rapidly saturated transport mechanism could be used at low concentrations, in parallel with a low affinity mechanism that would only saturate at high concentrations ({approx}mM). The latter could involve transporters used by sulphate and nitrates, as suggested by the inhibition of selenite uptake by those element. Se(IV) speciation changes with pH did not induce significant effect on bioavailability. On the basis of the relationship between Se concentration and maximal cell density achieved, an EC50 of 80 {mu}M ([64; 98]) was derived. No adaptation mechanism were observed as the same the same toxicity was quantified for Se-pre-exposed algae. Observations by TEM suggested chloroplasts as the first

  7. The cell-wall glycoproteins of the green alga Scenedesmus obliquus. The predominant cell-wall polypeptide of Scenedesmus obliquus is related to the cell-wall glycoprotein gp3 of Chlamydomonas reinhardtii.

    Science.gov (United States)

    Voigt, Jürgen; Stolarczyk, Adam; Zych, Maria; Malec, Przemysław; Burczyk, Jan

    2014-02-01

    The green alga Scenedesmus obliquus contains a multilayered cell wall, ultrastructurally similar to that of Chlamydomonas reinhardtii, although its proportion of hydroxyproline is considerably lower. Therefore, we have investigated the polypeptide composition of the insoluble and the chaotrope-soluble wall fractions of S. obliquus. The polypeptide pattern of the chaotrope-soluble wall fraction was strongly modified by chemical deglycosylation with anhydrous hydrogen fluoride (HF) in pyridine indicating that most of these polypeptides are glycosylated. Polypeptide constituents of the chaotrope-soluble cell-wall fraction with apparent molecular masses of 240, 270, 265, and 135 kDa cross-reacted with a polyclonal antibody raised against the 100 kDa deglycosylation product of the C. reinhardtii cell-wall glycoprotein GP3B. Chemical deglycosylation of the chaotrope-soluble wall fraction resulted in a 135 kDa major polypeptide and a 106 kDa minor component reacting with the same antibody. This antibody recognized specific peptide epitopes of GP3B. When the insoluble wall fraction of S. obliquus was treated with anhydrous HF/pyridine, three polypeptides with apparent molecular masses of 144, 135, and 65 kDa were solubilized, which also occured in the deglycosylated chaotrope-soluble wall fraction. These findings indicate that theses glycoproteins are cross-linked to the insoluble wall fraction via HF-sensitive bonds. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  8. Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell

    Directory of Open Access Journals (Sweden)

    Li-Fen Huang

    2015-08-01

    Full Text Available Ferredoxins (FDX are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC; power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC.

  9. Identification and molecular characterization of a Chlamydomonas reinhardtii mutant that shows a light intensity dependent progressive chlorophyll deficiency [v1; ref status: indexed, http://f1000r.es/1b6

    Directory of Open Access Journals (Sweden)

    Phillip B Grovenstein

    2013-06-01

    Full Text Available The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms. These tetrapyrroles are synthesized via a common branched pathway that involves mainly nuclear encoded enzymes. One of the enzymes in the pathway is Mg chelatase (MgChel which inserts Mg2+ into protoporphyrin IX (PPIX, proto to form Magnesium-protoporphyrin IX (MgPPIX, Mgproto, the first biosynthetic intermediate in the Chl branch. The GUN4 (genomes uncoupled 4 protein is not essential for the MgChel activity but has been shown to significantly stimulate its activity. We have isolated a light sensitive mutant, 6F14, by random DNA insertional mutagenesis. 6F14 cannot tolerate light intensities higher than 90-100 μmol photons m-2 s-1. It shows a light intensity dependent progressive photo-bleaching. 6F14 is incapable of photo-autotrophic growth under light intensity higher than 100 μmol photons m-2 s-1. PCR based analyses show that in 6F14 the insertion of the plasmid outside the GUN4 locus has resulted in a genetic rearrangement of the GUN4 gene and possible deletions in the genomic region flanking the GUN4 gene. Our gun4 mutant has a Chl content very similar to that in the wild type in the dark and is very sensitive to fluctuations in the light intensity in the environment unlike the earlier identified Chlamydomonas gun4 mutant. Complementation with a functional copy of the GUN4 gene restored light tolerance, Chl biosynthesis and photo-autotrophic growth under high light intensities in 6F14. 6F14 is the second gun4 mutant to be identified in C. reinhardtii. Additionally, we show that our two gun4 complements over-express the GUN4 protein and show a higher Chl content per cell compared to that in the wild type strain.

  10. A rapid live-cell ELISA for characterizing antibodies against cell surface antigens of Chlamydomonas reinhardtii and its use in isolating algae from natural environments with related cell wall components.

    Science.gov (United States)

    Jiang, Wenzhi; Cossey, Sarah; Rosenberg, Julian N; Oyler, George A; Olson, Bradley J S C; Weeks, Donald P

    2014-09-25

    Cell walls are essential for most bacteria, archaea, fungi, algae and land plants to provide shape, structural integrity and protection from numerous biotic and abiotic environmental factors. In the case of eukaryotic algae, relatively little is known of the composition, structure or mechanisms of assembly of cell walls in individual species or between species and how these differences enable algae to inhabit a great diversity of environments. In this paper we describe the use of camelid antibody fragments (VHHs) and a streamlined ELISA assay as powerful new tools for obtaining mono-specific reagents for detecting individual algal cell wall components and for isolating algae that share a particular cell surface component. To develop new microalgal bioprospecting tools to aid in the search of environmental samples for algae that share similar cell wall and cell surface components, we have produced single-chain camelid antibodies raised against cell surface components of the single-cell alga, Chlamydomonas reinhardtii. We have cloned the variable-region domains (VHHs) from the camelid heavy-chain-only antibodies and overproduced tagged versions of these monoclonal-like antibodies in E. coli. Using these VHHs, we have developed an accurate, facile, low cost ELISA that uses live cells as a source of antigens in their native conformation and that requires less than 90 minutes to perform. This ELISA technique was demonstrated to be as accurate as standard ELISAs that employ proteins from cell lysates and that generally require >24 hours to complete. Among the cloned VHHs, VHH B11, exhibited the highest affinity (EC50 algae sharing cell surface components with C. reinhardtii in water samples from natural environments. In addition, mCherry-tagged VHH B11 was used along with fluorescence activated cell sorting (FACS) to select individual axenic isolates of presumed wild relatives of C. reinhardtii and other Chlorphyceae from the same environmental samples. Camelid antibody

  11. Kinetic modeling of light limitation and sulfur deprivation effects in the induction of hydrogen production with Chlamydomonas reinhardtii. Part II: Definition of model-based protocols and experimental validation.

    Science.gov (United States)

    Degrenne, B; Pruvost, J; Titica, M; Takache, H; Legrand, J

    2011-10-01

    Photosynthetic hydrogen production under light by the green microalga Chlamydomonas reinhardtii was investigated in a torus-shaped PBR in sulfur-deprived conditions. Culture conditions, represented by the dry biomass concentration of the inoculum, sulfate concentration, and incident photon flux density (PFD), were optimized based on a previously published model (Fouchard et al., 2009. Biotechnol Bioeng 102:232-245). This allowed a strictly autotrophic production, whereas the sulfur-deprived protocol is usually applied in photoheterotrophic conditions. Experimental results combined with additional information from kinetic simulations emphasize effects of sulfur deprivation and light attenuation in the PBR in inducing anoxia and hydrogen production. A broad range of PFD was tested (up to 500 µmol photons m(-2) s(-1) ). Maximum hydrogen productivities were 1.0 ± 0.2 mL H₂ /h/L (or 25 ± 5 mL H₂ /m(2) h) and 3.1 mL ± 0.4 H₂ /h L (or 77.5 ± 10 mL H₂ /m(2) h), at 110 and 500 µmol photons m(-2) s(-1) , respectively. These values approached a maximum specific productivity of approximately 1.9 mL ± 0.4 H₂ /h/g of biomass dry weight, clearly indicative of a limitation in cell capacity to produce hydrogen. The efficiency of the process and further optimizations are discussed. Copyright © 2011 Wiley Periodicals, Inc.

  12. The BSD2 ortholog in Chlamydomonas reinhardtii is a polysome-associated chaperone that co-migrates on sucrose gradients with the rbcL transcript encoding the Rubisco large subunit.

    Science.gov (United States)

    Doron, Lior; Segal, Na'ama; Gibori, Hadas; Shapira, Michal

    2014-10-01

    The expression of the CO2 -fixation enzyme ribulose-bisphosphate carboxylase/oxygenase (Rubisco), which is affected by light, involves the cysteine-rich protein bundle-sheath defective-2 (BSD2) that was originally identified in maize bundle-sheath cells. We identified the BSD2 ortholog in Chlamydomonas reinhardtii as a small protein (17 kDa) localized to the chloroplast. The algal BSD2-ortholog contains four CXXCXGXG DnaJ-like elements, but lacks the other conserved domains of DnaJ. BSD2 co-migrated with the rbcL transcript on heavy polysomes, and both BSD2 and rbcL mRNA shifted to the lighter fractions under oxidizing conditions that repress the translation of the Rubisco large subunit (RbcL). This profile of co-migration supports the possibility that BSD2 is required for the de novo synthesis of RbcL. Furthermore, BSD2 co-migrated with the rbcL transcript in a C. reinhardtii premature-termination mutant that encodes the first 60 amino acids of RbcL. In both strains, BSD2 shared its migration profile with the rbcL transcript but not with psbA mRNA. The chaperone activity of BSD2 was exemplified by its ability to prevent the aggregation of both citrate synthase (CS) and RbcL in vitro following their chemical denaturation. This activity did not depend on the presence of the thiol groups on BSD2. In contrast, the activity of BSD2 in preventing the precipitation of reduced β-chains in vitro in the insulin turbidity assay was thiol-dependent. We conclude that BSD2 combines a chaperone 'holdase' function with the ability to interact with free thiols, with both activities being required to protect newly synthesized RbcL chains. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  13. Changes in the 5'-untranslated region of the rbcL gene accelerate transcript degradation more than 50-fold in the chloroplast of Chlamydomonas reinhardtii.

    Science.gov (United States)

    Salvador, Maria Luisa; Suay, Loreto; Anthonisen, Inger Lill; Klein, Uwe

    2004-03-01

    Using uidA (beta-glucuronidase; GUS) reporter gene constructs, the 5'-untranslated region (UTR) of the Chlamydomonas chloroplast rbcL gene was screened by deletion and mutational analysis for the presence of a promoter element that previous studies implied to reside within the first 63 base pairs of the UTR. Deleting a large segment of the rbcL 5'UTR in a 3'-->5' direction to position +36, changing the remaining 36 base pairs at the 5' end of the UTR, and increasing by five base pairs the distance between the rbcL 5'UTR and the basic promoter element located at position -10 did not abolish transcription from the basic rbcL promoter. It is concluded that the apparent loss of transcriptional activity found in earlier studies after deletion of sequences downstream of the transcription initiation site is due to the synthesis of very unstable transcripts that escape detection by Northern analysis and in vivo transcription assays. Chimeric rbcL:GUS transcripts containing changes in the beginning of the 5'UTR that affect RNA secondary structure are estimated to be at least 50 times less stable than rbcL:GUS transcripts containing the non-modified rbcL 5'UTR sequence.

  14. Influence of carbon dioxide, temperature, medium kind and light intensity on the growth of algae Chlamydomonas reinhardtii and Scenedesmus obliquus

    OpenAIRE

    Olejnik Przemysław Piotr; Lewicki Andrzej; Boniecki Piotr; Lewicka Aleksandra; Janczak Damian; Czekała Wojciech

    2016-01-01

    Microalgae attracts the attention of scientists because of the possibility of using in the energy industry as one of the substrates for the production of renewable energy. So far, the greatest emphasis was put on attempts to obtain strains, and technologies of their culturing, in order to efficiently acquire fat from cells and its further conversion to biodiesel using transesterification reaction. Increasingly, algae are considered also as an efficient biomass producer, which can be used as a...

  15. An improved ARS2-derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas

    DEFF Research Database (Denmark)

    Specht, Elizabeth A; Nour-Eldin, Hussam Hassan; Hoang, Kevin T D

    2015-01-01

    The model alga Chlamydomonas reinhardtii has been used to pioneer genetic engineering techniques for high-value protein and biofuel production from algae. To date, most studies of transgenic Chlamydomonas have utilized the chloroplast genome due to its ease of engineering, with a sizeable suite...

  16. Exploring the electron transfer pathways in photosystem I by high-time-resolution electron paramagnetic resonance: observation of the B-side radical pair P700(+)A1B(-) in whole cells of the deuterated green alga Chlamydomonas reinhardtii at cryogenic temperatures.

    Science.gov (United States)

    Berthold, Thomas; von Gromoff, Erika Donner; Santabarbara, Stefano; Stehle, Patricia; Link, Gerhard; Poluektov, Oleg G; Heathcote, Peter; Beck, Christoph F; Thurnauer, Marion C; Kothe, Gerd

    2012-03-28

    Crystallographic models of photosystem I (PS I) highlight a symmetrical arrangement of the electron transfer cofactors which are organized in two parallel branches (A, B) relative to a pseudo-C2 symmetry axis that is perpendicular to the membrane plane. Here, we explore the electron transfer pathways of PS I in whole cells of the deuterated green alga Chlamydomonas reinhardtii using high-time-resolution electron paramagnetic resonance (EPR) at cryogenic temperatures. Particular emphasis is given to quantum oscillations detectable in the tertiary radical pairs P700(+)A1A(-) and P700(+)A1B(-) of the electron transfer chain. Results are presented first for the deuterated site-directed mutant PsaA-M684H in which electron transfer beyond the primary electron acceptor A0A on the PsaA branch of electron transfer is impaired. Analysis of the quantum oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the geometry of the B-side radical pair. The orientation of the g tensor of P700(+) in an external reference system is adapted from a time-resolved multifrequency EPR study of deuterated and 15N-substituted cyanobacteria (Link, G.; Berthold, T.; Bechtold, M.; Weidner, J.-U.; Ohmes, E.; Tang, J.; Poluektov, O.; Utschig, L.; Schlesselman, S. L.; Thurnauer, M. C.; Kothe, G. J. Am. Chem. Soc. 2001, 123, 4211-4222). Thus, we obtain the three-dimensional structure of the B-side radical pair following photoexcitation of PS I in its native membrane. The new structure describes the position and orientation of the reduced B-side quinone A1B(-) on a nanosecond time scale after light-induced charge separation. Furthermore, we present results for deuterated wild-type cells of C. reinhardtii demonstrating that both radical pairs P700(+)A1A(-) and P700(+)A1B(-) participate in the electron transfer process according to a mole ratio of 0.71/0.29 in favor of P700(+)A1A(-). A detailed comparison reveals different orientations of A1A(-) and A1B(-) in their

  17. Influence of sulphate on the reduction of cadmium toxicity in the microalga Chlamydomonas moewusii.

    Science.gov (United States)

    Mera, Roi; Torres, Enrique; Abalde, Julio

    2016-06-01

    Cadmium is considered as one of the most hazardous metals for living organism and ecosystems. Environmental factors play an important role since they alter the toxicity of metals by varying the bioavailability of these elements for the organisms. The aim of the present study was to investigate, using the freshwater microalga Chlamydomonas moewusii, the existence of an interaction between cadmium and sulphate as a factor that varied the toxicity of this metal. Different cell parameters such as cell growth, content of chlorophylls and biosynthesis of phytochelatins (PCs) were determined. A two-way ANOVA showed that the interaction had a significant effect size of 21% (pmicroalga and around of a 6% on the content of chlorophylls/cell. The effect of this inhibition was that when the concentration of sulphate increased, a lower toxic effect of cadmium on the growth and on the content of chlorophylls was observed. In addition, the increase of sulphate concentration allowed the biosynthesis of a higher amount of PCs and/or PCs with higher chain length. This higher biosynthesis was responsible for the reduction of the toxic effect of cadmium and explained the interaction. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Manipulating the chloroplast genome of Chlamydomonas: Present realities and future prospects

    Energy Technology Data Exchange (ETDEWEB)

    Boynton, J.; Gillham, N.; Hauser, C.; Heifetz, P.; Lers, A.; Newman, S.; Osmond, B.

    1992-01-01

    Biotechnology is being applied in vitro modification and stable reintroduction of chloroplast genes in Chlamydomonas reinhardtii and Nicotiana tabacum by homologous recombination. We are attempting the function analyses of plastid encoded proteins involved in photosynthesis, characterization of sequences which regulate expression of plastid genes at the transcriptional and translational levels, targeted disruption of chloroplast genes and molecular analysis of processes involved in chloroplast recombination.

  19. Manipulating the chloroplast genome of Chlamydomonas: Present realities and future prospects

    Energy Technology Data Exchange (ETDEWEB)

    Boynton, J.; Gillham, N.; Hauser, C.; Heifetz, P.; Lers, A.; Newman, S.; Osmond, B.

    1992-12-31

    Biotechnology is being applied in vitro modification and stable reintroduction of chloroplast genes in Chlamydomonas reinhardtii and Nicotiana tabacum by homologous recombination. We are attempting the function analyses of plastid encoded proteins involved in photosynthesis, characterization of sequences which regulate expression of plastid genes at the transcriptional and translational levels, targeted disruption of chloroplast genes and molecular analysis of processes involved in chloroplast recombination.

  20. A 3' stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo.

    Science.gov (United States)

    Stern, D B; Radwanski, E R; Kindle, K L

    1991-01-01

    The Chlamydomonas reinhardtii chloroplast atpB mRNA contains sequences at its 3' end that can form a complex stem/loop structure. Deletions of part or all of this sequence in transformed C. reinhardtii cells led to decreased atpB mRNA accumulation, whereas transcription rates were unaffected. The reduction of mRNA to 20% to 35% of wild-type levels in transformants without 3' stem/loops was correlated with the accumulation of atpB mRNA that was heterogeneous in size. These results indicated that RNA secondary structures function both in mRNA stabilization and in 3' end formation in C. reinhardtii chloroplasts. Furthermore, deletion of the stem/loop resulted in a decrease in the steady-state level of the ATPase beta-subunit to approximately 60% of wild-type levels, suggesting that translational and/or post-translational mechanisms may influence the steady-state level of the atpB gene product. PMID:1840911

  1. The ARG9 Gene Encodes the Plastid-Resident N-Acetyl Ornithine Aminotransferase in the Green Alga Chlamydomonas reinhardtii▿

    OpenAIRE

    Remacle, Claire; Cline, Sara; Boutaffala, Layla; Gabilly, Stéphane; Larosa, Véronique; Barbieri, M. Rosario; Coosemans, Nadine; Hamel, Patrice P.

    2009-01-01

    Here we report the characterization of the Chlamydomonas reinhardtii gene ARG9, encoding the plastid resident N-acetyl ornithine aminotransferase, which is involved in arginine synthesis. Integration of an engineered ARG9 cassette in the plastid chromosome of the nuclear arg9 mutant restores arginine prototrophy. This suggests that ARG9 could be used as a new selectable marker for plastid transformation.

  2. Photosynthetic efficiency of Chlamydomonas reinhardtii in flashing light

    NARCIS (Netherlands)

    Vejrazka, C.; Janssen, M.G.J.; Streefland, M.; Wijffels, R.H.

    2011-01-01

    Efficient light to biomass conversion in photobioreactors is crucial for economically feasible microalgae production processes. It has been suggested that photosynthesis is enhanced in short light path photobioreactors by mixing-induced flashing light regimes. In this study, photosynthetic

  3. Metabolic responses to ethanol and butanol in Chlamydomonas reinhardtii

    OpenAIRE

    Jiang, Yongguang; Xiao, Peng; Shao, Qing; Qin, Huan; Hu, Zhangli; Lei, Anping; Wang, Jiangxin

    2017-01-01

    Background Microalgae have been demonstrated to be among the most promising phototrophic species for producing renewable biofuels and chemicals. Ethanol and butanol are clean energy sources with good chemical and physical properties as alternatives to gasoline. However, biosynthesis of these two biofuels has not been achieved due to low tolerance of algal cells to ethanol or butanol. Results With an eye to circumventing these problems in the future and engineering the robust alcohol-producing...

  4. UVI31+ is a DNA endonuclease that dynamically localizes to chloroplast pyrenoids in C. reinhardtii.

    Directory of Open Access Journals (Sweden)

    Manish Shukla

    Full Text Available UVI31+ is an evolutionarily conserved BolA family protein. In this study we examine the presence, localization and possible functions of this protein in the context of a unicellular alga, Chlamydomonas reinhardtii. UVI31+ in C. reinhardtii exhibits DNA endonuclease activity and is induced upon UV stress. Further, UVI31+ that normally localizes to the cell wall and pyrenoid regions gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV stress. The observed induction upon UV-stress as well as the endonuclease activity suggests plausible role of this protein in DNA repair. We have also observed that UV31+ is induced in C. reinhardtii grown in dark conditions, whereby the protein localization is enhanced in the pyrenoid. Biomolecular interaction between the purified pyrenoids and UVI31+ studied by NMR demonstrates the involvement of the disordered loop domain of the protein in its interaction.

  5. Total internal reflection fluorescence (TIRF) microscopy of Chlamydomonas flagella.

    Science.gov (United States)

    Engel, Benjamin D; Lechtreck, Karl-Ferdinand; Sakai, Tsuyoshi; Ikebe, Mitsuo; Witman, George B; Marshall, Wallace F

    2009-01-01

    The eukaryotic flagellum is host to a variety of dynamic behaviors, including flagellar beating, the motility of glycoproteins in the flagellar membrane, and intraflagellar transport (IFT), the bidirectional traffic of protein particles between the flagellar base and tip. IFT is of particular interest, as it plays integral roles in flagellar length control, cell signaling, development, and human disease. However, our ability to understand dynamic flagellar processes such as IFT is limited in large part by the fidelity with which we can image these behaviors in living cells. This chapter introduces the application of total internal reflection fluorescence (TIRF) microscopy to visualize the flagella of Chlamydomonas reinhardtii. The advantages and challenges of TIRF are discussed in comparison to confocal and differential interference contrast techniques. This chapter also reviews current IFT insights gleaned from TIRF microscopy of Chlamydomonas and provides an outlook on the future of the technique, with particular emphasis on combining TIRF with other emerging imaging technologies. 2009 Elsevier Inc. All rights reserved.

  6. MEETING: Chlamydomonas Annotation Jamboree - October 2003

    Energy Technology Data Exchange (ETDEWEB)

    Grossman, Arthur R

    2007-04-13

    Shotgun sequencing of the nuclear genome of Chlamydomonas reinhardtii (Chlamydomonas throughout) was performed at an approximate 10X coverage by JGI. Roughly half of the genome is now contained on 26 scaffolds, all of which are at least 1.6 Mb, and the coverage of the genome is ~95%. There are now over 200,000 cDNA sequence reads that we have generated as part of the Chlamydomonas genome project (Grossman, 2003; Shrager et al., 2003; Grossman et al. 2007; Merchant et al., 2007); other sequences have also been generated by the Kasuza sequence group (Asamizu et al., 1999; Asamizu et al., 2000) or individual laboratories that have focused on specific genes. Shrager et al. (2003) placed the reads into distinct contigs (an assemblage of reads with overlapping nucleotide sequences), and contigs that group together as part of the same genes have been designated ACEs (assembly of contigs generated from EST information). All of the reads have also been mapped to the Chlamydomonas nuclear genome and the cDNAs and their corresponding genomic sequences have been reassembled, and the resulting assemblage is called an ACEG (an Assembly of contiguous EST sequences supported by genomic sequence) (Jain et al., 2007). Most of the unique genes or ACEGs are also represented by gene models that have been generated by the Joint Genome Institute (JGI, Walnut Creek, CA). These gene models have been placed onto the DNA scaffolds and are presented as a track on the Chlamydomonas genome browser associated with the genome portal (http://genome.jgi-psf.org/Chlre3/Chlre3.home.html). Ultimately, the meeting grant awarded by DOE has helped enormously in the development of an annotation pipeline (a set of guidelines used in the annotation of genes) and resulted in high quality annotation of over 4,000 genes; the annotators were from both Europe and the USA. Some of the people who led the annotation initiative were Arthur Grossman, Olivier Vallon, and Sabeeha Merchant (with many individual

  7. The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions

    Energy Technology Data Exchange (ETDEWEB)

    Merchant, Sabeeha S

    2007-04-09

    Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

  8. The Chlamydomonas CO2 -concentrating mechanism and its potential for engineering photosynthesis in plants.

    Science.gov (United States)

    Mackinder, Luke C M

    2018-01-01

    Contents Summary I. Introduction 54 II. Recent advances in our understanding of the Chlamydomonas CCM 55 III. Current gaps in our understanding of the Chlamydomonas CCM 58 IV. Approaches to rapidly advance our understanding of the Chlamydomonas CCM 58 V. Engineering a CCM into higher plants 58 VI. Conclusion and outlook 59 Acknowledgements 60 References 60 SUMMARY: To meet the food demands of a rising global population, innovative strategies are required to increase crop yields. Improvements in plant photosynthesis by genetic engineering show considerable potential towards this goal. One prospective approach is to introduce a CO2 -concentrating mechanism into crop plants to increase carbon fixation by supplying the central carbon-fixing enzyme, Rubisco, with a higher concentration of its substrate, CO2 . A promising donor organism for the molecular machinery of this mechanism is the eukaryotic alga Chlamydomonas reinhardtii. This review summarizes the recent advances in our understanding of carbon concentration in Chlamydomonas, outlines the most pressing gaps in our knowledge and discusses strategies to transfer a CO2 -concentrating mechanism into higher plants to increase photosynthetic performance. © 2017 The Author. New Phytologist © 2017 New Phytologist Trust.

  9. An optimized, chemically regulated gene expression system for Chlamydomonas.

    Directory of Open Access Journals (Sweden)

    Paola Ferrante

    Full Text Available BACKGROUND: Chlamydomonas reinhardtii is a model system for algal and cell biology and is used for biotechnological applications, such as molecular farming or biological hydrogen production. The Chlamydomonas metal-responsive CYC6 promoter is repressed by copper and induced by nickel ions. However, induction by nickel is weak in some strains, poorly reversible by chelating agents like EDTA, and causes, at high concentrations, toxicity side effects on Chlamydomonas growth. Removal of these bottlenecks will encourage the wide use of this promoter as a chemically regulated gene expression system. METHODOLOGY: Using a codon-optimized Renilla luciferase as a reporter gene, we explored several strategies to improve the strength and reversibility of CYC6 promoter induction. Use of the first intron of the RBCS2 gene or of a modified TAP medium increases the strength of CYC6 induction up to 20-fold. In the modified medium, induction is also obtained after addition of specific copper chelators, like TETA. At low concentrations (up to 10 microM TETA is a more efficient inducer than Ni, which becomes a very efficient inducer at higher concentrations (50 microM. Neither TETA nor Ni show toxicity effects at the concentrations used. Unlike induction by Ni, induction by TETA is completely reversible by micromolar copper concentrations, thus resulting in a transient "wave" in luciferase activity, which can be repeated in subsequent growth cycles. CONCLUSIONS: We have worked out a chemically regulated gene expression system that can be finely tuned to produce temporally controlled "waves" in gene expression. The use of cassettes containing the CYC6 promoter, and of modified growth media, is a reliable and economically sustainable system for the temporally controlled expression of foreign genes in Chlamydomonas.

  10. Function of the chloroplast hydrogenase in the microalga Chlamydomonas: the role of hydrogenase and state transitions during photosynthetic activation in anaerobiosis.

    Directory of Open Access Journals (Sweden)

    Bart Ghysels

    Full Text Available Like a majority of photosynthetic microorganisms, the green unicellular alga Chlamydomonas reinhardtii may encounter O2 deprived conditions on a regular basis. In response to anaerobiosis or in a respiration defective context, the photosynthetic electron transport chain of Chlamydomonas is remodeled by a state transition process to a conformation that favours the photoproduction of ATP at the expense of reductant synthesis. In some unicellular green algae including Chlamydomonas, anoxia also triggers the induction of a chloroplast-located, oxygen sensitive hydrogenase, which accepts electrons from reduced ferredoxin to convert protons into molecular hydrogen. Although microalgal hydrogen evolution has received much interest for its biotechnological potential, its physiological role remains unclear. By using specific Chlamydomonas mutants, we demonstrate that the state transition ability and the hydrogenase function are both critical for induction of photosynthesis in anoxia. These two processes are thus important for survival of the cells when they are transiently placed in an anaerobic environment.

  11. The HSP70 chaperone machines of Chlamydomonas are induced by cold stress.

    Science.gov (United States)

    Maikova, Anna; Zalutskaya, Zhanneta; Lapina, Tatiana; Ermilova, Elena

    2016-10-01

    The responses of Chlamydomonas reinhardtii cells to low temperatures have not been extensively studied compared with other stresses. Like other organisms, this green alga has heat shock protein 70s (HSP70s) that are located in chloroplast, mitochondrion and cytosol. To test whether temperature downshifts affected HSP70s synthesis, we used real-time PCR and protein gel blot analysis. C. reinhardtii cells exposed to cold stress show increased HSP70s mRNA levels. Genes encoding other components of HSP70 chaperone machines (e.g. CGE1, CDJ1, HSP90C and HSP90A) are also up-regulated in response to decreased temperature. We demonstrated that the accumulation of all analyzed mRNA occur more slowly and with reduced amplitude in cells exposed to cold than in cells treated with heat. Furthermore, C. reinhardtii cells display the splicing of the CGE1 transcript that was dependent on low temperature. Finally, the transcription regulator of C. reinhardtii HSF1 is also cold-responsive, suggesting its role in the transcriptional regulation of HSP genes at low temperature. Copyright © 2016 Elsevier GmbH. All rights reserved.

  12. A Chlamydomonas-derived Human Papillomavirus 16 E7 vaccine induces specific tumor protection.

    Directory of Open Access Journals (Sweden)

    Olivia C Demurtas

    Full Text Available BACKGROUND: The E7 protein of the Human Papillomavirus (HPV type 16, being involved in malignant cellular transformation, represents a key antigen for developing therapeutic vaccines against HPV-related lesions and cancers. Recombinant production of this vaccine antigen in an active form and in compliance with good manufacturing practices (GMP plays a crucial role for developing effective vaccines. E7-based therapeutic vaccines produced in plants have been shown to be active in tumor regression and protection in pre-clinical models. However, some drawbacks of in whole-plant vaccine production encouraged us to explore the production of the E7-based therapeutic vaccine in Chlamydomonas reinhardtii, an organism easy to grow and transform and fully amenable to GMP guidelines. METHODOLOGY/PRINCIPAL FINDINGS: An expression cassette encoding E7GGG, a mutated, attenuated form of the E7 oncoprotein, alone or as a fusion with affinity tags (His6 or FLAG, under the control of the C. reinhardtii chloroplast psbD 5' UTR and the psbA 3' UTR, was introduced into the C. reinhardtii chloroplast genome by homologous recombination. The protein was mostly soluble and reached 0.12% of total soluble proteins. Affinity purification was optimized and performed for both tagged forms. Induction of specific anti-E7 IgGs and E7-specific T-cell proliferation were detected in C57BL/6 mice vaccinated with total Chlamydomonas extract and with affinity-purified protein. High levels of tumor protection were achieved after challenge with a tumor cell line expressing the E7 protein. CONCLUSIONS: The C. reinhardtii chloroplast is a suitable expression system for the production of the E7GGG protein, in a soluble, immunogenic form. The production in contained and sterile conditions highlights the potential of microalgae as alternative platforms for the production of vaccines for human uses.

  13. Radiocesium bioaccumulation in freshwater plankton: Influences of cation concentrations (K{sup +} and Na{sup +}) on direct uptake of {sup 137}Cs in Chlamydomonas, Scenedesmus and Daphnia. Food-chain transfer of {sup 137}Cs from Chlamydomonas to Daphnia at different K{sup +} concentrations

    Energy Technology Data Exchange (ETDEWEB)

    Hagstroem, J. [Uppsala Univ., Dept. of Limnology, Uppsala (Sweden)

    2002-04-01

    The influences of cation concentrations (K{sup +} and Na{sup +}) on radiocesium ({sup 137}Cs) bioaccumulation in two freshwater phytoplankton species (Scenedesmus quadricauda and Chlamydomonas noctigama) were systematically investigated in batch-cultures monitored during two weeks. Both species were cultured at 9 {mu}E M{sup -2} s{sup -1} constant illumination at 20 deg. C. The exponential growth phase lasted for more than 100 hours ({mu} {approx_equal} 0.02 h{sup -1} for C. noctigama and 0.03 h{sup -1} for S, quadricauda). Over cation concentration ranges encountered in natural fresh waters ([K{sup +}] from 0.1 {mu}M to 3 mM, [Na{sup +}] from 20 {mu}M to 3 mM), a more than three order of magnitude variation was found for both intake rate and observed bioconcentration factors (BCF) at apparent steady-state (from less than 10{sup 3} to 10{sup 6} L (kg C){sup -1}). For both species, the major effector on BCF and uptake rate was external [K{sup +}], which was inversely proportional to these parameters over wide ranges (1-1000 {mu}M for S. quadricauda and 0.1 to 300 {mu}M for C. noctigama). At concentrations above these ranges K{sup +} still reduced {sup 137} Cs bio-uptake, but less effectively. A minor influence of external [Na{sup +}] on {sup 137}Cs bioaccumulation was indicated for S. quadricauda, whereas no such influence was significant for C. noctigama. A biphasic pattern for {sup 137}Cs bioaccumulation was discovered in C. noctigama. A rapid 'quasi-steady state' with an effective equilibration time of less than 100 hours was approached during the exponential growth phase. A surge in the uptake occurred when exponential growth ceased, and this pattern was consistent over the range 30 {mu}M to 1.4 mM external [K{sup +}]. Since depletion of external [K{sup +}] was not detected for these treatments, this pattern can only be explained if there are at least two different cellular compartments involved. Although less certain, a second steady-state BCF

  14. Species-specific differences of the spectroscopic properties of P700 - Analysis of the influence of non-conserved amino acid residues by site-directed mutagenesis of photosystem I from Chlamydomonas reinhardtii

    NARCIS (Netherlands)

    Witt, H.; Bordignon, E.; Carbonera, D.; Dekker, J.P.; Karapetyan, N.; Teutloff, C.; Webber, A.; Lubitz, W.; Schlodder, E.

    2003-01-01

    We applied optical spectroscopy, magnetic resonance techniques, and redox titrations to investigate the properties of the primary electron donor P700 in photosystem I (PS I) core complexes from cyanobacteria (Thermosynechococcus elongatus, Spirulina platensis, and Synechocystis sp. PCC 6803), algae

  15. Carbon dioxide fixation and photoevolution of hydrogen and oxygen in a mutant of Chlamydomonas lacking Photosystem I

    Energy Technology Data Exchange (ETDEWEB)

    Greenbaum, E.; Lee, J.W.; Tevault, C.V. [Oak Ridge National Lab., TN (United States)] [and others

    1995-09-01

    Sustained photoassimilation of atmospheric CO{sub 2} and simultaneous photoevolution of molecular hydrogen and oxygen has been observed in a Photosystem I deficient mutant B4 of Chlamydomonas reinhardtii that contains only Photosystem II. The data indicate that Photosystem II alone is capable of spanning the potential difference between water oxidation/oxygen evolution and ferredoxin reduction. The rates of both CO{sub 2} fixation and hydrogen and oxygen evolution are similar in the mutant to that of the wild-type C. reinhardtii 137c containing both photosystems. The wild-type had stable photosynthetic activity, measured as CO{sub 2} fixation, under both air and anaerobic conditions, while the mutant was stable only under anaerobic conditions. The results are discussed in terms of the fundamental mechanisms and energetics of photosynthesis and possible implications for the evolution of oxygenic photosynthesis.

  16. Engineering the chloroplast targeted malarial vaccine antigens in Chlamydomonas starch granules.

    Directory of Open Access Journals (Sweden)

    David Dauvillée

    Full Text Available BACKGROUND: Malaria, an Anopheles-borne parasitic disease, remains a major global health problem causing illness and death that disproportionately affects developing countries. Despite the incidence of malaria, which remains one of the most severe infections of human populations, there is no licensed vaccine against this life-threatening disease. In this context, we decided to explore the expression of Plasmodium vaccine antigens fused to the granule bound starch synthase (GBSS, the major protein associated to the starch matrix in all starch-accumulating plants and algae such as Chlamydomonas reinhardtii. METHODS AND FINDINGS: We describe the development of genetically engineered starch granules containing plasmodial vaccine candidate antigens produced in the unicellular green algae Chlamydomonas reinhardtii. We show that the C-terminal domains of proteins from the rodent Plasmodium species, Plasmodium berghei Apical Major Antigen AMA1, or Major Surface Protein MSP1 fused to the algal granule bound starch synthase (GBSS are efficiently expressed and bound to the polysaccharide matrix. Mice were either immunized intraperitoneally with the engineered starch particles and Freund adjuvant, or fed with the engineered particles co-delivered with the mucosal adjuvant, and challenged intraperitoneally with a lethal inoculum of P. Berghei. Both experimental strategies led to a significantly reduced parasitemia with an extension of life span including complete cure for intraperitoneal delivery as assessed by negative blood thin smears. In the case of the starch bound P. falciparum GBSS-MSP1 fusion protein, the immune sera or purified immunoglobulin G of mice immunized with the corresponding starch strongly inhibited in vitro the intra-erythrocytic asexual development of the most human deadly plasmodial species. CONCLUSION: This novel system paves the way for the production of clinically relevant plasmodial antigens as algal starch-based particles

  17. Protein (Viridiplantae): 159463846 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 2036 predicted protein Chlamydomonas reinhardtii MRRLQVCACCAGAWRLVRHGGGWRLGVCQRAMKACASLFLHASTRTVSRCMPACVRPATQCDQSTGMHVNRKRDCAFIMYSKGSAGKSTARWGAARSRQAAHVYAALCLCRSELGPRPLTCCRGYRQTP ...

  18. Protein (Viridiplantae): 159468077 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available hypothetical protein CHLREDRAFT_171030 Chlamydomonas reinhardtii MWATKLEAQLQLMFMPTRLHRRPLHQGTCRNYSTAPGITGVIELTSAFYRMYPNATFVFNKETAAKGTYRGEEETAASWWLKHVGSKLEIYLSPLLGLWAMSPPGPSGAGTR ...

  19. Protein (Viridiplantae): 159470305 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available predicted protein Chlamydomonas reinhardtii MSSRPKRAASANMANVIAAEKANKAAALHAWPKMWATKLEAQLQLMFMPTRLHRRPLHQGTCRNYSTAPGITGVIELTSAFYRMYPNATFVFNKETAAKGTYRGEEETAASWWLKHVGSKLEIYLSPLRCRPEVSR ...

  20. Protein (Viridiplantae): 159472102 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 4474 predicted protein, partial Chlamydomonas reinhardtii PPSPAPPSPEPGSPPPSPAPPSPQPPSPAPPSPEPGSPPPSPAPPSPKPPSPAPPSPEQPGSPPPSPPPPRPQPPSPAPPSPEPGSPPPSPAPPSPQPPSPAPPSPEPGSPPPSPAPTQP ...

  1. Protein (Viridiplantae): 159488149 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 8105 predicted protein Chlamydomonas reinhardtii MSTSGLLFQRRSVTAATYKRSSNRQTRLNVVAFGGQQGAAPEHAARARTTPQASMAASTMPGPQGAELGNWLRQLDLFFSKSRDTRSLSEISDFNMSDEDHDDDHASHMYVSHLAARMAMEPLPGRE ...

  2. A transient receptor potential ion channel in Chlamydomonas shares key features with sensory transduction-associated TRP channels in mammals.

    Science.gov (United States)

    Arias-Darraz, Luis; Cabezas, Deny; Colenso, Charlotte K; Alegría-Arcos, Melissa; Bravo-Moraga, Felipe; Varas-Concha, Ignacio; Almonacid, Daniel E; Madrid, Rodolfo; Brauchi, Sebastian

    2015-01-01

    Sensory modalities are essential for navigating through an ever-changing environment. From insects to mammals, transient receptor potential (TRP) channels are known mediators for cellular sensing. Chlamydomonas reinhardtii is a motile single-celled freshwater green alga that is guided by photosensory, mechanosensory, and chemosensory cues. In this type of alga, sensory input is first detected by membrane receptors located in the cell body and then transduced to the beating cilia by membrane depolarization. Although TRP channels seem to be absent in plants, C. reinhardtii possesses genomic sequences encoding TRP proteins. Here, we describe the cloning and characterization of a C. reinhardtii version of a TRP channel sharing key features present in mammalian TRP channels associated with sensory transduction. In silico sequence-structure analysis unveiled the modular design of TRP channels, and electrophysiological experiments conducted on Human Embryonic Kidney-293T cells expressing the Cr-TRP1 clone showed that many of the core functional features of metazoan TRP channels are present in Cr-TRP1, suggesting that basic TRP channel gating characteristics evolved early in the history of eukaryotes. © 2015 American Society of Plant Biologists. All rights reserved.

  3. An improved ARS2-derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas.

    Science.gov (United States)

    Specht, Elizabeth A; Nour-Eldin, Hussam Hassan; Hoang, Kevin T D; Mayfield, Stephen P

    2015-03-01

    The model alga Chlamydomonas reinhardtii has been used to pioneer genetic engineering techniques for high-value protein and biofuel production from algae. To date, most studies of transgenic Chlamydomonas have utilized the chloroplast genome due to its ease of engineering, with a sizeable suite of reporters and well-characterized expression constructs. The advanced manipulation of algal nuclear genomes has been hampered by limited strong expression cassettes, and a lack of high-throughput reporters. We have improved upon an endogenous reporter gene - the ARS2 gene encoding an arylsulfatase enzyme - that was first cloned and characterized decades ago but has not been used extensively. The new construct, derived from ARS2 cDNA, expresses significantly higher levels of reporter protein and transforms more efficiently, allowing qualitative and quantitative screening using a rapid, inexpensive 96-well assay. The improved arylsulfatase expression cassette was used to screen a new transgene promoter from the ARG7 gene, and found that the ARG7 promoter can express the ARS2 reporter as strongly as the HSP70-RBCS2 chimeric promoter that currently ranks as the best available promoter, thus adding to the list of useful nuclear promoters. This enhanced arylsulfatase reporter construct improves the efficiency and ease of genetic engineering within the Chlamydomonas nuclear genome, with potential application to other algal strains. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Chlamydomonas as a model for biofuels and bio-products production.

    Science.gov (United States)

    Scranton, Melissa A; Ostrand, Joseph T; Fields, Francis J; Mayfield, Stephen P

    2015-05-01

    Developing renewable energy sources is critical to maintaining the economic growth of the planet while protecting the environment. First generation biofuels focused on food crops like corn and sugarcane for ethanol production, and soybean and palm for biodiesel production. Second generation biofuels based on cellulosic ethanol produced from terrestrial plants, has received extensive funding and recently pilot facilities have been commissioned, but to date output of fuels from these sources has fallen well short of what is needed. Recent research and pilot demonstrations have highlighted the potential of algae as one of the most promising sources of sustainable liquid transportation fuels. Algae have also been established as unique biofactories for industrial, therapeutic, and nutraceutical co-products. Chlamydomonas reinhardtii's long established role in the field of basic research in green algae has paved the way for understanding algal metabolism and developing genetic engineering protocols. These tools are now being utilized in C. reinhardtii and in other algal species for the development of strains to maximize biofuels and bio-products yields from the lab to the field. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  5. Photosynthetic efficiency and oxygen evolution of Chlamydomonas reinhardtii under continuous and flashing light.

    NARCIS (Netherlands)

    Vejrazka, C.; Janssen, M.; Benvenuti, G.; Streefland, M.; Wijffels, R.H.

    2013-01-01

    As a result of mixing and light attenuation in a photobioreactor (PBR), microalgae experience light/dark (L/D) cycles that can enhance PBR efficiency. One parameter which characterizes L/D cycles is the duty cycle; it determines the time fraction algae spend in the light. The objective of this study

  6. Purification of the second enzyme of chlorophyll biosynthesis from Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Krishnasamy, S.; Wang, Weiyeh (Univ. of Iowa, Iowa City (USA))

    1990-05-01

    Delta-aminolevulinic acid (ALA) is the universal precursor for the synthesis of chlorophylls and tetrapyrroles. The first enzyme of the pathway activates glutamate by ligating it to its cognate tRNA. The second enzyme is a dehydrogenase which converts the activated glutamate on glu-tRNA to glutamate-1-semialdehyde (GSA). We now report the purification of the second enzyme, GSA dehydrogenase by using ammonium sulfate fractionation and column chromatography with DEAE Sepharose, Blue Sepharose and 2{prime},5{prime}-ADP Agrose. The native molecular weight of this enzyme, as determined by HPLC, was 130 KD. We have developed a specific assay for the dehydrogenase by measuring the formation of labeled GSA from ({sup 3}H)-glu-tRNA. The efficiency of glu-tRNA to GSA conversion by the dehydrogenase was low. However, this rate could be increased many fold by the addition of glu-tRNA synthetase, glutamate and ATP. We think under in vivo conditions, the first two enzymes of ALA biosynthesis may form a complex which operates much more efficiently.

  7. Efficiency of light utilization of Chlamydomonas reinhardtii under medium-duration light/dark cycles

    NARCIS (Netherlands)

    Janssen, M.; Winter, de M.; Tramper, J.; Mur, L.R.; Snel, J.; Wijffels, R.H.

    2000-01-01

    The light regime inside a photobioreactor is characterized by a light gradient with full (sun)light at the light-exposed surface and darkness in the interior of the bioreactor. Consequently, depending on the mixing characteristics, algae will be exposed to certain light/dark cycles. In this study

  8. Efficient expression of nuclear transgenes in the green alga Chlamydomonas: synthesis of an HIV antigen and development of a new selectable marker.

    Science.gov (United States)

    Barahimipour, Rouhollah; Neupert, Juliane; Bock, Ralph

    2016-03-01

    The unicellular green alga Chlamydomonas reinhardtii has become an invaluable model system in plant biology. There is also considerable interest in developing this microalga into an efficient production platform for biofuels, pharmaceuticals, green chemicals and industrial enzymes. However, the production of foreign proteins in the nucleocytosolic compartment of Chlamydomonas is greatly hampered by the inefficiency of transgene expression from the nuclear genome. We have recently addressed this limitation by isolating mutant algal strains that permit high-level transgene expression and by determining the contributions of GC content and codon usage to gene expression efficiency. Here we have applied these new tools and explored the potential of Chlamydomonas to produce a recombinant biopharmaceutical, the HIV antigen P24. We show that a codon-optimized P24 gene variant introduced into our algal expression strains give rise to recombinant protein accumulation levels of up to 0.25% of the total cellular protein. Moreover, in combination with an expression strain, a resynthesized nptII gene becomes a highly efficient selectable marker gene that facilitates the selection of transgenic algal clones at high frequency. By establishing simple principles of successful transgene expression, our data open up new possibilities for biotechnological research in Chlamydomonas.

  9. CSL encodes a leucine-rich-repeat protein implicated in red/violet light signaling to the circadian clock in Chlamydomonas.

    Directory of Open Access Journals (Sweden)

    Ayumi Kinoshita

    2017-03-01

    Full Text Available The green alga Chlamydomonas reinhardtii shows various light responses in behavior and physiology. One such photoresponse is the circadian clock, which can be reset by external light signals to entrain its oscillation to daily environmental cycles. In a previous report, we suggested that a light-induced degradation of the clock protein ROC15 is a trigger to reset the circadian clock in Chlamydomonas. However, light signaling pathways of this process remained unclear. Here, we screened for mutants that show abnormal ROC15 diurnal rhythms, including the light-induced protein degradation at dawn, using a luciferase fusion reporter. In one mutant, ROC15 degradation and phase resetting of the circadian clock by light were impaired. Interestingly, the impairments were observed in response to red and violet light, but not to blue light. We revealed that an uncharacterized gene encoding a protein similar to RAS-signaling-related leucine-rich repeat (LRR proteins is responsible for the mutant phenotypes. Our results indicate that a previously uncharacterized red/violet light signaling pathway is involved in the phase resetting of circadian clock in Chlamydomonas.

  10. THE ECOLOGY AND GENETICS OF FITNESS IN CHLAMYDOMONAS. IV. THE PROPERTIES OF MIXTURES OF GENOTYPES OF THE SAME SPECIES.

    Science.gov (United States)

    Bell, Graham

    1991-06-01

    The properties of mixtures of genotypes of Chlamydomonas reinhardtii were investigated by growing them in monoculture and in all possible pairwise combinations in chemically defined axenic medium. Two sets of genotypes produced by crossing wild-type isolates were cultured in each of two physical environments. Mixtures were consistently more productive and less variable over environments than were their constituent monocultures. The average performance of a genotype in mixture was tightly correlated with its performance in monoculture. Reisolation of spores from mixtures at the end of growth showed that the mixtures became dominated by the component with the greater performance in monoculture, so that the properties of mixtures were attributable to replacement rather than to complementation. These results differ from those of similar trials using a range of different species of Chlamydomonas, where genetic interactions were found to be important. They are discussed in relation to theories of diversity and diversification, and related to the agronomic use of crop mixtures. © 1991 The Society for the Study of Evolution.

  11. Chlamydomonas as a model for biofuels and bio-products production

    Science.gov (United States)

    Scranton, Melissa A.; Ostrand, Joseph T.; Fields, Francis J.; Mayfield, Stephen P.

    2017-01-01

    SUMMARY Developing renewable energy sources is critical to maintaining the economic growth of the planet while protecting the environment. First generation biofuels focused on food crops like corn and sugarcane for ethanol production, and soybean and palm for biodiesel production. Second generation biofuels based on cellulosic ethanol produced from terrestrial plants, has received extensive funding and recently pilot facilities have been commissioned, but to date output of fuels from these sources has fallen well short of what is needed. Recent research and pilot demonstrations have highlighted the potential of algae as one of the most promising sources of sustainable liquid transportation fuels. Algae have also been established as unique biofactories for industrial, therapeutic, and nutraceutical co-products. Chlamydomonas reinhardtii’s long established role in the field of basic research in green algae has paved the way for understanding algal metabolism and developing genetic engineering protocols. These tools are now being utilized in C. reinhardtii and in other algal species for the development of strains to maximize biofuels and bio-products yields from the lab to the field. PMID:25641390

  12. Chlorophyll a is a favorable substrate for Chlamydomonas Mg-dechelatase encoded by STAY-GREEN.

    Science.gov (United States)

    Matsuda, Kaori; Shimoda, Yousuke; Tanaka, Ayumi; Ito, Hisashi

    2016-12-01

    Mg removal from chlorophyll by Mg-dechelatase is the first step of chlorophyll degradation. Recent studies showed that in Arabidopsis, Stay Green (SGR) encodes Mg-dechelatase. Though the Escherichia coli expression system is advantageous for investigating the properties of Mg-dechelatase, Arabidopsis Mg-dechelatase is not successfully expressed in E. coli. Chlamydomonas reinhardtii SGR (CrSGR) has a long, hydrophilic tail, suggesting that active CrSGR can be expressed in E. coli. After the incubation of chlorophyll a with CrSGR expressed in E. coli, pheophytin a accumulated, indicating that active CrSGR was expressed in E. coli. Substrate specificity of CrSGR against chlorophyll b and an intermediate molecule of the chlorophyll b degradation pathway was examined. CrSGR exhibited no activity against chlorophyll b and low activity against 7-hydroxymethyl chlorophyll a, consistent with the fact that chlorophyll b is degraded only after conversion to chlorophyll a. CrSGR exhibited low activity against divinyl chlorophyll a and chlorophyll a', and no activity against chlorophyllide a, protochlorophyll a, chlorophyll c2, and Zn-chlorophyll a. These observations indicate that chlorophyll a is the most favorable substrate for CrSGR. When CrSGR was expressed in Arabidopsis cells, the chlorophyll content decreased, further confirming that SGR has Mg-dechelating activity in chloroplasts. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  13. Efficient targeting of recombinant proteins to the thylakoid lumen in Chlamydomonas reinhardtii using a bacterial Tat signal peptide

    DEFF Research Database (Denmark)

    Zedler, Julie A Z; Mullineaux, Conrad W.; Robinson, Colin

    2016-01-01

    Interest in the exploitation of microalgae for biotechnological applications has increased over the last decade, and microalgae are now viewed as offering a sustainable alternative to traditionally used host chassis. A number of recombinant proteins have been expressed in genetically modified algal...

  14. Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii

    National Research Council Canada - National Science Library

    Rasala, Beth A; Muto, Machiko; Lee, Philip A; Jager, Michal; Cardoso, Rosa M F; Behnke, Craig A; Kirk, Peter; Hokanson, Craig A; Crea, Roberto; Mendez, Michael; Mayfield, Stephen P

    2010-01-01

    Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals...

  15. Mutations of Photosystem II D1 Protein That Empower Efficient Phenotypes of Chlamydomonas reinhardtii under Extreme Environment in Space

    OpenAIRE

    Maria Teresa Giardi; Giuseppina Rea; Lambreva, Maya D.; Amina Antonacci; Sandro Pastorelli; Ivo Bertalan; Udo Johanningmeier; Mattoo, Autar K.

    2013-01-01

    Space missions have enabled testing how microorganisms, animals and plants respond to extra-terrestrial, complex and hazardous environment in space. Photosynthetic organisms are thought to be relatively more prone to microgravity, weak magnetic field and cosmic radiation because oxygenic photosynthesis is intimately associated with capture and conversion of light energy into chemical energy, a process that has adapted to relatively less complex and contained environment on Earth. To study the...

  16. Mutations of photosystem II D1 protein that empower efficient phenotypes of Chlamydomonas reinhardtii under extreme environment in space

    National Research Council Canada - National Science Library

    Giardi, Maria Teresa; Rea, Giuseppina; Lambreva, Maya D; Antonacci, Amina; Pastorelli, Sandro; Bertalan, Ivo; Johanningmeier, Udo; Mattoo, Autar K

    2013-01-01

    .... To study the direct effect of the space environment on the fundamental process of photosynthesis, we sent into low Earth orbit space engineered and mutated strains of the unicellular green alga...

  17. MRL1, a conserved Pentatricopeptide repeat protein, is required for stabilization of rbcL mRNA in Chlamydomonas and Arabidopsis.

    Science.gov (United States)

    Johnson, Xenie; Wostrikoff, Katia; Finazzi, Giovanni; Kuras, Richard; Schwarz, Christian; Bujaldon, Sandrine; Nickelsen, Joerg; Stern, David B; Wollman, Francis-André; Vallon, Olivier

    2010-01-01

    We identify and functionally characterize MRL1, a conserved nuclear-encoded regulator of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. The nonphotosynthetic mrl1 mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase/oxygenase, and the resulting block in electron transfer is partially compensated by redirecting electrons toward molecular oxygen via the Mehler reaction. This allows continued electron flow and constitutive nonphotochemical quenching, enhancing cell survival during illumination in spite of photosystem II and photosystem I photoinhibition. The mrl1 mutant transcribes rbcL normally, but the mRNA is unstable. The molecular target of MRL1 is the 5 ' untranslated region of rbcL. MRL1 is located in the chloroplast stroma, in a high molecular mass complex. Treatment with RNase or deletion of the rbcL gene induces a shift of the complex toward lower molecular mass fractions. MRL1 is well conserved throughout the green lineage, much more so than the 10 other pentatricopeptide repeat proteins found in Chlamydomonas. Depending upon the organism, MRL1 contains 11 to 14 pentatricopeptide repeats followed by a novel MRL1-C domain. In Arabidopsis thaliana, MRL1 also acts on rbcL and is necessary for the production/stabilization of the processed transcript, presumably because it acts as a barrier to 5 ' >3 ' degradation. The Arabidopsis mrl1 mutant retains normal levels of the primary transcript and full photosynthetic capacity.

  18. MRL1, a Conserved Pentatricopeptide Repeat Protein, Is Required for Stabilization of rbcL mRNA in Chlamydomonas and Arabidopsis[C][W

    Science.gov (United States)

    Johnson, Xenie; Wostrikoff, Katia; Finazzi, Giovanni; Kuras, Richard; Schwarz, Christian; Bujaldon, Sandrine; Nickelsen, Joerg; Stern, David B.; Wollman, Francis-André; Vallon, Olivier

    2010-01-01

    We identify and functionally characterize MRL1, a conserved nuclear-encoded regulator of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. The nonphotosynthetic mrl1 mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase/oxygenase, and the resulting block in electron transfer is partially compensated by redirecting electrons toward molecular oxygen via the Mehler reaction. This allows continued electron flow and constitutive nonphotochemical quenching, enhancing cell survival during illumination in spite of photosystem II and photosystem I photoinhibition. The mrl1 mutant transcribes rbcL normally, but the mRNA is unstable. The molecular target of MRL1 is the 5 ′ untranslated region of rbcL. MRL1 is located in the chloroplast stroma, in a high molecular mass complex. Treatment with RNase or deletion of the rbcL gene induces a shift of the complex toward lower molecular mass fractions. MRL1 is well conserved throughout the green lineage, much more so than the 10 other pentatricopeptide repeat proteins found in Chlamydomonas. Depending upon the organism, MRL1 contains 11 to 14 pentatricopeptide repeats followed by a novel MRL1-C domain. In Arabidopsis thaliana, MRL1 also acts on rbcL and is necessary for the production/stabilization of the processed transcript, presumably because it acts as a barrier to 5 ′ >3 ′ degradation. The Arabidopsis mrl1 mutant retains normal levels of the primary transcript and full photosynthetic capacity. PMID:20097872

  19. Chlamydomonas carries out fatty acid β-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase.

    Science.gov (United States)

    Kong, Fantao; Liang, Yuanxue; Légeret, Bertrand; Beyly-Adriano, Audrey; Blangy, Stéphanie; Haslam, Richard P; Napier, Johnathan A; Beisson, Fred; Peltier, Gilles; Li-Beisson, Yonghua

    2017-04-01

    Peroxisomes are thought to have played a key role in the evolution of metabolic networks of photosynthetic organisms by connecting oxidative and biosynthetic routes operating in different compartments. While the various oxidative pathways operating in the peroxisomes of higher plants are fairly well characterized, the reactions present in the primitive peroxisomes (microbodies) of algae are poorly understood. Screening of a Chlamydomonas insertional mutant library identified a strain strongly impaired in oil remobilization and defective in Cre05.g232002 (CrACX2), a gene encoding a member of the acyl-CoA oxidase/dehydrogenase superfamily. The purified recombinant CrACX2 expressed in Escherichia coli catalyzed the oxidation of fatty acyl-CoAs into trans-2-enoyl-CoA and produced H2 O2 . This result demonstrated that CrACX2 is a genuine acyl-CoA oxidase, which is responsible for the first step of the peroxisomal fatty acid (FA) β-oxidation spiral. A fluorescent protein-tagging study pointed to a peroxisomal location of CrACX2. The importance of peroxisomal FA β-oxidation in algal physiology was shown by the impact of the mutation on FA turnover during day/night cycles. Moreover, under nitrogen depletion the mutant accumulated 20% more oil than the wild type, illustrating the potential of β-oxidation mutants for algal biotechnology. This study provides experimental evidence that a plant-type FA β-oxidation involving H2 O2 -producing acyl-CoA oxidation activity has already evolved in the microbodies of the unicellular green alga Chlamydomonas reinhardtii. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  20. Nickel and low CO2-controlled motility in Chlamydomonas through complementation of a paralyzed flagella mutant with chemically regulated promoters

    Directory of Open Access Journals (Sweden)

    Rosenbaum Joel L

    2011-01-01

    Full Text Available Abstract Background Chlamydomonas reinhardtii is a model system for the biology of unicellular green algae. Chemically regulated promoters, such as the nickel-inducible CYC6 or the low CO2-inducible CAH1 promoter, may prove useful for expressing, at precise times during its cell cycle, proteins with relevant biological functions, or complementing mutants in genes encoding such proteins. To this date, this has not been reported for the above promoters. Results We fused the CYC6 and CAH1 promoters to an HA-tagged RSP3 gene, encoding a protein of the flagellar radial spoke complex. The constructs were used for chemically regulated complementation of the pf14 mutant, carrying an ochre mutation in the RSP3 gene. 7 to 8% of the transformants showed cells with restored motility after induction with nickel or transfer to low CO2 conditions, but not in non-inducing conditions. Maximum complementation (5% motile cells was reached with very different kinetics (5-6 hours for CAH1, 48 hours for CYC6. The two inducible promoters drive much lower levels of RSP3 protein expression than the constitutive PSAD promoter, which shows almost complete rescue of motility. Conclusions To our knowledge, this is the first example of the use of the CYC6 or CAH1 promoters to perform a chemically regulated complementation of a Chlamydomonas mutant. Based on our data, the CYC6 and CAH1 promoters should be capable of fully complementing mutants in genes whose products exert their biological activity at low concentrations.

  1. Protein (Viridiplantae): 159465078 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 2449 hypothetical protein CHLREDRAFT_123720, partial Chlamydomonas reinhardtii MANRHSVSKCHHSQRTFCQRRVCVCVCVCVCVCVCVCVCVCVRMCVCVCRPS...LGLTQHDGQLCVSSSWQPSGACSVCVCCGKVCFWAWPACRPSLGLTQHDGQLCVSSSWQPSGACSVCVCCGKLCFRAWPACRPSLGLTQHDGQLCVSSSWQPSGACPVCVCCGEVC ...

  2. Protein (Viridiplantae): 159466610 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 2419 hypothetical protein CHLREDRAFT_123820, partial Chlamydomonas reinhardtii RVQCRLVDMPAPCLPPFLPTCPHKPRRIPMPCTDAH...ELVDMPAPCLPPFLPDNLPARAPQAPHAVTDAHECMQCRLVDMPAPCLPPFLPKCPHKPRRLPMPCTDAHECNMPAPCLPPFLPKCPHKPRRLPMPCTDAHECMQCRLVDMPAPCLPAFLPNCPHKPRRLPMPCTDAHECSAGW ...

  3. Gene : CBRC-CJAC-01-0078 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available protein [Chlamydomonas reinhardtii] 6e-13 39% MAAVLLCARDAEVAAVTRPAAITAVLLCARDAEVAAVIRPAAITAVLLCARDADVAAVIRPAAMAAV...WLCARDAEVAAAAGPAAMAAVWLCARDAEVAAAAGPAAMAAVLLCARDIDVAAVIRPAAITAVLLCARDMDVAAVIRPAAITAVLLCARDAEVAAAAGPAAMAAVLLCARDIDVAAVIRPAAITAVLLCARDIDVAAVIRPAAITAVLLCASSIAWNIIHRKQE ...

  4. Protein (Viridiplantae): 159468384 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 3436 hypothetical protein CHLREDRAFT_180911 Chlamydomonas reinhardtii MTTEEPLSCSKIRSWNITVYSFTLKGLPGCLEPSHSFWVKEREGEWGLKCLSETFSHELVENVPGREEVSNLLKKGGSSNKSQKGGWICCERNCFLCQHKKCQVLI ...

  5. Multiple stressor effects of predation by rotifers and herbicide pollution on different Chlamydomonas strains and potential impacts on population dynamics.

    Science.gov (United States)

    Fischer, Beat B; Roffler, Severin; Eggen, Rik I L

    2012-12-01

    Environmental factors can interact with the effects of chemical pollutants on natural systems by inducing multiple stressor effects in individual organisms as well as by altering selection pressure on tolerant strains in heterogeneous populations. Predation is a stressful environmental factor relevant for many species. Therefore, the impact of predation by the rotifer Brachionus calyciflorus on tolerance of eight genetically different strains of the green alga Chlamydomonas reinhardtii to simultaneous exposure to each of the three herbicides (diuron, paraquat, and S-metolachlor) was tested. Interactions of combined stressors were analyzed based on the independent action model; additive, synergistic, and antagonistic effects of the combined exposure could be detected depending on the herbicide and strain tested. If cultures were acclimated (pre-exposed) to one stressor, tolerance to the second stressor could be increased. This indicates that physiological changes can induce cotolerance of predation-exposed algae to herbicides and of herbicide-treated algae to predation depending on the combination of stressors. The strain-specific differences in multiple stressor effects also changed the correlation of strains' tolerances to individual stressors determined during combined and single-stressor exposure. Changes in cotolerance to stressors affect selection pressure and population dynamics during long-term exposure. This shows that predation stress can have adverse effects on the toxicity of chemical pollutants to microalgae on the organism and population levels. Copyright © 2012 SETAC.

  6. Gravity-dependent changes in bioconvection of Tetrahymena and Chlamydomonas during parabolic flight: increases in wave number induced by pre- and post-parabola hypergravity.

    Science.gov (United States)

    Kage, Azusa; Asato, Eriko; Chiba, Yoko; Wada, Yuuko; Katsu-Kimura, Yumiko; Kubota, Ayami; Sawai, Satoe; Niihori, Maki; Baba, Shoji A; Mogami, Yoshihiro

    2011-03-01

    Bioconvection emerges in a dense suspension of swimming protists as a consequence of their negative-gravitactic upward migration and later settling as a blob of density greater than that of water. Thus, gravity is an important parameter governing bioconvective pattern formation. However, inconsistencies are found in previous studies dealing with the response of bioconvection patterns to increased gravity acceleration (hypergravity); the wave number of the patterns has been reported to decrease during the hypergravity phases of parabolic aircraft flight, while it increases in centrifugal hypergravity. In this paper, we reassess the responses of bioconvection to altered gravity during parabolic flight on the basis of vertical and horizontal observations of the patterns formed by Tetrahymena thermophila and Chlamydomonas reinhardtii. Spatiotemporal analyses of the horizontal patterns revealed an increase in the pattern wave number in both pre- and post-parabola hypergravity. Vertical pattern analysis was generally in line with the horizontal pattern analysis, and further revealed that hypergravity-induced changes preceded at the top layer of the suspensions while microgravity-induced changes appeared to occur from the bottom part of the settling blobs. The responses to altered gravity were rather different between the two sample species: T. thermophila tended to drastically modify its bioconvection patterns in response to changes in gravity level, while the patterns of C. reinhardtii responded to a much lesser extent. This difference can be attributed to the distinct physical and physiological properties of the individual organisms, suggesting a significant contribution of the gyrotactic property to the swimming behavior of some protists.

  7. Identification and molecular characterization of the second Chlamydomonas gun4 mutant, gun4-II [v2; ref status: indexed, http://f1000r.es/1id

    Directory of Open Access Journals (Sweden)

    Phillip B Grovenstein

    2013-07-01

    Full Text Available The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study oxygenic photosynthesis. Chlorophyll (Chl and heme are major tetrapyrroles that play an essential role in photosynthesis and respiration. These tetrapyrroles are synthesized via a common branched pathway that involves mainly enzymes, encoded by nuclear genes. One of the enzymes in the pathway is Mg chelatase (MgChel. MgChel catalyzes insertion of Mg2+ into protoporphyrin IX (PPIX, proto to form Magnesium-protoporphyrin IX (MgPPIX, Mgproto, the first biosynthetic intermediate in the Chl branch. The GUN4 (genomes uncoupled 4 protein is not essential for the MgChel activity but has been shown to significantly stimulate its activity. We have isolated a light sensitive mutant, 6F14, by random DNA insertional mutagenesis. 6F14 cannot tolerate light intensities higher than 90-100 μmol photons m-2 s-1. It shows a light intensity dependent progressive photo-bleaching. 6F14 is incapable of photo-autotrophic growth under light intensity higher than 100 μmol photons m-2 s-1. PCR based analyses show that in 6F14 the insertion of the plasmid outside the GUN4 locus has resulted in a genetic rearrangement of the GUN4 gene and possible deletions in the genomic region flanking the GUN4 gene. Our gun4 mutant has a Chl content very similar to that in the wild type in the dark and is very sensitive to fluctuations in the light intensity in the environment unlike the earlier identified Chlamydomonas gun4 mutant. Complementation with a functional copy of the GUN4 gene restored light tolerance, Chl biosynthesis and photo-autotrophic growth under high light intensities in 6F14. 6F14 is the second gun4 mutant to be identified in C. reinhardtii. Additionally, we show that our two gun4 complements over-express the GUN4 protein and show a higher Chl content per cell compared to that in the wild type strain.

  8. NRT2.4 and NRT2.5 Are Two Half-Size Transporters from the Chlamydomonas NRT2 Family

    Directory of Open Access Journals (Sweden)

    Jose Javier Higuera

    2016-03-01

    Full Text Available The NRT2 transporters mediate High Affinity Nitrate/NitriteTransport (HAN/NiT, which are essential for nitrogen acquisition from these inorganic forms. The NRT2 proteins are encoded by a multigene family in plants, and contain 12 transmembrane-spanning domains. Chlamydomonas reinhardtii has six NRT2, two of which (NRT2.5 and NRT2.4 are located in Chromosome III, in tandem head to tail. cDNAs for these genes were isolated and their sequence revealed that they correspond to half-size NRT2 transporters each containing six transmembrane domains. NRT2.5 has long N- and C- termini sequences without known homology. NRT2.4 also contains long termini sequences but smaller than NRT2.5. Expression of both studied genes occurred at a very low level, slightly in darkness, and was not modified by the N or C source. Silencing of NRT2.4 by specific artificial miRNA resulted in the inhibition of nitrite transport in the absence of other HANNiT (NRT2.1/NAR2 in the cell genetic background. Nitrite transport activity in the Hansenula polymorpha Δynt::URA3 Leu2 mutant was restored by expressing CrNRT2.4. These results indicate that half-size NRT2 transporters are present in photosynthetic organisms and that NRT2.4 is a HANiT.

  9. Sulphate, more than a nutrient, protects the microalga Chlamydomonas moewusii from cadmium toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Mera, Roi; Torres, Enrique, E-mail: torres@udc.es; Abalde, Julio

    2014-03-01

    Highlights: • Sulphate effect on cadmium toxicity in the microalga Chlamydomonas moewusii Gerloff. • Cadmium increases the sulphur requirements in Chlamydomonas moewusii. • Kinetic coefficients for sulphate utilization and cadmium effect on them. • Sulphate and cadmium influence on the biosynthesis of low-molecular mass thiols. • Cadmium toxicity reduction by sulphate due to higher biosynthesis of thiols. - Abstract: Sulphur is an essential macroelement that plays important roles in living organisms. The thiol rich sulphur compounds, such as cysteine, γ-Glu–Cys, glutathione and phytochelatins participate in the tolerance mechanisms against cadmium toxicity. Plants, algae, yeasts and most prokaryotes cover their demand for reduced sulphur by reduction of inorganic sulphate. The aim of this study was to investigate, using a bifactorial experimental design, the effect of different sulphate concentrations in the nutrient solution on cadmium toxicity in the freshwater microalga Chlamydomonas moewusii. Cell growth, kinetic parameters of sulphate utilization and intracellular concentrations of low-molecular mass thiol compounds were determined. A mathematical model to describe the growth of this microalga based on the effects of sulphate and cadmium was obtained. An ANOVA revealed an interaction between them, 16% of the effect sizes was explained by this interaction. A higher amount of sulphate in the culture medium allowed a higher cadmium tolerance due to an increase in the thiol compound biosynthesis. The amount of low-molecular mass thiol compounds, mainly phytochelatins, synthesized by this microalga was significantly dependent on the sulphate and cadmium concentrations; the higher phytochelatin content was obtained in cultures with 4 mg Cd/L and 1 mM sulphate. The maximum EC{sub 50} value (based on nominal cadmium concentration) reached for this microalga was 4.46 ± 0.42 mg Cd/L when the sulphate concentration added to the culture medium was also 1 m

  10. The rhinoceros among Serpents: Comparative anatomy and experimental biophysics of Calabar burrowing python (Calabaria reinhardtii) skin.

    Science.gov (United States)

    Han, Dawei; Young, Bruce A

    2018-01-01

    The Calabar burrowing python (Calabaria reinhardtii) has a unique combination of marked thickness of the integumentary layers, a highly organized lamellate arrangement of the dermal collagen bundles, and a reduction in the size of the interscale hinge region of the integument. Biomechanical testing demonstrates that the skin of C. reinhardtii is more resistant to penetration than the skin of other snakes. The laminar arrangement of the collagen bundles provides for penetrative resistance, even while maintaining the flexibility characteristic of snake skin. Considering the life history of this species, it is hypothesized that the specialized integument of C. reinhardtii is a passive defensive mechanism against penetrative bites from maternal rodents and predators. © 2017 Wiley Periodicals, Inc.

  11. Final Report: Filling Knowledge Gaps in Biological Networks: Integrated Global Approaches to Understand H{sub 2} Metabolism in Chlamydomonas Reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Grossman, Arthur

    2012-05-01

    The major goal of our part of this project has been to generate mutants in fermentation metabolism and begin to decipher how lesions in the pathways associated with fermentation metabolism impact both H{sub 2} production and the production of other metabolites that accumulate as cells become anoxic. We are also trying to understand how metabolic pathways are regulated as O{sub 2} in the environment becomes depleted.

  12. Influences of TiO2nanoparticles on dietary metal uptake in Daphnia magna.

    Science.gov (United States)

    Tan, Cheng; Wang, Wen-Xiong

    2017-12-01

    Increasing applications of titanium dioxide nanoparticles (nano-TiO 2 ) have intensified the risk of environmental contamination. Since nano-TiO 2 can absorb metals and be consumed as 'food' by zooplankton but also can interact with phytoplankton, they could significantly disturb the existing metal assimilation patterns. In the present study, we quantified the dietary assimilation of Cd and Zn from nano-TiO 2 and algae (Chlamydomonas reinhardtii) at comparable particle concentrations as well as in complex food environment (variable food quality and quantity) in a freshwater zooplankton Daphnia magna using the radiotracer technique. For both nano-TiO 2 and algae as food, the feeding food quality and depuration food quantity significantly affected the assimilation efficiencies (AEs) of Cd and Zn. At feeding food quantity of 1 mg/L to 10 mg/L without food in depuration, the AEs of Cd and Zn from nano-TiO 2 were lower than those from algae. When food was added during depuration, the influences of nano-TiO 2 on metal AEs were variable due to the differential effects of food quantity on the gut passage of nano-TiO 2 and algae. Furthermore, mixed nano-TiO 2 and algae had the lowest metal AEs compared to sole nano-TiO 2 or algae as a result of interaction between nano-TiO 2 and algae during feeding. Overall, this study showed the distinguishing metal AEs between nano-TiO 2 and algae, and that nano-TiO 2 could significantly reduce the existing metal AEs from algae. More attention should be paid to the potential roles of nano-TiO 2 in disturbing metal assimilation in the environmental risk assessments of nanoparticles. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Protein (Viridiplantae): 714399 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 3051:329 ... 3052:329 ... 3055:329 ... predicted protein Chlamydomonas reinhardtii MAPAALPGRSVKSKQAHLLRTDAHRVKSKQAHLLRTDAHRVKSKQAHLLRTDA...HRVKSKQAHLLRTDAHRVKSKQAHLLRTDAHRVALTTLTGALSLFGGACTATSFVLQVSASAASYAASLRLSCPAVPSLTDVA

  14. Protein (Viridiplantae): 159482451 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 6194 hypothetical protein CHLREDRAFT_181148, partial Chlamydomonas reinhardtii GGAARAADDAVAATTTAGSGAAAGDAARAANTAAAATTAAGSGSAAGGAARA...ADDAAAATTAAGSGAAAGDAAPAANTAAAATTAGSGSAAGGAARAADDAAAATTAAGFGSAAGGAARAADDAAAATTAAGSGSAAGGAARA...ANDAVAATTAAGSGSAAGGAARAANTAAAATTAAGSGSAAGGAARAANDAAAATTAAGSGSAAGGAAPAANTAAAATTAGSGSAAGGAARA...ADDAVAATTAAGFGSAAGGAARAANTAAAATTAAGSGSAAGGAARAADDAVAATTAAGFGSAAGGAARAANTAAAATTAAGSGSAAGGAARAADDAVAATTAAGFGSAAGGAARA...ANTAAAATTAAGSGSAAGGAARAADDAAAATTAAGSGAAAGGAARAADDAAAATTAAGSGSAAGDAARAANTQRLPQRRALAPQQ

  15. Protein (Viridiplantae): 953258 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 3051:3557 ... 3052:3557 ... 3055:3557 ... predicted protein, partial Chlamydomonas reinhardtii MVCEMVELMGPANAVDLLSEVEGLAALLSGSHLPL...PPGLPPLANPLVSASPLAAALLAPLAPLRGPFAAFAAARALGPVLAPAVWRSVCYWQRTLPIMASYMRTTRRARALQRRGDWQGAEGLWAAQHEWGAGAIFDMLADLK

  16. Gene : CBRC-MMUR-01-0144 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available -C2 protein precursor [Chlamydomonas reinhardtii] 1e-100 63% MEQPSASAPPAALVSSPFSSPQPSPRPPPPSPPPSPTPSPPPPPSQP...SPSPPPPESLSSPPPPPQASPPPPPSSRPSPTPSPPPSLSSPPSPQPSPSPPPPPSLSSPPPSPQPSPPSQPSPTPSPPPPPSLSSPPPSPSPPPPPSPSSPPSPQPSPPPPPSSPQPSPPPSPSPPPPPS...SPSPPPSPSPPSQPSPQPSPTPSPPPPPSPQPSPSPPPLPSPQPSPTPSPPPPPSPQPSPSPPPPPSPQLSPLPSSPPSPTPSAPPSPPPP ...

  17. Biofiksasi CO2 Oleh Mikroalga Chlamydomonas sp dalam Photobioreaktor Tubular

    Directory of Open Access Journals (Sweden)

    Hadiyanto Hadiyanto

    2014-05-01

    Full Text Available Mikroalga memiliki potensi dalam membiofiksasi CO2 dan dapat dimanfaatkan untuk mengurangi kadar CO2 dalam gas pencemar. Pertumbuhan mikroalga sangat dipengaruhi oleh konsentrasi gas CO2 di dalam gas pencemar. Tujuan penelitian ini adalah untuk mengeetahui kemampuan mikroalga Chlamydomonas sp yang dikultivasi dalam photobioreaktor tubular dalam penyerapan gas CO2 serta untuk mengetahui konsentrasi maksimum gas CO2 dalam umpan untuk memproduksi biomasa mikroalga yang optimal. Percobaan dilakukan dnegan memvariasi laju alir dari 0.03 -0.071 L/menit dan konsentrasi CO2 dalam umpan 10-30%. Hasil penelitian menunjukkan bahwa biomasa mikroalga dapat diproduksi dengan maksimal dengan konsentrasi gas CO2 20% dengan laju alir 0.07 L/min. Semakin tinggi laju alir maka produksi biomasa alga semakin besar. Kecepatan pertumbuhan alga maksimum terjadi pada 0.31 /hari. Pada konsentrasi gas CO2 30%, terjadi substrate inhibition yang disebabkan carbon dalam bentuk ion bicarbonate tidak dapat dikonsumsi lagi di dalam kultur alga. Kata kunci : Mikroalga, chlamydomonas sp, biofiksasi CO2, biogas Abstract Microalgae have a potential for CO2 biofixation and therefore can be used to reduce the CO2 concentration in the gas pollutants. Moreover, microalgae growth is strongly affected by the concentration of CO2 in the exhaust gas pollutants. The objective of this research was to investigate the ability of microalgae Chlamydomonas sp which was cultivated in a tubular photobioreactor for CO2 absorption as well as to determine the maximum concentration of CO2 in the feed gas to obtain optimum microalgae biomass. The experiments were performed by varying the gas flow rate of 0.03 -0.071 L / min and the concentration of CO2 in the feed of 10-30%. The results showed that the maximum biomass of microalgae can be produced with CO2 concentration of 20% vol with a flow rate of 0.07 L / min. The result also showed that increasing the gas flow rate, the greater of the production of

  18. Structure-function analysis of small G proteins from Volvox and Chlamydomonas by complementation of Saccharomyces cerevisiae YPT/SEC mutations.

    Science.gov (United States)

    Fabry, S; Steigerwald, R; Bernklau, C; Dietmaier, W; Schmitt, R

    1995-05-10

    cDNAs representing nine small G protein genes encoding Ypt proteins from the green algae Volvox carteri (YptV) and Chlamydomonas reinhardtii (YptC) were tested for their ability to complement mutations in the YPT1, SEC4, and YPT7 genes of Saccharomyces cerevisiae strains defective in different steps of intracellular vesicle transport. None of the heterologously expressed algal genes was able to complement mutations in SEC4 or YPT7, but three of them, yptV1, yptC1, and yptV2, restored a YPT1 null mutation. On the amino acid sequence level, and particularly with respect to known small G protein specificity domains, YptV1p and YptC1p are the closest algal analogs of yeast Ypt1p, with 70% overall identity and identical effector regions, but YptV2p is only 55% identical to Ypt1p, and its effector domain resembles that of Sec4p. To define more precisely the regions that supply Ypt1p function, six chimeras were constructed by reciprocal exchange of 68/72-, 122/123-, and 162/163-amino acid segments of the C-terminal regions between YptV1p (complementing) and YptV3p (non-complementing). Segments containing 68 amino acids of the hypervariable C-terminal, and 41 residues of the N-terminal region including the effector region, of YptV1p could be replaced by the corresponding parts of YptV3p without loss of function in yeast, but exchanges within the central core destroyed the ability to rescue the YPT1 mutation. Sequence analysis of ypt1-complementing and -noncomplementing Ypt types suggests that surface loop3 represents a novel specificity domain of small G proteins.

  19. Redundant cis-acting determinants of 3' processing and RNA stability in the chloroplast rbcL mRNA of Chlamydomonas.

    Science.gov (United States)

    Goldschmidt-Clermont, Michel; Rahire, Michèle; Rochaix, Jean-David

    2008-02-01

    We have designed a screen for mutants affected in 3' maturation of the chloroplast rbcL mRNA in Chlamydomonas reinhardtii. We inserted a spectinomycin resistance cassette, 5'atpA::aadA::3'rbcL, in a peripheral domain of tscA, the gene for a small non-coding RNA involved in trans-splicing of psaA. Depending on the orientation of the cassette, a polar effect was observed which was due to processing at the 3'rbcL element: the chimeric tscA RNA was truncated and splicing of psaA was blocked. We selected phenotypic revertants of this insertion mutant that restored psaA splicing, which correlated with the presence of chimeric transcripts that regained the 3' part of tscA. We analyzed two nuclear and six chloroplast suppressors. Five chloroplast mutations altered a short element in the center of the second inverted repeat in the 3'rbcL (IR2), and one deleted a larger region including this element. These mutations revealed a cis-acting element in IR2 which is required for 3' processing. When the same mutations were inserted in the 3' untranslated region (UTR) of the native rbcL gene, the rbcL mRNA accumulated to normal levels, but in strong alleles its 3' end was located upstream, near the end of the first inverted repeat (IR1). Deletion of either IR1 or IR2 allowed stable accumulation of rbcL mRNA, but deletion of both resulted in its complete absence. This indicated that the two inverted repeats function as redundant mRNA stability determinants in the 3' UTR of rbcL.

  20. A mutant of Chlamydomonas without LHCSR maintains high rates of photosynthesis, but has reduced cell division rates in sinusoidal light conditions.

    Directory of Open Access Journals (Sweden)

    Michael Cantrell

    Full Text Available The LHCSR protein belongs to the light harvesting complex family of pigment-binding proteins found in oxygenic photoautotrophs. Previous studies have shown that this complex is required for the rapid induction and relaxation of excess light energy dissipation in a wide range of eukaryotic algae and moss. The ability of cells to rapidly regulate light harvesting between this dissipation state and one favoring photochemistry is believed to be important for reducing oxidative stress and maintaining high photosynthetic efficiency in a rapidly changing light environment. We found that a mutant of Chlamydomonas reinhardtii lacking LHCSR, npq4lhcsr1, displays minimal photoinhibition of photosystem II and minimal inhibition of short term oxygen evolution when grown in constant excess light compared to a wild type strain. We also investigated the impact of no LHCSR during growth in a sinusoidal light regime, which mimics daily changes in photosynthetically active radiation. The absence of LHCSR correlated with a slight reduction in the quantum efficiency of photosystem II and a stimulation of the maximal rates of photosynthesis compared to wild type. However, there was no reduction in carbon accumulation during the day. Another novel finding was that npq4lhcsr1 cultures underwent fewer divisions at night, reducing the overall growth rate compared to the wild type. Our results show that the rapid regulation of light harvesting mediated by LHCSR is required for high growth rates, but it is not required for efficient carbon accumulation during the day in a sinusoidal light environment. This finding has direct implications for engineering strategies directed at increasing photosynthetic productivity in mass cultures.

  1. Insecticides induced biochemical changes in freshwater microalga Chlamydomonas mexicana.

    Science.gov (United States)

    Kumar, Muthukannan Satheesh; Kabra, Akhil N; Min, Booki; El-Dalatony, Marwa M; Xiong, Jiuqiang; Thajuddin, Nooruddin; Lee, Dae Sung; Jeon, Byong-Hun

    2016-01-01

    The effect of insecticides (acephate and imidacloprid) on a freshwater microalga Chlamydomonas mexicana was investigated with respect to photosynthetic pigments, carbohydrate and protein contents, fatty acids composition and induction of stress indicators including proline, superoxide dismutase (SOD) and catalase (CAT). C. mexicana was cultivated with 1, 5, 10, 15, 20 and 25 mg L(-1) of acephate and imidacloprid. The microalga growth increased with increasing concentrations of both insecticides up to 15 mg L(-1), beyond which the growth declined compared to control condition (without insecticides). C. mexicana cultivated with 15 mg L(-1) of both insecticides for 12 days was used for further analysis. The accumulation of photosynthetic pigments (chlorophyll and carotenoids), carbohydrates and protein was decreased in the presence of both insecticides. Acephate and imidacloprid induced the activities of superoxide dismutase (SOD) and catalase (CAT) and increased the concentration of proline in the microalga, which play a defensive role against various environmental stresses. Fatty acid analysis revealed that the fraction of polyunsaturated fatty acids decreased on exposure to both insecticides. C. mexicana also promoted 25 and 21% removal of acephate and imidacloprid, respectively. The biochemical changes in C. mexicana on exposure to acephate and imidacloprid indicate that the microalga undergoes an adaptive change in response to the insecticide-induced oxidative stress.

  2. Hydrogenase-Mediated Activities in Isolated Chloroplasts of Chlamydomonas reinhardii.

    Science.gov (United States)

    Maione, T E; Gibbs, M

    1986-02-01

    Isolated intact chloroplasts of Chlamydomonas reinhardii were found to catalyze photoreduction of CO(2) in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when adapted under an atmosphere of H(2) demonstrating the association of a hydrogenase and anaerobic adaptation system with these plastids. The specific activity of photoreduction was approximately one third that detected in cells and protoplasts. Photoreduction was found to have a lower osmoticum optimum relative to aerobically maintained chloroplasts (50 millimolar versus 120 millimolar mannitol). 3-Phosphoglycerate (3-PGA) stimulated photoreduction up to a peak at 0.25 millimolar beyond which inhibition was observed. In the absence of 3-PGA, inorganic phosphate had no effect on photoreduction but in the presence of 3-PGA, inorganic phosphate also stimulated the reaction. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone inhibited photoreduction but inhibition by the former could be partially overcome by exogenously added ATP. The intact plastid can also catalyze photoevolution of H(2) while lysed chloroplast extracts catalyzed the reduction of methyl viologen by H(2). Both reactions occurred at rates approximately one-third of those found in cells. The oxyhydrogen reaction in the presence or absence of CO(2) was not detected.

  3. Mild environmental stress elicits mutations affecting fitness in Chlamydomonas.

    Science.gov (United States)

    Goho, S; Bell, G

    2000-01-01

    Cultures of Chlamydomonas were exposed to a range of relatively mild stresses for a period of 24 h. These stresses comprised high and low temperatures, osmotic stress, low pH, starvation and toxic stress. They were then allowed to recuperate for around ten vegetative generations under near-optimal conditions in unmodified minimal medium. Fitness was then assayed as the rate of division of isolated cells on agar. We found that there was a strong tendency for stressed cultures to have lower mean fitness and greater standardized variance in fitness than the negative controls which had been cultured throughout in unmodified minimal medium. The same tendency was shown, as expected, by positive controls which received mutagenic doses of ultraviolet irradiation. We concluded that the most reasonable interpretation of these observations is that mild stress increases the genomic rate of mutation. This appears to be the first time that this phenomenon has been noticed in eukaryotes. The response might be adaptive because lineages in which higher mutation rates are elicited by stress can be favourably selected through the production of a few mutants which are fortuitously well adapted to the stressful environment. Other interpretations are not excluded, however. Regardless of the mechanism involved, the elevation of mutation rates under stress will affect the rate of evolutionary response to environmental change and also the maintenance of sexuality. PMID:10687816

  4. An experimental study of the growth and hydrogen production of C. reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Tamburic, B.; Burgess, S.; Nixon, P.J.; Hellgardt, K. [Imperial College London (United Kingdom)

    2010-07-01

    Some unicellular green algae, such as C. reinhardtii, have the ability to photosynthetically produce molecular hydrogen under anaerobic conditions. They offer a biological route to renewable, carbon-neutral hydrogen production from two of nature's most plentiful resources - sunlight and water. This process provides the additional benefit of carbon dioxide sequestration and the option of deriving valuable products from algal biomass. The growth of dense and healthy algal biomass is a prerequisite for efficient hydrogen production. This study investigates the growth of C. reinhardtii under different cyclic light regimes and at various continuous light intensities. Algal growth is characterised in terms of the cell count, chlorophyll content and optical density of the culture. The consumption of critical nutrients such as acetate and sulphate is measured by chromatography techniques. C. reinhardtii wild-type CC-124 strain is analysed in a 3 litre tubular flow photobioreactor featuring a large surface-to-volume ratio and excellent light penetration through the culture. Key parameters of the hydrogen production process are continuously monitored and controlled; these include pH, pO{sub 2}, optical density, temperature, agitation and light intensity. Gas phase hydrogen production is determined by mass spectrometry. (orig.)

  5. Flagellar coordination in Chlamydomonas cells held on micropipettes.

    Science.gov (United States)

    Rüffer, U; Nultsch, W

    1998-01-01

    The two flagella of Chlamydomonas are known to beat synchronously: During breaststroke beating they are generally coordinated in a bilateral way while in shock responses during undulatory beating coordination is mostly parallel [Rüffer and Nultsch, 1995: Botanica Acta 108:169-276]. Analysis of a great number of shock responses revealed that in undulatory beats also periods of bilateral coordination are found and that the coordination type may change several times during a shock response, without concomitant changes of the beat envelope and the beat period. In normal wt cells no coordination changes are found during breaststroke beating, but only short temporary asynchronies: During 2 or 3 normal beats of the cis flagellum, the trans flagellum performs 3 or 4 flat beats with a reduced beat envelope and a smaller beat period, resulting in one additional trans beat. Long periods with flat beats of the same shape and beat period are found in both flagella of the non-phototactic mutant ptx1 and in defective wt 622E cells. During these periods, the coordination is parallel, the two flagella beat alternately. A correlation between normal asynchronous trans beats and the parallel-coordinated beats in the presumably cis defective cells and also the undulatory beats is discussed. In the cis defective cells, a perpetual spontaneous change between parallel beats with small beat periods (higher beat frequency) and bilateral beats with greater beat periods (lower beat frequency) are observed and render questionable the existence of two different intrinsic beat frequencies of the two flagella cis and trans. Asynchronies occur spontaneously but may also be induced by light changes, either step-up or step-down, but not by both stimuli in turn as breaststroke flagellar photoresponses (BFPRs). Asynchronies are not involved in phototaxis. They are independent of the BFPRs, which are supposed to be the basis of phototaxis. Both types of coordination must be assumed to be regulated

  6. Long-term experiment on physiological responses to synergetic effects of ocean acidification and photoperiod in the Antarctic sea ice algae Chlamydomonas sp. ICE-L.

    Science.gov (United States)

    Xu, Dong; Wang, Yitao; Fan, Xiao; Wang, Dongsheng; Ye, Naihao; Zhang, Xiaowen; Mou, Shanli; Guan, Zheng; Zhuang, Zhimeng

    2014-07-15

    Studies on ocean acidification have mostly been based on short-term experiments of low latitude with few investigations of the long-term influence on sea ice communities. Here, the combined effects of ocean acidification and photoperiod on the physiological response of the Antarctic sea ice microalgae Chlamydomonas sp. ICE-L were examined. There was a general increase in growth, PSII photosynthetic parameters, and N and P uptake in continuous light, compared to those exposed to regular dark and light cycles. Elevated pCO2 showed no consistent effect on growth rate (p=0.8) and N uptake (p=0.38) during exponential phrase, depending on the photoperiod but had a positive effect on PSII photosynthetic capacity and P uptake. Continuous dark reduced growth, photosynthesis, and nutrient uptake. Moreover, intracellular lipid, mainly in the form of PUFA, was consumed at 80% and 63% in low and high pCO2 in darkness. However, long-term culture under high pCO2 gave a more significant inhibition of growth and Fv/Fm to high light stress. In summary, ocean acidification may have significant effects on Chlamydomonas sp. ICE-L survival in polar winter. The current study contributes to an understanding of how a sea ice algae-based community may respond to global climate change at high latitudes.

  7. Identification and sequence analysis of six new members of the NIMA-related kinase family in Chlamydomonas.

    Science.gov (United States)

    Bradley, Brian A; Wagner, James J D; Quarmby, Lynne M

    2004-01-01

    The NIMA kinases are an evolutionarily conserved protein family with enigmatic roles in the regulation of mitosis. We report six new members of this family in Chlamydomonas, in addition to the previously identified NIMA-related kinase, Fa2p. Chlamydomonas NIMA-related kinases (CNKs) 1-6 were sequenced from subclones generated by RT-PCR using information from EST libraries and the recently sequenced Chlamydomonas genome. Phylogenetic and bioinformatic approaches were used to determine the relationships of the six new members with known members of the NIMA-related kinase family. Although humans express at least eleven NIMA-related kinases, the eukaryotic microbes that have been studied to date express only one or two members of the family. Thus, the discovery that Chlamydomonas expresses a total of at least seven NIMA-related kinases is intriguing. Our analyses suggest that members of this family may play roles in the assembly and function of cilia.

  8. Comparison of steam gasification reactivity of algal and lignocellulosic biomass: influence of inorganic elements.

    Science.gov (United States)

    Hognon, Céline; Dupont, Capucine; Grateau, Maguelone; Delrue, Florian

    2014-07-01

    This study aims at comparing the steam gasification behaviour of two species of algal biomass (Chlamydomonas reinhardtii and Arthrospira platensis) and three species of lignocellulosic biomass (miscanthus, beech and wheat straw). Isothermal experiments were carried out in a thermobalance under chemical regime. Samples had very different contents in inorganic elements, which resulted in different reactivities, with about a factor of 5 between samples. For biomasses with ratio between potassium content and phosphorus and silicon content K/(Si+P) higher than one, the reaction rate was constant during most of the reaction and then slightly increased at high conversion. On the contrary, for biomasses with ratio K/(Si+P) lower than one, the reaction rate decreased along conversion. A simple kinetic model was proposed to predict these behaviours. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Development of lipid productivities under different CO2 conditions of marine microalgae Chlamydomonas sp. JSC4.

    Science.gov (United States)

    Nakanishi, Akihito; Aikawa, Shimpei; Ho, Shih-Hsin; Chen, Chun-Yen; Chang, Jo-Shu; Hasunuma, Tomohisa; Kondo, Akihiko

    2014-01-01

    Biodiesel production from microalgae has become a popular research topic. In this study, Chlamydomonas sp. JSC4 isolated from the southern coast of Taiwan was selected for a detailed study on cell growth and lipid accumulation under marine salinity (3.5% sea salt). Proper CO2 was supplied as the improvement of lipid productivity. Under the optimal condition, the highest lipid productivity was 169.1mg/L/d, which was significantly higher than those reported in current studies for marine green algae. To date, only very few studies have reported a marine algae strain with both high cell growth and lipid productivity. This study demonstrated that a newly isolated marine green alga Chlamydomonas sp. JSC4 would be a feasible oil producer due to its high biomass production and lipid productivity under marine salinity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. A NIMA-related kinase, Cnk2p, regulates both flagellar length and cell size in Chlamydomonas.

    Science.gov (United States)

    Bradley, Brian A; Quarmby, Lynne M

    2005-08-01

    The cycle of ciliogenesis and ciliary disassembly is coordinated with cell division. In the unicellular alga Chlamydomonas, the two flagella are maintained at constant and equal length during interphase, and are reabsorbed prior to mitosis. We report that the NIMA-related kinase, Cnk2p, is an axonemal protein that affects flagellar length via effects on disassembly rate and also plays a role in the cellular assessment of size prior to committing to mitosis. This is the second NIMA-related kinase shown to affect ciliary function and cell cycle progression in Chlamydomonas. We speculate that members of the NIMA family have evolved nuanced roles to coordinate cilia/cell cycle regulation.

  11. Protein (Viridiplantae): 232868 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 3051:4703 ... 3052:4703 ... 3055:4703 ... hypothetical protein CHLREDRAFT_120274, partial Chlamydomonas reinhardtii PPGCRCSSAPPGCRC...SSAPPGCRCSSAPPGCRCSSAPPGCRCSSAPPGCRCSSAPPGCRCSSAPPGCRCSSAPPGCRCSSAPPGCRCSSAPPGCRCSSAPPGCRCSSAPPGCRCS

  12. Protein (Viridiplantae): 588569 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 3051:512 ... 3052:512 ... 3055:512 ... cyclin dependent kinase Chlamydomonas reinhardtii MAPGFGNFATADDASTSSGYQDQGPLARLLSKLRQFKALAADKDLAN...SELAPLIRALPLDTELQQLLAKFHWYPGHNFTTTVDLANLKGALQKYKYIKIGQLGSGSYGVVHKAINRETNELLAIKKVVHS...ANSIMHRDLKPQNVLVGVHSGNVKITDFGLARCFLPNEDRAYTERVVTLYYRAPELLLGAQHYTSAVDLWSVGCIMAEMVNFEPLFRSDSEIGLLFRMFEQLGTPTPDAWHELSGLAYYSENFPRFVPRRFEDMVPRLANDAVGLDLLRRMLCYDPRQRITASEALVHPWFNDVVV

  13. Protein (Viridiplantae): 785127 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 3051:843 ... 3052:843 ... 3055:843 ... hypothetical protein CHLREDRAFT_180936, partial Chlamydomonas reinhardtii MAAALMLDPC...CPPPAAPQRLHRQRLNRSRRFHQPWLLSSQALMLDPCCPPPAAPQRLHRQRLNRQQAFPPALAWDWVLKMRAVRGGGAGSHAGPMLPPTGGTPAAA...GGTPEAAQAAAQPQQAVPPVLAWDWVLKMRAALMLDPCCPPPAAPQRLHRQRLNRQQAFPPALAWDWVLKMRAVGGGGAGSHAGPMLPPTGGTPAAAQAAAQPQQAVP...PALAWDWVLKMRAAMAAVVAGTHAGPVLPPTGGTPTAAQAAAQPQQAVPPALGPWLLSSPALKLDPCCPPPAAPQRLHRQP

  14. Protein (Viridiplantae): 840646 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 3051:4281 ... 3052:4281 ... 3055:4281 ... predicted protein Chlamydomonas reinhardtii MPPPLMPISGCGPLRLSISGCMPLPLVPTSGCGPLQLSISGCMPPPL...VPTSGCGPLQLSISGCMPPPLVPTSGCGPLQLSISGCMPPPLVPTSGCGPLQLSISGCMPPPLMPISGCGPLQLSISGCVPPPLVPTSGCGPLQLSISGCMPPPL...VPTSGCGPLQLSISGCMPPPLVPTSGCGPLQLSISGCMPPPLVPTSGCGPRQLSNSGCMPPPLVPTSGCGPLQLSISGCVPPPL...VPTSGCGPLQLSISGCVPPPLVPTSGCGPLQLSISGCVPPPLVPTSGCGPRQLSNSGCMPPPLVPTSGCGPLSISGCVPPPLVPTSGCGPLSISGCVPPPLVPL

  15. Gene : CBRC-PHAM-01-1088 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available -C2 protein precursor [Chlamydomonas reinhardtii] 4e-84 47% MTDTLVDQGRGTPLFPWRSHPPSPWLWRQLSSAAPCHGILPGTSGWLP...MPSPIPASQKVPAILVTTFSFPWLPPSPPTPALPARPPPSPPTPALPARPPPSPPTPALPARPPPSPPTPALPARPPPSALTPALPARPPPSALTPALPACPPPSALTPALPAWPPPSALTPALPAWPPPS...APTPALPAWPPPSAPTPALPAWPPPSPPTPALPAWPPPSPPTPALPAWPPPSPPTPALPAWPPPSPPTPALPAWPPPS...ALTPALPAWPSPSALTPALPAWPPPSALTPALPAWPPPSALTPALPACPPPSALTPALPVCPPSSALTPALPARPPPSPPTPALSAQPLLSPPTPALPVWPPPS...PPTPALPACPPPSALTPALPACPPPSALTPALPAWPSPSALTPALPAWPLLTSPAHPSSLASSRDAQGHGSASVTRKVCNVHTKQVLLKLKWAGGHQASPGLPRPVCGKLCGHPVQPCSSPGHFALQLSPGCWLCTGLPFPSRITNSKASSHLV ...

  16. Biofixation of Carbon dioxide by Chlamydomonas sp. in a Tubular Photobioreactor

    Directory of Open Access Journals (Sweden)

    H Hadiyanto

    2012-02-01

    Full Text Available The biogas production from anaerobic digestion is a potential fuel for power generators application, if biogas can be upgraded to the same standards as fossil natural gas by CO2, H2S, and other non-combustible component removal. Microalgae Chlamydomonas sp. has potency to biofix the carbon dioxide and can be used as an additional food ingredient. The variations of flow rate and carbon dioxide concentration in the process resulting different value of biomass production and carbon dioxide biofixation. Biomass production at 40% carbon dioxide concentration obtained 5.685 gr/dm3 at 10% carbon dioxide concentration obtained 4.892 gr/dm3. The greatest value of carbon dioxide absorption occurs at a 40% concentration amounting to 12.09%. The rate of growth and productivity of microalgae tend to rise in 10% and 20% (%v carbon dioxide concentration, but began started a constant at 30% and 40% (%v carbon dioxide concentration. Biomass production tends to increase in light conditions while a constant in dark conditions. This study used Chlamydomonas sp. as media culture and performed on bubble column and tubular reactor with 6 litres of culture medium at a temperature of 28oC and atmospheric pressure.

  17. Biofixation of Carbon dioxide by Chlamydomonas sp. in a Tubular Photobioreactor

    Directory of Open Access Journals (Sweden)

    H Hadiyanto

    2012-04-01

    Full Text Available The biogas production from anaerobic digestion is a potential fuel for power generators application, if biogas can be upgraded to the same standards as fossil natural gas by CO2, H2S, and other non-combustible component removal. Microalgae Chlamydomonas sp. has potency to biofix the carbon dioxide and can be used as an additional food ingredient. The variations of flow rate and carbon dioxide concentration in the process resulting different value of biomass production and carbon dioxide biofixation. Biomass production at 40% carbon dioxide concentration obtained 5.685 gr/dm3 at 10% carbon dioxide concentration obtained 4.892 gr/dm3. The greatest value of carbon dioxide absorption occurs at a 40% concentration amounting to 12.09%. The rate of growth and productivity of microalgae tend to rise in 10% and 20% (%v carbon dioxide concentration, but began started a constant at 30% and 40% (%v carbon dioxide concentration. Biomass production tends to increase in light conditions while a constant in dark conditions. This study used Chlamydomonas sp. as media culture and performed on bubble column and tubular reactor with 6 litres of culture medium at a temperature of 28oC and atmospheric pressure.

  18. The BSD2 ortholog in Chlamydomonas reinhardtii is a polysome‐associated chaperone that co‐migrates on sucrose gradients with the rbcL transcript encoding the Rubisco large subunit

    National Research Council Canada - National Science Library

    Doron, Lior; Segal, Na'ama; Gibori, Hadas; Shapira, Michal

    2014-01-01

    ...‐migrated with the rbcL transcript on heavy polysomes, and both BSD 2 and rbcL m RNA shifted to the lighter fractions under oxidizing conditions that repress the translation of the Rubisco large subunit (RbcL). This profile of co...

  19. Adhesion of Chlamydomonas microalgae to surfaces is switchable by light

    Science.gov (United States)

    Kreis, Christian Titus; Le Blay, Marine; Linne, Christine; Makowski, Marcin Michal; Bäumchen, Oliver

    2018-01-01

    Microalgae are photoactive microbes that live in liquid-infused environments, such as soil, temporary pools and rocks, where they encounter and colonize a plethora of surfaces. Their photoactivity manifests itself in a variety of processes, including light-directed motility (phototaxis), the growth of microalgal populations, and their photosynthetic machinery. Although microbial responses to light have been widely recognized, any influence of light on cell-surface interactions remains elusive. Here, we reveal that the unspecific adhesion of microalgae to surfaces can be reversibly switched on and off by light. Using a micropipette force spectroscopy technique, we measured in vivo single-cell adhesion forces and show that the microalga's flagella provide light-switchable adhesive contacts with the surface. This light-induced adhesion to surfaces is an active and completely reversible process that occurs on a timescale of seconds. Our results suggest that light-switchable adhesiveness is a natural functionality of microalgae to regulate the transition between the planktonic and the surface-associated state, which yields an adhesive adaptation to optimize the photosynthetic efficiency in conjunction with phototaxis.

  20. Dynamic curvature regulation accounts for the symmetric and asymmetric beats of Chlamydomonas flagella

    CERN Document Server

    Sartori, Pablo; Scholich, Andre; Jülicher, Frank; Howard, Jonathon

    2015-01-01

    Axonemal dyneins are the molecular motors responsible for the beating of cilia and flagella. These motors generate sliding forces between adjacent microtubule doublets within the axoneme, the motile cytoskeletal structure inside the flagellum. To create regular, oscillatory beating patterns, the activities of the axonemal dyneins must be coordinated both spatially and temporally. It is thought that coordination is mediated by stresses or strains that build up within the moving axoneme, but it is not known which components of stress or strain are involved, nor how they feed back on the dyneins. To answer this question, we used isolated, reactivate axonemes of the unicellular alga Chlamydomonas as a model system. We derived a theory for beat regulation in a two-dimensional model of the axoneme. We then tested the theory by measuring the beat waveforms of wild type axonemes, which have asymmetric beats, and mutant axonemes, in which the beat is nearly symmetric, using high-precision spatial and temporal imaging....

  1. Cell types and their status in Chlamydomonas-like algae (Chlorophyceae on agar medium culture

    Directory of Open Access Journals (Sweden)

    M.М. Pavlovska

    2014-04-01

    Full Text Available The classification of cell types under agar culture was proposed. Six cell morphotypes were allocated. The statuses were identified depending on the reduction of monade attributes of cells. The variants of transition from one cell morphotype to another under dissolving mucilage were shown. The monade, cocciod, palmeloid and gloeocysta morphotypes approximately equally represented in all clades. The asterococcus and mucogleocysta morphotypes presented only in Reinhardtinia аnd Oogamochlamydinia clades. Any morphotype isn’t typical for all clades of Chlamydomonas-like algae at once. The most of morphotypes numbers (5 from 6 are presented in Reinhardtinia clade. This demonstrates the diversity of the Reinhardtinia clade species. There are only one morphotype presented in Polytominia and Monadinia clades. There are four morphotypes presented in Oogamochlamydinia clade, three – in Moewusinia, two morphotypes – in Chloromonadinia.

  2. Inhomogeneous distribution of Chlamydomonas in a cylindrical container with a bubble plume

    Directory of Open Access Journals (Sweden)

    Yuki Nonaka

    2016-02-01

    Full Text Available Swimming microalgae show various taxes, such as phototaxis and gravitaxis, which sometimes result in the formation of a cell-rich layer or a patch in a suspension. Despite intensive studies on the effects of shear flow and turbulence on the inhomogeneous distribution of microalgae, the effect of a bubble plume has remained unclear. In this study, we used Chlamydomonas as model microalgae, and investigated the spatial distribution of cells in a cylindrical container with a bubble plume. The results illustrate that cells become inhomogeneously distributed in the suspension due to their motility and photo-responses. A vortical ring distribution was observed below the free surface when the bubble flow rate was sufficiently small. We performed a scaling analysis on the length scale of the vortical ring, which captured the main features of the experimental results. These findings are important in understanding transport phenomena in a microalgae suspension with a bubble plume.

  3. Chlamydomonas fla mutants reveal a link between deflagellation and intraflagellar transport.

    Science.gov (United States)

    Parker, Jeremy David Kirk; Quarmby, Lynne Marie

    2003-08-20

    Cilia and flagella are often lost in anticipation of mitosis or in response to stress. There are two ways that a cell can lose its flagella: resorption or deflagellation. Deflagellation involves active severing of the axoneme at the base of the flagellum; this process is defective in Chlamydomonas fa mutants. In contrast, resorption has been thought to occur as a consequence of constitutive disassembly at the tip in the absence of continued assembly, which requires intraflagellar transport (IFT). Chlamydomonas fla mutants are unable to build and maintain flagella due to defects in IFT. fla10 cells, which are defective in kinesin-II, the anterograde IFT motor, resorb their flagella at the restrictive temperature (33 degrees C), as previously reported. We find that in standard media containing approximately 300 microM calcium, fla10 cells lose flagella by deflagellation at 33 degrees C. This temperature-induced deflagellation of a fla mutant is not predicted by the IFT-based model for flagellar length control. Other fla mutants behave similarly, losing their flagella by deflagellation instead of resorption, if adequate calcium is available. These data suggest a new model whereby flagellar resorption involves active disassembly at the base of the flagellum via a mechanism with components in common with the severing machinery of deflagellation. As predicted by this model, we discovered that deflagellation stimuli induce resorption if deflagellation is blocked either by mutation in a FA gene or by lack of calcium. Further support for this model comes from our discovery that fla10-fa double mutants resorb their flagella more slowly than fla10 mutants. Deflagellation of the fla10 mutant at the restrictive temperature is indicative of an active disassembly signal, which can manifest as either resorption or deflagellation. We propose that when IFT is halted by either an inactivating mutation or a cellular signal, active flagellar disassembly is initiated. This active

  4. Survival and proliferation characteristics of the microalga Chlamydomonas sp. ICE-L after hypergravitational stress pretreatment

    Science.gov (United States)

    Gao, Zhengquan; Li, Demao; Meng, Chunxiao; Xu, Dong; Zhang, Xiaowen; Ye, Naihao

    2013-09-01

    Seeking extraterrestrial life, transferring between planets, even migrating to other planets attracts more and more attention of public and scientists. However, to make it clear for the ability to survive the forces studies is prerequisite to enable the speculations by natural means. Gravity is a critical force involved in all the life on Earth and, possibly, others planets. Organisms have been grown in microgravity habitats and in centrifuges to characterize the biological response to a range of gravitational forces and radiation levels in space and on Earth. However, little is known about the profiles of eukaryotic life under conditions of hyperacceleration attributable to extreme gravities. In this study, a eukaryotic extremophile, the Antarctic green microalga Chlamydomonas sp. ICE-L, showed amazing proliferation capacity during and after hypergravitational stress for 30 min to 48 h at 110,200, 423,400, and 670,800g. These extreme gravities also had profound effects on viability, reproduction rate, photosynthesis efficiency, and gene transcriptional expression of this microalga. Most notably, all three supergravities efficiently stimulated algal cell division, but the greater the centrifugal force and the longer the duration of treatment, the lower the viable rate and breeding potential of samples in the following incubation. These results illustrated Chlamydomonas sp. ICE-L is a useful eukaryotic model system candidate for space research. Further studies could provide new insight into the physical limits of life and its evolution and enhance the possibility for interstellar space travel and the quest for extraterrestrial life according to panspermia theory. Also, it indicated that life come from the outer space is not always prokaryotes but may be eukaryotes.

  5. Tolerance to cadmium in Chlamydomonas sp. (Chlorophyta) strains isolated from an extreme acidic environment, the Tinto River (SW, Spain)

    Energy Technology Data Exchange (ETDEWEB)

    Aguilera, Angeles [Centro de Astrobiologia, Instituto Nacional de Tecnica Aeroespacial, Carretera de Ajalvir Km 4, Torrejon de Ardoz, 28850 Madrid (Spain)]. E-mail: aaguilera@cbm.uam.es; Amils, Ricardo [Centro de Astrobiologia, Instituto Nacional de Tecnica Aeroespacial, Carretera de Ajalvir Km 4, Torrejon de Ardoz, 28850 Madrid (Spain); Centro de Biologia Molecular (UAM-CSIC), Universidad Autonoma de Madrid, Canto Blanco, 28049 Madrid (Spain)

    2005-11-30

    The effects of selected concentrations of Cd on the growth and ultrastructure of three strains of Chlamydomonas sp. isolated from a highly acidic river, Rio Tinto (SW Spain) were examined. The river is characterized by its extreme physico-chemical conditions in terms of low pH, mean 2.2 and high concentrations of heavy metals. Growth, Cd accumulation, chlorophyll a, influence of Fe in Cd toxicity and ultrastructural localization were determined. The strains were cultured in both, artificial chemically defined media as well as in natural water from the river. Since iron is the main component of the river water, the effect of different concentrations of this element in relation with Cd toxicity was also analysed. The three strains analysed showed comparable growth and ultrastructural changes. Cd concentration corresponding to 50% growth inhibition (EC{sub 5}) was 0.2 mM when cells were grown in artificial media. When cells were grown in natural water, no significant differences were found between the controls and the Cd supplemented media even at the highest concentration of 0.8 mM. At an inhibitory level of 0.1 mM of Cd, increasing the concentration of iron up to 90 or 180 mM resulted in a dramatic recovery in algal growth rates in artificial media, reaching normal growth curves. The accumulation of Cd depended on dose and time in the artificial media. The maximal accumulation of Cd was reached after 3 days for all Cd doses, and remained almost unchanged in the subsequent period of time. Chlorophyll a amount depended on dose but not on time in the artificial growth media. At the ultrastructural level, an increase in the periplasmalemmal space was observed due to the presence of a large number of vacuoles, together with a decrease in the relative volume of the nucleus when the cells were incubated in the presence of Cd. Pyrenoid and starch granules were observed and accumulation of spherical electron-dense bodies were also detected. X-ray spectra of these bodies for

  6. Renewable Bio-Solar Hydrogen Production: The Second Generation (Part B)

    Science.gov (United States)

    2015-03-20

    Growth of Chlamydomonas reinhardtii in acetate-free medium when co-cultured with alginate - encapsulated strains of Synechococcus sp. PCC 7002...2014. Growth of Chlamydomonas reinhardtii in acetate-free medium when co-cultured with alginate - encapsulated strains of Synechococcus sp. PCC 7002

  7. NCBI nr-aa BLAST: CBRC-FRUB-02-0434 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-FRUB-02-0434 gb|ABM66085.1| putative ferric chelate reductase [Chlamydomonas r...einhardtii] gb|EDP04183.1| ferric-chelate reductase/ oxidoreductase [Chlamydomonas reinhardtii] ABM66085.1 0.57 29% ...

  8. NCBI nr-aa BLAST: CBRC-PHAM-01-1025 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PHAM-01-1025 ref|XP_001697359.1| magnesium chelatase subunit H [Chlamydomonas ...reinhardtii] gb|EDP00299.1| magnesium chelatase subunit H [Chlamydomonas reinhardtii] XP_001697359.1 7e-05 47% ...

  9. The salvage/turnover/repair (STOR) model for uniparental inheritance in Chlamydomonas: DNA as a source of sustenance.

    Science.gov (United States)

    Sears, B B; VanWinkle-Swift, K

    1994-01-01

    The non-Mendelian inheritance of chloroplast genes in Chlamydomonas has engaged researchers for decades and has prompted numerous debates regarding molecular mechanisms and evolutionary significance. The hallmarks of chloroplast inheritance in Chlamydomonas are reviewed here, including observations on vegetative haploid cells, somatic hybrids, meiotic zygospores, and vegetative zygotes resulting from sexual reproduction. Models invoked to explain the typical uniparental maternal inheritance of chloroplast genes, and which center upon the presumed existence of sex-specific protectors and destroyers of chloroplast genomes, are briefly discussed. In an effort to bring together the diverse observations on chloroplast gene inheritance in somatic as well as sexual cells, a model is proposed that focuses on organelle DNA turnover as a source of sustenance for the cell during periods of starvation. The salvage/turnover/repair (STOR) model for chloroplast inheritance in Chlamydomonas proposes that as a consequence of the high ploidy of the chloroplast genome, many copies are dispensable; their degradation would provide nucleotides for recombination, repair, RNA synthesis and cell metabolism. The STOR model offers an alternative view of uniparental inheritance as a phenomenon of direct selective benefit to the organism rather than simply being of selfish benefit to the chloroplast genome. These concepts may also have application to other lower eukaryotes that have sexual reproduction coupled with an extended dormancy.

  10. Chlamydomonas fla mutants reveal a link between deflagellation and intraflagellar transport

    Directory of Open Access Journals (Sweden)

    Quarmby Lynne

    2003-08-01

    Full Text Available Abstract Background Cilia and flagella are often lost in anticipation of mitosis or in response to stress. There are two ways that a cell can lose its flagella: resorption or deflagellation. Deflagellation involves active severing of the axoneme at the base of the flagellum; this process is defective in Chlamydomonas fa mutants. In contrast, resorption has been thought to occur as a consequence of constitutive disassembly at the tip in the absence of continued assembly, which requires intraflagellar transport (IFT. Chlamydomonas fla mutants are unable to build and maintain flagella due to defects in IFT. Results fla10 cells, which are defective in kinesin-II, the anterograde IFT motor, resorb their flagella at the restrictive temperature (33°C, as previously reported. We find that in standard media containing ~300 microM calcium, fla10 cells lose flagella by deflagellation at 33°C. This temperature-induced deflagellation of a fla mutant is not predicted by the IFT-based model for flagellar length control. Other fla mutants behave similarly, losing their flagella by deflagellation instead of resorption, if adequate calcium is available. These data suggest a new model whereby flagellar resorption involves active disassembly at the base of the flagellum via a mechanism with components in common with the severing machinery of deflagellation. As predicted by this model, we discovered that deflagellation stimuli induce resorption if deflagellation is blocked either by mutation in a FA gene or by lack of calcium. Further support for this model comes from our discovery that fla10-fa double mutants resorb their flagella more slowly than fla10 mutants. Conclusions Deflagellation of the fla10 mutant at the restrictive temperature is indicative of an active disassembly signal, which can manifest as either resorption or deflagellation. We propose that when IFT is halted by either an inactivating mutation or a cellular signal, active flagellar disassembly

  11. Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella*

    Science.gov (United States)

    Lin, Jianfeng; Tritschler, Douglas; Song, Kangkang; Barber, Cynthia F.; Cobb, Jennifer S.; Porter, Mary E.; Nicastro, Daniela

    2011-01-01

    The directional flow generated by motile cilia and flagella is critical for many processes, including human development and organ function. Normal beating requires the control and coordination of thousands of dynein motors, and the nexin-dynein regulatory complex (N-DRC) has been identified as an important regulatory node for orchestrating dynein activity. The nexin link appears to be critical for the transformation of dynein-driven, linear microtubule sliding to flagellar bending, yet the molecular composition and mechanism of the N-DRC remain largely unknown. Here, we used proteomics with special attention to protein phosphorylation to analyze the composition of the N-DRC and to determine which subunits may be important for signal transduction. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry of WT and mutant flagellar axonemes from Chlamydomonas identified 12 N-DRC-associated proteins, including all seven previously observed N-DRC components. Sequence and PCR analyses identified the mutation responsible for the phenotype of the sup-pf-4 strain, and biochemical comparison with a radial spoke mutant revealed two components that may link the N-DRC and the radial spokes. Phosphoproteomics revealed eight proteins with phosphorylated isoforms for which the isoform patterns changed with the genotype as well as two components that may play pivotal roles in N-DRC function through their phosphorylation status. These data were assembled into a model of the N-DRC that explains aspects of its regulatory function. PMID:21700706

  12. Production of biodiesel from microalgae Chlamydomonas polypyrenoideum grown on dairy industry wastewater.

    Science.gov (United States)

    Kothari, Richa; Prasad, Ravindra; Kumar, Virendra; Singh, D P

    2013-09-01

    This study involves a process of phyco-remediation of dairy industry wastewater by algal strain Chlamydomonas polypyrenoideum. The results of selected algal strain indicated that dairy industry wastewater was good nutrient supplement for algal growth in comparable with BG-11 growth medium. Alga grown on dairy industry wastewater reduced the pollution load of nitrate (90%), nitrite (74%), phosphate (70%), chloride (61%), fluoride (58%), and ammonia (90%) on 10th day of its growth as compared to that of uninoculated wastewater. The lipid content of algal biomass grown on dairy wastewater on 10th day (1.6g) and 15th day (1.2 g) of batch experiment was found to be higher than the lipid content of algal biomass grown in BG-11 growth medium on 10th day (1.27 g) and 15th day (1.0 g) of batch experiment. The results on FTIR analysis of the extracted bio-oil through transesterification reaction was comparable with bio-oil obtained from other sources. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. The Antarctic Chlamydomonas raudensis: an emerging model for cold adaptation of photosynthesis.

    Science.gov (United States)

    Dolhi, Jenna M; Maxwell, Denis P; Morgan-Kiss, Rachael M

    2013-09-01

    Permanently cold habitats dominate our planet and psychrophilic microorganisms thrive in cold environments. Environmental adaptations unique to psychrophilic microorganisms have been thoroughly described; however, the vast majority of studies to date have focused on cold-adapted bacteria. The combination of low temperatures in the presence of light is one of the most damaging environmental stresses for a photosynthetic organism: in order to survive, photopsychrophiles (i.e. photosynthetic organisms adapted to low temperatures) balance temperature-independent reactions of light energy capture/transduction with downstream temperature-dependent metabolic processes such as carbon fixation. Here, we review research on photopsychrophiles with a focus on an emerging model organism, Chlamydomonas raudensis UWO241 (UWO241). UWO241 is a psychrophilic green algal species and is a member of the photosynthetic microbial eukaryote community that provides the majority of fixed carbon for ice-covered lake ecosystems located in the McMurdo Dry Valleys, Antarctica. The water column exerts a range of environmental stressors on the phytoplankton community that inhabits this aquatic ecosystem, including low temperatures, extreme shade of an unusual spectral range (blue-green), high salinity, nutrient deprivation and extremes in seasonal photoperiod. More than two decades of work on UWO241 have produced one of our most comprehensive views of environmental adaptation in a cold-adapted, photosynthetic microbial eukaryote.

  14. Ciprofloxacin toxicity and its co-metabolic removal by a freshwater microalga Chlamydomonas mexicana.

    Science.gov (United States)

    Xiong, Jiu-Qiang; Kurade, Mayur B; Kim, Jung Rae; Roh, Hyun-Seog; Jeon, Byong-Hun

    2017-02-05

    This study evaluated the toxicity and cellular stresses of ciprofloxacin (CIP) and its co-metabolic removal in a freshwater microalga Chlamydomonas mexicana. The toxicological effects of CIP on C. mexicana were assessed by studying the growth and biochemical characteristics of the microalga including total chlorophyll, carotenoid content, malondialdehyde (MDA) and superoxide dismutase (SOD) activity. The calculated effective concentration (EC50) of CIP on C. mexicana was 65±4mgL(-1) at 96h. The growth of C. mexicana was significantly inhibited at increased concentrations of CIP, showing 36±1, 75±3. and 88±3% inhibition at 40, 60 and 100mgL(-1) CIP, respectively, compared to the control after 11days of cultivation. The total chlorophyll, carotenoid, MDA and SOD activity were significantly increased as a result of relatively high concentrations of CIP stress. C. mexicana showed 13±1% removal of CIP (2mgL(-1)) after 11days of cultivation; however, the addition of an electron donor (sodium acetate, 4gL(-1)) highly enhanced the removal of CIP (2mgL(-1)) by>3-fold after 11days. Kinetic studies showed that removal of CIP followed a first-order model (R(2) 0.94-0.97) with the apparent rate constants (k) ranging from 0.0121 to 0.079 d(-1). Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Toxicity of atrazine and its bioaccumulation and biodegradation in a green microalga, Chlamydomonas mexicana.

    Science.gov (United States)

    Kabra, Akhil N; Ji, Min-Kyu; Choi, Jaewon; Kim, Jung Rae; Govindwar, Sanjay P; Jeon, Byong-Hun

    2014-11-01

    This study evaluated the toxicity of herbicide atrazine, along with its bioaccumulation and biodegradation in the green microalga Chlamydomonas mexicana. At low concentration (10 μg L(-1)), atrazine had no profound effect on the microalga, while higher concentrations (25, 50, and 100 μg L(-1)) imposed toxicity, leading to inhibition of cell growth and chlorophyll a accumulation by 22 %, 33 %, and 36 %, and 13 %, 24 %, and 27 %, respectively. Atrazine 96-h EC50 for C. mexicana was estimated to be 33 μg L(-1). Microalga showed a capability to accumulate atrazine in the cell and to biodegrade the cell-accumulated atrazine resulting in 14-36 % atrazine degradation at 10-100 μg L(-1). Increasing atrazine concentration decreased the total fatty acids (from 102 to 75 mg g(-1)) and increased the unsaturated fatty acid content in the microalga. Carbohydrate content increased gradually with the increase in atrazine concentration up to 15 %. This study shows that C. mexicana has the capability to degrade atrazine and can be employed for the remediation of atrazine-contaminated streams.

  16. Hydrogenase-Mediated Activities in Isolated Chloroplasts of Chlamydomonas reinhardii1

    Science.gov (United States)

    Maione, Theodore E.; Gibbs, Martin

    1986-01-01

    Isolated intact chloroplasts of Chlamydomonas reinhardii were found to catalyze photoreduction of CO2 in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when adapted under an atmosphere of H2 demonstrating the association of a hydrogenase and anaerobic adaptation system with these plastids. The specific activity of photoreduction was approximately one third that detected in cells and protoplasts. Photoreduction was found to have a lower osmoticum optimum relative to aerobically maintained chloroplasts (50 millimolar versus 120 millimolar mannitol). 3-Phosphoglycerate (3-PGA) stimulated photoreduction up to a peak at 0.25 millimolar beyond which inhibition was observed. In the absence of 3-PGA, inorganic phosphate had no effect on photoreduction but in the presence of 3-PGA, inorganic phosphate also stimulated the reaction. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone inhibited photoreduction but inhibition by the former could be partially overcome by exogenously added ATP. The intact plastid can also catalyze photoevolution of H2 while lysed chloroplast extracts catalyzed the reduction of methyl viologen by H2. Both reactions occurred at rates approximately one-third of those found in cells. The oxyhydrogen reaction in the presence or absence of CO2 was not detected. PMID:16664626

  17. Biosíntesis de Glutamato en "Chlamydomonas reinhardii" purificación y propiedades de las encimas con actividad Glutamato Sintasa

    OpenAIRE

    Márquez Cabeza, Antonio José

    1985-01-01

    De forma resumida, los puntos más importantes que aporta este trabajo son los siguientes: 1. Puesta a punto t caracterización de un ensayo adecuado para medir la actividad GOGAT en Chlamydomonas reinhardii, tanto in situ como in vitro. 2. Estudios in vivo de asimilación de amonio, utilizando distintas estirpes de Chlamydomonas reinhardii, y efecto de distintas condiciones metabólicas sobre el nivel de las actividades NADH- y Fd-GOGAT. 3. Purificación parcial de la NADH-GOGAT y estudio físi...

  18. Tying Down Loose Ends in the Chlamydomonas Genome: Functional Significance of Abundant Upstream Open Reading Frames

    Directory of Open Access Journals (Sweden)

    Frederick R. Cross

    2016-02-01

    Full Text Available The Chlamydomonas genome has been sequenced, assembled, and annotated to produce a rich resource for genetics and molecular biology in this well-studied model organism. The annotated genome is very rich in open reading frames upstream of the annotated coding sequence (‘uORFs’: almost three quarters of the assigned transcripts have at least one uORF, and frequently more than one. This is problematic with respect to the standard ‘scanning’ model for eukaryotic translation initiation. These uORFs can be grouped into three classes: class 1, initiating in-frame with the coding sequence (CDS (thus providing a potential in-frame N-terminal extension; class 2, initiating in the 5′ untranslated sequences (5UT and terminating out-of-frame in the CDS; and class 3, initiating and terminating within the 5UT. Multiple bioinformatics criteria (including analysis of Kozak consensus sequence agreement and BLASTP comparisons to the closely related Volvox genome, and statistical comparison to cds and to random sequence controls indicate that of ∼4000 class 1 uORFs, approximately half are likely in vivo translation initiation sites. The proposed resulting N-terminal extensions in many cases will sharply alter the predicted biochemical properties of the encoded proteins. These results suggest significant modifications in ∼2000 of the ∼20,000 transcript models with respect to translation initiation and encoded peptides. In contrast, class 2 uORFs may be subject to purifying selection, and the existent ones (surviving selection are likely inefficiently translated. Class 3 uORFs are found in more than half of transcripts, frequently multiple times per transcript; however, they are remarkably similar to random sequence expectations with respect to size, number, and composition, and therefore may in most cases be selectively neutral.

  19. Sulphate, more than a nutrient, protects the microalga Chlamydomonas moewusii from cadmium toxicity.

    Science.gov (United States)

    Mera, Roi; Torres, Enrique; Abalde, Julio

    2014-03-01

    Sulphur is an essential macroelement that plays important roles in living organisms. The thiol rich sulphur compounds, such as cysteine, γ-Glu-Cys, glutathione and phytochelatins participate in the tolerance mechanisms against cadmium toxicity. Plants, algae, yeasts and most prokaryotes cover their demand for reduced sulphur by reduction of inorganic sulphate. The aim of this study was to investigate, using a bifactorial experimental design, the effect of different sulphate concentrations in the nutrient solution on cadmium toxicity in the freshwater microalga Chlamydomonas moewusii. Cell growth, kinetic parameters of sulphate utilization and intracellular concentrations of low-molecular mass thiol compounds were determined. A mathematical model to describe the growth of this microalga based on the effects of sulphate and cadmium was obtained. An ANOVA revealed an interaction between them, 16% of the effect sizes was explained by this interaction. A higher amount of sulphate in the culture medium allowed a higher cadmium tolerance due to an increase in the thiol compound biosynthesis. The amount of low-molecular mass thiol compounds, mainly phytochelatins, synthesized by this microalga was significantly dependent on the sulphate and cadmium concentrations; the higher phytochelatin content was obtained in cultures with 4 mg Cd/L and 1mM sulphate. The maximum EC50 value (based on nominal cadmium concentration) reached for this microalga was 4.46 ± 0.42 mg Cd/L when the sulphate concentration added to the culture medium was also 1mM. An increase in the sulphate concentration, in deficient environments, could alleviate the toxic effect of this metal; however, a relative excess is also negative. The results obtained showed a substrate inhibition for this nutrient. An uncompetitive model for sulphate was chosen to establish the mathematical model that links both factors. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Preventive effect of the microalga Chlamydomonas debaryana on the acute phase of experimental colitis in rats.

    Science.gov (United States)

    Avila-Román, Javier; Talero, Elena; Alcaide, Antonio; Reyes, Carolina de Los; Zubía, Eva; García-Mauriño, Sofía; Motilva, Virginia

    2014-10-14

    Inflammatory bowel diseases (IBD) are characterised by chronic uncontrolled inflammation of intestinal mucosa. Diet and nutritional factors have emerged as possible interventions for IBD. Microalgae are rich sources of n-3 PUFA and derived oxylipins. Oxylipins are lipid mediators involved in the resolution of many inflammatory disorders. The aim of the present study was to investigate the effects of the oxylipin-containing biomass of the microalga Chlamydomonas debaryana and its major oxylipin constituent, (9Z,11E,13S,15Z)-13-hydroxyoctadeca-9,11,15-trienoic acid ((13S)-HOTE), on acute 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats. Lyophilised microalgal biomass and (13S)-HOTE were administered by oral route 48, 24 and 1 h before the induction of colitis and 24 h later, and the rats were killed after 48 h. The treatment with the lyophilised microalga and (13S)-HOTE improved body-weight loss and colon shortening, as well as attenuated the extent of colonic damage and increased mucus production. Cellular neutrophil infiltration, with the subsequent increase in myeloperoxidase levels induced by TNBS, were also reduced after the administration of the lyophilised microalga or (13S)-HOTE. The anti-inflammatory effects of these treatments were confirmed by the inhibition of colonic TNF-α production. Moreover, lyophilised microalga or (13S)-HOTE down-regulated cyclo-oxygenase-2 and inducible nitric oxide synthase expression. The present study was the first to show the prophylactic effects of a lyophilised biomass sample of the microalga C. debaryana and the oxylipin (13S)-HOTE on TNBS-induced acute colitis in rats. Our findings suggest that the microalga C. debaryana or derived oxylipins could be used as nutraceuticals in the treatment of the active phase of IBD.

  1. Productivity and selective accumulation of carotenoids of the novel extremophile microalga Chlamydomonas acidophila grown with different carbon sources in batch systems

    NARCIS (Netherlands)

    Cuaresma, M.; Casal, C.; Forján, E.; Vílchez, C.

    2011-01-01

    Cultivation of extremophile microorganisms has attracted interest due to their ability to accumulate highvalue compounds. Chlamydomonas acidophila is an acidophile green microalga isolated by our group from Tinto River, an acidic river that flows down from the mining area in Huelva, Spain. This

  2. Chlamydomonas DYX1C1/PF23 is essential for axonemal assembly and proper morphology of inner dynein arms.

    Directory of Open Access Journals (Sweden)

    Ryosuke Yamamoto

    2017-09-01

    Full Text Available Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA as well as a fraction of the outer dynein arms (ODA. A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly.

  3. Effect of Temperature and light intensity on growth and Photosynthetic Activity of Chlamydomonas reinhard II; Efecto de la temperatura e intensidad luminosa sobre el crecimiento y actividad fotosintetica del alga Chlamydomonas Reinhardt II

    Energy Technology Data Exchange (ETDEWEB)

    Alfonsel Jaen, M.; Fernandez Gonzalez, J.

    1985-07-01

    The effect of five temperatures (15,20,25,30 and 35 degree centigree) and two levels of illumination on growth and photosynthetic activity of Chlamydomonas reinhard II has been studied. The growth of the cultures was evaluated by optical density. Photosynthetic activity has been carried out studying either the assimilation rate of C0{sub 2} labelled with C-14 or the oxygen evolution by means of polarographic measurements. The maximum photosynthetic rate has been obtained at 25 degree centigree for the lower level of illumination (2400 lux) and at 35 degree centigree for the higher one (13200 lux) and at 35 degree centigree for the higher ono (13200 lux). These results suggest an interaction of temperature and illumination on photosynthetic activity. (Author) 37 refs.

  4. Association of the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardii with Photoreduction and the Oxyhydrogen Reaction 1

    Science.gov (United States)

    Maione, Theodore E.; Gibbs, Martin

    1986-01-01

    The hydrogenase-dependent processes, photoreduction and the dark oxyhydrogen reaction, both of which can support CO2 assimilation, were compared with aerobic photosynthesis and respiration for their sensitivity to electron transport inhibitors in cells and intact chloroplasts of Chlamydomonas reinhardii 11-32/6. Photoreduction but not photosynthesis was inhibited in chloroplasts and the oxyhydrogen reaction detected only in cells was inhibited up to 75 and 90%, respectively, by 150 micromolar rotenone, indicating the involvement of a NAD(P)H-plastoquinone oxidoreductase in the hydrogen utilizing pathways. The oxyhydrogen reaction coupled to CO2 fixation was inhibited more than 95% by 10 micromolar 2,5 - dibromo - 3 - methyl - 6 - isopropyl - p - benzoquinone (DBMIB), a concentration which did not affect respiratory activity. In cells, both photoreduction and the oxyhydrogen reaction exhibited a similar sensitivity to salicylhydroxamic acid (SHAM) showing approximately 90% inhibition by 7 millimolar concentration. Photosynthesis was inhibited only 30% by the same concentration of SHAM. Antimycin A (18 micromolar, 10 micrograms per milliliter) inhibited both photoreduction (80%) and the oxyhydrogen reaction (92%) in cells with the oxyhydrogen reaction being approximately 10 times more sensitive to lower concentrations of the inhibitor. Antimycin A at 18 micromolar concentration did not inhibit photosynthetic CO2 fixation unless the cells were adapted to an atmosphere of N2 and the reaction conducted anaerobically. Photosynthesis, photoreduction, and the oxyhydrogen reaction coupled to CO2 fixation were all inhibited greater than 90% by 10 micromolar carbonylcyanide-p-trifluoromethoxyphenylhydrazone. ATP added to chloroplasts adapted to an atmosphere of H2 could support CO2 uptake in the dark. These results are interpreted as evidence that photoreduction and the oxyhydrogen reaction involve some common components of thylakoidal electron transport pathways in

  5. Association of the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardii with Photoreduction and the Oxyhydrogen Reaction.

    Science.gov (United States)

    Maione, T E; Gibbs, M

    1986-02-01

    The hydrogenase-dependent processes, photoreduction and the dark oxyhydrogen reaction, both of which can support CO(2) assimilation, were compared with aerobic photosynthesis and respiration for their sensitivity to electron transport inhibitors in cells and intact chloroplasts of Chlamydomonas reinhardii 11-32/6. Photoreduction but not photosynthesis was inhibited in chloroplasts and the oxyhydrogen reaction detected only in cells was inhibited up to 75 and 90%, respectively, by 150 micromolar rotenone, indicating the involvement of a NAD(P)H-plastoquinone oxidoreductase in the hydrogen utilizing pathways. The oxyhydrogen reaction coupled to CO(2) fixation was inhibited more than 95% by 10 micromolar 2,5 - dibromo - 3 - methyl - 6 - isopropyl - p - benzoquinone (DBMIB), a concentration which did not affect respiratory activity. In cells, both photoreduction and the oxyhydrogen reaction exhibited a similar sensitivity to salicylhydroxamic acid (SHAM) showing approximately 90% inhibition by 7 millimolar concentration. Photosynthesis was inhibited only 30% by the same concentration of SHAM. Antimycin A (18 micromolar, 10 micrograms per milliliter) inhibited both photoreduction (80%) and the oxyhydrogen reaction (92%) in cells with the oxyhydrogen reaction being approximately 10 times more sensitive to lower concentrations of the inhibitor. Antimycin A at 18 micromolar concentration did not inhibit photosynthetic CO(2) fixation unless the cells were adapted to an atmosphere of N(2) and the reaction conducted anaerobically. Photosynthesis, photoreduction, and the oxyhydrogen reaction coupled to CO(2) fixation were all inhibited greater than 90% by 10 micromolar carbonylcyanide-p-trifluoromethoxyphenylhydrazone. ATP added to chloroplasts adapted to an atmosphere of H(2) could support CO(2) uptake in the dark. These results are interpreted as evidence that photoreduction and the oxyhydrogen reaction involve some common components of thylakoidal electron transport

  6. Copper Status of Exposed Microorganisms Influences Susceptibility to Metallic Nanoparticles

    Science.gov (United States)

    Reyes, Vincent C.; Spitzmiller, Melissa R.; Hong-Hermesdorf, Anne; Kropat, Janette; Damoiseaux, Robert D.; Merchant, Sabeeha S.; Mahendra, Shaily

    2017-01-01

    Although interactions of metallic nanoparticles (NP) with various microorganisms have been previously explored, few studies have examined how metal sensitivity impacts NP toxicity. Herein, we investigated the effects of copper nanoparticles’ (Cu-NPs) exposure to the model alga, Chlamydomonas reinhardtii, in the presence and absence of the essential micronutrient copper. The toxic ranges for Cu-NPs and the ionic control, CuCl2, were determined using a high-throughput ATP-based fluorescence assay. Cu-NPs caused similar mortality in copper-replete and copper-deplete cells (IC50: 14–16 mg/L), but were less toxic than the ionic control, CuCl2 (IC50: 7 mg/L). Using this concentration range, we assessed Cu-NP impacts to cell morphology, copper accumulation, chlorophyll content, and expression of stress genes under both copper supply states. Osmotic swelling, membrane damage, and chloroplast and organelle disintegration were observed by transmission electron microscopy at both conditions. Despite these similarities, copper-deplete cells showed greater accumulation of loosely bound and tightly bound copper after exposure to Cu-NPs. Furthermore, copper-replete cells experienced greater loss of chlorophyll content, 19 % for Cu-NPs, compared to only an 11% net decrease in copper-deplete cells. The tightly bound copper was bioavailable as assessed by reverse-transcriptase quantitative PCR analysis of CYC6, a biomarker for Cu-deficiency. The increased resistance of copper-deplete cells to Cu-NPs suggests that these cells potentially metabolize excess Cu-NPs or better manage sudden influxes of ions. Our findings recommend that toxicity assessments must account for the nutritional status of impacted organisms and use toxicity models based on estimations of the bioavailable fractions. PMID:26387648

  7. Copper status of exposed microorganisms influences susceptibility to metallic nanoparticles.

    Science.gov (United States)

    Reyes, Vincent C; Spitzmiller, Melissa R; Hong-Hermesdorf, Anne; Kropat, Janette; Damoiseaux, Robert D; Merchant, Sabeeha S; Mahendra, Shaily

    2016-05-01

    Although interactions of metallic nanoparticles (NPs) with various microorganisms have been previously explored, few studies have examined how metal sensitivity impacts NP toxicity. The present study investigated the effects of copper NPs (Cu-NP) exposure on the model alga Chlamydomonas reinhardtii in the presence and absence of the essential micronutrient copper. The toxic ranges for Cu-NPs and the ionic control, CuCl2 , were determined using a high-throughput adenosine triphosphate (ATP)-based fluorescence assay. The Cu-NPs caused similar mortality in copper-replete and copper-deplete cells (median inhibitory concentration [IC50]: 14-16 mg/L) but were less toxic than the ionic control, CuCl2 (IC50: 7 mg/L). Using this concentration range, the Cu-NP impacts on cell morphology, copper accumulation, chlorophyll content, and expression of stress genes under both copper supply states were assessed. Osmotic swelling, membrane damage, and chloroplast and organelle disintegration were observed by transmission electron microscopy at both conditions. Despite these similarities, copper-deplete cells showed greater accumulation of loosely bound and tightly bound copper after exposure to Cu-NPs. Furthermore, copper-replete cells experienced greater loss of chlorophyll content, 19% for Cu-NPs, compared with only an 11% net decrease in copper-deplete cells. The tightly bound copper was bioavailable as assessed by reverse-transcriptase quantitative polymerase chain reaction analysis of CYC6, a biomarker for Cu deficiency. The increased resistance of copper-deplete cells to Cu-NPs suggests that these cells potentially metabolize excess Cu-NPs or better manage sudden influxes of ions. The results suggest that toxicity assessments must account for the nutritional status of impacted organisms and use toxicity models based on estimations of the bioavailable fractions. © 2015 SETAC.

  8. Águas com predominância de Eutreptia lanowi steuer e Chlamydomonas reinhardi dangeard no plancton, na enseada de Inhauma, Baía de Guanabara

    Directory of Open Access Journals (Sweden)

    Lejeune P. H. de Oliveira

    1962-03-01

    Full Text Available In brackish waters of a creek of Guanabara Bay, the author points by the first time the presence of Chlamydomonas reinhardi, Eutreptia lanowi, Oscillatoria putrida, O. limosa, O. chlorina that were unknown in our waters; such biologic indicators proved themselves pollutional conditions, so bad a stark-mesosaprobic regime. Other news are plankton analysis by the Standar methods, of two most expressive samples of water masses;also the mobility of the plankters are measured in micra by second.

  9. A NIMA-related kinase, Fa2p, localizes to a novel site in the proximal cilia of Chlamydomonas and mouse kidney cells.

    Science.gov (United States)

    Mahjoub, Moe R; Qasim Rasi, M; Quarmby, Lynne M

    2004-11-01

    Polycystic kidney disease and related syndromes involve dysregulation of cell proliferation in conjunction with ciliary defects. The relationship between cilia and cell cycle is enigmatic, but it may involve regulation by the NIMA-family of kinases (Neks). We previously showed that the Nek Fa2p is important for ciliary function and cell cycle in Chlamydomonas. We now show that Fa2p localizes to an important regulatory site at the proximal end of cilia in both Chlamydomonas and a mouse kidney cell line. Fa2p also is associated with the proximal end of centrioles. Its localization is dynamic during the cell cycle, following a similar pattern in both cell types. The cell cycle function of Fa2p is kinase independent, whereas its ciliary function is kinase dependent. Mice with mutations in Nek1 or Nek8 have cystic kidneys; therefore, our discovery that a member of this phylogenetic group of Nek proteins is localized to the same sites in Chlamydomonas and kidney epithelial cells suggests that Neks play conserved roles in the coordination of cilia and cell cycle progression.

  10. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

    acer

    cells of Chlamydomonas reinhardtii CC-124. mitochondria (Merchant et ... hemocytometer under a light microscope .... (Table 1). For further investigation of the 12 colonies by ... is bombarded with the full brunt of solar UV (ultraviolet) radiation ...

  11. Life history consequences for Daphnia pulex feeding on nutrient-limited phytoplankton

    NARCIS (Netherlands)

    Lürling, M.; Van Donk, E.

    1997-01-01

    1. The growth and feeding of Daphnia pulex De Geer on different algal species was examined. The green algae Chlamydomonas reinhardtii Dangeard and Scenedesmus acutus Meyen, the diatom Synedra tenuissima Kutzing, the cryptophyte Cryptomonas pyrenoidifera Geitler and the cyanobacterium Microcystis

  12. Serpins in unicellular Eukarya, Archaea, and Bacteria:

    DEFF Research Database (Denmark)

    Roberts, T.H.; Hejgaard, Jørn; Saunders, N.F.W

    2004-01-01

    in unicellular eukaryotes: the green alga Chlamydomonas reinhardtii, the dinoflagellate Alexandrium tamarense, and the human pathogens Entamoeba spp., Eimera tenella, Toxoplasma gondii, and Giardia lamblia. We compare these sequences to others, particularly those in the complete genome sequences of Archaea...

  13. Biodegradation of carbamazepine using freshwater microalgae Chlamydomonas mexicana and Scenedesmus obliquus and the determination of its metabolic fate.

    Science.gov (United States)

    Xiong, Jiu-Qiang; Kurade, Mayur B; Abou-Shanab, Reda A I; Ji, Min-Kyu; Choi, Jaeyoung; Kim, Jong Oh; Jeon, Byong-Hun

    2016-04-01

    This study evaluated the toxicity and cellular stresses of carbamazepine (CBZ) on Chlamydomonas mexicana and Scenedesmus obliquus, and its biodegradation by both microalgal species. The growth of both microalgal species decreased with increase of CBZ concentration. The growth of S. obliquus was significantly inhibited (97%) at 200 mg CBZ L(-1), as compared to the control after 10days; whereas, C. mexicana showed 30% inhibition at the same experimental conditions. Biochemical characteristics including total chlorophyll, carotenoid contents and enzyme activities (SOD and CAT) for both species were affected by CBZ at relatively high concentration. C. mexicana and S. obliquus could achieve a maximum of 35% and 28% biodegradation of CBZ, respectively. Two metabolites (10,11-dihydro-10,11-expoxycarbamazepine and n-hydroxy-CBZ) were identified by UPLC-MS, as a result of CBZ biodegradation by C. mexicana. This study demonstrated that C. mexicana was more tolerant to CBZ and could be used for treatment of CBZ contaminated wastewater. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Effects of sodium sulfate on the freshwater microalga Chlamydomonas moewusii: implications for the optimization of algal culture media.

    Science.gov (United States)

    Mera, Roi; Torres, Enrique; Abalde, Julio

    2016-02-01

    The study of the microalgal growth kinetics is an indispensable tool in all fields of phycology. Knowing the optimal nutrient concentration is an important issue that will help to develop efficient growth systems for these microorganisms. Although nitrogen and phosphorus are well studied for this purpose, sulfur seems to be less investigated. Sulfate is a primary sulfur source used by microalgae; moreover, the concentration of this compound is increasing in freshwater systems due to pollution. The aim of this study was to investigate the effects of different sodium sulfate concentrations in the culture medium on growth and growth kinetics of the freshwater microalga Chlamydomonas moewusii. Production of biomass, chl content, kinetic equations, and a mathematical model that describe the microalgal growth in relation with the concentration of sodium sulfate were obtained. The lowest concentration of sodium sulfate allowing optimal growth was 0.1 mM. Concentrations higher than 3 mM generated a toxic effect. This work demonstrates that this toxic effect was not directly due to the excess of sulfate ion but by the elevation of the ionic strength. An inhibition model was successfully used to simulate the relationship between specific growth rate and sodium sulfate in this microalga. © 2015 Phycological Society of America.

  15. Assessing Effects and interactions among key variables affecting the growth of mixotrophic microalgae: pH, inoculum volume, and growth medium composition

    DEFF Research Database (Denmark)

    Ale, Marcel Tutor; Pinelo, Manuel; Meyer, Anne S.

    2014-01-01

    A 2(3) + 3 full factorial experimental design was used to evaluate growth rate and biomass productivity of four selected, high-biomass-yielding microalgae species,namely, Chlorella vulgaris (CV), Scenedesmus acutus (SA), Chlamydomonas reinhardtii (CR), and Chlamydomonas debaryana (CD), in mixtures...... of CV and CR slowed down in mixtures with high proportions of wastewater. However, the biomass productivity of SA was dependent on pH, while the growth of the other microalgae was independent of pH (7-9). When evaluating the influence of pH and proportion of medium, CD appeared most robust among...... text] ratio. Both SA and CV reduced ∼20-25% of the chemical oxygen demand (COD) contained in the wastewater. This study shows the remarkable influence of certain variables that are often ignored in the search for optimal conditions of microalgal growth and also reveals the importance of considering...

  16. Association of the chloroplastic respiratory and photosynthetic electron transport chains of Chlamydomonas reinhardii with photoreduction and the oxyhydrogen reaction

    Energy Technology Data Exchange (ETDEWEB)

    Maione, T.E.; Gibbs, M.

    1986-02-01

    The hydrogenase-dependent processes, photoreduction and the dark oxyhydrogen reaction, both of which can support CO/sub 2/ assimilation, were compared with aerobic photosynthesis and respiration for their sensitivity to electron transport inhibitors in cells and intact chloroplasts of Chlamydomonas reinhardii 11-32/6. Photoreduction but no photosynthesis was inhibited in chloroplasts and the oxyhydrogen reaction detected only in cells was inhibited up to 75 and 90%, respectively, by 150 micromolar rotenone, indicating the involvement of a NAD(P)H-plastoquinone oxidoreductase in the hydrogen utilizing pathways. The oxyhydrogen reaction coupled to CO/sub 2/ fixation was inhibited more than 95% by 10 micromolar 2,5 - dibromo - 3 - methyl - 6 - isopropyl - p - benzoquinone (DBMIB), a concentration which did not affect respiratory activity. In cells, both photoreduction and the oxyhydrogen reaction exhibited a similar sensitivity to salicylhydroxamic acid (SHAM) showing approximately 90% inhibition by 7 millimolar concentration. Photosynthesis was inhibited only 30% by the same concentration of SHAM. Antimycin A (18 micromolar, 10 micrograms per milliliter) inhibited both photoreduction (80%) and the oxyhydrogen reaction (92%) in cells with the oxyhydrogen reaction being approximately 10 times more sensitive to lower concentrations of the inhibitor. Antimycin A at 18 micromolar concentration did not inhibit photosynthetic CO/sub 2/ fixation unless the cells were adapted to an atmosphere of N/sub 2/ and the reaction conducted anaerobically. Photosynthesis, photoreduction, and the oxyhydrogen reaction coupled to CO/sub 2/ fixation were all inhibited greater than 90% by 10 micromolar carbonylcyanide-p-trifluoromethoxyphenylhydrazone. ATP added to chloroplasts adapted to an atmosphere of H/sub 2/ could support CO/sub 2/ uptake in the dark.

  17. Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double-strand break in the genome.

    Science.gov (United States)

    Odom, Obed W; Baek, Kwang-Hyun; Dani, Radhika N; Herrin, David L

    2008-03-01

    Certain group I introns insert into intronless DNA via an endonuclease that creates a double-strand break (DSB). There are two models for intron homing in phage: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR). The Cr.psbA4 intron homes efficiently from a plasmid into the chloroplast psbA gene in Chlamydomonas, but little is known about the mechanism. Analysis of co-transformants selected using a spectinomycin-resistant 16S gene (16S(spec)) provided evidence for both pathways. We also examined the consequences of the donor DNA having only one-sided or no homology with the psbA gene. When there was no homology with the donor DNA, deletions of up to 5 kb involving direct repeats that flank the psbA gene were obtained. Remarkably, repeats as short as 15 bp were used for this repair, which is consistent with the single-strand annealing (SSA) pathway. When the donor had one-sided homology, the DSB in most co-transformants was repaired using two DNAs, the donor and the 16S(spec) plasmid, which, coincidentally, contained a region that is repeated upstream of psbA. DSB repair using two separate DNAs provides further evidence for the SDSA pathway. These data show that the chloroplast can repair a DSB using short dispersed repeats located proximally, distally, or even on separate molecules relative to the DSB. They also provide a rationale for the extensive repertoire of repeated sequences in this genome.

  18. A Forward Genetic Screen and Whole Genome Sequencing Identify Deflagellation Defective Mutants in Chlamydomonas, Including Assignment of ADF1 as a TRP Channel

    Directory of Open Access Journals (Sweden)

    Laura K. Hilton

    2016-10-01

    Full Text Available With rare exception, ciliated cells entering mitosis lose their cilia, thereby freeing basal bodies to serve as centrosomes in the formation of high-fidelity mitotic spindles. Cilia can be lost by shedding or disassembly, but either way, it appears that the final release may be via a coordinated severing of the nine axonemal outer doublet microtubules linking the basal body to the ciliary transition zone. Little is known about the mechanism or regulation of this important process. The stress-induced deflagellation response of Chlamydomonas provides a basis to identifying key players in axonemal severing. In an earlier screen we uncovered multiple alleles for each of three deflagellation genes, ADF1, FA1, and FA2. Products of the two FA genes localize to the site of axonemal severing and encode a scaffolding protein and a member of the NIMA-related family of ciliary-cell cycle kinases. The identity of the ADF1 gene remained elusive. Here, we report a new screen using a mutagenesis that yields point mutations in Chlamydomonas, an enhanced screening methodology, and whole genome sequencing. We isolated numerous new alleles of the three known genes, and one or two alleles each of at least four new genes. We identify ADF1 as a TRP ion channel, which we suggest may reside at the flagellar transition zone.

  19. EFFECT OF TREATED DOMESTIC WASTEWATER USED AS CULTURE MEDIUM ON THE GROWTH AND PRODUCTIVITY OF Chlamydomonas sp. STRAIN ISOLATED FROM LANDFILL LEACHATE

    Directory of Open Access Journals (Sweden)

    Fábio de Farias Neves

    2013-07-01

    Full Text Available Microalgae have been culturing to fix carbon and produce biofuels from the biomass. However, it is important to develop low cost strategies for microalgae production in orther to make it a viable alternative of renewable energy. The present research studied the effect of treated wastewater used as an alternative culture medium for growth and productivity of a Chlamydomonas sp. strain isolated from landfills leachate of a treatment pond located in Southern Brazil. Three culture media were evaluated, the control consisted of synthetic TAP medium, other, consisting of 50% TAP medium and 50% wastewater, and another consisting of 100% wastewater. The growth parameters do not have significant difference among the three culture media. Also, productivity do not have significant difference among the cultures with TAP medium and with 100% wastewater, resulting in dry weight values of 1,4±0,14g/L and 1,3±0,19g/L respectively. The culture with 50% TAP medium and 50% wastewater showed the highest productivity, showing an average dry weight value of 1,7±0,07g/L. The results indicate that treated wastewater can be used as an alternative culture medium for Chlamydomonas sp. strain without negative effects on growth and productivity, and possible leading to a decrease in production costs.

  20. Dynamic metabolic profiling of the marine microalga Chlamydomonas sp. JSC4 and enhancing its oil production by optimizing light intensity.

    Science.gov (United States)

    Ho, Shih-Hsin; Nakanishi, Akihito; Ye, Xiaoting; Chang, Jo-Shu; Chen, Chun-Yen; Hasunuma, Tomohisa; Kondo, Akihiko

    2015-01-01

    Marine microalgae are among the most promising lipid sources for biodiesel production because they can be grown on nonarable land without the use of potable water. Marine microalgae also harvest solar energy efficiently with a high growth rate, converting CO2 into lipids stored in the cells. Both light intensity and nitrogen availability strongly affect the growth, lipid accumulation, and fatty acid composition of oleaginous microalgae. However, very few studies have systematically examined how to optimize lipid productivity by adjusting irradiance intensity, and the metabolic dynamics that may lead to improved lipid accumulation in microalgae have not been elucidated. Little is known about the mechanism of lipid synthesis regulation in microalgae. Moreover, few studies have assessed the potential of using marine microalgae as oil producers. In this work, a newly isolated marine microalga, Chlamydomonas sp. JSC4, was selected as a potential lipid producer, and the effect of photobioreactor operations on cell growth and lipid production was investigated. The combined effects of light intensity and nitrogen depletion stresses on growth and lipid accumulation were further explored in an effort to markedly improve lipid production and quality. The optimal lipid productivity and content attained were 312 mg L(-1) d(-1) and 43.1% per unit dry cell weight, respectively. This lipid productivity is the highest ever reported for marine microalgae. Metabolic intermediates were profiled over time to observe transient changes during lipid accumulation triggered by combined stresses. Finally, metabolite turnover was also assessed using an in vivo (13)C-labeling technique to directly measure the flow of carbon during lipid biosynthesis under stress associated with light intensity and nitrogen deficiency. This work demonstrates the synergistic integration of cultivation and dynamic metabolic profiling technologies to develop a simple and effective strategy for enhancing oil

  1. Calmodulin regulates a TRP channel (ADF1) and phospholipase C (PLC) to mediate elevation of cytosolic calcium during acidic stress that induces deflagellation in Chlamydomonas.

    Science.gov (United States)

    Wu, Qiong; Gao, Kang; Zheng, Shuzhi; Zhu, Xin; Liang, Yinwen; Pan, Junmin

    2018-01-29

    Calcium has been implicated in the motility, assembly, disassembly, and deflagellation of the eukaryotic flagellum or cilium (exchangeable terms). Calmodulin (CaM) is known to be critical for flagellar motility; however, it is unknown whether and how CaM is involved in other flagella-related activities. We have studied CaM in Chlamydomonas, a widely used organism for ciliary studies. CaM is present in the cell body and the flagellum, with enrichment in the basal body region. Loss of CaM causes shortening of the nucleus basal body connector and impairs flagellar motility and assembly but not flagellar disassembly. Moreover, the cam mutant is defective in pH shock-induced deflagellation. The mutant deflagellates, however, upon mechanical shearing and treatment with mastoparan or detergent undergo permeabilization in the presence of calcium, indicating the cam mutant is defective in elevations of cytosolic calcium induced by pH shock, rather than by the deflagellation machinery. Indeed, the cam mutant fails to produce inositol 1,4,5-trisphosphate. Biochemical and genetic analysis showed that CaM does not directly activate PLC. Furthermore, CaM interacts with ADF1, a transient receptor channel that functions in acid-induced calcium entry. Thus, CaM is a critical regulator of flagellar activities especially those involved in modulating calcium homeostasis during acidic stress.-Wu, Q., Gao, K., Zheng, S., Zhu, X., Liang, Y., Pan, J. Calmodulin regulates a TRP channel (ADF1) and phospholipase C (PLC) to mediate elevation of cytosolic calcium during acidic stress that induces deflagellation in Chlamydomonas.

  2. A two dimensional clinostat experiment for microalgae cultures - basic work for bio- regenerativ life support systems

    Science.gov (United States)

    Harting, Benjamin; Slenzka, Klaus

    2012-07-01

    To investigate the influence of microgravity environments on photosynthetic organisms we designed a 2 dimensional clinostatexperiment for a suspended cell culture of Chlamydomonas reinhardtii. A novel approach of online measurments concerning relevant parameters important for the clasification of photosynthesis was obtained. To adress the photosynthesis rate we installed and validated an optical mesurement system to monitor the evolution and consumption of dissolved oxygen. Simultaneously a PAM sensor to analyse the flourescence quantum yield of the photochemical reaction was integarted. Thus it was possible to directly classify important parameters of the phototrophic metabolism during clinorotation. The experiment design including well suited light conditions and further biochemical analysis were directly performed for microalgal cell cultures. Changes in the photosynthetic efficiancy of phototrophic cyanobacteria has been observed during parabolic flight campaign but the cause is already not understood. Explenations could be the dependency of gravitaxis by intracellular ionconcentartion or the existance of mechanosensitive ionchannels for example associated in chloroplasts of Chlamydomonas reinhardtii. The purpuse of the microalgal clinostat are studies in a qasi microgravity environment for the process design of future bioregenerative life suport systems in spaceflight missions. First results has indicated the need for special nourishment of the cell culture during microgravity experiments. Further data will be presented during the assembly.

  3. Productivity and selective accumulation of carotenoids of the novel extremophile microalga Chlamydomonas acidophila grown with different carbon sources in batch systems.

    Science.gov (United States)

    Cuaresma, María; Casal, Carlos; Forján, Eduardo; Vílchez, Carlos

    2011-01-01

    Cultivation of extremophile microorganisms has attracted interest due to their ability to accumulate high-value compounds. Chlamydomonas acidophila is an acidophile green microalga isolated by our group from Tinto River, an acidic river that flows down from the mining area in Huelva, Spain. This microalga accumulates high concentrations of lutein, a very well-known natural antioxidant. The aim of this study is to assess use of different carbon sources (CO(2), glucose, glycerol, starch, urea, and glycine) for efficient growth of and carotenoid production by C. acidophila. Our results reveal that growth of the microalga on different carbon sources resulted in different algal biomass productivities, urea being as efficient as CO(2) when used as sole carbon source (~20 g dry biomass m(-2) day(-1)). Mixotrophic growth on glucose was also efficient in terms of biomass production (~14 g dry biomass m(-2) day(-1)). In terms of carotenoid accumulation, mixotrophic growth on urea resulted in even higher productivity of carotenoids (mainly lutein, probably via α-carotene) than obtained with photoautotrophic cultures (70% versus 65% relative abundance of lutein, respectively). The accumulated lutein concentrations of C. acidophila reported in this work (about 10 g/kg dry weight, produced in batch systems) are among the highest reported for a microalga. Glycerol and glycine seem to enhance β-carotene biosynthesis, and when glycine is used as carbon source, zeaxanthin becomes the most accumulated carotenoid in the microalga. Strategies for production of lutein and zeaxanthin are suggested based on the obtained results.

  4. Global Metabolic Regulation of the Snow Alga Chlamydomonas nivalis in Response to Nitrate or Phosphate Deprivation by a Metabolome Profile Analysis

    Directory of Open Access Journals (Sweden)

    Na Lu

    2016-05-01

    Full Text Available In the present work, Chlamydomonas nivalis, a model species of snow algae, was used to illustrate the metabolic regulation mechanism of microalgae under nutrient deprivation stress. The seed culture was inoculated into the medium without nitrate or phosphate to reveal the cell responses by a metabolome profile analysis using gas chromatography time-of-flight mass spectrometry (GC/TOF-MS. One hundred and seventy-one of the identified metabolites clustered into five groups by the orthogonal partial least squares discriminant analysis (OPLS-DA model. Among them, thirty of the metabolites in the nitrate-deprived group and thirty-nine of the metabolites in the phosphate-deprived group were selected and identified as “responding biomarkers” by this metabolomic approach. A significant change in the abundance of biomarkers indicated that the enhanced biosynthesis of carbohydrates and fatty acids coupled with the decreased biosynthesis of amino acids, N-compounds and organic acids in all the stress groups. The up- or down-regulation of these biomarkers in the metabolic network provides new insights into the global metabolic regulation and internal relationships within amino acid and fatty acid synthesis, glycolysis, the tricarboxylic acid cycle (TCA and the Calvin cycle in the snow alga under nitrate or phosphate deprivation stress.

  5. A horizontally acquired group II intron in the chloroplast psbA gene of a psychrophilic Chlamydomonas: in vitro self-splicing and genetic evidence for maturase activity.

    Science.gov (United States)

    Odom, Obed W; Shenkenberg, David L; Garcia, Joshua A; Herrin, David L

    2004-07-01

    The majority of known group II introns are from chloroplast genomes, yet the first self-splicing group II intron from a chloroplast gene was reported only recently, from the psbA gene of the euglenoid, Euglena myxocylindracea. Herein, we describe a large (2.6-kb) group II intron from the psbA gene (psbA1) of a psychrophilic Chlamydomonas sp. from Antarctica that self-splices accurately in vitro. Remarkably, this intron, which also encodes an ORF with putative reverse transcriptase, maturase, and endonuclease domains, is in the same location, and is related to the E. myxocylindracea intron, as well as to group IIB2 introns from cyanobacteria. In vitro self-splicing of Chs.psbA1 occurred via a lariat, and required Mg(2+) (>12 mM) and NH(4)(+). Self-splicing was improved by deleting most of the ORF and by using pre-RNAs directly from transcription reactions, suggestive of a role for folding during transcription. Self-splicing of Chs.psbA1 pre-RNAs showed temperature optima of ~44 degrees C, but with a broad shoulder on the low side of the peak; splicing was nearly absent at 50 degrees C, indicative of thermolability. Splicing of wild-type Chs.psbA1 also occurred in Escherichia coli, but not when the ORF was disrupted by mutations, providing genetic evidence that it has maturase activity. This work provides the first description of a ribozyme from a psychrophilic organism. It also appears to provide a second instance of interkingdom horizontal transfer of this group IIB2 intron (or a close relative) from cyanobacteria to chloroplasts.

  6. Phycoremediation of landfill leachate with the chlorophyte Chlamydomonas sp. SW15aRL and evaluation of toxicity pre and post treatment.

    Science.gov (United States)

    Paskuliakova, Andrea; McGowan, Ted; Tonry, Steve; Touzet, Nicolas

    2018-01-01

    Landfill leachate treatment is an ongoing challenge in the wastewater management of existing sanitary landfill sites due to the complex nature of leachates and their heavy pollutant load. There is a continuous interest in treatment biotechnologies with expected added benefits for resource recovery; microalgal bioremediation is seen as promising in this regard. Toxicity reduction of landfill leachate subsequent to phycoremediation was investigated in this study. The treatment eventuated from the growth of the ammonia tolerant microalgal strain Chlamydomonas sp. SW15aRL using a N:P ratio adjustment in diluted leachate for facilitating the process. Toxicity tests ranging over a number of trophic levels were applied, including bacterial-yeast (MARA), protistean (microalgae growth inhibition test), crustacean (daphnia, rotifer) and higher plant (monocot, dicot) assays. Ammonia nitrogen in the diluted landfill leachate containing up to 158mgl(-1) NH4(+)-N (60% dilution of the original) was reduced by 83% during the microalgal treatment. Testing prior to remediation indicated the highest toxicity in the crustacean assays Daphnia magna and Brachionus calyciflorus with EC50s at 24h of ~ 35% and 40% leachate dilution, respectively. A major reduction in toxicity was achieved with both bioassays post microalgal treatment with effects well below the EC20s. The microalgae inhibition test on the other hand indicated increased stimulation of growth after treatment as a result of toxicity reduction but also the presence of residual nutrients. Several concurrent processes of both biotic and abiotic natures contributed to pollutant reduction during the treatment. Modifying phosphate dosage especially seems to require further attention. As a by-product of the remediation process, up to 1.2gl(-1) of microalgal biomass was obtained with ~ 18% DW lipid content. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Morphological and ultrastructural characterization of the acidophilic and lipid-producer strain Chlamydomonas acidophila LAFIC-004 (Chlorophyta) under different culture conditions.

    Science.gov (United States)

    Souza, Luana Dos S; Simioni, Carmen; Bouzon, Zenilda L; Schneider, Rosana de Cassia da S; Gressler, Pablo; Miotto, Maria Cecília; Rossi, Marcio J; Rörig, Leonardo R

    2017-05-01

    Chlamydomonas acidophila LAFIC-004 is an acidophilic strain of green microalgae isolated from coal mining drainage. In the present work, this strain was cultivated in acidic medium (pH 3.6) under phototrophic, mixotrophic, and heterotrophic regimes to determine the best condition for growth and lipid production, simultaneously assessing possible morphological and ultrastructural alterations in the cells. For heterotrophic and mixotrophic treatments, two organic carbon sources were tested: 1 % glucose and 1 % sodium acetate. Lipid content and fatty acid profiles were only determined in phototrophic condition. The higher growth rates were achieved in phototrophic conditions, varying from 0.18 to 0.82 day-1. Glucose did not result in significant growth increase in either mixotrophic or heterotrophic conditions, and acetate proved to be toxic to the strain in both conditions. Oil content under phototrophic condition was 15.9 % at exponential growth phase and increased to 54.63 % at stationary phase. Based on cell morphology (flow cytometry and light microscopy) and ultrastructure (transmission electron microscopy), similar characteristics were observed between phototrophic and mixotrophic conditions with glucose evidencing many lipid bodies, starch granules, and intense fluorescence. Under the tested conditions, mixotrophic and heterotrophic modes did not result in increased neutral lipid fluorescence. It can be concluded that the strain is a promising lipid producer when grown until stationary phase in acidic medium and under a phototrophic regime, presenting a fatty acid profile suitable for biodiesel production. The ability to grow this strain in acidic mining residues suggests a potential for bioremediation with production of useful biomass.

  8. Flagellar Kinematics and Swimming of Algal Cells in Viscoelastic Fluids

    Science.gov (United States)

    Qin, B.; Gopinath, A.; Yang, J.; Gollub, J. P.; Arratia, P. E.

    2015-03-01

    The motility of microorganisms is influenced greatly by their hydrodynamic interactions with the fluidic environment they inhabit. We show by direct experimental observation of the bi-flagellated alga Chlamydomonas reinhardtii that fluid elasticity and viscosity strongly influence the beating pattern - the gait - and thereby control the propulsion speed. The beating frequency and the wave speed characterizing the cyclical bending are both enhanced by fluid elasticity. Despite these enhancements, the net swimming speed of the alga is hindered for fluids that are sufficiently elastic. The origin of this complex response lies in the interplay between the elasticity-induced changes in the spatial and temporal aspects of the flagellar cycle and the buildup and subsequent relaxation of elastic stresses during the power and recovery strokes.

  9. Entrainment dominates the interaction of microalgae with micron-sized objects

    CERN Document Server

    Jeanneret, Raphaël; Polin, Marco

    2016-01-01

    The incessant activity of swimming microorganisms has a direct physical effect on surrounding microscopic objects, leading to enhanced diffusion far beyond the level of Brownian motion with possible influences on the spatial distribution of non-motile planktonic species and particulate drifters. Here we study in detail the effect of eukaryotic flagellates, represented by the green microalga Chlamydomonas reinhardtii, on microparticles. Macro- and micro-scopic experiments reveal that microorganism-colloid interactions are dominated by rare close encounters leading to large displacements through direct entrainment. Simulations and theoretical modelling show that the ensuing particle dynamics can be understood in terms of a simple jump-diffusion process, combining standard diffusion with Poisson-distributed jumps. This heterogeneous dynamics is likely to depend on generic features of the near-field of swimming microorganisms with front-mounted flagella.

  10. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Biosciences. Basuthkar J Rao. Articles written in Journal of Biosciences. Volume 26 Issue 5 December 2001 pp 583-594 Articles. Local repeat sequence organization of an intergenic spacer in the chloroplast genome of Chlamydomonas reinhardtii leads to DNA expansion and sequence ...

  11. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

    The induced mutagenesis method for deriving pigment mutants of a green microalga, Chlamydomonas reinhardtii CC-124 and their pigment composition as well as ability to assess mutability of contaminated aquatic ecosystems were studied. In the present study, 14086 mutants (colonies) were obtained by exposure of the ...

  12. International Hydrogenase Conference (7th) Held at the University of Reading on August 24th to 29th 2004.

    Science.gov (United States)

    2004-08-19

    only hydrogenase and for the heterologous over-expressions of the Chlamydomonas reinhardtii and Scenedesmus obliquus HydAl Fe-only hydrogenases. The...unfolding the enzyme with urea in the presence mercaptoethanol [1 ]. The purified cofactor contained iron which remained bound even in the presence of 50

  13. Drosophila ABC Transporter DmHMT-1 Confers Tolerance to Cadmium.

    Science.gov (United States)

    Half molecule ATP-binding cassette transporters of the HMT1(heavy metal tolerance factor 1)subfamily are required for Cd2+ tolerance in Schizosaccharomyces pombe, Caenorhabditis elegans and Chlamydomonas reinhardtii, and have homologs in other species, including plants and humans. Based on studies i...

  14. Environmental, genetic and cellular toxicity of tenuazonic acid ...

    African Journals Online (AJOL)

    Alternaria alternata, an important pathogen of many plants, produces tenuazonic acid (TeA) with bioactivity to microbes, plants and animals. TeA is one of the main mycotoxin to humans and other organisms. Using Chlamydomonas reinhardtii, Vicia faba root tip and three mammalian normal cell lines as target materials, ...

  15. Scale-up aspects of photobioreactors : effects of mixing-induced light/dark cycles

    NARCIS (Netherlands)

    Janssen, M.; Bresser, de L.; Baijens, T.; Tramper, J.; Mur, L.R.; Snel, J.F.H.; Wijffels, R.H.

    2000-01-01

    The green micro-algae Chlamydomonas reinhardtii and Dunaliella tertiolecta were cultivated under medium-duration square-wave light/dark cycles with a cycle time of 15 s. These cycles were used to simulate the light regime experienced by micro-algae in externally-illuminated (sunlight) air-lift loop

  16. Ship Integration of Energy Scavenging Technology for Sea Base Operations

    Science.gov (United States)

    2009-07-01

    ocean and wastewater so it does not affect freshwater organisms.12 Algal fuels are the biological equivalent of solar panels in harvesting solar...for hydrogen production requires control of nutrient levels. New strains of mutant microalgae , like Chlamydomonas reinhardtii, can be used because

  17. Final technical report [Molecular genetic analysis of biophotolytic hydrogen production in green algae

    Energy Technology Data Exchange (ETDEWEB)

    Mets, Laurens

    2000-12-31

    The principal objective of this project was to identify genes necessary for biophotolytic hydrogen production in green algae, using Chlamydomonas reinhardtii as an experimental organism. The main strategy was to isolate mutants that are selectively deficient in hydrogen production and to genetically map, physically isolate, and ultimately sequence the affected genes.

  18. Environmental, genetic and cellular toxicity of tenuazonic acid ...

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... Alternaria alternata, an important pathogen of many plants, produces tenuazonic acid (TeA) with bioactivity to microbes, plants and animals. TeA is one of the main mycotoxin to humans and other organisms. Using Chlamydomonas reinhardtii, Vicia faba root tip and three mammalian normal cell lines.

  19. H2 production pathways in nutrient-replete mixotrophic Chlamydomonas cultures under low light. Response to the commentary article "On the pathways feeding the H2 production process in nutrient-replete, hypoxic conditions," by Alberto Scoma and Szilvia Z. Tóth.

    Science.gov (United States)

    González-Ballester, David; Jurado-Oller, Jose Luis; Galván, Aurora; Fernández, Emilio; Dubini, Alexandra

    2017-01-01

    A recent Commentary article entitled "On the pathways feeding the H2 production process in nutrient-replete, hypoxic conditions" by Dr. Scoma and Dr. Tóth, Biotechnology for Biofuels (2017), opened a very interesting debate about the H2 production photosynthetic-linked pathways occurring in Chlamydomonas cultures grown in acetate-containing media and incubated under hypoxia/anoxia conditions. This Commentary article mainly focused on the results of our previous article "Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures," by Jurado-Oller et al., Biotechnology for Biofuels (7, 2015; 8:149). Here, we review some previous knowledge about the H2 production pathways linked to photosynthesis in Chlamydomonas, especially focusing on the role of the PSII-dependent and -independent pathways in acetate-containing nutrient-replete cultures. The potential contributions of these pathways to H2 production under anoxia/hypoxia are discussed. Despite the fact that the PSII inhibitor DCMU is broadly used to discern between the two different photosynthetic pathways operating under H2 production conditions, its use may lead to distinctive conclusions depending on the growth conditions. The different potential sources of reductive power needed for the PSII-independent H2 production in mixotrophic nutrient-replete cultures are a matter of debate and conclusive evidences are still missing.

  20. Efeito do uso de efluente doméstico tratado, como meio de cultura, sobre o crescimento e produtividade no cultivo de chlamydomonas sp. Isolada de lixiviado de aterro sanitário

    Directory of Open Access Journals (Sweden)

    Fábio de Farias Neves

    2013-01-01

    Full Text Available É crescente a aplicação do cultivo de microalgas no campo da Biotecnologia Ambiental, buscando fixação de dióxido de carbono (CO2 e obtenção de energia da biomassa. Entretanto, para essas aplicações se tornarem economicamente viáveis, é necessáriaa adoção de estratégias para baixar o custo de produção de microalgas. A presente pesquisa avaliou o efeito do uso de efluentedoméstico tratado como meio de cultura alternativo de baixo custo sobre o crescimento e a produtividade do cultivo de Chlamydomonas sp. isolada de uma lagoa de tratamento de lixiviados de aterro sanitário, situada na região sul do Brasil. Três tratamentos foram testados: um controle utilizando o meio de cultura sintético TAP, outro com 50% do meio TAP e 50% do efluente e o terceiro com 100% do efluente. Não houve diferença significativa dos parâmetros de crescimento entre os tratamentos, assim como entre a produtividade alcançada nos cultivos com meio TAP e 100% efluente, atingindo valores de massa seca após 10 dias de cultivo de 1,4 ± 0,14g L-1 e 1,3 ± 0,19 g L-1 respectivamente. Já o cultivo em meio TAP com adição de 50% do efluente apresentou a maior produtividade, atingindo um valor de massa seca médio após 10 dias de cultivo de 1,7 ± 0,07 g L-1. Os resultados demonstram que o efluente doméstico tratado tem potencial para ser utilizado como meio de cultura para o cultivo das cepas de Chlamydomonas sp. sem prejudicar o crescimento e a produtividade Abstract Microalgae have been cultured increasingly in order to fix carbon dioxide and produce biofuels from the biomass. However, it is important to develop low cost strategies for microalgae production in order to turn this into a viable alternative of renewable energy. The present investigation studied the effect of treated wastewater used as an alternative culture medium for growth and productivity of a Chlamydomonas sp. strain isolated from landfills leachate of a treatment pond located in

  1. Transcriptome-Based Identification of the Desiccation Response Genes in Marine Red Algae Pyropia tenera (Rhodophyta) and Enhancement of Abiotic Stress Tolerance by PtDRG2 in Chlamydomonas.

    Science.gov (United States)

    Im, Sungoh; Lee, Ha-Nul; Jung, Hyun Shin; Yang, Sunghwan; Park, Eun-Jeong; Hwang, Mi Sook; Jeong, Won-Joong; Choi, Dong-Woog

    2017-06-01

    Pyropia tenera (Kjellman) are marine red algae that grow in the intertidal zone and lose more than 90% of water during hibernal low tides every day. In order to identify the desiccation response gene (DRG) in P. tenera, we generated 1,444,210 transcriptome sequences using the 454-FLX platform from the gametophyte under control and desiccation conditions. De novo assembly of the transcriptome reads generated 13,170 contigs, covering about 12 Mbp. We selected 1160 differentially expressed genes (DEGs) in response to desiccation stress based on reads per kilobase per million reads (RPKM) expression values. As shown in green higher plants, DEGs under desiccation are composed of two groups of genes for gene regulation networks and functional proteins for carbohydrate metabolism, membrane perturbation, compatible solutes, and specific proteins similar to higher plants. DEGs that show no significant homology with known sequences in public databases were selected as DRGs in P. tenera. PtDRG2 encodes a novel polypeptide of 159 amino acid residues locating chloroplast. When PtDRG2 was overexpressed in Chlamydomonas, the PtDRG2 confer mannitol and salt tolerance in transgenic cells. These results suggest that Pyropia may possess novel genes that differ from green plants, although the desiccation tolerance mechanism in red algae is similar to those of higher green plants. These transcriptome sequences will facilitate future studies to understand the common processes and novel mechanisms involved in desiccation stress tolerance in red algae.

  2. Development of a biosensor for environmental monitoring based on microalgae immobilized in silica hydrogels.

    Science.gov (United States)

    Ferro, Yannis; Perullini, Mercedes; Jobbagy, Matias; Bilmes, Sara A; Durrieu, Claude

    2012-12-06

    A new biosensor was designed for the assessment of aquatic environment quality. Three microalgae were used as toxicity bioindicators: Chlorella vulgaris, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii. These microalgae were immobilized in alginate and silica hydrogels in a two step procedure. After studying the growth rate of entrapped cells, chlorophyll fluorescence was measured after exposure to (3-(3,4-dichlorophenyl)-1,1-dimethylurea) (DCMU) and various concentrations of the common herbicide atrazine. Microalgae are very sensitive to herbicides and detection of fluorescence enhancement with very good efficiency was realized. The best detection limit was 0.1 µM, obtained with the strain C. reinhardtii after 40 minutes of exposure.

  3. Rationales and Approaches for Studying Metabolism in Eukaryotic Microalgae

    Directory of Open Access Journals (Sweden)

    Daniel Veyel

    2014-04-01

    Full Text Available The generation of efficient production strains is essential for the use of eukaryotic microalgae for biofuel production. Systems biology approaches including metabolite profiling on promising microalgal strains, will provide a better understanding of their metabolic networks, which is crucial for metabolic engineering efforts. Chlamydomonas reinhardtii represents a suited model system for this purpose. We give an overview to genetically amenable microalgal strains with the potential for biofuel production and provide a critical review of currently used protocols for metabolite profiling on Chlamydomonas. We provide our own experimental data to underpin the validity of the conclusions drawn.

  4. "Vision" in single-celled algae.

    Science.gov (United States)

    Kateriya, Suneel; Nagel, Georg; Bamberg, Ernst; Hegemann, Peter

    2004-06-01

    Photosynthetic unicellular algae have a unique visual system. In Chlamydomonas reinhardtii, the pigmented eye comprises the optical system and at least five different rhodopsin photoreceptors. Two of them, the channelrhodopsins, are rhodopsin-ion channel hybrids switched between closed and open states by photoisomerization of the attached retinal chromophore. They promise to become a useful tool for noninvasive control of membrane potential and intracellular ion concentrations.

  5. Development of a Biosensor for Environmental Monitoring Based on Microalgae Immobilized in Silica Hydrogels

    OpenAIRE

    Ferro, Yannis; Perullini, Mercedes; Jobbagy, Matias; Bilmes, Sara A.; Durrieu, Claude

    2012-01-01

    International audience; A new biosensor was designed for the assessment of aquatic environment quality. Three microalgae were used as toxicity bioindicators: Chlorella vulgaris, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii. These microalgae were immobilized in alginate and silica hydrogels in a two step procedure. After studying the growth rate of entrapped cells, chlorophyll fluorescence was measured after exposure to (3-(3,4-dichlorophenyl)-1,1-dimethylurea) (DCMU) and vari...

  6. Analytical approaches to photobiological hydrogen production in unicellular green algae

    OpenAIRE

    Hemschemeier, Anja; Melis, Anastasios; Happe, Thomas

    2009-01-01

    Several species of unicellular green algae, such as the model green microalga Chlamydomonas reinhardtii, can operate under either aerobic photosynthesis or anaerobic metabolism conditions. A particularly interesting metabolic condition is that of “anaerobic oxygenic photosynthesis”, whereby photosynthetically generated oxygen is consumed by the cell’s own respiration, causing anaerobiosis in the culture in the light, and induction of the cellular “hydrogen metabolism” process. The latter enta...

  7. [Technical support in the testing of microoganisms for their ability to accumulate strontium and cesium from aqueous solutions]. Final reports, Task order No. 2

    Energy Technology Data Exchange (ETDEWEB)

    1987-06-15

    This report describes the binding of cesium and strontium ions from aqueous solution in a variety of microorganisms. Data is provided on the absorption by Ashbya gossyppi, Chlorella pyrenoidosa, Candida sp. Ml13, Saccharomyces cerevisiae, Scenedesmus obliqus, Streptococcus mutans, Anabaena flosaquae, Escherichia coli, Streptomyces viridochromogenes, Chlamydomonas reinhardtii, Rhizopus oryzae, Bacillus megaterium, Micrococcus luteus, Zoogloea ramigera, Coelastrum proboscideum, Pseudomonas aeruginosa, Citrobacter freundii, Paecilomyces marquandi, and Caulobacter fusiformis.

  8. Alga-based HPV16 E7 vaccine elicits specific immune response in mice

    Czech Academy of Sciences Publication Activity Database

    Vlasák, Josef; Bříza, Jindřich; Ryba, Š.; Ludvíková, V.

    2013-01-01

    Roč. 34, č. 1 (2013), s. 141-148 ISSN 2249-7412 R&D Projects: GA AV ČR IAA500960903 Institutional support: RVO:60077344 Keywords : Chlamydomonas reinhardtii * chloroplast transformation * human papillomaviruses * E7 oncogene Subject RIV: EB - Genetics ; Molecular Biology http://pelagiaresearchlibrary.com/asian-journal-of-plant-science/vol3-iss1/AJPSR-2013-3-1-141-148.pdf

  9. Assessing Effects and interactions among key variables affecting the growth of mixotrophic microalgae: pH, inoculum volume, and growth medium composition.

    Science.gov (United States)

    Ale, M T; Pinelo, M; Meyer, A S

    2014-01-01

    A 2(3) + 3 full factorial experimental design was used to evaluate growth rate and biomass productivity of four selected, high-biomass-yielding microalgae species,namely, Chlorella vulgaris (CV), Scenedesmus acutus (SA), Chlamydomonas reinhardtii (CR), and Chlamydomonas debaryana (CD), in mixtures of growth medium (MWC) and wastewater at different proportions (from 20 to 50% of MWC) and at different pH (from 7 to 9). Multilinear regression analysis of the biomass productivity data showed that for SA and CD the biomass productivity was independent of the proportion of medium (MWC), while the growth of CV and CR slowed down in mixtures with high proportions of wastewater. However, the biomass productivity of SA was dependent on pH, while the growth of the other microalgae was independent of pH (7-9). When evaluating the influence of pH and proportion of medium, CD appeared most robust among the algae species, despite its lower biomass productivity. All the four species reduced 80-90% of the nitrate [Formula: see text] and 60-70% of the ammonia [Formula: see text] initially present in the wastewater:medium mixture, although the extent of the reduction was dependent on the initial [Formula: see text] ratio. Both SA and CV reduced ∼20-25% of the chemical oxygen demand (COD) contained in the wastewater. This study shows the remarkable influence of certain variables that are often ignored in the search for optimal conditions of microalgal growth and also reveals the importance of considering interactions among growth variables in potential applications at large scale, particularly in the field of bioremediation.

  10. AcEST: DK955670 [AcEST

    Lifescience Database Archive (English)

    Full Text Available rosophila mojavens... 39 0.31 tr|A9V1G6|A9V1G6_MONBE Predicted protein OS=Monosiga brevicollis... 37 0.46 tr|Q764P7|Q764P7_CHLRE Basa...l body protein OS=Chlamydomonas reinh... 35 2.6 tr|A8ID55|A8ID55_CHLRE Basal body p... HTHTHTH 105 >tr|Q764P7|Q764P7_CHLRE Basal body protein OS=Chlamydomonas reinhard...REEEARA 805 >tr|A8ID55|A8ID55_CHLRE Basal body protein OS=Chlamydomonas reinhardtii GN=BLD10 PE=4 SV=1 Lengt

  11. Unusual catalysts from molasses: synthesis, properties and application in obtaining biofuels from algae.

    Science.gov (United States)

    Samorì, Chiara; Torri, Cristian; Fabbri, Daniele; Falini, Giuseppe; Faraloni, Cecilia; Galletti, Paola; Spera, Silvia; Tagliavini, Emilio; Torzillo, Giuseppe

    2012-08-01

    Acid catalysts were prepared by sulfonation of carbon materials obtained from the pyrolysis of sugar beet molasses, a cheap, viscous byproduct in the processing of sugar beets into sugar. Conditions for the pyrolysis of molasses (temperature and time) influenced catalyst performance; the best combination came from pyrolysis at low temperature (420 °C) for a relatively long time (8-15 h), which ensured better stability of the final material. The most effective molasses catalyst was highly active in the esterification of fatty acids with methanol (100 % yield after 3 h) and more active than common solid acidic catalysts in the transesterification of vegetable oils with 25-75 wt % of acid content (55-96 % yield after 8 h). A tandem process using a solid acid molasses catalyst and potassium hydroxide in methanol was developed to de-acidificate and transesterificate algal oils from Chlamydomonas reinhardtii, Nannochloropsis gaditana, and Phaeodactylum tricornutum, which contain high amounts of free fatty acids. The amount of catalyst required for the de-acidification step was influenced by the chemical composition of the algal oil, thus operational conditions were determined not only in relation to free fatty acids content in the oil, but according to the composition of the lipid extract of each algal species. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Mono- and dichromatic LED illumination leads to enhanced growth and energy conversion for high-efficiency cultivation of microalgae for application in space.

    Science.gov (United States)

    Wagner, Ines; Steinweg, Christian; Posten, Clemens

    2016-08-01

    Illumination with red and blue photons is known to be efficient for cultivation of higher plants. For microalgae cultivation, illumination with specific wavelengths rather than full spectrum illumination can be an alternative where there is a lack of knowledge about achievable biomass yields. This study deals with the usage of color LED illumination to cultivate microalgae integrated into closed life support systems for outer space. The goal is to quantify biomass yields using color illumination (red, blue, green and mixtures) compared to white light. Chlamydomonas reinhardtii was cultivated in plate reactors with color compared to white illumination regarding PCE, specific pigment concentration and cell size. Highest PCE values were achieved under low PFDs with a red/blue illumination (680 nm/447 nm) at a 90 to 10% molar ratio. At higher PFDs saturation effects can be observed resulting from light absorption characteristics and the linear part of PI curve. Cell size and aggregation are also influenced by the applied light color. Red/blue color illumination is a promising option applicable for microalgae-based modules of life support systems under low to saturating light intensities and double-sided illumination. Results of higher PCE with addition of blue photons to red light indicate an influence of sensory pigments. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Trust and Influence

    Science.gov (United States)

    2013-03-06

    Influence effects • Psychological /Behavioral Effects of Novel Weaponry BRI •Cognitive mechanisms for influence •Socio-digital Influence BRI...Officer AFOSR/RTC Air Force Research Laboratory Trust and Influence Date:6 MAR 2013 Report Documentation Page Form ApprovedOMB No. 0704-0188 Public...00-2013 4. TITLE AND SUBTITLE Trust and Influence 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT

  14. Improving Influence Operations by Defining Influence and Influence Operations

    Science.gov (United States)

    2013-12-10

    the aforementioned lack of definition, and media-spun uncertainty, then Secretary of Defense Robert Gates attempted to reduce the stigma of one...despite the great expenditure of financial, intellectual , and human resources, these so-called influence efforts have at times been very ineffectual...objective, this form of influence is the focus of American operational art, the process whereby U.S. land forces locate, destroy, or otherwise disable an

  15. Effective viscosity of a suspension of flagellar-beating microswimmers: Three-dimensional modeling.

    Science.gov (United States)

    Jibuti, Levan; Zimmermann, Walter; Rafaï, Salima; Peyla, Philippe

    2017-11-01

    Micro-organisms usually can swim in their liquid environment by flagellar or ciliary beating. In this numerical work, we analyze the influence of flagellar beating on the orbits of a swimming cell in a shear flow. We also calculate the effect of the flagellar beating on the rheology of a dilute suspension of microswimmers. A three-dimensional model is proposed for Chlamydomonas Reinhardtii swimming with a breaststroke-like beating of two anterior flagella modeled by two counter-rotating fore beads. The active swimmer model reveals unusual angular orbits in a linear shear flow. Namely, the swimmer sustains orientations transiently across the flow. Such behavior is a result of the interplay between shear flow and the swimmer's periodic beating motion of flagella, which exert internal torques on the cell body. This peculiar behavior has some significant consequences on the rheological properties of the suspension. We calculate Einstein's viscosity of the suspension composed of such isolated modeled microswimmers (dilute case) in a shear flow. We use numerical simulations based on a Rotne-Prager-like approximation for hydrodynamic interaction between simplified flagella and the cell body. The results show an increased intrinsic viscosity for active swimmer suspensions in comparison to nonactive ones as well as a shear thinning behavior in accordance with previous experimental measurements [Phys. Rev. Lett. 104, 098102 (2010)10.1103/PhysRevLett.104.098102].

  16. Zeaxanthin Binds to Light-Harvesting Complex Stress-Related Protein to Enhance Nonphotochemical Quenching in Physcomitrella patens[W

    Science.gov (United States)

    Pinnola, Alberta; Dall’Osto, Luca; Gerotto, Caterina; Morosinotto, Tomas; Bassi, Roberto; Alboresi, Alessandro

    2013-01-01

    Nonphotochemical quenching (NPQ) dissipates excess energy to protect the photosynthetic apparatus from excess light. The moss Physcomitrella patens exhibits strong NPQ by both algal-type light-harvesting complex stress-related (LHCSR)–dependent and plant-type S subunit of Photosystem II (PSBS)-dependent mechanisms. In this work, we studied the dependence of NPQ reactions on zeaxanthin, which is synthesized under light stress by violaxanthin deepoxidase (VDE) from preexisting violaxanthin. We produced vde knockout (KO) plants and showed they underwent a dramatic reduction in thermal dissipation ability and enhanced photoinhibition in excess light conditions. Multiple mutants (vde lhcsr KO and vde psbs KO) showed that zeaxanthin had a major influence on LHCSR-dependent NPQ, in contrast with previous reports in Chlamydomonas reinhardtii. The PSBS-dependent component of quenching was less dependent on zeaxanthin, despite the near-complete violaxanthin to zeaxanthin exchange in LHC proteins. Consistent with this, we provide biochemical evidence that native LHCSR protein binds zeaxanthin upon excess light stress. These findings suggest that zeaxanthin played an important role in the adaptation of modern plants to the enhanced levels of oxygen and excess light intensity of land environments. PMID:24014548

  17. Adaptation and Influence

    DEFF Research Database (Denmark)

    Paster, Thomas

    on influence. These two dimensions - adaptation and influence - result in four ideal types: business-dominated social compromise, imposed social compromise, business dominance, and political confrontation. Examples from German welfare state history illustrate these four types. The paper suggests...

  18. Peer Influence and Recovery

    Science.gov (United States)

    Collier, Crystal; Simpson, Shelly; Najera, John; Weiner, Lauren

    2012-01-01

    Research has shown that peer influence can be negative, by increasing the likelihood that a youth will engage in high-risk behaviors and make risky decisions. However, peer influence can also be positive and protect a youth from these same high-risk activities. This article examines the extent of peer influence and then describes the Alternative…

  19. Social influence and obesity.

    Science.gov (United States)

    Hammond, Ross A

    2010-10-01

    To review a selection of research published in the last 12 months on the role of social influence in the obesity epidemic. Recent papers add evidence to previous work linking social network structures and obesity. Social norms, both eating norms and body image norms, are identified as one major source of social influence through networks. Social capital and social stress are additional types of social influence. There is increasing evidence that social influence and social network structures are significant factors in obesity. Deeper understanding of the mechanisms of action and dynamics of social influence, and its link with other factors involved in the obesity epidemic, is an important goal for further research.

  20. Testimonial a influencer marketing

    OpenAIRE

    Kúdelková, Andrea

    2017-01-01

    The topic of the diploma thesis is testimonial and influencer marketing. The aim of this work is to find out whether influencer increases the likelihood of purchasing a healthy nutritional product for Slovak women aged 20-40 years, and also whether Peter Sagana as a testimonial enhances brand credibility. The theoretical part deals with general communication and presentation of testimonial and marketing influence, their categories and examples. The practical part of the thesis is set into the...

  1. Noah Webster's Linguistic Influences.

    Science.gov (United States)

    Kreidler, Charles W.

    1998-01-01

    Examines ways in which Noah Webster's linguistic theories and work on dictionaries influenced North American English lexicography, arguing that his impact on American education was great because his spellers and dictionaries monopolized a rapidly growing market, and influence on lexicography was substantial because he insisted on the validity of…

  2. Can Television Influence People?

    Science.gov (United States)

    Comstock, George

    1985-01-01

    Discusses simultaneous airing by three commercial networks and influence on viewers' attitudes of The Great American Values Test, a 30-minute informational program designed to affect viewers' values about environmental issues, racial equality, and sexual equality. The program's effectiveness at influencing behavior was proven by a field…

  3. THE INFLUENCED FLOW REGIMES

    Directory of Open Access Journals (Sweden)

    Gavril PANDI

    2011-03-01

    Full Text Available The influenced flow regimes. The presence and activities ofhumanity influences the uniform environmental system, and in this context, therivers water resources. In concordance with this, the natural runoff regime suffersbigger and deeper changes. The nature of these changes depending on the type anddegree of water uses. The multitude of the use cause different types of influence,whit different quantitative aspects. In the same time, the influences havequalitative connotations, too, regarding to the modifications of the yearly watervolume runoff. So the natural runoff regime is modified. After analyzing thedistribution laws of the monthly runoff, there have been differenced four types ofinfluenced runoff regimes. In the excess type the influenced runoff is bigger thanthe natural, continuously in the whole year. The deficient type is characterized byinverse rapports like the first type, in the whole year. In the sinusoidal type, theinfluenced runoff is smaller than the natural in the period when the water isretained in the lake reservoirs, and in the depletion period the situation inverts. Atthe irregular type the ratio between influenced and natural runoff is changeable ina random meaner monthly. The recognition of the influenced regime and the gradeof influence are necessary in the evaluation and analysis of the usable hydrologicalriver resources, in the flood defence activities, in the complex scheme of thehydrographic basins, in the environment design and so on.

  4. Social Influence for Security

    Directory of Open Access Journals (Sweden)

    Florin Iftode

    2014-08-01

    Full Text Available The main aim of this work marks the reveling of scientific premises intended to structure the issue of social influence for security. The approach has as aim the identification of those elements that define and characterize the social influence in order to manage conflict, from the perspective of public communication. The proposed approach establishes some synthetic, clear boundaries through the method of research and analysis of the concept of security, social influence, revealing the specifics of public communication in conflict management.

  5. Sequence of the Gonium pectorale Mating Locus Reveals a Complex and Dynamic History of Changes in Volvocine Algal Mating Haplotypes

    Directory of Open Access Journals (Sweden)

    Takashi Hamaji

    2016-05-01

    Full Text Available Sex-determining regions (SDRs or mating-type (MT loci in two sequenced volvocine algal species, Chlamydomonas reinhardtii and Volvox carteri, exhibit major differences in size, structure, gene content, and gametolog differentiation. Understanding the origin of these differences requires investigation of MT loci from related species. Here, we determined the sequences of the minus and plus MT haplotypes of the isogamous 16-celled volvocine alga, Gonium pectorale, which is more closely related to the multicellular V. carteri than to C. reinhardtii. Compared to C. reinhardtii MT, G. pectorale MT is moderately larger in size, and has a less complex structure, with only two major syntenic blocs of collinear gametologs. However, the gametolog content of G. pectorale MT has more overlap with that of V. carteri MT than with C. reinhardtii MT, while the allelic divergence between gametologs in G. pectorale is even lower than that in C. reinhardtii. Three key sex-related genes are conserved in G. pectorale MT: GpMID and GpMTD1 in MT–, and GpFUS1 in MT+. GpFUS1 protein exhibited specific localization at the plus-gametic mating structure, indicating a conserved function in fertilization. Our results suggest that the G. pectorale–V. carteri common ancestral MT experienced at least one major reformation after the split from C. reinhardtii, and that the V. carteri ancestral MT underwent a subsequent expansion and loss of recombination after the divergence from G. pectorale. These data begin to polarize important changes that occurred in volvocine MT loci, and highlight the potential for discontinuous and dynamic evolution in SDRs.

  6. "Implementation and Social Influence"

    OpenAIRE

    2008-01-01

    This paper incorporates social psychology into implementation theory. Real individuals care not only about their material benefits but also about their social influence in terms of obedience and conformity. Using a continuous time horizon, we demonstrate a method of manipulating the decision-making process, according to which, an uninformed principal utilizes her/his power of social influence to incentivize multiple informed agents to make honest announcements. Following this method, we show ...

  7. Addiction and network influence

    OpenAIRE

    Popiel, Michał Ksawery

    2014-01-01

    Social networks are an important component in understanding the decision to consume addictive substances. They capture the role of limited access, peer influence, and social acceptance and tolerance. However, despite the empirical evidence of their role, they have been absent from theoretical models. This paper proposes a mechanism through which agents can influence each other in their decision to consume an addictive good. An agent's decision is sensitive to her state of addiction as well as...

  8. Social media influencer marketing

    OpenAIRE

    Isosuo, Heli

    2016-01-01

    The marketing field is changing simultaneously with the digital world. Social media is getting more and more important to marketers, and there is a need to stand out in the social media noise. Social media influencer marketing could be a good alternative to other types of marketing. A need from the consignor and the interest of the author were the motivations for conducting the study. Sääskilahti Consulting has a social media influencer network Somevaikuttajat, which is offering social media ...

  9. Trust and Influence

    Science.gov (United States)

    2012-03-05

    strikes, messaging, developmental activities) •Understanding the cognitive mechanisms that drive influence effects – identify the avenues of influence...competencies – trust building AF Tech Horizon‟s 2010 “In the near to mid-term, developing methods for establishing „certifiable trust in...Stokes – Dynamic Trust Model PI: Charlene Stokes (AFRL) Lab Task Drs. Lin ( Sunway U) and Chen (NICTA) Objective: Examine contextual factors

  10. Development of a Biosensor for Environmental Monitoring Based on Microalgae Immobilized in Silica Hydrogels

    Directory of Open Access Journals (Sweden)

    Claude Durrieu

    2012-12-01

    Full Text Available A new biosensor was designed for the assessment of aquatic environment quality. Three microalgae were used as toxicity bioindicators: Chlorella vulgaris, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii. These microalgae were immobilized in alginate and silica hydrogels in a two step procedure. After studying the growth rate of entrapped cells, chlorophyll fluorescence was measured after exposure to (3-(3,4-dichlorophenyl-1,1-dimethylurea (DCMU and various concentrations of the common herbicide atrazine. Microalgae are very sensitive to herbicides and detection of fluorescence enhancement with very good efficiency was realized. The best detection limit was 0.1 µM, obtained with the strain C. reinhardtii after 40 minutes of exposure.

  11. Expanding Fungal Diets Through Synthetic Algal-Fungal Mutualism

    Science.gov (United States)

    Sharma, Alaisha; Galazka, Jonathan (Editor)

    2015-01-01

    Fungi can synthesize numerous molecules with important properties, and could be valuable production platforms for space exploration and colonization. However, as heterotrophs, fungi require reduced carbon. This limits their efficiency in locations such as Mars, where reduced carbon is scarce. We propose a system to induce mutualistic symbiosis between the green algae Chlamydomonas reinhardtii and the filamentous fungi Neurospora crassa. This arrangement would mimic natural algal-fungal relationships found in lichens, but have added advantages including increased growth rate and genetic tractability. N. crassa would metabolize citrate (C6H5O7 (sup -3)) and release carbon dioxide (CO2) that C. reinhardtii would assimilate into organic sugars during photosynthesis. C. reinhardtii would metabolize nitrate (NO3-) and release ammonia (NH3) as a nitrogen source for N. crassa. A N. crassa mutant incapable of reducing nitrate will be used to force this interaction. This system eliminates the need to directly supply its participants with carbon dioxide and ammonia. Furthermore, the release of oxygen by C. reinhardtii via photosynthesis would enable N. crassa to respire. We hope to eventually create a system closer to lichen, in which the algae transfers not only nitrogen but reduced carbon, as organic sugars, to the fungus for growth and production of valuable compounds.

  12. The effect of rapamycin on biodiesel-producing protist Euglena gracilis.

    Science.gov (United States)

    Mukaida, Shiho; Ogawa, Takumi; Ohishi, Kazuko; Tanizawa, Yasuhiro; Ohta, Daisaku; Arita, Masanori

    2016-06-01

    Rapamycin induces autophagy with lipid remodeling in yeast and mammalian cells. To investigate the lipid biosynthesis of Euglena gracilis, rapamycin was supplemented in comparison with two model algae, Chlamydomonas reinhardtii and Cyanidioschyzon merolae. In Euglena, rapamycin induced the reduction of chlorophylls and the accumulation of neutral lipids without deterring its cell proliferation. Its lipidomic profile revealed that the fatty acid composition did not alter by supplementing rapamycin. In Chlamydomonas, however, rapamycin induced serious growth inhibition as reported elsewhere. With a lower concentration of rapamycin, the alga accumulated neutral lipids without reducing chlorophylls. In Cyanidioschyzon, rapamycin did not increase neutral lipids but reduced its chlorophyll content. We also tested fatty acid elongase inhibitors such as pyroxasulfone or flufenacet in Euglena with no significant change in its neutral lipid contents. In summary, controlled supplementation of rapamycin can increase the yield of neutral lipids while the scheme is not always applicable for other algal species.

  13. Evolution of an Expanded Sex Determining Locus in Volvox

    Science.gov (United States)

    Ferris, Patrick; Olson, Bradley J.S.C.; De Hoff, Peter L.; Douglass, Stephen; Diaz-Cano, David Casero; Prochnik, Simon; Geng, Sa; Rai, Rhitu; Grimwood, Jane; Schmutz, Jeremy; Nishii, Ichiro; Hamaji, Takashi; Nozaki, Hisayoshi; Pellegrini, Matteo; Umen, James G.

    2010-01-01

    Although dimorphic sexes have evolved repeatedly in multicellular eukaryotes, their origins are unknown. The mating locus (MT) of the sexually dimorphic multicellular green alga, Volvox carteri, specifies the production of eggs and sperm and has undergone a remarkable expansion and divergence relative to MT from Chlamydomonas reinhardtii, a closely related unicellular species that has equal-sized gametes. Transcriptome analysis revealed a rewired gametic expression program for Volvox MT genes relative to Chlamydomonas, and identified multiple gender-specific and sex-regulated transcripts. The retinoblastoma tumor suppressor homolog MAT3 is a Volvox MT gene that displays sexually regulated alternative splicing and evidence of gender-specific selection, both indicative of cooption into the sexual cycle. Thus, sex-determining loci impact the evolution of both sex-related and non-sex-related genes. PMID:20395508

  14. Resonance Raman and FTIR spectroscopic characterization of the closed and open states of channelrhodopsin-1.

    Science.gov (United States)

    Muders, Vera; Kerruth, Silke; Lórenz-Fonfría, Víctor A; Bamann, Christian; Heberle, Joachim; Schlesinger, Ramona

    2014-06-27

    Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a light-activated cation channel, which is a promising optogenetic tool. We show by resonance Raman spectroscopy and retinal extraction followed by high pressure liquid chromatography (HPLC) that the isomeric ratio of all-trans to 13-cis of solubilized channelrhodopsin-1 is with 70:30 identical to channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Critical frequency shifts in the retinal vibrations are identified in the Raman spectrum upon transition to the open (conductive P2(380)) state. Fourier transform infrared spectroscopy (FTIR) spectra indicate different structures of the open states in the two channelrhodopsins as reflected by the amide I bands and the protonation pattern of acidic amino acids. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Highly Efficient Sex Chromosome Interchanges Produced By I-CreI Expression in Drosophila

    OpenAIRE

    Maggert, Keith A; Golic, Kent G

    2005-01-01

    The homing endonuclease I-CreI recognizes a site in the gene encoding the 23S rRNA of Chlamydomonas reinhardtii. A very similar sequence is present in the 28S rRNA genes that are located on the X and Y chromosomes of Drosophila melanogaster. In this work we show that I-CreI expression in Drosophila is capable of causing induced DNA damage and eliciting cell cycle arrest. Expression also caused recombination between the X and Y chromosomes in the heterochromatic regions where the rDNA is locat...

  16. A Method for Microalgae Proteomics Analysis Based on Modified Filter-Aided Sample Preparation.

    Science.gov (United States)

    Li, Song; Cao, Xupeng; Wang, Yan; Zhu, Zhen; Zhang, Haowei; Xue, Song; Tian, Jing

    2017-11-01

    With the fast development of microalgal biofuel researches, the proteomics studies of microalgae increased quickly. A filter-aided sample preparation (FASP) method is widely used proteomics sample preparation method since 2009. Here, a method of microalgae proteomics analysis based on modified filter-aided sample preparation (mFASP) was described to meet the characteristics of microalgae cells and eliminate the error caused by over-alkylation. Using Chlamydomonas reinhardtii as the model, the prepared sample was tested by standard LC-MS/MS and compared with the previous reports. The results showed mFASP is suitable for most of occasions of microalgae proteomics studies.

  17. Hydrodynamics Versus Intracellular Coupling in the Synchronization of Eukaryotic Flagella

    NARCIS (Netherlands)

    Quaranta, G.; Aubin, M.E.; Tam, D.S.W.

    2015-01-01

    The influence of hydrodynamic forces on eukaryotic flagella synchronization is investigated by triggering phase locking between a controlled external flow and the flagella of C. reinhardtii. Hydrodynamic forces required for synchronization are over an order of magnitude larger than hydrodynamic

  18. Status, Numbers and Influence

    Science.gov (United States)

    Melamed, David; Savage, Scott V.

    2013-01-01

    We develop a theoretical model of social influence in n-person groups. We argue that disagreement between group members introduces uncertainty into the social situation, and this uncertainty motivates people to use status characteristics to evaluate the merits of a particular opinion. Our model takes the numerical distribution of opinions and the…

  19. Color: An Unsuspected Influence.

    Science.gov (United States)

    Scargall, Hollie

    1999-01-01

    Discusses the appropriate use of colors in school libraries. Highlights include how colors affect students' learning and behavior; influences on users' moods; users' ages; the use of colors to bring out the best physical attributes; and the use of color for floor coverings, window treatments, furnishings, and accessories. (LRW)

  20. Emotion is for influence

    NARCIS (Netherlands)

    van Kleef, G.A.; van Doorn, E.A.; Heerdink, M.W.; Koning, L.F.

    2011-01-01

    Functional approaches to emotion are rapidly gaining in popularity. Thus far the functions of emotions have been conceptualised and studied mainly at the intrapersonal level of analysis, the key question being how individuals are influenced by the emotions they experience. Relatively little is known

  1. The Drive to Influence

    Science.gov (United States)

    Rodriguez, Diego

    2017-01-01

    At the heart of the educational vocation is a drive to influence, to meaningfully affect the learning and development of others. For adult educators working in higher education, daily activities--from teaching classes to supervising student research to attending faculty meetings to sitting on advisory boards--are full of opportunities to…

  2. Purchase influence attempts

    NARCIS (Netherlands)

    Buijzen, M.A.; Valkenburg, P.M.

    2007-01-01

    Children have an important influence on a variety of purchasing decisions, with respect to both child-related purchases such as toys, snacks, or sweets and everyday household purchases such as breakfast products and desserts. As children grow older, they even gain a say in their parents' choice of

  3. Microbiologically Influenced Corrosion

    Science.gov (United States)

    2015-11-05

    inCluding oil and gas, from to 2011 [2]. Over that period of time approximately of all releases were attributed to corrosion . National ;sociati<Jn...c>fComJsicmEngineers (NACE) International [3] the cost of corrosion for onshore gas and liquid nsn1ission pipelines was $7 billion. However, there...are no statistics related to microbiologically influenced (MIC) of low alloy steel pipelines. Russian inves- [4] estimated that 30% of the corrosion

  4. Does Observation Influence Learning?

    OpenAIRE

    Olivier Armantier

    2001-01-01

    A common value auction experiment is run to compare the relative influence of observation and experience on learning. It is shown that the ex-post observation of opponents' actions and payoffs homogenizes behavior and accelerates learning toward the Nash equilibrium. Besides, experiential and observational learning are both relevant and of comparable magnitude. A general reinforcement model for continuous strategies, encompassing choice reinforcement learning, direction learning and payoff de...

  5. Factors influencing on lactation

    OpenAIRE

    M. V. Gmoshinskaya

    2013-01-01

    All factors influencing on lactation can be divided into 4 groups: organizational, medical, psychological and social. Among the last ones family support, solving of social problems in the family, formation of comfort conditions for breast-feeding, psychological state of the mother and her ability to relax during breast-feeding have the main significance. Formed maternity instinct and developed lactation dominant during pregnancy, understanding of the significance of breast-feeding among the f...

  6. Influence of flowing water

    Science.gov (United States)

    Wirtz, S.; Zell, A.; Wagner, C.; Seeger, M.; Ries, J. B.

    2009-04-01

    In literature authors often state that turbulence is an important or even "the critical" factor for soil erosion. But in erosion modelling the influence of turbulence is largely disregarded until now. The Reynolds number is often used as value of turbulence for a flowing liquid. In technical physics for example it is used to test the viscoelastic behavior of diluted polymer solutions and their elastic instabilities, in hydraulic engineering to characterise flow processes. Single factors that are included into the calculation of the Reynolds number (flow length, flow velocity and liqiud density) are used in many models but not the viscosity. In a study of rill erosion dynamics in Andalusia the Reynolds number is calculated for the collected samples. The scientific question reads as follow: Is there a correlation between turbulence (Reynolds number) and soil erosion (sediment concentration)? And what kind of influence has the turbulence to soil erosion? The factors for calculation of the Reynolds number are the liquid's density, a characteristic flow velocity, a characteristic flow length and the (dynamic) viscosity. The density of the sample is calculated using sediment concentration and the grain density, the flow velocity has been measured in the field experiments and the viscosity is measured by a cone-plate rheometer. For the flow length, a value of 1 meter is used following the assumption that the processes leading to the measured sediment concentrations mainly take place in the last meter before the sampling point. In the study it was ascertained that 1) high flow velocities cause high sediment concentrations and 2) viscosity increase with increasing sediment concentration. So there should be an influence on Reynolds number. Due to the fact that flow velocity is in the numerator and viscosity in the denominator of the Reynolds equation it could be expected that there are two different sections: In one section the Reynolds number increases with increasing

  7. INFLUENCERS :The Role of Social Influence in Marketing

    NARCIS (Netherlands)

    C. Du Plessis (Christilene)

    2017-01-01

    markdownabstractSocial influence is the corner stone of consumer psychology. In fact, in the last decade of the 19th century the study of consumer psychology emerged from an interest in advertising and its influence on people. Traditionally research on social influence has focused on understanding

  8. influence of gravity

    Directory of Open Access Journals (Sweden)

    Animesh Mukherjee

    1991-01-01

    Full Text Available Based upon Biot's [1965] theory of initial stresses of hydrostatic nature produced by the effect of gravity, a study is made of surface waves in higher order visco-elastic media under the influence of gravity. The equation for the wave velocity of Stonely waves in the presence of viscous and gravitational effects is obtained. This is followed by particular cases of surface waves including Rayleigh waves and Love waves in the presence of viscous and gravity effects. In all cases the wave-velocity equations are found to be in perfect agreement with the corresponding classical results when the effects of gravity and viscosity are neglected.

  9. Role of the Rubisco Small Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Spreitzer, Robert Joseph [Univ. of Nebraska, Lincoln, NE (United States)

    2016-11-05

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. However, it is a slow enzyme, and O2 competes with CO2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO2. If carboxylation could be increased or oxygenation decreased, an increase in net CO2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO2/O2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

  10. Markov Influence Diagrams.

    Science.gov (United States)

    Díez, Francisco J; Yebra, Mar; Bermejo, Iñigo; Palacios-Alonso, Miguel A; Calleja, Manuel Arias; Luque, Manuel; Pérez-Martín, Jorge

    2017-02-01

    Markov influence diagrams (MIDs) are a new type of probabilistic graphical model that extends influence diagrams in the same way that Markov decision trees extend decision trees. They have been designed to build state-transition models, mainly in medicine, and perform cost-effectiveness analyses. Using a causal graph that may contain several variables per cycle, MIDs can model various patient characteristics without multiplying the number of states; in particular, they can represent the history of the patient without using tunnel states. OpenMarkov, an open-source tool, allows the decision analyst to build and evaluate MIDs-including cost-effectiveness analysis and several types of deterministic and probabilistic sensitivity analysis-with a graphical user interface, without writing any code. This way, MIDs can be used to easily build and evaluate complex models whose implementation as spreadsheets or decision trees would be cumbersome or unfeasible in practice. Furthermore, many problems that previously required discrete event simulation can be solved with MIDs; i.e., within the paradigm of state-transition models, in which many health economists feel more comfortable.

  11. Vehicular road influence areas

    Science.gov (United States)

    Huertas, María E.; Huertas, José I.; Valencia, Alexander

    2017-02-01

    Vehicle operation over paved and unpaved roads is an emission source that significantly contributes to air pollution. Emissions are derived from vehicle exhaust pipes and re-suspension of particulate matter generated by wind erosion and tire to road surface interactions. Environmental authorities require a methodology to evaluate road impact areas, which enable managers to initiate counter-measures, particularly under circumstances where historic meteorological and/or air quality data is unavailable. The present study describes an analytical and experimental work developed to establish a simplified methodology to estimate the area influenced by vehicular roads. AERMOD was chosen to model pollutant dispersion generated by two roads of common attributes (straight road over flat terrain) under the effects of several arbitrary chosen weather conditions. The resulting pollutant concentration vs. Distance curves collapsed into a single curve when concentration and distance were expressed as dimensionless numbers and this curve can be described by a beta distribution function. This result implied that average concentration at a given distance was proportional to emission intensity and that it showed minor sensitivity to meteorological conditions. Therefore, road influence was defined by the area adjacent to the road limited by distance at which the beta distribution function equaled the limiting value specified by the national air quality standard for the pollutant under consideration.

  12. Purification and photobiochemical profile of photosystem 1 from a high-salt tolerant, oleaginous Chlorella (Trebouxiophycaea, Chlorophyta).

    Science.gov (United States)

    McConnell, Michael D; Lowry, David; Rowan, Troy N; van Dijk, Karin; Redding, Kevin E

    2015-06-01

    The eukaryotic green alga Chlamydomonas reinhardtii has been studied extensively within the biofuel industry as a model organism, as researchers look towards algae to provide chemical feedstocks (i.e., lipids) for the production of liquid transportation fuels. C. reinhardtii, however, is unsuitable for high-level production of such precursors due to its relatively poor lipid accumulation and fresh-water demand. In this study we offer insight into the primary light harvesting and electron transfer reactions that occur during phototropic growth in a high-salt tolerant strain of Chlorella (a novel strain introduced here as NE1401), a single-celled eukaryotic algae also in the phylum Chlorophyta. Under nutrient starvation many eukaryotic algae increase dramatically the amount of lipids stored in lipid bodies within their cell interiors. Microscopy and lipid analyses indicate that Chlorella sp. NE1401 may become a superior candidate for algal biofuels production. We have purified highly active Photosystem 1 (PS1) complexes to study in vitro, so that we may understand further the photobiochemisty of this promising biofuel producer and how its characteristics compare and contrast with that of the better understood C. reinhardtii. Our findings suggest that the PS1 complex from Chlorella sp. NE1401 demonstrates similar characteristics to that of C. reinhardtii with respect to light-harvesting and electron transfer reactions. We also illustrate that the relative extent of the light state transition performed by Chlorella sp. NE1401 is smaller compared to C. reinhardtii, although they are triggered by the same dynamic light stresses.

  13. French influence on Britain.

    Science.gov (United States)

    Walgate, Robert

    1985-02-21

    Jérôme Lejeune, the French geneticist who discovered the cause of Down's syndrome, is reported to have influenced British Members of Parliament in their vote to ban research on human embryos. Lejeune, brought to Britain to address Parliament by the anti-abortion lobby, said that embryonic research was unnecessary and would not add to knowledge about congenital defects, as argued in the Warnock Committee's report. Lejeune, who is a member of the French pro-life lobby, believes that embryos are persons and should not be subjected to experimentation. His current research concerns the chemical causes of congenital defects, which he predicts may be treatable with drugs in the future.

  14. Monitoring Microbially Influenced Corrosion

    DEFF Research Database (Denmark)

    Hilbert, Lisbeth Rischel

    Abstract Microbially influenced corrosion (MIC) of carbon steel may occur in media with microbiological activity of especially sulphate-reducing bacteria (SRB). The applicability and reliability of a number of corrosion monitoring techniques for monitoring MIC has been evaluated in experiments...... and diffusional effects and unreliable corrosion rates, when biofilm and ferrous sulphide corrosion products cover the steel surface. Corrosion rates can be overestimated by a factor of 10 to 100 by electrochemical techniques. Weight loss coupons and ER are recommended as necessary basic monitoring techniques....... EIS might be used for detection of MIC as the appearance of very large capacitances can be attributed to the combined ferrous sulphide and biofilm formation. Capacitance correlates directly with sulphide concentration in sterile sulphide media. Keywords: Corrosion monitoring, carbon steel, MIC, SRB...

  15. Developing Global Nurse Influencers.

    Science.gov (United States)

    Spies, Lori A

    2016-01-01

    How can universities create engaged citizens and global leaders? Each year, a select group of advanced practice nursing students at Baylor University Louise Herrington School of Nursing travel to Africa for a month-long clinical mission experience. Students work alongside local and missionary healthcare providers in a comprehensive Christian outreach to the community at a high-volume clinic. Creating rich learning experiences in a global setting in significant and sustainable ways is difficult, but intentionally focusing on what we are called to do and who we serve provides ballast for faculty and students. The success of the trip in preparing students to be global influencers is evident by the work graduates elect to do around the world, following graduation.

  16. Common Influence Join

    DEFF Research Database (Denmark)

    Yiu, Man Lung; Mamoulis, Nikos; Karras, Panagiotis

    2008-01-01

    We identify and formalize a novel join operator for two spatial pointsets P and Q. The common influence join (CIJ) returns the pairs of points (p,q),p isin P,q isin Q, such that there exists a location in space, being closer to p than to any other point in P and at the same time closer to q than...... to any other point in Q. In contrast to existing join operators between pointsets (i.e., e-distance joins and fc-closest pairs), CIJ is parameter- free, providing a natural join result that finds application in marketing and decision support. We propose algorithms for the efficient evaluation of CIJ......-demand, is very efficient in practice, incurring only slightly higher I/O cost than the theoretical lower bound cost for the problem....

  17. Assessing Undue Influence.

    Science.gov (United States)

    Plotkin, Daniel A; Spar, James E; Horwitz, Howard L

    2016-09-01

    A claim of undue influence (UI) often figures prominently in will and trust contests and in other legal matters. Mental health professionals (MHPs) are frequently asked to provide expert opinions on UI, but the task is challenging, because of the lack of a clear definition of UI, and conflicting and contradictory recommendations in the literature on the specific conduct of the MHP in rendering opinions on UI. Recently, however, a California statutory scheme on UI applicable in will, trust, conservatorship, and financial elder abuse cases was adopted, bringing greater clarity to the meaning of the construct. The clarification provides an opportunity to review the concern, and to recommend a set of principles to guide MHPs in the role of expert in such litigation. © 2016 American Academy of Psychiatry and the Law.

  18. Evolution of Daily Gene Co-expression Patterns from Algae to Plants

    Directory of Open Access Journals (Sweden)

    Pedro de los Reyes

    2017-07-01

    Full Text Available Daily rhythms play a key role in transcriptome regulation in plants and microalgae orchestrating responses that, among other processes, anticipate light transitions that are essential for their metabolism and development. The recent accumulation of genome-wide transcriptomic data generated under alternating light:dark periods from plants and microalgae has made possible integrative and comparative analysis that could contribute to shed light on the evolution of daily rhythms in the green lineage. In this work, RNA-seq and microarray data generated over 24 h periods in different light regimes from the eudicot Arabidopsis thaliana and the microalgae Chlamydomonas reinhardtii and Ostreococcus tauri have been integrated and analyzed using gene co-expression networks. This analysis revealed a reduction in the size of the daily rhythmic transcriptome from around 90% in Ostreococcus, being heavily influenced by light transitions, to around 40% in Arabidopsis, where a certain independence from light transitions can be observed. A novel Multiple Bidirectional Best Hit (MBBH algorithm was applied to associate single genes with a family of potential orthologues from evolutionary distant species. Gene duplication, amplification and divergence of rhythmic expression profiles seems to have played a central role in the evolution of gene families in the green lineage such as Pseudo Response Regulators (PRRs, CONSTANS-Likes (COLs, and DNA-binding with One Finger (DOFs. Gene clustering and functional enrichment have been used to identify groups of genes with similar rhythmic gene expression patterns. The comparison of gene clusters between species based on potential orthologous relationships has unveiled a low to moderate level of conservation of daily rhythmic expression patterns. However, a strikingly high conservation was found for the gene clusters exhibiting their highest and/or lowest expression value during the light transitions.

  19. Cross-kingdom comparison of transcriptomic adjustments to low-oxygen stress highlights conserved and plant-specific responses.

    Science.gov (United States)

    Mustroph, Angelika; Lee, Seung Cho; Oosumi, Teruko; Zanetti, Maria Eugenia; Yang, Huijun; Ma, Kelvin; Yaghoubi-Masihi, Arbi; Fukao, Takeshi; Bailey-Serres, Julia

    2010-03-01

    High-throughput technology has facilitated genome-scale analyses of transcriptomic adjustments in response to environmental perturbations with an oxygen deprivation component, such as transient hypoxia or anoxia, root waterlogging, or complete submergence. We showed previously that Arabidopsis (Arabidopsis thaliana) seedlings elevate the levels of hundreds of transcripts, including a core group of 49 genes that are prioritized for translation across cell types of both shoots and roots. To recognize low-oxygen responses that are evolutionarily conserved versus species specific, we compared the transcriptomic reconfiguration in 21 organisms from four kingdoms (Plantae, Animalia, Fungi, and Bacteria). Sorting of organism proteomes into clusters of putative orthologs identified broadly conserved responses associated with glycolysis, fermentation, alternative respiration, metabolite transport, reactive oxygen species amelioration, chaperone activity, and ribosome biogenesis. Differentially regulated genes involved in signaling and transcriptional regulation were poorly conserved across kingdoms. Strikingly, nearly half of the induced mRNAs of Arabidopsis seedlings encode proteins of unknown function, of which over 40% had up-regulated orthologs in poplar (Populus trichocarpa), rice (Oryza sativa), or Chlamydomonas reinhardtii. Sixteen HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes, including four that are Arabidopsis specific, were ectopically overexpressed and evaluated for their effect on seedling tolerance to oxygen deprivation. This allowed the identification of HUPs coregulated with genes associated with anaerobic metabolism and other processes that significantly enhance or reduce stress survival when ectopically overexpressed. These findings illuminate both broadly conserved and plant-specific low-oxygen stress responses and confirm that plant-specific HUPs with limited phylogenetic distribution influence low-oxygen stress endurance.

  20. Cross-Kingdom Comparison of Transcriptomic Adjustments to Low-Oxygen Stress Highlights Conserved and Plant-Specific Responses1[W][OA

    Science.gov (United States)

    Mustroph, Angelika; Lee, Seung Cho; Oosumi, Teruko; Zanetti, Maria Eugenia; Yang, Huijun; Ma, Kelvin; Yaghoubi-Masihi, Arbi; Fukao, Takeshi; Bailey-Serres, Julia

    2010-01-01

    High-throughput technology has facilitated genome-scale analyses of transcriptomic adjustments in response to environmental perturbations with an oxygen deprivation component, such as transient hypoxia or anoxia, root waterlogging, or complete submergence. We showed previously that Arabidopsis (Arabidopsis thaliana) seedlings elevate the levels of hundreds of transcripts, including a core group of 49 genes that are prioritized for translation across cell types of both shoots and roots. To recognize low-oxygen responses that are evolutionarily conserved versus species specific, we compared the transcriptomic reconfiguration in 21 organisms from four kingdoms (Plantae, Animalia, Fungi, and Bacteria). Sorting of organism proteomes into clusters of putative orthologs identified broadly conserved responses associated with glycolysis, fermentation, alternative respiration, metabolite transport, reactive oxygen species amelioration, chaperone activity, and ribosome biogenesis. Differentially regulated genes involved in signaling and transcriptional regulation were poorly conserved across kingdoms. Strikingly, nearly half of the induced mRNAs of Arabidopsis seedlings encode proteins of unknown function, of which over 40% had up-regulated orthologs in poplar (Populus trichocarpa), rice (Oryza sativa), or Chlamydomonas reinhardtii. Sixteen HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes, including four that are Arabidopsis specific, were ectopically overexpressed and evaluated for their effect on seedling tolerance to oxygen deprivation. This allowed the identification of HUPs coregulated with genes associated with anaerobic metabolism and other processes that significantly enhance or reduce stress survival when ectopically overexpressed. These findings illuminate both broadly conserved and plant-specific low-oxygen stress responses and confirm that plant-specific HUPs with limited phylogenetic distribution influence low-oxygen stress endurance. PMID:20097791

  1. Characterization of a subunit of the outer dynein arm docking complex necessary for correct flagellar assembly in Leishmania donovani.

    Directory of Open Access Journals (Sweden)

    Simone Harder

    Full Text Available BACKGROUND: In order to proceed through their life cycle, Leishmania parasites switch between sandflies and mammals. The flagellated promastigote cells transmitted by the insect vector are phagocytized by macrophages within the mammalian host and convert into the amastigote stage, which possesses a rudimentary flagellum only. During an earlier proteomic study of the stage differentiation of the parasite we identified a component of the outer dynein arm docking complex, a structure of the flagellar axoneme. The 70 kDa subunit of the outer dynein arm docking complex consists of three subunits altogether and is essential for the assembly of the outer dynein arm onto the doublet microtubule of the flagella. According to the nomenclature of the well-studied Chlamydomonas reinhardtii complex we named the Leishmania protein LdDC2. METHODOLOGY/PRINCIPAL FINDINGS: This study features a characterization of the protein over the life cycle of the parasite. It is synthesized exclusively in the promastigote stage and localizes to the flagellum. Gene replacement mutants of lddc2 show reduced growth rates and diminished flagellar length. Additionally, the normally spindle-shaped promastigote parasites reveal a more spherical cell shape giving them an amastigote-like appearance. The mutants lose their motility and wiggle in place. Ultrastructural analyses reveal that the outer dynein arm is missing. Furthermore, expression of the amastigote-specific A2 gene family was detected in the deletion mutants in the absence of a stage conversion stimulus. In vitro infectivity is slightly increased in the mutant cell line compared to wild-type Leishmania donovani parasites. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the correct assembly of the flagellum has a great influence on the investigated characteristics of Leishmania parasites. The lack of a single flagellar protein causes an aberrant morphology, impaired growth and altered infectiousness of the parasite.

  2. Influence at work and the desire for more influence

    DEFF Research Database (Denmark)

    Markey, Raymond; Ravenswood, Katherine; Webber, Don J.

    2013-01-01

    What determines whether workers want more influence in their workplace? Much of the literature on employee voice assumes that employees desire a say in how they do their work, and that where they lack influence they are more likely to desire a greater say. This econometric study of 536 Danish...... and New Zealand employees in four industries indicates that workers’ desire for more influence was not dependent on how much influence they thought they already had. What mattered was age, length of service and specific organisational characteristics. Those who wanted more influence were not learning new...... things and did not feel that they received sufficient information about the workplace, and those who felt appreciated by management did not desire more influence. The results support human resource management literature that suggests the importance of integrated and mutually supportive ‘bundles...

  3. Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

    Science.gov (United States)

    Mayfield, Stephen P.

    2010-03-16

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

  4. Dissecting the molecular mechanisms of intraflagellar transport in Chlamydomonas

    DEFF Research Database (Denmark)

    Pedersen, L. B.; Geimer, S.; Rosenbaum, J. L.

    2006-01-01

    and show that during retrograde IFT, kinesin-2 can exit the flagella independent of the cDynein1b light intermediate chain (LIC) and IFT particles. Furthermore, using biochemical approaches, we find that IFT complex B can associate with cDynein1b independent of complex A and cDynein1b LIC. Finally, using...... electron microscopy, we show that the IFT tip turnaround point most likely is localized distal to the plus end of the outer-doublet B MTs. Conclusion Our results support a model for IFT in which tip turnaround involves (1) dissociation of IFT complexes A and B and release of inactive cDynein1b from complex...

  5. Improving the optimum yield and growth of Chlamydomonas ...

    African Journals Online (AJOL)

    Both strains had a quicker growth rate in just 35 mM of acetate and 10 mM glycerol although feed of algal cells on 35 mM acetate produced more and quicker biomass. In use of 10 mM acetate in micro plate and tissue culture flasks, CW15 had a maximum growth rate of 5.3×104 and 1.3×104 cells/hour; while on use of 35 ...

  6. Improving the optimum yield and growth of Chlamydomonas ...

    African Journals Online (AJOL)

    N.T

    use in biofuel processing it plays a key role to convert triglycerides (TGA) into biodiesel through a mechanism called transesterification. Introducing the methanol into algal system for instance (0.01-718 mM) compared to other carbon sources (glucose, acetate glycerol) has an advantage as it can sterilize the media and ...

  7. Fast pesticide detection inside microfluidic device with integrated optical pH, oxygen sensors and algal fluorescence.

    Science.gov (United States)

    Tahirbegi, Islam Bogachan; Ehgartner, Josef; Sulzer, Philipp; Zieger, Silvia; Kasjanow, Alice; Paradiso, Mirco; Strobl, Martin; Bouwes, Dominique; Mayr, Torsten

    2017-02-15

    The necessities of developing fast, portable, cheap and easy to handle pesticide detection platforms are getting attention of scientific and industrial communities. Although there are some approaches to develop microchip based pesticide detection platforms, there is no compact microfluidic device for the complementary, fast, cheap, reusable and reliable analysis of different pesticides. In this work, a microfluidic device is developed for in-situ analysis of pesticide concentration detected via metabolism/photosynthesis of Chlamydomonas reinhardtii algal cells (algae) in tap water. Algae are grown in glass based microfluidic chip, which contains integrated optical pH and oxygen sensors in a portable system for on-site detection. In addition, intrinsic algal fluorescence is detected to analyze the pesticide concentration in parallel to pH and oxygen sensors with integrated fluorescence detectors. The response of the algae under the effect of different concentrations of pesticides is evaluated and complementary inhibition effects depending on the pesticide concentration are demonstrated. The three different sensors allow the determination of various pesticide concentrations in the nanomolar concentration range. The miniaturized system provides the fast quantification of pesticides in less than 10min and enables the study of toxic effects of different pesticides on Chlamydomonas reinhardtii green algae. Consequently, the microfluidic device described here provides fast and complementary detection of different pesticides with algae in a novel glass based microfluidic device with integrated optical pH, oxygen sensors and algal fluorescence. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Sexual reproduction and sex determination in green algae.

    Science.gov (United States)

    Sekimoto, Hiroyuki

    2017-05-01

    The sexual reproductive processes of some representative freshwater green algae are reviewed. Chlamydomonas reinhardtii is a unicellular volvocine alga having two mating types: mating type plus (mt+) and mating type minus (mt-), which are controlled by a single, complex mating-type locus. Sexual adhesion between the gametes is mediated by sex-specific agglutinin molecules on their flagellar membranes. Cell fusion is initiated by an adhesive interaction between the mt+ and mt- mating structures, followed by localized membrane fusion. The loci of sex-limited genes and the conformation of sex-determining regions have been rearranged during the evolution of volvocine algae; however, the essential function of the sex-determining genes of the isogamous unicellular Chlamydomonas reinhardtii is conserved in the multicellular oogamous Volvox carteri. The sexual reproduction of the unicellular charophycean alga, Closterium peracerosum-strigosum-littorale complex, is also focused on here. The sexual reproductive processes of heterothallic strains are controlled by two multifunctional sex pheromones, PR-IP and PR-IP Inducer, which independently promote multiple steps in conjugation at the appropriate times through different induction mechanisms. The molecules involved in sexual reproduction and sex determination have also been characterized.

  9. Influencing holistic health policy.

    Science.gov (United States)

    Bell, Erica

    2007-09-17

    Beliefs that health policy-making is an inherently 'ideological' or 'irrational' process appear to have worked to prevent researchers from developing better understandings of the kind of evidence that does work to influence policy. Without a model of policy-making that positions policy decision-makers as capable of being informed by specific forms of evidence that speak to policy contexts, it is difficult for research to begin to shape health policy. Recent years have seen the development of a research industry that focuses on developing and describing research approaches for shaping health and social services policy. This analysis paper offers a highly selective overview of generic features of policy-relevant research for holistic health. It aims to support efforts to develop better evidence for health policy by exploring elements of the genre of policy-relevant research, particularly as it applies to the challenges of holistic health policy-making. First, it offers a conceptual definition of holistic health policy-making, as well as research evidence for this kind of policy making, identifying some of the generic features of policy-relevant research. Second, it outlines some of the key practices for delivering sound evidence for health policy, in ways that highlight the salient differences between doing research for holistic health policy, and doing academic research in health. The paper concludes with directions for developing better evidence for holistic health policy-making that question the assumptions of quality which often inform elite funding agencies, calling for their diversification.

  10. Social Influence as Reinforcement Learning

    Science.gov (United States)

    2016-01-13

    SECURITY CLASSIFICATION OF: This project examined a reinforcement learning model of conformity and social influence. Under this model, individuals...Oct-2015 Approved for Public Release; Distribution Unlimited Final Report: Social Influence as Reinforcement Learning The views, opinions and/or...Research Triangle Park, NC 27709-2211 W911NF-14-1-0001 - Final Report - Social Influence as Reinforcement Learning REPORT DOCUMENTATION PAGE 11. SPONSOR

  11. MEDIA INFLUENCE ON OPERATIVE SITUATION

    OpenAIRE

    Mikhail M. Ardavov

    2016-01-01

    The problems regarding the influence of the media on various aspects of society are viewed in the article. Historical facts of the media influence on large groups of people are given. Concerning the discussed problem views on the concept of operative situation are set out. Analysis of factors affecting the operative situation is given. Based on the analysis of publications devoted to the problem of the media influence on the processes in society, a generalization of areas of their impact on t...

  12. Judicial Influence on Policy Outputs?

    DEFF Research Database (Denmark)

    Martinsen, Dorte Sindbjerg

    2015-01-01

    to override unwanted jurisprudence. In this debate, the Court of Justice of the European Union (CJEU) has become famous for its central and occasionally controversial role in European integration. This article examines to what extent and under which conditions judicial decisions influence European Union (EU......) social policy outputs. A taxonomy of judicial influence is constructed, and expectations of institutional and political conditions on judicial influence are presented. The analysis draws on an extensive novel data set and examines judicial influence on EU social policies over time, that is, between 1958...

  13. Diverse pathways of phosphatidylcholine biosynthesis in algae as estimated by labeling studies and genomic sequence analysis.

    Science.gov (United States)

    Sato, Naoki; Mori, Natsumi; Hirashima, Takashi; Moriyama, Takashi

    2016-08-01

    Phosphatidylcholine (PC) is an almost ubiquitous phospholipid in eukaryotic algae and plants but is not found in a few species, for example Chlamydomonas reinhardtii. We recently found that some species of the genus Chlamydomonas possess PC. In the universal pathway, PC is synthesized de novo by methylation of phosphatidylethanolamine (PE) or transfer of phosphocholine from cytidine diphosphate (CDP)-choline to diacylglycerol. Phosphocholine, the direct precursor to CDP-choline, is synthesized either by methylation of phosphoethanolamine or phosphorylation of choline. Here we analyzed the mechanism of PC biosynthesis in two species of Chlamydomonas (asymmetrica and sphaeroides) as well as in a red alga, Cyanidioschyzon merolae. Comparative genomic analysis of enzymes involved in PC biosynthesis indicated that C. merolae possesses only the PE methylation pathway. Radioactive tracer experiments using [(32) P]phosphate showed delayed labeling of PC with respect to PE, which was consistent with the PE methylation pathway. In Chlamydomonas asymmetrica, labeling of PC was detected from the early time of incubation with [(32) P]phosphate, suggesting the operation of phosphoethanolamine methylation pathway. Genomic analysis indeed detected the genes for the phosphoethanolamine methylation pathway. In contrast, the labeling of PC in C. sphaeroides was slow, suggesting that the PE methylation pathway was at work. These results as well as biochemical and computational results uncover an unexpected diversity of the mechanisms for PC biosynthesis in algae. Based on these results, we will discuss plausible mechanisms for the scattered distribution of the ability to biosynthesize PC in the genus Chlamydomonas. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  14. Fermentation metabolism and its evolution in algae

    Directory of Open Access Journals (Sweden)

    Claudia eCatalanotti

    2013-05-01

    Full Text Available Fermentation or anoxic metabolism allows unicellular organisms to colonize environments that become anoxic. Free-living unicellular algae capable of a photoautotrophic lifestyle can also use a range of metabolic circuitry associated with different branches of fermentation metabolism. While algae that perform mixed-acid fermentation are widespread, the use of anaerobic respiration is more typical of eukaryotic heterotrophs. The occurrence of a core set of fermentation pathways among the algae provides insights into the evolutionary origins of these pathways, which were likely derived from a common ancestral eukaryote. Based on genomic, transcriptomic, and biochemical studies, anaerobic energy metabolism has been examined in more detail in Chlamydomonas reinhardtii (Chlamydomonas than in any other photosynthetic protist. This green alga is metabolically flexible and can sustain energy generation and maintain cellular redox balance under a variety of different environmental conditions. Fermentation metabolism in Chlamydomonas appears to be highly controlled, and the flexible use of the different branches of fermentation metabolism has been demonstrated in studies of various metabolic mutants. Additionally, when Chlamydomonas ferments polysaccharides, it has the ability to eliminate part of the reductant (to sustain glycolysis through the production of H2, a molecule that can be developed as a source of renewable energy. To date, little is known about the specific role(s of the different branches of fermentation metabolism, how photosynthetic eukaryotes sense changes in environmental O2 levels, and the mechanisms involved in controlling these responses, at both the transcriptional and post-transcriptional levels. In this review, we focus on fermentation metabolism in Chlamydomonas and other protists, with only a brief discussion of plant fermentation when relevant, since it is thoroughly discussed in other articles in this volume.

  15. Power, Influence Tactics, and Influence Processes in Virtual Teams

    Science.gov (United States)

    Boughton, Marla

    2011-01-01

    Current studies of power, influence tactics, and influence processes in virtual teams assume that these constructs operate in a similar manner as they do in the face-to-face (FtF) environment. However, the virtual context differs from the FtF environment on a variety of dimensions, such as the availability of status cues. The differences between…

  16. Influencing Holistic Health Policy

    Directory of Open Access Journals (Sweden)

    Erica Bell

    2007-01-01

    Full Text Available Beliefs that health policy-making is an inherently ‘ideological’ or ‘irrational’ process appear to have worked to prevent researchers from developing better understandings of the kind of evidence that does work to influence policy. Without a model of policy-making that positions policy decision-makers as capable of being informed by specific forms of evidence that speak to policy contexts, it is difficult for research to begin to shape health policy. Recent years have seen the development of a research industry that focuses on developing and describing research approaches for shaping health and social services policy. This analysis paper offers a highly selective overview of generic features of policy-relevant research for holistic health. It aims to support efforts to develop better evidence for health policy by exploring elements of the genre of policy-relevant research, particularly as it applies to the challenges of holistic health policy-making. First, it offers a conceptual definition of holistic health policy-making, as well as research evidence for this kind of policy making, identifying some of the generic features of policy-relevant research. Second, it outlines some of the key practices for delivering sound evidence for health policy, in ways that highlight the salient differences between doing research for holistic health policy, and doing academic research in health. The paper concludes with directions for developing better evidence for holistic health policy-making that question the assumptions of quality which often inform elite funding agencies, calling for their diversification.

  17. Interdependent Sampling and Social Influence

    Science.gov (United States)

    Denrell, Jerker; Le Mens, Gael

    2007-01-01

    Most explanations of social influence focus on why individuals might want to agree with the opinions or attitudes of others. The authors propose a different explanation that assumes the attitudes of others influence only the activities and objects individuals are exposed to. For example, individuals are likely to be exposed to activities that…

  18. Solar influence on Earth's climate

    DEFF Research Database (Denmark)

    Marsh, N.; Svensmark, Henrik

    2003-01-01

    An increasing number of studies indicate that variations in solar activity have had a significant influence on Earth's climate. However, the mechanisms responsible for a solar influence are still not known. One possibility is that atmospheric transparency is influenced by changing cloud propertie...... and thereby influence the radiative properties of clouds. If the GCR-Cloud link is confirmed variations in galactic cosmic ray flux, caused by changes in solar activity and the space environment, could influence Earth's radiation budget.......An increasing number of studies indicate that variations in solar activity have had a significant influence on Earth's climate. However, the mechanisms responsible for a solar influence are still not known. One possibility is that atmospheric transparency is influenced by changing cloud properties...... via cosmic ray ionisation (the latter being modulated by solar activity). Support for this idea is found from satellite observations of cloud cover. Such data have revealed a striking correlation between the intensity of galactic cosmic rays (GCR) and low liquid clouds (

  19. Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses.

    Science.gov (United States)

    Rasala, Beth A; Mayfield, Stephen P

    2015-03-01

    Recombinant proteins are widely used for industrial, nutritional, and medical applications. Green microalgae have attracted considerable attention recently as a biomanufacturing platform for the production of recombinant proteins for a number of reasons. These photosynthetic eukaryotic microorganisms are safe, scalable, easy to genetically modify through transformation, mutagenesis, or breeding, and inexpensive to grow. Many microalgae species are genetically transformable, but the green alga Chlamydomonas reinhardtii is the most widely used host for recombinant protein expression. An extensive suite of molecular genetic tools has been developed for C. reinhardtii over the last 25 years, including a fully sequenced genome, well-established methods for transformation, mutagenesis and breeding, and transformation vectors for high levels of recombinant protein accumulation and secretion. Here, we review recent successes in the development of C. reinhardtii as a biomanufacturing host for recombinant proteins, including antibodies and immunotoxins, hormones, industrial enzymes, an orally-active colostral protein for gastrointestinal health, and subunit vaccines. In addition, we review the biomanufacturing potential of other green algae from the genera Dunaliella and Chlorella.

  20. Alternating Current-Dielectrophoresis Collection and Chaining of Phytoplankton on Chip: Comparison of Individual Species and Artificial Communities

    Directory of Open Access Journals (Sweden)

    Coralie Siebman

    2017-01-01

    Full Text Available The capability of alternating current (AC dielectrophoresis (DEP for on-chip capture and chaining of the three species representative of freshwater phytoplankton was evaluated. The effects of the AC field intensity, frequency and duration on the chaining efficiency and chain lengths of green alga Chlamydomonas reinhardtii, cyanobacterium Synechocystis sp. and diatom Cyclotella meneghiniana were characterized systematically. C. reinhardtii showed an increase of the chaining efficiency from 100 Hz to 500 kHz at all field intensities; C. meneghiniana presented a decrease of chaining efficiency from 100 Hz to 1 kHz followed by a significant increase from 1 kHz to 500 kHz, while Synechocystis sp. exhibited low chaining tendency at all frequencies and all field intensities. The experimentally-determined DEP response and cell alignment of each microorganism were in agreement with their effective polarizability. Mixtures of cells in equal proportion or 10-times excess of Synechocystis sp. showed important differences in terms of chaining efficiency and length of the chains compared with the results obtained when the cells were alone in suspension. While a constant degree of chaining was observed with the mixture of C. reinhardtii and C. meneghiniana, the presence of Synechocystis sp. in each mixture suppressed the formation of chains for the two other phytoplankton species. All of these results prove the potential of DEP to discriminate different phytoplankton species depending on their effective polarizability and to enable their manipulation, such as specific collection or separation in freshwater.

  1. Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies.

    Science.gov (United States)

    Dutta, Soumita; Avasthi, Prachee

    2017-01-01

    The unicellular green alga Chlamydomonas reinhardtii is an ideal model organism for studies of ciliary function and assembly. In assays for biological and biochemical effects of various factors on flagellar structure and function, synchronous culture is advantageous for minimizing variability. Here, we have characterized a method in which 100% synchronization is achieved with respect to flagellar length but not with respect to the cell cycle. The method requires inducing flagellar regeneration by amputation of the entire cell population and limiting regeneration time. This results in a maximally homogeneous distribution of flagellar lengths at 3 h postamputation. We found that time-limiting new protein synthesis during flagellar synchronization limits variability in the unassembled pool of limiting flagellar protein and variability in flagellar length without affecting the range of cell volumes. We also found that long- and short-flagella mutants that regenerate normally require longer and shorter synchronization times, respectively. By minimizing flagellar length variability using a simple method requiring only hours and no changes in media, flagellar synchronization facilitates the detection of small changes in flagellar length resulting from both chemical and genetic perturbations in Chlamydomonas. This method increases our ability to probe the basic biology of ciliary size regulation and related disease etiologies. IMPORTANCE Cilia and flagella are highly conserved antenna-like organelles that found in nearly all mammalian cell types. They perform sensory and motile functions contributing to numerous physiological and developmental processes. Defects in their assembly and function are implicated in a wide range of human diseases ranging from retinal degeneration to cancer. Chlamydomonas reinhardtii is an algal model system for studying mammalian cilium formation and function. Here, we report a simple synchronization method that allows detection of small

  2. Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

    Science.gov (United States)

    Hirashima, Takashi; Toyoshima, Masakazu; Moriyama, Takashi; Sato, Naoki

    2018-01-12

    Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

  3. Social influence: compliance and conformity.

    Science.gov (United States)

    Cialdini, Robert B; Goldstein, Noah J

    2004-01-01

    This review covers recent developments in the social influence literature, focusing primarily on compliance and conformity research published between 1997 and 2002. The principles and processes underlying a target's susceptibility to outside influences are considered in light of three goals fundamental to rewarding human functioning. Specifically, targets are motivated to form accurate perceptions of reality and react accordingly, to develop and preserve meaningful social relationships, and to maintain a favorable self-concept. Consistent with the current movement in compliance and conformity research, this review emphasizes the ways in which these goals interact with external forces to engender social influence processes that are subtle, indirect, and outside of awareness.

  4. Influence of Mercury

    Science.gov (United States)

    Tackley, P. J.; Aurnou, J. M.; Aubert, J.

    2009-04-01

    Due to the absence of an atmosphere and proximity to the Sun, Mercury's surface temperature varies laterally by several 100s K, even when averaged over long time periods. The dominant variation in time-averaged surface T occurs from pole to equator (~225 K) [1]. The resonant relationship between Mercury's orbit and rotation results in a smaller longitudinal variation (~100 K) [1]. Here we demonstrate, using models of mantle convection in a 3-D spherical shell, that this stationary lateral variation in surface temperature has a small but significant influence on mantle convection and on the lateral variation of heat flux across the core-mantle boundary (CMB). We evaluate the possible observational signature of this laterally-varying convection in terms of boundary topography, stress distribution, gravity and moment of inertia tensor. We furthermore test whether the lateral variation in CMB flux is capable of driving a thermal wind dynamo, i.e., weak dynamo action with no internally-driven core convective motions. For Mercury's mantle we assume a dry olivine rheology including both diffusion creep and disclocation creep with rheological parameters such as activation energy and volume taken from the synthesis of [2]. We assume decaying radiogenic heat sources with the same concentration as in the bulk silicate Earth, and a parameterised model of core cooling. The models are run for 4.5 Ga from a relatively hot initial state with random initial perturbations. We use the code StagYY, which uses a finite-volume discretization on a spherical yin-yang grid and a multigrid solver [3]. Results in spherical axisymmetric geometry, compare a case with constant surface temperature to one with a latitude-dependent surface temperature. The system forms about 3 convection cells from pole to equator. Although the results look similar to first order, in the latitude-dependent case the convection is noticably more sluggish and colder towards the pole. In CMB flux, both cases display

  5. Bayesian Networks and Influence Diagrams

    DEFF Research Database (Denmark)

    Kjærulff, Uffe Bro; Madsen, Anders Læsø

     Probabilistic networks, also known as Bayesian networks and influence diagrams, have become one of the most promising technologies in the area of applied artificial intelligence, offering intuitive, efficient, and reliable methods for diagnosis, prediction, decision making, classification...

  6. Increasing work-time influence

    DEFF Research Database (Denmark)

    Nabe-Nielsen, Kirsten; Garde, Anne Helene; Aust, Birgit

    2012-01-01

    This quasi-experimental study investigated how an intervention aiming at increasing eldercare workers' influence on their working hours affected the flexibility, variability, regularity and predictability of the working hours. We used baseline (n = 296) and follow-up (n = 274) questionnaire data......-operation with colleagues. In subgroup B and C, the participants ended up discussing the potential consequences of more work-time influence without actually implementing any changes. PRACTITIONER SUMMARY: Employee work-time influence may buffer the adverse effects of shift work. However, our intervention study suggested...... that while increasing the individual flexibility, increasing work-time influence may also result in decreased regularity of the working hours and less continuity in the care of clients and co-operation with colleagues....

  7. Peer Influence on Managerial Honesty

    DEFF Research Database (Denmark)

    Brunner, Markus; Ostermaier, Andreas

    2018-01-01

    We investigate peer influence on managerial honesty under varying levels of transparency. In a laboratory experiment, managers report their costs to a superior to request budget. We manipulate whether the managers learn each other’s report and cost (full transparency) or the report but not the cost...... (partial transparency). The results show, first, that managers are susceptible to peer influence, as they join peers in reporting honestly and dishonestly both under full and partial transparency. Second, however, the effect of peer influence is asymmetric. While managers’ dishonesty increases much when...... peers’ reports are higher than they have expected, the opposite is not true. Third, partial transparency reinforces this asymmetry in peer influence. Unlike full transparency, it allows managers to substitute self-serving assumptions for missing information and to thus justify their own dishonesty more...

  8. How Technology Influences Interior Design.

    Science.gov (United States)

    McDavitt, Tish

    1999-01-01

    Examines telecommunication technology's influences on interior school design and effective learning, and discusses how to implement this technology into the school. Building the infrastructure to support telecommunications in an educational setting and the importance of effective lighting are discussed. (GR)

  9. Interaction between social influence and payoff transparency.

    Science.gov (United States)

    Zhou, Xinyue; Xie, Wenwen; Ye, Maolin

    2014-02-01

    Social influence and payoff transparency interact with each other to influence decision making. Social influence masks payoff transparency, and lacking transparency drives people to seek social influence. Moreover, our survey supports our claim by showing that social influence and payoff transparency correlate with each other (r(53) = -.71). Bentley et al.'s model can be revised to accommodate the covariance.

  10. Analysis of interactions between intraflagellar transport proteins.

    Science.gov (United States)

    Behal, Robert H; Cole, Douglas G

    2013-01-01

    Intraflagellar transport (IFT) involves the movement of large proteinaceous particles or trains along the length of ciliary and flagellar axonemal microtubules. The particles contain multiple copies of two protein complexes. As isolated from the flagellated model organism, Chlamydomonas reinhardtii, IFT A contains 6 distinct gene products while IFT B contains at least 13 distinct gene products. To better understand the architecture of these two complexes, a multifaceted approach has been employed to identify subcomplexes and specific protein-protein interactions. The high biochemical yields afforded with Chlamydomonas preparations have allowed traditional biochemical approaches including chemical cross-linking and disruption of native complexes, which, in the case of IFT B, have revealed a core subcomplex retaining nine of the B subunits. Complementing these results are molecular approaches including two-hybrid screenings and heterologous expression that have identified specific protein-protein interactions. Lastly, genetic approaches utilizing Chlamydomonas IFT mutants have shown how the loss of specific subunits perturb the complexes and, in the case of IFT A, they have revealed a core subcomplex containing half of the A subunits. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Intergenerational influence on adolescents' proenvironmental behavior

    National Research Council Canada - National Science Library

    Liu, Chunlin; Li, Jianan

    2016-01-01

    .... By differentiating 2 types of social influence (i.e., informational and normative influence), we found that adolescents' proenvironmental behavior was positively related to both informational and normative influence in Chinese families...

  12. Thinking aloud influences perceived time.

    Science.gov (United States)

    Hertzum, Morten; Holmegaard, Kristin Due

    2015-02-01

    We investigate whether thinking aloud influences perceived time. Thinking aloud is widely used in usability evaluation, yet it is debated whether thinking aloud influences thought and behavior. If thinking aloud is restricted to the verbalization of information to which a person is already attending, there is evidence that thinking aloud does not influence thought and behavior. In an experiment, 16 thinking-aloud participants and 16 control participants solved a code-breaking task 24 times each. Participants estimated task duration. The 24 trials involved two levels of time constraint (timed, untimed) and resulted in two levels of success (solved, unsolved). The ratio of perceived time to clock time was lower for thinking-aloud than control participants. Participants overestimated time by an average of 47% (thinking aloud) and 94% (control). The effect of thinking aloud on time perception also held separately for timed, untimed, solved, and unsolved trials. Thinking aloud (verbalization at Levels 1 and 2) influences perceived time. Possible explanations of this effect include that thinking aloud may require attention, cause a processing shift that overshadows the perception of time, or increase mental workload. For usability evaluation, this study implies that time estimates made while thinking aloud cannot be compared with time estimates made while not thinking aloud, that ratings of systems experienced while thinking aloud may be inaccurate (because the experience of time influences other experiences), and that it may therefore be considered to replace concurrent thinking aloud with retrospective thinking aloud when evaluations involve time estimation.

  13. Leadership's influence on job satisfaction.

    Science.gov (United States)

    Watson, Liana M

    2009-01-01

    To assess the leadership styles of frontline medical imaging supervisors and examine the relationship between leadership behaviors and motivational factors that influence job satisfaction of medical imaging staff in acute care facilities. Three hundred fifty nine staff technologists completed a survey on their supervisors' leadership behaviors and the intrinsic and extrinsic motivating factors that influence job satisfaction and organizational commitment. This study indicates that there are strong, positive relationships between supervisors' transformational and contingent reward behaviors and intrinsic esteem motivators that influence staff members' job satisfaction. The behaviors with the strongest relationship to intrinsic esteem motivators were individualized consideration and contingent rewards. The results of this study provide insight into what employees need from their leader and the organization that employs them. This information can be used to help develop strategies to meet those needs through work redesign and leadership behavior development.

  14. Thinking Aloud Influences Perceived Time

    DEFF Research Database (Denmark)

    Hertzum, Morten; Holmegaard, Kristin Due

    2015-01-01

    Objective: We investigate whether thinking aloud influences perceived time. Background: Thinking aloud is widely used in usability evaluation, yet it is debated whether thinking aloud influences thought and behavior. If thinking aloud is restricted to the verbalization of information to which...... a person is already attending, there is evidence that thinking aloud does not influence thought and behavior. Method: In an experiment, 16 thinking-aloud participants and 16 control participants solved a code-breaking task 24 times each. Participants estimated task duration. The 24 trials involved two...... levels of time constraint (timed, untimed) and resulted in two levels of success (solved, unsolved). Results: The ratio of perceived time to clock time was lower for thinking-aloud than control participants. Participants overestimated time by an average of 47% (thinking aloud) and 94% (control...

  15. Multi-currency Influence Diagrams

    DEFF Research Database (Denmark)

    Nielsen, Søren Holbech; Nielsen, Thomas Dyhre; Jensen, Finn V.

    2007-01-01

    When using the influence diagrams framework for solving a decision problem with several different quantitative utilities, the traditional approach has been to convert the utilities into one common currency. This conversion is carried out using a tacit transformation, under the assumption that the......When using the influence diagrams framework for solving a decision problem with several different quantitative utilities, the traditional approach has been to convert the utilities into one common currency. This conversion is carried out using a tacit transformation, under the assumption...... that the converted problem is equivalent to the original one. In this paper we present an extension of the influence diagram framework. The extension allows for these decision problems to be modelled in their original form. We present an algorithm that, given a linear conversion function between the currencies...

  16. Executive control influences linguistic representations.

    Science.gov (United States)

    Lev-Ari, Shiri; Keysar, Boaz

    2014-02-01

    Although it is known that words acquire their meanings partly from the contexts in which they are used, we proposed that the way in which words are processed can also influence their representation. We further propose that individual differences in the way that words are processed can consequently lead to individual differences in the way that they are represented. Specifically, we showed that executive control influences linguistic representations by influencing the coactivation of competing and reinforcing terms. Consequently, people with poorer executive control perceive the meanings of homonymous terms as being more similar to one another, and those of polysemous terms as being less similar to one another, than do people with better executive control. We also showed that bilinguals with poorer executive control experience greater cross-linguistic interference than do bilinguals with better executive control. These results have implications for theories of linguistic representation and language organization.

  17. Advertising influences on young children's food choices and parental influence.

    Science.gov (United States)

    Ferguson, Christopher J; Muñoz, Monica E; Medrano, Maria R

    2012-03-01

    To evaluate whether advertising for food influences choices made by children, the strength of these influences, and whether they might be easily undone by parental influences. Children between 3 and 8 years of age (n=75) were randomized to watch a series of programs with embedded commercials. Some children watched a commercial for a relatively healthy food item, the other children watched a commercial for a less healthy item, both from the same fast-food company. Children were also randomized either to receive parental encouragement to choose the healthy item or to choose whichever item they preferred. Results indicated that children were more likely to choose the advertised item, despite parental input. Parental input only slightly moderated this influence. Although advertising impact on children's food choices is moderate in size, it appears resilient to parental efforts to intervene. Food advertisements directed at children may have a small but meaningful effect on their healthy food choices. Copyright © 2012 Mosby, Inc. All rights reserved.

  18. The Anxiety of Influence and the Influence of Anxiety.

    Science.gov (United States)

    Kountz, Carol

    A composition researcher collected stories from students with writing anxiety, using qualitative research tools of interview and interpretation. In literary theory it is not unusual to speak of anxiety of influence when referring to the torment of proving one is equal to a revered author. The critic Harold Bloom presented it as his theory of the…

  19. Marketing Logics, Ambidexterity and Influence

    DEFF Research Database (Denmark)

    Tollin, Karin; Schmidt, Marcus

    2012-01-01

    in four CMOs have taken on this challenge, or adopted a marketing logic which could be referred to as ambidextrous. Furthermore, the study shows that this logic exerts a stronger impact on marketing's influence, compared to logics related to assuring brand consistency and measuring the performance...... of marketing processes. Three other ways to enact marketing management were also revealed, namely: an innovation; a communication; and a supporting marketing logic. This leads us to conclude that the influence of companies' marketing functions show up a heterogeneous picture within which the marketing logics...

  20. Peer influence on adolescent snacking

    DEFF Research Database (Denmark)

    Nørgaard, Maria Kümpel; Hansen, Kathrine Nørgaard; Grunert, Klaus G

    2013-01-01

    show that the youngest adolescents and the girls perceived the highest influence from peers, and that peer social influence has more effect on what adolescents perceive as important snack attributes as compared to more personal factors. The focus group results show that adolescents purchase and consume...... to marketers developing social marketing campaigns aiming at reducing bullying among adolescents or promoting healthy snacking. Social implications – Moreover, the results may help generate societal emphasis on the importance of social and self-image aspects in consumption settings when it comes to adolescent...

  1. Influencing the future of AGU

    Science.gov (United States)

    McPhaden, Michael; Finn, Carol; McEntee, Chris

    2012-01-01

    Steve Jobs, visionary cofounder of Apple, Inc., once said, “Everyone here has the sense that right now is one of those moments when we are influencing the future.” This statement aptly describes AGU at this time as the Board of Directors and the Council continue to influence the future in exciting ways by advancing our strategic plan (http://www.agu.org/about/mission.shtml). Both governing bodies held meetings in San Francisco immediately preceding the 2011 AGU Fall Meeting. The agendas for both meetings, along with the key outcomes, are posted on AGU's Web site (http://www.agu.org/about/governance/).

  2. A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65

    Directory of Open Access Journals (Sweden)

    Maxwell Denis

    2008-11-01

    Full Text Available Abstract Background Human glutamic acid decarboxylase 65 (hGAD65 is a key autoantigen in type 1 diabetes, having much potential as an important marker for the prediction and diagnosis of type 1 diabetes, and for the development of novel antigen-specific therapies for the treatment of type 1 diabetes. However, recombinant production of hGAD65 using conventional bacterial or mammalian cell culture-based expression systems or nuclear transformed plants is limited by low yield and low efficiency. Chloroplast transformation of the unicellular eukaryotic alga Chlamydomonas reinhardtii may offer a potential solution. Results A DNA cassette encoding full-length hGAD65, under the control of the C. reinhardtii chloroplast rbcL promoter and 5'- and 3'-UTRs, was constructed and introduced into the chloroplast genome of C. reinhardtii by particle bombardment. Integration of hGAD65 DNA into the algal chloroplast genome was confirmed by PCR. Transcriptional expression of hGAD65 was demonstrated by RT-PCR. Immunoblotting verified the expression and accumulation of the recombinant protein. The antigenicity of algal-derived hGAD65 was demonstrated with its immunoreactivity to diabetic sera by ELISA and by its ability to induce proliferation of spleen cells from NOD mice. Recombinant hGAD65 accumulated in transgenic algae, accounts for approximately 0.25–0.3% of its total soluble protein. Conclusion Our results demonstrate the potential value of C. reinhardtii chloroplasts as a novel platform for rapid mass production of immunologically active hGAD65. This demonstration opens the future possibility for using algal chloroplasts as novel bioreactors for the production of many other biologically active mammalian therapeutic proteins.

  3. Influence

    Directory of Open Access Journals (Sweden)

    M.A. Hegazy

    2013-12-01

    Full Text Available The synthesized cationic surfactant N-(2-(2-mercaptoacetoxy ethyl-N,N-dimethyldodecan-1-aminium bromide (QSH was used to prepare colloidal copper nanoparticles (CuNPs in water through the chemical reduction method. The obtained copper nanoparticles were characterized by FTIR spectrum and transmission electron microscopy (TEM. The corrosion performance was evaluated using potentiodynamic polarization and electrochemical impedance spectroscopy (EIS measurements in addition to the salt spray test. The results obtained from these methods were in good agreement. Results showed that the modified coating provide a good coverage and an additional corrosion protection of the carbon steel.

  4. INFLUENCE OF SUPERPLASTICIZER AND VARYING ...

    African Journals Online (AJOL)

    This paper presents the results of the study on the influence of superplasticizer and varying aggregate size on the drying shrinkage and compressive strength of laterised concrete. Four different samples of laterised concrete were made from prescribed mix ratio of 1:1:2 which include; two control specimens made with ...

  5. Individual Influence on Model Selection

    Science.gov (United States)

    Sterba, Sonya K.; Pek, Jolynn

    2012-01-01

    Researchers in psychology are increasingly using model selection strategies to decide among competing models, rather than evaluating the fit of a given model in isolation. However, such interest in model selection outpaces an awareness that one or a few cases can have disproportionate impact on the model ranking. Though case influence on the fit…

  6. Institutional Influences on Succession Intentions

    DEFF Research Database (Denmark)

    Boyd, Britta; Fietze, Simon

    ) and the Comparative Welfare Entitlements Dataset (CWED). With this study we fill a research gap by analyzing the influence of social policy measured by the total generosity index on SI of students. The regression results show that SI among other variables depends on the welfare model in certain institutional contexts...

  7. Harnessing Aquatic Physicochemical Parameters Influencing ...

    African Journals Online (AJOL)

    The management-oriented background for harnessing aquatic physicochemical parameters influencing macro invertebrate fauna of Anambra River basin for sustainable fish productivity was studied. The intra seasonal variability in the water quality of the river revealed mean transparency of 1.79 cm, Conductivity of 28.81 ...

  8. Influencing the online consumer's behavior

    NARCIS (Netherlands)

    Constantinides, Efthymios

    2004-01-01

    Addresses one of the fundamental issues of e-marketing: how to attract and win over the consumer in the highly competitive Internet marketplace. Analyses the factors affecting the online consumer's behavior and examines how e-marketers can influence the outcome of the virtual interaction and buying

  9. Influence Maximization in Ising Networks

    Science.gov (United States)

    Lynn, Christopher; Lee, Daniel

    In the analysis of social networks, a fundamental problem is influence maximization: Which individuals should be influenced to maximally impact the collective opinions of an entire population? Traditionally, influence maximization has been studied in the context of contagion models and irreversible processes. However, by including stochastic noise in the opinion formation process, repeated interactions between individuals give rise to complex macroscopic patterns that are observed, for example, in the formation of political opinions. Here we map influence maximization in the presence of stochastic noise onto the Ising model, and the resulting problem has a natural physical interpretation as maximizing the magnetization given a budget of external magnetic field. Using the susceptibility matrix, we provide a gradient ascent algorithm for calculating optimal external fields in real-world social networks. Remarkably, we find that the optimal external field solutions undergo a phase transition from intuitively focusing on high-degree individuals at high temperatures to counterintuitively focusing on low-degree individuals at low temperatures, a feature previously neglected under the viral paradigm. We acknowledge support from the U.S. National Science Foundation, the Air Force Office of Scientific Research, and the Department of Transportation.

  10. The Influences of Rudolf Laban.

    Science.gov (United States)

    Foster, John

    Rudolf Laban has influenced the introduction and development of modern educational dance as well as general education. Much of the available material concerning Laban is arts oriented; it is concerned with dance as an art form and is not focused towards the educational uses of Laban's ideas. This volume attempts to present materials relevant to…

  11. The Influence of Marketing Education.

    Science.gov (United States)

    Leventhal, Jerome I.

    2002-01-01

    A look at the history of marketing education shows how it has influenced the education of students and how its concepts are still being used in education today. Marketing educators were pioneers in educational opportunities for women, performance-based education, cooperative education, and leadership development. (JOW)

  12. Phonological Awareness: Factors of Influence

    Science.gov (United States)

    Frohlich, Linda Paulina; Petermann, Franz; Metz, Dorothee

    2013-01-01

    Early child development is influenced by various genetic and environmental factors. This study aims to identify factors that affect the phonological awareness of preschool and first grade children. Based on a sample of 330 German-speaking children (mean age = 6.2 years) the following domains were evaluated: Parent factors, birth and pregnancy,…

  13. Bayesian Networks and Influence Diagrams

    DEFF Research Database (Denmark)

    Kjærulff, Uffe Bro; Madsen, Anders Læsø

    Bayesian Networks and Influence Diagrams: A Guide to Construction and Analysis, Second Edition, provides a comprehensive guide for practitioners who wish to understand, construct, and analyze intelligent systems for decision support based on probabilistic networks. This new edition contains six new...

  14. Adolescents' Susceptibility to Maternal and Peer Influence

    OpenAIRE

    Thomas, April Gile

    2017-01-01

    Adolescence is a time of heightened risk taking and vulnerability to social influence. Research has demonstrated the effects of social influence on adolescents’ risk taking; however, most research has focused on the effects of peer influence, with less known about the direct effects of parental influence on adolescent risk. The present study uses an experimental design to examine the effect of indirect and direct maternal and peer influence on adolescents’ risk taking on two behavioral tasks....

  15. Understanding Robert Lucas (1967-1981: his influence and influences

    Directory of Open Access Journals (Sweden)

    Alexandre F.S. Andrada

    2017-05-01

    Full Text Available This paper analyzes Robert Lucas's contribution to economic theory between 1967 (year of his first solo publication and 1981 (the year before the emergence of Real Business Cycle approach, and it has two parts. The first one, using citation data from three different sources, we try to answer two questions: (i What are Lucas's most influential papers currently? (ii How has this influence changed through time? We show, for instance, that according to two of those three sources, Lucas's most influential paper today is not from his business cycle research agenda, which gave him his Nobel Prize in 1995. Moreover, it is clear the loss of influence of Lucas's macroeconomic theory since the early 1980s. In the second part, by cataloging all the works that Lucas had used as bibliographical references in his papers and separating them in two categories (positive and negative, we try to understand who exerted influence on him. We show that the author that Lucas most cited in a positive context were John Muth, Milton Friedman and Edmund Phelps. The authors more often cited in a negative context were John M. Keynes and A. W. Phillips. We discuss the reasons behind this data.

  16. Deliberate change without hierarchical influence?

    DEFF Research Database (Denmark)

    Nørskov, Sladjana; Kesting, Peter; Ulhøi, John Parm

    2017-01-01

    Purpose This paper aims to present that deliberate change is strongly associated with formal structures and top-down influence. Hierarchical configurations have been used to structure processes, overcome resistance and get things done. But is deliberate change also possible without formal...... reveals that deliberate change is indeed achievable in a non-hierarchical collaborative OSS community context. However, it presupposes the presence and active involvement of informal change agents. The paper identifies and specifies four key drivers for change agents’ influence. Originality....../value The findings contribute to organisational analysis by providing a deeper understanding of the importance of leadership in making deliberate change possible in non-hierarchical settings. It points to the importance of “change-by-conviction”, essentially based on voluntary behaviour. This can open the door...

  17. Bayesian Networks and Influence Diagrams

    DEFF Research Database (Denmark)

    Kjærulff, Uffe Bro; Madsen, Anders Læsø

     Probabilistic networks, also known as Bayesian networks and influence diagrams, have become one of the most promising technologies in the area of applied artificial intelligence, offering intuitive, efficient, and reliable methods for diagnosis, prediction, decision making, classification......, and exercises are included for the reader to check his/her level of understanding. The techniques and methods presented for knowledge elicitation, model construction and verification, modeling techniques and tricks, learning models from data, and analyses of models have all been developed and refined......, troubleshooting, and data mining under uncertainty. Bayesian Networks and Influence Diagrams: A Guide to Construction and Analysis provides a comprehensive guide for practitioners who wish to understand, construct, and analyze intelligent systems for decision support based on probabilistic networks. Intended...

  18. Bayesian Networks and Influence Diagrams

    DEFF Research Database (Denmark)

    Kjærulff, Uffe Bro; Madsen, Anders Læsø

    , troubleshooting, and data mining under uncertainty. Bayesian Networks and Influence Diagrams: A Guide to Construction and Analysis provides a comprehensive guide for practitioners who wish to understand, construct, and analyze intelligent systems for decision support based on probabilistic networks. Intended...... Probabilistic networks, also known as Bayesian networks and influence diagrams, have become one of the most promising technologies in the area of applied artificial intelligence, offering intuitive, efficient, and reliable methods for diagnosis, prediction, decision making, classification......, and exercises are included for the reader to check his/her level of understanding. The techniques and methods presented for knowledge elicitation, model construction and verification, modeling techniques and tricks, learning models from data, and analyses of models h