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Sample records for chlamydomonas sp ice-l

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

    NASA Astrophysics Data System (ADS)

    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.

  2. Expression of fatty acid desaturase genes and fatty acid accumulation in Chlamydomonas sp. ICE-L under salt stress.

    PubMed

    An, Meiling; Mou, Shanli; Zhang, Xiaowen; Zheng, Zhou; Ye, Naihao; Wang, Dongsheng; Zhang, Wei; Miao, Jinlai

    2013-12-01

    The Antarctic ice microalgae Chlamydomonas sp. ICE-L which is highly resistant to salt stress holds promise in providing an alternative species for the production of microalgal oil. We studied the effects of the alga in confrontation with NaCl stress on the growth, oil yield and expression of fatty acid desaturase genes. The growth rate of Chlamydomonas sp. ICE-L decreased with the gradual increase in NaCl concentration. Interestingly, we found that the highest lipid content was achieved at 16‰ NaCl, reaching 23% (w/w). Meanwhile, the expression of Δ9ACPCiFAD increased rapidly while Δ12CiFAD, ω3CiFAD2 and Δ6CiFAD showed a delayed elevation in response to altered salt stress. C18:3 was the dominant PUFA, which account for about 75% TFA in Chlamydomonas sp. ICE-L. Under 96‰ and 128‰ NaCl stress, the content of C20:5 almost approached that of C18:3. In contrast, low salinity enhanced the dominance of C18:3 at the expense of C20:3 and C20:5. PMID:24084208

  3. Transcriptome-wide analysis of DEAD-box RNA helicase gene family in an Antarctic psychrophilic alga Chlamydomonas sp. ICE-L.

    PubMed

    Liu, Chenlin; Huang, Xiaohang

    2015-09-01

    DEAD-box RNA helicase family proteins have been identified in almost all living organisms. Some of them play a crucial role in adaptation to environmental changes and stress response, especially in the low-temperature acclimation in different kinds of organisms. Compared with the full swing study in plants and bacteria, the characters and functions of DEAD-box family proteins had not been surveyed in algae. To identify genes critical for freezing acclimation in algae, we screened DEAD-box RNA helicase genes from the transcriptome sequences of a psychrophilic microalga Chlamydomonas sp. ICE-L which was isolated from Antarctic sea ice. Totally 39 DEAD-box RNA helicase genes had been identified. Most of the DEAD-box RNA helicase have 1:1 homologous relationships in Chlamydomonas reinhardtii and Chlamydomonas sp. ICE-L with several exceptions. The homologous proteins in ICE-L to the helicases critical for cold or freezing tolerance in Arabidopsis thaliana had been identified based on phylogenetic comparison studies. The response of these helicase genes is not always identical in the Chlamydomonas sp. ICE-L and Arabidopsis under the same low-temperature treatment. The expression of several DEAD-box RNA helicase genes including CiRH5, CiRH25, CiRH28, and CiRH55 were significantly up-regulated under freezing treatment of ICE-L and their function in freezing acclimation of ICE-L deserved further investigation. PMID:26174530

  4. Temperature regulates fatty acid desaturases at a transcriptional level and modulates the fatty acid profile in the Antarctic microalga Chlamydomonas sp. ICE-L.

    PubMed

    An, Meiling; Mou, Shanli; Zhang, Xiaowen; Ye, Naihao; Zheng, Zhou; Cao, Shaona; Xu, Dong; Fan, Xiao; Wang, Yitao; Miao, Jinlai

    2013-04-01

    Chlamydomonas sp. ICE-L which can thrive in extreme environments of the Antarctic is a major biomass producer. The FAD genes in Chlamydomonas sp. ICE-L were obtained and sequence alignment showed that these genes are homologous to known FADs with conserved histidine motifs. In this study, we analyzed the transcription of five FADs and FA compositions at different temperatures. The results showed that the expressions of Δ9CiFAD, ω3CiFAD1 and ω3CiFAD2 were apparently up-regulated at 0°C, however, the up-regulation of Δ6CiFAD intensified with rising temperature. Meanwhile, analysis of the FA compositions showed that PUFAs were dominant compositions, accounting for more than 75% TFA in Chlamydomonas sp. ICE-L. Furthermore, PUFAs were significantly increased at 0 and 5°C, which may be attributed to higher proportions of C18:3 and C20:3. Moreover, PUFAs were significantly decreased at 15°C whereas SFAs were significantly increased. PMID:23500572

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

    PubMed

    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. PMID:24922067

  6. Chlamydomonas sajao nov. sp. (Chlorophyta, Volvocales)

    NASA Astrophysics Data System (ADS)

    Lewin, Ralph A.

    1984-06-01

    A new species of Chlamydomonas, namely, C. sajao nov. sp. of the Volvocales, Chlorophyta was isolated from a duckweed growing near a ricefield in the vicinity of Guangzhou, China. This interesting unicellular green alga, similar to C. mexicana from Mexico, secretes quantities of extracellular mucilaginous polysaccharides, and may be employed in improving soil quality. The new species resembles C. waldenburgensis Moewus in most characteristics but differs in three important features.

  7. Azolla filiculoides Nitrogenase Activity Decrease Induced by Inoculation with Chlamydomonas sp.

    PubMed

    Habte, M

    1986-11-01

    Experiments were conducted to determine the influence of Chlamydomonas sp. on nitrogen fixation (C(2)H(2) --> C(2)H(4)) in Azolla filiculoides and on the nitrogen fixation and growth of free-living Anabaena azollae 2B organisms. Inoculation of azolla medium with Chlamydomonas sp. was associated with decreased nitrogenase activity in A. filiculoides and with increases in the density of a fungal population identified as Acremonium sp. Subsequent inoculation of azolla medium with this fungus was also accompanied by a significant decrease in nitrogenase activity of A. filiculoides. However, the extent of depression of nitrogenase activity was significantly higher when azolla medium was inoculated with Chlamydomonas sp. than when it was inoculated with Acremonium sp. Inoculation of nitrogen-free Stanier medium with either Acremonium sp. or Chlamydomonas sp. did not adversely affect the growth or nitrogenase activity of free-living A. azollae. Decreased nitrogenase activity in A. filiculoides is apparently related to the adverse influence of the green alga and the fungus on the macrosymbiont. The mechanisms that might be involved are discussed. PMID:16347211

  8. Azolla filiculoides Nitrogenase Activity Decrease Induced by Inoculation with Chlamydomonas sp. †

    PubMed Central

    Habte, Mitiku

    1986-01-01

    Experiments were conducted to determine the influence of Chlamydomonas sp. on nitrogen fixation (C2H2 → C2H4) in Azolla filiculoides and on the nitrogen fixation and growth of free-living Anabaena azollae 2B organisms. Inoculation of azolla medium with Chlamydomonas sp. was associated with decreased nitrogenase activity in A. filiculoides and with increases in the density of a fungal population identified as Acremonium sp. Subsequent inoculation of azolla medium with this fungus was also accompanied by a significant decrease in nitrogenase activity of A. filiculoides. However, the extent of depression of nitrogenase activity was significantly higher when azolla medium was inoculated with Chlamydomonas sp. than when it was inoculated with Acremonium sp. Inoculation of nitrogen-free Stanier medium with either Acremonium sp. or Chlamydomonas sp. did not adversely affect the growth or nitrogenase activity of free-living A. azollae. Decreased nitrogenase activity in A. filiculoides is apparently related to the adverse influence of the green alga and the fungus on the macrosymbiont. The mechanisms that might be involved are discussed. PMID:16347211

  9. Validation of housekeeping genes for gene expression studies in an ice alga Chlamydomonas during freezing acclimation.

    PubMed

    Liu, Chenlin; Wu, Guangting; Huang, Xiaohang; Liu, Shenghao; Cong, Bailin

    2012-05-01

    Antarctic ice alga Chlamydomonas sp. ICE-L can endure extreme low temperature and high salinity stress under freezing conditions. To elucidate the molecular acclimation mechanisms using gene expression analysis, the expression stabilities of ten housekeeping genes of Chlamydomonas sp. ICE-L during freezing stress were analyzed. Some discrepancies were detected in the ranking of the candidate reference genes between geNorm and NormFinder programs, but there was substantial agreement between the groups of genes with the most and the least stable expression. RPL19 was ranked as the best candidate reference genes. Pairwise variation (V) analysis indicated the combination of two reference genes was sufficient for qRT-PCR data normalization under the experimental conditions. Considering the co-regulation between RPL19 and RPL32 (the most stable gene pairs given by geNorm program), we propose that the mean data rendered by RPL19 and GAPDH (the most stable gene pairs given by NormFinder program) be used to normalize gene expression values in Chlamydomonas sp. ICE-L more accurately. The example of FAD3 gene expression calculation demonstrated the importance of selecting an appropriate category and number of reference genes to achieve an accurate and reliable normalization of gene expression during freeze acclimation in Chlamydomonas sp. ICE-L. PMID:22527038

  10. Acclimation of Antarctic Chlamydomonas to the sea-ice environment: a transcriptomic analysis.

    PubMed

    Liu, Chenlin; Wang, Xiuliang; Wang, Xingna; Sun, Chengjun

    2016-07-01

    The Antarctic green alga Chlamydomonas sp. ICE-L was isolated from sea ice. As a psychrophilic microalga, it can tolerate the environmental stress in the sea-ice brine, such as freezing temperature and high salinity. We performed a transcriptome analysis to identify freezing stress responding genes and explore the extreme environmental acclimation-related strategies. Here, we show that many genes in ICE-L transcriptome that encoding PUFA synthesis enzymes, molecular chaperon proteins, and cell membrane transport proteins have high similarity to the gens from Antarctic bacteria. These ICE-L genes are supposed to be acquired through horizontal gene transfer from its symbiotic microbes in the sea-ice brine. The presence of these genes in both sea-ice microalgae and bacteria indicated the biological processes they involved in are possibly contributing to ICE-L success in sea ice. In addition, the biological pathways were compared between ICE-L and its closely related sister species, Chlamydomonas reinhardtii and Volvox carteri. In ICE-L transcripome, many sequences homologous to the plant or bacteria proteins in the post-transcriptional, post-translational modification, and signal-transduction KEGG pathways, are absent in the nonpsychrophilic green algae. These complex structural components might imply enhanced stress adaptation capacity. At last, differential gene expression analysis at the transcriptome level of ICE-L indicated that genes that associated with post-translational modification, lipid metabolism, and nitrogen metabolism are responding to the freezing treatment. In conclusion, the transcriptome of Chlamydomonas sp. ICE-L is very useful for exploring the mutualistic interaction between microalgae and bacteria in sea ice; and discovering the specific genes and metabolism pathways responding to the freezing acclimation in psychrophilic microalgae. PMID:27161450

  11. Fatty acid patterns in Chlamydomonas sp. as a marker for nutritional regimes and temperature under extremely acidic conditions.

    PubMed

    Poerschmann, J; Spijkerman, E; Langer, U

    2004-07-01

    Fatty acid profiles were used to characterize nutritional pathways in Chlamydomonas sp. isolated from an acidic mining lake (pH 2.7). Surprisingly, profiles of Chlamydomonas sp. grown in the lab under photoautotrophic, mixotrophic, and heterotrophic conditions at in situ deep strata lake water temperatures (8 degrees C) were very similar, polyunsaturated fatty acids including alpha-linolenic acid (18:3omega3) and 16:4omega3 along with palmitic acid (16:0) being most abundant. Therefore, heterotrophic growth of Chlamydomonas sp. at low temperatures can result in high concentrations of polyunsaturated fatty acids, as previously only described for some psychrophilic bacteria. By contrast, the cultivation of isolated Chlamydomonas sp. at 20 degrees C, reflecting surface water temperatures, provided fatty acid patterns characteristic of the nutrition strategy applied: the concentration of polyunsaturated fatty acids decreased when the growth pathway changed from photoautotrophic via mixotrophic to heterotrophic. Total fatty acid concentration also diminished in this order. Principal component analysis confirmed the significance of FA profiling to mirror nutritional pathways. Lake-water analysis revealed low concentrations of dissolved organic carbon, mainly consisting of polymeric fulvic acids that are unable to support heterotrophic growth of Chlamydomonas sp. Polymeric fulvic acids present in the deeper strata of the lake turned out to be formed in situ on the basis of organic monomers including reduced sulfur-containing ones, as revealed by thermochemolysis and pyrolysis. Growth of Chlamydomonas sp. in the deep chlorophyll maximum is therefore assumed to mainly result from photosynthesis, despite very low photon densities. Phytol-including metabolites proved to be significant biomarkers to indicate the nutritional pathway of Chlamydomonas sp. alpha, omega-Dicarboxylic acids-light-induced degradation products of unsaturated fatty acids-appeared to be good indicators

  12. Resolving the phylogenetic relationship between Chlamydomonas sp. UWO 241 and Chlamydomonas raudensis sag 49.72 (Chlorophyceae) with nuclear and plastid DNA sequences.

    PubMed

    Possmayer, Marc; Gupta, Rajesh K; Szyszka-Mroz, Beth; Maxwell, Denis P; Lachance, Marc-André; Hüner, Norman P A; Smith, David Roy

    2016-04-01

    The Antarctic psychrophilic green alga Chlamy-domonas sp. UWO 241 is an emerging model for studying microbial adaptation to polar environments. However, little is known about its evolutionary history and its phylogenetic relationship with other chlamydomonadalean algae is equivocal. Here, we attempt to clarify the phylogenetic position of UWO 241, specifically with respect to Chlamydomonas rau-densis SAG 49.72. Contrary to a previous report, we show that UWO 241 is a distinct species from SAG 49.72. Our phylogenetic analyses of nuclear and plastid DNA sequences reveal that UWO 241 represents a unique lineage within the Moewusinia clade (sensu Nakada) of the Chlamydomonadales (Chlorophyceae, Chlorophyta), closely affiliated to the marine species Chlamydomonas parkeae SAG 24.89. PMID:27037594

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

    PubMed

    Hung, Chun-Hsien; Endo, Kaichiro; Kobayashi, Koichi; Nakamura, Yuki; Wada, Hajime

    2015-01-01

    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. PMID:26379630

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

    PubMed Central

    Hung, Chun-Hsien; Endo, Kaichiro; Kobayashi, Koichi; Nakamura, Yuki; Wada, Hajime

    2015-01-01

    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. PMID:26379630

  15. Ecophysiology, secondary pigments and ultrastructure of Chlainomonas sp. (Chlorophyta) from the European Alps compared with Chlamydomonas nivalis forming red snow

    PubMed Central

    Remias, Daniel; Pichrtová, Martina; Pangratz, Marion; Lütz, Cornelius; Holzinger, Andreas

    2016-01-01

    Red snow is a well-known phenomenon caused by microalgae thriving in alpine and polar regions during the melting season. The ecology and biodiversity of these organisms, which are adapted to low temperatures, high irradiance and freeze–thaw events, are still poorly understood. We compared two different snow habitats containing two different green algal genera in the European Alps, namely algae blooming in seasonal rock-based snowfields (Chlamydomonas nivalis) and algae dominating waterlogged snow bedded over ice (Chlainomonas sp.). Despite the morphological similarity of the red spores found at the snow surface, we found differences in intracellular organization investigated by light and transmission electron microscopy and in secondary pigments investigated by chromatographic analysis in combination with mass spectrometry. Spores of Chlainomonas sp. show clear differences from Chlamydomonas nivalis in cell wall arrangement and plastid organization. Active photosynthesis at ambient temperatures indicates a high physiological activity, despite no cell division being present. Lipid bodies containing the carotenoid astaxanthin, which produces the red color, dominate cells of both species, but are modified differently. While in Chlainomonas sp. astaxanthin is mainly esterified with two fatty acids and is more apolar, in Chamydomonas nivalis, in contrast, less apolar monoesters prevail. PMID:26884467

  16. Interactions between marine facultative epiphyte Chlamydomonas sp. (Chlamydomonadales, Chlorophyta) and ceramiaceaen algae (Rhodophyta).

    PubMed

    Klochkova, Tatyana A; Cho, Ga Youn; Boo, Sung Min; Chung, Ki Wha; Kim, Song Ja; Kim, Gwang Hoon

    2008-07-01

    Previously unrecorded marine Chlamydomonas that grew epiphytic on ceramiaceaen algae was collected from the western coast of Korea and isolated into a unialgal culture. The isolate was subjected to 18S rDNA phylogenetic analysis as well as ultrastructure and life cycle studies. It had an affinity with the marine Chlamydomonas species and was less related to freshwater/terrestrial representatives of this genus. It had flagella shorter than the cell body two-layered cell wall with striated outer surface and abundant mucilaginous material beneath the innermost layer and no contractile vacuoles. This alga grew faster in mixed cultures with ceramiaceaen algae rather than in any tested unialgal culture condition; the cells looked healthier and zoosporangia and motile flagellated vegetative cells appeared more often. These results suggested that this Chlamydomonas might be a facultative epiphyte benefiting from its hosts. Several ceramiaceaen algae were tested as host plants. Meanwhile, cell deformation or collapse of the whole thallus was caused to Aglaothamnion byssoides, and preliminary study suggested that a substance released from Chlamydomonas caused the response. This is first report on harmful epiphytic interactions between Chlamydomonas species and red ceramiaceaen algae. PMID:19195375

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

    DOE PAGESBeta

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

    2014-10-18

    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. We demonstrate the growth of C. reinhardtii on acetate provided by mutant strains of the cyanobacterium Synechococcus sp. PCC7002.

  18. The Antarctic Psychrophile Chlamydomonas sp. UWO 241 Preferentially Phosphorylates a Photosystem I-Cytochrome b6/f Supercomplex1[OPEN

    PubMed Central

    Szyszka-Mroz, Beth; Pittock, Paula; Ivanov, Alexander G.; Lajoie, Gilles; Hüner, Norman P.A.

    2015-01-01

    Chlamydomonas sp. UWO 241 (UWO 241) is a psychrophilic green alga isolated from Antarctica. A unique characteristic of this algal strain is its inability to undergo state transitions coupled with the absence of photosystem II (PSII) light-harvesting complex protein phosphorylation. We show that UWO 241 preferentially phosphorylates specific polypeptides associated with an approximately 1,000-kD pigment-protein supercomplex that contains components of both photosystem I (PSI) and the cytochrome b6/f (Cyt b6/f) complex. Liquid chromatography nano-tandem mass spectrometry was used to identify three major phosphorylated proteins associated with this PSI-Cyt b6/f supercomplex, two 17-kD PSII subunit P-like proteins and a 70-kD ATP-dependent zinc metalloprotease, FtsH. The PSII subunit P-like protein sequence exhibited 70.6% similarity to the authentic PSII subunit P protein associated with the oxygen-evolving complex of PSII in Chlamydomonas reinhardtii. Tyrosine-146 was identified as a unique phosphorylation site on the UWO 241 PSII subunit P-like polypeptide. Assessment of PSI cyclic electron transport by in vivo P700 photooxidation and the dark relaxation kinetics of P700+ indicated that UWO 241 exhibited PSI cyclic electron transport rates that were 3 times faster and more sensitive to antimycin A than the mesophile control, Chlamydomonas raudensis SAG 49.72. The stability of the PSI-Cyt b6/f supercomplex was dependent upon the phosphorylation status of the PsbP-like protein and the zinc metalloprotease FtsH as well as the presence of high salt. We suggest that adaptation of UWO 241 to its unique low-temperature and high-salt environment favors the phosphorylation of a PSI-Cyt b6/f supercomplex to regulate PSI cyclic electron transport rather than the regulation of state transitions through the phosphorylation of PSII light-harvesting complex proteins. PMID:26169679

  19. The Antarctic Psychrophile Chlamydomonas sp. UWO 241 Preferentially Phosphorylates a Photosystem I-Cytochrome b6/f Supercomplex.

    PubMed

    Szyszka-Mroz, Beth; Pittock, Paula; Ivanov, Alexander G; Lajoie, Gilles; Hüner, Norman P A

    2015-09-01

    Chlamydomonas sp. UWO 241 (UWO 241) is a psychrophilic green alga isolated from Antarctica. A unique characteristic of this algal strain is its inability to undergo state transitions coupled with the absence of photosystem II (PSII) light-harvesting complex protein phosphorylation. We show that UWO 241 preferentially phosphorylates specific polypeptides associated with an approximately 1,000-kD pigment-protein supercomplex that contains components of both photosystem I (PSI) and the cytochrome b₆/f (Cyt b₆/f) complex. Liquid chromatography nano-tandem mass spectrometry was used to identify three major phosphorylated proteins associated with this PSI-Cyt b₆/f supercomplex, two 17-kD PSII subunit P-like proteins and a 70-kD ATP-dependent zinc metalloprotease, FtsH. The PSII subunit P-like protein sequence exhibited 70.6% similarity to the authentic PSII subunit P protein associated with the oxygen-evolving complex of PSII in Chlamydomonas reinhardtii. Tyrosine-146 was identified as a unique phosphorylation site on the UWO 241 PSII subunit P-like polypeptide. Assessment of PSI cyclic electron transport by in vivo P700 photooxidation and the dark relaxation kinetics of P700(+) indicated that UWO 241 exhibited PSI cyclic electron transport rates that were 3 times faster and more sensitive to antimycin A than the mesophile control, Chlamydomonas raudensis SAG 49.72. The stability of the PSI-Cyt b₆/f supercomplex was dependent upon the phosphorylation status of the PsbP-like protein and the zinc metalloprotease FtsH as well as the presence of high salt. We suggest that adaptation of UWO 241 to its unique low-temperature and high-salt environment favors the phosphorylation of a PSI-Cyt b₆/f supercomplex to regulate PSI cyclic electron transport rather than the regulation of state transitions through the phosphorylation of PSII light-harvesting complex proteins. PMID:26169679

  20. Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy

    PubMed Central

    2014-01-01

    Background Biodiesel production from marine microalgae has received much attention as microalgae can be cultivated on non-arable land without the use of potable water, and with the additional benefits of mitigating CO2 emissions and yielding biomass. However, there is still a lack of effective operational strategies to promote lipid accumulation in marine microalgae, which are suitable for making biodiesel since they are mainly composed of saturated and monounsaturated fatty acids. Moreover, the regulatory mechanisms involved in lipid biosynthesis in microalgae under environmental stress are not well understood. Results In this work, the combined effects of salinity and nitrogen depletion stresses on lipid accumulation of a newly isolated marine microalga, Chlamydomonas sp. JSC4, were explored. Metabolic intermediates were profiled over time to observe transient changes during the lipid accumulation triggered by the combination of the two stresses. An innovative cultivation strategy (denoted salinity-gradient operation) was also employed to markedly improve the lipid accumulation and lipid quality of the microalga, which attained an optimal lipid productivity of 223.2 mg L-1 d-1 and a lipid content of 59.4% per dry cell weight. This performance is significantly higher than reported in most related studies. Conclusions This work demonstrated the synergistic integration of biological and engineering technologies to develop a simple and effective strategy for the enhancement of oil production in marine microalgae. PMID:25002905

  1. Ecophysiology, secondary pigments and ultrastructure of Chlainomonas sp. (Chlorophyta) from the European Alps compared with Chlamydomonas nivalis forming red snow.

    PubMed

    Remias, Daniel; Pichrtová, Martina; Pangratz, Marion; Lütz, Cornelius; Holzinger, Andreas

    2016-04-01

    Red snow is a well-known phenomenon caused by microalgae thriving in alpine and polar regions during the melting season. The ecology and biodiversity of these organisms, which are adapted to low temperatures, high irradiance and freeze-thaw events, are still poorly understood. We compared two different snow habitats containing two different green algal genera in the European Alps, namely algae blooming in seasonal rock-based snowfields (Chlamydomonas nivalis) and algae dominating waterlogged snow bedded over ice (Chlainomonassp.). Despite the morphological similarity of the red spores found at the snow surface, we found differences in intracellular organization investigated by light and transmission electron microscopy and in secondary pigments investigated by chromatographic analysis in combination with mass spectrometry. Spores ofChlainomonassp. show clear differences fromChlamydomonas nivalisin cell wall arrangement and plastid organization. Active photosynthesis at ambient temperatures indicates a high physiological activity, despite no cell division being present. Lipid bodies containing the carotenoid astaxanthin, which produces the red color, dominate cells of both species, but are modified differently. While inChlainomonassp. astaxanthin is mainly esterified with two fatty acids and is more apolar, inChamydomonas nivalis, in contrast, less apolar monoesters prevail. PMID:26884467

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

    SciTech Connect

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

    2014-10-18

    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. We demonstrate the growth of C. reinhardtii on acetate provided by mutant strains of the cyanobacterium Synechococcus sp. PCC7002.

  3. The Chlamydomonas Cell Cycle

    PubMed Central

    Cross, Frederick R.; Umen, James G.

    2015-01-01

    The position of Chlamydomonas within the eukaryotic phylogeny makes it a unique model in at least two important ways: as a representative of the critically important, early-diverging lineage leading to plants, and as a microbe retaining important features of the last eukaryotic common ancestor (LECA) that have been lost in the highly studied yeast lineages. Its cell biology has been studied for many decades, and it has well-developed experimental genetic tools, both classical (Mendelian) and molecular. Unlike land plants, it is a haploid with very few gene duplicates, making it ideal for loss-of-function genetic studies. The Chlamydomonas cell cycle has a striking temporal and functional separation between cell growth and rapid cell divisions, probably connected to the interplay between diurnal cycles that drive photosynthetic cell growth with the cell division cycle; it also exhibits a highly choreographed interaction between the cell cycle and its centriole/basal body/flagellar cycle. Here we review the current status of studies of the Chlamydomonas cell cycle. We begin with an overview of cell cycle control in the well-studied yeast and animal systems, which has yielded a canonical, well-supported model. We discuss briefly what is known about similarities and differences in plant cell cycle control compared to this model. We next review the cytology and cell biology of the multiple fission cell cycle of Chlamydomonas. Lastly we review recent genetic approaches and insights into Chlamydomonas cell cycle regulation that have been enabled by a new generation of genomics-based tools. PMID:25690512

  4. Chlamydomonas: A Model Green Plant.

    ERIC Educational Resources Information Center

    Sheffield, E.

    1985-01-01

    Discusses the instructional potential of Chlamydomonas in providing a basis for a range of experimental investigations to illustrate basic biological phenomena. Describes the use of this algae genus in studies of population growth, photosynthesis, and mating behavior. Procedures for laboratory exercises are included. (ML)

  5. 13th International Conference on Chlamydomonas

    SciTech Connect

    Silflow, Carolyn D.

    2014-03-11

    The 13th International Conference on Chlamydomonas (EMBO Workshop on the Cell and Molecular Biology of Chlamydomonas) was held May 27 to June 1, 2008 in Hyeres, France. The conference was the biennial meeting for all researchers studying the green algal systems Chlamydomonas and Volvox. The conference brought together approximately 200 investigators from around the world (North America, Asia, Europe and Australia) representing different fields and disciplines (cell biology, genetics, biochemistry, biophysics, plant physiology, genomics). It provided an opportunity for investigators from different countries to share methodologies and to discuss recent results with a focus on the Chlamydomonas experimental system.

  6. Iron economy in Chlamydomonas reinhardtii

    PubMed Central

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

    2013-01-01

    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. PMID:24032036

  7. The Chlamydomonas heat stress response.

    PubMed

    Schroda, Michael; Hemme, Dorothea; Mühlhaus, Timo

    2015-05-01

    Heat waves occurring at increased frequency as a consequence of global warming jeopardize crop yield safety. One way to encounter this problem is to genetically engineer crop plants toward increased thermotolerance. To identify entry points for genetic engineering, a thorough understanding of how plant cells perceive heat stress and respond to it is required. Using the unicellular green alga Chlamydomonas reinhardtii as a model system to study the fundamental mechanisms of the plant heat stress response has several advantages. Most prominent among them is the suitability of Chlamydomonas for studying stress responses system-wide and in a time-resolved manner under controlled conditions. Here we review current knowledge on how heat is sensed and signaled to trigger temporally and functionally grouped sub-responses termed response elements to prevent damage and to maintain cellular homeostasis in plant cells. PMID:25754362

  8. Cell and molecular biology of Chlamydomonas

    SciTech Connect

    Not Available

    1988-01-01

    This document contains only the abstracts of 92 presentations on the biology of Chlamydomonas. Topics include gene transformations, gene regulation, biosynthetic pathways, cell surfaces, circadian clocks, and the development and structure of the flagellar apparatus. (TEM)

  9. The basal bodies of Chlamydomonas reinhardtii.

    PubMed

    Dutcher, Susan K; O'Toole, Eileen T

    2016-01-01

    The unicellular green alga, Chlamydomonas reinhardtii, is a biflagellated cell that can swim or glide. C. reinhardtii cells are amenable to genetic, biochemical, proteomic, and microscopic analysis of its basal bodies. The basal bodies contain triplet microtubules and a well-ordered transition zone. Both the mother and daughter basal bodies assemble flagella. Many of the proteins found in other basal body-containing organisms are present in the Chlamydomonas genome, and mutants in these genes affect the assembly of basal bodies. Electron microscopic analysis shows that basal body duplication is site-specific and this may be important for the proper duplication and spatial organization of these organelles. Chlamydomonas is an excellent model for the study of basal bodies as well as the transition zone. PMID:27252853

  10. Photomixing of chlamydomonas rheinhardtii suspensions

    NASA Astrophysics Data System (ADS)

    Dervaux, Julien; Capellazzi Resta, Marina; Abou, Bérengère; Brunet, Philippe

    2014-11-01

    Chlamydomonas rheinhardtii is a fast swimming unicellular alga able to bias its swimming direction in gradients of light intensity, an ability know as phototaxis. We have investigated experimentally both the swimming behavior of individual cells and the macroscopic response of shallow suspensions of these micro-organisms in response to a localized light source. At low light intensity, algae exhibit positive phototaxis and accumulate beneath the excitation light. In weakly concentrated thin layers, the balance between phototaxis and cell motility results in steady symmetrical patterns compatible with a purely diffusive model using effective diffusion coefficients extracted from the analysis of individual cell trajectories. However, at higher cell density and layer depth, collective effects induce convective flows around the light source. These flows disturb the cell concentration patterns which spread and may then becomes unstable. Using large passive tracer particles, we have characterized the velocity fields associated with this forced bioconvection and their dependence on the cell density and layer depth. By tuning the light distribution, this mechanism of photo-bioconvection allows a fine control over the local fluid flows, and thus the mixing efficiency, in algal suspensions.

  11. Effect of Triacontanol on Chlamydomonas1

    PubMed Central

    Houtz, Robert L.; Ries, Stanley K.; Tolbert, N. E.

    1985-01-01

    Treatment of Chlamydomonas reinhardtii cells, cultured at 5% CO2, with 1 to 1000 micrograms triacontanol (TRIA) per liter resulted in 21 to 35% increases in cell density, 7 to 31% increases in total chlorophyll, and 20 to 100% increases in photosynthetic CO2 assimilation. The increase in CO2 fixation with TRIA treatment occurred before, and was independent of, increases in total chlorophyll or cell number. Chlamydomonas cells responded to a broad range of TRIA concentrations that were at least one order of magnitude above the optimum concentration established for higher plants. The necessity for larger concentrations of TRIA may be due to destabilizing effects of Ca2+ and K+ present in the Chlamydomonas growth medium. These ions caused flocculation of the colloidally dispersed TRIA in apparent competition with binding of [14C]TRIA to Chlamydomonas cells. Octacosanol inhibited the effect of TRIA on photosynthetic CO2 assimilation. TRIA treatment did not alter the distribution of 14C-label among photosynthetic products. The effect of TRIA on photosynthetic CO2 assimilation increased with time after treatment up to 3 days. Chlamydomonas cells that had been grown at low-CO2 (air) did not respond to TRIA, and transfer of high-CO2 (5%) grown cells that had responded to TRIA to a low-CO2 atmosphere resulted in a loss of the effect of TRIA. The effect of pH on photosynthetic CO2 assimilation indicated that CO2 is probably the species of inorganic carbon utilized by control and TRIA-treated Chlamydomonas cells. PMID:16664414

  12. A steering mechanism for phototaxis in Chlamydomonas.

    PubMed

    Bennett, Rachel R; Golestanian, Ramin

    2015-03-01

    Chlamydomonas shows both positive and negative phototaxis. It has a single eyespot near its equator, and as the cell rotates during the forward motion, the light signal received by the eyespot varies. We use a simple mechanical model of Chlamydomonas that couples the flagellar beat pattern to the light intensity at the eyespot to demonstrate a mechanism for phototactic steering that is consistent with observations. The direction of phototaxis is controlled by a parameter in our model, and the steering mechanism is robust to noise. Our model shows switching between directed phototaxis when the light is on and run-and-tumble behaviour in the dark. PMID:25589576

  13. A steering mechanism for phototaxis in Chlamydomonas

    PubMed Central

    Bennett, Rachel R.; Golestanian, Ramin

    2015-01-01

    Chlamydomonas shows both positive and negative phototaxis. It has a single eyespot near its equator, and as the cell rotates during the forward motion, the light signal received by the eyespot varies. We use a simple mechanical model of Chlamydomonas that couples the flagellar beat pattern to the light intensity at the eyespot to demonstrate a mechanism for phototactic steering that is consistent with observations. The direction of phototaxis is controlled by a parameter in our model, and the steering mechanism is robust to noise. Our model shows switching between directed phototaxis when the light is on and run-and-tumble behaviour in the dark. PMID:25589576

  14. Fermentative metabolism of Chlamydomonas reinhardtii

    SciTech Connect

    Gfeller, R.P.; Gibbs, M.

    1984-05-01

    The anaerobic starch breakdown into end-products in the green alga Chlamydomonas reinhardtii F-60 has been investigated in the dark and in the light. The effects of 3-(3,4-dicholorophenyl)-1,1-dimethylurea (DCMU) and carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) on the fermentation in the light have also been investigated. Anaerobic starch breakdown rate (13.1 +/- 3.5 micromoles C per milligram chlorophyll per hour) is increased 2-fold by FCCP in the dark. Light (100 watts per square meter) decreases up to 4-fold the dark rate, an inhibition reversed by FCCP. In the dark, formate, acetate, and ethanol are formed in the ratios of 2.07:1.07:0.91, and account for roughly 100% of the C from the starch. H/sub 2/ production is 0.43 mole per mole glucose in the starch. Glycerol, D-lactate, and CO/sub 2/ have been detected in minor amounts. In the light, with DCMU and FCCP present, acetate is produced in a 1:1 ratio to formate, and H/sub 2/ evolution is 2.13 moles per mole glucose. When FCCP only is present, acetate production is lower, and CO/sub 2/ and H/sub 2/ evolutions is 1.60 and 4.73 moles per mole glucose, respectively. When DCMU alone is present, CO/sub 2/ and H/sub 2/ photoevolution is higher than in the dark. Without DCMU, CO/sub 2/ and H/sub 2/ evolution is about 100% higher than in its presence. In both conditions, acetate is not formed. In all conditions in the light, ethanol is a minor product. Formate production is least affected by light. The stoichiometry in the dark indicates that starch is degraded via the glycolytic pathway, and pyruvate is broken down into acetyl-CoA and formate. Acetyl-CoA is further dissimilated into acetate and ethanol. In the light, acetate is produced only in the presence of FCCP and, when photophosphorylation is possible, it is used in unidentified reactions. Ethanol formation is inhibited by the light in all conditions. 30 references, 5 figures, 2 tables.

  15. A steering mechanism for phototaxis in Chlamydomonas

    NASA Astrophysics Data System (ADS)

    Bennett, Rachel; Golestanian, Ramin

    2015-03-01

    Chlamydomonas shows both positive and negative phototaxis. It has a single eyespot near its equator and as the cell rotates during forward motion the light signal received by the eyespot varies. We use a simple mechanical model of Chlamydomonas that couples the flagellar beat pattern to the light intensity at the eyespot to demonstrate a mechanism for phototactic steering that is consistent with observations. The direction of phototaxis is controlled by a parameter in our model and the steering mechanism is robust to noise. In the dark, our model shows emergent run-and-tumble behavior and we see switching between directed phototaxis and run-and-tumble when we switch the light on and off.

  16. The Dynein Gene Family in Chlamydomonas Reinhardtii

    PubMed Central

    Porter, M. E.; Knott, J. A.; Myster, S. H.; Farlow, S. J.

    1996-01-01

    To correlate dynein heavy chain (Dhc) genes with flagellar mutations and gain insight into the function of specific dynein isoforms, we placed eight members of the Dhc gene family on the genetic map of Chlamydomonas. Using a PCR-based strategy, we cloned 11 Dhc genes from Chlamydomonas. Comparisons with other Dhc genes indicate that two clones correspond to genes encoding the alpha and beta heavy chains of the outer dynein arm. Alignment of the predicted amino acid sequences spanning the nucleotide binding site indicates that the remaining nine clones can be subdivided into three groups that are likely to include representatives of the inner-arm Dhc isoforms. Gene-specific probes reveal that each clone represents a single-copy gene that is expressed as a transcript of the appropriate size (>13 kb) sufficient to encode a high molecular weight Dhc polypeptide. The expression of all nine genes is upregulated in response to deflagellation, suggesting a role in axoneme assembly or motility. Restriction fragment length polymorphisms between divergent C. reinhardtii strains have been used to place each Dhc gene on the genetic map of Chlamydomonas. These studies lay the groundwork for correlating defects in different Dhc genes with specific flagellar mutations. PMID:8889521

  17. Effect of Triacontanol on Chlamydomonas1

    PubMed Central

    Houtz, Robert L.; Ries, Stanley K.; Tolbert, N. E.

    1985-01-01

    Increased photosynthetic CO2 assimilation by Chlamydomonas reinhardtii cells treated with triacontanol (TRIA) was not due to changes in glycolate excretion, CO2 compensation point, or the sensitivity of photosynthetic CO2 assimilation to O2. Kinetic analysis of TRIA-treated cells showed that the increase in photosynthetic CO2 assimilation was a result of an increase in the apparent Vmax for intact cells. The total activity of ribulose-P2 carboxylase/oxygenase was higher in cell lysates from TRIA-treated cells. However quantification of this enzyme concentration by binding of [14C]carboxyarabinitol-P2 did not show an increase in TRIA-treated cells. Thus, there was an increase in the specific activity of ribulose-P2 carboxylase/oxygenase extracted from Chlamydomonas cells treated with TRIA. TRIA alone had no effect on the activity of the enzyme in cell lysates from Chlamydomonas or purified from spinach (Spinacia oleracea L.) leaves. The ribulose-P2 pool was 50 to 60% higher in cells treated with TRIA that were assayed for photosynthetic CO2 assimilation at high- and low-CO2. TRIA also increased ribulose-P2 levels in the absence of CO2 in the light with atmospheres of N2 or N2 with 21% O2. PMID:16664415

  18. MEETING: Chlamydomonas Annotation Jamboree - October 2003

    SciTech Connect

    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

  19. Chlamydomonas Flavodiiron Proteins Facilitate Acclimation to Anoxia During Sulfur Deprivation

    PubMed Central

    Jokel, Martina; Kosourov, Sergey; Battchikova, Natalia; Tsygankov, Anatoly A.; Aro, Eva Mari; Allahverdiyeva, Yagut

    2015-01-01

    The flavodiiron proteins (FDPs) are involved in the detoxification of oxidative compounds, such as nitric oxide (NO) or O2 in Archaea and Bacteria. In cyanobacteria, the FDPs Flv1 and Flv3 are essential in the light-dependent reduction of O2 downstream of PSI. Phylogenetic analysis revealed that two genes (flvA and flvB) in the genome of Chlamydomonas reinhardtii show high homology to flv1 and flv3 genes of the cyanobacterium Synechocystis sp. PCC 6803. The physiological role of these FDPs in eukaryotic green algae is not known, but it is of a special interest since these phototrophic organisms perform oxygenic photosynthesis similar to higher plants, which do not possess FDP homologs. We have analyzed the levels of flvA and flvB transcripts in C. reinhardtii cells under various environmental conditions and showed that these genes are highly expressed under ambient CO2 levels and during the early phase of acclimation to sulfur deprivation, just before the onset of anaerobiosis and the induction of efficient H2 photoproduction. Importantly, the increase in transcript levels of the flvA and flvB genes was also corroborated by protein levels. These results strongly suggest the involvement of FLVA and FLVB proteins in alternative electron transport. PMID:26063391

  20. Chlamydomonas Flavodiiron Proteins Facilitate Acclimation to Anoxia During Sulfur Deprivation.

    PubMed

    Jokel, Martina; Kosourov, Sergey; Battchikova, Natalia; Tsygankov, Anatoly A; Aro, Eva Mari; Allahverdiyeva, Yagut

    2015-08-01

    The flavodiiron proteins (FDPs) are involved in the detoxification of oxidative compounds, such as nitric oxide (NO) or O(2) in Archaea and Bacteria. In cyanobacteria, the FDPs Flv1 and Flv3 are essential in the light-dependent reduction of O(2) downstream of PSI. Phylogenetic analysis revealed that two genes (flvA and flvB) in the genome of Chlamydomonas reinhardtii show high homology to flv1 and flv3 genes of the cyanobacterium Synechocystis sp. PCC 6803. The physiological role of these FDPs in eukaryotic green algae is not known, but it is of a special interest since these phototrophic organisms perform oxygenic photosynthesis similar to higher plants, which do not possess FDP homologs. We have analyzed the levels of flvA and flvB transcripts in C. reinhardtii cells under various environmental conditions and showed that these genes are highly expressed under ambient CO(2) levels and during the early phase of acclimation to sulfur deprivation, just before the onset of anaerobiosis and the induction of efficient H(2) photoproduction. Importantly, the increase in transcript levels of the flvA and flvB genes was also corroborated by protein levels. These results strongly suggest the involvement of FLVA and FLVB proteins in alternative electron transport. PMID:26063391

  1. Regulation of Flagellar Length in Chlamydomonas

    PubMed Central

    Wilson, Nedra F.; Iyer, Janaki Kannan; Buchheim, Julie A.; Meek, William

    2008-01-01

    Chlamydomonas reinhardtii has two apically localized flagella that are maintained at an equal and appropriate length. Assembly and maintenance of flagella requires a microtubule-based transport system known as intraflagellar transport (IFT). During IFT, proteins destined for incorporation into or removal from a flagellum are carried along doublet microtubules via IFT particles. Regulation of IFT activity therefore is pivotal in determining the length of a flagellum. Reviewed is our current understanding of the role of IFT and signal transduction pathways in the regulation of flagellar length. PMID:18692148

  2. Light stress and photoprotection in Chlamydomonas reinhardtii.

    PubMed

    Erickson, Erika; Wakao, Setsuko; Niyogi, Krishna K

    2015-05-01

    Plants and algae require light for photosynthesis, but absorption of too much light can lead to photo-oxidative damage to the photosynthetic apparatus and sustained decreases in the efficiency and rate of photosynthesis (photoinhibition). Light stress can adversely affect growth and viability, necessitating that photosynthetic organisms acclimate to different environmental conditions in order to alleviate the detrimental effects of excess light. The model unicellular green alga, Chlamydomonas reinhardtii, employs diverse strategies of regulation and photoprotection to avoid, minimize, and repair photo-oxidative damage in stressful light conditions, allowing for acclimation to different and changing environments. PMID:25758978

  3. [An experiment with Chlamydomonas reinhardtii on the Kosmos-2044 biosatellite].

    PubMed

    Gavrilova, O V; Gabova, A V; Goriainova, L N; Filatova, E V

    1992-01-01

    Space experiment with Chlamydomonas reinhardtii demonstrated that the microgravity effects were noted in Chlamydomonas at both cellular and population levels: in space the cell size is increased, stage of active growth of the culture is extended, it contains the juvenile vegetative motile cells in greater quantities. Ultrastructural analysis indicated that in microgravity the changes in shape, structure and distribution of intracellular organelles and in volume ratio of organelles and cytoplasma are absent. Chlamydomonas data are in line with the results of the Infusoria and Chlorella experiments. PMID:1307032

  4. Nucleotide-metabolizing enzymes in Chlamydomonas flagella.

    PubMed

    Watanabe, T; Flavin, M

    1976-01-10

    Nucleotides have at least two functions in eukaryotic cilia and flagella. ATP, originating in the cells, is utilized for motility by energy-transducing protein(s) called dynein, and the binding of guanine nucleotides to tubulin, and probably certain transformations of the bound nucleotides, are prerequisites for the assembly of microtubules. Besides dynein, which can be solubulized from Chlamydomonas flagella as a heterogeneous, Mg2+ or Ca2+-activated ATPase, we have purified and characterized five other flagellar enzymes involved in nucleotide transformations. A homogeneous, low molecular weight, Ca2+-specific adenosine triphosphatase was isolated, which was inhibited by Mg2+ and was not specific for ATP. This enzyme was not formed by treating purified dynein with proteases. It was absent from extracts of Tetrahymena cilia. Its function might be an auxiliary energy transducer, or in steering or tactic responses. Two species of adenylate kinase were isolated, one of which was much elevated in regenerating flagella; the latter was also present in cell bodies. A large part of flagellar nucleoside diphosphokinase activity could not be solubilized. Two soluble enzyme species were identified, one of which was also present in cell bodies. Since these enzymes are of interest because they might function in microtubule assembly, we studied the extent to which brain nucleoside diphosphokinase co-polymerizes with tubulin purified by repeated cycles of polymerization. Arginine kinase was not detected in Chlamydomonas flagellar extracts. PMID:397

  5. International Conference on the Cell and Molecular Biology of Chlamydomonas

    SciTech Connect

    Dr. Stephen Miller

    2010-06-10

    The 2010 Conference on the Cell and Molecular Biology of Chlamydomonas was held June 6-10 near Boston, MA, and attracted a record 273 participants, 146 from US labs, 10 from Canada, and the remainder from 18 other countries. The single-celled algal protist Chlamydomonas is a key research organism for many investigators, including those who study photosynthesis, cell motility, adaptation to environmental stresses, the evolution of multicellularity, and the production of biofuels. Chlamydomonas researchers gather every two years at a research conference to exchange methods, develop collaborative efforts, disseminate recent findings, and plan large-scale studies to improve the usefulness of this unique research organism. This conference provides the only opportunity for Chlamydomonas scientists who work on different research problems to meet face to face, and greatly speeds progress in their respective fields. An important function of these Chlamydomonas conferences is to promote and showcase the work of younger scientists, and to attract new investigators into the Chlamydomonas community. DOE award SC0004085 was used to offset the travel and registration costs for 18 young investigators, 9 of whom were women, including one African American. Most of these scientists would not have been able to attend the conference without DOE support. A total of 208 research presentations were made at the meeting, 80 talks (63 presented by students, postdocs, and pre-tenured faculty) and 128 posters. Cell motility and biofuels/metabolism were the best-represented research areas, with a total of 77 presentations. This fact underscores the growing importance of Chlamydomonas as a research and production tool in the rapidly expanding world of biofuels research. A total of 28 talks and posters were presented on the topics of photosynthesis and stress responses, which were among the next best-represented research areas. As at several recent Chlamydomonas meetings, important advances were

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

    NASA Technical Reports Server (NTRS)

    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.

  7. Swimming of Chlamydomonas reinhardtii in weakly elastic fluids

    NASA Astrophysics Data System (ADS)

    Yang, Jing; Gollub, Jerry; Arratia, Paulo

    2012-11-01

    The swimming behavior of the algae Chlamydomonas reinhardtii in weakly elastic fluids is investigated in experiments using microscopy and tracking methods. The effects of fluid viscosity and elasticity on the swimming speed, flagellar shape, beating frequency, and efficiency are examined. Here, the fluid viscosity is varied using water and sucrose solutions, while fluid elasticity is introduced by adding flexible polymer CMC (carboxymethyl cellulose) to the buffer solution. Swimming experiments are performed in a thin-film apparatus equipped with a microscope and high-speed camera. We find that even small amounts of fluid elasticity can have a significant effect on the swimming kinematics and dynamics of Chlamydomonas because of the relatively high beating frequency of its flagella (50-60 Hz). For example, the Chlamydomonas swimming speed is hindered by fluid elasticity compared to Newtonian fluids. In addition, the algae swimming speed decreases as the fluid elasticity is increased. This research is supported by the NSF through grant DMR-1104705.

  8. Studies on flagellar shortening in Chlamydomonas reinhardtii

    SciTech Connect

    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.

  9. Patching Holes in the Chlamydomonas Genome.

    PubMed

    Tulin, Frej; Cross, Frederick R

    2016-01-01

    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. However, the current reference genome contains ∼1000 blocks of unknown sequence ('N-islands'), which are frequently placed in introns of annotated gene models. We developed a strategy to search for previously unknown exons hidden within such blocks, and determine the sequence, and exon/intron boundaries, of such exons. These methods are based on assembly and alignment of short cDNA and genomic DNA reads, completely independent of prior reference assembly or annotation. Our evidence indicates that a substantial proportion of the annotated intronic N-islands contain hidden exons. For most of these, our algorithm recovers full exonic sequence with associated splice junctions and exon-adjacent intronic sequence. These new exons represent de novo sequence generally present nowhere in the assembled genome, and the added sequence improves evolutionary conservation of the predicted encoded peptides. PMID:27175017

  10. Patching Holes in the Chlamydomonas Genome

    PubMed Central

    Tulin, Frej; Cross, Frederick R.

    2016-01-01

    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. However, the current reference genome contains ∼1000 blocks of unknown sequence (‘N-islands’), which are frequently placed in introns of annotated gene models. We developed a strategy to search for previously unknown exons hidden within such blocks, and determine the sequence, and exon/intron boundaries, of such exons. These methods are based on assembly and alignment of short cDNA and genomic DNA reads, completely independent of prior reference assembly or annotation. Our evidence indicates that a substantial proportion of the annotated intronic N-islands contain hidden exons. For most of these, our algorithm recovers full exonic sequence with associated splice junctions and exon-adjacent intronic sequence. These new exons represent de novo sequence generally present nowhere in the assembled genome, and the added sequence improves evolutionary conservation of the predicted encoded peptides. PMID:27175017

  11. Analysis of Axonemal Assembly During Ciliary Regeneration in Chlamydomonas.

    PubMed

    Hunter, Emily L; Sale, Winfield S; Alford, Lea M

    2016-01-01

    Chlamydomonas reinhardtii is an outstanding model genetic organism for study of assembly of cilia. Here, methods are described for synchronization of ciliary regeneration in Chlamydomonas to analyze the sequence in which ciliary proteins assemble. In addition, the methods described allow analysis of the mechanisms involved in regulation of ciliary length, the proteins required for ciliary assembly, and the temporal expression of genes encoding ciliary proteins. Ultimately, these methods can contribute to discovery of conserved genes that when defective lead to abnormal ciliary assembly and human disease. PMID:27514926

  12. Systemic Cold Stress Adaptation of Chlamydomonas reinhardtii*

    PubMed Central

    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

  13. Manganese encrustation of zygospores of a chlamydomonas (chlorophyta: volvocales).

    PubMed

    Schulz-Baldes, M; Lewin, R A

    1975-06-13

    In media containing normal trace-element supplements, but not in manganese-deficient media, zygospores of a new species of Chlamydomonas (isolated from soil) become encrusted with a dark brown mineral coating. Staining with benzidine indicates that the encrustation is rich in manganese. This has been confirmed by x-ray analysis in combination with a scanning electron microscope. PMID:17798436

  14. The uni chromosome of Chlamydomonas: histone genes and nucleosome structure.

    PubMed

    Walther, Z; Hall, J L

    1995-09-25

    The uni linkage group (ULG) of Chlamydomonas reinhardtii contains many genes involved in the basal body-flagellar system. Recent evidence suggests that the corresponding uni chromosome is located in close proximity to the basal body complex. In the course of studies into its molecular organization, we have found a cluster of four histone genes on the ULG. The genes are arranged as divergently-transcribed pairs: H3-H4 and H2B-H2A. Genomic sequencing reveals that these genes lack introns and contain characteristic 3' palindromes similar to those of animals. The predicted amino acid sequences are highly conserved across species, with greatest similarities to the histone genes of Volvox. Southern analysis shows that each histone gene is present in 15-20 copies in Chlamydomonas and suggests a dispersed genomic organization. Northern analysis of mitotically-synchronized cells shows that, like the replication-dependent histones of higher eukaryotes, Chlamydomonas histone genes are expressed during S-phase. Using a gene-specific probe on Northern blots, we provide evidence that the ULG H4 gene is regulated in the same manner as other Chlamydomonas histone genes. Finally, micrococcal nuclease protection experiments show that the uni chromosome itself associates with histone proteins and displays a conventional nucleosomal banding pattern. PMID:7479007

  15. UV-B Perception and Acclimation in Chlamydomonas reinhardtii.

    PubMed

    Tilbrook, Kimberley; Dubois, Marine; Crocco, Carlos D; Yin, Ruohe; Chappuis, Richard; Allorent, Guillaume; Schmid-Siegert, Emanuel; Goldschmidt-Clermont, Michel; Ulm, Roman

    2016-04-01

    Plants perceive UV-B, an intrinsic component of sunlight, via a signaling pathway that is mediated by the photoreceptor UV RESISTANCE LOCUS8 (UVR8) and induces UV-B acclimation. To test whether similar UV-B perception mechanisms exist in the evolutionarily distant green alga Chlamydomonas reinhardtii, we identified Chlamydomonas orthologs of UVR8 and the key signaling factor CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). Cr-UVR8 shares sequence and structural similarity to Arabidopsis thaliana UVR8, has conserved tryptophan residues for UV-B photoreception, monomerizes upon UV-B exposure, and interacts with Cr-COP1 in a UV-B-dependent manner. Moreover, Cr-UVR8 can interact with At-COP1 and complement the Arabidopsis uvr8 mutant, demonstrating that it is a functional UV-B photoreceptor. Chlamydomonas shows apparent UV-B acclimation in colony survival and photosynthetic efficiency assays. UV-B exposure, at low levels that induce acclimation, led to broad changes in the Chlamydomonas transcriptome, including in genes related to photosynthesis. Impaired UV-B-induced activation in the Cr-COP1 mutant hit1 indicates that UVR8-COP1 signaling induces transcriptome changes in response to UV-B. Also, hit1 mutants are impaired in UV-B acclimation. Chlamydomonas UV-B acclimation preserved the photosystem II core proteins D1 and D2 under UV-B stress, which mitigated UV-B-induced photoinhibition. These findings highlight the early evolution of UVR8 photoreceptor signaling in the green lineage to induce UV-B acclimation and protection. PMID:27020958

  16. Developing molecular tools for Chlamydomonas reinhardtii

    NASA Astrophysics Data System (ADS)

    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

  17. Three light-inducible heat shock genes of Chlamydomonas reinhardtii.

    PubMed Central

    von Gromoff, E D; Treier, U; Beck, C F

    1989-01-01

    Genomic clones representing three Chlamydomonas reinhardtii genes homologous to the Drosophila hsp70 heat shock gene were isolated. The mRNAs of genes hsp68, hsp70, and hsp80 could be translated in vitro into proteins of Mr 68,000, 70,000, and 80,000, respectively. Transcription of these genes increased dramatically upon heat shock, and the corresponding mRNAs rapidly accumulated, reaching a peak at around 30 min after a shift to the elevated temperature. Light also induced the accumulation of the mRNAs encoded by these heat shock genes. A shift of dark-grown cells to light resulted in a drastic increase in mRNA levels, which reached a maximum at around 1 h after the shift. Thus, in Chlamydomonas, expression of hsp70-homologous heat shock genes appears to be regulated by thermal stress and light. Images PMID:2779571

  18. Salinity affects the photoacclimation of Chlamydomonas raudensis Ettl UWO241.

    PubMed

    Takizawa, Kenji; Takahashi, Shinichiro; Hüner, Norman P A; Minagawa, Jun

    2009-03-01

    Chlamydomonas raudensis Ettl UWO241, a natural variant of C. raudensis, is deficient in state transitions. Its habitat, the deepest layer of Lake Bonney in Antarctica, features low irradiance, low temperature, and high salinity. Although psychrophily and low-light acclimation of this green alga has been described, very little information is available on the effect of salinity. Here, we demonstrate that this psychrophile is halotolerant, not halophilic, and it shows energy redistribution between photosystem I and II based on energy spillover under low-salt conditions. Furthermore, we revealed that C. raudensis exhibits higher non-photochemical quenching in comparison with the mesophile Chlamydomonas reinhardtii, when grown with low-salt, which is due to the lower proton conductivity across the thylakoid membrane. Significance of the C. raudensis UWO241 traits found in the low salinity culture are implicated with their natural habitats, including the high salinity and extremely stable light environments. PMID:19137412

  19. Nonphotochemical quenching of chlorophyll fluorescence in Chlamydomonas reinhardtii.

    PubMed

    Finazzi, Giovanni; Johnson, Giles N; Dall'Osto, Luca; Zito, Francesca; Bonente, Giulia; Bassi, Roberto; Wollman, Francis-André

    2006-02-01

    Unlike plants, Chlamydomonas reinhardtii shows a restricted ability to develop nonphotochemical quenching upon illumination. Most of this limited quenching is due to state transitions instead of DeltapH-driven high-energy state quenching, qE. The latter could only be observed when the ability of the cells to perform photosynthesis was impaired, either by lowering temperature to approximately 0 degrees C or in mutants lacking RubisCO activity. Two main features were identified that account for the low level of qE in Chlamydomonas. On one hand, the electrochemical proton gradient generated upon illumination is apparently not sufficient to promote fluorescence quenching. On the other hand, the capacity to transduce the presence of a DeltapH into a quenching response is also intrinsically decreased in this alga, when compared to plants. The possible mechanism leading to these differences is discussed. PMID:16445291

  20. Relevance of nutrient media composition for hydrogen production in Chlamydomonas.

    PubMed

    Gonzalez-Ballester, David; Jurado-Oller, Jose Luis; Fernandez, Emilio

    2015-09-01

    Microalgae are capable of biological H2 photoproduction from water, solar energy, and a variety of organic substrates. Acclimation responses to different nutrient regimes finely control photosynthetic activity and can influence H2 production. Hence, nutrient stresses are an interesting scenario to study H2 production in photosynthetic organisms. In this review, we mainly focus on the H2-production mechanisms in Chlamydomonas reinhardtii and the physiological relevance of the nutrient media composition when producing H2. PMID:25952745

  1. D-lactate metabolism in the alga, Chlamydomonas Reinhardtii

    SciTech Connect

    Husic, D.W.; Tolbert, N.E.

    1986-05-01

    (/sup 14/C)D-lactate rapidly accumulates in Chlamydomonas cells under anaerobic conditions from the sugar-phosphate pools which are labeled during photosynthesis with /sup 14/CO/sub 2/. A soluble D-lactate dehydrogenase (30 ..mu..mol NADH oxidized/h/mg Chl), which functions only in the direction of pyruvate reduction, has been partially purified and characterized. The D-lactate is reoxidized in Chlamydomonas by a mitochondrial membrane-bound dehydrogenase. This enzyme is known in the plant literature as glycolate dehydrogenase, an enzyme of the oxidative photosynthetic carbon (C/sub 2/) cycle. This dehydrogenase may be linked to the mitochondrial electron transport chain, although the direct electron acceptor is unknown. Therefore, D-lactate accumulation may be, in part, due to the shut down of electron transport during anaerobiosis. In vivo chase experiments have shown that the D-lactate turns over rapidly when algal cells, which have been grown with air levels of CO/sub 2/ (0.04%), are returned to aerobic conditions in the light. Such turnover is not observed in cells which had been grown with 1 to 5% CO/sub 2/. Cells grown with high CO/sub 2/ have lower levels of glycolate dehydrogenase activity. They are currently using mutants of Chlamydomonas deficient in mitochondrial respiration to study the role of D-lactate oxidation in these algae.

  2. Forward and reverse genetic analysis of microtubule motors in Chlamydomonas.

    PubMed

    Pazour, G J; Witman, G B

    2000-12-01

    The ability to integrate biochemical, cell biological, and genetic approaches makes Chlamydomonas reinhardtii the premier model organism for studies of the eukaryotic flagellum and its associated molecular motors. Hundreds of motility mutations have been identified in Chlamydomonas, including many that affect dyneins and kinesins. These mutations have yielded much information on the structure and function of the motors as well as the roles of individual subunits within the motors. The development of insertional mutagenesis has opened the door to powerful new approaches for genetic analysis in Chlamydomonas. Insertional mutants are created by transforming cells with DNA-containing selectable markers. The DNA is randomly integrated throughout the genome and usually deletes part of the chromosome at the site of insertion, thereby creating mutations that are marked by the integrated DNA. These mutations can be used for forward genetic approaches where one characterizes a mutant phenotype and then clones the relevant gene using the integrated DNA as a tag. The insertional mutants also may be used in a reverse genetic approach in which mutants lacking a gene of interest are identified by DNA hybridization. We describe methods to generate and characterize insertional mutants, using mutations that affect the outer dynein arm as examples. PMID:11133235

  3. N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

    PubMed Central

    Chen, Kai; Deng, Xin; Yu, Miao; Han, Dali; Hao, Ziyang; Liu, Jianzhao; Lu, Xingyu; Dore, Louis C; Weng, Xiaocheng; Ji, Quanjiang; Mets, Laurens; He, Chuan

    2015-01-01

    SUMMARY N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. PMID:25936837

  4. Activation of Autophagy by Metals in Chlamydomonas reinhardtii

    PubMed Central

    Pérez-Martín, Marta; Blaby-Haas, Crysten E.; Pérez-Pérez, María Esther; Andrés-Garrido, Ascensión; Blaby, Ian K.; Merchant, Sabeeha S.

    2015-01-01

    Autophagy is an intracellular self-degradation pathway by which eukaryotic cells recycle their own material in response to specific stress conditions. Exposure to high concentrations of metals causes cell damage, although the effect of metal stress on autophagy has not been explored in photosynthetic organisms. In this study, we investigated the effect of metal excess on autophagy in the model unicellular green alga Chlamydomonas reinhardtii. We show in cells treated with nickel an upregulation of ATG8 that is independent of CRR1, a global regulator of copper signaling in Chlamydomonas. A similar effect on ATG8 was observed with copper and cobalt but not with cadmium or mercury ions. Transcriptome sequencing data revealed an increase in the abundance of the protein degradation machinery, including that responsible for autophagy, and a substantial overlap of that increased abundance with the hydrogen peroxide response in cells treated with nickel ions. Thus, our results indicate that metal stress triggers autophagy in Chlamydomonas and suggest that excess nickel may cause oxidative damage, which in turn activates degradative pathways, including autophagy, to clear impaired components and recover cellular homeostasis. PMID:26163317

  5. Activation of Autophagy by Metals in Chlamydomonas reinhardtii.

    PubMed

    Pérez-Martín, Marta; Blaby-Haas, Crysten E; Pérez-Pérez, María Esther; Andrés-Garrido, Ascensión; Blaby, Ian K; Merchant, Sabeeha S; Crespo, José L

    2015-09-01

    Autophagy is an intracellular self-degradation pathway by which eukaryotic cells recycle their own material in response to specific stress conditions. Exposure to high concentrations of metals causes cell damage, although the effect of metal stress on autophagy has not been explored in photosynthetic organisms. In this study, we investigated the effect of metal excess on autophagy in the model unicellular green alga Chlamydomonas reinhardtii. We show in cells treated with nickel an upregulation of ATG8 that is independent of CRR1, a global regulator of copper signaling in Chlamydomonas. A similar effect on ATG8 was observed with copper and cobalt but not with cadmium or mercury ions. Transcriptome sequencing data revealed an increase in the abundance of the protein degradation machinery, including that responsible for autophagy, and a substantial overlap of that increased abundance with the hydrogen peroxide response in cells treated with nickel ions. Thus, our results indicate that metal stress triggers autophagy in Chlamydomonas and suggest that excess nickel may cause oxidative damage, which in turn activates degradative pathways, including autophagy, to clear impaired components and recover cellular homeostasis. PMID:26163317

  6. Actin is required for IFT regulation in Chlamydomonas reinhardtii

    PubMed Central

    Avasthi, Prachee; Onishi, Masayuki; Karpiak, Joel; Yamamoto, Ryosuke; Mackinder, Luke; Jonikas, Martin C.; Sale, Winfield S.; Shoichet, Brian; Pringle, John R.; Marshall, Wallace F.

    2014-01-01

    Summary Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Since actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here, we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation, and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor suggesting actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length. PMID:25155506

  7. Actin is required for IFT regulation in Chlamydomonas reinhardtii.

    PubMed

    Avasthi, Prachee; Onishi, Masayuki; Karpiak, Joel; Yamamoto, Ryosuke; Mackinder, Luke; Jonikas, Martin C; Sale, Winfield S; Shoichet, Brian; Pringle, John R; Marshall, Wallace F

    2014-09-01

    Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length. PMID:25155506

  8. Tools for regulated gene expression in the chloroplast of Chlamydomonas.

    PubMed

    Rochaix, Jean-David; Surzycki, Raymond; Ramundo, Silvia

    2014-01-01

    The green unicellular alga Chlamydomonas reinhardtii has emerged as a very attractive model system for chloroplast genetic engineering. Algae can be transformed readily at the chloroplast level through bombardment of cells with a gene gun, and transformants can be selected using antibiotic resistance or phototrophic growth. An inducible chloroplast gene expression system could be very useful for several reasons. First, it could be used to elucidate the function of essential chloroplast genes required for cell growth and survival. Second, it could be very helpful for expressing proteins which are toxic to the algal cells. Third, it would allow for the reversible depletion of photosynthetic complexes thus making it possible to study their biogenesis in a controlled fashion. Fourth, it opens promising possibilities for hydrogen production in Chlamydomonas. Here we describe an inducible/repressible chloroplast gene expression system in Chlamydomonas in which the copper-regulated Cyc6 promoter drives the expression of the nuclear Nac2 gene encoding a protein which is targeted to the chloroplast where it acts specifically on the chloroplast psbD 5'-untranslated region and is required for the stable accumulation of the psbD mRNA and photosystem II. The system can be used for any chloroplast gene or transgene by placing it under the control of the psbD 5'-untranslated region. PMID:24599871

  9. Nucleated assembly of Chlamydomonas and Volvox cell walls.

    PubMed

    Adair, W S; Steinmetz, S A; Mattson, D M; Goodenough, U W; Heuser, J E

    1987-11-01

    The Chlamydomonas reinhardtii cell wall is made up of hydroxyproline-rich glycoproteins, arranged in five distinct layers. The W6 (crystalline) layer contains three major glycoproteins (GP1, GP2, GP3), selectively extractable with chaotropic agents, that self-assemble into crystals in vitro. A system to study W6 assembly in a quantitative fashion was developed that employs perchlorate-extracted Chlamydomonas cells as nucleating agents. Wall reconstitution by biotinylated W6 monomers was monitored by FITC-streptavidin fluorescence and quick-freeze/deep-etch electron microscopy. Optimal reconstitution was obtained at monomer concentrations (0.2-0.3 mg/ml) well below those required for nonnucleated assembly. Assembly occurred from multiple nucleation sites, and faithfully reflected the structure of the intact W6 layer. Specificity of nucleated assembly was demonstrated using two cell-wall mutants (cw-2 and cw-15); neither served as a substrate for assembly of wild-type monomers. In addition, W6 sublayers were assembled from purified components: GP2 and GP3 coassembled to form the inner (W6A) sublayer; this then served as a substrate for self-assembly of GP1 into the outer (W6B) sublayer. Finally, evolutionary relationships between C. reinhardtii and two additional members of the Volvocales (Chlamydomonas eugametos and Volvox carteri) were explored by performing interspecific reconstitutions. Hybrid walls were obtained between C. reinhardtii and Volvox but not with C. eugametos, confirming taxonomic assignments based on structural criteria. PMID:3680387

  10. Lumped pathway metabolic model of organic carbon accumulation and mobilization by the alga Chlamydomonas reinhardtii.

    PubMed

    Guest, Jeremy S; van Loosdrecht, Mark C M; Skerlos, Steven J; Love, Nancy G

    2013-04-01

    Phototrophic microorganisms have significant potential as bioenergy feedstocks, but the sustainability of large-scale cultivation will require the use of wastewater as a renewable resource. A key barrier to this advancement is a lack of bioprocess understanding that would enable the design and implementation of efficient and resilient mixed community, naturally lit cultivation systems. In this study, a lumped pathway metabolic model (denoted the phototrophic process model or PPM) was developed for mixed phototrophic communities subjected to day/night cycling. State variables included functional biomass (XCPO), stored carbohydrates (XCH), stored lipids (XLI), nitrate (SNO), phosphate (SP), and others. PPM metabolic reactions and stoichiometry were based on Chlamydomonas reinhardtii , but experiments for model calibration and validation were performed in flat panel photobioreactors (PBRs) originally inoculated with biomass from a phototrophic system at a wastewater treatment plant. PBRs were operated continuously as cyclostats to poise cells for intrinsic kinetic parameter estimation in batch studies, which included nutrient-available conditions in light and dark as well as nitrogen-starved and phosphorus-starved conditions in light. The model was calibrated and validated and was shown to be a reasonable predictor of growth, lipid and carbohydrate storage, and lipid and carbohydrate mobilization by a mixed microbial community. PMID:23452258

  11. Regulation of cellular manganese and manganese transport rates in the unicellular alga Chlamydomonas

    SciTech Connect

    Sunda, W.G.; Huntsman, S.A.

    1985-01-01

    The cellular accumulation and uptake kinetics of manganese by Chlamydomonas sp. were studied in model chelate buffer systems. Cellular manganese concentrations and uptake rates were related to the computed free manganese ion concentration and were independent of the total or chelated manganese concentration. Cellular manganese was constant at about 1 mmol liter/sup -1/ of cellular volume at free manganese ion concentrations of 10/sup -7/ /sup 6/-10/sup -6/ /sup 3/ mol liter/sup -1/ and decreased below this range. Manganese uptake rates followed saturation kinetics and V/sub max/, but not K/sub s/, varied with the free manganese ion concentration in the growth medium. V/sub max/ appeared to be under negative feedback control and increased with decreasing manganese ion concentration. Variations of up to 30-fold in this parameter seemed to be instrumental in limiting the variation in cellular manganese to a sixfold range despite a 1000-fold variation in free manganese ion concentration in the growth medium.

  12. Individual Flagellar Waveform Affects Collective Behavior of Chlamydomonas reinhardtii.

    PubMed

    Kage, Azusa; Mogami, Yoshihiro

    2015-08-01

    Bioconvection is a form of collective motion that occurs spontaneously in the suspension of swimming microorganisms. In a previous study, we quantitatively described the "pattern transition," a phase transition phenomenon that so far has exclusively been observed in bioconvection of the unicellular green alga Chlamydomonas. We suggested that the transition could be induced by changes in the balance between the gravitational and shear-induced torques, both of which act to determine the orientation of the organism in the shear flow. As both of the torques should be affected by the geometry of the Chlamydomonas cell, alteration in the flagellar waveform might change the extent of torque generation by altering overall geometry of the cell. Based on this working hypothesis, we examined bioconvection behavior of two flagellar mutants of Chlamydomonas reinhardtii, ida1 and oda2, making reference to the wild type. Flagella of ida1 beat with an abnormal waveform, while flagella of oda2 show a normal waveform but lower beat frequency. As a result, both mutants had swimming speed of less than 50% of the wild type. ida1 formed bioconvection patterns with smaller spacing than those of wild type and oda2. Two-axis view revealed the periodic movement of the settling blobs of ida1, while oda2 showed qualitatively similar behavior to that of wild type. Unexpectedly, ida1 showed stronger negative gravitaxis than did wild type, while oda2 showed relatively weak gravitaxis. These findings suggest that flagellar waveform, not swimming speed or beat frequency, strongly affect bioconvection behavior in C. reinhardtii. PMID:26245228

  13. Katanin Localization Requires Triplet Microtubules in Chlamydomonas reinhardtii

    PubMed Central

    Esparza, Jessica M.; O’Toole, Eileen; Li, Linya; Giddings, Thomas H.; Kozak, Benjamin; Albee, Alison J.; Dutcher, Susan K.

    2013-01-01

    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. PMID:23320108

  14. Effective viscosity of non-gravitactic Chlamydomonas Reinhardtii microswimmer suspensions

    NASA Astrophysics Data System (ADS)

    Mussler, Matthias; Rafaï, Salima; Peyla, Philippe; Wagner, Christian

    2013-03-01

    Active microswimmers are known to affect the macroscopic viscosity of suspensions in a more complex manner than passive particles. For puller-like microswimmers an increase in the viscosity has been observed. It has been suggested that the persistence of the orientation of the microswimmers hinders the rotation that is normally caused by the vorticity. It was previously shown that some sorts of algae are bottom-heavy swimmers, i.e., their centre of mass is not located in the centre of the body. In this way, the algae affect the vorticity of the flow when they are perpendicularly oriented to the axis of gravity. This orientation of gravity to vorticity is given in a rheometer that is equipped with a cone-plate geometry. Here we present measurements of the viscosity both in a cone-plate and a Taylor-Couette cell. The two set-ups yielded the same increase in viscosity although the axis of gravitation in the Taylor-Couette cell is parallel to the direction of vorticity. In a complementary experiment we tested the orientation of the direction of swimming through microscopic observation of single Chlamydomonas reinhardtii and could not identify a preferred orientation, i.e., our specific strain of Chlamydomonas reinhardtii are not bottom-heavy swimmers. We thus conclude that bottom heaviness is not a prerequisite for the increase of viscosity and that the effect of gravity on the rheology of our strain of Chlamydomonas reinhardtii is negligible. This finding reopens the question of whether the origin of persistence in the orientation of cells is actually responsible for the increased viscosity of the suspension.

  15. Production of therapeutic proteins in the chloroplast of Chlamydomonas reinhardtii

    PubMed Central

    2014-01-01

    Chloroplast transformation in the photosynthetic alga Chlamydomonas reinhardtii has been used to explore the potential to use it as an inexpensive and easily scalable system for the production of therapeutic recombinant proteins. Diverse proteins, such as bacterial and viral antigens, antibodies and, immunotoxins have been successfully expressed in the chloroplast using endogenous and chimeric promoter sequences. In some cases, proteins have accumulated to high level, demonstrating that this technology could compete with current production platforms. This review focuses on the works that have engineered the chloroplast of C. reinhardtii with the aim of producing recombinant proteins intended for therapeutical use in humans or animals. PMID:25136510

  16. Flagellar force production during regeneration in Chlamydomonas reinhardtii

    NASA Astrophysics Data System (ADS)

    Yukich, John N.; Clodfelter, Catherine; Bernd, Karen K.

    2009-11-01

    Several respiratory, digestive, and reproductive disorders originate with motional dysfunction of cilia and flagella. The usefulness of cilia and flagella is understood, but the internal mechanism for creating their breast stroke-like motion is not. This study reports on standardization of calibration, trapping and cell movement recording methods. Our techniques permit us to measure the flagellar swimming force of Chlamydomonas during flagella regeneration. We find that as flagella length increases, the flagellar force is maximized after 50% of full length is achieved except for a significant dip at 75% of full length. These results raise many questions regarding the flagella infrastructure.

  17. Production of therapeutic proteins in the chloroplast of Chlamydomonas reinhardtii.

    PubMed

    Almaraz-Delgado, Alma Lorena; Flores-Uribe, José; Pérez-España, Víctor Hugo; Salgado-Manjarrez, Edgar; Badillo-Corona, Jesús Agustín

    2014-01-01

    Chloroplast transformation in the photosynthetic alga Chlamydomonas reinhardtii has been used to explore the potential to use it as an inexpensive and easily scalable system for the production of therapeutic recombinant proteins. Diverse proteins, such as bacterial and viral antigens, antibodies and, immunotoxins have been successfully expressed in the chloroplast using endogenous and chimeric promoter sequences. In some cases, proteins have accumulated to high level, demonstrating that this technology could compete with current production platforms. This review focuses on the works that have engineered the chloroplast of C. reinhardtii with the aim of producing recombinant proteins intended for therapeutical use in humans or animals. PMID:25136510

  18. Identification of Global Ferredoxin Interaction Networks in Chlamydomonas reinhardtii*

    PubMed Central

    Peden, Erin A.; Boehm, Marko; Mulder, David W.; Davis, ReAnna; Old, William M.; King, Paul W.; Ghirardi, Maria L.; Dubini, Alexandra

    2013-01-01

    Ferredoxins (FDXs) can distribute electrons originating from photosynthetic water oxidation, fermentation, and other reductant-generating pathways to specific redox enzymes in different organisms. The six FDXs identified in Chlamydomonas reinhardtii are not fully characterized in terms of their biological function. In this report, we present data from the following: (a) yeast two-hybrid screens, identifying interaction partners for each Chlamydomonas FDX; (b) pairwise yeast two-hybrid assays measuring FDX interactions with proteins from selected biochemical pathways; (c) affinity pulldown assays that, in some cases, confirm and even expand the interaction network for FDX1 and FDX2; and (d) in vitro NADP+ reduction and H2 photo-production assays mediated by each FDX that verify their role in these two pathways. Our results demonstrate new potential roles for FDX1 in redox metabolism and carbohydrate and fatty acid biosynthesis, for FDX2 in anaerobic metabolism, and possibly in state transition. Our data also suggest that FDX3 is involved in nitrogen assimilation, FDX4 in glycolysis and response to reactive oxygen species, and FDX5 in hydrogenase maturation. Finally, we provide experimental evidence that FDX1 serves as the primary electron donor to two important biological pathways, NADPH and H2 photo-production, whereas FDX2 is capable of driving these reactions at less than half the rate observed for FDX1. PMID:24100040

  19. Chloroplast Genetics of Chlamydomonas. III. Closing the Circle

    PubMed Central

    Singer, Burt; Sager, Ruth; Ramanis, Zenta

    1976-01-01

    A novel mapping procedure is presented for organelle genes or any other genetic system exhibiting a measurable frequency of exchanges occurring at a constant rate over a measurable time interval. For a set of markers in a multiply-marked cross, the exchange rates measure relative map distances from a centromere-like attachment point. With this method, we present mapping data and a linear map of genes in the chlcroplast genome of Chlamydomonas. The data are plotted as log (percent remaining heterozygotes) against time and map distances are taken as proportional to slope. A statistical method which is an adaptation of jackknife methodology to a regression problem was developed to estimate slope values. A single line is fitted to pooled data for each marker from several crosses, and then lines are re-fit to a series of pooled data sets in each of which the observations from a single cross have been omitted. From these data sets a final summary slope is computed as well as a statement of its variability. The relative positions of new markers present in single crosses can then be estimated utilizing data from many crosses. The method does not distinguish between one-armed and two-armed linear or circular maps. However, evaluation of this map in conjunction with cosegregation frequency data (Sager and Ramanis 1976b) provides unambiguous evidence of the genetic circularity of the Chlamydomonas chloroplast genome. PMID:17248718

  20. Molecular techniques to interrogate and edit the Chlamydomonas nuclear genome.

    PubMed

    Jinkerson, Robert E; Jonikas, Martin C

    2015-05-01

    The success of the green alga Chlamydomonas reinhardtii as a model organism is to a large extent due to the wide range of molecular techniques that are available for its characterization. Here, we review some of the techniques currently used to modify and interrogate the C. reinhardtii nuclear genome and explore several technologies under development. Nuclear mutants can be generated with ultraviolet (UV) light and chemical mutagens, or by insertional mutagenesis. Nuclear transformation methods include biolistic delivery, agitation with glass beads, and electroporation. Transforming DNA integrates into the genome at random sites, and multiple strategies exist for mapping insertion sites. A limited number of studies have demonstrated targeted modification of the nuclear genome by approaches such as zinc-finger nucleases and homologous recombination. RNA interference is widely used to knock down expression levels of nuclear genes. A wide assortment of transgenes has been successfully expressed in the Chlamydomonas nuclear genome, including transformation markers, fluorescent proteins, reporter genes, epitope tagged proteins, and even therapeutic proteins. Optimized expression constructs and strains help transgene expression. Emerging technologies such as the CRISPR/Cas9 system, high-throughput mutant identification, and a whole-genome knockout library are being developed for this organism. We discuss how these advances will propel future investigations. PMID:25704665

  1. Propulsive Forces on the Flagellum during Locomotion of Chlamydomonas reinhardtii

    PubMed Central

    Bayly, P.V.; Lewis, B.L.; Ranz, E.C.; Okamoto, R.J.; Pless, R.B.; Dutcher, S.K.

    2011-01-01

    The distributed propulsive forces exerted on the flagellum of the swimming alga Chlamydomonas reinhardtii by surrounding fluid were estimated from experimental image data. Images of uniflagellate mutant Chlamydomonas cells were obtained at 350 frames/s with 125-nm spatial resolution, and the motion of the cell body and the flagellum were analyzed in the context of low-Reynolds-number fluid mechanics. Wild-type uniflagellate cells, as well as uniflagellate cells lacking inner dynein arms (ida3) or outer dynein arms (oda2) were studied. Ida3 cells exhibit stunted flagellar waveforms, whereas oda2 cells beat with lower frequency. Image registration and sorting algorithms provided high-resolution estimates of the motion of the cell body, as well as detailed kinematics of the flagellum. The swimming cell was modeled as an ellipsoid in Stokes flow, propelled by viscous forces on the flagellum. The normal and tangential components of force on the flagellum (fN and fT) were related by resistive coefficients (CN and CT) to the corresponding components of velocity (VN and VT).The values of these coefficients were estimated by satisfying equilibrium requirements for force and torque on the cell. The estimated values of the resistive coefficients are consistent among all three genotypes and similar to theoretical predictions. PMID:21641317

  2. Potassium Fluxes in Chlamydomonas reinhardtii (II. Compartmental Analysis).

    PubMed Central

    Malhotra, B.; Glass, ADM.

    1995-01-01

    42K+ and 86Rb+ were used to determine the subcellular distribution of potassium in Chlamydomonas reinhardtii by compartmental analysis. In both wild type and a mutant strain, three distinct compartments (referred to as I, II, and III) were apparent. Using 42K+, we found that these had half-lives for K+ exchange of 1.07 min, 12.8 min, and 2.9 h, respectively, in wild-type cells and 0.93 min, 14.7 min, and 9.8 h, respectively, for the mutants. Half-lives were not significantly different when 86Rb+ was used to trace K+. Compartments I and II probably correspond to the cell wall and cytoplasm, respectively. Based on the lack of a large central vacuole in Chlamydomonas, the effect of a dark pretreatment on the kinetic properties of compartment III and the similarity between the [K+] of compartment III and that of isolated chloroplasts, this slowly exchanging compartment was identified as the chloroplast. Growth of wild-type cells at 100 [mu]M (instead of 10 mM K+) caused no change of cytoplasmic [K+] but reduced chloroplast [K+] very substantially. The mutants failed to grow at 100 [mu]M K+. PMID:12228560

  3. Metabolism of acyl-lipids in Chlamydomonas reinhardtii.

    PubMed

    Li-Beisson, Yonghua; Beisson, Fred; Riekhof, Wayne

    2015-05-01

    Microalgae are emerging platforms for production of a suite of compounds targeting several markets, including food, nutraceuticals, green chemicals, and biofuels. Many of these products, such as biodiesel or polyunsaturated fatty acids (PUFAs), derive from lipid metabolism. A general picture of lipid metabolism in microalgae has been deduced from well characterized pathways of fungi and land plants, but recent advances in molecular and genetic analyses of microalgae have uncovered unique features, pointing out the necessity to study lipid metabolism in microalgae themselves. In the past 10 years, in addition to its traditional role as a model for photosynthetic and flagellar motility processes, Chlamydomonas reinhardtii has emerged as a model organism to study lipid metabolism in green microalgae. Here, after summarizing data on total fatty acid composition, distribution of acyl-lipid classes, and major acyl-lipid molecular species found in C. reinhardtii, we review the current knowledge on the known or putative steps for fatty acid synthesis, glycerolipid desaturation and assembly, membrane lipid turnover, and oil remobilization. A list of characterized or putative enzymes for the major steps of acyl-lipid metabolism in C. reinhardtii is included, and subcellular localizations and phenotypes of associated mutants are discussed. Biogenesis and composition of Chlamydomonas lipid droplets and the potential importance of lipolytic processes in increasing cellular oil content are also highlighted. PMID:25660108

  4. Analysis of the ciliary/flagellar beating of Chlamydomonas.

    PubMed

    Foster, Kenneth W

    2009-01-01

    Eukaryotic flagella and cilia are alternative names, for the slender cylindrical protrusions of a cell (240nm diameter, approximately 12,800nm-long in Chlamydomonas reinhardtii) that propel a cell or move fluid. Cilia are extraordinarily successful complex organelles abundantly found in animals performing many tasks. They play a direct or developmental role in the sensors of fluid flow, light, sound, gravity, smells, touch, temperature, and taste in mammals. The failure of cilia can lead to hydrocephalus, infertility, and blindness. However, in spite of their large role in human function and pathology, there is as yet no consensus on how cilia beat and perform their many functions, such as moving fluids in brain ventricles and lungs and propelling and steering sperm, larvae, and many microorganisms. One needs to understand and analyze ciliary beating and its hydrodynamic interactions. This chapter provides a guide for measuring, analyzing, and interpreting ciliary behavior in various contexts studied in the model system of Chlamydomonas. It describes: (1) how cilia work as self-organized beating structures (SOBSs), (2) the overlaid control in the cilia that optimizes the SOBS to achieve cell dispersal, phototaxis steering, and avoidance of obstacles, (3) the assay of a model intracellular signal processing system that responds to multiple external and internal inputs, choosing mode of behavior and then controlling the cilia, (4) how cilia sense their environment, and (5) potentially an assay of ciliary performance for toxicology or medical assessment. PMID:20409788

  5. Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue.

    PubMed

    Warakanont, Jaruswan; Tsai, Chia-Hong; Michel, Elena J S; Murphy, George R; Hsueh, Peter Y; Roston, Rebecca L; Sears, Barbara B; Benning, Christoph

    2015-12-01

    In plants, lipids of the photosynthetic membrane are synthesized by parallel pathways associated with the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Lipids derived from the two pathways are distinguished by their acyl-constituents. Following this plant paradigm, the prevalent acyl composition of chloroplast lipids suggests that Chlamydomonas reinhardtii (Chlamydomonas) does not use the ER pathway; however, the Chlamydomonas genome encodes presumed plant orthologues of a chloroplast lipid transporter consisting of TGD (TRIGALACTOSYLDIACYLGLYCEROL) proteins that are required for ER-to-chloroplast lipid trafficking in plants. To resolve this conundrum, we identified a mutant of Chlamydomonas deleted in the TGD2 gene and characterized the respective protein, CrTGD2. Notably, the viability of the mutant was reduced, showing the importance of CrTGD2. Galactoglycerolipid metabolism was altered in the tgd2 mutant with monogalactosyldiacylglycerol (MGDG) synthase activity being strongly stimulated. We hypothesize this to be a result of phosphatidic acid accumulation in the chloroplast outer envelope membrane, the location of MGDG synthase in Chlamydomonas. Concomitantly, increased conversion of MGDG into triacylglycerol (TAG) was observed. This TAG accumulated in lipid droplets in the tgd2 mutant under normal growth conditions. Labeling kinetics indicate that Chlamydomonas can import lipid precursors from the ER, a process that is impaired in the tgd2 mutant. PMID:26496373

  6. Blue Light Regulation of Cell Division in Chlamydomonas reinhardtii 1

    PubMed Central

    Münzner, Petra; Voigt, Jürgen

    1992-01-01

    A delay in cell division was observed when synchronized cultures of the unicellular green alga Chlamydomonas reinhardtii growing under heterotrophic conditions were exposed to white light during the second half of the growth period. This effect was also observed when photosynthesis was blocked by addition of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Light pulses of 10 minutes were sufficient to induce a delay in cell division in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. A delay in cell division was induced by blue light but not by illumination with red or far-red light. The equal intensity action spectrum revealed two peaks at 400 and 500 nm. PMID:16669046

  7. Effects of light on gravitaxis and velocity in Chlamydomonas reinhardtii.

    PubMed

    Sineshchekov, O; Lebert, M; Hader, D P

    2000-09-01

    The effects of light on gravitaxis and velocity in the bi-flagellated green alga Chlamydomonas reinhardtii were investigated using a real time automatic tracking system. Three distinct light effects on gravitaxis and velocity with parallel kinetics were found. Photosynthetically active continuous red light reversibly enhances the swimming velocity and increases or decreases the precision of gravitaxis, depending on its initial level. Blue light flashes induce fast transient increases in velocity immediately after the photophobic response, and transiently decrease or even reverse negative gravitaxis. The calcium dependence of this response, its fluence-response curve and its spectral characteristics strongly suggest the participation of chlamy-rhodopsin in this effect. The third response, a prolonged activation of velocity and gravitaxis, is also induced by blue light flashes, which can be observed even in calcium-free medium. PMID:12090268

  8. Antiphase Synchronization in a Flagellar-Dominance Mutant of Chlamydomonas

    NASA Astrophysics Data System (ADS)

    Leptos, Kyriacos C.; Wan, Kirsty Y.; Polin, Marco; Tuval, Idan; Pesci, Adriana I.; Goldstein, Raymond E.

    2013-10-01

    Groups of beating flagella or cilia often synchronize so that neighboring filaments have identical frequencies and phases. A prime example is provided by the unicellular biflagellate Chlamydomonas reinhardtii, which typically displays synchronous in-phase beating in a low-Reynolds number version of breaststroke swimming. We report the discovery that ptx1, a flagellar-dominance mutant of C. reinhardtii, can exhibit synchronization in precise antiphase, as in the freestyle swimming stroke. High-speed imaging shows that ptx1 flagella switch stochastically between in-phase and antiphase states, and that the latter has a distinct waveform and significantly higher frequency, both of which are strikingly similar to those found during phase slips that stochastically interrupt in-phase beating of the wild-type. Possible mechanisms underlying these observations are discussed.

  9. Mechanosensitive physiology of chlamydomonas reinhardtii under direct membrane distortion

    PubMed Central

    Min, Seul Ki; Yoon, Gwang Heum; Joo, Jung Hyun; Sim, Sang Jun; Shin, Hwa Sung

    2014-01-01

    Cellular membrane distortion invokes variations in cellular physiology. However, lack of an appropriate system to control the stress and facilitate molecular analyses has hampered progress of relevant studies. In this study, a microfluidic system that finely manipulates membrane distortion of Chlamydomonas reinhardtii (C. reinhardtii) was developed. The device facilitated a first-time demonstration that directs membrane distortion invokes variations in deflagellation, cell cycle, and lipid metabolism. C. reinhardtii showed a prolonged G1 phase with an extended total cell cycle time, and upregulated Mat3 regulated a cell size and cell cycle. Additionally, increased TAG compensated for the loss of cell mass. Overall, this study suggest that cell biology that requires direct membrane distortion can be realized using this system, and the implication of cell cycle with Mat3 expression of C. reinhardtii was first demonstrated. Finally, membrane distortion can be an attractive inducer for biodiesel production since it is reliable and robust. PMID:24728350

  10. Bioaccessibility of carotenoids from Chlorella vulgaris and Chlamydomonas reinhardtii.

    PubMed

    Gille, Andrea; Trautmann, Andreas; Posten, Clemens; Briviba, Karlis

    2015-08-01

    Microalgae can contribute to a balanced diet because of their composition. Beside numerous essential nutrients, carotenoids are in the focus for food applications. The bioavailability of carotenoids from photoautotrophic-cultivated Chlorella vulgaris (C. vulgaris) and Chlamydomonas reinhardtii (C. reinhardtii) was compared. An in vitro digestion model was used to investigate carotenoid bioaccessibility. Furthermore, the effect of sonication on bioaccessibility was assessed. Lutein was the main carotenoid in both species. C. reinhardtii showed higher amounts of lutein and β-carotene than C. vulgaris. In contrast to C. reinhardtii, no β-carotene and only 7% of lutein were bioaccessible in nonsonicated C. vulgaris. Sonication increased the bioaccessibility of carotenoids from C. vulgaris to a level comparable with C. reinhardtii (β-carotene: ≥ 10%; lutein: ≥ 15%). Thus, C. reinhardtii represents a good carotenoid source for potential use in foods without processing, while the application of processing methods, like sonication, is necessary for C. vulgaris. PMID:27146695

  11. Glucose respiration in the intact chloroplast of Chlamydomonas reinhardtii

    SciTech Connect

    Changguo Chen; Gibbs, M. )

    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.

  12. Establishing Chlamydomonas reinhardtii as an industrial biotechnology host

    PubMed Central

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

    2015-01-01

    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. Significance Statement Chlamydomonas reinhardtii offers potential as a host for the production of high value compounds for industrial biotechnology. Synthetic biology provides a mechanism to generate generic, well characterised tools for application in the rational genetic manipulation of organisms: if synthetic biology principles were adopted for manipulation of C. reinhardtii, development of this microalga as an industrial biotechnology platform would be expedited. PMID:25641561

  13. O2 Uptake in the Light in Chlamydomonas

    PubMed Central

    Peltier, Gilles; Thibault, Pierre

    1985-01-01

    The nature of the process responsible for the stationary O2 uptake occurring in the light under saturating CO2 concentration in Chlamydomonas reinhardii has been investigated. For this purpose, a mass spectrometer with a membrane inlet system was used to measure O2 uptake and evolution in the algal suspension. First, we observed that the O2 uptake rate was constant (about 0.5 micromoles of O2 per milligram chlorophyll per minute) during a light to dark transition and was not affected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Salicylhydroxamic acid had no effect on O2 uptake in the dark or in the light, but was found to have the same inhibitory effect either in the dark or in the light when added to cyanide-treated algae. The stimulation of the O2 uptake rate due to the uncoupling effect of carbonyl cyanide m-chlorophenylhydrazone was about the same in the dark or in the light. From these results, we conclude that mitochondrial respiration is maintained during illumination and therefore is not inhibited by high ATP levels. Another conclusion is that in conditions where photorespiration is absent, no other light-dependent O2 uptake process occurs. If Mehler reactions are involved, in Chlamydomonas, under conditions where both photosynthetic carbon oxidation and reduction cycles cannot operate (as in cyanide-treated algae), their occurrence in photosynthesizing algae either under saturating CO2 concentration or at the CO2 compensation point appears very unlikely. The comparison with the situation previously reported in Scenedesmus (R. J. Radmer and B. Kok 1976 Plant Physiol 58: 336-340) suggests that different O2 uptake processes might be present in these two algal species. PMID:16664375

  14. Cytochrome f from the Antarctic psychrophile, Chlamydomonas raudensis UWO 241: structure, sequence, and complementation in the mesophile, Chlamydomonas reinhardtii.

    PubMed

    Gudynaite-Savitch, Loreta; Gretes, Michael; Morgan-Kiss, Rachael M; Savitch, Leonid V; Simmonds, John; Kohalmi, Susanne E; Hüner, Norman P A

    2006-04-01

    Although cytochrome f from the Antarctic psychrophile, Chlamydomonas raudensis UWO 241, exhibits a lower apparent molecular mass (34 kD) than that of the mesophile C. reinhardtii (41 kD) based on SDS-PAGE, both proteins are comparable in calculated molecular mass and show 79% identity in amino acid sequence. The difference in apparent molecular mass was maintained after expression of petA from both Chlamydomonas species in either E. coli or a C. reinhardtii DeltapetA mutant and after substitution of a unique third cysteine-292 to phenylalanine in the psychrophilic cytochrome f. Moreover, the heme of the psychrophilic form of cytochrome f was less stable upon heating than that of the mesophile. In contrast to C. raudensis, a C. reinhardtii DeltapetA mutant transformed with petA from C. raudensis exhibited the ability to undergo state transitions and a capacity for intersystem electron transport comparable to that of C. reinhardtii wild type. However, the C. reinhardtii petA transformants accumulated lower levels of cytochrome b ( 6 ) /f complexes and exhibited lower light saturated rates of O(2) evolution than C. reinhardtii wild type. We show that the presence of an altered form of cytochrome f in C. raudensis does not account for its inability to undergo state transitions or its impaired capacity for intersystem electron transport as previously suggested. A combined survey of the apparent molecular mass, thermal stability and amino acid sequences of cytochrome f from a broad range of mesophilic species shows unequivocally that the observed differences in cytochrome f structure are not related to psychrophilly. Thus, caution must be exercised in relating differences in amino acid sequence and thermal stability to adaptation to cold environments. PMID:16425016

  15. Functional Specificity of Cardiolipin Synthase Revealed by the Identification of a Cardiolipin Synthase CrCLS1 in Chlamydomonas reinhardtii

    PubMed Central

    Hung, Chun-Hsien; Kobayashi, Koichi; Wada, Hajime; Nakamura, Yuki

    2016-01-01

    Phosphatidylglycerol (PG) and cardiolipin (CL) are two essential classes of phospholipid in plants and algae. Phosphatidylglycerophosphate synthase (PGPS) and cardiolipin synthase (CLS) involved in the biosynthesis of PG and CL belong to CDP-alcohol phosphotransferase and share overall amino acid sequence homology. However, it remains elusive whether PGPS and CLS are functionally distinct in vivo. Here, we report identification of a gene encoding CLS in Chlamydomonas reinhardtii, CrCLS1, and its functional compatibility. Whereas CrCLS1 did not complement the growth phenotype of a PGPS mutant of Synechocystis sp. PCC 6803, it rescued the temperature-sensitive growth phenotype, growth profile with different carbon sources, phospholipid composition and enzyme activity of Δcrd1, a CLS mutant of Saccharomyces cerevisiae. These results suggest that CrCLS1 encodes a functional CLS of C. reinhardtii as the first identified algal CLS, whose enzyme function is distinct from that of PGPSs from C. reinhardtii. Comparison of CDP-alcohol phosphotransferase motif between PGPS and CLS among different species revealed a possible additional motif that might define the substrate specificity of these closely related enzymes. PMID:26793177

  16. Treatment with NaHSO3 greatly enhances photobiological H2 production in the green alga Chlamydomonas reinhardtii.

    PubMed

    Ma, Weimin; Chen, Ming; Wang, Lianjun; Wei, Lanzhen; Wang, Quanxi

    2011-09-01

    Treatment with NaHSO3 induces a 10-fold increase in H2 photoproduction in the filamentous N2-fixing cyanobacterium Anabaena sp. strain PCC 7120. However, it is unclear whether this treatment also increases H2 photoproduction in green alga. In this study, treatment with 13 mM NaHSO3 resulted in about a 200-fold increase in H2 production in Chlamydomonas reinhardtii, and this increase was most probably the result of reduced O2 content and enhanced hydrogenase activity. Compared to the conventional strategy of sulfur deprivation, NaHSO3 treatment results in a higher maximum rate of H2 photoproduction, greater efficiency of conversion of light energy into H2, shorter half-time to produce the maximum accumulated H2 levels, and reduced costs because no centrifugation is involved. We therefore conclude that NaHSO3 treatment is an efficient, rapid, and economic strategy for improving photobiological H2 production in the green alga C. reinhardtii. PMID:21489780

  17. Inhibition of Target of Rapamycin Signaling and Stress Activate Autophagy in Chlamydomonas reinhardtii1[W

    PubMed Central

    Pérez-Pérez, María Esther; Florencio, Francisco J.; Crespo, José L.

    2010-01-01

    Autophagy is a catabolic membrane-trafficking process whereby cells recycle cytosolic proteins and organelles under stress conditions or during development. This degradative process is mediated by autophagy-related (ATG) proteins that have been described in yeast, animals, and more recently in plants. In this study, we report the molecular characterization of autophagy in the unicellular green alga Chlamydomonas reinhardtii. We demonstrate that the ATG8 protein from Chlamydomonas (CrATG8) is functionally conserved and may be used as a molecular autophagy marker. Like yeast ATG8, CrATG8 is cleaved at the carboxyl-terminal conserved glycine and is associated with membranes in Chlamydomonas. Cell aging or different stresses such as nutrient limitation, oxidative stress, or the accumulation of misfolded proteins in the endoplasmic reticulum caused an increase in CrATG8 abundance as well as the detection of modified forms of this protein, both landmarks of autophagy activation. Furthermore, rapamycin-mediated inhibition of the Target of Rapamycin signaling pathway, a major regulator of autophagy in eukaryotes, results in identical effects on CrATG8 and a relocalization of this protein in Chlamydomonas cells similar to the one observed upon nutrient limitation. Thus, our findings indicate that Chlamydomonas cells may respond to stress conditions by inducing autophagy via Target of Rapamycin signaling modulation. PMID:20107021

  18. Metabolism of D-lactate and structurally related organic acids in Chlamydomonas reinhardtii

    SciTech Connect

    Husic, D.W.

    1986-01-01

    During the initial minutes of anaerobiosis, /sup 14/C-labeled D-lactate, derived from the photosynthetic sugar phosphate pool, accumulated in the unicellular green alga, Chlamydomonas reinhardtii. The production of the D-isomer of lactate by algae is in contrast to plant and mammalian cells in which L-lactate is formed. After initial lactate formation, Chlamydomonas exhibits a mixed-acid type fermentation, thereby avoiding lactate accumulation and enabling the cells to tolerate extended periods of anaerobiosis. A pyruvate reductase which catalyzes the formation of D-lactate in Chlamydomonas was partially purified and characterized. Lactate produced anaerobically was metabolized only when Chlamydomonas cells were returned to aerobic conditions, and reoxidation of the D-lactate was apparently catalyzed by a mitochondrial membrane-bound dehydrogenase, rather than by the soluble pyruvate reductase. Mutants of Chlamydomonas, deficient in mitochondrial respiration, were used to demonstrate that lactate metabolism was linked to the mitochondrial electron transport chain. In addition, the oxidation of glycolate, a structural analog of lactate, was also linked to mitochondrial electron transport in vivo.

  19. Activation of a chloroplast type of fructose bisphosphatase from Chlamydomonas reinhardtii by light-mediated agents

    NASA Technical Reports Server (NTRS)

    Huppe, H. C.; Buchanan, B. B.

    1989-01-01

    A chloroplast type of fructose-1,6-bisphosphatase, a central regulatory enzyme of photosynthetic carbon metabolism, has been partially purified from Chlamydomonas reinhardtii. Unlike its counterpart from spinach chloroplasts, the algal FBPase showed a strict requirement for a dithiol reductant irrespective of Mg2+ concentration. The enzymes from the two sources resembled each other immunologically, in subunit molecular mass and response to pH. In the presence of dithiothreitol, the pH optimum for both the algal and spinach enzymes shifted from 8.5 to a more physiologic value of 8.0 as the Mg2+ concentration was increased from 1 to 16 mM. At 1 mM Mg2+, a concentration estimated to be close to physiological, the Chlamydomonas FBPase was active only in the presence of reduced thioredoxin and was most active with Chlamydomonas thioredoxin f. Under these conditions, the enzyme showed a pH optimum of 8.0. The data suggest that the Chlamydomonas enzyme resembles its spinach counterpart in most respects, but it has a stricter requirement for reduction and less strict reductant specificity. A comparison of the properties of the FBPases from Chlamydomonas and spinach will be helpful for elucidating the mechanism of the reductive activation of this enzyme.

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

    PubMed Central

    Merchant, Sabeeha S.; Prochnik, Simon E.; Vallon, Olivier; Harris, Elizabeth H.; Karpowicz, Steven J.; Witman, George B.; Terry, Astrid; Salamov, Asaf; Fritz-Laylin, Lillian K.; Maréchal-Drouard, Laurence; Marshall, Wallace F.; Qu, Liang-Hu; Nelson, David R.; Sanderfoot, Anton A.; Spalding, Martin H.; Kapitonov, Vladimir V.; Ren, Qinghu; Ferris, Patrick; Lindquist, Erika; Shapiro, Harris; Lucas, Susan M.; Grimwood, Jane; Schmutz, Jeremy; Cardol, Pierre; Cerutti, Heriberto; Chanfreau, Guillaume; Chen, Chun-Long; Cognat, Valérie; Croft, Martin T.; Dent, Rachel; Dutcher, Susan; Fernández, Emilio; Ferris, Patrick; Fukuzawa, Hideya; González-Ballester, David; González-Halphen, Diego; Hallmann, Armin; Hanikenne, Marc; Hippler, Michael; Inwood, William; Jabbari, Kamel; Kalanon, Ming; Kuras, Richard; Lefebvre, Paul A.; Lemaire, Stéphane D.; Lobanov, Alexey V.; Lohr, Martin; Manuell, Andrea; Meier, Iris; Mets, Laurens; Mittag, Maria; Mittelmeier, Telsa; Moroney, James V.; Moseley, Jeffrey; Napoli, Carolyn; Nedelcu, Aurora M.; Niyogi, Krishna; Novoselov, Sergey V.; Paulsen, Ian T.; Pazour, Greg; Purton, Saul; Ral, Jean-Philippe; Riaño-Pachón, Diego Mauricio; Riekhof, Wayne; Rymarquis, Linda; Schroda, Michael; Stern, David; Umen, James; Willows, Robert; Wilson, Nedra; Zimmer, Sara Lana; Allmer, Jens; Balk, Janneke; Bisova, Katerina; Chen, Chong-Jian; Elias, Marek; Gendler, Karla; Hauser, Charles; Lamb, Mary Rose; Ledford, Heidi; Long, Joanne C.; Minagawa, Jun; Page, M. Dudley; Pan, Junmin; Pootakham, Wirulda; Roje, Sanja; Rose, Annkatrin; Stahlberg, Eric; Terauchi, Aimee M.; Yang, Pinfen; Ball, Steven; Bowler, Chris; Dieckmann, Carol L.; Gladyshev, Vadim N.; Green, Pamela; Jorgensen, Richard; Mayfield, Stephen; Mueller-Roeber, Bernd; Rajamani, Sathish; Sayre, Richard T.; Brokstein, Peter; Dubchak, Inna; Goodstein, David; Hornick, Leila; Huang, Y. Wayne; Jhaveri, Jinal; Luo, Yigong; Martínez, Diego; Ngau, Wing Chi Abby; Otillar, Bobby; Poliakov, Alexander; Porter, Aaron; Szajkowski, Lukasz; Werner, Gregory; Zhou, Kemin; Grigoriev, Igor V.; Rokhsar, Daniel S.; Grossman, Arthur R.

    2010-01-01

    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. PMID:17932292

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

    SciTech Connect

    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.

  2. Isolation, growth, ultrastructure, and metal tolerance of the green alga, Chlamydomonas acidophila (Chlorophyta).

    PubMed

    Nishikawa, K; Tominaga, N

    2001-12-01

    An acidophilic volvocine flagellate, Chlamydomonas acidophila (Volvocales) that was isolated from an acid lake, Katanuma, in Miyagi prefecture, Japan was studied for growth, ultrastructural characterization, and metal tolerance. Chlamydomonas acidophila is obligately photoautotrophic, and did not grow in the cultures containing acetate or citrate even in the light. The optimum pH for growth was 3.5-4.5. To characterize metal tolerance, the toxic effects of Cd, Co, Cu, and Zn on this alga were also studied. Effective metal concentrations, which limited the growth by 50%, EC50 were measured, after 72 h of static exposure. EC50s were 14.4 microM Cd2+, 81.3 microM Co2+, 141 microM Cu2+, and 1.16 mM Zn2+ for 72 h of exposure. Thus, this alga had stronger tolerance to these metals than other species in the genus Chlamydomonas. PMID:11826960

  3. Phase-dependent forcing and synchronization in the three-sphere model of Chlamydomonas

    NASA Astrophysics Data System (ADS)

    Bennett, Rachel R.; Golestanian, Ramin

    2013-07-01

    The green alga Chlamydomonas swims with synchronized beating of its two flagella, and is experimentally observed to exhibit run-and-tumble behaviour similar to bacteria. Recently, we studied a simple hydrodynamic three-sphere model of Chlamydomonas with a phase-dependent driving force that can produce run-and-tumble behaviour when intrinsic noise is added, due to the nonlinear mechanics of the system. Here, we consider the noiseless case and explore numerically the parameter space in the driving force profiles, which determine whether or not the synchronized state evolves from a given initial condition, as well as the stability of the synchronized state. We find that phase-dependent forcing, or a beat pattern, is necessary for stable synchronization in the geometry we work with. The phase-dependent forcing allows this simple model of Chlamydomonas to produce a rich variety of behaviours.

  4. Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation

    PubMed Central

    Karpowicz, Steven J.; Heinnickel, Mark; Dewez, David; Hamel, Blaise; Dent, Rachel; Niyogi, Krishna K.; Johnson, Xenie; Alric, Jean; Wollman, Francis-André; Li, Huiying; Merchant, Sabeeha S.

    2010-01-01

    Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus. PMID:20490922

  5. Transport and metabolism of glycolic acid by Chlamydomonas reinhardtii

    SciTech Connect

    Wilson, B.J.

    1987-01-01

    In order to understand the excretion of glycolate from Chlamydomonas reinhardtii, the conditions affecting glycolate synthesis and metabolism were investigated. Although glycolate is synthesized only in the light, the metabolism occurs in the light and dark with greater metabolism in the light due to refixation of photorespiratory CO/sub 2/. The amount of internal glycolate will affect the metabolism of externally added glycolate. When glycolate synthesis exceeds the metabolic capacity, glycolate is excreted from the cell. The transport of glycolate into the cells occurs very rapidly. Equilibrium is achieved at 4/sup 0/C within the time cells are pelleted by the silicone oil centrifugation technique through a layer of (/sup 14/C) glycolate. Glycolate uptake does not show the same time, temperature and pH dependencies as diffusion of benzoate. Uptake can be inhibited by treatment of cells with N-ethylmaleimide and stimulated in the presence of valino-mycin/KCl. Acetate and lactate are taken up as quickly as glycolate. The hypothesis was made that glycolate is transported by a protein carrier that transports monocarboxylic acids. The equilibrium concentration of glycolate is dependent on the cell density, implying that there may be a large number of transporter sites and that uptake is limited by substrate availability.

  6. Activation and de novo synthesis of hydrogenase in chlamydomonas.

    PubMed

    Roessler, P G; Lien, S

    1984-12-01

    Two distinct processes are involved in the formation of active hydrogenase during anaerobic adaptation of Chlamydomonas reinhardtii cells. In the first 30 minutes of anaerobiosis, nearly all of the hydrogenase activity can be attributed to activation of a constituitive polypeptide precursor, based on the insensitivity of the process to treatment with cycloheximide (15 micrograms per milliliter). This concentration of cycloheximide inhibits protein synthesis by greater than 98%. After the initial activation period, de novo protein synthesis plays a critical role in the adaptation process since cycloheximide inhibits the expression of hydrogense in maximally adapted cells by 70%. Chloramphenicol (500 micrograms per milliliter) has a much lesser effect on the adaptation process.Incubation of cell-free extracts under anaerobic conditions in the presence of dithionite, dithiothreitol, NADH, NADP, ferredoxin, ATP, Mg(2+), Ca(2+), and iron does not lead to active hydrogenase formation. Futhermore, in vivo reactivation of oxygen-inactivated hydrogenase does not appear to take place.The adaptation process is very sensitive to the availability of iron. Iron-deficient cultures lose the ability to form active hydrogenase before growth, photosynthesis, and respiration are significantly affected. Preincubation of iron-deficient cells with iron 2 hours prior to the adaptation period fully restores the capacity of the cells to synthesize functional hydrogenase. PMID:16663954

  7. Identification of the Elusive Pyruvate Reductase of Chlamydomonas reinhardtii Chloroplasts.

    PubMed

    Burgess, Steven J; Taha, Hussein; Yeoman, Justin A; Iamshanova, Oksana; Chan, Kher Xing; Boehm, Marko; Behrends, Volker; Bundy, Jacob G; Bialek, Wojciech; Murray, James W; Nixon, Peter J

    2016-01-01

    Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various fermentation pathways leading to the creation of formate, acetate, ethanol and small amounts of other metabolites including d-lactate and hydrogen. Progress has been made in identifying the enzymes involved in these pathways and their subcellular locations; however, the identity of the enzyme involved in reducing pyruvate to d-lactate has remained unclear. Based on sequence comparisons, enzyme activity measurements, X-ray crystallography, biochemical fractionation and analysis of knock-down mutants, we conclude that pyruvate reduction in the chloroplast is catalyzed by a tetrameric NAD(+)-dependent d-lactate dehydrogenase encoded by Cre07.g324550. Its expression during aerobic growth supports a possible function as a 'lactate valve' for the export of lactate to the mitochondrion for oxidation by cytochrome-dependent d-lactate dehydrogenases and by glycolate dehydrogenase. We also present a revised spatial model of fermentation based on our immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the cytoplasm. PMID:26574578

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

    PubMed

    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. PMID:23346954

  9. Singlet oxygen production in Chlamydomonas reinhardtii under heat stress.

    PubMed

    Prasad, Ankush; Ferretti, Ursula; Sedlářová, Michaela; Pospíšil, Pavel

    2016-01-01

    In the current study, singlet oxygen formation by lipid peroxidation induced by heat stress (40 °C) was studied in vivo in unicellular green alga Chlamydomonas reinhardtii. Primary and secondary oxidation products of lipid peroxidation, hydroperoxide and malondialdehyde, were generated under heat stress as detected using swallow-tailed perylene derivative fluorescence monitored by confocal laser scanning microscopy and high performance liquid chromatography, respectively. Lipid peroxidation was initiated by enzymatic reaction as inhibition of lipoxygenase by catechol and caffeic acid prevented hydroperoxide formation. Ultra-weak photon emission showed formation of electronically excited species such as triplet excited carbonyl, which, upon transfer of excitation energy, leads to the formation of either singlet excited chlorophyll or singlet oxygen. Alternatively, singlet oxygen is formed by direct decomposition of hydroperoxide via Russell mechanisms. Formation of singlet oxygen was evidenced by the nitroxyl radical 2,2,6,6-tetramethylpiperidine-1-oxyl detected by electron paramagnetic resonance spin-trapping spectroscopy and the imaging of green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. Suppression of singlet oxygen formation by lipoxygenase inhibitors indicates that singlet oxygen may be formed via enzymatic lipid peroxidation initiated by lipoxygenase. PMID:26831215

  10. Isolation and characterization of glutamine synthetase genes in Chlamydomonas reinhardtii.

    PubMed

    Chen, Q; Silflow, C D

    1996-11-01

    To elucidate the role of glutamine synthetase (GS) in nitrogen assimilation in the green alga Chlamydomonas reinhardtii we used maize GS1 (the cytosolic form) and GS2 (the chloroplastic form) cDNAs as hybridization probes to isolate C. reinhardtii cDNA clones. The amino acid sequences derived from the C. reinhardtii clones have extensive homology with GS enzymes from higher plants. A putative amino-terminal transit peptide encoded by the GS2 cDNA suggests that the protein localizes to the chloroplast. Genomic DNA blot analysis indicated that GS1 is encoded by a single gene, whereas two genomic fragments hybridized to the GS2 cDNA probe. All GS2 cDNA clones corresponded to only one of the two GS2 genomic sequences. We provide evidence that ammonium, nitrate, and light regulate GS transcript accumulation in green algae. Our results indicate that the level of GS1 transcripts is repressed by ammonium but induced by nitrate. The level of GS2 transcripts is not affected by ammonium or nitrate. Expression of both GS1 and GS2 genes is regulated by light, but perhaps through different mechanisms. Unlike in higher plants, no decreased level of GS2 transcripts was detected when cells were grown under conditions that repress photorespiration. Analysis of GS transcript levels in mutants with defects in the nitrate assimilation pathway show that nitrate assimilation and ammonium assimilation are regulated independently. PMID:8938407

  11. Acclimation of Chlamydomonas reinhardtii to Different Growth Irradiances*

    PubMed Central

    Bonente, Giulia; Pippa, Sara; Castellano, Stefania; Bassi, Roberto; Ballottari, Matteo

    2012-01-01

    We report on the changes the photosynthetic apparatus of Chlamydomonas reinhardtii undergoes upon acclimation to different light intensity. When grown in high light, cells had a faster growth rate and higher biomass production compared with low and control light conditions. However, cells acclimated to low light intensity are indeed able to produce more biomass per photon available as compared with high light-acclimated cells, which dissipate as heat a large part of light absorbed, thus reducing their photosynthetic efficiency. This dissipative state is strictly dependent on the accumulation of LhcSR3, a protein related to light-harvesting complexes, responsible for nonphotochemical quenching in microalgae. Other changes induced in the composition of the photosynthetic apparatus upon high light acclimation consist of an increase of carotenoid content on a chlorophyll basis, particularly zeaxanthin, and a major down-regulation of light absorption capacity by decreasing the chlorophyll content per cell. Surprisingly, the antenna size of both photosystem I and II is not modulated by acclimation; rather, the regulation affects the PSI/PSII ratio. Major effects of the acclimation to low light consist of increased activity of state 1 and 2 transitions and increased contributions of cyclic electron flow. PMID:22205699

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

    PubMed Central

    Yang, Dawei; Song, Donghui; Kind, Tobias; Ma, Yan; Hoefkens, Jens; Fiehn, Oliver

    2015-01-01

    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. PMID:26375463

  13. Activation and de novo synthesis of hydrogenase in Chlamydomonas

    SciTech Connect

    Roessler, P.G.; Lien, S.

    1984-12-01

    Two distinct processes are involved in the formation of active hydrogenase during anaerobic adaptation of Chlamydomonas reinhardtii cells. In the first 30 minutes of anaerobiosis, nearly all of the hydrogenase activity can be attributed to activation of constituitive polypeptide precursor, based on the insensitivity of the process to treatment with cycloheximide (15 micrograms per milliliter). This concentration of cycloheximide inhibits protein synthesis by greater than 98%. After the initial activation period, de novo protein synthesis plays a critical role in the adaptation process since cycloheximide inhibits the expression of hydrogenase in maximally adapted cells by 70%. Chloramphenicol (500 micrograms per milliliter) has a much lesser effect on the adaptation process. Incubation of cell-free extracts under anaerobic conditions in the presence of dithionite, dithiothreitol, NADH, NADP, ferredoxin, ATP, Mg/sup 2 +/, Ca/sup 2 +/, and iron does not lead to active hydrogenase formation. Furthermore, in vivo reactivation of oxygen-inactivated hydrogenase does not appear to take place. The adaptation process is very sensitive to the availability of iron. Iron-deficient cultures lose the ability to form active hydrogenase before growth, photosynthesis, and respiration are significantly affected. Preincubation of iron-deficient cells with iron 2 hours prior to the adaptation period fully restores the capacity of the cells to synthesize functional hydrogenase.

  14. Targeted disruption of the NIT8 gene in Chlamydomonas reinhardtii.

    PubMed Central

    Nelson, J A; Lefebvre, P A

    1995-01-01

    We have used homologous recombination to disrupt the nuclear gene NIT8 in Chlamydomonas reinhardtii. This is the first report of targeted gene disruption of an endogenous locus in C. reinhardtii and only the second for a photosynthetic eukaryote. NIT8 encodes a protein necessary for nitrate and nitrite assimilation by C. reinhardtii. A disruption vector was constructed by placing the CRY1-1 selectable marker gene, which confers emetine resistance, within the NIT8 coding region. nit8 mutants are unable to grow on nitrate as their sole nitrogen source (Nit-) and are resistant to killing by chlorate. One of 2,000 transformants obtained after selection on emetine-chlorate medium contained a homologous insertion of five copies of the disruption plasmid into the NIT8 gene, producing an emetine-resistant, chlorate-resistant Nit- phenotype. The mutant phenotype was rescued by the wild-type NIT8 gene upon transformation. Seven other mutations at the nit8 locus, presumably resulting from homologous recombination with the disruption plasmid, were identified but were shown to be accompanied by deletions of the surrounding genomic region. PMID:7565729

  15. The pH tolerance of Chlamydomonas applanata (Volvocales, Chlorophyta).

    PubMed

    Visviki, I; Santikul, D

    2000-02-01

    The effects of hydrogen ions on the growth and ultrastructure of Chlamydomonas applanata Pringsheim were examined. This species exhibits wide tolerance growing at pH values ranging from 3.4 to 8.4, with optimum growth obtained at 7.4. Growth is noticeably depressed at pH 4.4 and 3.4. At the ultrastructural level, exposure to pH 4.4 results in a 10% decrease in cell volume of single vegetative cells, an increase in pyrenoidal volume, and reduction of starch reserves. Palmelloid colonies also appear. pH 3.4 induces excessive production of mucilage and leads to the preponderance of palmelloid colonies. Cell death of both colony and single cells is seen, as well as loss of motility and abnormal cell division. Surviving single cells are significantly larger than controls, with thicker cell walls, smaller chloroplasts, and larger vacuome. Such cells entering dormancy ensure the survival of the species in times of stress. PMID:10629274

  16. Insight into Protein S-nitrosylation in Chlamydomonas reinhardtii

    PubMed Central

    Morisse, Samuel; Zaffagnini, Mirko; Gao, Xing-Huang

    2014-01-01

    Abstract Aims: Protein S-nitrosylation, a post-translational modification (PTM) consisting of the covalent binding of nitric oxide (NO) to a cysteine thiol moiety, plays a major role in cell signaling and is recognized to be involved in numerous physiological processes and diseases in mammals. The importance of nitrosylation in photosynthetic eukaryotes has been less studied. The aim of this study was to expand our knowledge on protein nitrosylation by performing a large-scale proteomic analysis of proteins undergoing nitrosylation in vivo in Chlamydomonas reinhardtii cells under nitrosative stress. Results: Using two complementary proteomic approaches, 492 nitrosylated proteins were identified. They participate in a wide range of biological processes and pathways, including photosynthesis, carbohydrate metabolism, amino acid metabolism, translation, protein folding or degradation, cell motility, and stress. Several proteins were confirmed in vitro by western blot, site-directed mutagenesis and activity measurements. Moreover, 392 sites of nitrosylation were also identified. These results strongly suggest that S-nitrosylation could constitute a major mechanism of regulation in C. reinhardtii under nitrosative stress conditions. Innovation: This study constitutes the largest proteomic analysis of protein nitrosylation reported to date. Conclusion: The identification of 381 previously unrecognized targets of nitrosylation further extends our knowledge on the importance of this PTM in photosynthetic eukaryotes. The data have been deposited to the ProteomeXchange repository with identifier PXD000569. Antioxid. Redox Signal. 21, 1271–1284. PMID:24328795

  17. Ammonium removal from anaerobically treated effluent by Chlamydomonas acidophila.

    PubMed

    Escudero, Ania; Blanco, Fernando; Lacalle, Arrate; Pinto, Miriam

    2014-02-01

    Several batch culture studies were carried out to evaluate an anaerobically treated effluent as a low-cost growth medium for the microalga Chlamydomonas acidophila and to study the effectiveness of the microalga in removing NH4-N from the effluent. An initial decrease in the effluent pH to 3 was required for adequate growth of C. acidophila and removal of NH4-N. Growth of the microalgae was inhibited at high light intensity (224μmolphotonsm(-2)s(-1) at the surface of the vessels). However, the growth was not greatly affected by the high solid content and turbidity of the effluent. The microalga was able to grow in media containing NH4-N at concentrations of up to 1000mgL(-1) (50% of effluent) and to remove 88mg of NH4-NL(-1) in 10days. C. acidophila therefore appears a promising agent for the removal of NH4-N from anaerobically treated effluents. PMID:24342946

  18. Characterizing the anaerobic response of Chlamydomonas reinhardtii by quantitative proteomics.

    PubMed

    Terashima, Mia; Specht, Michael; Naumann, Bianca; Hippler, Michael

    2010-07-01

    The versatile metabolism of the green alga Chlamydomonas reinhardtii is reflected in its complex response to anaerobic conditions. The anaerobic response is also remarkable in the context of renewable energy because C. reinhardtii is able to produce hydrogen under anaerobic conditions. To identify proteins involved during anaerobic acclimation as well as to localize proteins and pathways to the powerhouses of the cell, chloroplasts and mitochondria from C. reinhardtii in aerobic and anaerobic (induced by 8 h of argon bubbling) conditions were isolated and analyzed using comparative proteomics. A total of 2315 proteins were identified. Further analysis based on spectral counting clearly localized 606 of these proteins to the chloroplast, including many proteins of the fermentative metabolism. Comparative quantitative analyses were performed with the chloroplast-localized proteins using stable isotopic labeling of amino acids ([(13)C(6)]arginine/[(12)C(6)]arginine in an arginine auxotrophic strain). The quantitative data confirmed proteins previously characterized as induced at the transcript level as well as identified several new proteins of unknown function induced under anaerobic conditions. These proteins of unknown function provide new candidates for further investigation, which could bring insights for the engineering of hydrogen-producing alga strains. PMID:20190198

  19. Singlet oxygen production in Chlamydomonas reinhardtii under heat stress

    PubMed Central

    Prasad, Ankush; Ferretti, Ursula; Sedlářová, Michaela; Pospíšil, Pavel

    2016-01-01

    In the current study, singlet oxygen formation by lipid peroxidation induced by heat stress (40 °C) was studied in vivo in unicellular green alga Chlamydomonas reinhardtii. Primary and secondary oxidation products of lipid peroxidation, hydroperoxide and malondialdehyde, were generated under heat stress as detected using swallow-tailed perylene derivative fluorescence monitored by confocal laser scanning microscopy and high performance liquid chromatography, respectively. Lipid peroxidation was initiated by enzymatic reaction as inhibition of lipoxygenase by catechol and caffeic acid prevented hydroperoxide formation. Ultra-weak photon emission showed formation of electronically excited species such as triplet excited carbonyl, which, upon transfer of excitation energy, leads to the formation of either singlet excited chlorophyll or singlet oxygen. Alternatively, singlet oxygen is formed by direct decomposition of hydroperoxide via Russell mechanisms. Formation of singlet oxygen was evidenced by the nitroxyl radical 2,2,6,6-tetramethylpiperidine-1-oxyl detected by electron paramagnetic resonance spin-trapping spectroscopy and the imaging of green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. Suppression of singlet oxygen formation by lipoxygenase inhibitors indicates that singlet oxygen may be formed via enzymatic lipid peroxidation initiated by lipoxygenase. PMID:26831215

  20. Calcium titration of Chlamydomonas reinhardtii centrin and its structural changes

    NASA Astrophysics Data System (ADS)

    Ocaña, Wanda; Pastrana-Ríos, Belinda

    2014-07-01

    Chlamydomonas reinhardtii centrin is a highly conserved calcium binding protein belonging to the EF-hand superfamily. Centrin, like other calcium binding proteins, changes conformation upon calcium binding. In addition, the calcium binding sites are comprised mainly of aspartates and glutamates which would serve as probes for a calcium binding event. 2D IR correlation spectroscopy has proven to be a valuable technique to determine the differences in the molecular behavior of the EF-hand domains within centrin. Moreover, the differences in affinity for calcium displayed by these domains were correlated to differences in the molecular behavior of these EF-hand domains when compared with each other and the full-length protein. We were able to confirm the nature of the two independent domains within centrin. Furthermore, we established the mechanism of aggregation was self-association due to adsorption of centrin to the ZnSe ATR crystal and estimated the extent of aggregation of the full-length protein.

  1. Transport of urea at low concentrations in Chlamydomonas reinhardi.

    PubMed

    Williams, S K; Hodson, R C

    1977-04-01

    Urea transport into the unicellular green alga Chlamydomonas reinhardi was investigated to further our understanding of controls operating on urea catabolism in this organism. Transport into cells grown with acetate and deprived of ammonia is a saturable process, mediated by at least two systems operating maximally at different external urea concentrations. The lower concentration system, with an apparent Km for urea of 5.1 micron, was the object of detailed study. Transport of urea from a saturating concentration (57 micron) into ammonia- and acetate-grown cells freshly suspended in ammonia-limited medium was not detected. Upon further culturing in the absence of ammonia, derepression occurred with transport ability, first appearing at about 1 h , reaching a maximum at about 2 h, and maintaining this maximum at least 5 h. In contrast to this, CO2-grown cells became derepressed more slowly, and maximum transport ability was not maintained. Addition of ammonia or methylamine (5 mM) during nitrogen deprivation prevented further increases in transport ability and caused loss of previously acquired transport ability. Cycloheximide (10 microng/ml) had a similar effect. Energy uncouplers or dark, anaerobic conditions depressed transport. By these criteria, transport from low urea concentrations is mediated by a process that requires protein synthesis and activation by cellular energy, and the process has a rapid rate of turnover and of deactivation by ammonia. PMID:856784

  2. Identification of the Elusive Pyruvate Reductase of Chlamydomonas reinhardtii Chloroplasts

    PubMed Central

    Burgess, Steven J.; Taha, Hussein; Yeoman, Justin A.; Iamshanova, Oksana; Chan, Kher Xing; Boehm, Marko; Behrends, Volker; Bundy, Jacob G.; Bialek, Wojciech; Murray, James W.; Nixon, Peter J.

    2016-01-01

    Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various fermentation pathways leading to the creation of formate, acetate, ethanol and small amounts of other metabolites including d-lactate and hydrogen. Progress has been made in identifying the enzymes involved in these pathways and their subcellular locations; however, the identity of the enzyme involved in reducing pyruvate to d-lactate has remained unclear. Based on sequence comparisons, enzyme activity measurements, X-ray crystallography, biochemical fractionation and analysis of knock-down mutants, we conclude that pyruvate reduction in the chloroplast is catalyzed by a tetrameric NAD+-dependent d-lactate dehydrogenase encoded by Cre07.g324550. Its expression during aerobic growth supports a possible function as a ‘lactate valve’ for the export of lactate to the mitochondrion for oxidation by cytochrome-dependent d-lactate dehydrogenases and by glycolate dehydrogenase. We also present a revised spatial model of fermentation based on our immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the cytoplasm. PMID:26574578

  3. MLT1 links cytoskeletal asymmetry to organelle placement in Chlamydomonas

    PubMed Central

    Mittelmeier, Telsa M.; Thompson, Mark D.; Lamb, Mary Rose; Lin, Huawen; Dieckmann, Carol L.

    2015-01-01

    Asymmetric placement of the photosensory eyespot organelle in Chlamydomonas is patterned by mother-daughter differences between the two basal bodies, which template the anterior flagella. Each basal body is associated with two bundled microtubule rootlets, one with two microtubules and one with four, forming a cruciate pattern. In wild type cells, the single eyespot is positioned at the equator in close proximity to the plus end of the daughter rootlet comprising four microtubules, the D4. Here we identify mutations in two linked loci, MLT1 and MLT2, which cause multiple eyespots. Antiserum raised against MLT1 localized the protein along the D4 rootlet microtubules, from the basal bodies to the eyespot. MLT1 associates immediately with the new D4 as it extends during cell division, before microtubule acetylation. MLT1 is a low-complexity protein of over 300,000 daltons. The expression or stability of MLT1 is dependent on MLT2, predicted to encode a second large, low-complexity protein. MLT1 was not restricted to the D4 rootlet in cells with the vfl2-220 mutation in the gene encoding the basal body-associated protein centrin. The cumulative data highlight the role of mother-daughter basal body differences in establishing asymmetry in associated rootlets, and suggest that eyespot components are directed to the correct location by MLT1 on the D4 microtubules. PMID:25809438

  4. Site-specific basal body duplication in Chlamydomonas.

    PubMed

    O'Toole, Eileen T; Dutcher, Susan K

    2014-02-01

    Correct centriole/basal body positioning is required for numerous biological processes, yet how the cell establishes this positioning is poorly understood. Analysis of centriolar/basal body duplication provides a key to understanding basal body positioning and function. Chlamydomonas basal bodies contain structural features that enable specific triplet microtubules to be specified. Electron tomography of cultures enriched in mitotic cells allowed us to follow basal body duplication and identify a specific triplet at which duplication occurs. Probasal bodies elongate in prophase, assemble transitional fibers (TF) and are segregated with a mature basal body near the poles of the mitotic spindle. A ring of nine-singlet microtubules is initiated at metaphase, orthogonal to triplet eight. At telophase/cytokinesis, triplet microtubule blades assemble first at the distal end, rather than at the proximal cartwheel. The cartwheel undergoes significant changes in length during duplication, which provides further support for its scaffolding role. The uni1-1 mutant contains short basal bodies with reduced or absent TF and defective transition zones, suggesting that the UNI1 gene product is important for coordinated probasal body elongation and maturation. We suggest that this site-specific basal body duplication ensures the correct positioning of the basal body to generate landmarks for intracellular patterning in the next generation. PMID:24166861

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

    SciTech Connect

    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/.

  6. Effect of Triacontanol on Chlamydomonas: I. Stimulation of Growth and Photosynthetic CO(2) Assimilation.

    PubMed

    Houtz, R L; Ries, S K; Tolbert, N E

    1985-10-01

    Treatment of Chlamydomonas reinhardtii cells, cultured at 5% CO(2), with 1 to 1000 micrograms triacontanol (TRIA) per liter resulted in 21 to 35% increases in cell density, 7 to 31% increases in total chlorophyll, and 20 to 100% increases in photosynthetic CO(2) assimilation. The increase in CO(2) fixation with TRIA treatment occurred before, and was independent of, increases in total chlorophyll or cell number. Chlamydomonas cells responded to a broad range of TRIA concentrations that were at least one order of magnitude above the optimum concentration established for higher plants. The necessity for larger concentrations of TRIA may be due to destabilizing effects of Ca(2+) and K(+) present in the Chlamydomonas growth medium. These ions caused flocculation of the colloidally dispersed TRIA in apparent competition with binding of [(14)C]TRIA to Chlamydomonas cells. Octacosanol inhibited the effect of TRIA on photosynthetic CO(2) assimilation. TRIA treatment did not alter the distribution of (14)C-label among photosynthetic products. The effect of TRIA on photosynthetic CO(2) assimilation increased with time after treatment up to 3 days. Chlamydomonas cells that had been grown at low-CO(2) (air) did not respond to TRIA, and transfer of high-CO(2) (5%) grown cells that had responded to TRIA to a low-CO(2) atmosphere resulted in a loss of the effect of TRIA. The effect of pH on photosynthetic CO(2) assimilation indicated that CO(2) is probably the species of inorganic carbon utilized by control and TRIA-treated Chlamydomonas cells. PMID:16664414

  7. Functional study of diacylglycerol acyltransferase type 2 family in Chlamydomonas reinhardtii.

    PubMed

    Hung, Chun-Hsien; Ho, Ming-Yang; Kanehara, Kazue; Nakamura, Yuki

    2013-08-01

    Algal triacylglycerol biosynthesis is of increasing interest for potential biodiesel production. A model microalga, Chlamydomonas, has multiple isoforms of diacylglycerol acyltransferase type 2 (DGTT) catalyzing the final step of triacylglycerol biosynthesis; however, the functions of the isoforms are poorly understood. Here, we performed heterologous complementation assay of Chlamydomonas DGTT1 to 4 in a yeast mutant defective in triacylglycerol biosynthesis. DGTT1, 2 and 3 but not 4 complemented the phenotype, including triacylglycerol levels. Interestingly, complementation by DGTT2 increased triacylglycerol content by 9-fold. PMID:23770092

  8. Biosynthesis and intracellular processing of carbonic anhydrase in Chlamydomonas reinhardtii.

    PubMed

    Toguri, T; Muto, S; Miyachi, S

    1986-08-01

    Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the CO2 concentration during photoautotrophic growth was decreased to air level. After decreasing the CO2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall-less mutant CW-15, which secretes CA into the culture medium, were pulse-labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti-CA serum. A 42-kDa polypeptide with isoelectric point (pI) of 7.1-7.3 was first synthesized. Within 5 min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)-rich RNA in a cell-free reticulocyte lysate system from a rabbit was a 38-kDa polypeptide. This was cotranslationally converted into the 42-kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42-kDa precursor had a high affinity to concanavalin A, it was assumed to have a high-mannose-type oligosaccharide. The mature enzyme had a pI of 6.1-6.2 and was composed of more than two isoforms, which had a complex-type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis. PMID:2874027

  9. Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production.

    PubMed

    Pinto, T S; Malcata, F X; Arrabaça, J D; Silva, J M; Spreitzer, R J; Esquível, M G

    2013-06-01

    Molecular hydrogen (H2) is an ideal fuel characterized by high enthalpy change and lack of greenhouse effects. This biofuel can be released by microalgae via reduction of protons to molecular hydrogen catalyzed by hydrogenases. The main competitor for the reducing power required by the hydrogenases is the Calvin cycle, and rubisco plays a key role therein. Engineered Chlamydomonas with reduced rubisco levels, activity and stability was used as the basis of this research effort aimed at increasing hydrogen production. Biochemical monitoring in such metabolically engineered mutant cells proceeded in Tris/acetate/phosphate culture medium with S-depletion or repletion, both under hypoxia. Photosynthetic activity, maximum photochemical efficiency, chlorophyll and protein levels were all measured. In addition, expression of rubisco, hydrogenase, D1 and Lhcb were investigated, and H2 was quantified. At the beginning of the experiments, rubisco increased followed by intense degradation. Lhcb proteins exhibited monomeric isoforms during the first 24 to 48 h, and D1 displayed sensitivity under S-depletion. Rubisco mutants exhibited a significant decrease in O2 evolution compared with the control. Although the S-depleted medium was much more suitable than its complete counterpart for H2 production, hydrogen release was observed also in sealed S-repleted cultures of rubisco mutated cells under low-moderate light conditions. In particular, the rubisco mutant Y67A accounted for 10-15-fold higher hydrogen production than the wild type under the same conditions and also displayed divergent metabolic parameters. These results indicate that rubisco is a promising target for improving hydrogen production rates in engineered microalgae. PMID:23649352

  10. Carbonic anhydrase activity in isolated chloroplasts of chlamydomonas reinhardtii

    SciTech Connect

    Katzman, G.; Togasaki, R.K. ); Marcus, Y. ); Moroney, J.V. )

    1989-04-01

    In a new assay of carbonic anhydrase, NaH{sup 14}CO{sub 3} solution at the bottom of a sealed vessel releases {sup 14}CO{sub 3} which diffuses to the top of the vessel to be assimilated by actively photosynthesizing Chlamydomonas cells. The assay is initiated by illuminating cells and stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid stable radioactivity above the uncatalyzed background level. With bovine carbonic anhydrase, 1.5 Wilbur Anderson Unit (WAU) can be consistantly measured at 5-6 fold above background. Sonicated whole cells of air adapted wild type (+)gave 741.1 {plus minus} 12.4 WAU/mg chl. Intact washed cells of mixotrophically grown wall-less mutant CWD(-) and a high CO2 requiring wall-less double mutant CIA-3/CW15 (-) gave 7.1 {plus minus} 1.9 and 2.8 {plus minus} 7.8 WAU/mg chl respectively. Chloroplasts isolated from CWD and CIA-3/CW15 and subsequently disrupted gave 64.0 {plus minus} 14.7 and 2.8 {plus minus} 3.2 WAU/mg chl respectively. Chloroplast sonicate from another wall-less mutant CW15(-) gave activity comparable to CWD. Thus on a chlorophyll basis, enzyme activity in chloroplasts from mixotrophically grown cells is about 1/10th of the level found in air adapted wild type cells. CIA-3 seems to lack this activity.

  11. Functional and Spectroscopic Characterization of Chlamydomonas reinhardtii Truncated Hemoglobins

    PubMed Central

    Droghetti, Enrica; Tundo, Grazia R.; Sanz-Luque, Emanuel; Polticelli, Fabio; Visca, Paolo; Smulevich, Giulietta; Ascenzi, Paolo; Coletta, Massimo

    2015-01-01

    The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO2− binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO2− concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO2−binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles. PMID:25993270

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

    PubMed Central

    Baselga-Cervera, Beatriz; Costas, Eduardo; Bustillo-Avendaño, Estéfano

    2016-01-01

    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-resistant genotype

  13. Functional and Spectroscopic Characterization of Chlamydomonas reinhardtii Truncated Hemoglobins.

    PubMed

    Ciaccio, Chiara; Ocaña-Calahorro, Francisco; Droghetti, Enrica; Tundo, Grazia R; Sanz-Luque, Emanuel; Polticelli, Fabio; Visca, Paolo; Smulevich, Giulietta; Ascenzi, Paolo; Coletta, Massimo

    2015-01-01

    The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO2- binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO2- concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO2-binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles. PMID:25993270

  14. Photosynthetic H2 metabolism in Chlamydomonas reinhardtii (unicellular green algae).

    PubMed

    Melis, Anastasios

    2007-10-01

    Unicellular green algae have the ability to operate in two distinctly different environments (aerobic and anaerobic), and to photosynthetically generate molecular hydrogen (H2). A recently developed metabolic protocol in the green alga Chlamydomonas reinhardtii permitted separation of photosynthetic O2-evolution and carbon accumulation from anaerobic consumption of cellular metabolites and concomitant photosynthetic H2-evolution. The H2 evolution process was induced upon sulfate nutrient deprivation of the cells, which reversibly inhibits photosystem-II and O2-evolution in their chloroplast. In the absence of O2, and in order to generate ATP, green algae resorted to anaerobic photosynthetic metabolism, evolved H2 in the light and consumed endogenous substrate. This study summarizes recent advances on green algal hydrogen metabolism and discusses avenues of research for the further development of this method. Included is the mechanism of a substantial tenfold starch accumulation in the cells, observed promptly upon S-deprivation, and the regulated starch and protein catabolism during the subsequent H2-evolution. Also discussed is the function of a chloroplast envelope-localized sulfate permease, and the photosynthesis-respiration relationship in green algae as potential tools by which to stabilize and enhance H2 metabolism. In addition to potential practical applications of H2, approaches discussed in this work are beginning to address the biochemistry of anaerobic H2 photoproduction, its genes, proteins, regulation, and communication with other metabolic pathways in microalgae. Photosynthetic H2 production by green algae may hold the promise of generating a renewable fuel from nature's most plentiful resources, sunlight and water. The process potentially concerns global warming and the question of energy supply and demand. PMID:17721788

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

    PubMed

    Baselga-Cervera, Beatriz; Costas, Eduardo; Bustillo-Avendaño, Estéfano; García-Balboa, Camino

    2016-01-01

    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-resistant genotype suggest

  16. Appearance of Novel Glucose-6-Phosphate Dehydrogenase Isoforms in Chlamydomonas reinhardtii during Growth on Nitrate.

    PubMed Central

    Huppe, H. C.; Turpin, D. H.

    1996-01-01

    Extractable glucose-6-phosphate dehydrogenase activity is higher from N-limited Chlamydomonas reinhardtii cells than from N-sufficient cells. Native gels reveal that the isoform complexity varies depending on the form of N supplied. The isoforms associated with NO3- growth appear within 2 h of switching cells from NH4+ to NO3-. PMID:12226271

  17. Similar relative mutation rates in the three genetic compartments of Mesostigma and Chlamydomonas.

    PubMed

    Hua, Jimeng; Smith, David Roy; Borza, Tudor; Lee, Robert W

    2012-01-01

    Levels of nucleotide substitution at silent sites in organelle versus nuclear DNAs have been used to estimate relative mutation rates among these compartments and explain lineage-specific features of genome evolution. Synonymous substitution divergence values in animals suggest that the rate of mutation in the mitochondrial DNA is 10-50 times higher than that of the nuclear DNA, whereas overall data for most seed plants support relative mutation rates in mitochondrial, plastid, and nuclear DNAs of 1:3:10. Little is known about relative mutation rates in green algae, as substitution rate data is limited to only the mitochondrial and nuclear genomes of the chlorophyte Chlamydomonas. Here, we measure silent-site substitution rates in the plastid DNA of Chlamydomonas and the three genetic compartments of the streptophyte green alga Mesostigma. In contrast to the situation in animals and land plants, our results support similar relative mutation rates among the three genetic compartments of both Chlamydomonas and Mesostigma. These data are discussed in relation to published intra-species genetic diversity data for the three genetic compartments of Chlamydomonas and are ultimately used to address contemporary hypotheses on the organelle genome evolution. To guide future work, we describe evolutionary divergence data of all publically available Mesostigma viride strains and identify, for the first time, three distinct lineages of Mesostigma. PMID:21621456

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

    SciTech Connect

    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. Missense mutation in the Chlamydomonas chloroplast gene that encodes the Rubisco large subunit

    SciTech Connect

    Spreitzer, R.J.; Brown, T.; Chen, Zhixiang; Zhang, Donghong; Al-Abed, S.R. )

    1988-04-01

    The 69-12Q mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase activity, but retains holoenzyme protein. It results from a mutation in the chloroplast large-subunit gene that causes an isoleucine-for-threonine substitution at amino-acid residue 173. Considering that lysine-175 is involved in catalysis, it appears that mutations cluster at the active site.

  20. Utilizing the green alga Chlamydomonas reinhardtii for microbial electricity generation: a living solar cell.

    PubMed

    Rosenbaum, Miriam; Schröder, Uwe; Scholz, Fritz

    2005-10-01

    By employing living cells of the green alga Chlamydomonas reinhardtii, we demonstrate the possibility of direct electricity generation from microbial photosynthetic activity. The presented concept is based on an in situ oxidative depletion of hydrogen, photosynthetically produced by C. reinhardtii under sulfur-deprived conditions, by polymer-coated electrocatalytic electrodes. PMID:15696280

  1. Identification and Regulation of Plasma Membrane Sulfate Transporters in Chlamydomonas1[W][OA

    PubMed Central

    Pootakham, Wirulda; Gonzalez-Ballester, David; Grossman, Arthur R.

    2010-01-01

    Chlamydomonas (Chlamydomonas reinhardtii) exhibits several responses following exposure to sulfur (S)-deprivation conditions, including an increased efficiency of import and assimilation of the sulfate anion (SO42−). Aspects of SO42− transport during S-replete and S-depleted conditions were previously studied, although the transporters had not been functionally identified. We employed a reverse genetics approach to identify putative SO42− transporters, examine their regulation, establish their biogenesis and subcellular locations, and explore their functionality. Upon S starvation of wild-type Chlamydomonas cells, the accumulation of transcripts encoding the putative SO42− transporters SLT1 (for SAC1-like transporter 1), SLT2, and SULTR2 markedly increased, suggesting that these proteins function in high-affinity SO42− transport. The Chlamydomonas sac1 and snrk2.1 mutants (defective for acclimation to S deprivation) exhibited much less of an increase in the levels of SLT1, SLT2, and SULTR2 transcripts and their encoded proteins in response to S deprivation compared with wild-type cells. All three transporters were localized to the plasma membrane, and their rates of turnover were significantly impacted by S availability; the turnover of SLT1 and SLT2 was proteasome dependent, while that of SULTR2 was proteasome independent. Finally, mutants identified for each of the S-deprivation-responsive transporters were used to establish their critical role in the transport of SO42− into S-deprived cells. PMID:20498339

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

    SciTech Connect

    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.

  3. Procedures for the Generation of Mature Chlamydomonas reinhardtii Zygotes for Molecular and Biochemical Analyses 1

    PubMed Central

    Wegener, Dorothee; Treier, Ulrike; Beck, Christoph F.

    1989-01-01

    Zygotes represent an important stage in the sexual cycle of the unicellular green alga Chlamydomonas reinhardtii. To study zygote germination at a molecular level, a protocol was elaborated for the generation of zygotes in large quantities and a method was developed for the extraction from zygotes of RNA that could be translated in vitro. Images Figure 1 Figure 3 PMID:16666800

  4. UV-B Perception and Acclimation in Chlamydomonas reinhardtii[OPEN

    PubMed Central

    Chappuis, Richard; Allorent, Guillaume

    2016-01-01

    Plants perceive UV-B, an intrinsic component of sunlight, via a signaling pathway that is mediated by the photoreceptor UV RESISTANCE LOCUS8 (UVR8) and induces UV-B acclimation. To test whether similar UV-B perception mechanisms exist in the evolutionarily distant green alga Chlamydomonas reinhardtii, we identified Chlamydomonas orthologs of UVR8 and the key signaling factor CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). Cr-UVR8 shares sequence and structural similarity to Arabidopsis thaliana UVR8, has conserved tryptophan residues for UV-B photoreception, monomerizes upon UV-B exposure, and interacts with Cr-COP1 in a UV-B-dependent manner. Moreover, Cr-UVR8 can interact with At-COP1 and complement the Arabidopsis uvr8 mutant, demonstrating that it is a functional UV-B photoreceptor. Chlamydomonas shows apparent UV-B acclimation in colony survival and photosynthetic efficiency assays. UV-B exposure, at low levels that induce acclimation, led to broad changes in the Chlamydomonas transcriptome, including in genes related to photosynthesis. Impaired UV-B-induced activation in the Cr-COP1 mutant hit1 indicates that UVR8-COP1 signaling induces transcriptome changes in response to UV-B. Also, hit1 mutants are impaired in UV-B acclimation. Chlamydomonas UV-B acclimation preserved the photosystem II core proteins D1 and D2 under UV-B stress, which mitigated UV-B-induced photoinhibition. These findings highlight the early evolution of UVR8 photoreceptor signaling in the green lineage to induce UV-B acclimation and protection. PMID:27020958

  5. Chlamydomonas reinhardtii PsbS Protein Is Functional and Accumulates Rapidly and Transiently under High Light.

    PubMed

    Tibiletti, Tania; Auroy, Pascaline; Peltier, Gilles; Caffarri, Stefano

    2016-08-01

    Photosynthetic organisms must respond to excess light in order to avoid photo-oxidative stress. In plants and green algae the fastest response to high light is non-photochemical quenching (NPQ), a process that allows the safe dissipation of the excess energy as heat. This phenomenon is triggered by the low luminal pH generated by photosynthetic electron transport. In vascular plants the main sensor of the low pH is the PsbS protein, while in the green alga Chlamydomonas reinhardtii LhcSR proteins appear to be exclusively responsible for this role. Interestingly, Chlamydomonas also possesses two PsbS genes, but so far the PsbS protein has not been detected and its biological function is unknown. Here, we reinvestigated the kinetics of gene expression and PsbS and LhcSR3 accumulation in Chlamydomonas during high light stress. We found that, unlike LhcSR3, PsbS accumulates very rapidly but only transiently. In order to determine the role of PsbS in NPQ and photoprotection in Chlamydomonas, we generated transplastomic strains expressing the algal or the Arabidopsis psbS gene optimized for plastid expression. Both PsbS proteins showed the ability to increase NPQ in Chlamydomonas wild-type and npq4 (lacking LhcSR3) backgrounds, but no clear photoprotection activity was observed. Quantification of PsbS and LhcSR3 in vivo indicates that PsbS is much less abundant than LhcSR3 during high light stress. Moreover, LhcSR3, unlike PsbS, also accumulates during other stress conditions. The possible role of PsbS in photoprotection is discussed. PMID:27329221

  6. High-Throughput Genetics Strategies for Identifying New Components of Lipid Metabolism in the Green Alga Chlamydomonas reinhardtii.

    PubMed

    Li, Xiaobo; Jonikas, Martin C

    2016-01-01

    Microalgal lipid metabolism is of broad interest because microalgae accumulate large amounts of triacylglycerols (TAGs) that can be used for biodiesel production (Durrett et al Plant J 54(4):593-607, 2008; Hu et al Plant J 54(4):621-639, 2008). Additionally, green algae are close relatives of land plants and serve as models to understand conserved lipid metabolism pathways in the green lineage. The green alga Chlamydomonas reinhardtii (Chlamydomonas hereafter) is a powerful model organism for understanding algal lipid metabolism. Various methods have been used to screen Chlamydomonas mutants for lipid amount or composition, and for identification of the mutated loci in mutants of interest. In this chapter, we summarize the advantages and caveats for each of these methods with a focus on screens for mutants with perturbed TAG content. We also discuss technical opportunities and new tools that are becoming available for screens of mutants altered in TAG content or perturbed in other processes in Chlamydomonas. PMID:27023238

  7. Sustained hydrogen photoproduction by Chlamydomonas reinhardtii: Effects of culture parameters.

    PubMed

    Kosourov, Sergey; Tsygankov, Anatoly; Seibert, Michael; Ghirardi, Maria L

    2002-06-30

    The green alga, Chlamydomonas reinhardtii, is capable of sustained H(2) photoproduction when grown under sulfur-deprived conditions. This phenomenon is a result of the partial deactivation of photosynthetic O(2)-evolution activity in response to sulfur deprivation. At these reduced rates of water-oxidation, oxidative respiration under continuous illumination can establish an anaerobic environment in the culture. After 10-15 hours of anaerobiosis, sulfur-deprived algal cells induce a reversible hydrogenase and start to evolve H(2) gas in the light. Using a computer-monitored photobioreactor system, we investigated the behavior of sulfur-deprived algae and found that: (1) the cultures transition through five consecutive phases: an aerobic phase, an O(2)-consumption phase, an anaerobic phase, a H(2)-production phase and a termination phase; (2) synchronization of cell division during pre-growth with 14:10 h light:dark cycles leads to earlier establishment of anaerobiosis in the cultures and to earlier onset of the H(2)-production phase; (3) re-addition of small quantities of sulfate (12.5-50 microM MgSO(4), final concentration) to either synchronized or unsynchronized cell suspensions results in an initial increase in culture density, a higher initial specific rate of H(2) production, an increase in the length of the H(2)-production phase, and an increase in the total amount of H(2) produced; and (4) increases in the culture optical density in the presence of 50 microM sulfate result in a decrease in the initial specific rates of H(2) production and in an earlier start of the H(2)-production phase with unsynchronized cells. We suggest that the effects of sulfur re-addition on H(2) production, up to an optimal concentration, are due to an increase in the residual water-oxidation activity of the algal cells. We also demonstrate that, in principle, cells synchronized by growth under light:dark cycles can be used in an outdoor H(2)-production system without loss of

  8. Hydrogen evolution as a consumption mode of reducing equivalents in green algal fermentation. [Chlamydomonas reinhardii; Chlorella pyrenoidosa; Chlorococcum minutum

    SciTech Connect

    Ohta, S.; Miyamoto, K.; Miura, Y.

    1987-04-01

    Dark anaerobic fermentation in the green algae Chlamydomonas MGA 161, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Chlorococcum minutum was studied. Their isolate, Chlamydomonas MGA 161, was unusual in having high H/sub 2/ but almost no formate. The fermentation pattern in Chlamydomonas MGA 161 was altered by changes in the NaCl or NH/sub 4/Cl concentration. Glycerol formation increased at low (0.1%) and high (7%) NaCl concentrations starch degradation, and formation of ethanol, H/sub 2/, and CO/sub 2/ increased with the addition of NH/sub 4/Cl to above 5 millimolar in N-deficient cells. C. reinhardtii and C.pyrenoidosa exhibited a very similar anaerobic metabolism, forming formate, acetate and ethanol in a ratio of about 2:2:1. C. minimum was also unusual in forming acetate, glycerol, and CO/sub 2/ as its main products, with H/sub 2/, formate, and ethanol being formed in negligible amounts. In the presence of CO, ethanol formation increased twofold in Chlamydomonas MGA 161 and C. reinhardtii, but the fermentation pattern in C. minimum did not change. An experiment with hypophosphite addition showed that dark H/sub 2/ evolution of the Escherichia coli type could be ruled out in Chlamydomonas MGA 161 and C. reinhardtii. Among the green algae investigated, three fermentation types were identified by the distribution pattern of the end products, which reflected the consumption model of reducing equivalents in the cells.

  9. Plasmids in Frankia sp.

    PubMed

    Normand, P; Simonet, P; Butour, J L; Rosenberg, C; Moiroud, A; Lalonde, M

    1983-07-01

    A method to achieve cell lysis and isolate Frankia sp. plasmid DNA was developed. A screening of Frankia sp. strains belonging to different host compatibility groups (Alnus sp., Elaeagnus sp., Ceanothus sp.) showed that, of 39 strains tested, 4 (strains Cp11, ARgN22d, ArI3, and EUN1f) possessed plasmids ranging in size from 7.1 to 32.2 kilobase pairs as estimated from agarose gel electrophoresis and electron microscopy. A total of 11 plasmids were detected. PMID:6863219

  10. Rescue of a paralyzed-flagella mutant of Chlamydomonas by transformation

    SciTech Connect

    Diener, D.R.; Curry, A.M.; Johnson, K.A.; Williams, B.D.; Rosenbaum, J.L. ); Lefebvre, P.A. ); Kindle, K.L. )

    1990-08-01

    The biflagellate alga Chlamydomonas has been used extensively in the genetic and biochemical analysis of flagellar assembly and motility. The authors have restored motility to a paralyzed-flagella mutant of Chlamydomonas by transforming with the corresponding wild-type gene. A nitrate reductase-deficient paralyzed-flagella strain, nit1-305 pf-14, carrying mutations in the genes for nitrate reductase and radial spoke protein 3, was transformed with wild-type copies of both genes. Two-thirds of the cells that survived nitrate selection also regained motility, indicating that they had been transformed with both the nitrate reductase and radial spoke protein 3 genes. Transformants typically contained multiple copies of both genes, genetically linked to each other, but not linked to the original mutant loci. Complementation of paralyzed-flagella mutants by transformation is a powerful tool for investigating flagellar assembly and function.

  11. Antimicrobial cocktails to control bacterial and fungal contamination in Chlamydomonas reinhardtii cultures.

    PubMed

    Wang, Liang; Yang, Fengyuan; Chen, Huiyi; Fan, Zhiyue; Zhou, Yongkang; Lu, Jun; Zheng, Yuanlin

    2016-01-01

    Chlamydomonas reinhardtii is a unicellular green alga widely used for research in photosynthesis, cell cycle regulation, ciliary biogenesis, and other physiological processes. Sterile cultures are needed for these studies, but contamination from bacteria and fungi occurs frequently. Although the One-shot Solution cocktail consisting of carbendazim, ampicillin, and cefotaxime has been developed for removing these contaminants from algal cultures, it is not always effective. Here we report two new antimicrobial cocktails for treating mixed bacterial and fungal contamination of Chlamydomonas cultures. A combination of the bactericide nalidixic acid with one of two fungicides, azoxystrobin or tebuconazole, was more effective than the One-shot Solution cocktail. In some of our tests, we find that alternating use of our new cocktails with One-shot Solution is needed to remove obstinate contaminants. PMID:26956093

  12. Limited photosynthetic electron flow but no CO2 fixation in Chlamydomonas mutants lacking photosystem I.

    PubMed

    Cournac, L; Redding, K; Bennoun, P; Peltier, G

    1997-10-13

    By measuring O2 and CO2 exchange in mutants of the green alga Chlamydomonas reinhardtii in which genes encoding the reaction center of photosystem I (psaA or psaB) have been deleted, we found that a photosystem II-dependent electron flow using O2 as the final acceptor can be sustained in the light. However, in contrast with recent reports using other Chlamydomonas mutants (B4 and F8), we show here that CO2 fixation does not occur in the absence of photosystem I. By deleting the psaA gene in both B4 and F8 strains, we conclude that the ability of these mutants to fix CO2 in the light is due to the presence of residual amounts of photosystem I. PMID:9369234

  13. Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis

    PubMed Central

    Mitra, Mautusi

    2013-01-01

    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 Mg 2+ 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. PMID:24555064

  14. Prolongation of H2 Photoproduction by Immobilized, Sulfur-Limited Chlamydomonas reinhardtii Cultures

    SciTech Connect

    Laurinavichene, T. V.; Kosourov, S. N.; Ghirardi, M. L.; Seibert, M.; Tsygankov, A. A.

    2008-04-30

    Two approaches to prolong the duration of hydrogen production by immobilized, sulfur-limited Chlamydomonas reinhardtii cells are examined. The results demonstrate that continuous H{sub 2} photoproduction can occur for at least 90 days under constant flow of TAP medium containing micromolar sulfate concentrations. Furthermore, it is also possible to prolong the duration of H{sub 2} production by cycling immobilized cells between minus and plus sulfate conditions.

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

    PubMed Central

    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

  16. Plectin-like proteins are present in cells of Chlamydomonas eugametos (Volvocales).

    PubMed

    Hendrychová, J; Vítová, M; Bisová, K; Wiche, G; Zachleder, V

    2002-01-01

    Using both monoclonal and polyclonal antibodies against mammalian plectin (multifunctional protein cross-linking cytoskeletal structures, mainly intermediate filaments, in mammalian cells), several putative isoforms of plectin-like proteins were found in protein extracts from the green alga Chlamydomonas eugametos (Volvocales). Immunofluorescence and immunoblotting revealed that some of the plectin-like proteins were present in perinuclear region or localized near the cell wall, probably being attached to the cytoplasmic membrane. PMID:12503400

  17. A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii.

    PubMed

    Bertalan, Ivo; Munder, Matthias C; Weiß, Caroline; Kopf, Judith; Fischer, Dirk; Johanningmeier, Udo

    2015-02-10

    In search of alternative expression platforms heterologous protein production in microalgae has gained increasing importance in the last years. Particularly, the chloroplast of the green alga Chlamydomonas reinhardtii has been adopted to successfully express foreign proteins like vaccines and antibodies. However, when compared with other expression systems, the development of the algal chloroplast to a powerful production platform for recombinant proteins is still in its early stages. In an effort to further improve methods for a reliable and rapid generation of transplastomic Chlamydomonas strains we constructed the key plasmid pMM2 containing the psbA gene and a multiple cloning site for foreign gene insertion. The psbA gene allows a marker-free selection procedure using as a recipient the Fud7 strain of Chlamydomonas, which grows on media containing acetate as a carbon source, but is unable to grow photoautotrophically due to the lack of an intact psbA gene. Biolistic transformation of Fud7 with vectors containing this gene restores photoautotrophic growth and thus permits selection in the light on media without carbon sources and antibiotics. The multiple cloning site with a BsaI recognition sequence allows type IIs restriction enzyme-based modular cloning which rapidly generates new gene constructs without sequences, which could influence the expression and characteristics of the foreign protein. In order to demonstrate the feasibility of this approach, a codon optimized version of the gene for the bacterial protein MPT64 has been integrated into the plastome. Several strains with different promoter/UTR combinations show a stable expression of the HA tagged MPT64 protein in Chlamydomonas chloroplasts. PMID:25554634

  18. Production of Recombinant Proteins in the Chloroplast of the Green Alga Chlamydomonas reinhardtii.

    PubMed

    Guzmán-Zapata, Daniel; Macedo-Osorio, Karla Soledad; Almaraz-Delgado, Alma Lorena; Durán-Figueroa, Noé; Badillo-Corona, Jesus Agustín

    2016-01-01

    Chloroplast transformation in the green algae Chlamydomonas reinhardtii can be used for the production of valuable recombinant proteins. Here, we describe chloroplast transformation of C. reinhardtii followed by protein detection. Genes of interest integrate stably by homologous recombination into the chloroplast genome following introduction by particle bombardment. Genes are inherited and expressed in lines recovered after selection in the presence of an antibiotic. Recombinant proteins can be detected by conventional techniques like immunoblotting and purified from liquid cultures. PMID:26614282

  19. Chlamydomonas reinhardtii, a model system for functional validation of abiotic stress responsive genes.

    PubMed

    Hema, R; Senthil-Kumar, M; Shivakumar, S; Chandrasekhara Reddy, P; Udayakumar, M

    2007-08-01

    Stress tolerance is a multigenic character and there are many stress responsive genes, which are stress specific. Although many of these have been cloned, their functional significance remains fragmentary. Hence it is important to identify the relevant stress genes involved in altering the metabolism for adaptation. Overexpression is one of the several approaches and Chlamydomonas is a suitable system to study the functional relevance of stress genes. Stress responses can only be assessed on prior exposure to sublethal induction stress. In this study the acclimation response of Chlamydomonas was assessed for different abiotic stresses using physiological screens like chlorophyll stability, membrane damage, cell viability, accumulation of free radicals, survival and recovery growth. We demonstrate that Chlamydomonas responds to diverse stresses and is a potential system to study the relevance of stress genes. The relevance of choline oxidase A (codA), a key enzyme in glycinebetaine biosynthesis, was examined by developing transformants expressing codA gene from Arthrobacter globiformis. Southern positive transformants showed enhanced accumulation of glycinebetaine. The transformants also showed enhanced growth under salinity, high light coupled with methylviologen-induced oxidative stress, high temperature and cold stress. However the transgenics were not tolerant to PEG-mediated simulated osmotic stress, LiCl, menadione and UV stress. Increased cell survival and decreased chlorophyll degradation in transformants under acclimated conditions further confirmed the relevance of codA in imparting stress tolerance. Our results indicated that the relevance of stress responsive genes can be efficiently validated for diverse abiotic stresses using Chlamydomonas system. PMID:17431668

  20. Purification and cDNA isolation of chloroplastic phosphoglycerate kinase from Chlamydomonas reinhardtii.

    PubMed Central

    Kitayama, M; Togasaki, R K

    1995-01-01

    Chloroplastic phosphoglycerate kinase (PGK) was purified to homogeneity from a soluble fraction of chloroplasts of a cell-wall-deficient mutant strain of Chlamydomonas reinhardtii (cw-15) using ammonium sulfate fractionation, Reactive Blue-72 column chromatography, and native polyacrylamide gel electrophoresis. PGK activity was attributed to a single polypeptide with a molecular mass of 42 kD. Relative purity and identity of the isolated enzyme was confirmed by N-terminal amino acid sequence determination. Antiserum against this enzyme was raised and a western blot analysis of whole-cell lysate from cw-15 cells using this anti-chloroplastic PGK serum detected a single polypeptide with a molecular mass of 42 kD. The cDNA clone corresponding to the Chlamydomonas chloroplastic PGK was isolated from a Chlamydomonas cDNA expression library using the anti-PGK serum. The cDNA sequence was determined and apparently codes for the entire precursor peptide, which consists of 461 codons. The results from Southern and northern blot analyses suggest that the chloroplastic PGK gene exists as a single copy in the nuclear genome of C. reinhardtii and is expressed as a 1.8-kb transcript. The C. reinhardtii chloroplastic PGK cDNA has 71 and 66% homology with wheat chloroplastic PGK and spinach chloroplastic PGK, respectively. Based on the deduced amino acid sequence, the chloroplastic PGK of C. reinhardtii has more similarity to plant PGKs than to other PGKs, having both prokaryotic and eukaryotic features. PMID:7724671

  1. Chlamydomonas Xanthophyll Cycle Mutants Identified by Video Imaging of Chlorophyll Fluorescence Quenching.

    PubMed Central

    Niyogi, K. K.; Bjorkman, O.; Grossman, A. R.

    1997-01-01

    The photosynthetic apparatus in plants is protected against oxidative damage by processes that dissipate excess absorbed light energy as heat within the light-harvesting complexes. This dissipation of excitation energy is measured as nonphotochemical quenching of chlorophyll fluorescence. Nonphotochemical quenching depends primarily on the [delta]pH that is generated by photosynthetic electron transport, and it is also correlated with the amounts of zeaxanthin and antheraxanthin that are formed from violaxanthin by the operation of the xanthophyll cycle. To perform a genetic dissection of nonphotochemical quenching, we have isolated npq mutants of Chlamydomonas by using a digital video-imaging system. In excessive light, the npq1 mutant is unable to convert violaxanthin to antheraxanthin and zeaxanthin; this reaction is catalyzed by violaxanthin de-epoxidase. The npq2 mutant appears to be defective in zeaxanthin epoxidase activity, because it accumulates zeaxanthin and completely lacks antheraxanthin and violaxanthin under all light conditions. Characterization of these mutants demonstrates that a component of nonphotochemical quenching that develops in vivo in Chlamydomonas depends on the accumulation of zeaxanthin and antheraxanthin via the xanthophyll cycle. However, observation of substantial, rapid, [delta]pH-dependent nonphotochemical quenching in the npq1 mutant demonstrates that the formation of zeaxanthin and antheraxanthin via violaxanthin de-epoxidase activity is not required for all [delta]pH-dependent nonphotochemical quenching in this alga. Furthermore, the xanthophyll cycle is not required for survival of Chlamydomonas in excessive light. PMID:12237386

  2. Mutants of Chlamydomonas: tools to study thylakoid membrane structure, function and biogenesis.

    PubMed

    de Vitry, C; Vallon, O

    1999-06-01

    The unicellular green alga Chlamydomonas reinhardtii is a model system for the study of photosynthesis and chloroplast biogenesis. C. reinhardtii has a photosynthesis apparatus similar to that of higher plants and it grows at rapid rate (generation time about 8 h). It is a facultative phototroph, which allows the isolation of mutants unable to perform photosynthesis and its sexual cycle allows a variety of genetic studies. Transformation of the nucleus and chloroplast genomes is easily performed. Gene transformation occurs mainly by homologous recombination in the chloroplast and heterologous recombination in the nucleus. Mutants are precious tools for studies of thylakoid membrane structure, photosynthetic function and assembly. Photosynthesis mutants affected in the biogenesis of a subunit of a protein complex usually lack the entire complex; this pleiotropic effect has been used in the identification of the other subunits, in the attribution of spectroscopic signals and also as a 'genetic cleaning' process which facilitates both protein complex purification, absorption spectroscopy studies or freeze-fracture analysis. The cytochrome b6f complex is not required for the growth of C. reinhardtii, unlike the case of photosynthetic prokaryotes in which the cytochrome complex is also part of the respiratory chain, and can be uniquely studied in Chlamydomonas by genetic approaches. We describe in greater detail the use of Chlamydomonas mutants in the study of this complex. PMID:10433117

  3. Detection and characterization of phosphatidylcholine in various strains of the genus Chlamydomonas (Volvocales, Chlorophyceae).

    PubMed

    Sakurai, Kenta; Mori, Natsumi; Sato, Naoki

    2014-09-01

    The laboratory strains of Chlamydomonas reinhardtii have been reported to contain no phosphatidylcholine (PC), which is considered to be replaced by another zwitterionic lipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). According to the recently published classification, the strains belonged to the subgroup Reinhardtinia. Screening for PC in 13 selected strains of Chlamydomonas in the NIES Algal Collection, which are different in habitats and belong to different phylogenetic subgroups in the genus, revealed the presence of PC in four strains: a strain in the subgroup Polytominia, and three strains in Reinhardtinia. PC was not detected in three other strains of Reinhardtinia analyzed. The presence/absence of PC was not related to the phylogenetic relationship based on 18S rRNA. DGTS was detected in all the strains analyzed. The rare isomer of linolenic acid, 18:3(5,9,12), which has been found in the DGTS of C. reinhardtii, was found in the PC of the two strains and in the DGTS of the five strains. The occurrence of this fatty acid seems limited to a branch of Reinhardtinia. Acquisition and loss of PC in various strains of Chlamydomonas are discussed from the viewpoint of evolution of PC biosynthetic pathway. PMID:24947506

  4. Recharacterization of Chlamydomonas reinhardtii and its relatives with new isolates from Japan.

    PubMed

    Nakada, Takashi; Shinkawa, Haruka; Ito, Takuro; Tomita, Masaru

    2010-01-01

    Chlamydomonas reinhardtii P. A. Dang. (Volvocales, Chlorophyceae) is one of the most intensely studied algae, and its whole genome was sequenced. Although this species was originally described based on materials from France and is often referred to as a cosmopolitan species, all culture strains available today have been isolated from eastern North America. The distinctions with similar and/or closely related species, such as Chlamydomonas globosa J. Snow and Chlamydomonas orbicularis E. G. Pringsh., are also contentious. In this study, new strains of C. reinhardtii and C. globosa were isolated from Japan and compared with several strains similar to C. reinhardtii. Based on the morphological, genealogical, phylogenetical, and mating studies including the new Japanese strains, the circumscription of C. reinhardtii was clarified. C. reinhardtii was most closely related to C. globosa, and they were shown to be different species. Although C. reinhardtii was similar to C. orbicularis, the authentic strain of C. orbicularis was morphologically distinguishable and phylogenetically distant from C. reinhardtii. Discovery of the Japanese strains of C. reinhardtii supports the cosmopolitan distribution of this species. Based on Japanese strains and/or strains from other countries, emended descriptions of C. reinhardtii, C. globosa, and C. orbicularis are given. PMID:19882207

  5. Isolation of the Chlamydomonas Regulatory Gene Nit2 by Transposon Tagging

    PubMed Central

    Schnell, R. A.; Lefebvre, P. A.

    1993-01-01

    Genetic evidence suggests that the NIT2 gene of Chlamydomonas reinhardtii encodes a positive regulator of the nitrate-assimilation pathway. To learn more about the function of the NIT2 gene product, we isolated the gene using a transposon-tagging strategy. A nit2 mutation caused by the insertion of a transposon was identified by testing spontaneous nit2 mutants for the presence of new copies of Gulliver or TOC1, transposable elements that have been identified in Chlamydomonas. In 2 of the 14 different mutants that were analyzed, a Gulliver element was found to be genetically and phenotypically associated with the nit2 mutation. Using the Gulliver element as a probe, one of the transposon-induced nit2 alleles was isolated, and a sequence adjoining the transposon was used to isolate the corresponding wild-type locus. The NIT2 gene was delimited by mapping DNA rearrangements associated with nit2 mutations and mutant rescue by genetic transformation. The NIT2 gene encodes a 6-kb transcript that was not detected in cells grown in the presence of ammonium. Likewise, NIT2-dependent genes are repressed in ammonium-grown cells. These results suggest that repression of the NIT2 gene may mediate metabolite repression of the nitrate assimilation pathway in Chlamydomonas. PMID:8394263

  6. Anaerobic acclimation in Chlamydomonas reinhardtii: anoxic gene expression, hydrogenase induction, and metabolic pathways.

    PubMed

    Mus, Florence; Dubini, Alexandra; Seibert, Michael; Posewitz, Matthew C; Grossman, Arthur R

    2007-08-31

    Both prokaryotic and eukaryotic photosynthetic microbes experience conditions of anoxia, especially during the night when photosynthetic activity ceases. In Chlamydomonas reinhardtii, dark anoxia is characterized by the activation of an extensive set of fermentation pathways that act in concert to provide cellular energy, while limiting the accumulation of potentially toxic fermentative products. Metabolite analyses, quantitative PCR, and high density Chlamydomonas DNA microarrays were used to monitor changes in metabolite accumulation and gene expression during acclimation of the cells to anoxia. Elevated levels of transcripts encoding proteins associated with the production of H2, organic acids, and ethanol were observed in congruence with the accumulation of fermentation products. The levels of over 500 transcripts increased significantly during acclimation of the cells to anoxic conditions. Among these were transcripts encoding transcription/translation regulators, prolyl hydroxylases, hybrid cluster proteins, proteases, transhydrogenase, catalase, and several putative proteins of unknown function. Overall, this study uses metabolite, genomic, and transcriptome data to provide genome-wide insights into the regulation of the complex metabolic networks utilized by Chlamydomonas under the anaerobic conditions associated with H2 production. PMID:17565990

  7. A simple, low-cost method for chloroplast transformation of the green alga Chlamydomonas reinhardtii.

    PubMed

    Economou, Chloe; Wannathong, Thanyanan; Szaub, Joanna; Purton, Saul

    2014-01-01

    The availability of routine techniques for the genetic manipulation of the chloroplast genome of Chlamydomonas reinhardtii has allowed a plethora of reverse-genetic studies of chloroplast biology using this alga as a model organism. These studies range from fundamental investigations of chloroplast gene function and regulation to sophisticated metabolic engineering programs and to the development of the algal chloroplast as a platform for producing high-value recombinant proteins. The established method for delivering transforming DNA into the Chlamydomonas chloroplast involves microparticle bombardment, with the selection of transformant lines most commonly involving the use of antibiotic resistance markers. In this chapter we describe a simpler and cheaper delivery method in which cell/DNA suspensions are agitated with glass beads: a method that is more commonly used for nuclear transformation of Chlamydomonas. Furthermore, we highlight the use of an expression vector (pASapI) that employs an endogenous gene as a selectable marker, thereby avoiding the contentious issue of antibiotic resistance determinants in transgenic lines. PMID:24599870

  8. A new method to identify flanking sequence tags in chlamydomonas using 3’-RACE

    PubMed Central

    2012-01-01

    Background The green alga Chlamydomonas reinhardtii, although a premier model organism in biology, still lacks extensive insertion mutant libraries with well-identified Flanking Sequence Tags (FSTs). Rapid and efficient methods are needed for FST retrieval. Results Here, we present a novel method to identify FSTs in insertional mutants of Chlamydomonas. Transformants can be obtained with a resistance cassette lacking a 3’ untranslated region (UTR), suggesting that the RNA that is produced from the resistance marker terminates in the flanking genome when it encounters a cleavage/polyadenylation signal. We have used a robust 3’-RACE method to specifically amplify such chimeric cDNAs. Out of 38 randomly chosen transformants, 27 (71%) yielded valid FSTs, of which 23 could be unambiguously mapped to the genome. Eighteen of the mutants lie within a predicted gene. All but two of the intragenic insertions occur in the sense orientation with respect to transcription, suggesting a bias against situations of convergent transcription. Among the 14 insertion sites tested by genomic PCR, 12 could be confirmed. Among these are insertions in genes coding for PSBS3 (possibly involved in non-photochemical quenching), the NimA-related protein kinase CNK2, the mono-dehydroascorbate reductase MDAR1, the phosphoglycerate mutase PGM5 etc.. Conclusion We propose that our 3’-RACE FST method can be used to build large scale FST libraries in Chlamydomonas and other transformable organisms. PMID:22735168

  9. Construction and evaluation of a whole genome microarray of Chlamydomonas reinhardtii

    PubMed Central

    2011-01-01

    Background Chlamydomonas reinhardtii is widely accepted as a model organism regarding photosynthesis, circadian rhythm, cell mobility, phototaxis, and biotechnology. The complete annotation of the genome allows transcriptomic studies, however a new microarray platform was needed. Based on the completed annotation of Chlamydomonas reinhardtii a new microarray on an Agilent platform was designed using an extended JGI 3.1 genome data set which included 15000 transcript models. Results In total 44000 probes were determined (3 independent probes per transcript model) covering 93% of the transcriptome. Alignment studies with the recently published AUGUSTUS 10.2 annotation confirmed 11000 transcript models resulting in a very good coverage of 70% of the transcriptome (17000). Following the estimation of 10000 predicted genes in Chlamydomonas reinhardtii our new microarray, nevertheless, covers the expected genome by 90-95%. Conclusions To demonstrate the capabilities of the new microarray, we analyzed transcript levels for cultures grown under nitrogen as well as sulfate limitation, and compared the results with recently published microarray and RNA-seq data. We could thereby confirm previous results derived from data on nutrient-starvation induced gene expression of a group of genes related to protein transport and adaptation of the metabolism as well as genes related to efficient light harvesting, light energy distribution and photosynthetic electron transport. PMID:22118351

  10. An energy balance from absorbed photons to new biomass for Chlamydomonas reinhardtii and Chlamydomonas acidophila under neutral and extremely acidic growth conditions.

    PubMed

    Langner, Uwe; Jakob, Torsten; Stehfest, Katja; Wilhelm, Christian

    2009-03-01

    Chlamydomonas is one of the most well-studied photosynthetic organisms that had important biotechnological potential for future bioproductions of biofuels. However, an energy balance from incident photons to the energy stored in the new biomass is still lacking. In this study, we applied a recently developed system to measure the energy balance for steady state growth of Chlamydomonas reinhardtii grown at pH 6.5, and C. acidophila that was grown at pH 6.5 and 2.6. Energy use efficiency was quantified on the basis of light absorption, photosynthetic quantum yield, photosynthetic and respiratory quotient, and electron partitioning into proteins, carbohydrates and lipids. The results showed that lower growth rates of C. acidophila under both pH conditions were not caused by the differences in the photosynthetic quantum yield or in alternative electron cycling, but rather by differences in the efficiency of light absorption and increased dark respiration. Analysis of the macromolecular composition of the cells during the light phase showed that C. acidophila uses biosynthetic electrons preferentially for carbohydrate synthesis but not for synthesis of lipids. This led to a strong diurnal cycle of the C/N ratio and could explain the higher dark respiration of C. acidophila compared with C. reinhardtii. PMID:19054351

  11. The SEC6 protein is required for contractile vacuole function in Chlamydomonas reinhardtii.

    PubMed

    Komsic-Buchmann, Karin; Stephan, Lisa Marie; Becker, Burkhard

    2012-06-15

    Contractile vacuoles (CVs) are essential for osmoregulation in many protists. To investigate the mechanism of CV function in Chlamydomonas, we isolated novel osmoregulatory mutants. Four of the isolated mutant cell lines carried the same 33,641 base deletion, rendering the cell lines unable to grow under strong hypotonic conditions. One mutant cell line (Osmo75) was analyzed in detail. The CV morphology was variable in mutant cells, and most cells had multiple small CVs. In addition, one or two enlarged CVs or no visible CVs at all, were observed by light microscopy. These findings suggest that the mutant is impaired in homotypic vacuolar and exocytotic membrane fusion. Furthermore the mutants had long flagella. One of the affected genes is the only SEC6 homologue in Chlamydomonas (CreSEC6). The SEC6 protein is a component of the exocyst complex that is required for efficient exocytosis. Transformation of the Osmo75 mutant with a CreSEC6-GFP construct rescued the mutant completely (osmoregulation and flagellar length). Rescued strains overexpressed CreSEC6 (as a GFP-tagged protein) and displayed a modified CV activity. CVs were larger, whereas the CV contraction interval remained unchanged, leading to increased water efflux rates. Electron microscopy analysis of Osmo75 cells showed that the mutant is able to form the close contact zones between the plasma membrane and the CV membrane observed during late diastole and systole. These results indicate that CreSEC6 is essential for CV function and required for homotypic vesicle fusion during diastole and water expulsion during systole. In addition, CreSEC6 is not only necessary for CV function, but possibly influences the CV cycle in an indirect manner and flagellar length in Chlamydomonas. PMID:22427688

  12. Analysis of the high-affinity iron uptake system at the Chlamydomonas reinhardtii plasma membrane.

    PubMed

    Terzulli, Alaina; Kosman, Daniel J

    2010-05-01

    Multicopper ferroxidases play a vital role in iron metabolism in bacteria, fungi, algae, and mammals. Saccharomyces cerevisiae utilizes a channeling mechanism to couple the ferroxidase activity of Fet3p to Fe(3+) transport into the cell by Ftr1p. In contrast, the mechanisms by which mammals couple the ferroxidase reaction to iron trafficking is unclear. The human ferroxidases ceruloplasmin and hephaestin are twice the size of Fet3p and interact with proteins that are not expressed in fungi. Chlamydomonas FOX1 is a homolog of the human ferroxidases but likely supports iron uptake in a manner similar to that of yeast, since Chlamydomonas reinhardtii expresses a ferric iron permease homolog, FTR1. The results presented support this hypothesis. We show that FOX1 is trafficked to the plasma membrane and is oriented with its multicopper oxidase/ferroxidase domain in the exocytoplasmic space. Our analysis of FTR1 indicates its topology is similar to that of S. cerevisiae Ftr1p, with a potential exocytoplasmic iron channeling motif and two potential iron permeation motifs in membrane-spanning regions. We demonstrate that high-affinity iron uptake is dependent on FOX1 and the copper status of the cell. Kinetic inhibition of high-affinity iron uptake by a ferric iron chelator does not reflect the strength of the chelator, supporting a ferric iron channeling mechanism for high-affinity iron uptake in Chlamydomonas. Last, recombinant FOX1 (rFOX1) has been isolated in a partially holo form that exhibits the UV-visible absorbance spectrum of a multicopper oxidase and the catalytic activity of a ferroxidase. PMID:20348389

  13. The Hsp70 and Hsp40 Chaperones Influence Microtubule Stability in Chlamydomonas

    PubMed Central

    Silflow, Carolyn D.; Sun, Xiaoqing; Haas, Nancy A.; Foley, Joseph W.; Lefebvre, Paul A.

    2011-01-01

    Mutations at the APM1 and APM2 loci in the green alga Chlamydomonas reinhardtii confer resistance to phosphorothioamidate and dinitroaniline herbicides. Genetic interactions between apm1 and apm2 mutations suggest an interaction between the gene products. We identified the APM1 and APM2 genes using a map-based cloning strategy. Genomic DNA fragments containing only the DNJ1 gene encoding a type I Hsp40 protein rescue apm1 mutant phenotypes, conferring sensitivity to the herbicides and rescuing a temperature-sensitive growth defect. Lesions at five apm1 alleles include missense mutations and nucleotide insertions and deletions that result in altered proteins or very low levels of gene expression. The HSP70A gene, encoding a cytosolic Hsp70 protein known to interact with Hsp40 proteins, maps near the APM2 locus. Missense mutations found in three apm2 alleles predict altered Hsp70 proteins. Genomic fragments containing the HSP70A gene rescue apm2 mutant phenotypes. The results suggest that a client of the Hsp70–Hsp40 chaperone complex may function to increase microtubule dynamics in Chlamydomonas cells. Failure of the chaperone system to recognize or fold the client protein(s) results in increased microtubule stability and resistance to the microtubule-destabilizing effect of the herbicides. The lack of redundancy of genes encoding cytosolic Hsp70 and Hsp40 type I proteins in Chlamydomonas makes it a uniquely valuable system for genetic analysis of the function of the Hsp70 chaperone complex. PMID:21940683

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

    PubMed

    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. PMID:22002656

  15. The Purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features

    PubMed Central

    Derrien, Benoît; Majeran, Wojciech; Effantin, Grégory; Ebenezer, Joseph; Friso, Giulia; van Wijk, Klaas J.; Steven, Alasdair C.; Maurizi, Michael R.; Vallon, Olivier

    2012-01-01

    The ClpP peptidase is a major constituent of the proteolytic machinery of bacteria and organelles. The chloroplast ClpP complex is unusual, in that it associates a large number of subunits, one of which (ClpP1) is encoded in the chloroplast, the others in the nucleus. The complexity of these large hetero-oligomeric complexes has been a major difficulty in their overproduction and biochemical characterization. In this paper, we describe the purification of native chloroplast ClpP complex from the green alga Chlamydomonas reinhardtii, using a strain that carries the Strep-tag II at the C-terminus of the ClpP1 subunit. Similar to land plants, the algal complex comprises active and inactive subunits (3 ClpP and 5 ClpR, respectively). Evidence is presented that a sub-complex can be produced by dissociation, comprising ClpP1 and ClpR1, 2, 3 and 4, similar to the ClpR-ring described in land plants. Our Chlamydomonas ClpP preparation also contains two ClpT subunits, ClpT3 and ClpT4, which like the land plant ClpT1 and ClpT2 show 2 Clp-N domains. ClpTs are believed to function in substrate binding and/or assembly of the two heptameric rings. Phylogenetic analysis indicates that ClpT subunits have appeared independently in Chlorophycean algae, in land plants and in dispersed cyanobacterial genomes. Negative staining electron microscopy shows that the Chlamydomonas complex retains the barrel-like shape of homo-oligomeric ClpPs, with 4 additional peripheral masses that we speculate represent either the additional IS1 domain of ClpP1 (a feature unique to algae) or ClpTs or extensions of ClpR subunits PMID:22772861

  16. Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures

    SciTech Connect

    Jurado-Oller, Jose Luis; Dubini, Alexandra; Galvan, Aurora; Fernandez, Emilio; Gonzalez-Ballester, David

    2015-09-17

    Currently, hydrogen fuel is derived mainly from fossil fuels, but there is an increasing interest in clean and sustainable technologies for hydrogen production. In this context, the ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen is a promising alternative for renewable, clean-energy production. Among a diverse array of photosynthetic microorganisms able to produce hydrogen, the green algae Chlamydomonas reinhardtii is the model organism widely used to study hydrogen production. Furthermore, the well-known fact that acetate-containing medium enhances hydrogen production in this algae, little is known about the precise role of acetate during this process.

  17. Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures

    DOE PAGESBeta

    Jurado-Oller, Jose Luis; Dubini, Alexandra; Galvan, Aurora; Fernandez, Emilio; Gonzalez-Ballester, David

    2015-09-17

    Currently, hydrogen fuel is derived mainly from fossil fuels, but there is an increasing interest in clean and sustainable technologies for hydrogen production. In this context, the ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen is a promising alternative for renewable, clean-energy production. Among a diverse array of photosynthetic microorganisms able to produce hydrogen, the green algae Chlamydomonas reinhardtii is the model organism widely used to study hydrogen production. Furthermore, the well-known fact that acetate-containing medium enhances hydrogen production in this algae, little is known about the precise role of acetate during this process.

  18. Novel insights into the function of LHCSR3 in Chlamydomonas reinhardtii

    PubMed Central

    Xue, Huidan; Bergner, Sonja Verena; Scholz, Martin; Hippler, Michael

    2015-01-01

    Light is essential for photosynthesis but excess light is hazardous as it may lead to the formation of reactive oxygen species. Photosynthetic organisms struggle to optimize light utilization and photosynthesis while minimizing photo-oxidative damage. Hereby light to heat dissipation via specialized proteins is a potent mechanism to acclimate toward excess light. In the green alga Chlamydomonas reinhardtii the expression of an ancient light-harvesting protein LHCSR3 enables cells to dissipate harmful excess energy. Herein we summarize newest insights into the function of LHCSR3 from C. reinhardtii. PMID:26237677

  19. Multiple facets of anoxic metabolism and hydrogen production in the unicellular green alga Chlamydomonas reinhardtii.

    PubMed

    Grossman, Arthur R; Catalanotti, Claudia; Yang, Wenqiang; Dubini, Alexandra; Magneschi, Leonardo; Subramanian, Venkataramanan; Posewitz, Matthew C; Seibert, Michael

    2011-04-01

    Many microbes in the soil environment experience micro-oxic or anoxic conditions for much of the late afternoon and night, which inhibit or prevent respiratory metabolism. To sustain the production of energy and maintain vital cellular processes during the night, organisms have developed numerous pathways for fermentative metabolism. This review discusses fermentation pathways identified for the soil-dwelling model alga Chlamydomonas reinhardtii, its ability to produce molecular hydrogen under anoxic conditions through the activity of hydrogenases, and the molecular flexibility associated with fermentative metabolism that has only recently been revealed through the analysis of specific mutant strains. PMID:21563367

  20. Ciliary kinematics of Chlamydomonas reinhardtii in Complex Fluids: Role of viscosity

    NASA Astrophysics Data System (ADS)

    Gopinath, Arvind; Qin, Boyang; Arratia, Paulo

    2014-11-01

    The motility behavior of microorganisms can be significantly affected by the rheology of their fluidic environment. Guided by our experiments on the swimming gait of Chlamydomonas reinhardtii in viscoelastic fluids, we focus on ciliary waveforms in Newtonian fluids and systematically study the effect of increasing viscosity. We find that the beat frequency as well as the wave speed are both strongly influenced by fluid viscosity. Interestingly, ciliary waveforms at low viscosity show a larger influence of the cell body than waveforms at higher viscosity. We use slender body theory and principal component analysis to elucidate the role of fluid viscosity in regulating the kinematics of the swimming process.

  1. Emergent Run-and-Tumble Behavior in a Simple Model of Chlamydomonas with Intrinsic Noise

    NASA Astrophysics Data System (ADS)

    Bennett, Rachel R.; Golestanian, Ramin

    2013-04-01

    Recent experiments on the green alga Chlamydomonas that swims using synchronized beating of a pair of flagella have revealed that it exhibits a run-and-tumble behavior similar to that of bacteria such as E. coli. Using a simple purely hydrodynamic model that incorporates a stroke cycle and an intrinsic Gaussian white noise, we show that a stochastic run-and-tumble behavior could emerge due to the nonlinearity of the combined synchronization-rotation-translation dynamics. Our study suggests that nonlinear mechanics could be a significant contributing factor to how the trajectories of the microorganism are selected.

  2. Regulation of Chlamydomonas flagella and ependymal cell motile cilia by ceramide-mediated translocation of GSK3

    PubMed Central

    Kong, Ji Na; Hardin, Kara; Dinkins, Michael; Wang, Guanghu; He, Qian; Mujadzic, Tarik; Zhu, Gu; Bielawski, Jacek; Spassieva, Stefka; Bieberich, Erhard

    2015-01-01

    Cilia are important organelles formed by cell membrane protrusions; however, little is known about their regulation by membrane lipids. We characterize a novel activation mechanism for glycogen synthase kinase-3 (GSK3) by the sphingolipids phytoceramide and ceramide that is critical for ciliogenesis in Chlamydomonas and murine ependymal cells, respectively. We show for the first time that Chlamydomonas expresses serine palmitoyl transferase (SPT), the first enzyme in (phyto)ceramide biosynthesis. Inhibition of SPT in Chlamydomonas by myriocin led to loss of flagella and reduced tubulin acetylation, which was prevented by supplementation with the precursor dihydrosphingosine. Immunocytochemistry showed that (phyto)ceramide was colocalized with phospho–Tyr-216-GSK3 (pYGSK3) at the base and tip of Chlamydomonas flagella and motile cilia in ependymal cells. The (phyto)ceramide distribution was consistent with that of a bifunctional ceramide analogue UV cross-linked and visualized by click-chemistry–mediated fluorescent labeling. Ceramide depletion, by myriocin or neutral sphingomyelinase deficiency (fro/fro mouse), led to GSK3 dephosphorylation and defective flagella and cilia. Motile cilia were rescued and pYGSK3 localization restored by incubation of fro/fro ependymal cells with exogenous C24:1 ceramide, which directly bound to pYGSK3. Our findings suggest that (phyto)ceramide-mediated translocation of pYGSK into flagella and cilia is an evolutionarily conserved mechanism fundamental to the regulation of ciliogenesis. PMID:26446842

  3. Action Spectrum of the Activity of Acifluorfen-methyl, a Diphenyl Ether Herbicide, in Chlamydomonas eugametos1

    PubMed Central

    Ensminger, Michael P.; Hess, F. Dan

    1985-01-01

    Light is required for the herbicide activity of diphenyl ether herbicides. An action spectrum of acifluorfen-methyl activity with Chlamydomonas eugametos (Moewus) determined that cell death occurred at two peaks of light; 450 and 670 nanometers. These data indicate both chlorophyll and carotenoids, but not riboflavin, are involved in herbicide toxicity. PMID:16664086

  4. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

    SciTech Connect

    Catalanotti, C.; Dubini, A.; Subramanian, V.; Yang, W. Q.; Magneschi, L.; Mus, F.; Seibert, M.; Posewitz, M. C.; Grossman, A. R.

    2012-02-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. 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 it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

  5. Complementation cloning and sequence analysis of the Chlamydomonas reinhardtii hemL gene encoding glutamate-1-semialdehyde aminotransferase

    SciTech Connect

    Matters, G.L.; Beale, S.I. )

    1993-05-01

    Glutamate-1-semialdehyde amino-transferase (GSAT) catalyzes formation of the tetrapyrrole precursor, [delta]-aminolevulinic acid. GSAT is encoded by the hemL gene. A Chlamydomonas reinhardtii hemL cDNA was selected from a vegetative stage expression library by complementation of Escherichia coli hemL mutant GE 1377. In vitro GSAT activity was ten-fold higher in an extract of the complemented hemL cells than in an extract of uncomplemented mutant cells. The complementing cDNA is 2010 bp long and includes 591 bp of 3' noncoding DNA and an estimated 27 bp of 5' noncoding DNA. The coding region includes the sequence for a putative 30-amino acid chloroplast transit peptide and a 433-amino acid mature protein. The mature protein deduced from the Chlamydomonas cDNA sequence has a molecular weight of 45,880, compared to the value of 43,000 reported for purified Chlamydomonas GSAT (d. Jahn et al., 1991, J. Biol. Chem. 266:161-167). The deduced peptide is 74% identical to Synechococcus GSAT, 70% identical to barley GSAT and 66% identical to tobacco GSAT. The putative pyridoxal binding region has the sequence TTMGKVIGG, which differs somewhat from those reported for other aminotransferases. The deduced putative chloroplast transit peptide has recognizable similarity to barley GSAT transit peptide. Southern analysis of genomic DNA from Chlamydomonas strain CC124, using the cDNA as a probe, indicates that GSAT is probably encoded by a single gene.

  6. The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes

    PubMed Central

    Liu, Yanjie; Tewari, Rita; Ning, Jue; Blagborough, Andrew M.; Garbom, Sara; Pei, Jimin; Grishin, Nick V.; Steele, Robert E.; Sinden, Robert E.; Snell, William J.; Billker, Oliver

    2008-01-01

    The cellular and molecular mechanisms that underlie species-specific membrane fusion between male and female gametes remain largely unknown. Here, by use of gene discovery methods in the green alga Chlamydomonas, gene disruption in the rodent malaria parasite Plasmodium berghei, and distinctive features of fertilization in both organisms, we report discovery of a mechanism that accounts for a conserved protein required for gamete fusion. A screen for fusion mutants in Chlamydomonas identified a homolog of HAP2, an Arabidopsis sterility gene. Moreover, HAP2 disruption in Plasmodium blocked fertilization and thereby mosquito transmission of malaria. HAP2 localizes at the fusion site of Chlamydomonas minus gametes, yet Chlamydomonas minus and Plasmodium hap2 male gametes retain the ability, using other, species-limited proteins, to form tight prefusion membrane attachments with their respective gamete partners. Membrane dye experiments show that HAP2 is essential for membrane merger. Thus, in two distantly related eukaryotes, species-limited proteins govern access to a conserved protein essential for membrane fusion. PMID:18367645

  7. Overexpression of Ferredoxin, PETF, Enhances Tolerance to Heat Stress in Chlamydomonas reinhardtii

    PubMed Central

    Lin, Yi-Hsien; Pan, Kui-You; Hung, Ching-Hui; Huang, Hsiang-En; Chen, Ching-Lian; Feng, Teng-Yung; Huang, Li-Fen

    2013-01-01

    Reactive oxygen species (ROS) produced by plants in adverse environments can cause damage to organelles and trigger cell death. Removal of excess ROS can be achieved through the ascorbate scavenger pathway to prevent plant cell death. The amount of this scavenger can be regulated by ferredoxin (FDX). Chloroplastic FDXs are electron transfer proteins that perform in distributing photosynthetic reducing power. In this study, we demonstrate that overexpression of the endogenous photosynthetic FDX gene, PETF, in Chlamydomonas reinhardtii could raise the level of reduced ascorbate and diminish H2O2 levels under normal growth conditions. Furthermore, the overexpressing PETF transgenic Chlamydomonas lines produced low levels of H2O2 and exhibited protective effects that were observed through decreased chlorophyll degradation and increased cell survival under heat-stress conditions. The findings of this study suggest that overexpression of PETF can increase the efficiency of ROS scavenging in chloroplasts to confer heat tolerance. The roles of PETF in the downregulation of the ROS level offer a method for potentially improving the tolerance of crops against heat stress. PMID:24141188

  8. Experimental Genome-Wide Determination of RNA Polyadenylation in Chlamydomonas reinhardtii

    PubMed Central

    Bell, Stephen A.; Shen, Chi; Brown, Alishea; Hunt, Arthur G.

    2016-01-01

    The polyadenylation of RNA is a near-universal feature of RNA metabolism in eukaryotes. This process has been studied in the model alga Chlamydomonas reinhardtii using low-throughput (gene-by-gene) and high-throughput (transcriptome sequencing) approaches that recovered poly(A)-containing sequence tags which revealed interesting features of this critical process in Chlamydomonas. In this study, RNA polyadenylation has been studied using the so-called Poly(A) Tag Sequencing (PAT-Seq) approach. Specifically, PAT-Seq was used to study poly(A) site choice in cultures grown in four different media types—Tris-Phosphate (TP), Tris-Phosphate-Acetate (TAP), High-Salt (HS), and High-Salt-Acetate (HAS). The results indicate that: 1. As reported before, the motif UGUAA is the primary, and perhaps sole, cis-element that guides mRNA polyadenylation in the nucleus; 2. The scope of alternative polyadenylation events with the potential to change the coding sequences of mRNAs is limited; 3. Changes in poly(A) site choice in cultures grown in the different media types are very few in number and do not affect protein-coding potential; 4. Organellar polyadenylation is considerable and affects primarily ribosomal RNAs in the chloroplast and mitochondria; and 5. Organellar RNA polyadenylation is a dynamic process that is affected by the different media types used for cell growth. PMID:26730730

  9. A Gamete-specific, Sex-limited Homeodomain Protein in Chlamydomonas

    PubMed Central

    Kurvari, Venkatesh; Grishin, Nick V.; Snell, William J.

    1998-01-01

    During fertilization in Chlamydomonas, gametes of opposite mating types interact with each other through sex-specific adhesion molecules on their flagellar surfaces. Flagellar adhesion brings the cell bodies of the gametes into close contact and initiates a signal transduction pathway in preparation for cell–cell fusion. We have identified a cDNA, gsp1, whose transcript levels are upregulated during flagellar adhesion. The GSP1 polypeptide is a novel, gamete-specific homeodomain protein, the first to be identified in an alga. Its homeodomain shows significant identity with several higher plant homeodomain proteins. Although encoded by a single copy gene present in cells of both mating types, immunoblot analysis showed that GSP1 was expressed in mating type (mt)+ gametes, but was not detectable in mt− gametes or in vegetative cells of either mating type. Moreover, GSP1 appeared late during gametogenesis, suggesting that it may function during adhesion with mt− gametes or after zygote formation. GSP1 is expressed in imp11, mt− mutant gametes, which have a lesion in the mid gene involved in sex determination and exhibit many phenotypic characteristics of mt+ gametes. Thus, gsp1 is negatively regulated by mid and is the first molecule to be identified in Chlamydomonas that shows sex-limited expression. PMID:9864368

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

    PubMed

    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. PMID:25217463

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

    PubMed

    Kim, Hanul; Jang, Sunghoon; Kim, Sangwoo; Yamaoka, Yasuyo; Hong, Daewoong; Song, Won-Yong; Nishida, Ikuo; Li-Beisson, Yonghua; Lee, Youngsook

    2015-01-01

    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 fourfold 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 twofold 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. PMID:25759683

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

    PubMed Central

    Kim, Hanul; Jang, Sunghoon; Kim, Sangwoo; Yamaoka, Yasuyo; Hong, Daewoong; Song, Won-Yong; Nishida, Ikuo; Li-Beisson, Yonghua; Lee, Youngsook

    2015-01-01

    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 fourfold 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 twofold 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. PMID:25759683

  13. Spontaneous Dominant Mutations in Chlamydomonas Highlight Ongoing Evolution by Gene Diversification

    PubMed Central

    Boulouis, Alix; Drapier, Dominique; Razafimanantsoa, Hélène; Wostrikoff, Katia; Tourasse, Nicolas J.; Pascal, Kevin; Girard-Bascou, Jacqueline; Vallon, Olivier; Wollman, Francis-André; Choquet, Yves

    2015-01-01

    We characterized two spontaneous and dominant nuclear mutations in the unicellular alga Chlamydomonas reinhardtii, ncc1 and ncc2 (for nuclear control of chloroplast gene expression), which affect two octotricopeptide repeat (OPR) proteins encoded in a cluster of paralogous genes on chromosome 15. Both mutations cause a single amino acid substitution in one OPR repeat. As a result, the mutated NCC1 and NCC2 proteins now recognize new targets that we identified in the coding sequences of the chloroplast atpA and petA genes, respectively. Interaction of the mutated proteins with these targets leads to transcript degradation; however, in contrast to the ncc1 mutation, the ncc2 mutation requires on-going translation to promote the decay of the petA mRNA. Thus, these mutants reveal a mechanism by which nuclear factors act on chloroplast mRNAs in Chlamydomonas. They illustrate how diversifying selection can allow cells to adapt the nuclear control of organelle gene expression to environmental changes. We discuss these data in the wider context of the evolution of regulation by helical repeat proteins. PMID:25804537

  14. The Contractile Vacuole as a Key Regulator of Cellular Water Flow in Chlamydomonas reinhardtii

    PubMed Central

    Komsic-Buchmann, Karin; Wöstehoff, Luisa

    2014-01-01

    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. PMID:25217463

  15. CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii

    PubMed Central

    Shin, Sung-Eun; Lim, Jong-Min; Koh, Hyun Gi; Kim, Eun Kyung; Kang, Nam Kyu; Jeon, Seungjib; Kwon, Sohee; Shin, Won-Sub; Lee, Bongsoo; Hwangbo, Kwon; Kim, Jungeun; Ye, Sung Hyeok; Yun, Jae-Young; Seo, Hogyun; Oh, Hee-Mock; Kim, Kyung-Jin; Kim, Jin-Soo; Jeong, Won-Joong; Chang, Yong Keun; Jeong, Byeong-ryool

    2016-01-01

    Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including “safe harboring” techniques shown in other organisms. PMID:27291619

  16. The Chlamydomonas mutant pf27 reveals novel features of ciliary radial spoke assembly

    PubMed Central

    Alford, Lea M.; Mattheyses, Alexa L.; Hunter, Emily L.; Lin, Huawen; Dutcher, Susan K.; Sale, Winfield S.

    2014-01-01

    To address the mechanisms of ciliary radial spoke assembly, we took advantage of the Chlamydomonas pf27 mutant. The radial spokes that assemble in pf27 are localized to the proximal quarter of the axoneme, but otherwise are fully assembled into 20S radial spoke complexes competent to bind spokeless axonemes in vitro. Thus, pf27 is not defective in radial spoke assembly or docking to the axoneme. Rather, our results suggest that pf27 is defective in the transport of spoke complexes. During ciliary regeneration in pf27, radial spoke assembly occurs asynchronously from other axonemal components. In contrast, during ciliary regeneration in wild-type Chlamydomonas, radial spokes and other axonemal components assemble concurrently as the axoneme grows. Complementation in temporary dikaryons between wild-type and pf27 reveals rescue of radial spoke assembly that begins at the distal tip, allowing further assembly to proceed from tip to base of the axoneme. Notably, rescued assembly of radial spokes occurred independently of the established proximal radial spokes in pf27 axonemes in dikaryons. These results reveal that 20S radial spokes can assemble proximally in the pf27 cilium but as the cilium lengthens, spoke assembly requires transport. We postulate that PF27 encodes an adaptor or modifier protein required for radial spoke – IFT interaction. PMID:24124175

  17. CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii.

    PubMed

    Shin, Sung-Eun; Lim, Jong-Min; Koh, Hyun Gi; Kim, Eun Kyung; Kang, Nam Kyu; Jeon, Seungjib; Kwon, Sohee; Shin, Won-Sub; Lee, Bongsoo; Hwangbo, Kwon; Kim, Jungeun; Ye, Sung Hyeok; Yun, Jae-Young; Seo, Hogyun; Oh, Hee-Mock; Kim, Kyung-Jin; Kim, Jin-Soo; Jeong, Won-Joong; Chang, Yong Keun; Jeong, Byeong-Ryool

    2016-01-01

    Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including "safe harboring" techniques shown in other organisms. PMID:27291619

  18. Overexpression of ferredoxin, PETF, enhances tolerance to heat stress in Chlamydomonas reinhardtii.

    PubMed

    Lin, Yi-Hsien; Pan, Kui-You; Hung, Ching-Hui; Huang, Hsiang-En; Chen, Ching-Lian; Feng, Teng-Yung; Huang, Li-Fen

    2013-01-01

    Reactive oxygen species (ROS) produced by plants in adverse environments can cause damage to organelles and trigger cell death. Removal of excess ROS can be achieved through the ascorbate scavenger pathway to prevent plant cell death. The amount of this scavenger can be regulated by ferredoxin (FDX). Chloroplastic FDXs are electron transfer proteins that perform in distributing photosynthetic reducing power. In this study, we demonstrate that overexpression of the endogenous photosynthetic FDX gene, PETF, in Chlamydomonas reinhardtii could raise the level of reduced ascorbate and diminish H2O2 levels under normal growth conditions. Furthermore, the overexpressing PETF transgenic Chlamydomonas lines produced low levels of H2O2 and exhibited protective effects that were observed through decreased chlorophyll degradation and increased cell survival under heat-stress conditions. The findings of this study suggest that overexpression of PETF can increase the efficiency of ROS scavenging in chloroplasts to confer heat tolerance. The roles of PETF in the downregulation of the ROS level offer a method for potentially improving the tolerance of crops against heat stress. PMID:24141188

  19. An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii.

    PubMed

    Li, Xiaobo; Zhang, Ru; Patena, Weronika; Gang, Spencer S; Blum, Sean R; Ivanova, Nina; Yue, Rebecca; Robertson, Jacob M; Lefebvre, Paul A; Fitz-Gibbon, Sorel T; Grossman, Arthur R; Jonikas, Martin C

    2016-02-01

    The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids. PMID:26764374

  20. High-Level Accumulation of Triacylglycerol and Starch in Photoautotrophically Grown Chlamydomonas debaryana NIES-2212.

    PubMed

    Toyoshima, Masakazu; Sato, Naoki

    2015-12-01

    Microalgae have the potential to produce triacylglycerol (TAG) and starch, which provide alternative sources of biofuel. A problem in using Chlamydomonas reinhardtii as a model for TAG production has been that this alga lacks phosphatidylcholine (PC), which is thought to be important for TAG synthesis in plants. We found that C. debaryana is one of the rare species of Chlamydomonas having PC. Here we show that this strain, grown under complete photoautotrophic conditions, accumulated TAG and starch up to 20 and 250 pg per cell, respectively, during the stationary phase without nutrient deprivation. Addition of nutrients in this state did not cause loss of TAG, which was found in dilution with fresh medium. The photosynthetically produced TAG contained a high level of monounsaturated fatty acids, which is a preferred property as a material for biodiesel. The oil bodies were present in the cytoplasm, either between the cytoplasmic membrane and the chloroplast or between the chloroplast and the nucleus, whereas the starch granules were present within the chloroplast. Oil bodies were also deposited as a broad layer in the peripheral space of the cytoplasm outside the chloroplast, and might be easily released from the cells by genetic, chemical or mechanical manipulation. These results suggest that C. debaryana is a promising seed organism for developing a good biofuel producer. PMID:26542110

  1. Atomic Resolution Modeling of the Ferredoxin:[FeFe] Hydrogenase Complex from Chlamydomonas reinhardtii

    PubMed Central

    Chang, Christopher H.; King, Paul W.; Ghirardi, Maria L.; Kim, Kwiseon

    2007-01-01

    The [FeFe] hydrogenases HydA1 and HydA2 in the green alga Chlamydomonas reinhardtii catalyze the final reaction in a remarkable metabolic pathway allowing this photosynthetic organism to produce H2 from water in the chloroplast. A [2Fe-2S] ferredoxin is a critical branch point in electron flow from Photosystem I toward a variety of metabolic fates, including proton reduction by hydrogenases. To better understand the binding determinants involved in ferredoxin:hydrogenase interactions, we have modeled Chlamydomonas PetF1 and HydA2 based on amino-acid sequence homology, and produced two promising electron-transfer model complexes by computational docking. To characterize these models, quantitative free energy calculations at atomic resolution were carried out, and detailed analysis of the interprotein interactions undertaken. The protein complex model we propose for ferredoxin:HydA2 interaction is energetically favored over the alternative candidate by 20 kcal/mol. This proposed model of the electron-transfer complex between PetF1 and HydA2 permits a more detailed view of the molecular events leading up to H2 evolution, and suggests potential mutagenic strategies to modulate electron flow to HydA2. PMID:17660315

  2. Photoinduced electric currents in carotenoid-deficient Chlamydomonas mutants reconstituted with retinal and its analogs.

    PubMed Central

    Sineshchekov, O A; Govorunova, E G; Dér, A; Keszthelyi, L; Nultsch, W

    1994-01-01

    Reconstitution of the photoelectric responses involved in photosensory transduction in "blind" cells of Chlamydomonas reinhardtii carotenoid-deficient mutants was studied by means of a recently developed population method. Both the photoreceptor current and the regenerative response can be restored by addition of all-trans-retinal, 9-demethyl-retinal, or dimethyl-octatrienal, while the retinal analogs prevented from 13-cis/trans isomerization, 13-demethyl-retinal and citral, are not effective. Fluence dependence, spectral sensitivity, and effect of hydroxylamine treatment on retinal-induced photoelectric responses are similar to those found earlier in green strains of Chlamydomonas, although an alternative mechanism of antenna directivity in white cells of reconstituted "blind" mutants (likely based on the focusing effect of the transparent cell bodies) leads to the reversed sign of phototaxis in mutant cells under the same conditions. The results obtained indicate that both photoreceptor current and regenerative response are initiated by the same or similar rhodopsins with arhaebacterial-like chromophore(s) and prove directly the earlier suggested identity of the photoreceptor pigment(s) involved in photomotile and photoelectric responses in flagellated algae. PMID:8075341

  3. Subunit Interactions and Organization of the Chlamydomonas reinhardtii Intraflagellar Transport Complex A Proteins*

    PubMed Central

    Behal, Robert H.; Miller, Mark S.; Qin, Hongmin; Lucker, Ben F.; Jones, Alexis; Cole, Douglas G.

    2012-01-01

    Chlamydomonas reinhardtii intraflagellar transport (IFT) particles can be biochemically resolved into two smaller assemblies, complexes A and B, that contain up to six and 15 protein subunits, respectively. We provide here the proteomic and immunological analyses that verify the identity of all six Chlamydomonas A proteins. Using sucrose density gradient centrifugation and antibody pulldowns, we show that all six A subunits are associated in a 16 S complex in both the cell bodies and flagella. A significant fraction of the cell body IFT43, however, exhibits a much slower sedimentation of ∼2 S and is not associated with the IFT A complex. To identify interactions between the six A proteins, we combined exhaustive yeast-based two-hybrid analysis, heterologous recombinant protein expression in Escherichia coli, and analysis of the newly identified complex A mutants, ift121 and ift122. We show that IFT121 and IFT43 interact directly and provide evidence for additional interactions between IFT121 and IFT139, IFT121 and IFT122, IFT140 and IFT122, and IFT140 and IFT144. The mutant analysis further allows us to propose that a subset of complex A proteins, IFT144/140/122, can form a stable 12 S subcomplex that we refer to as the IFT A core. Based on these results, we propose a model for the spatial arrangement of the six IFT A components. PMID:22170070

  4. VMP1-deficient Chlamydomonas exhibits severely aberrant cell morphology and disrupted cytokinesis

    PubMed Central

    2014-01-01

    Background The versatile Vacuole Membrane Protein 1 (VMP1) has been previously investigated in six species. It has been shown to be essential in macroautophagy, where it takes part in autophagy initiation. In addition, VMP1 has been implicated in organellar biogenesis; endo-, exo- and phagocytosis, and protein secretion; apoptosis; and cell adhesion. These roles underly its proven involvement in pancreatitis, diabetes and cancer in humans. Results In this study we analyzed a VMP1 homologue from the green alga Chlamydomonas reinhardtii. CrVMP1 knockdown lines showed severe phenotypes, mainly affecting cell division as well as the morphology of cells and organelles. We also provide several pieces of evidence for its involvement in macroautophagy. Conclusion Our study adds a novel role to VMP1's repertoire, namely the regulation of cytokinesis. Though the directness of the observed effects and the mechanisms underlying them remain to be defined, the protein's involvement in macroautophagy in Chlamydomonas, as found by us, suggests that CrVMP1 shares molecular characteristics with its animal and protist counterparts. PMID:24885763

  5. Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

    PubMed Central

    Newman, S. M.; Boynton, J. E.; Gillham, N. W.; Randolph-Anderson, B. L.; Johnson, A. M.; Harris, E. H.

    1990-01-01

    Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas. PMID:1981764

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

    PubMed

    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. PMID:15802504

  7. Spontaneous Mutations in the Ammonium Transport Gene AMT4 of Chlamydomonas reinhardtii

    PubMed Central

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

    2005-01-01

    Evidence in several microorganisms indicates that Amt proteins are gas channels for NH3 and CH3NH2, 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 [14C]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 [14C]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 NH3 and CO2, respectively. PMID:15802504

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

    PubMed

    Ghysels, Bart; Godaux, Damien; Matagne, René F; Cardol, Pierre; Franck, Fabrice

    2013-01-01

    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. PMID:23717558

  9. 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

    PubMed Central

    ODOM, OBED W.; SHENKENBERG, DAVID L.; GARCIA, JOSHUA A.; HERRIN, DAVID L.

    2004-01-01

    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 Mg2+ (>12 mM) and NH4+. 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°C, but with a broad shoulder on the low side of the peak; splicing was nearly absent at 50°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. PMID:15208445

  10. Pseudomonas kuykendallii sp. nov.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This is a submission to the list of microorganisms with standing in nomenclature maintained by the International Journal of Systematic and Evolutionary Microbiology. We wish to have Pseudomonas kuykendallii sp. nov. added to the list as a valid species belonging to the genus Pseudomonas. Three str...

  11. Dispensability of a sulfolipid for photoautotrophic cell growth and photosynthesis in a marine cyanobacterium, Synechococcus sp. PCC 7002.

    PubMed

    Sato, Norihiro; Kamimura, Ryohei; Tsuzuki, Mikio

    2016-09-01

    Sulfoquinovosyl diacylglycerol, which mainly comprises thylakoid membranes in oxygenic photosynthetic organisms, plays species-dependent roles in freshwater microbes. In this study, a sulfoquinovosyl-diacylglycerol deficient mutant was generated in a cyanobacterium, Synechococcus sp. PCC 7002, for the first time among marine microbes to gain more insight into its physiological significance. The mutation had little deleterious impact on photoautotrophic cell growth, and functional and structural properties of the photosystem II complex. These findings were similar to previous observations for a freshwater cyanobacterium, Synechococcus elongatus PCC 7942, but were distinct from those for another freshwater cyanobacterium, Synechocystis sp. PCC 6803, and a green alga, Chlamydomonas reinhardtii, both of which require sulfoquinovosyl diacylglycerol for cell growth and/or photosystem II. Therefore, the functionality of PSII to dispense with sulfoquinovosyl diacylglycerol in Synechococcus sp. PCC 7002, similar to that in Synechococcus elongatus PCC 7942, seemed to have been excluded from the evolution of the PSII complex from cyanobacteria to green algal chloroplasts. Meanwhile, sulfoquinovosyl diacylglycerol was found to contribute to photoheterotrophic growth of Synechococcus sp. PCC 7002, which revealed a novel species-dependent strategy for utilizing SQDG in physiological processes. PMID:27372425

  12. Comparison of denitrification between Paracoccus sp. and Diaphorobacter sp.

    PubMed

    Chakravarthy, Srinandan S; Pande, Samay; Kapoor, Ashish; Nerurkar, Anuradha S

    2011-09-01

    Denitrification was compared between Paracoccus sp. and Diaphorobacter sp. in this study, both of which were isolated from activated sludge of a denitrifying reactor. Denitrification of both isolates showed contrasting patterns, where Diaphorobacter sp. showed accumulation of nitrite in the medium while Paracoccus sp. showed no accumulation. The nitrate reduction rate was 1.5 times more than the nitrite reduction in Diaphorobacter sp., as analyzed by the resting state denitrification kinetics. Increasing the nitrate concentration in the medium increased the nitrite accumulation in Diaphorobacter sp., but not in Paracoccus sp., indicating a branched electron transfer during denitrification. Diaphorobacter sp. was unable to denitrify efficiently at high nitrate concentrations from 1 M, but Paracoccus sp. could denitrify even up to 2 M nitrate. Paracoccus sp. was found to be an efficient denitrifier with insignificant amounts of nitrite accumulation, and it could also denitrify high amounts of nitrate up to 2 M. Efficient denitrification without accumulation of intermediates like nitrite is desirable in the removal of high nitrates from wastewaters. Paracoccus sp. is shown to suffice this demand and could be a potential organism to remove high nitrates effectively. PMID:21509603

  13. Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas.

    PubMed Central

    Cerutti, H; Johnson, A M; Gillham, N W; Boynton, J E

    1997-01-01

    The unstable expression of introduced genes poses a serious problem for the application of transgenic technology in plants. In transformants of the unicellular green alga Chlamydomonas reinhardtii, expression of a eubacterial aadA gene, conferring spectinomycin resistance, is transcriptionally suppressed by a reversible epigenetic mechanism(s). Variations in the size and frequency of colonies surviving on different concentrations of spectinomycin as well as the levels of transcriptional activity of the introduced transgene(s) suggest the existence of intermediate expression states in genetically identical cells. Gene silencing does not correlate with methylation of the integrated DNA and does not involve large alterations in its chromatin structure, as revealed by digestion with restriction endonucleases and DNase I. Transgene repression is enhanced by lower temperatures, similar to position effect variegation in Drosophila. By analogy to epigenetic phenomena in several eukaryotes, our results suggest a possible role for (hetero)chromatic chromosomal domains in transcriptional inactivation. PMID:9212467

  14. Induction of triacylglycerol production in Chlamydomonas reinhardtii: comparative analysis of different element regimes.

    PubMed

    Çakmak, Zeynep E; Ölmez, Tolga T; Çakmak, Turgay; Menemen, Yusuf; Tekinay, Turgay

    2014-03-01

    In this study, impacts of different element absence (nitrogen, sulfur, phosphorus and magnesium) and supplementation (nitrogen and zinc) on element uptake and triacylglycerol production was followed in wild type Chlamydomonas reinhardtii CC-124 strain. Macro- and microelement composition of C. reinhardtii greatly differed under element regimes studied. In particular, heavy metal quotas of the microalgae increased strikingly under zinc supplementation. Growth was suppressed, cell biovolume, carbohydrate, total neutral lipid and triacylglycerol levels increased when microalgae were incubated under these element regimes. Most of the intracellular space was occupied by lipid bodies under all nutrient starvations, as observed by confocal microscopy and transmission electron micrographs. Results suggest that sulfur, magnesium and phosphorus deprivations are superior to nitrogen deprivation for the induction triacylglycerol production in C. reinhardtii. On the other hand, FAME profiles of the nitrogen, sulfur and phosphorus deprived cells were found to meet the requirements of international standards for biodiesel. PMID:24472680

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

    PubMed

    Sartori, Pablo; Geyer, Veikko F; Scholich, Andre; Jülicher, Frank; Howard, Jonathon

    2016-01-01

    Cilia and flagella are model systems for studying how mechanical forces control morphology. The periodic bending motion of cilia and flagella is thought to arise from mechanical feedback: dynein motors generate sliding forces that bend the flagellum, and bending leads to deformations and stresses, which feed back and regulate the motors. Three alternative feedback mechanisms have been proposed: regulation by the sliding forces, regulation by the curvature of the flagellum, and regulation by the normal forces that deform the cross-section of the flagellum. In this work, we combined theoretical and experimental approaches to show that the curvature control mechanism is the one that accords best with the bending waveforms of Chlamydomonas flagella. We make the surprising prediction that the motors respond to the time derivative of curvature, rather than curvature itself, hinting at an adaptation mechanism controlling the flagellar beat. PMID:27166516

  16. Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism

    PubMed Central

    Wittkopp, Tyler M.; Warakanont, Jaruswan; Dubini, Alexandra; Catalanotti, Claudia; Kim, Rick G.; Nowack, Eva C. M.; Mackinder, Luke C. M.; Aksoy, Munevver; Page, Mark Dudley; D’Adamo, Sarah; Saroussi, Shai; Heinnickel, Mark; Johnson, Xenie; Richaud, Pierre; Alric, Jean; Boehm, Marko; Jonikas, Martin C.; Benning, Christoph; Merchant, Sabeeha S.; Posewitz, Matthew C.; Grossman, Arthur R.

    2015-01-01

    Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions. PMID:26627249

  17. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

    NASA Astrophysics Data System (ADS)

    Wu, Jinxia; Hu, Zhangli; Wang, Chaogang; Li, Shuangfei; Lei, Anping

    2008-08-01

    To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

  18. Measurement of swimming force generation during flagella regeneration in Chlamydomonas reinhardtii

    NASA Astrophysics Data System (ADS)

    Yukich, John N.; Shaban, Mona; Clodfelter, Catherine; Bernd, Karen

    2007-11-01

    The green alga Chlamydomonas reinhardtii has been at the forefront of many studies investigating the establishment and function of flagella in facilitating cellular motility. Previously we reported an intriguing pattern during flagella regeneration in which increases in force do not always correspond with increase in flagella length. That work made direct measurement of maximum flagellar swimming force by measuring the cell's ability to escape from an optical trap (optical tweezers). Here, we report on optimization and automation of the force measurement using power spectral density calibration of the trap and distance of periodic displacement from the trap center. This process yields an average value for the swimming force. The intriguing pattern described for maximum swimming force is also evident in the average swimming force data, suggesting that the phenomenon reflects a change in flagella functionality during regeneration.

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

    PubMed

    Nonaka, Yuki; Kikuchi, Kenji; Numayama-Tsuruta, Keiko; Kage, Azusa; Ueno, Hironori; Ishikawa, Takuji

    2016-01-01

    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. PMID:26787679

  20. Quantification of phytochelatins in Chlamydomonas reinhardtii using ferrocene-based derivatization.

    PubMed

    Bräutigam, Anja; Bomke, Susanne; Pfeifer, Thorben; Karst, Uwe; Krauss, Gerd-Joachim; Wesenberg, Dirk

    2010-08-01

    A method for the identification and quantification of canonic and isoforms of phytochelatins (PCs) from Chlamydomonas reinhardtii was developed. After disulfide reduction with tris(2-carboxyethyl)phosphine (TCEP) PCs were derivatized with ferrocenecarboxylic acid (2-maleimidoyl)ethylamide (FMEA) in order to avoid oxidation of the free thiol functions during analysis. Liquid chromatography (LC) coupled to electrospray mass spectrometry (ESI-MS) and inductively coupled plasma-mass spectrometry (ICP-MS) was used for rapid and quantitative analysis of the precolumn derivatized PCs. PC(2-4), CysGSH, CysPC(2-4), CysPC(2)desGly, CysPC(2)Glu and CysPC(2)Ala were determined in the algal samples depending on the exposure of the cells to cadmium ions. PMID:21072341

  1. Antagonistic and synergistic effects of light irradiation on the effects of copper on Chlamydomonas reinhardtii.

    PubMed

    Cheloni, Giulia; Cosio, Claudia; Slaveykova, Vera I

    2014-10-01

    The present study showed the important role of light intensity and spectral composition on Cu uptake and effects on green alga Chlamydomonas reinhardtii. High-intenisty light (HL) increased cellular Cu concentrations, but mitigated the Cu-induced decrease in chlorophyll fluorescence, oxidative stress and lipid peroxidation at high Cu concentrations, indicating that Cu and HL interact in an antagonistic manner. HL up-regulated the transcription of genes involved in the antioxidant response in C. reinhardtii and thus reduced the oxidative stress upon exposure to Cu and HL. Combined exposure to Cu and UVBR resulted in an increase of cellular Cu contents and caused severe oxidative damage to the cells. The observed effects were higher than the sum of the effects corresponding to exposure to UVBR or Cu alone suggesting a synergistic interaction. PMID:25072593

  2. Inhibition of Plastocyanin to P700+ Electron Transfer in Chlamydomonas reinhardtii by Hyperosmotic Stress1

    PubMed Central

    Cruz, Jeffrey A.; Salbilla, Brian A.; Kanazawa, Atsuko; Kramer, David M.

    2001-01-01

    Oxygen electrode and fluorescence studies demonstrate that linear electron transport in the freshwater alga Chlamydomonas reinhardtii can be completely abolished by abrupt hyperosmotic shock. We show that the most likely primary site of inhibition of electron transfer by hyperosmotic shock is a blockage of electron transfer between plastocyanin (PC) or cytochrome c6 and P700. The effects on this reaction were reversible upon dilution of the osmolytes and the stability of plastocyanin or photosystem (PS) I was unaffected. Electron micrographs of osmotically shocked cells showed a significant decrease in the thylakoid lumen volume. Comparison of estimated lumenal width with the x-ray structures of plastocyanin and PS I suggest that lumenal space contracts during HOS so as to hinder the movement of docking to PS I of plastocyanin or cytochrome c6. PMID:11706196

  3. Some observations on the carbon dioxide burst in chlorella and chlamydomonas.

    PubMed

    Bunt, J S

    1970-02-01

    In the course of mass spectrometer studies with the algae Chlorella and Chlamydomonas, data were obtained which indicate that the CO(2) burst and gulp are sensitive to oxygen. Furthermore, the CO(2) burst was found to be strongly suppressed when wave lengths shorter than 460 nanometers were blocked at intensities adequate to saturate photosynthesis. Under appropriate conditions at 30 degrees , the CO(2) burst was interrupted by a brief CO(2) gulp and the post illumination gulp by a brief burst of CO(2). The post illumination gulp of CO(2) could be induced during illumination by interposition of a filter blocking wave lengths shorter than 460 nanometers. These data are discussed in relation to earlier reports of the phenomenon and briefly as they affect the detection of photorespiration. PMID:16657291

  4. Membrane-Associated Polypeptides Induced in Chlamydomonas by Limiting CO(2) Concentrations.

    PubMed

    Spalding, M H; Jeffrey, M

    1989-01-01

    Chlamydomonas reinhardtii and other unicellular green algae have a high apparent affinity for CO(2), little O(2) inhibition of photosynthesis, and reduced photorespiration. These characteristics result from operation of a CO(2)-concentrating system. The CO(2)-concentrating system involves active inorganic carbon transport and is under environmental control. Cells grown at limiting CO(2) concentrations have inorganic carbon transport activity, but cells grown at 5% CO(2) do not. Four membrane-associated polypeptides (M(r) 19, 21, 35, and 36 kilodaltons) have been identified which either appear or increase in abundance during adaptation to limiting CO(2) concentrations. The appearance of two of the polypeptides occurs over roughly the same time course as the appearance of the CO(2)-concentrating system activity in response to CO(2) limitation. PMID:16666503

  5. Identification and characterization of a cis-regulatory element for zygotic gene expression in Chlamydomonas reinhardtii

    DOE PAGESBeta

    Hamaji, Takashi; Lopez, David; Pellegrini, Matteo; Umen, James

    2016-03-26

    Upon fertilization Chlamydomonas reinhardtii zygotes undergo a program of differentiation into a diploid zygospore that is accompanied by transcription of hundreds of zygote-specific genes. We identified a distinct sequence motif we term a zygotic response element (ZYRE) that is highly enriched in promoter regions of C. reinhardtii early zygotic genes. A luciferase reporter assay was used to show that native ZYRE motifs within the promoter of zygotic gene ZYS3 or intron of zygotic gene DMT4 are necessary for zygotic induction. A synthetic luciferase reporter with a minimal promoter was used to show that ZYRE motifs introduced upstream are sufficient tomore » confer zygotic upregulation, and that ZYRE-controlled zygotic transcription is dependent on the homeodomain transcription factor GSP1. Furthermore, we predict that ZYRE motifs will correspond to binding sites for the homeodomain proteins GSP1-GSM1 that heterodimerize and activate zygotic gene expression in early zygotes.« less

  6. Experimental evolution of an alternating uni- and multicellular life cycle in Chlamydomonas reinhardtii

    PubMed Central

    Ratcliff, William C.; Herron, Matthew D.; Howell, Kathryn; Pentz, Jennifer T.; Rosenzweig, Frank; Travisano, Michael

    2013-01-01

    The transition to multicellularity enabled the evolution of large, complex organisms, but early steps in this transition remain poorly understood. Here we show that multicellular complexity, including development from a single cell, can evolve rapidly in a unicellular organism that has never had a multicellular ancestor. We subject the alga Chlamydomonas reinhardtii to conditions that favour multicellularity, resulting in the evolution of a multicellular life cycle in which clusters reproduce via motile unicellular propagules. While a single-cell genetic bottleneck during ontogeny is widely regarded as an adaptation to limit among-cell conflict, its appearance very early in this transition suggests that it did not evolve for this purpose. Instead, we find that unicellular propagules are adaptive even in the absence of intercellular conflict, maximizing cluster-level fecundity. These results demonstrate that the unicellular bottleneck, a trait essential for evolving multicellular complexity, can arise rapidly via co-option of the ancestral unicellular form. PMID:24193369

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

    SciTech Connect

    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.

  8. Membrane-associated polypeptides induced in Chlamydomonas by limiting CO sub 2 concentrations

    SciTech Connect

    Spalding, M.H.; Jeffrey, M. )

    1989-01-01

    Chlamydomonas reinhardtii and other unicellular green algae have a high apparent affinity for CO{sub 2}, little O{sub 2} inhibition of photosynthesis, and reduced photorespiration. These characteristics result from operation of a CO{sub 2}-concentrating system. The CO{sub 2}-concentrating system involves active inorganic carbon transport and is under environmental control. Cells grown at limiting CO{sub 2} concentrations have inorganic carbon transport activity, but cells grown at 5% CO{sub 2} do not. Four membrane-associated polypeptides (M{sub r}, 19, 21, 35, and 36 kilodaltons) have been identified which either appear or increase in abundance during adaptation to limiting CO{sub 2} concentrations. The appearance of two of the polypeptides occurs over roughly the same time course as the appearance of the CO{sub 2}-concentrating system activity in response to CO{sub 2} limitation.

  9. Lack of mutagenic activity of crude and refined oils in the unicellular alga Chlamydomonas reinhardtii

    SciTech Connect

    Vandermeulen, J.H.; Lee, R.W.

    1986-02-01

    Over the past several years, an increasing number of studies have presented evidence for the mutagenicity and/or carcinogenic potential of petroleum-derived hydrocarbons. These most usually were obtained with individual hydrocarbons, and using either specialized bacterial strains (e.g. Ames' strains) or mammalian tissue preparations. While providing important insights into mutagenic mechanisms involving xenobiotic compounds, the relevance of these studies to the natural aquatic environment is not always evident. This applies especially to the mutagenic potential of water-soluble fractions of hydrocarbon mixtures, as in whole oils or in complex distillate fractions, and involving typical marine biota. Accordingly, the authors have examined the mutagenic potential of the water-soluble fractions of four oils (two crude oils and two refined oils) using the unicellular haploid alga Chlamydomonas reinhardtii.

  10. Structural Insight into the Complex of Ferredoxin and [FeFe] Hydrogenase from Chlamydomonas reinhardtii.

    PubMed

    Rumpel, Sigrun; Siebel, Judith F; Diallo, Mamou; Farès, Christophe; Reijerse, Edward J; Lubitz, Wolfgang

    2015-07-27

    The transfer of photosynthetic electrons by the ferredoxin PetF to the [FeFe] hydrogenase HydA1 in the microalga Chlamydomonas reinhardtii is a key step in hydrogen production. Electron delivery requires a specific interaction between PetF and HydA1. However, because of the transient nature of the electron-transfer complex, a crystal structure remains elusive. Therefore, we performed protein-protein docking based on new experimental data from a solution NMR spectroscopy investigation of native and gallium-substituted PetF. This provides valuable information about residues crucial for complex formation and electron transfer. The derived complex model might help to pinpoint residue substitution targets for improved hydrogen production. PMID:26010059

  11. An ISFET-algal (Chlamydomonas) hybrid provides a system for eco-toxicological tests.

    PubMed

    Schubnell, D; Lehmann, M; Baumann, W; Rott, F G; Wolf, B; Beck, C F

    1999-05-31

    A cellular sensoring system was designed in which metabolism-dedicated pH-ISFETs and the unicellular green alga Chlamydomonas reinhardtii as a biological component, were combined. The system permits on-line detection of pH changes caused by the metabolic and photosynthetic activities of the cells. Photosynthetic activity results in a basification of the medium caused by uptake of CO2. In darkness, an acidification of the medium, resulting from the production of CO2 by degradation of starch was observed. Both, acidification and basification, are sensitive indicators for the physiological activity of the alga. Experiments using inhibitors of energy metabolism or photosynthesis illustrate the utility of this system for an on-line monitoring of substances of eco-toxicological importance. PMID:10451914

  12. Brownian dynamics and molecular dynamics study of the association between hydrogenase and ferredoxin from Chlamydomonas reinhardtii.

    PubMed

    Long, Hai; Chang, Christopher H; King, Paul W; Ghirardi, Maria L; Kim, Kwiseon

    2008-10-01

    The [FeFe] hydrogenase from the green alga Chlamydomonas reinhardtii can catalyze the reduction of protons to hydrogen gas using electrons supplied from photosystem I and transferred via ferredoxin. To better understand the association of the hydrogenase and the ferredoxin, we have simulated the process over multiple timescales. A Brownian dynamics simulation method gave an initial thorough sampling of the rigid-body translational and rotational phase spaces, and the resulting trajectories were used to compute the occupancy and free-energy landscapes. Several important hydrogenase-ferredoxin encounter complexes were identified from this analysis, which were then individually simulated using atomistic molecular dynamics to provide more details of the hydrogenase and ferredoxin interaction. The ferredoxin appeared to form reasonable complexes with the hydrogenase in multiple orientations, some of which were good candidates for inclusion in a transition state ensemble of configurations for electron transfer. PMID:18621810

  13. Growth of the green algae Chlamydomonas reinhardtii under red and blue lasers

    NASA Astrophysics Data System (ADS)

    Kuwahara, Sara S.; Cuello, Joel L.; Myhre, Graham; Pau, Stanley

    2011-03-01

    Red and blue lasers, holding promise as an electric light source for photosynthetic systems on account of being true monochromatic, high-power, and having high electrical-conversion efficiency, were employed in growing a green alga, Chlamydomonas reinhardtii. The laser treatments tested included: 655-nm Red; 680-nm Red; 655-nm Red+474-nm Blue and 680-nm Red+474-nm Blue. A white cold cathode lamp with spectral output similar to that of white fluorescent lamp served as control. C. reinhardtii successfully grew and divided under the 655 and 680-nm red lasers as well as under the white-light control. Supplementing either red with blue laser, however, resulted in increased algae cell count that significantly exceeded those under both red lasers and the white-light control on average by 241%.

  14. Recombinant Reconstitution and Purification of the IFT-B Core Complex from Chlamydomonas reinhardtii.

    PubMed

    Taschner, Michael; Lorentzen, Esben

    2016-01-01

    Eukaryotic cilia and flagella are assembled and maintained by intraflagellar transport (IFT), the bidirectional transport of proteins between the ciliary base and tip. IFT is mediated by the multi-subunit IFT complex, which simultaneously binds cargo proteins and the ciliary motors. So far 22 subunits of the IFT complex have been identified, but insights into the biochemical architecture and especially the three-dimensional structure of this machinery are only starting to emerge because of difficulties in obtaining homogeneous material suitable for structural analysis. Here, we describe a protocol for the purification and reconstitution of a complex containing nine Chlamydomonas reinhardtii IFT proteins, commonly known as the IFT-B core complex. In our hands, this protocol routinely yields several milligrams of pure complex suitable for structural analysis by X-ray crystallography and single-particle cryo-electron microscopy. PMID:27514916

  15. Identification and Characterization of a cis-Regulatory Element for Zygotic Gene Expression in Chlamydomonas reinhardtii.

    PubMed

    Hamaji, Takashi; Lopez, David; Pellegrini, Matteo; Umen, James

    2016-01-01

    Upon fertilization Chlamydomonas reinhardtii zygotes undergo a program of differentiation into a diploid zygospore that is accompanied by transcription of hundreds of zygote-specific genes. We identified a distinct sequence motif we term a zygotic response element (ZYRE) that is highly enriched in promoter regions of C reinhardtii early zygotic genes. A luciferase reporter assay was used to show that native ZYRE motifs within the promoter of zygotic gene ZYS3 or intron of zygotic gene DMT4 are necessary for zygotic induction. A synthetic luciferase reporter with a minimal promoter was used to show that ZYRE motifs introduced upstream are sufficient to confer zygotic upregulation, and that ZYRE-controlled zygotic transcription is dependent on the homeodomain transcription factor GSP1. We predict that ZYRE motifs will correspond to binding sites for the homeodomain proteins GSP1-GSM1 that heterodimerize and activate zygotic gene expression in early zygotes. PMID:27172209

  16. DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins.

    PubMed

    Baek, Kwangryul; Kim, Duk Hyoung; Jeong, Jooyeon; Sim, Sang Jun; Melis, Anastasios; Kim, Jin-Soo; Jin, EonSeon; Bae, Sangsu

    2016-01-01

    Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity. PMID:27466170

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

    PubMed Central

    Sartori, Pablo; Geyer, Veikko F; Scholich, Andre; Jülicher, Frank; Howard, Jonathon

    2016-01-01

    Cilia and flagella are model systems for studying how mechanical forces control morphology. The periodic bending motion of cilia and flagella is thought to arise from mechanical feedback: dynein motors generate sliding forces that bend the flagellum, and bending leads to deformations and stresses, which feed back and regulate the motors. Three alternative feedback mechanisms have been proposed: regulation by the sliding forces, regulation by the curvature of the flagellum, and regulation by the normal forces that deform the cross-section of the flagellum. In this work, we combined theoretical and experimental approaches to show that the curvature control mechanism is the one that accords best with the bending waveforms of Chlamydomonas flagella. We make the surprising prediction that the motors respond to the time derivative of curvature, rather than curvature itself, hinting at an adaptation mechanism controlling the flagellar beat. DOI: http://dx.doi.org/10.7554/eLife.13258.001 PMID:27166516

  18. Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism.

    PubMed

    Yang, Wenqiang; Wittkopp, Tyler M; Li, Xiaobo; Warakanont, Jaruswan; Dubini, Alexandra; Catalanotti, Claudia; Kim, Rick G; Nowack, Eva C M; Mackinder, Luke C M; Aksoy, Munevver; Page, Mark Dudley; D'Adamo, Sarah; Saroussi, Shai; Heinnickel, Mark; Johnson, Xenie; Richaud, Pierre; Alric, Jean; Boehm, Marko; Jonikas, Martin C; Benning, Christoph; Merchant, Sabeeha S; Posewitz, Matthew C; Grossman, Arthur R

    2015-12-01

    Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions. PMID:26627249

  19. Identification and Characterization of a cis-Regulatory Element for Zygotic Gene Expression in Chlamydomonas reinhardtii

    PubMed Central

    Hamaji, Takashi; Lopez, David; Pellegrini, Matteo; Umen, James

    2016-01-01

    Upon fertilization Chlamydomonas reinhardtii zygotes undergo a program of differentiation into a diploid zygospore that is accompanied by transcription of hundreds of zygote-specific genes. We identified a distinct sequence motif we term a zygotic response element (ZYRE) that is highly enriched in promoter regions of C. reinhardtii early zygotic genes. A luciferase reporter assay was used to show that native ZYRE motifs within the promoter of zygotic gene ZYS3 or intron of zygotic gene DMT4 are necessary for zygotic induction. A synthetic luciferase reporter with a minimal promoter was used to show that ZYRE motifs introduced upstream are sufficient to confer zygotic upregulation, and that ZYRE-controlled zygotic transcription is dependent on the homeodomain transcription factor GSP1. We predict that ZYRE motifs will correspond to binding sites for the homeodomain proteins GSP1-GSM1 that heterodimerize and activate zygotic gene expression in early zygotes. PMID:27172209

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

    PubMed Central

    Nonaka, Yuki; Kikuchi, Kenji; Numayama-Tsuruta, Keiko; Kage, Azusa; Ueno, Hironori; Ishikawa, Takuji

    2016-01-01

    ABSTRACT 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. PMID:26787679

  1. [LIGHT-DEPENDENT SYNTHESIS OF CELL MEMBRANES IN THE Brc-1 MUTANT OF CHLAMYDOMONAS REINHARDTII].

    PubMed

    Semenova, G A; Chekunova, E M; Ladygin, V G

    2015-01-01

    The structural organization of cells of the Brc-1 mutant of the unicellular green algae Chlamydomonas reinhardtii grown in the light and in the dark has been studied. The Brc-1 mutant contains the brc-1 mutation in the nucleus gene LTS3. In the light, all membrane structures in mutant cells form normally and are well developed. In the dark under heterotrophic conditions, the mutant cells grew and divided well, however, all its cell membranes: plasmalemma, tonoplast, mitochondrial membranes, membranes of the nucleus shell and chloroplast, thylakoids, and the membranes of dictiosomes of the Golgi apparatus were not detected. In the dark under heterotrophic conditions, mutant cells well grow and divide. It were shown that a short-term (1-10 min) exposure of Brc-1 mutant cells to light leads to the restoration of all above-mentioned membrane structures. Possible reasons for the alterations of membrane structures are discussed. PMID:26281212

  2. Successful expression of heterologous egfp gene in the mitochondria of a photosynthetic eukaryote Chlamydomonas reinhardtii.

    PubMed

    Hu, Zhangli; Zhao, Zhonglin; Wu, Zhihua; Fan, Zhun; Chen, Jun; Wu, Jinxia; Li, Jiancheng

    2011-09-01

    The efficient expression of exogenous gene in mitochondria of photosynthetic organism has been an insurmountable problem. In this study, the pBsLPNCG was constructed by inserting the egfp gene into a site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA of Chlamydomonas reinhardtii CC-124 and introduced into the mitochondria of respiratory deficient dum-1 mutation of C. reinhardtii CC-2654. Sequencing and DNA Southern analyses revealed that egfp gene had been integrated into the mitochondrial genome of transgenic algae as expected and no other copy of egfp existed in their nucleic genome. Both the fluorescence detection and Western blot analysis confirmed the presence of eGFP protein in the transgenic algae; it indicated that the egfp gene was successfully expressed in the mitochondria of C. reinhardtii. PMID:21664493

  3. A TRP conductance modulates repolarization after sensory-dependent depolarization in Chlamydomonas reinhardtii

    PubMed Central

    Arias-Darraz, Luis; Colenso, Charlotte K; Veliz, Luis A; Vivar, Juan P; Cardenas, Sylvana; Brauchi, Sebastian

    2015-01-01

    Sensory integration is vital for motile organisms constantly exposed to changing surroundings. Chlamydomonas reinhardtii is a single-celled green alga found swimming in freshwater. 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. Many components of the machinery associated with sensory integration in C. reinhardtii, such as chemoreceptors and repolarization-associated channels, are yet uncharacterized. TRP channels are known mediators for cellular sensing in animal cells and it has been suggested that the C. reinhardtii genome encodes for a set of TRP proteins. Here, by combining behavioral studies with electrophysiological experiments conducted on both population and single alga, we test whether TRP channel blockers affect algal swimming behavior. Our results suggest that a TRP conductance is associated to the repolarization that follows a depolarizing receptor potential, highlighting a primitive function of TRP proteins. PMID:26186626

  4. DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins

    PubMed Central

    Baek, Kwangryul; Kim, Duk Hyoung; Jeong, Jooyeon; Sim, Sang Jun; Melis, Anastasios; Kim, Jin-Soo; Jin, EonSeon; Bae, Sangsu

    2016-01-01

    Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity. PMID:27466170

  5. Metabolic network reconstruction of Chlamydomonas offers insight into light-driven algal metabolism

    PubMed Central

    Chang, Roger L; Ghamsari, Lila; Manichaikul, Ani; Hom, Erik F Y; Balaji, Santhanam; Fu, Weiqi; Shen, Yun; Hao, Tong; Palsson, Bernhard Ø; Salehi-Ashtiani, Kourosh; Papin, Jason A

    2011-01-01

    Metabolic network reconstruction encompasses existing knowledge about an organism's metabolism and genome annotation, providing a platform for omics data analysis and phenotype prediction. The model alga Chlamydomonas reinhardtii is employed to study diverse biological processes from photosynthesis to phototaxis. Recent heightened interest in this species results from an international movement to develop algal biofuels. Integrating biological and optical data, 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. PMID:21811229

  6. Zinc Deficiency Impacts CO2 Assimilation and Disrupts Copper Homeostasis in Chlamydomonas reinhardtii*

    PubMed Central

    Malasarn, Davin; Kropat, Janette; Hsieh, Scott I.; Finazzi, Giovanni; Casero, David; Loo, Joseph A.; Pellegrini, Matteo; Wollman, Francis-André; Merchant, Sabeeha S.

    2013-01-01

    Zinc is an essential nutrient because of its role in catalysis and in protein stabilization, but excess zinc is deleterious. We distinguished four nutritional zinc states in the alga Chlamydomonas reinhardtii: toxic, replete, deficient, and limited. Growth is inhibited in zinc-limited and zinc-toxic cells relative to zinc-replete cells, whereas zinc deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition-responsive changes in gene expression. We identified genes encoding zinc-handling components, including ZIP family transporters and candidate chaperones. Additionally, we noted an impact on two other regulatory pathways, the carbon-concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc deficiency, probably due to reduced carbonic anhydrase activity, validated by quantitative proteomics and immunoblot analysis of Cah1, Cah3, and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in zinc-limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. The Crr1 regulon responds to copper limitation and is turned on in zinc deficiency, and Crr1 is required for growth in zinc-limiting conditions. Zinc-deficient cells are functionally copper-deficient, although they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester copper in a biounavailable form, perhaps to prevent mismetallation of critical zinc sites. PMID:23439652

  7. Potassium Fluxes in Chlamydomonas reinhardtii (I.Kinetics and Electrical Potentials).

    PubMed Central

    Malhotra, B.; Glass, ADM.

    1995-01-01

    Potassium influx and cellular [K+] were measured in the unicellular green alga Chlamydomonas reinhardtii after pretreatment in either 10 or 0 mM external K+ ([K]0). K+ (42K+ or 86Rb+) influx was mediated by a saturable, high-affinity transport system (HATS) at low [K+]0 and a linear, low-affinity transport system at high [K+]o. The HATS was typically more sensitive to metabolic inhibition (and darkness) than the low-affinity transport system. Membrane electrical potentials were determined by measuring the equilibrium distribution of tetraphenylphosphonium. These values, together with estimates of cytoplasmic [K+] (B. Malhotra and A.D.M. Glass [1995] Plant Physiol 108: 1537-1545), demonstrated that at 0.1 mM [K+]0 K+ uptake must be active. At higher [K+]0 (>0.3 mM) K+ influx appeared to be passive and possibly channel mediated. When cells were deprived of K+ for 24 h, the Vmax for the HATS increased from 50 x 10-6 to 85 x 10-6 nmol h-1 cell-1 and the Km value decreased from 0.25 to 0.162 mM. Meanwhile, cellular [K+] declined from 24 x 10-6 to 9 x 10-6 nmol cell-1. During this period influx increased exponentially, reaching its peak value after 18 h of K+ deprivation. This increase of K+ influx was not expressed when cells were exposed to inhibitors of protein synthesis. The use of 42K+ and 86Rb+ in parallel experiments demonstrated that Chlamydomonas discriminated in favor of K+ over Rb+, and this effect increased with the duration of K+ deprivation. PMID:12228559

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

    PubMed

    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. PMID:21309872

  9. O(2) uptake in the light in chlamydomonas: evidence for persistent mitochondrial respiration.

    PubMed

    Peltier, G; Thibault, P

    1985-09-01

    The nature of the process responsible for the stationary O(2) uptake occurring in the light under saturating CO(2) concentration in Chlamydomonas reinhardii has been investigated. For this purpose, a mass spectrometer with a membrane inlet system was used to measure O(2) uptake and evolution in the algal suspension. First, we observed that the O(2) uptake rate was constant (about 0.5 micromoles of O(2) per milligram chlorophyll per minute) during a light to dark transition and was not affected by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Salicylhydroxamic acid had no effect on O(2) uptake in the dark or in the light, but was found to have the same inhibitory effect either in the dark or in the light when added to cyanide-treated algae. The stimulation of the O(2) uptake rate due to the uncoupling effect of carbonyl cyanide m-chlorophenylhydrazone was about the same in the dark or in the light. From these results, we conclude that mitochondrial respiration is maintained during illumination and therefore is not inhibited by high ATP levels. Another conclusion is that in conditions where photorespiration is absent, no other light-dependent O(2) uptake process occurs. If Mehler reactions are involved, in Chlamydomonas, under conditions where both photosynthetic carbon oxidation and reduction cycles cannot operate (as in cyanide-treated algae), their occurrence in photosynthesizing algae either under saturating CO(2) concentration or at the CO(2) compensation point appears very unlikely. The comparison with the situation previously reported in Scenedesmus (R. J. Radmer and B. Kok 1976 Plant Physiol 58: 336-340) suggests that different O(2) uptake processes might be present in these two algal species. PMID:16664375

  10. How Chlamydomonas keeps track of the light once it has reached the right phototactic orientation.

    PubMed Central

    Schaller, K; David, R; Uhl, R

    1997-01-01

    By using a real-time assay that allows measurement of the phototactic orientation of the unicellular alga Chlamydomonas with millisecond time resolution, it can be shown that single photons not only induce transient direction changes but that fluence rates as low as 1 photon cell(-1) s(-1) can already lead to a persistent orientation. Orientation is a binary variable, i.e., in a partially oriented population some organisms are fully oriented while the rest are still at random. Action spectra reveal that the response to a pulsed stimulus follows the Dartnall-nomogram for a rhodopsin while the response to a persistent stimulus falls off more rapidly toward the red end of the spectrum. Thus light of 540 nm, for which chlamy-rhodopsin is equally sensitive as for 440-nm light, induces no measurable persistent orientation while 440-nm light does. A model is presented which explains not only this behavior, but also how Chlamydomonas can track the light direction and switches between a positive and negative phototaxis. According to the model the ability to detect the direction of light, to make the right turn and to stay oriented, is a direct consequence of the helical path of the organism, the orientation of its eyespot relative to the helix-axis, and the special shielding properties of eyespot and cell body. The model places particular emphasis on the fact that prolonged swimming into the correct direction not only requires making a correct turn initially, but also avoiding further turns once the right direction has been reached. Images FIGURE 1 FIGURE 4 FIGURE 6 FIGURE 7 FIGURE 8 PMID:9284323

  11. Toxicity assessment of manufactured nanomaterials using the unicellular green alga Chlamydomonas reinhardtii.

    PubMed

    Wang, Jiangxin; Zhang, Xuezhi; Chen, Yongsheng; Sommerfeld, Milton; Hu, Qiang

    2008-10-01

    With the rapid development of nanotechnology, there is an increasing risk of human and environmental exposure to nanotechnology-based materials and products. As water resources are particularly vulnerable to direct and indirect contamination of nonomaterials (NMs), the potential toxicity and environmental implication of NMs to aquatic organisms must be evaluated. In this study, we assessed potential toxicity of two commercially used NMs, titanium dioxide (TiO(2)) and quantum dots (QDs), using the unicellular green alga Chlamydomonas reinhartii as a model system. The response of the organism to NMs was assessed at physiological, biochemical, and molecular genetic levels. Growth kinetics showed that growth inhibition occurred during the first two to three days of cultivation in the presence of TiO(2) or QDs. Measurements of lipid peroxidation measurement indicated that oxidative stress of the cells occurred as early as 6 h after exposure to TiO(2) or QDs. The transcriptional expression profiling of four stress response genes (sod1, gpx, cat, and ptox2) revealed that transient up-regulation of these genes occurred in cultures containing as low as 1.0 mg L(-1) of TiO(2) or 0.1 mg L(-1) of QDs, and the maximum transcripts of cat, sod1, gpx, and ptox2 occurred at 1.5, 3, 3, and 6 h, respectively, and were proportional to the initial concentration of the NMs. As the cultures continued, recovery in growth was observed and the extent of recovery, as indicated by the final cell concentration, was dosage-dependent. QDs were found to be more toxic to Chlamydomonas cells than TiO(2) under our experimental conditions. PMID:18768203

  12. A comparison of hydrogen photoproduction by sulfur-deprived Chlamydomonas reinhardtii under different growth conditions.

    PubMed

    Kosourov, Sergey; Patrusheva, Elena; Ghirardi, Maria L; Seibert, Michael; Tsygankov, Anatoly

    2007-03-10

    Continuous photoproduction of H(2) by the green alga, Chlamydomonas reinhardtii, is observed after incubating the cultures for about a day in the absence of sulfate and in the presence of acetate. Sulfur deprivation causes the partial and reversible inactivation of photosynthetic O(2) evolution in algae, resulting in the light-induced establishment of anaerobic conditions in sealed photobioreactors, expression of two [FeFe]-hydrogenases in the cells, and H(2) photoproduction for several days. We have previously demonstrated that sulfur-deprived algal cultures can produce H(2) gas in the absence of acetate, when appropriate experimental protocols were used (Tsygankov, A.A., Kosourov, S.N., Tolstygina, I.V., Ghirardi, M.L., Seibert, M., 2006. Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions. Int. J. Hydrogen Energy 31, 1574-1584). We now report the use of an automated photobioreactor system to compare the effects of photoautotrophic, photoheterotrophic and photomixotrophic growth conditions on the kinetic parameters associated with the adaptation of the algal cells to sulfur deprivation and H(2) photoproduction. This was done under the experimental conditions outlined in the above reference, including controlled pH. From this comparison we show that both acetate and CO(2) are required for the most rapid inactivation of photosystem II and the highest yield of H(2) gas production. Although, the presence of acetate in the system is not critical for the process, H(2) photoproduction under photoautotrophic conditions can be increased by optimizing the conditions for high starch accumulation. These results suggest ways of engineering algae to improve H(2) production, which in turn may have a positive impact on the economics of applied systems for H(2) production. PMID:17275940

  13. Photo-oxidative stress in a xanthophyll-deficient mutant of Chlamydomonas.

    PubMed

    Baroli, Irene; Gutman, Benjamin L; Ledford, Heidi K; Shin, Jai W; Chin, Brian L; Havaux, Michel; Niyogi, Krishna K

    2004-02-20

    When there is an imbalance between the light energy absorbed by a photosynthetic organism and that which can be utilized in photosynthesis, photo-oxidative stress can damage pigments, proteins, lipids, and nucleic acids. In this work we compared the wild type and a xanthophyll-deficient mutant of Chlamydomonas reinhardtii in their response to high amounts of light. Wild-type Chlamydomonas cells were able to acclimate to high amounts of light following transfer from low light conditions. In contrast, the npq1 lor1 double mutant, which lacks protective xanthophylls (zeaxanthin and lutein) in the chloroplast, progressively lost viability and photosynthetic capacity along with destruction of thylakoid membrane protein-pigment complexes and accumulation of reactive oxygen species and membrane lipid peroxides. Loss of viability was partially rescued by lowered oxygen tension, suggesting that the high sensitivity of the mutant to light stress is caused by the production of reactive oxygen species in the chloroplast. Cell death was not prevented by the addition of an organic carbon source to the growth medium, demonstrating that the photo-oxidative damage can target other essential chloroplast processes besides photosynthesis. From the differential sensitivity of the mutant to exogenously added pro-oxidants, we infer that the reactive oxygen species produced during light stress in npq1 lor1 may be singlet oxygen and/or superoxide but not hydrogen peroxide. The bleaching phenotype of npq1 lor1 was not due to enhanced photodamage to photosystem II but rather to a less localized phenomenon of accumulation of photo-oxidation products in chloroplast membranes. PMID:14665619

  14. Combined intracellular nitrate and NIT2 effects on storage carbohydrate metabolism in Chlamydomonas

    PubMed Central

    Vigeolas, H.

    2014-01-01

    Microalgae are receiving increasing attention as alternative production systems for renewable energy such as biofuel. The photosynthetic alga Chlamydomonas reinhardtii is widely recognized as the model system to study all aspects of algal physiology, including the molecular mechanisms underlying the accumulation of starch and triacylglycerol (TAG), which are the precursors of biofuel. All of these pathways not only require a carbon (C) supply but also are strongly dependent on a source of nitrogen (N) to sustain optimal growth rate and biomass production. In order to gain a better understanding of the regulation of C and N metabolisms and the accumulation of storage carbohydrates, the effect of different N sources (NH4NO3 and ) on primary metabolism using various mutants impaired in either NIA1, NIT2 or both loci was performed by metabolic analyses. The data demonstrated that, using NH4NO3, nia1 strain displayed the most striking phenotype, including an inhibition of growth, accumulation of intracellular nitrate, and strong starch and TAG accumulation. The measurements of the different C and N intermediate levels (amino, organic, and fatty acids), together with the determination of acetate and remaining in the medium, clearly excluded the hypothesis of a slower and acetate assimilation in this mutant in the presence of NH4NO3. The results provide evidence of the implication of intracellular nitrate and NIT2 in the control of C partitioning into different storage carbohydrates under mixotrophic conditions in Chlamydomonas. The underlying mechanisms and implications for strategies to increase biomass yield and storage product composition in oleaginous algae are discussed. PMID:24187418

  15. The Antarctic psychrophile, Chlamydomonas subcaudata, is deficient in state I-state II transitions.

    PubMed

    Morgan-Kiss, Rachael M; Ivanov, Alexander G; Huner, Norman P A

    2002-01-01

    State I-State II transitions were monitored in vivo and in vitro in the Antarctic, psychrophillic, green alga, Chlamydomonas subcaudata, as changes in the low-temperature (77 K) chlorophyll fluorescence emission maxima at 722 nm (F722) relative to 699 nm (F699). As expected, the control mesophillic species, Chlamydomonas reinhardtii, was able to modulate the light energy distribution between photosystem II and photosystem I in response to exposure to four different conditions: (i) dark/anaerobic conditions, (ii) a change in Mg2+ concentration, (iii) red light, and (iv) increased incubation temperature. This was correlated with the ability to phosphorylate both of its major light-harvesting polypeptides. In contrast, exposure of C. subcaudata to the same four conditions induced minimum alterations in the 77 K fluorescence emission spectra, which was correlated with the ability to phosphorylate only one of its major light-harvesting polypeptides. Thus, C. subcaudata appears to be deficient in the ability to undergo a State I-State II transition. Functionally, this is associated with alterations in the apparent redox status of the intersystem electron transport chain and with higher rates of photosystem I cyclic electron transport in the psychrophile than in the mesophile, based on in vivo P700 measurements. Structurally, this deficiency is associated with reduced levels of Psa A/B relative to D1, the absence of specific photosystem I light-harvesting polypeptides [R.M. Morgan et al. (1998) Photosynth Res 56:303-314] and a cytochrome b6/f complex that exhibits a form of cytochrome f that is approximately 7 kDa smaller than that observed in C. reinhardtii. We conclude that the Antarctic psychrophile, C. subcaudata, is an example of a natural variant deficient in State I-State II transitions. PMID:11859846

  16. Ferric reduction by iron-limited Chlamydomonas cells interacts with both photosynthesis and respiration.

    PubMed

    Weger, H G; Espie, G S

    2000-04-01

    Iron limitation led to a large increase in extracellular ferricyanide (Fe[III]) reductase activity in cells of the green alga Chlamydomonas reinhardtii Dangeard. Mass-spectrometric measurement of gas exchange indicated that ferricyanide reduction in the dark resulted in a stimulation of respiratory CO2 production without affecting the rate of respiratory O2 consumption, consistent with the previously postulated activation of the oxidative pentose phosphate pathway in support of Fe(III) reduction by iron-limited Chlamydomonas cells (X. Xue et al., 1998, J. Phycol. 34: 939-944). At saturating irradiance, the rate of ferricyanide reduction was stimulated almost 3-fold, and this stimulation was inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea. Ferricyanide reduction during photosynthesis resulted in approximately a 50% inhibition of photosynthetic CO2 fixation at saturating irradiance, and almost 100% inhibition of CO2 fixation at sub-saturating irradiance. Photosynthesis by iron-sufficient cells was not affected by ferricyanide addition. Addition of 250 microM ferricyanide to iron-limited cells in which photosynthesis was inhibited (either by the presence of glycolaldehyde, or by maintaining the cells at the CO2 compensation point) resulted in a stimulation in the rate of gross photosynthetic O2 evolution. Chlorophyll a fluorescence measurements indicated a large increase in non-photochemical quenching during ferricyanide reduction in the light; the increase in nonphotochemical quenching was abolished by the addition of nigericin. These results suggest that reduction of extracellular ferricyanide (mediated at the plasma membrane) interacts with both photosynthesis and respiration, and that both of these processes contribute NADPH in the light. PMID:10805449

  17. Metabolic Flexibility of the Green Alga Chlamydomonas reinhardtii as Revealed by the Link between State Transitions and Cyclic Electron Flow.

    PubMed

    Finazzi, Giovanni; Forti, Giorgio

    2004-01-01

    In this Review we focus on the conversion of linear photosynthetic electron transport from water to NADP to the cyclic pathway around Photosystem I in the green alga Chlamydomonas reinhardtii. We discuss the strict relationship that exists between the changes in pathways of electron transport and state transitions, i.e., the reversible functional association of light harvesting proteins with one of the two photosystems of oxygenic photosynthesis. Such a link has not been reported in the case of other photosynthetic organisms, where the state transitions do not affect the pathway of electron transport. Rather, they provide a tool to optimise the rate of linear flow. We propose a kinetic-structural model that explains the mechanism of this particular relationship in Chlamydomonas, and discuss the advantages that this peculiar situation gives to the energetic metabolism of this alga. PMID:16143844

  18. Stable isotope fractionation in photosynthesis: Analysis of autotrophic competence following transformation of the chloroplast genome of Chlamydomonas

    SciTech Connect

    Boynton, J.E.; Gillham, N.W.; Osmond, C.B.

    1991-06-15

    Isotopic techniques needed to assess the interactions between photosynthesis and respiration in Chlamydomonas have been devised for {sup 13}C, using plate and liquid cultures. The effectiveness of various transformation strategies for the chloroplast psbA gene has been evaluated with respect to their utility in constructing and characterizing strains homoplasmic for site-directed mutations in an otherwise isogenic background. Our analysis of the first site-directed change in the D-1 protein of Chlamydomonas indicates that a second site mutation (arg{sub 238} > lys) in the loop between transmembrane helices IV -- V can partially compensate for the reduced photosynthetic performance that accompanies the atrazine resistant mutation (ser{sub 264} > ala/gly) in this alga and in higher plants grown under high light intensities. 31 refs., 2 figs.

  19. The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii: identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components

    NASA Technical Reports Server (NTRS)

    Huppe, H. C.; de Lamotte-Guery, F.; Buchanan, B. B.

    1990-01-01

    The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (Mrs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa ("similar") subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa ("variable") subunit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.

  20. Cell-to-Cell Diversity in a Synchronized Chlamydomonas Culture As Revealed by Single-Cell Analyses

    PubMed Central

    Garz, Andreas; Sandmann, Michael; Rading, Michael; Ramm, Sascha; Menzel, Ralf; Steup, Martin

    2012-01-01

    In a synchronized photoautotrophic culture of Chlamydomonas reinhardtii, cell size, cell number, and the averaged starch content were determined throughout the light-dark cycle. For single-cell analyses, the relative cellular starch was quantified by measuring the second harmonic generation (SHG). In destained cells, amylopectin essentially represents the only biophotonic structure. As revealed by various validation procedures, SHG signal intensities are a reliable relative measure of the cellular starch content. During photosynthesis-driven starch biosynthesis, synchronized Chlamydomonas cells possess an unexpected cell-to-cell diversity both in size and starch content, but the starch-related heterogeneity largely exceeds that of size. The cellular volume, starch content, and amount of starch/cell volume obey lognormal distributions. Starch degradation was initiated by inhibiting the photosynthetic electron transport in illuminated cells or by darkening. Under both conditions, the averaged rate of starch degradation is almost constant, but it is higher in illuminated than in darkened cells. At the single-cell level, rates of starch degradation largely differ but are unrelated to the initial cellular starch content. A rate equation describing the cellular starch degradation is presented. SHG-based three-dimensional reconstructions of Chlamydomonas cells containing starch granules are shown. PMID:23009858

  1. Alternative acetate production pathways in Chlamydomonas reinhardtii during dark anoxia and the dominant role of chloroplasts in fermentative acetate production.

    PubMed

    Yang, Wenqiang; Catalanotti, Claudia; D'Adamo, Sarah; Wittkopp, Tyler M; Ingram-Smith, Cheryl J; Mackinder, Luke; Miller, Tarryn E; Heuberger, Adam L; Peers, Graham; Smith, Kerry S; Jonikas, Martin C; Grossman, Arthur R; Posewitz, Matthew C

    2014-11-01

    Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes. PMID:25381350

  2. Characterization of a Mutant Deficient for Ammonium and Nitric Oxide Signalling in the Model System Chlamydomonas reinhardtii

    PubMed Central

    Sanz-Luque, Emanuel; Ocaña-Calahorro, Francisco; Galván, Aurora; Fernández, Emilio; de Montaigu, Amaury

    2016-01-01

    The ubiquitous signalling molecule Nitric Oxide (NO) is characterized not only by the variety of organisms in which it has been described, but also by the wealth of biological processes that it regulates. In contrast to the expanding repertoire of functions assigned to NO, however, the mechanisms of NO action usually remain unresolved, and genes that work within NO signalling cascades are seldom identified. A recent addition to the list of known NO functions is the regulation of the nitrogen assimilation pathway in the unicellular alga Chlamydomonas reinhardtii, a well-established model organism for genetic and molecular studies that offers new possibilities in the search for mediators of NO signalling. By further exploiting a collection of Chlamydomonas insertional mutant strains originally isolated for their insensitivity to the ammonium (NH4+) nitrogen source, we found a mutant which, in addition to its ammonium insensitive (AI) phenotype, was not capable of correctly sensing the NO signal. Similarly to what had previously been described in the AI strain cyg56, the expression of nitrogen assimilation genes in the mutant did not properly respond to treatments with various NO donors. Complementation experiments showed that NON1 (NO Nitrate 1), a gene that encodes a protein containing no known functional domain, was the gene underlying the mutant phenotype. Beyond the identification of NON1, our findings broadly demonstrate the potential for Chlamydomonas reinhardtii to be used as a model system in the search for novel components of gene networks that mediate physiological responses to NO. PMID:27149516

  3. Whole-Genome Resequencing Reveals Extensive Natural Variation in the Model Green Alga Chlamydomonas reinhardtii[OPEN

    PubMed Central

    Hazzouri, Khaled M.; Rosas, Ulises; Bahmani, Tayebeh; Nelson, David R.; Abdrabu, Rasha; Harris, Elizabeth H.; Salehi-Ashtiani, Kourosh; Purugganan, Michael D.

    2015-01-01

    We performed whole-genome resequencing of 12 field isolates and eight commonly studied laboratory strains of the model organism Chlamydomonas reinhardtii to characterize genomic diversity and provide a resource for studies of natural variation. Our data support previous observations that Chlamydomonas is among the most diverse eukaryotic species. Nucleotide diversity is ∼3% and is geographically structured in North America with some evidence of admixture among sampling locales. Examination of predicted loss-of-function mutations in field isolates indicates conservation of genes associated with core cellular functions, while genes in large gene families and poorly characterized genes show a greater incidence of major effect mutations. De novo assembly of unmapped reads recovered genes in the field isolates that are absent from the CC-503 assembly. The laboratory reference strains show a genomic pattern of polymorphism consistent with their origin as the recombinant progeny of a diploid zygospore. Large duplications or amplifications are a prominent feature of laboratory strains and appear to have originated under laboratory culture. Extensive natural variation offers a new source of genetic diversity for studies of Chlamydomonas, including naturally occurring alleles that may prove useful in studies of gene function and the dissection of quantitative genetic traits. PMID:26392080

  4. High-Resolution Profiling of a Synchronized Diurnal Transcriptome from Chlamydomonas reinhardtii Reveals Continuous Cell and Metabolic Differentiation[OPEN

    PubMed Central

    2015-01-01

    The green alga Chlamydomonas reinhardtii is a useful model organism for investigating diverse biological processes, such as photosynthesis and chloroplast biogenesis, flagella and basal body structure/function, cell growth and division, and many others. We combined a highly synchronous photobioreactor culture system with frequent temporal sampling to characterize genome-wide diurnal gene expression in Chlamydomonas. Over 80% of the measured transcriptome was expressed with strong periodicity, forming 18 major clusters. Genes associated with complex structures and processes, including cell cycle control, flagella and basal bodies, ribosome biogenesis, and energy metabolism, all had distinct signatures of coexpression with strong predictive value for assigning and temporally ordering function. Importantly, the frequent sampling regime allowed us to discern meaningful fine-scale phase differences between and within subgroups of genes and enabled the identification of a transiently expressed cluster of light stress genes. Coexpression was further used both as a data-mining tool to classify and/or validate genes from other data sets related to the cell cycle and to flagella and basal bodies and to assign isoforms of duplicated enzymes to their cognate pathways of central carbon metabolism. Our diurnal coexpression data capture functional relationships established by dozens of prior studies and are a valuable new resource for investigating a variety of biological processes in Chlamydomonas and other eukaryotes. PMID:26432862

  5. Evidence that an internal carbonic anhydrase is present in 5% CO/sub 2/-grown and air-grown Chlamydomonas. [Chlamydomonas reinhardtii

    SciTech Connect

    Moroney, J.V.; Togasaki, R.K.; Husic, H.D.; Tolbert, N.E.

    1987-07-01

    Inorganic carbon (C/sub i/) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO/sub 2/. Both air-grown cells, that have a CO/sub 2/ concentrating system, and 5% CO/sub 2/-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C/sub i/) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO/sub 2/-grown cells also accumulated some C/sub i/, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO/sub 2/ fixation by high CO/sub 2/-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO/sub 2/-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.

  6. Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves

    PubMed Central

    2011-01-01

    Background When cultivated under stress conditions, many microalgae species accumulate both starch and oil (triacylglycerols). The model green microalga Chlamydomonas reinhardtii has recently emerged as a model to test genetic engineering or cultivation strategies aiming at increasing lipid yields for biodiesel production. Blocking starch synthesis has been suggested as a way to boost oil accumulation. Here, we characterize the triacylglycerol (TAG) accumulation process in Chlamydomonas and quantify TAGs in various wild-type and starchless strains. Results In response to nitrogen deficiency, Chlamydomonas reinhardtii produced TAGs enriched in palmitic, oleic and linoleic acids that accumulated in oil-bodies. Oil synthesis was maximal between 2 and 3 days following nitrogen depletion and reached a plateau around day 5. In the first 48 hours of oil deposition, a ~80% reduction in the major plastidial membrane lipids occurred. Upon nitrogen re-supply, mobilization of TAGs started after starch degradation but was completed within 24 hours. Comparison of oil content in five common laboratory strains (CC124, CC125, cw15, CC1690 and 11-32A) revealed a high variability, from 2 μg TAG per million cell in CC124 to 11 μg in 11-32A. Quantification of TAGs on a cell basis in three mutants affected in starch synthesis (cw15sta1-2, cw15sta6 and cw15sta7-1) showed that blocking starch synthesis did not result in TAG over-accumulation compared to their direct progenitor, the arginine auxotroph strain 330. Moreover, no significant correlation was found between cellular oil and starch levels among the twenty wild-type, mutants and complemented strains tested. By contrast, cellular oil content was found to increase steeply with salt concentration in the growth medium. At 100 mM NaCl, oil level similar to nitrogen depletion conditions could be reached in CC124 strain. Conclusion A reference basis for future genetic studies of oil metabolism in Chlamydomonas is provided. Results

  7. Group I introns interrupt the chloroplast psaB and psbC and the mitochondrial rrnL gene in Chlamydomonas.

    PubMed Central

    Turmel, M; Mercier, J P; Côté, M J

    1993-01-01

    The polymerase chain reaction was used to identify novel IAI subgroup introns in cpDNA-enriched preparations from the interfertile green algae Chlamydomonas eugametos and Chlamydomonas moewusii. These experiments along with sequence analysis disclosed the presence, in both green algae, of a single IA1 intron in the psaB gene and of two group I introns (IA2 and IA1) in the psbC gene. In addition, two group I introns (IA1 and IB4) were found in the peptidyltransferase region of the mitochondrial large subunit rRNA gene at the same positions as previously reported Chlamydomonas chloroplast introns. The 188 bp segment preceding the first mitochondrial intron revealed extensive sequence similarity to the distantly spaced rRNA-coding modules L7 and L8 in the Chlamydomonas reinhardtii mitochondrial DNA, indicating that these two modules have undergone rearrangements in Chlamydomonas. The IA1 introns in psaB and psbC were found to be related in sequence to the first intron in the C. moewusii chloroplast psbA gene. The similarity between the former introns extends to the immediate 5' flanking exon sequence, suggesting that group I intron transposition occurred from one of the two genes to the other through reverse splicing. PMID:7504814

  8. SP mountain data analysis

    NASA Technical Reports Server (NTRS)

    Rawson, R. F.; Hamilton, R. E.; Liskow, C. L.; Dias, A. R.; Jackson, P. L.

    1981-01-01

    An analysis of synthetic aperture radar data of SP Mountain was undertaken to demonstrate the use of digital image processing techniques to aid in geologic interpretation of SAR data. These data were collected with the ERIM X- and L-band airborne SAR using like- and cross-polarizations. The resulting signal films were used to produce computer compatible tapes, from which four-channel imagery was generated. Slant range-to-ground range and range-azimuth-scale corrections were made in order to facilitate image registration; intensity corrections were also made. Manual interpretation of the imagery showed that L-band represented the geology of the area better than X-band. Several differences between the various images were also noted. Further digital analysis of the corrected data was done for enhancement purposes. This analysis included application of an MSS differencing routine and development of a routine for removal of relief displacement. It was found that accurate registration of the SAR channels is critical to the effectiveness of the differencing routine. Use of the relief displacement algorithm on the SP Mountain data demonstrated the feasibility of the technique.

  9. Retinal chromophore structure and Schiff base interactions in red-shifted channelrhodopsin-1 from Chlamydomonas augustae.

    PubMed

    Ogren, John I; Mamaev, Sergey; Russano, Daniel; Li, Hai; Spudich, John L; Rothschild, Kenneth J

    2014-06-24

    Channelrhodopsins (ChRs), which form a distinct branch of the microbial rhodopsin family, control phototaxis in green algae. Because ChRs can be expressed and function in neuronal membranes as light-gated cation channels, they have rapidly become an important optogenetic tool in neurobiology. While channelrhodopsin-2 from the unicellular alga Chlamydomonas reinhardtii (CrChR2) is the most commonly used and extensively studied optogenetic ChR, little is known about the properties of the diverse group of other ChRs. In this study, near-infrared confocal resonance Raman spectroscopy along with hydrogen-deuterium exchange and site-directed mutagenesis were used to study the structure of red-shifted ChR1 from Chlamydomonas augustae (CaChR1). These measurements reveal that (i) CaChR1 has an all-trans-retinal structure similar to those of the light-driven proton pump bacteriorhodopsin (BR) and sensory rhodopsin II but different from that of the mixed retinal composition of CrChR2, (ii) lowering the pH from 7 to 2 or substituting neutral residues for Glu169 or Asp299 does not significantly shift the ethylenic stretch frequency more than 1-2 cm(-1) in contrast to BR in which a downshift of 7-9 cm(-1) occurs reflecting neutralization of the Asp85 counterion, and (iii) the CaChR1 protonated Schiff base (SB) has stronger hydrogen bonding than BR. A model is proposed to explain these results whereby at pH 7 the predominant counterion to the SB is Asp299 (the homologue to Asp212 in BR) while Glu169 (the homologue to Asp85 in BR) exists in a neutral state. We observe an unusual constancy of the resonance Raman spectra over the broad range from pH 9 to 2 and discuss its implications. These results are in accord with recent visible absorption and current measurements of CaChR1 [Sineshchekov, O. A., et al. (2013) Intramolecular proton transfer in channelrhodopsins. Biophys. J. 104, 807-817; Li, H., et al. (2014) Role of a helix B lysine residue in the photoactive site in

  10. Laser sculpting of atomic sp, sp(2) , and sp(3) hybrid orbitals.

    PubMed

    Liu, Chunmei; Manz, Jörn; Yang, Yonggang

    2015-01-12

    Atomic sp, sp(2) , and sp(3) hybrid orbitals were introduced by Linus Pauling to explain the nature of the chemical bond. Quantum dynamics simulations show that they can be sculpted by means of a selective series of coherent laser pulses, starting from the 1s orbital of the hydrogen atom. Laser hybridization generates atoms with state-selective electric dipoles, opening up new possibilities for the study of chemical reaction dynamics and heterogeneous catalysis. PMID:25257703

  11. Partial purification of the chloroplast ATP synthase from Chlamydomonas reinhardtii and the cloning and sequencing of a cDNA encoding the gamma subunit

    SciTech Connect

    Yu, L.M.

    1988-01-01

    The chloroplast ATP synthase was partially purified from the green alga Chlamydomonas reinhardtii by extracting membranes with deoxycholate and KCl, followed by centrifugation and ammonium sulfate fractionation of the supernatant. The enzyme assay involved the reconstitution of such fractions with bacteriorhodopsin and soybean phospholipids to form vesicles capable of light-dependent ({sup 32}P)-phosphate esterification. A cDNA for the gamma subunit from Chlamydomonas was isolated, expressed in vitro and sequenced. It contains the entire coding region for the gamma subunit precursor. A 35 amino acid long transit peptide resides at the NH{sub 2}-terminus of a 323 amino acid long mature peptide that is 77% similar to the spinach gamma subunit. Six cysteines were found; three were conserved in Chlamydomonas and spinach.

  12. Corynebacterium appendicis sp. nov.

    PubMed

    Yassin, A F; Steiner, U; Ludwig, W

    2002-07-01

    A lipophilic, coryneform bacterium isolated from a human clinical specimen was characterized by phenotypic and molecular-taxonomic methods. Chemotaxonomic investigations revealed the presence of cell-wall chemotype IV and short-chain mycolic acids consistent with the genus Corynebacterium. The isolate could be distinguished from other members of the genus Corynebacterium by positive urease and catalase tests as well as its failure to produce acid from carbohydrates. Comparative 16S rRNA gene sequencing showed that this isolate constitutes a distinct subline within the genus Corynebacterium, displaying >3.0% sequence divergence from other known Corynebacterium species. Based on both phenotypic and phylogenetic evidence, it is proposed that this isolate be classified as a novel species, Corynebacterium appendicis sp. nov., represented by strain IMMIB R-3491T (= DSM 44531T = NRRL B-24151T). PMID:12148623

  13. Acetobacter intermedius, sp. nov.

    PubMed

    Boesch, C; Trcek, J; Sievers, M; Teuber, M

    1998-03-01

    Strains of a new species in the genus Acetobacter, for which we propose the name A. intermedius sp. nov., were isolated and characterized in pure culture from different sources (Kombucha beverage, cider vinegar, spirit vinegar) and different countries (Switzerland, Slovenia). The isolated strains grow in media with 3% acetic acid and 3% ethanol as does A. europaeus, do, however, not require acetic acid for growth. These characteristics phenotypically position A. intermedius between A. europaeus and A. xylinus, DNA-DNA hybridizations of A. intermedius-DNA with DNA of the type strains of Acetobacter europaeus, A. xylinus, A. aceti, A. hansenii, A. liquefaciens, A. methanolicus, A. pasteurianus, A. diazotrophicus, Gluconobacter oxydans and Escherichia coli HB 101 indicated less than 60% DNA similarity. The important features of the new species are described. Acetobacter intermedius strain TF2 (DSM11804) isolated from the liquid phase of a tea fungus beverage (Kombucha) is the type strain. PMID:13678040

  14. DADiSP processing guide

    NASA Technical Reports Server (NTRS)

    Rogers, Melissa J. B.

    1993-01-01

    A guide for DADiSP software, intended for use by the Lambda Point Experiment (LPE) Team during and after the United States Microgravity Payload (USMP)-1 mission, is presented. DADiSP is a Data Analysis and Display Software developed and marketed by DSP Development Corporation, Cambridge, Massachusetts. This guide is intended to be used in addition to the DADiSP Worksheet User Manual and Reference Manual which are supplied by the company with the software. Technical support for DADiSP is available from DSP at (617) 577-1133. Access to DADiSP on Acceleration Characterization and Analysis Project (ACAP) EGSE is being provided to the LPE team during USMP-1 for off-line processing of SAMS data.

  15. A Dual Strategy to Cope with High Light in Chlamydomonas reinhardtii[W

    PubMed Central

    Allorent, Guillaume; Tokutsu, Ryutaro; Roach, Thomas; Peers, Graham; Cardol, Pierre; Girard-Bascou, Jacqueline; Seigneurin-Berny, Daphné; Petroutsos, Dimitris; Kuntz, Marcel; Breyton, Cécile; Franck, Fabrice; Wollman, Francis-André; Niyogi, Krishna K.; Krieger-Liszkay, Anja; Minagawa, Jun; Finazzi, Giovanni

    2013-01-01

    Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition–deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II. PMID:23424243

  16. Modeling light-induced currents in the eye of Chlamydomonas reinhardtii.

    PubMed

    Gradmann, D; Ehlenbeck, S; Hegemann, P

    2002-09-15

    Rhodopsin-mediated electrical events in green algae have been recorded in the past from the eyes of numerous micro-algae like Haematococcus pluvialis, Chlamydomonas reinhardtii and Volvox carteri. However, the electrical data gathered by suction-pipette techniques could be interpreted in qualitative terms only. Here we present two models that allow a quantitative analysis of such results: First, an electrical analog circuit for the cell in suction pipette configuration is established. Applying this model to experimental data from unilluminated cells of C. reinhardtii yields a membrane conductance of about 3 Sm(-2). Furthermore, an analog circuit allows the determination of the photocurrent fraction that is recorded under experimental conditions. Second, a reaction scheme of a rhodopsin-type photocycle with an early Ca(2+) conductance and a later H(+) conductance is presented. The combination of both models provides good fits to light-induced currents recorded from C. reinhardtii. Finally, it allowed the calculation of the impact of each model parameter on the time courses of observable photocurrent and of inferred transmembrane voltage. The reduction of the flash-to-peak times at increasing light intensities are explained by superposition of two kinetically distinct rhodopsins and by assuming that the Ca(2+)-conducting state decays faster at more positive membrane voltages. PMID:12235485

  17. X-Ray structure of a truncated form of cytochrome f from chlamydomonas Reinhardtii

    SciTech Connect

    Chi, Young-In; Huang, Li-Shar; Zhang, Zhaolei; Fernando-Velasquez, Javier G.; Berry, E. A.

    2000-03-01

    A truncated form of cytochrome f from Chlamydomonas Reinhardtii (an important eukaryotic model organism for photosynthetic electron transfer studies) has been crystallized (space group P212121; 3 molecules/ asymmetric unit) and its structure determined to 2.0 Angstrom by molecular replacement using the coordinates of a truncated turnip cytochrome f as a model. The structure displays the same folding and detailed features as turnip cytochrome f including: (a) an unusual heme Fe ligation by alpha-amino group of tyrosine 1, (b) a cluster of lysine residues (proposed docking site of plastocyanin), and (c) the presence of a chain of 7 water molecules bound to conserved residues and extending between the heme pocket and K58 and K66 at the lysine cluster. For this array of waters we propose a structural role. Two cytochrome f molecules are related by a non-crystallographic symmetry operator which is a distorted proper 2-fold rotation. This may represent the dimeric relation of the monomers in situ, however the heme orientation suggested by this model is not consistent with previous epr measurements on oriented membranes.

  18. Nitrogen and sulfur deprivation differentiate lipid accumulation targets of Chlamydomonas reinhardtii.

    PubMed

    Cakmak, Turgay; Angun, Pinar; Ozkan, Alper D; Cakmak, Zeynep; Olmez, Tolga T; Tekinay, Turgay

    2012-01-01

    Nitrogen (N) and sulfur (S) have inter-related and distinct impacts on microalgal metabolism; with N starvation having previously been reported to induce elevated levels of the biodiesel feedstock material triacylglycerol (TAG), while S deprivation is extensively studied for its effects on biohydrogen production in microalgae. ( 1) (,) ( 2) We have previously demonstrated that N- and S-starved cells of Chlamydomonas reinhardtii display different metabolic trends, suggesting that different response mechanisms exist to compensate for the absence of those two elements. ( 3) We used C. reinhardtii CC-124 mt(-) and CC-125 mt(+) strains to test possible metabolic changes related to TAG accumulation in response to N and S deprivation, considering that gamete differentiation in this organism is mainly regulated by N. ( 4) Our findings contribute to the understanding of microalgal response to element deprivation and potential use of element deprivation for biodiesel feedstock production using microalgae, but much remains to be elucidated on the precise contribution of both N and S starvation on microalgal metabolism. PMID:22892589

  19. Nitrogen and sulfur deprivation differentiate lipid accumulation targets of Chlamydomonas reinhardtii

    PubMed Central

    Cakmak, Turgay; Angun, Pinar; Ozkan, Alper D.; Cakmak, Zeynep; Olmez, Tolga T.; Tekinay, Turgay

    2012-01-01

    Nitrogen (N) and sulfur (S) have inter-related and distinct impacts on microalgal metabolism; with N starvation having previously been reported to induce elevated levels of the biodiesel feedstock material triacylglycerol (TAG), while S deprivation is extensively studied for its effects on biohydrogen production in microalgae.1,2 We have previously demonstrated that N- and S-starved cells of Chlamydomonas reinhardtii display different metabolic trends, suggesting that different response mechanisms exist to compensate for the absence of those two elements.3 We used C. reinhardtii CC-124 mt(-) and CC-125 mt(+) strains to test possible metabolic changes related to TAG accumulation in response to N and S deprivation, considering that gamete differentiation in this organism is mainly regulated by N.4 Our findings contribute to the understanding of microalgal response to element deprivation and potential use of element deprivation for biodiesel feedstock production using microalgae, but much remains to be elucidated on the precise contribution of both N and S starvation on microalgal metabolism. PMID:22892589

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

    PubMed

    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. PMID:24754449

  1. Using Natural Selection to Explore the Adaptive Potential of Chlamydomonas reinhardtii

    PubMed Central

    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. PMID:24658261

  2. Functional Organization of the Chlorophyll-Containing Complexes of Chlamydomonas reinhardi1

    PubMed Central

    Gershoni, Jonathan M.; Shochat, Susana; Malkin, Shmuel; Ohad, Itzhak

    1982-01-01

    The stepwise synthesis and assembly of photosynthetic membrane components in the y-1 mutant of Chlamydomonas reinhardi have been previously demonstrated (Ohad 1975 In Membrane Biogenesis, Mitochondria, Chloroplasts and Bacteria, Plenum, pp 279-350). This experimental system was used here in order to investigate the process of formation and interconnection of the energy collecting chlorophylls with the reaction centers of both photosystems I and II. The following measurements were carried out: photosynthetic electron flow at various light intensities, including parts or the entire electron transfer chain; analysis of the kinetics of fluorescence emission at room temperature and fluorescence emission spectra at 77 K, and electrophoretic separation of membrane polypeptides and chlorophyll protein complexes. Based on the data obtained it is concluded that: (a) each photosystem (PSI and PSII) contains, in addition to the reaction center, an interconnecting antenna and a main or light harvesting antenna complex; (b) the formation of the light harvesting complex, interconnecting antenna, and reaction centers for each photosystem can occur independently. (c) the interconnecting antennae link the light harvesting complexes with the respective reaction centers. In their absence, energy transfer between the light harvesting chlorophylls and the reaction centers is inefficient. The formation of the interconnecting antennae and efficient assembly of photosystem components occur simultaneously with the de novo synthesis of chlorophyll and at least three polypeptides, one translated in the cytoplasm and two translated in the chloroplast. The synthesis of these polypeptides was found to be light dependent. Images Fig. 1 Fig. 3 Fig. 6 PMID:16662548

  3. The Unicellular Green Alga Chlamydomonas reinhardtii as an Experimental System to Study Chloroplast RNA Metabolism

    NASA Astrophysics Data System (ADS)

    Nickelsen, J.; Kück, U.

    Chloroplasts are typical organelles of photoautotrophic eukaryotic cells which drive a variety of functions, including photosynthesis. For many years the unicellular green alga Chlamydomonas reinhardtii has served as an experimental organism for studying photosynthetic processes. The recent development of molecular tools for this organism together with efficient methods of genetic analysis and the availability of many photosynthesis mutants has now made this alga a powerful model system for the analysis of chloroplast biogenesis. For example, techniques have been developed to transfer recombinant DNA into both the nuclear and the chloroplast genome. This allows both complementation tests and analyses of gene functions in vivo. Moreover, site-specific DNA recombinations in the chloroplast allow targeted gene disruption experiments which enable a "reverse genetics" to be performed. The potential of the algal system for the study of chloroplast biogenesis is illustrated in this review by the description of regulatory systems of gene expression involved in organelle biogenesis. One example concerns the regulation of trans-splicing of chloroplast mRNAs, a process which is controlled by both multiple nuclear- and chloroplast-encoded factors. The second example involves the stabilization of chloroplast mRNAs. The available data lead us predict distinct RNA elements, which interact with trans-acting factors to protect the RNA against nucleolytic attacks.

  4. Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production.

    PubMed

    Karpagam, R; Preeti, R; Ashokkumar, B; Varalakshmi, P

    2015-11-01

    Lipid from microalgae is one of the putative oil resources to facilitate the biodiesel production during this era of energy dissipation and environmental pollution. In this study, the key parameters such as biomass productivity, lipid productivity and lipid content were evaluated at the early stationary phase of Chlamydomonas reinhardtii, CC1010 cultivated in nutrient starved (nitrogen, phosphorous), glucose (0.05%, 0.1%, 0.15% and 0.2%) and vitamin B12 supplementation (0.001%, 0.002% and 0.003%) in Tris-Acetate-Phosphate (TAP) medium. The lipid content in nitrogen starved media was 61% which is 2.34 folds higher than nutrient sufficient TAP medium. Glucose supplementation has lead to proportional increase in biomass productivity with the increasing concentration of glucose whereas vitamin B12 supplementations had not shown any influence in lipid and biomass production. Further, fatty acid methyl ester (FAME) profiling of C. reinhardtii, CC 1010 has revealed more than 80% of total SFA (saturated fatty acid) and MUFA (mono unsaturated fatty acid) content. Quality checking parameters of biodiesel like cetane number, saponification value, iodine number and degree of unsaturation were analyzed and the biodiesel fuel properties were found to be appropriate as per the international standards, EN 14214 and ASTM D6751. Conclusively, among all the treatments, nitrogen starvation with 0.1% glucose supplementation had yielded high lipid content in C. reinhardtii, CC 1010. PMID:25838071

  5. Phytotoxicity Evaluation of Type B Trichothecenes Using a Chlamydomonas reinhardtii Model System

    PubMed Central

    Suzuki, Tadahiro; Iwahashi, Yumiko

    2014-01-01

    Type B trichothecenes, which consist of deoxynivalenol (DON) and nivalenol (NIV) as the major end products, are produced by phytotoxic fungi, such as the Fusarium species, and pollute arable fields across the world. The DON toxicity has been investigated using various types of cell systems or animal bioassays. The evaluation of NIV toxicity, however, has been relatively restricted because of its lower level compared with DON. In this study, the Chlamydomonas reinhardtii testing system, which has been reported to have adequate NIV sensitivity, was reinvestigated under different mycotoxin concentrations and light conditions. The best concentration of DON and NIV, and their derivatives, for test conditions was found to be 25 ppm (2.5 × 10−2 mg/mL). In all light test conditions, DON, NIV, and fusarenon-X (FusX) indicated significant growth inhibition regardless of whether a light source existed, or under differential wavelength conditions. FusX growth was also influenced by changes in photon flux density. These results suggest that C. reinhardtii is an appropriate evaluation system for type B trichothecenes. PMID:24476708

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

    PubMed Central

    Yang, Duanpeng; Li, Weiqi

    2016-01-01

    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. PMID:26731741

  7. Characterization of the Major Light-Harvesting Complexes (LHCBM) of the Green Alga Chlamydomonas reinhardtii

    PubMed Central

    Natali, Alberto; Croce, Roberta

    2015-01-01

    Nine genes (LHCBM1-9) encode the major light-harvesting system of Chlamydomonas reinhardtii. Transcriptomic and proteomic analyses have shown that those genes are all expressed albeit in different amounts and some of them only in certain conditions. However, little is known about the properties and specific functions of the individual gene products because they have never been isolated. Here we have purified several complexes from native membranes and/or we have reconstituted them in vitro with pigments extracted from C. reinhardtii. It is shown that LHCBM1 and -M2/7 represent more than half of the LHCBM population in the membrane. LHCBM2/7 forms homotrimers while LHCBM1 seems to be present in heterotrimers. Trimers containing only type I LHCBM (M3/4/6/8/9) were also observed. Despite their different roles, all complexes have very similar properties in terms of pigment content, organization, stability, absorption, fluorescence and excited-state lifetimes. Thus the involvement of LHCBM1 in non-photochemical quenching is suggested to be due to specific interactions with other components of the membrane and not to the inherent quenching properties of the complex. Similarly, the overexpression of LHCBM9 during sulfur deprivation can be explained by its low sulfur content as compared with the other LHCBMs. Considering the highly conserved biochemical and spectroscopic properties, the major difference between the complexes may be in their capacity to interact with other components of the thylakoid membrane. PMID:25723534

  8. Independent Control of the Static and Dynamic Components of the Chlamydomonas Flagellar Beat.

    PubMed

    Geyer, Veikko F; Sartori, Pablo; Friedrich, Benjamin M; Jülicher, Frank; Howard, Jonathon

    2016-04-25

    When the green alga Chlamydomonas reinhardtii swims, it uses the breaststroke beat of its two flagella to pull itself forward [1]. The flagellar waveform can be decomposed into a static component, corresponding to an asymmetric time-averaged shape, and a dynamic component, corresponding to the time-varying wave [2]. Extreme lightening conditions photoshock the cell, converting the breaststroke beat into a symmetric sperm-like beat, which causes a reversal of the direction of swimming [3]. Waveform conversion is achieved by a reduction in magnitude of the static component, whereas the dynamic component remains unchanged [2]. The coupling between static and dynamic components, however, is poorly understood, and it is not known whether the static component requires the dynamic component or whether it can exist independently. We used isolated and reactivated axonemes [4] to investigate the relation between the two beat components. We discovered that, when reactivated in the presence of low ATP concentrations, axonemes displayed the static beat component in absence of the dynamic component. Furthermore, we found that the amplitudes of the two components depend on ATP in qualitatively different ways. These results show that the decomposition into static and dynamic components is not just a mathematical concept but that the two components can independently control different aspects of cell motility: the static component controls swimming direction, whereas the dynamic component provides propulsion. PMID:27040779

  9. Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography

    PubMed Central

    Engel, Benjamin D; Schaffer, Miroslava; Kuhn Cuellar, Luis; Villa, Elizabeth; Plitzko, Jürgen M; Baumeister, Wolfgang

    2015-01-01

    Chloroplast function is orchestrated by the organelle's intricate architecture. By combining cryo-focused ion beam milling of vitreous Chlamydomonas cells with cryo-electron tomography, we acquired three-dimensional structures of the chloroplast in its native state within the cell. Chloroplast envelope inner membrane invaginations were frequently found in close association with thylakoid tips, and the tips of multiple thylakoid stacks converged at dynamic sites on the chloroplast envelope, implicating lipid transport in thylakoid biogenesis. Subtomogram averaging and nearest neighbor analysis revealed that RuBisCO complexes were hexagonally packed within the pyrenoid, with ∼15 nm between their centers. Thylakoid stacks and the pyrenoid were connected by cylindrical pyrenoid tubules, physically bridging the sites of light-dependent photosynthesis and light-independent carbon fixation. Multiple parallel minitubules were bundled within each pyrenoid tubule, possibly serving as conduits for the targeted one-dimensional diffusion of small molecules such as ATP and sugars between the chloroplast stroma and the pyrenoid matrix. DOI: http://dx.doi.org/10.7554/eLife.04889.001 PMID:25584625

  10. Cell adhesion-dependent inactivation of a soluble protein kinase during fertilization in Chlamydomonas.

    PubMed Central

    Zhang, Y; Luo, Y; Emmett, K; Snell, W J

    1996-01-01

    Within seconds after the flagella of mt+ and mt- Chlamydomonas gametes adhere during fertilization, their flagellar adenylyl cyclase is activated several fold and preparation for cell fusion is initiated. Our previous studies indicated that early events in this pathway, including control of adenylyl cyclase, are regulated by phosphorylation and dephosphorylation. Here, we describe a soluble, flagellar protein kinase activity that is regulated by flagellar adhesion. A 48-kDa, soluble flagellar protein was consistently phosphorylated in an in vitro assay in flagella isolated from nonadhering mt+ and mt- gametes, but not in flagella isolated from mt+ and mt- gametes that had been adhering for 1 min. Although the 48-kDa protein was present in the flagella isolated from adhering gametes, we demonstrate that its protein kinase was inactivated by flagellar adhesion. Immunoblot analysis and inhibitor studies indicate that the 48-kDa protein in nonadhering gametes is phosphorylated by a protein tyrosine kinase. In vivo experiments showing that the protein tyrosine phosphatase inhibitor sodium orthovanadate inhibits fertilization suggest that protein dephosphorylation may be required for signal transduction. The 48-kDa protein and its protein kinase may be among the first elements of a novel signalling pathway that couples interaction of flagellar adhesion molecules to gamete activation. Images PMID:8730096

  11. Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas

    DOE PAGESBeta

    Hong-Hermesdorf, Anne; Miethke, Marcus; Gallaher, Sean D.; Kropat, Janette; Dodani, Sheel C.; Chan, Jefferson; Barupala, Dulmini; Domaille, Dylan W.; Shirasaki, Dyna I.; Loo, Joseph A.; et al

    2014-10-26

    Here we identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu+ accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labelingmore » demonstrated that sequestered Cu+ became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.« less

  12. Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas

    SciTech Connect

    Hong-Hermesdorf, Anne; Miethke, Marcus; Gallaher, Sean D.; Kropat, Janette; Dodani, Sheel C.; Chan, Jefferson; Barupala, Dulmini; Domaille, Dylan W.; Shirasaki, Dyna I.; Loo, Joseph A.; Weber, Peter K.; Pett-Ridge, Jennifer; Stemmler, Timothy L.; Chang, Christopher J.; Merchant, Sabeeha S.

    2014-10-26

    Here we identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu+ accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu+ became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

  13. Identification of regulatory network hubs that control lipid metabolism in Chlamydomonas reinhardtii.

    PubMed

    Gargouri, Mahmoud; Park, Jeong-Jin; Holguin, F Omar; Kim, Min-Jeong; Wang, Hongxia; Deshpande, Rahul R; Shachar-Hill, Yair; Hicks, Leslie M; Gang, David R

    2015-08-01

    Microalgae-based biofuels are promising sources of alternative energy, but improvements throughout the production process are required to establish them as economically feasible. One of the most influential improvements would be a significant increase in lipid yields, which could be achieved by altering the regulation of lipid biosynthesis and accumulation. Chlamydomonas reinhardtii accumulates oil (triacylglycerols, TAG) in response to nitrogen (N) deprivation. Although a few important regulatory genes have been identified that are involved in controlling this process, a global understanding of the larger regulatory network has not been developed. In order to uncover this network in this species, a combined omics (transcriptomic, proteomic and metabolomic) analysis was applied to cells grown in a time course experiment after a shift from N-replete to N-depleted conditions. Changes in transcript and protein levels of 414 predicted transcription factors (TFs) and transcriptional regulators (TRs) were monitored relative to other genes. The TF and TR genes were thus classified by two separate measures: up-regulated versus down-regulated and early response versus late response relative to two phases of polar lipid synthesis (before and after TAG biosynthesis initiation). Lipidomic and primary metabolite profiling generated compound accumulation levels that were integrated with the transcript dataset and TF profiling to produce a transcriptional regulatory network. Evaluation of this proposed regulatory network led to the identification of several regulatory hubs that control many aspects of cellular metabolism, from N assimilation and metabolism, to central metabolism, photosynthesis and lipid metabolism. PMID:26022256

  14. The regulation of photosynthetic structure and function during nitrogen deprivation in Chlamydomonas reinhardtii.

    PubMed

    Juergens, Matthew T; Deshpande, Rahul R; Lucker, Ben F; Park, Jeong-Jin; Wang, Hongxia; Gargouri, Mahmoud; Holguin, F Omar; Disbrow, Bradley; Schaub, Tanner; Skepper, Jeremy N; Kramer, David M; Gang, David R; Hicks, Leslie M; Shachar-Hill, Yair

    2015-02-01

    The accumulation of carbon storage compounds by many unicellular algae after nutrient deprivation occurs despite declines in their photosynthetic apparatus. To understand the regulation and roles of photosynthesis during this potentially bioenergetically valuable process, we analyzed photosynthetic structure and function after nitrogen deprivation in the model alga Chlamydomonas reinhardtii. Transcriptomic, proteomic, metabolite, and lipid profiling and microscopic time course data were combined with multiple measures of photosynthetic function. Levels of transcripts and proteins of photosystems I and II and most antenna genes fell with differing trajectories; thylakoid membrane lipid levels decreased, while their proportions remained similar and thylakoid membrane organization appeared to be preserved. Cellular chlorophyll (Chl) content decreased more than 2-fold within 24 h, and we conclude from transcript protein and (13)C labeling rates that Chl synthesis was down-regulated both pre- and posttranslationally and that Chl levels fell because of a rapid cessation in synthesis and dilution by cellular growth rather than because of degradation. Photosynthetically driven oxygen production and the efficiency of photosystem II as well as P700(+) reduction and electrochromic shift kinetics all decreased over the time course, without evidence of substantial energy overflow. The results also indicate that linear electron flow fell approximately 15% more than cyclic flow over the first 24 h. Comparing Calvin-Benson cycle transcript and enzyme levels with changes in photosynthetic (13)CO2 incorporation rates also pointed to a coordinated multilevel down-regulation of photosynthetic fluxes during starch synthesis before the induction of high triacylglycerol accumulation rates. PMID:25489023

  15. The response of Chlamydomonas reinhardtii to nitrogen deprivation: a systems biology analysis.

    PubMed

    Park, Jeong-Jin; Wang, Hongxia; Gargouri, Mahmoud; Deshpande, Rahul R; Skepper, Jeremy N; Holguin, F Omar; Juergens, Matthew T; Shachar-Hill, Yair; Hicks, Leslie M; Gang, David R

    2015-02-01

    Drastic alteration in macronutrients causes large changes in gene expression in the photosynthetic unicellular alga Chlamydomonas reinhardtii. Preliminary data suggested that cells follow a biphasic response to this change hinging on the initiation of lipid accumulation, and we hypothesized that drastic repatterning of metabolism also followed this biphasic modality. To test this hypothesis, transcriptomic, proteomic, and metabolite changes that occur under nitrogen (N) deprivation were analyzed. Eight sampling times were selected covering the progressive slowing of growth and induction of oil synthesis between 4 and 6 h after N deprivation. Results of the combined, systems-level investigation indicated that C. reinhardtii cells sense and respond on a large scale within 30 min to a switch to N-deprived conditions turning on a largely gluconeogenic metabolic state, which then transitions to a glycolytic stage between 4 and 6 h after N depletion. This nitrogen-sensing system is transduced to carbon- and nitrogen-responsive pathways, leading to down-regulation of carbon assimilation and chlorophyll biosynthesis, and an increase in nitrogen metabolism and lipid biosynthesis. For example, the expression of nearly all the enzymes for assimilating nitrogen from ammonium, nitrate, nitrite, urea, formamide/acetamide, purines, pyrimidines, polyamines, amino acids and proteins increased significantly. Although arginine biosynthesis enzymes were also rapidly up-regulated, arginine pool size changes and isotopic labeling results indicated no increased flux through this pathway. PMID:25515814

  16. Sub-cellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas

    PubMed Central

    Hong-Hermesdorf, Anne; Miethke, Marcus; Gallaher, Sean D; Kropat, Janette; Dodani, Sheel C; Chan, Jefferson; Barupala, Dulmini; Domaille, Dylan W; Shirasaki, Dyna I; Loo, Joseph A; Weber, Peter K; Pett-Ridge, Jennifer; Stemmler, Timothy L; Chang, Christopher J; Merchant, Sabeeha S

    2014-01-01

    We identified a Cu accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulated Cu, dependent on the nutritional Cu sensor CRR1, but was functionally Cu-deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. NanoSIMS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy (XAS) was consistent with Cu+ accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu+ became bio-available for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mis-metallation during Zn deficiency and enabling efficient cuproprotein (re)-metallation upon Zn resupply. PMID:25344811

  17. The sac mutants of Chlamydomonas reinhardtii reveal transcriptional and posttranscriptional control of cysteine biosynthesis.

    PubMed

    Ravina, Cristina G; Chang, Chwenn-In; Tsakraklides, George P; McDermott, Jeffery P; Vega, Jose M; Leustek, Thomas; Gotor, Cecilia; Davies, John P

    2002-12-01

    Algae and vascular plants are cysteine (Cys) prototrophs. They are able to import, reduce, and assimilate sulfate into Cys, methionine, and other organic sulfur-containing compounds. Characterization of genes encoding the enzymes required for Cys biosynthesis from the unicellular green alga Chlamydomonas reinhardtii reveals that transcriptional and posttranscriptional mechanisms regulate the pathway. The derived amino acid sequences of the C. reinhardtii genes encoding 5'-adenylylsulfate (APS) reductase and serine (Ser) acetyltransferase are orthologous to sequences from vascular plants. The Cys biosynthetic pathway of C. reinhardtii is regulated by sulfate availability. The steady-state level of transcripts and activity of ATP sulfurylase, APS reductase, Ser acetyltransferase, and O-acetyl-Ser (thiol) lyase increase when cells are deprived of sulfate. The sac1 mutation, which impairs C. reinhardtii ability to acclimate to sulfur-deficient conditions, prevents the increase in accumulation of the transcripts encoding these enzymes and also prevents the increase in activity of all the enzymes except APS reductase. The sac2 mutation, which does not affect accumulation of APS reductase transcripts, blocks the increase in APS reductase activity. These results suggest that APS reductase activity is regulated posttranscriptionally in a SAC2-dependent process. PMID:12481091

  18. Extensive de novo mutation rate variation between individuals and across the genome of Chlamydomonas reinhardtii

    PubMed Central

    Ness, Rob W.; Morgan, Andrew D.; Vasanthakrishnan, Radhakrishnan B.; Colegrave, Nick; Keightley, Peter D.

    2015-01-01

    Describing the process of spontaneous mutation is fundamental for understanding the genetic basis of disease, the threat posed by declining population size in conservation biology, and much of evolutionary biology. Directly studying spontaneous mutation has been difficult, however, because new mutations are rare. Mutation accumulation (MA) experiments overcome this by allowing mutations to build up over many generations in the near absence of natural selection. Here, we sequenced the genomes of 85 MA lines derived from six genetically diverse strains of the green alga Chlamydomonas reinhardtii. We identified 6843 new mutations, more than any other study of spontaneous mutation. We observed sevenfold variation in the mutation rate among strains and that mutator genotypes arose, increasing the mutation rate approximately eightfold in some replicates. We also found evidence for fine-scale heterogeneity in the mutation rate, with certain sequence motifs mutating at much higher rates, and clusters of multiple mutations occurring at closely linked sites. There was little evidence, however, for mutation rate heterogeneity between chromosomes or over large genomic regions of 200 kbp. We generated a predictive model of the mutability of sites based on their genomic properties, including local GC content, gene expression level, and local sequence context. Our model accurately predicted the average mutation rate and natural levels of genetic diversity of sites across the genome. Notably, trinucleotides vary 17-fold in rate between the most and least mutable sites. Our results uncover a rich heterogeneity in the process of spontaneous mutation both among individuals and across the genome. PMID:26260971

  19. Identification of Novel Mitochondrial Protein Components of Chlamydomonas reinhardtii. A Proteomic Approach1

    PubMed Central

    van Lis, Robert; Atteia, Ariane; Mendoza-Hernández, Guillermo; González-Halphen, Diego

    2003-01-01

    Pure mitochondria of the photosynthetic alga Chlamydomonas reinhardtii were analyzed using blue native-polyacrylamide gel electrophoresis (BN-PAGE). The major oxidative phosphorylation complexes were resolved: F1F0-ATP synthase, NADH-ubiquinone oxidoreductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase. The oligomeric states of these complexes were determined. The F1F0-ATP synthase runs exclusively as a dimer, in contrast to the C. reinhardtii chloroplast enzyme, which is present as a monomer and subcomplexes. The sequence of a 60-kD protein, associated with the mitochondrial ATP synthase and with no known counterpart in any other organism, is reported. This protein may be related to the strong dimeric character of the algal F1F0-ATP synthase. The oxidative phosphorylation complexes resolved by BN-PAGE were separated into their subunits by second dimension sodium dodecyl sulfate-PAGE. A number of polypeptides were identified mainly on the basis of their N-terminal sequence. Core I and II subunits of complex III were characterized, and their proteolytic activities were predicted. Also, the heterodimeric nature of COXIIA and COXIIB subunits in cytochrome c oxidase was demonstrated. Other mitochondrial proteins like the chaperone HSP60, the alternative oxidase, the aconitase, and the ADP/ATP carrier were identified. BN-PAGE was also used to approach the analysis of the major chloroplast protein complexes of C. reinhardtii. PMID:12746537

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

    PubMed

    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. PMID:25641390

  1. The Proteome of Copper, Iron, Zinc, and Manganese Micronutrient Deficiency in Chlamydomonas reinhardtii*

    PubMed Central

    Hsieh, Scott I.; Castruita, Madeli; Malasarn, Davin; Urzica, Eugen; Erde, Jonathan; Page, M. Dudley; Yamasaki, Hiroaki; Casero, David; Pellegrini, Matteo; Merchant, Sabeeha S.; Loo, Joseph A.

    2013-01-01

    Trace metals such as copper, iron, zinc, and manganese play important roles in several biochemical processes, including respiration and photosynthesis. Using a label-free, quantitative proteomics strategy (MSE), we examined the effect of deficiencies in these micronutrients on the soluble proteome of Chlamydomonas reinhardtii. We quantified >103 proteins with abundances within a dynamic range of 3 to 4 orders of magnitude and demonstrated statistically significant changes in ∼200 proteins in each metal-deficient growth condition relative to nutrient-replete media. Through analysis of Pearson's coefficient, we also examined the correlation between protein abundance and transcript abundance (as determined via RNA-Seq analysis) and found moderate correlations under all nutritional states. Interestingly, in a subset of transcripts known to significantly change in abundance in metal-replete and metal-deficient conditions, the correlation to protein abundance is much stronger. Examples of new discoveries highlighted in this work include the accumulation of O2 labile, anaerobiosis-related enzymes (Hyd1, Pfr1, and Hcp2) in copper-deficient cells; co-variation of Cgl78/Ycf54 and coprogen oxidase; the loss of various stromal and lumenal photosynthesis-related proteins, including plastocyanin, in iron-limited cells; a large accumulation (from undetectable amounts to over 1,000 zmol/cell) of two COG0523 domain-containing proteins in zinc-deficient cells; and the preservation of photosynthesis proteins in manganese-deficient cells despite known losses in photosynthetic function in this condition. PMID:23065468

  2. The selective breeding of the freshwater microalga Chlamydomonas reinhardtii for growth in salinity.

    PubMed

    Takouridis, Simon J; Tribe, David E; Gras, Sally L; Martin, Gregory J O

    2015-05-01

    The potential for Chlamydomonas reinhardtii to be utilized for biofuel production was strengthened by developing it for growth in elevated salinity via the selective breeding method of genome shuffling. A population was constructed via random mutagenesis and subjected to multiple rounds of sex and growth in increasing salinity. This sexual line was capable of growth in up to 700 mM NaCl, unlike its progenitor, which could only grow in 300 mM NaCl. An asexual control line was capable of growth in 500 mM NaCl. Palmelloid aggregations increased in size and the concentration of final biomass decreased as a function of NaCl concentration, which poses considerations for future strain development. The sexual line maintained sexual efficiencies of up to 50% over the course of selection. This investigation achieved significant strain improvement of C. reinhardtii and demonstrated the clear advantage of its ability to participate in laboratory controlled and reproducible high efficiency sex. PMID:25466995

  3. The Global Phosphoproteome of Chlamydomonas reinhardtii Reveals Complex Organellar Phosphorylation in the Flagella and Thylakoid Membrane *

    PubMed Central

    Wang, Hongxia; Gau, Brian; Slade, William O.; Juergens, Matthew; Li, Ping; Hicks, Leslie M.

    2014-01-01

    Chlamydomonas reinhardtii is the most intensively-studied and well-developed model for investigation of a wide-range of microalgal processes ranging from basic development through understanding triacylglycerol production. Although proteomic technologies permit interrogation of these processes at the protein level and efforts to date indicate phosphorylation-based regulation of proteins in C. reinhardtii is essential for its underlying biology, characterization of the C. reinhardtii phosphoproteome has been limited. Herein, we report the richest exploration of the C. reinhardtii proteome to date. Complementary enrichment strategies were used to detect 4588 phosphoproteins distributed among every cellular component in C. reinhardtii. Additionally, we report 18,160 unique phosphopeptides at <1% false discovery rate, which comprise 15,862 unique phosphosites - 98% of which are novel. Given that an estimated 30% of proteins in a eukaryotic cell are subject to phosphorylation, we report the majority of the phosphoproteome (23%) of C. reinhardtii. Proteins in key biological pathways were phosphorylated, including photosynthesis, pigment production, carbon assimilation, glycolysis, and protein and carbohydrate metabolism, and it is noteworthy that hyperphosphorylation was observed in flagellar proteins. This rich data set is available via ProteomeXchange (ID: PXD000783) and will significantly enhance understanding of a range of regulatory mechanisms controlling a variety of cellular process and will serve as a critical resource for the microalgal community. PMID:24917610

  4. Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures*

    PubMed Central

    Mathieu-Rivet, Elodie; Scholz, Martin; Arias, Carolina; Dardelle, Flavien; Schulze, Stefan; Le Mauff, François; Teo, Gavin; Hochmal, Ana Karina; Blanco-Rivero, Amaya; Loutelier-Bourhis, Corinne; Kiefer-Meyer, Marie-Christine; Fufezan, Christian; Burel, Carole; Lerouge, Patrice; Martinez, Flor; Bardor, Muriel; Hippler, Michael

    2013-01-01

    Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-O-methylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of 18O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants. PMID:23912651

  5. Isolation of Chlamydomonas reinhardtii mutants with altered mitochondrial respiration by chlorophyll fluorescence measurement.

    PubMed

    Massoz, Simon; Larosa, Véronique; Horrion, Bastien; Matagne, René F; Remacle, Claire; Cardol, Pierre

    2015-12-10

    The unicellular green alga Chlamydomonas reinhardtii is a model organism for studying energetic metabolism. Most mitochondrial respiratory-deficient mutants characterized to date have been isolated on the basis of their reduced ability to grow in heterotrophic conditions. Mitochondrial deficiencies are usually partly compensated by adjustment of photosynthetic activity and more particularly by transition to state 2. In this work, we explored the opportunity to select mutants impaired in respiration and/or altered in dark metabolism by measuring maximum photosynthetic efficiency by chlorophyll fluorescence analyses (FV/FM). Out of about 2900 hygromycin-resistant insertional mutants generated from wild type or from a mutant strain deficient in state transitions (stt7 strain), 22 were found to grow slowly in heterotrophic conditions and 8 of them also showed a lower FV/FM value. Several disrupted coding sequences were identified, including genes coding for three different subunits of respiratory-chain complex I (NUO9, NUOA9, NUOP4) or for isocitrate lyase (ICL1). Overall, the comparison of respiratory mutants obtained in wild-type or stt7 genetic backgrounds indicated that the FV/FM value can be used to isolate mutants severely impaired in dark metabolism. PMID:26022424

  6. Phosphoprotein SAK1 is a regulator of acclimation to singlet oxygen in Chlamydomonas reinhardtii

    PubMed Central

    Wakao, Setsuko; Chin, Brian L; Ledford, Heidi K; Dent, Rachel M; Casero, David; Pellegrini, Matteo; Merchant, Sabeeha S; Niyogi, Krishna K

    2014-01-01

    Singlet oxygen is a highly toxic and inevitable byproduct of oxygenic photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is capable of acclimating specifically to singlet oxygen stress, but the retrograde signaling pathway from the chloroplast to the nucleus mediating this response is unknown. Here we describe a mutant, singlet oxygen acclimation knocked-out 1 (sak1), that lacks the acclimation response to singlet oxygen. Analysis of genome-wide changes in RNA abundance during acclimation to singlet oxygen revealed that SAK1 is a key regulator of the gene expression response during acclimation. The SAK1 gene encodes an uncharacterized protein with a domain conserved among chlorophytes and present in some bZIP transcription factors. The SAK1 protein is located in the cytosol, and it is induced and phosphorylated upon exposure to singlet oxygen, suggesting that it is a critical intermediate component of the retrograde signal transduction pathway leading to singlet oxygen acclimation. DOI: http://dx.doi.org/10.7554/eLife.02286.001 PMID:24859755

  7. Eyespot-dependent determination of the phototactic sign in Chlamydomonas reinhardtii.

    PubMed

    Ueki, Noriko; Ide, Takahiro; Mochiji, Shota; Kobayashi, Yuki; Tokutsu, Ryutaro; Ohnishi, Norikazu; Yamaguchi, Katsushi; Shigenobu, Shuji; Tanaka, Kan; Minagawa, Jun; Hisabori, Toru; Hirono, Masafumi; Wakabayashi, Ken-Ichi

    2016-05-10

    The biflagellate green alga Chlamydomonas reinhardtii exhibits both positive and negative phototaxis to inhabit areas with proper light conditions. It has been shown that treatment of cells with reactive oxygen species (ROS) reagents biases the phototactic sign to positive, whereas that with ROS scavengers biases it to negative. Taking advantage of this property, we isolated a mutant, lts1-211, which displays a reduction-oxidation (redox) dependent phototactic sign opposite to that of the wild type. This mutant has a single amino acid substitution in phytoene synthase, an enzyme that functions in the carotenoid-biosynthesis pathway. The eyespot contains large amounts of carotenoids and is crucial for phototaxis. Most lts1-211 cells have no detectable eyespot and reduced carotenoid levels. Interestingly, the reversed phototactic-sign phenotype of lts1-211 is shared by other eyespot-less mutants. In addition, we directly showed that the cell body acts as a convex lens. The lens effect of the cell body condenses the light coming from the rear onto the photoreceptor in the absence of carotenoid layers, which can account for the reversed-phototactic-sign phenotype of the mutants. These results suggest that light-shielding property of the eyespot is essential for determination of phototactic sign. PMID:27122315

  8. Induction and Segregation of Chloroplast Mutations in Vegetative Cell Cultures of Chlamydomonas Reinhardtii

    PubMed Central

    Lee, Robert W.; Haughn, George W.

    1980-01-01

    The single chloroplast of the alga Chlamydomonas reinhardtii contains at least 100 copies of the chloroplast chromosome. It is not known how the chloroplast (or cell) becomes homoplasmic for a mutation that arises in one of these copies. Under suitable selection conditions, clones with chloroplast mutations for streptomycin resistance induced by methyl methanesulfonate can be recovered with direct plating after mutagenesis. Using an adaptation of the Luria-Delbrück fluctuation test, mutagenized cultures grown on nonselective liquid medium for seven to nine doublings show negligible proliferation of cells capable of forming such mutant colonies. In contrast, cells among the same cultures with reduced nuclear mutations conferring streptomycin resistance reveal considerable clonal propagation prior to plating on selection medium. Reconstruction growth-rate experiments show no reduced growth of cells with chloroplast mutations relative to either wild-type cells or to those with nuclear mutations. We propose that newly arising chloroplast mutations and their copies are usually transmitted to only one daughter cell for several cell generations by reductional divisions of the chloroplast genome. In the absence of recombination and mixing, such a reductional partition of chloroplast alleles would readily permit the formation of homoplasmic lines without the need for selection. PMID:17249064

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

    PubMed

    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% (p<0.001) for the growth of this microalga 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. PMID:26963118

  10. The Awesome Power of Dikaryons for Studying Flagella and Basal Bodies in Chlamydomonas reinhardtii

    PubMed Central

    Dutcher, Susan K.

    2014-01-01

    Cilia/flagella and basal bodies/centrioles play key roles in human health and homeostasis. Among the organisms used to study these microtubule-based organelles, the green alga Chlamydomonas reinhardtii has several advantages. One is the existence of a temporary phase of the life cycle, termed the dikaryon. These cells are formed during mating when the cells fuse and the behavior of flagella from two genetically distinguishable parents can be observed. During this stage, the cytoplasms mix allowing for a defect in the flagella of one parent to be rescued by proteins from the other parent. This offers the unique advantage of adding back wild-type gene product or labeled protein at endogenous levels that can used to monitor various flagellar and basal body phenotypes. Mutants that show rescue and ones that fail to show rescue are both informative about the nature of the flagella and basal body defects. When rescue occurs, it can be used to determine the mutant gene product and to follow the temporal and spatial patterns of flagellar assembly. This review describes many examples of insights into basal body and flagellar proteins’ function and assembly that have been discovered using dikaryons and discusses the potential for further analyses. PMID:24272949

  11. Rubisco small-subunit α-helices control pyrenoid formation in Chlamydomonas

    PubMed Central

    Meyer, Moritz T.; Genkov, Todor; Skepper, Jeremy N.; Jouhet, Juliette; Mitchell, Madeline C.; Spreitzer, Robert J.; Griffiths, Howard

    2012-01-01

    The pyrenoid is a subcellular microcompartment in which algae sequester the primary carboxylase, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The pyrenoid is associated with a CO2-concentrating mechanism (CCM), which improves the operating efficiency of carbon assimilation and overcomes diffusive limitations in aquatic photosynthesis. Using the model alga Chlamydomonas reinhardtii, we show that pyrenoid formation, Rubisco aggregation, and CCM activity relate to discrete regions of the Rubisco small subunit (SSU). Specifically, pyrenoid occurrence was shown to be conditioned by the amino acid composition of two surface-exposed α-helices of the SSU: higher plant-like helices knock out the pyrenoid, whereas native algal helices establish a pyrenoid. We have also established that pyrenoid integrity was essential for the operation of an active CCM. With the algal CCM being functionally analogous to the terrestrial C4 pathway in higher plants, such insights may offer a route toward transforming algal and higher plant productivity for the future. PMID:23112177

  12. Effect of Chlamydomonas plastid terminal oxidase 1 expressed in tobacco on photosynthetic electron transfer.

    PubMed

    Feilke, Kathleen; Streb, Peter; Cornic, Gabriel; Perreau, François; Kruk, Jerzy; Krieger-Liszkay, Anja

    2016-01-01

    The plastid terminal oxidase PTOX is a plastohydroquinone:oxygen oxidoreductase that is important for carotenoid biosynthesis and plastid development. Its role in photosynthesis is controversially discussed. Under a number of abiotic stress conditions, the protein level of PTOX increases. PTOX is thought to act as a safety valve under high light protecting the photosynthetic apparatus against photodamage. However, transformants with high PTOX level were reported to suffer from photoinhibition. To analyze the effect of PTOX on the photosynthetic electron transport, tobacco expressing PTOX-1 from Chlamydomonas reinhardtii (Cr-PTOX1) was studied by chlorophyll fluorescence, thermoluminescence, P700 absorption kinetics and CO2 assimilation. Cr-PTOX1 was shown to compete very efficiently with the photosynthetic electron transport for PQH2 . High pressure liquid chromatography (HPLC) analysis confirmed that the PQ pool was highly oxidized in the transformant. Immunoblots showed that, in the wild-type, PTOX was associated with the thylakoid membrane only at a relatively alkaline pH value while it was detached from the membrane at neutral pH. We present a model proposing that PTOX associates with the membrane and oxidizes PQH2 only when the oxidation of PQH2 by the cytochrome b6 f complex is limiting forward electron transport due to a high proton gradient across the thylakoid membrane. PMID:26663146

  13. Phytohormone supplementation significantly increases growth of Chlamydomonas reinhardtii cultivated for biodiesel production.

    PubMed

    Park, Won-Kun; Yoo, Gursong; Moon, Myounghoon; Kim, Chul Woong; Choi, Yoon-E; Yang, Ji-Won

    2013-11-01

    Cultivation is the most expensive step in the production of biodiesel from microalgae, and substantial research has been devoted to developing more cost-effective cultivation methods. Plant hormones (phytohormones) are chemical messengers that regulate various aspects of growth and development and are typically active at very low concentrations. In this study, we investigated the effect of different phytohormones on microalgal growth and biodiesel production in Chlamydomonas reinhardtii and their potential to lower the overall cost of commercial biofuel production. The results indicated that all five of the tested phytohormones (indole-3-acetic acid, gibberellic acid, kinetin, 1-triacontanol, and abscisic acid) promoted microalgal growth. In particular, hormone treatment increased biomass production by 54 to 69 % relative to the control growth medium (Tris-acetate-phosphate, TAP). Phytohormone treatments also affected microalgal cell morphology but had no effect on the yields of fatty acid methyl esters (FAMEs) as a percent of biomass. We also tested the effect of these phytohormones on microalgal growth in nitrogen-limited media by supplementation in the early stationary phase. Maximum cell densities after addition of phytohormones were higher than in TAP medium, even when the nitrogen source was reduced to 40 % of that in TAP medium. Taken together, our results indicate that phytohormones significantly increased microalgal growth, particularly in nitrogen-limited media, and have potential for use in the development of efficient microalgal cultivation for biofuel production. PMID:23881782

  14. The Regulation of Photosynthetic Structure and Function during Nitrogen Deprivation in Chlamydomonas reinhardtii1[OPEN

    PubMed Central

    Juergens, Matthew T.; Deshpande, Rahul R.; Lucker, Ben F.; Park, Jeong-Jin; Wang, Hongxia; Gargouri, Mahmoud; Holguin, F. Omar; Disbrow, Bradley; Schaub, Tanner; Skepper, Jeremy N.; Kramer, David M.; Gang, David R.; Hicks, Leslie M.; Shachar-Hill, Yair

    2015-01-01

    The accumulation of carbon storage compounds by many unicellular algae after nutrient deprivation occurs despite declines in their photosynthetic apparatus. To understand the regulation and roles of photosynthesis during this potentially bioenergetically valuable process, we analyzed photosynthetic structure and function after nitrogen deprivation in the model alga Chlamydomonas reinhardtii. Transcriptomic, proteomic, metabolite, and lipid profiling and microscopic time course data were combined with multiple measures of photosynthetic function. Levels of transcripts and proteins of photosystems I and II and most antenna genes fell with differing trajectories; thylakoid membrane lipid levels decreased, while their proportions remained similar and thylakoid membrane organization appeared to be preserved. Cellular chlorophyll (Chl) content decreased more than 2-fold within 24 h, and we conclude from transcript protein and 13C labeling rates that Chl synthesis was down-regulated both pre- and posttranslationally and that Chl levels fell because of a rapid cessation in synthesis and dilution by cellular growth rather than because of degradation. Photosynthetically driven oxygen production and the efficiency of photosystem II as well as P700+ reduction and electrochromic shift kinetics all decreased over the time course, without evidence of substantial energy overflow. The results also indicate that linear electron flow fell approximately 15% more than cyclic flow over the first 24 h. Comparing Calvin-Benson cycle transcript and enzyme levels with changes in photosynthetic 13CO2 incorporation rates also pointed to a coordinated multilevel down-regulation of photosynthetic fluxes during starch synthesis before the induction of high triacylglycerol accumulation rates. PMID:25489023

  15. A Simple and Non-Invasive Method for Nuclear Transformation of Intact-walled Chlamydomonas reinhardtii

    PubMed Central

    Kim, Sora; Lee, Young-Chul; Cho, Dae-Hyun; Lee, Hyun Uk; Huh, Yun Suk; Kim, Geun-Joong; Kim, Hee-Sik

    2014-01-01

    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(CH2)3]8Si8Mg6O12(OH)4), 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 (5.03×102 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. PMID:24988123

  16. Oxidative stress contributes to autophagy induction in response to endoplasmic reticulum stress in Chlamydomonas reinhardtii.

    PubMed

    Pérez-Martín, Marta; Pérez-Pérez, María Esther; Lemaire, Stéphane D; Crespo, José L

    2014-10-01

    The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) results in the activation of stress responses, such as the unfolded protein response or the catabolic process of autophagy to ultimately recover cellular homeostasis. ER stress also promotes the production of reactive oxygen species, which play an important role in autophagy regulation. However, it remains unknown whether reactive oxygen species are involved in ER stress-induced autophagy. In this study, we provide evidence connecting redox imbalance caused by ER stress and autophagy activation in the model unicellular green alga Chlamydomonas reinhardtii. Treatment of C. reinhardtii cells with the ER stressors tunicamycin or dithiothreitol resulted in up-regulation of the expression of genes encoding ER resident endoplasmic reticulum oxidoreductin1 oxidoreductase and protein disulfide isomerases. ER stress also triggered autophagy in C. reinhardtii based on the protein abundance, lipidation, cellular distribution, and mRNA levels of the autophagy marker ATG8. Moreover, increases in the oxidation of the glutathione pool and the expression of oxidative stress-related genes were detected in tunicamycin-treated cells. Our results revealed that the antioxidant glutathione partially suppressed ER stress-induced autophagy and decreased the toxicity of tunicamycin, suggesting that oxidative stress participates in the control of autophagy in response to ER stress in C. reinhardtii In close agreement, we also found that autophagy activation by tunicamycin was more pronounced in the C. reinhardtii sor1 mutant, which shows increased expression of oxidative stress-related genes. PMID:25143584

  17. Critical Function of a Chlamydomonas reinhardtii Putative Polyphosphate Polymerase Subunit during Nutrient Deprivation[C][W

    PubMed Central

    Aksoy, Munevver; Pootakham, Wirulda; Grossman, Arthur R.

    2014-01-01

    Forward genetics was used to isolate Chlamydomonas reinhardtii mutants with altered abilities to acclimate to sulfur (S) deficiency. The ars76 mutant has a deletion that eliminates several genes, including VACUOLAR TRANSPORTER CHAPERONE1 (VTC1), which encodes a component of a polyphosphate polymerase complex. The ars76 mutant cannot accumulate arylsulfatase protein or mRNA and shows marked alterations in levels of many transcripts encoded by genes induced during S deprivation. The mutant also shows little acidocalcisome formation compared with wild-type, S-deprived cells and dies more rapidly than wild-type cells following exposure to S-, phosphorus-, or nitrogen (N)-deficient conditions. Furthermore, the mutant does not accumulate periplasmic l-amino acid oxidase during N deprivation. Introduction of the VTC1 gene specifically complements the ars76 phenotypes, suggesting that normal acidocalcisome formation in cells deprived of S requires VTC1. Our data also indicate that a deficiency in acidocalcisome function impacts trafficking of periplasmic proteins, which can then feed back on the transcription of the genes encoding these proteins. These results and the reported function of vacuoles in degradation processes suggest a major role of the acidocalcisome in reshaping the cell during acclimation to changing environmental conditions. PMID:25281687

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

    PubMed

    Jamers, An; Blust, Ronny; De Coen, Wim; Griffin, Julian L; Jones, Oliver A H

    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 μM 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 μM 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 μM 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. PMID:23063003

  19. The metabolome of Chlamydomonas reinhardtii following induction of anaerobic H2 production by sulfur depletion.

    PubMed

    Matthew, Timmins; Zhou, Wenxu; Rupprecht, Jens; Lim, Lysha; Thomas-Hall, Skye R; Doebbe, Anja; Kruse, Olaf; Hankamer, Ben; Marx, Ute C; Smith, Steven M; Schenk, Peer M

    2009-08-28

    The metabolome of the model species Chlamydomonas reinhardtii has been analyzed during 120 h of sulfur depletion to induce anaerobic hydrogen (H(2)) production, using NMR spectroscopy, gas chromatography coupled to mass spectrometry, and TLC. The results indicate that these unicellular green algae consume freshly supplied acetate in the medium to accumulate energy reserves during the first 24 h of sulfur depletion. In addition to the previously reported accumulation of starch, large amounts of triacylglycerides were deposited in the cells. During the early 24- to 72-h time period fermentative energy metabolism lowered the pH, H(2) was produced, and amino acid levels generally increased. In the final phase from 72 to 120 h, metabolism slowed down leading to a stabilization of pH, even though some starch and most triacylglycerides remained. We conclude that H(2) production does not slow down due to depletion of energy reserves but rather due to loss of essential functions resulting from sulfur depletion or due to a build-up of the toxic fermentative products formate and ethanol. PMID:19478077

  20. Identification of regulatory network hubs that control lipid metabolism in Chlamydomonas reinhardtii

    PubMed Central

    Gargouri, Mahmoud; Park, Jeong-Jin; Holguin, F. Omar; Kim, Min-Jeong; Wang, Hongxia; Deshpande, Rahul R.; Shachar-Hill, Yair; Hicks, Leslie M.; Gang, David R.

    2015-01-01

    Microalgae-based biofuels are promising sources of alternative energy, but improvements throughout the production process are required to establish them as economically feasible. One of the most influential improvements would be a significant increase in lipid yields, which could be achieved by altering the regulation of lipid biosynthesis and accumulation. Chlamydomonas reinhardtii accumulates oil (triacylglycerols, TAG) in response to nitrogen (N) deprivation. Although a few important regulatory genes have been identified that are involved in controlling this process, a global understanding of the larger regulatory network has not been developed. In order to uncover this network in this species, a combined omics (transcriptomic, proteomic and metabolomic) analysis was applied to cells grown in a time course experiment after a shift from N-replete to N-depleted conditions. Changes in transcript and protein levels of 414 predicted transcription factors (TFs) and transcriptional regulators (TRs) were monitored relative to other genes. The TF and TR genes were thus classified by two separate measures: up-regulated versus down-regulated and early response versus late response relative to two phases of polar lipid synthesis (before and after TAG biosynthesis initiation). Lipidomic and primary metabolite profiling generated compound accumulation levels that were integrated with the transcript dataset and TF profiling to produce a transcriptional regulatory network. Evaluation of this proposed regulatory network led to the identification of several regulatory hubs that control many aspects of cellular metabolism, from N assimilation and metabolism, to central metabolism, photosynthesis and lipid metabolism. PMID:26022256

  1. Biogenesis of Thylakoid Membranes in Chlamydomonas reinhardtii y1 (A Kinetic Study of Initial Greening).

    PubMed Central

    White, R. A.; Hoober, J. K.

    1994-01-01

    Initiation of thylakoid membrane assembly was examined in degreened cells of Chlamydomonas reinhardtii y1 cells depleted of thylakoid membranes and photosynthetic activity by growth in the dark for 3 to 4 d. Photoreductive activities of photosystem II (PSII) and photosystem I (PSI) increased with no apparent lag when degreened cells were exposed to light at 38[deg]C. However, fluorescence transients induced by actinic light, which reflect the functional state of PSII, changed only slightly during the first 2 h of greening. When these cells were treated with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) or saturating light, fluorescence increased commensurate with the cellular content of chlorophyll. In similar experiments with greening cells of C. reinhardtii CC-2341 (ac-u-g-2.3), a PSI-minus strain, fluorescence increased with chlorophyll without treatment with DCMU. These data suggested that fluorescence of initial PSII centers in greening y1 cells was quenched by activity of PSI. Continuous monitoring of fluorescence in the presence or absence of DCMU showed that assembly of quenched PSII centers occurred within seconds after exposure of y1 cells to light. These results are consistent with initial assembly of PSI and PSII within localized domains, where their proximity allows efficient energy coupling. PMID:12232351

  2. Genetic analysis of suppressors of the PF10 mutation in Chlamydomonas reinhardtii

    SciTech Connect

    Dutcher, S.K.; Gibbons, W.; Inwood, W.B.

    1988-12-01

    A mutation at the PF10 locus of the unicellular green alga Chlamydomonas reinhardtii leads to abnormal cell motility. The asymmetric form of the ciliary beat stroke characteristic of wild-type flagella is modified by this mutation to a nearly symmetric beat. We report here that this abnormal motility is a conditional phenotype that depends on light intensity. In the absence of light or under low light intensities, the motility is more severely impaired than at higher light intensities. By UV mutagenesis we obtained 11 intragenic and 70 extragenic strains that show reversion of the pf10 motility phenotype observed in low light. The intragenic events reverted the motility phenotype of the pf10 mutation completely. The extragenic events define at least seven suppressor loci; these map to linkage groups IV, VII, IX, XI, XII and XVII. Suppressor mutations at two of the seven loci (LIS1 and LIS2) require light for their suppressor activity. Forty-eight of the 70 extragenic suppressors were examined in heterozygous diploid cells; 47 of these mutants were recessive to the wild-type allele and one mutant (bop5-1) was dominant to the wild-type allele. Complementation analysis of the 47 recessive mutants showed unusual patterns. Most mutants within a recombinationally defined group failed to complement one another, although there were pairs that showed intra-allelic complementation. Additionally, some of the mutants at each recombinationally defined locus failed to complement mutants at other loci. They define dominant enhancers of one another.

  3. Adaptation of Chlamydomonas reinhardtii high-CO sub 2 -requiring mutants to limiting CO sub 2

    SciTech Connect

    Suzuki, K.; Spalding, M.H. )

    1989-07-01

    Photosynthetic characteristics of four high-CO{sub 2}-requiring mutants of Chlamydomonas reinhardtii were compared to those of wild type before and after a 24-hour exposure to limiting CO{sub 2} concentrations. The four mutants represent two loci involved in the CO{sub 2}-concentrating system of this unicellular alga. All mutants had a lower photosynthetic affinity for inorganic carbon than did the wild type when grown at an elevated CO{sub 2} concentration, indicating that the genetic lesion in each is expressed even at elevated CO{sub 2} concentrations. Wild type and all four mutants exhibited adaptive responses to limiting CO{sub 2} characteristic of the induction of the CO{sub 2}-concentrating system, resulting in an increased affinity for inorganic carbon only in wild type. Although other components of the CO{sub 2}-concentrating system were induced in these mutants, the defective component in each was sufficient to prevent any increase in the affinity for inorganic carbon. It was concluded that the genes corresponding to the ca-1 and pmp-1 loci exhibit at least partially constitutive expression and that all components of the CO{sub 2}-concentrating system may be required to significantly affect the photosynthetic affinity for inorganic carbon.

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

    SciTech Connect

    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.

  5. Volatile fractions of landfill leachates and their effect on Chlamydomonas reinhardtii: In vivo chlorophyll a fluorescence

    SciTech Connect

    Brack, W.; Rottler, H.; Frank, H.

    1998-10-01

    Volatile organic compounds such as short-chain halogenated hydrocarbons and alkylated benzenes are widely used as solvents or as intermediates in the chemical industry, and some of them are fuel components. Dichloromethane, trichloroethene, 1,1,1-trichloroethane, and tetrachloroethene have been produced in amounts of 500,000 to 1 million t/year, 80 to 100% of which are released to the environment. The production of toluene, a major component of fuels for internal combustion engines, amounts to about 30 million t/year. A method for identification of toxic volatile constituents of landfill leachates is presented that combines bioassay-compatible sample preparation, chemical analysis, and a bioassay based on in vivo chlorophyll a fluorescence of the green alga Chlamydomonas reinhardtii. Two major pathways of toxicity were identified by comparing fluorescence patterns: specific toxicity of hydrogen sulfide, and narcotic action of nonreactive organic compounds. For quantification, the contributions of identified compounds were calculated using toxic units. The ecotoxicologic relevance of volatile fractions from hazardous waste leachates was shown.

  6. The UVS9 gene of Chlamydomonas encodes an XPG homolog with a new conserved domain.

    PubMed

    Deitsch, Erin; Hibbard, Erin M; Petersen, Jason L

    2016-01-01

    Nucleotide excision repair (NER) is a key pathway for removing DNA damage that destabilizes the DNA double helix. During NER a protein complex coordinates to cleave the damaged DNA strand on both sides of the damage. The resulting lesion-containing oligonucleotide is displaced from the DNA and a replacement strand is synthesized using the undamaged strand as template. Ultraviolet (UV) light is known to induce two primary forms of DNA damage, the cyclobutane pyrimidine dimer and the 6-4 photoproduct, both of which destabilize the DNA double helix. The uvs9 strain of Chlamydomonas reinhardtii was isolated based on its sensitivity to UV light and was subsequently shown to have a defect in NER. In this work, the UVS9 gene was cloned through molecular mapping and shown to encode a homolog of XPG, the structure-specific nuclease responsible for cleaving damaged DNA strands 3' to sites of damage during NER. 3' RACE revealed that the UVS9 transcript is alternatively polyadenylated. The predicted UVS9 protein is nearly twice as long as other XPG homologs, primarily due to an unusually long spacer region. Despite this difference, amino acid sequence alignment of UVS9p with XPG homologs revealed a new conserved domain involved in TFIIH interaction. PMID:26658142

  7. Variations in the alternative oxidase in chlamydomonas grown in air or high CO sub 2

    SciTech Connect

    Goyal, A.; Tolbert, N.E. )

    1989-03-01

    Chlamydomonas in the resting phase of growth has an equal capacity of about 15 micromole O{sub 2} uptake per hour per milligram of chlorophyll for both the cytochrome c, CN-sensitive respiration, and for the alternative, salicylhydroxamic acid-sensitive respiration. Alternative respiration capacity was measured as salicylhydroxamic acid inhibited O{sub 2} uptake in the presence of CN, and cytochrome c respiration capacity as CN inhibition of O{sub 2} uptake in the presence of salicylhydroxamic acid. Measured total respiration was considerably less than the combined capacities for respiration. During the log phase of growth on high (2-5%) CO{sub 2}, the alternative respiration capacity decreased about 90% but returned as the culture entered the lag phase. When the alternative oxidase capacity was low, addition of salicylic acid or cyanide induced its reappearance. When cells were grown on low (air-level) CO{sub 2}, which induced a CO{sub 2} concentrating mechanism, the alternative oxidase capacity did not decrease during the growth phase. Attempts to measure in vivo distribution of respiration between the two pathways with either CN or salicylhydroxamic acid alone were inconclusive.

  8. Improved automated monitoring and new analysis algorithm for circadian phototaxis rhythms in Chlamydomonas

    PubMed Central

    Gaskill, Christa; Forbes-Stovall, Jennifer; Kessler, Bruce; Young, Mike; Rinehart, Claire A.; Jacobshagen, Sigrid

    2010-01-01

    Automated monitoring of circadian rhythms is an efficient way of gaining insight into oscillation parameters like period and phase for the underlying pacemaker of the circadian clock. Measurement of the circadian rhythm of phototaxis (swimming towards light) exhibited by the green alga Chlamydomonas reinhardtii has been automated by directing a narrow and dim light beam through a culture at regular intervals and determining the decrease in light transmittance due to the accumulation of cells in the beam. In this study, the monitoring process was optimized by constructing a new computer-controlled measuring machine that limits the test beam to wavelengths reported to be specific for phototaxis and by choosing an algal strain, which does not need background illumination between test light cycles for proper expression of the rhythm. As a result, period and phase of the rhythm are now unaffected by the time a culture is placed into the machine. Analysis of the rhythm data was also optimized through a new algorithm, whose robustness was demonstrated using virtual rhythms with various noises. The algorithm differs in particular from other reported algorithms by maximizing the fit of the data to a sinusoidal curve that dampens exponentially. The algorithm was also used to confirm the reproducibility of rhythm monitoring by the machine. Machine and algorithm can now be used for a multitude of circadian clock studies that require unambiguous period and phase determinations such as light pulse experiments to identify the photoreceptor(s) that reset the circadian clock in C. reinhardtii. PMID:20116270

  9. Metabolomic analysis of the green microalga Chlamydomonas reinhardtii cultivated under day/night conditions.

    PubMed

    Willamme, Rémi; Alsafra, Zouheir; Arumugam, Rameshkumar; Eppe, Gauthier; Remacle, Françoise; Levine, R D; Remacle, Claire

    2015-12-10

    Biomass composition of Chlamydomonas reinhardtii was studied during two consecutive cycles of 12h light/12h dark. As in our experimental conditions the two synchronized divisions were separated by 20h, it was possible to show that accumulation of dry weight, proteins, chlorophyll and fatty acids mainly depends on cell division, whereas starch accumulation depends on a circadian rhythm as reported previously. Our metabolomics analyses also revealed that accumulation of five (Ser, Val, Leu, Ile and Thr) of the nine free amino acids detected displayed rhythmicity, depending on cell division while Glu was 20-50 times more abundant than the other ones probably because this free amino acid serves not only for protein synthesis but also for biosynthesis of nitrogen compounds. In addition, we performed a thermodynamic-motivated theoretical approach known as 'surprisal analysis'. The results from this analysis showed that cells were close to a steady state all along the 48h of the experiment. In addition, calculation of free energy of cellular metabolites showed that the transition point, i.e. the state which immediately precedes cell division, corresponds to the most unstable stage of the cell cycle and that division is identified as the greatest drop in the free energy of metabolites. PMID:25941156

  10. Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas.

    PubMed

    Hong-Hermesdorf, Anne; Miethke, Marcus; Gallaher, Sean D; Kropat, Janette; Dodani, Sheel C; Chan, Jefferson; Barupala, Dulmini; Domaille, Dylan W; Shirasaki, Dyna I; Loo, Joseph A; Weber, Peter K; Pett-Ridge, Jennifer; Stemmler, Timothy L; Chang, Christopher J; Merchant, Sabeeha S

    2014-12-01

    We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply. PMID:25344811

  11. The Involvement of hybrid cluster protein 4, HCP4, in Anaerobic Metabolism in Chlamydomonas reinhardtii.

    PubMed

    Olson, Adam C; Carter, Clay J

    2016-01-01

    The unicellular green algae Chlamydomonas reinhardtii has long been studied for its unique fermentation pathways and has been evaluated as a candidate organism for biofuel production. Fermentation in C. reinhardtii is facilitated by a network of three predominant pathways producing four major byproducts: formate, ethanol, acetate and hydrogen. Previous microarray studies identified many genes as being highly up-regulated during anaerobiosis. For example, hybrid cluster protein 4 (HCP4) was found to be one of the most highly up-regulated genes under anoxic conditions. Hybrid cluster proteins have long been studied for their unique spectroscopic properties, yet their biological functions remain largely unclear. To probe its role during anaerobiosis, HCP4 was silenced using artificial microRNAs (ami-hcp4) followed by extensive phenotypic analyses of cells grown under anoxic conditions. Both the expression of key fermentative enzymes and their respective metabolites were significantly altered in ami-hcp4, with nitrogen uptake from the media also being significantly different than wild-type cells. The results strongly suggest a role for HCP4 in regulating key fermentative and nitrogen utilization pathways. PMID:26930496

  12. Effect of aluminum on cellular division and photosynthetic electron transport in Euglena gracilis and Chlamydomonas acidophila.

    PubMed

    Perreault, François; Dewez, David; Fortin, Claude; Juneau, Philippe; Diallo, Amirou; Popovic, Radovan

    2010-04-01

    The present study investigated aluminum's effect on cellular division and the photosynthetic processes in Euglena gracilis and Chlamydomonas acidophila at pH 3.0, at which Al is present mostly as Al(3+), AlSO(4) (+), and Al(SO(4))(2) (-). These algal species were exposed to 100, 188, and 740 microM Al, and after 24 h cell-bound Al was significantly different from control only for the highest concentration tested. However, very different effects of Al on algal cellular division, biomass per cell, and photosynthetic activity were found. Aluminum stimulated cell division but decreased at some level biomass per cell in C. acidophila. Primary photochemistry of photosynthesis, as Photosystem II quantum yield, and energy dissipation via nonphotochemical activity were slightly affected. However, for E. gracilis, under the same conditions, Al did not show a stimulating effect on cellular division or photosynthetic activity. Primary photochemical activity was diminished, and energy dissipation via nonphotochemical pathways was strongly increased. Therefore, when Al is highly available in aquatic ecosystems, these effects may indicate very different response mechanisms that are dependent on algal species. PMID:20821518

  13. New insights into Chlamydomonas reinhardtii hydrogen production processes by combined microarray/RNA-seq transcriptomics.

    PubMed

    Toepel, Jörg; Illmer-Kephalides, Maike; Jaenicke, Sebastian; Straube, Jasmin; May, Patrick; Goesmann, Alexander; Kruse, Olaf

    2013-08-01

    Hydrogen production with Chlamydomonas reinhardtii induced by sulphur starvation is a multiphase process while the cell internal metabolism is completely remodelled. The first cellular response is characterized by induction of genes with regulatory functions, followed by a total remodelling of the metabolism to provide reduction equivalents for cellular processes. We were able to characterize all major processes that provide energy and reduction equivalents during hydrogen production. Furthermore, C. reinhardtii showed a strong transcript increase for gene models responsible for stress response and detoxification of oxygen radicals. Finally, we were able to determine potential bottlenecks and target genes for manipulation to increase hydrogen production or to prolong the hydrogen production phase. The investigation of transcriptomic changes during the time course of hydrogen production in C. reinhardtii with microarrays and RNA-seq revealed new insights into the regulation and remodelling of the cell internal metabolism. Both methods showed a good correlation. The microarray platform can be used as a reliable standard tool for routine gene expression analysis. RNA-seq additionally allowed a detailed time-dependent study of gene expression and determination of new genes involved in the hydrogen production process. PMID:23551401

  14. Process development for hydrogen production with Chlamydomonas reinhardtii based on growth and product formation kinetics.

    PubMed

    Lehr, Florian; Morweiser, Michael; Rosello Sastre, Rosa; Kruse, Olaf; Posten, Clemens

    2012-11-30

    Certain strains of microalgae are long known to produce hydrogen under anaerobic conditions. In Chlamydomonas reinhardtii the oxygen-sensitive hydrogenase enzyme recombines electrons from the chloroplast electron transport chain with protons to form molecular hydrogen directly inside the chloroplast. A sustained hydrogen production can be obtained under low sulfur conditions in C. reinhardtii, reducing the net oxygen evolution by reducing the photosystem II activity and thereby overcoming the inhibition of the hydrogenases. The development of specially adapted hydrogen production strains led to higher yields and optimized biological process preconditions. So far sustainable hydrogen production required a complete exchange of the growth medium to establish sulfur-deprived conditions after biomass growth. In this work we demonstrate the transition from the biomass growth phase to the hydrogen production phase in a single batch culture only by exact dosage of sulfur. This eliminates the elaborate and energy intensive solid-liquid separation step and establishes a process strategy to proceed further versus large scale production. This strategy has been applied to determine light dependent biomass growth and hydrogen production kinetics to assess the potential of H₂ production with C. reinhardtii as a basis for scale up and further process optimization. PMID:22750091

  15. Alternative photosynthetic electron transport pathways during anaerobiosis in the green alga Chlamydomonas reinhardtii.

    PubMed

    Hemschemeier, Anja; Happe, Thomas

    2011-08-01

    Oxygenic photosynthesis uses light as energy source to generate an oxidant powerful enough to oxidize water into oxygen, electrons and protons. Upon linear electron transport, electrons extracted from water are used to reduce NADP(+) to NADPH. The oxygen molecule has been integrated into the cellular metabolism, both as the most efficient electron acceptor during respiratory electron transport and as oxidant and/or "substrate" in a number of biosynthetic pathways. Though photosynthesis of higher plants, algae and cyanobacteria produces oxygen, there are conditions under which this type of photosynthesis operates under hypoxic or anaerobic conditions. In the unicellular green alga Chlamydomonas reinhardtii, this condition is induced by sulfur deficiency, and it results in the production of molecular hydrogen. Research on this biotechnologically relevant phenomenon has contributed largely to new insights into additional pathways of photosynthetic electron transport, which extend the former concept of linear electron flow by far. This review summarizes the recent knowledge about various electron sources and sinks of oxygenic photosynthesis besides water and NADP(+) in the context of their contribution to hydrogen photoproduction by C. reinhardtii. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts. PMID:21376011

  16. A dual strategy to cope with high light in Chlamydomonas reinhardtii.

    PubMed

    Allorent, Guillaume; Tokutsu, Ryutaro; Roach, Thomas; Peers, Graham; Cardol, Pierre; Girard-Bascou, Jacqueline; Seigneurin-Berny, Daphné; Petroutsos, Dimitris; Kuntz, Marcel; Breyton, Cécile; Franck, Fabrice; Wollman, Francis-André; Niyogi, Krishna K; Krieger-Liszkay, Anja; Minagawa, Jun; Finazzi, Giovanni

    2013-02-01

    Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition-deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II. PMID:23424243

  17. An organelle K+ channel is required for osmoregulation in Chlamydomonas reinhardtii.

    PubMed

    Xu, Feifei; Wu, Xiaoan; Jiang, Lin-Hua; Zhao, Hucheng; Pan, Junmin

    2016-08-01

    Fresh water protozoa and algae face hypotonic challenges in their living environment. Many of them employ a contractile vacuole system to uptake excessive water from the cytoplasm and expel it to the environment to achieve cellular homeostasis. K(+), a major osmolyte in contractile vacuole, is predicted to create higher osmolarity for water influx. Molecular mechanisms for K(+) permeation through the plasma membrane have been well studied. However, how K(+) permeates organelles such as the contractile vacuole is not clear. Here, we show that the six-transmembrane K(+) channel KCN11 in Chlamydomonas is exclusively localized to contractile vacuole. Ectopic expression of KCN11 in HEK293T cells results in voltage-gated K(+) channel activity. Disruption of the gene or mutation of key residues for K(+) permeability of the channel leads to dysfunction of cell osmoregulation in very hypotonic conditions. The contractile cycle is inhibited in the mutant cells with a slower rate of contractile vacuole swelling, leading to cell death. These data demonstrate a new role for six-transmembrane K(+) channels in contractile vacuole functioning and provide further insights into osmoregulation mediated by the contractile vacuole. PMID:27311484

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

    PubMed

    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. PMID:27395008

  19. The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas.

    PubMed

    Reck, Jaimee; Schauer, Alexandria M; VanderWaal Mills, Kristyn; Bower, Raqual; Tritschler, Douglas; Perrone, Catherine A; Porter, Mary E

    2016-08-01

    The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms. PMID:27251063

  20. High-resolution crystal structure and redox properties of chloroplastic triosephosphate isomerase from Chlamydomonas reinhardtii.

    PubMed

    Zaffagnini, Mirko; Michelet, Laure; Sciabolini, Chiara; Di Giacinto, Nastasia; Morisse, Samuel; Marchand, Christophe H; Trost, Paolo; Fermani, Simona; Lemaire, Stéphane D

    2014-01-01

    Triosephosphate isomerase (TPI) catalyzes the interconversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. Photosynthetic organisms generally contain two isoforms of TPI located in both cytoplasm and chloroplasts. While the cytoplasmic TPI is involved in the glycolysis, the chloroplastic isoform participates in the Calvin-Benson cycle, a key photosynthetic process responsible for carbon fixation. Compared with its cytoplasmic counterpart, the functional features of chloroplastic TPI have been poorly investigated and its three-dimensional structure has not been solved. Recently, several studies proposed TPI as a potential target of different redox modifications including dithiol/disulfide interchanges, glutathionylation, and nitrosylation. However, neither the effects on protein activity nor the molecular mechanisms underlying these redox modifications have been investigated. Here, we have produced recombinantly and purified TPI from the unicellular green alga Chlamydomonas reinhardtii (Cr). The biochemical properties of the enzyme were delineated and its crystallographic structure was determined at a resolution of 1.1 Å. CrTPI is a homodimer with subunits containing the typical (β/α)8-barrel fold. Although no evidence for TRX regulation was obtained, CrTPI was found to undergo glutathionylation by oxidized glutathione and trans-nitrosylation by nitrosoglutathione, confirming its sensitivity to multiple redox modifications. PMID:24157611

  1. Metabolic control of urea catabolism in Chlamydomonas reinhardi and Chlorella pyrenoidosa.

    PubMed

    Hodson, R C; Williams, S K; Davidson, W R

    1975-03-01

    In the unicellular green alga Chlamydomonas reinhardi (strain y-1), synthesis of the enzymes required for urea hydrolysis is under substrate induction control by urea and under end product repression control by ammonia. Hydrolysis of urea if effected by the sequential action of the discrete enzymes urea carboxylase and allophanate lyase, collectively called urea amidolyase. The carboxylase converts urea to allophanate in a reaction requiring biotin, adenosine 5'-triphosphate, and Mg2+. The lyase hydrolzyes allophanate to ammonium ions and bicarbonate. Neither activity is present in more than trace amounts when cultures are grown with ammonia or urea plus ammonia, or when they are starved for nitrogen for 8 h. Urea in the absence of ammonia induces both activities 10 to 100 times the basal levels. Addition of ammonia to an induced culture causes complete cessation of carboxylase accumulation and an 80% depression of lyase accumulation. Ammonia does not reduce urea uptake by repressed cells, so it does not prevent induction by the mechanism of inducer exclusion. The unicellular green alga Chlorella pyrenoidosa (strain 3 Emerson) also has discrete carboxylase and lyase enzymes, but only the carboxylase exhibits metabolic control. PMID:1116994

  2. Nitrogen-Sparing Mechanisms in Chlamydomonas Affect the Transcriptome, the Proteome, and Photosynthetic Metabolism[W

    PubMed Central

    Schmollinger, Stefan; Mühlhaus, Timo; Boyle, Nanette R.; Blaby, Ian K.; Casero, David; Mettler, Tabea; Moseley, Jeffrey L.; Kropat, Janette; Sommer, Frederik; Strenkert, Daniela; Hemme, Dorothea; Pellegrini, Matteo; Grossman, Arthur R.; Stitt, Mark; Schroda, Michael; Merchant, Sabeeha S.

    2014-01-01

    Nitrogen (N) is a key nutrient that limits global primary productivity; hence, N-use efficiency is of compelling interest in agriculture and aquaculture. We used Chlamydomonas reinhardtii as a reference organism for a multicomponent analysis of the N starvation response. In the presence of acetate, respiratory metabolism is prioritized over photosynthesis; consequently, the N-sparing response targets proteins, pigments, and RNAs involved in photosynthesis and chloroplast function over those involved in respiration. Transcripts and proteins of the Calvin-Benson cycle are reduced in N-deficient cells, resulting in the accumulation of cycle metabolic intermediates. Both cytosolic and chloroplast ribosomes are reduced, but via different mechanisms, reflected by rapid changes in abundance of RNAs encoding chloroplast ribosomal proteins but not cytosolic ones. RNAs encoding transporters and enzymes for metabolizing alternative N sources increase in abundance, as is appropriate for the soil environmental niche of C. reinhardtii. Comparison of the N-replete versus N-deplete proteome indicated that abundant proteins with a high N content are reduced in N-starved cells, while the proteins that are increased have lower than average N contents. This sparing mechanism contributes to a lower cellular N/C ratio and suggests an approach for engineering increased N-use efficiency. PMID:24748044

  3. The Involvement of hybrid cluster protein 4, HCP4, in Anaerobic Metabolism in Chlamydomonas reinhardtii

    PubMed Central

    Olson, Adam C.; Carter, Clay J.

    2016-01-01

    The unicellular green algae Chlamydomonas reinhardtii has long been studied for its unique fermentation pathways and has been evaluated as a candidate organism for biofuel production. Fermentation in C. reinhardtii is facilitated by a network of three predominant pathways producing four major byproducts: formate, ethanol, acetate and hydrogen. Previous microarray studies identified many genes as being highly up-regulated during anaerobiosis. For example, hybrid cluster protein 4 (HCP4) was found to be one of the most highly up-regulated genes under anoxic conditions. Hybrid cluster proteins have long been studied for their unique spectroscopic properties, yet their biological functions remain largely unclear. To probe its role during anaerobiosis, HCP4 was silenced using artificial microRNAs (ami-hcp4) followed by extensive phenotypic analyses of cells grown under anoxic conditions. Both the expression of key fermentative enzymes and their respective metabolites were significantly altered in ami-hcp4, with nitrogen uptake from the media also being significantly different than wild-type cells. The results strongly suggest a role for HCP4 in regulating key fermentative and nitrogen utilization pathways. PMID:26930496

  4. Effect of O₂:CO₂ ratio on the primary metabolism of Chlamydomonas reinhardtii.

    PubMed

    Kliphuis, Anna M J; Martens, Dirk E; Janssen, Marcel; Wijffels, René H

    2011-10-01

    High oxygen:carbon dioxide ratios may have a negative effect on growth and productivity of microalgae. To investigate the effect of O₂ and CO₂ concentrations and the ratio between these on the metabolism of Chlamydomonas reinhardtii we performed turbidostat experiments at different O₂:CO₂ ratios. These experiments showed that elevated O₂ concentrations and the corresponding increase in the ratio of O₂:CO₂ common in photobioreactors led to a reduction of growth and biomass yield on light with 20-30%. This is most probably related to the oxygenase activity of Rubisco and the resulting process of photorespiration. Using metabolic flux modeling with measured rates for each experiment we were able to quantify the ratio of the oxygenase reaction to the carboxylase reaction of Rubisco and could demonstrate that photorespiration indeed can cause the reduction in biomass yield on light. The calculated ratio of the oxygenase reaction to the carboxylase reaction was 16.6% and 20.5% for air with 2% CO₂ and 1% CO₂, respectively. Thus photorespiration has a significant impact on the biomass yield on light already at conditions common in photobioreactors (air with 2% CO₂). PMID:21538341

  5. Expression of bkt and bch genes from Haematococcus pluvialis in transgenic Chlamydomonas.

    PubMed

    Zheng, KaiJing; Wang, ChaoGang; Xiao, Ming; Chen, Jun; Li, JianCheng; Hu, ZhangLi

    2014-10-01

    β-carotene ketolase and β-carotene hydroxylase encoded by bkt and bch, respectively, are key enzymes required for astaxanthin biosynthesis in Haematococcu pluvialis 34-1n. Two expression vectors containing cDNA sequences of bkt and bch were constructed and co-transformed into cell-wall-deficient Chlamydomonas reinhardtii CC-849. Transgenic algae were screened on TAP agar plates containing 10 μg mL(-1) Zeomycin. PCR-Southern analysis showed that bkt and bch were integrated into the genomes of C. reinhardtii. Transcripts of bkt and bch were further confirmed by RT-PCR-Southern analysis. Compared with the wild type, transgenic algae produced 29.04% and 30.27% more carotenoids and xanthophylls, respectively. Moreover, the transgenic algae could accumulate 34% more astaxanthin than wild type. These results indicate that foreign bkt and bch genes were successfully translated into β-carotene ketolase and β-carotene hydroxylase, which were responsible for catalyzing the biosynthesis of astaxanthin in transgenic algae. PMID:25209726

  6. Effects of Light Intensity and Oxidized Nitrogen Sources on Hydrogen Production by Chlamydomonas reinhardii1

    PubMed Central

    Aparicio, Pedro J.; Azuara, María P.; Ballesteros, Antonio; Fernández, Victor M.

    1985-01-01

    Chlamydomonas reinhardii cells, after a period of dark anaerobic adaptation, evolve H2 not only in the dark but also in the light. Our results show that high irradiances impair prolonged H2 evolution, while under low irradiances or darkness H2 evolution proceeds for more than 50 hours. NO3− and NO2− suppress H2 evolution both in the dark or under low irradiance. Apparently the cells prefer these oxidized nitrogen sources to protons as electron acceptors, since both NO3− and NO2− become reduced to NH4+, which is excreted to the culture medium in high amounts. H2 evolution started once these oxidized anions were largely depleted from the medium. Moreover, H2 evolution was consistently associated with NH4+ excretion even if NH4+ was already present in high amounts in the medium. This observation indicates that the cells utilize not only their carbohydrate but also their protein reserves as sources of reducing power for H2 evolution. This conclusion was supported by the observation that when nitrogen-starved cells were made anaerobic in a nitrogen-free medium, they not only evolved H2 at very high rates but excreted concomitantly NH4+ up to concentrations in the millimolar range. PMID:16664329

  7. Controlling expression of genes in the unicellular alga Chlamydomonas reinhardtii with a vitamin-repressible riboswitch.

    PubMed

    Ramundo, Silvia; Rochaix, Jean-David

    2015-01-01

    Chloroplast genomes of land plants and algae contain generally between 100 and 150 genes. These genes are involved in plastid gene expression and photosynthesis and in various other tasks. The function of some chloroplast genes is still unknown and some of them appear to be essential for growth and survival. Repressible and reversible expression systems are highly desirable for functional and biochemical characterization of these genes. We have developed a genetic tool that allows one to regulate the expression of any coding sequence in the chloroplast genome of the unicellular alga Chlamydomonas reinhardtii. Our system is based on vitamin-regulated expression of the nucleus-encoded chloroplast Nac2 protein, which is specifically required for the expression of any plastid gene fused to the psbD 5'UTR. With this approach, expression of the Nac2 gene in the nucleus and, in turn, that of the chosen chloroplast gene artificially driven by the psbD 5'UTR, is controlled by the MetE promoter and Thi4 riboswitch, which can be inactivated in a reversible way by supplying vitamin B12 and thiamine to the growth medium, respectively. This system opens interesting possibilities for studying the assembly and turnover of chloroplast multiprotein complexes such as the photosystems, the ribosome, and the RNA polymerase. It also provides a way to overcome the toxicity often associated with the expression of proteins of biotechnological interest in the chloroplast. PMID:25605390

  8. The Antarctic Chlamydomonas raudensis: an emerging model for cold adaptation of photosynthesis.

    PubMed

    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. PMID:23903324

  9. Evidence for phenotypic plasticity in the Antarctic extremophile Chlamydomonas raudensis Ettl. UWO 241.

    PubMed

    Pocock, Tessa; Vetterli, Adrien; Falk, Stefan

    2011-01-01

    Life in extreme environments poses unique challenges to photosynthetic organisms. The ability for an extremophilic green alga and its genetic and mesophilic equivalent to acclimate to changes in their environment was examined to determine the extent of their phenotypic plasticities. The Antarctic extremophile Chlamydomonas raudensis Ettl. UWO 241 (UWO) was isolated from an ice-covered lake in Antarctica, whereas its mesophilic counterpart C. raudensis Ettl. SAG 49.72 (SAG) was isolated from a meadow pool in the Czech Republic. The effects of changes in temperature and salinity on growth, morphology, and photochemistry were examined in the two strains. Differential acclimative responses were observed in UWO which include a wider salinity range for growth, and broader temperature- and salt-induced fluctuations in F(v)/F(m), relative to SAG. Furthermore, the redox state of the photosynthetic electron transport chain, measured as 1-q(P), was modulated in the extremophile whereas this was not observed in the mesophile. Interestingly, it is shown for the first time that SAG is similar to UWO in that it is unable to undergo state transitions. The different natural histories of these two strains exert different evolutionary pressures and, consequently, different abilities for acclimation, an important component of phenotypic plasticity. In contrast to SAG, UWO relied on a redox sensing and signalling system under the growth conditions used in this study. It is proposed that growth and adaptation of UWO under a stressful and extreme environment poises this extremophile for better success under changing environmental conditions. PMID:21041369

  10. Proteomic Analysis of a Fraction with Intact Eyespots of Chlamydomonas reinhardtii and Assignment of Protein Methylation

    PubMed Central

    Eitzinger, Nicole; Wagner, Volker; Weisheit, Wolfram; Geimer, Stefan; Boness, David; Kreimer, Georg; Mittag, Maria

    2015-01-01

    Flagellate green algae possess a visual system, the eyespot. In Chlamydomonas reinhardtii it is situated at the edge of the chloroplast and consists of two carotenoid rich lipid globule layers subtended by thylakoid membranes (TM) that are attached to both chloroplast envelope membranes and a specialized area of the plasma membrane (PM). A former analysis of an eyespot fraction identified 203 proteins. To increase the understanding of eyespot related processes, knowledge of the protein composition of the membranes in its close vicinity is desirable. Here, we present a purification procedure that allows isolation of intact eyespots. This gain in intactness goes, however, hand in hand with an increase of contaminants from other organelles. Proteomic analysis identified 742 proteins. Novel candidates include proteins for eyespot development, retina-related proteins, ion pumps, and membrane-associated proteins, calcium sensing proteins as well as kinases, phosphatases and 14-3-3 proteins. Methylation of proteins at Arg or Lys is known as an important posttranslational modification involved in, e.g., signal transduction. Here, we identify several proteins from eyespot fractions that are methylated at Arg and/or Lys. Among them is the eyespot specific SOUL3 protein that influences the size and position of the eyespot and EYE2, a protein important for its development. PMID:26697039

  11. Color tuning in binding pocket models of the chlamydomonas-type channelrhodopsins.

    PubMed

    Welke, Kai; Frähmcke, Jan S; Watanabe, Hiroshi C; Hegemann, Peter; Elstner, Marcus

    2011-12-22

    We examined the shift of absorption maxima between the chlamydomonas-type channelrhodopsins (ChRs) and bacteriorhodopsin (BR). Starting from the BR X-ray structure, we modeled the color tuning in the binding pockets of the ChRs by mutating up to 28 amino acids in the vicinity of the chromophore. By applying the efficient self-consistent charge density functional tight binding (SCC-DFTB) method in a quantum mechanical/molecular mechanical (QM/MM) framework, including explicit polarization and calculating excitation energies with the semiempirical OM2/MRCI method and the ab initio SORCI method, we have shown that multiple mutations in the binding pocket of BR causes large hypsochromic shifts that are of the same order as the experimentally observed shifts of the absorption maxima between BR and the ChRs. This study further demonstrates that mutations in the proximity of the Schiff base and complex counterion lead to a stronger but more flexible interaction with the retinal, which could serve as a possible explanation for the spectral patterns found in the ChRs. PMID:22077286

  12. Salt-Sensitive Mutants of Chlamydomonas reinhardtii Isolated after Insertional Tagging.

    PubMed Central

    Prieto, R.; Pardo, J. M.; Niu, X.; Bressan, R. A.; Hasegawa, P. M.

    1996-01-01

    We describe the isolation of salt-sensitive Chlamydomonas reinhardtii mutants by insertional mutagenesis using the nitrate reductase (Nit1) gene. The plasmid pMN24, containing Nit1, was used for transformation of 305CW15 (nit1 cw15 mt+), and transformants were selected for complementation of the nit- phenotype. From 6875 nit+ colonies, four transformants (S4, S18, S46, and S66) were isolated that exhibited both Na+ and Li+ sensitivity (sod-), and another transformant (S33) was selected that exhibited sensitivity to Li+ but not Na+ (lit-) based on relative growth comparisons with the wild-type strain. S33, S46, and S66 were no more growth inhibited by sorbitol than was 305CW15. In comparison, S4 and S18 exhibited substantial growth inhibition in medium supplemented with sorbitol. Genetic analyses indicated that the salt-sensitive mutants were each defective in a single recessive gene. The mutant genes in S4 (sod1), S33 (lit1), and S66 (sod3) are linked to a functional copy of Nit1 and are presumably tagged with a pMN24 insertion. PMID:12226377

  13. Cellular internalization and intracellular biotransformation of silver nanoparticles in Chlamydomonas reinhardtii.

    PubMed

    Wang, Songshan; Lv, Jitao; Ma, Jingyuan; Zhang, Shuzhen

    2016-10-01

    It is necessary to elucidate cellular internalization and intracellular biotransformation in order to accurately assess the toxicity and fate of nanoparticles after interaction with organisms. Therefore, this work employed a combination of high resolution imaging and in situ detection spectroscopic techniques to systematically investigate the intracellular localization, morphology and chemical speciation of silver in the cells of Chlamydomonas reinhardtii, a unicellular freshwater green alga, after exposure to AgNPs coated with polyvinylpyrrolidone at a concentration of 2.0 mg/L. High resolution secondary ion mass spectrometry and high-angle annular dark field scanning transmission electron microscopy together with energy dispersive spectroscopy and selected area electron diffraction collectively confirmed that after 48 h of exposure, AgNPs entered the periplasmic space after cellular internalization into the algal cells. Silver was also found to coexist with sulfur inside the cytoplasm in both crystalline and amorphous forms, which were further identified as β-Ag2S and silver thiolates with synchrotron X-ray absorption spectroscopy. In combination, these analyses demonstrated that silver inside algae could be attributed to the uptake and sequestration of Ag(+) ion released from AgNPs, which was further sequestrated into cellular compartments. This study provides solid evidence for particle internalization and biotransformation of AgNPs after interaction with algae. PMID:27098098

  14. Evidence for phenotypic plasticity in the Antarctic extremophile Chlamydomonas raudensis Ettl. UWO 241

    PubMed Central

    Pocock, Tessa; Vetterli, Adrien; Falk, Stefan

    2011-01-01

    Life in extreme environments poses unique challenges to photosynthetic organisms. The ability for an extremophilic green alga and its genetic and mesophilic equivalent to acclimate to changes in their environment was examined to determine the extent of their phenotypic plasticities. The Antarctic extremophile Chlamydomonas raudensis Ettl. UWO 241 (UWO) was isolated from an ice-covered lake in Antarctica, whereas its mesophilic counterpart C. raudensis Ettl. SAG 49.72 (SAG) was isolated from a meadow pool in the Czech Republic. The effects of changes in temperature and salinity on growth, morphology, and photochemistry were examined in the two strains. Differential acclimative responses were observed in UWO which include a wider salinity range for growth, and broader temperature- and salt-induced fluctuations in Fv/Fm, relative to SAG. Furthermore, the redox state of the photosynthetic electron transport chain, measured as 1–qP, was modulated in the extremophile whereas this was not observed in the mesophile. Interestingly, it is shown for the first time that SAG is similar to UWO in that it is unable to undergo state transitions. The different natural histories of these two strains exert different evolutionary pressures and, consequently, different abilities for acclimation, an important component of phenotypic plasticity. In contrast to SAG, UWO relied on a redox sensing and signalling system under the growth conditions used in this study. It is proposed that growth and adaptation of UWO under a stressful and extreme environment poises this extremophile for better success under changing environmental conditions. PMID:21041369

  15. Loss of phylloquinone in Chlamydomonas affects plastoquinone pool size and photosystem II synthesis.

    PubMed

    Lefebvre-Legendre, Linnka; Rappaport, Fabrice; Finazzi, Giovanni; Ceol, Mauro; Grivet, Chantal; Hopfgartner, Gérard; Rochaix, Jean-David

    2007-05-01

    Phylloquinone functions as the electron transfer cofactor at the A(1) site of photosystem I. We have isolated and characterized a mutant of Chlamydomonas reinhardtii, menD1, that is deficient in MenD, which encodes 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase, an enzyme that catalyzes the first specific step of the phylloquinone biosynthetic pathway. The mutant is photosynthetically active but light-sensitive. Analysis of total pigments by mass spectrometry reveals that phylloquinone is absent in menD1, but plastoquinone levels are not affected. This is further confirmed by the rescue of menD1 by addition of phylloquinone to the growth medium. Analysis of electron transfer by absorption spectroscopy indicates that plastoquinone replaces phylloquinone in photosystem I and that electron transfer from A(1) to the iron-sulfur centers is slowed down at least 40-fold. Consistent with a replacement of phylloquinone by plastoquinone, the size of the free plastoquinone pool of menD1 is reduced by 20-30%. In contrast to cyanobacterial MenD-deficient mutants, photosystem I accumulates normally in menD1, whereas the level of photosystem II declines. This decrease is because of reduced synthesis of the photosystem II core subunits. The relationship between plastoquinone occupancy of the A(1) site in photosystem I and the reduced accumulation of photosystem II is discussed. PMID:17339322

  16. Isolation and characterization of a mutant defective in triacylglycerol accumulation in nitrogen-starved Chlamydomonas reinhardtii.

    PubMed

    Hung, Chun-Hsien; Kanehara, Kazue; Nakamura, Yuki

    2016-09-01

    Triacylglycerol (TAG), a major source of biodiesel production, accumulates in nitrogen-starved Chlamydomonas reinhardtii. However, the metabolic pathway of starch-to-TAG conversion remains elusive because an enzyme that affects the starch degradation is unknown. Here, we isolated a new class of mutant bgal1, which expressed an overaccumulation of starch granules and defective photosynthetic growth. The bgal1 was a null mutant of a previously uncharacterized β-galactosidase-like gene (Cre02.g119700), which decreased total β-galactosidase activity 40% of the wild type. Upon nitrogen starvation, the bgal1 mutant showed decreased TAG accumulation mainly due to the reduced flux of de novo TAG biosynthesis evidenced by increased unsaturation of fatty acid composition in TAG and reduced TAG accumulation by additional supplementation of acetate to the culture media. Metabolomic analysis of the bgal1 mutant showed significantly reduced levels of metabolites following the hydrolysis of starch and substrates for TAG accumulation, whereas metabolites in TCA cycle were unaffected. Upon nitrogen starvation, while levels of glucose 6-phosphate, fructose 6-phosphate and acetyl-CoA remained lower, most of the other metabolites in glycolysis were increased but those in the TCA cycle were decreased, supporting TAG accumulation. We suggest that BGAL1 may be involved in the degradation of starch, which affects TAG accumulation in nitrogen-starved C. reinhardtii. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. PMID:27060488

  17. Toxicological effects of nanometer titanium dioxide (nano-TiO2) on Chlamydomonas reinhardtii.

    PubMed

    Chen, Lanzhou; Zhou, Lina; Liu, Yongding; Deng, Songqiang; Wu, Hao; Wang, Gaohong

    2012-10-01

    The toxicological effects of nanometer titanium dioxide (nano-TiO2) on a unicellular green alga Chlamydomonas reinhardtii were assessed by investigating the changes of the physiology and cyto-ultrastructure of this species under treatment. We found that nano-TiO2 inhibited photosynthetic efficiency and cell growth, but the content of chlorophyll a content in algae did not change, while carotenoid and chlorophyll b contents increased. Malondialdehyde (MDA) content reached maximum values after 8h exposure and then decreased to a moderately low level at 72 h. Electron microscopy images indicated that as concentrations of nano-TiO2 increased, a large number of C. reinhardtii cells were noted to be damaged: the number of chloroplasts declined, various other organelles were degraded, plasmolysis occurred, and TiO2 nanoparticles were found to be located inside cell wall and membrane. It was also noted that cell surface was surrounded by TiO2 particles, which could present an obstacle to the exchange of substances between the cell and its surrounding environment. To sum up, the effect of nano-TiO2 on C. reinhardtii included cell surface aggregation, photosynthesis inhibition, lipid peroxidation and new protein synthesis, while the response of C. reinhardtii to nano-TiO2 was a rapid process which occurs during 24 h after exposing and may relate to physiological stress system to mitigate damage. PMID:22883605

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

    PubMed

    Yang, Duanpeng; Li, Weiqi

    2016-01-01

    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. PMID:26731741

  19. SPONTANEOUS MUTATION ACCUMULATION IN MULTIPLE STRAINS OF THE GREEN ALGA, CHLAMYDOMONAS REINHARDTII

    PubMed Central

    Morgan, Andrew D; Ness, Rob W; Keightley, Peter D; Colegrave, Nick

    2014-01-01

    Estimates of mutational parameters, such as the average fitness effect of a new mutation and the rate at which new genetic variation for fitness is created by mutation, are important for the understanding of many biological processes. However, the causes of interspecific variation in mutational parameters and the extent to which they vary within species remain largely unknown. We maintained multiple strains of the unicellular eukaryote Chlamydomonas reinhardtii, for approximately 1000 generations under relaxed selection by transferring a single cell every ∼10 generations. Mean fitness of the lines tended to decline with generations of mutation accumulation whereas mutational variance increased. We did not find any evidence for differences among strains in any of the mutational parameters estimated. The overall change in mean fitness per cell division and rate of input of mutational variance per cell division were more similar to values observed in multicellular organisms than to those in other single-celled microbes. However, after taking into account differences in genome size among species, estimates from multicellular organisms and microbes, including our new estimates from C. reinhardtii, become substantially more similar. Thus, we suggest that variation in genome size is an important determinant of interspecific variation in mutational parameters. PMID:24826801

  20. Examination of chlorophyll fluorescence decay kinetics in sulfur deprived algae Chlamydomonas reinhardtii.

    PubMed

    Volgusheva, A A; Zagidullin, V E; Antal, T K; Korvatovsky, B N; Krendeleva, T E; Paschenko, V Z; Rubin, A B

    2007-06-01

    Chlorophyll fluorescence decay kinetics was measured in sulfur deprived cells of green alga Chlamydomonas reinhardtii with a home made picosecond fluorescence laser spectrometer. The measurements were carried out on samples either shortly adapted to the dark ('Fo conditions') or treated to reduce Qa ('Fm conditions'). Bi-exponential fitting of decay kinetics was applied to distinguish two components one of them related to energy trapping (fast component) and the other to charge stabilization and recombination in PS 2 reaction centers (slow component). It was found that the slow component yield increased by 2.0 and 1.2 times when measured under 'Fo' and 'Fm conditions', respectively, in sulfur deprived cells as compared to control ones. An additional rapid rise of the slow component yield was observed when incubation was carried out in a sealed bioreactor and cell culture turned to anaerobic conditions. The obtained results strongly indicate the existence of the redox control of PS 2 activity during multiphase adaptation of C. reinhardtii to sulfur deficiency stress. Probable mechanisms responsible for the observed increased recombinant fluorescence yield in starved cells are discussed. PMID:17543273

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

    PubMed

    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. PMID:25756227

  2. Building Blocks of the Nexin-Dynein Regulatory Complex in Chlamydomonas Flagella*

    PubMed Central

    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

  3. Cadmium exposure and phosphorus limitation increases metal content in the freshwater alga Chlamydomonas reinhardtii.

    PubMed

    Webster, Rachel E; Dean, Andrew P; Pittman, Jon K

    2011-09-01

    The characteristics of metal accumulation in freshwater microalgae are important to elucidate for a full understanding of metal cycling and toxicity in a freshwater system. This study has utilized an elemental profiling approach to investigate the impacts of Cd exposure and phosphorus (P) availability on metal accumulation after 7 days in batch culture-grown Chlamydomonas reinhardtii. Multivariate statistical analysis of the elemental data demonstrated distinct responses between both stresses. Sublethal concentrations of Cd (up to 15 μM) caused increased accumulation of Co. Cu, Fe, and Zn content also increased in response to enhanced Cd concentrations but only when P availability was low. While Cd exposure effected the accumulation of a few specific metals, P limitation increased the accumulation of all essential trace metals and macronutrients analyzed including Co, Fe, K, Na, and Zn but not Mn. The accumulation of Cd also markedly increased in response to P limitation. The impact of P availability on essential metal accumulation was the same when either inorganic P or an organic P source (glycerophosphate) was used. These results highlight the potential risks of metal toxicity for freshwater microalgae and aquatic food chains when P availability is limiting and which can be exacerbated by Cd pollution. PMID:21809879

  4. Stable nuclear transformation of Chlamydomonas reinhardtii by using a C. reinhardtii gene as the selectable marker.

    PubMed Central

    Mayfield, S P; Kindle, K L

    1990-01-01

    We have developed a stable nuclear transformation system for the unicellular green alga Chlamydomonas reinhardtii. Transformation was accomplished by introducing the cloned C. reinhardtii oxygen-evolving enhancer protein 1 (OEE1) gene into C. reinhardtii cells by bombardment with DNA-coated tungsten particles. The recipient strain was an OEE1-deficient, nonphotosynthetic, acetate-requiring mutant, which recovered photosynthetic competence after transformation, and was therefore able to grow in the absence of acetate. Analysis of several transformants indicates that transformation has proceeded via second-site integration of the cloned gene, leaving the endogenous mutant gene intact. In genetic crosses of transformants with wild type, both mutant and wild-type phenotypes were recovered, showing that the photosynthetic competence of transformants was due not to reversion of the original locus but rather to expression of the introduced gene. We suggest that the success of the present system is largely due to using a homologous C. reinhardtii gene, leading to stable maintenance and expression of the gene. Transformation with heterologous genes may be problematic because of poor expression due to an unusual codon bias in C. reinhardtii. Images PMID:2179948

  5. Bacillus herbersteinensis sp. nov.

    PubMed

    Wieser, Monika; Worliczek, Hanna; Kämpfer, Peter; Busse, Hans-Jürgen

    2005-09-01

    Two bacterial strains, designated D-1,5a(T) and D-1,5b, were isolated from a medieval wall painting in the chapel of Castle Herberstein, Styria (Austria). The Gram-positive, heterotrophic, aerobic, spore-forming rods showed nearly identical whole-cell protein patterns, identical genomic fingerprints and identical physiological profiles, demonstrating their relationship at the species level. Both strains contained meso-diaminopimelic acid in their peptidoglycan, possessed a quinone system comprising menaquinone MK-7 and had fatty acid profiles in which C(15:0) iso and C(15:0) anteiso were predominant. The 16S rRNA gene sequence of D-1,5a(T) showed the highest similarity (99.5%) to the sequence of Bacillus sp. LMG 20243, and Bacillus flexus IFO 15715(T) was the next most closely related established species (96.5%). Other type strains, such as Bacillus fastidiosus DSM 91(T), Bacillus indicus SD/3(T), Bacillus cibi JG-30(T), Bacillus megaterium IAM 13418(T), Bacillus cohnii DSM 6308(T), Bacillus bataviensis LMG 21833(T) and Bacillus soli LMG 21838(T), shared 96.0-96.1% 16S rRNA gene sequence similarity with D-1,5a(T). The combination of physiological and chemotaxonomic traits distinguishes the two strains from those species sharing the highest sequence similarities (96.0-96.5%). On the basis of these characteristics and the phylogenetic position of strain D-1,5a(T) (=DSM 16534(T)=CCM 7228(T)), this strain is assigned as the type strain of a novel species of the genus Bacillus, for which the name Bacillus herbersteinensis sp. nov. is proposed. PMID:16166719

  6. Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp. Its mitochondrial respiratory chain lacks a plant-like alternative oxidase.

    PubMed

    Reyes-Prieto, Adrián; El-Hafidi, Mohammed; Moreno-Sánchez, Rafael; González-Halphen, Diego

    2002-07-01

    The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, alpha-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives. PMID:12160990

  7. Functional specialization of Chlamydomonas reinhardtii cytosolic thioredoxin h1 in the response to alkylation-induced DNA damage.

    PubMed

    Sarkar, Nandita; Lemaire, Stéphane; Wu-Scharf, Danxia; Issakidis-Bourguet, Emmanuelle; Cerutti, Heriberto

    2005-02-01

    DNA damage occurs as a by-product of intrinsic cellular processes, like DNA replication, or as a consequence of exposure to genotoxic agents. Organisms have evolved multiple mechanisms to avoid, tolerate, or repair DNA lesions. To gain insight into these processes, we have isolated mutants hypersensitive to DNA-damaging agents in the green alga Chlamydomonas reinhardtii. One mutant, Ble-1, showed decreased survival when it was treated with methyl methanesulfonate (MMS), bleomycin, or hydrogen peroxide (H2O2) but behaved like the wild type when it was exposed to UVC irradiation. Ble-1 carries an extensive chromosomal deletion that includes the gene encoding cytosolic thioredoxin h1 (Trxh1). Transformation of Ble-1 with a wild-type copy of Trxh1 fully corrected the MMS hypersensitivity and partly restored the tolerance to bleomycin. Trxh1 also complemented a defect in the repair of MMS-induced DNA strand breaks and alkali-labile sites. In addition, a Trxh1-beta-glucuronidase fusion protein translocated to the nucleus in response to treatment with MMS. However, somewhat surprisingly, Trxh1 failed to correct the Ble-1 hypersensitivity to H2O2. Moreover, Trxh1 suppression by RNA interference in a wild-type strain resulted in enhanced sensitivity to MMS and DNA repair defects but no increased cytotoxicity to H2O2. Thioredoxins have been implicated in oxidative-stress responses in many organisms. Yet our results indicate a specific role of Chlamydomonas Trxh1 in the repair of MMS-induced DNA damage, whereas it is dispensable for the response to H2O2. These observations also suggest functional specialization among cytosolic thioredoxins since another Chlamydomonas isoform (Trxh2) does not compensate for the lack of Trxh1. PMID:15701788

  8. Identification of AGO3-Associated miRNAs and Computational Prediction of Their Targets in the Green Alga Chlamydomonas reinhardtii

    PubMed Central

    Voshall, Adam; Kim, Eun-Jeong; Ma, Xinrong; Moriyama, Etsuko N.; Cerutti, Heriberto

    2015-01-01

    The unicellular green alga Chlamydomonas reinhardtii harbors many types of small RNAs (sRNAs) but little is known about their role(s) in the regulation of endogenous genes and cellular processes. To define functional microRNAs (miRNAs) in Chlamydomonas, we characterized sRNAs associated with an argonaute protein, AGO3, by affinity purification and deep sequencing. Using a stringent set of criteria for canonical miRNA annotation, we identified 39 precursor miRNAs, which produce 45 unique, AGO3-associated miRNA sequences including 13 previously reported miRNAs and 32 novel ones. Potential miRNA targets were identified based on the complementarity of miRNAs with candidate binding sites on transcripts and classified, depending on the extent of complementarity, as being likely to be regulated through cleavage or translational repression. The search for cleavage targets identified 74 transcripts. However, only 6 of them showed an increase in messenger RNA (mRNA) levels in a mutant strain almost devoid of sRNAs. The search for translational repression targets, which used complementarity criteria more stringent than those empirically required for a reduction in target protein levels, identified 488 transcripts. However, unlike observations in metazoans, most predicted translation repression targets did not show appreciable changes in transcript abundance in the absence of sRNAs. Additionally, of three candidate targets examined at the protein level, only one showed a moderate variation in polypeptide amount in the mutant strain. Our results emphasize the difficulty in identifying genuine miRNA targets in Chlamydomonas and suggest that miRNAs, under standard laboratory conditions, might have mainly a modulatory role in endogenous gene regulation in this alga. PMID:25769981

  9. Measurement of ethanol formation in single living cells of Chlamydomonas reinhardtii using synchrotron Fourier Transform Infrared spectromicroscopy

    NASA Astrophysics Data System (ADS)

    Goff, Kira L.; Quaroni, Luca; Pedersen, Tor; Wilson, Kenneth E.

    2010-02-01

    We demonstrate the capability of Fourier-Transform Infra-Red (FITR) spectroscopy to detect metabolite formation by the unicellular algae Chlamydomonas reinhardtii in solution. We show that using a synchrotron source in the microscopy configuration provides a sufficient s/n ratio to detect small molecular species accumulating at a single cell, allowing an increased sensitivity relative to measurements of bulk cultures. The formation of small molecular species, including ethanol and at least one carbonyl containing compound, can be detected with a time resolution of the order of one minute.

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

    SciTech Connect

    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

  11. Measurement of ethanol formation in single living cells of Chlamydomonas reinhardtii using synchrotron Fourier Transform Infrared spectromicroscopy

    SciTech Connect

    Goff, Kira L.; Quaroni, Luca; Pedersen, Tor; Wilson, Kenneth E.

    2010-02-03

    We demonstrate the capability of Fourier-Transform Infra-Red (FITR) spectroscopy to detect metabolite formation by the unicellular algae Chlamydomonas reinhardtii in solution. We show that using a synchrotron source in the microscopy configuration provides a sufficient s/n ratio to detect small molecular species accumulating at a single cell, allowing an increased sensitivity relative to measurements of bulk cultures. The formation of small molecular species, including ethanol and at least one carbonyl containing compound, can be detected with a time resolution of the order of one minute.

  12. 76 FR 56876 - Proposed Collection; Comment Request for Forms 9779, 9779(SP), 9783, 9783(SP), 9787, 9787(SP...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-14

    ... Internal Revenue Service Proposed Collection; Comment Request for Forms 9779, 9779(SP), 9783, 9783(SP), 9787, 9787(SP), 9789 and 9789(SP) AGENCY: Internal Revenue Service (IRS), Treasury. ACTION: Notice and... Reduction Act of 1995, Public Law 104-13 (44 U.S.C. 3506(c)(2)(A)). Currently, the IRS is...

  13. SP-100 Advanced Technology Program

    NASA Technical Reports Server (NTRS)

    Sovie, Ronald J.

    1987-01-01

    The goal of the triagency SP-100 Program is to develop long-lived, compact, lightweight, survivable nuclear reactor space power systems for application to the power range 50 kWe to 1 MWe. The successful development of these systems should enable or significantly enhance many of the future NASA civil and commercial missions. The NASA SP-100 Advanced Technology Program strongly augments the parallel SP-100 Ground Engineering System Development program and enhances the chances for success of the overall SP-100 program. The purpose of this paper is to discuss the key technical elements of the Advanced Technology Program and the progress made in the initial year and a half of the project.

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

    PubMed

    Korkaric, Muris; Xiao, Mao; Behra, Renata; Eggen, Rik I L

    2015-10-01

    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 herbicides paraquat and diuron, suggesting that the fast physiological and biochemical defence mechanisms that conferred tolerance of algae towards higher UVR levels were related to singlet oxygen defence. The presented study suggests that knowledge of the molecular toxicity mechanisms of chemicals, rather than their general physiological target, is needed in order to predict co-tolerance between environmental and chemical stressors. PMID:26349947

  15. Chloroplast remodeling during state transitions in Chlamydomonas reinhardtii as revealed by noninvasive techniques in vivo

    PubMed Central

    Nagy, Gergely; Ünnep, Renáta; Zsiros, Ottó; Tokutsu, Ryutaro; Takizawa, Kenji; Porcar, Lionel; Moyet, Lucas; Petroutsos, Dimitris; Garab, Győző; Finazzi, Giovanni; Minagawa, Jun

    2014-01-01

    Plants respond to changes in light quality by regulating the absorption capacity of their photosystems. These short-term adaptations use redox-controlled, reversible phosphorylation of the light-harvesting complexes (LHCIIs) to regulate the relative absorption cross-section of the two photosystems (PSs), commonly referred to as state transitions. It is acknowledged that state transitions induce substantial reorganizations of the PSs. However, their consequences on the chloroplast structure are more controversial. Here, we investigate how state transitions affect the chloroplast structure and function using complementary approaches for the living cells of Chlamydomonas reinhardtii. Using small-angle neutron scattering, we found a strong periodicity of the thylakoids in state 1, with characteristic repeat distances of ∼200 Å, which was almost completely lost in state 2. As revealed by circular dichroism, changes in the thylakoid periodicity were paralleled by modifications in the long-range order arrangement of the photosynthetic complexes, which was reduced by ∼20% in state 2 compared with state 1, but was not abolished. Furthermore, absorption spectroscopy reveals that the enhancement of PSI antenna size during state 1 to state 2 transition (∼20%) is not commensurate to the decrease in PSII antenna size (∼70%), leading to the possibility that a large part of the phosphorylated LHCIIs do not bind to PSI, but instead form energetically quenched complexes, which were shown to be either associated with PSII supercomplexes or in a free form. Altogether these noninvasive in vivo approaches allow us to present a more likely scenario for state transitions that explains their molecular mechanism and physiological consequences. PMID:24639515

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

    PubMed

    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. PMID:26471875

  17. Photoproduction of Hydrogen by Sulfur-Deprived Chlamydomonas reinhardtii Mutants with Impaired Photosystem II Photochemical Activity

    SciTech Connect

    Makarova, V. V.; Kosourov, S.; Krendeleva, T. E.; Semin, B. K.; Kukarskikh, G. P.; Rubin, A. B.; Sayre, R. T.; Ghirardi, M. L.; Seibert, M.

    2007-01-01

    Photoproduction of H2 was examined in a series of sulfur-deprived Chlamydomonas reinhardtii D1-R323 mutants with progressively impaired PSII photochemical activity. In the R323H, R323D, and R323E D1 mutants, replacement of arginine affects photosystem II (PSII) function, as demonstrated by progressive decreases in O2-evolving activity and loss of PSII photochemical activity. Significant changes in PSII activity were found when the arginine residue was replaced by negatively charged amino acid residues (R323D and R323E). However, the R323H (positively charged or neutral, depending on the ambient pH) mutant had minimal changes in PSII activity. The R323H, R323D, and R323E mutants and the pseudo-wild-type (pWt) with restored PSII function were used to study the effects of sulfur deprivation on H2-production activity. All of these mutants exhibited significant changes in the normal parameters associated with the H2-photoproduction process, such as a shorter aerobic phase, lower accumulation of starch, a prolonged anaerobic phase observed before the onset of H2-production, a shorter duration of H2-production, lower H2 yields compared to the pWt control, and slightly higher production of dark fermentation products such as acetate and formate. The more compromised the PSII photochemical activity, the more dramatic was the effect of sulfur deprivation on the H2-production process, which depends both on the presence of residual PSII activity and the amount of stored starch.

  18. Preventive effect of the microalga Chlamydomonas debaryana on the acute phase of experimental colitis in rats.

    PubMed

    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. PMID:25192306

  19. Characterization of lead induced metal-phytochelatin complexes in Chlamydomonas reinhardtii.

    PubMed

    Scheidegger, Christian; Sigg, Laura; Behra, Renata

    2011-11-01

    Accumulation of Pb and induction of phytochelatin synthesis were observed in Chlamydomonas reinhardtii upon Pb(II) exposure. Our aim was to examine whether Pb(II) is bound by phytochelatins (PCs) in C. reinhardtii and to examine formed complexes for their stoichiometry and composition. Metal-phytochelatin (Me-PC) complexes induced by Pb were isolated by size-exclusion chromatography in 13 collected fractions, which were analyzed for their PC and metal content by high-performance liquid chromatography and inductively coupled plasma mass spectrometry. A recovery of more than 90% of Pb from standard Pb-PC₂ complexes within the total volume of the size-exclusion column indicated the adequacy of the method for Pb-PC(n) complex separation and characterization. Phytochelatins were detected mainly in a molecular weight ranging from 1,000 to 5,300 daltons (Da), indicating the formation of complexes with various stoichiometries. Approximately 72% of total PC₂ eluted in the range from 1,000 to 1,600 Da, and 80% of total PC₃ eluted in the molecular weight range from 1,600 to 2,300 Da. The distribution of Cu, Zn, and Pb showed that more than 70% of these metals were associated with the high-molecular-weight fractions. Copper, zinc, and lead were also observed in PC-containing fractions, suggesting the formation of various Me-PC complexes. The results of the present study indicate that the role of PCs in Pb detoxification is minor, because only 13% of total Pb was associated with PCs. PMID:21898554

  20. Dissecting the Sequential Assembly and Localization of Intraflagellar Transport Particle Complex B in Chlamydomonas

    PubMed Central

    Richey, Elizabeth A.; Qin, Hongmin

    2012-01-01

    Intraflagellar transport (IFT), the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact complex B, but such complex is highly unstable and prone to degradation. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, in ift88 mutant cells, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies. In addition, in bld2 mutant, the IFT complex B proteins still localize to the proximal ends of defective centrioles which completely lack transition fibers. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism. PMID:22900094

  1. DNA repair in Chlamydomonas reinhardtii induced by heat shock and gamma radiation.

    PubMed

    Boreham, D R; Mitchel, R E

    1993-09-01

    Saccharomyces cerevisiae and Chlamydomonas reinhardtii respond to a sublethal exposure of ionizing radiation by increasing their resistance to killing by a second exposure. We demonstrate here that the two lower eukaryotes apparently achieve this by different mechanisms. We have shown that induced radioresistance in yeast results from increased capacity for recombinational repair, which we believe to occur in G2-phase haploid cells by recombination between homologous chromosomes. This is not possible in G1-phase haploid cells, which lack a second copy of DNA. Haploid C. reinhardtii cells, however, show induced resistance when irradiated asynchronously or in the G1 phase of the cell cycle. We have shown previously that the development of radiation resistance in yeast is proportional to the magnitude of the inducing dose and clearly demonstrates an oxygen effect. There was no oxygen effect for induced radiation resistance in C. reinhardtii cells, but induction remained proportional to dose. In yeast we have reported that both increased radioresistance and thermotolerance are inducible by a heat shock. Here, C. reinhardtii showed induced thermotolerance but no induced radioresistance in response to a heat stress. We have also determined previously that the induced recombinational DNA repair system in yeast recognizes alkylation lesions and therefore confers increased resistance to mutation by MNNG. In these experiments, C. reinhardtii induced for radioresistance were not more resistant to MNNG mutagenesis. These data indicate that haploid C. reinhardtii has a unique DSB repair mechanism. We propose that one possible mechanism may involve chloroplast DNA in a cooperative chloroplast/nuclear recombinational repair process. PMID:8378529

  2. Transcriptional program for nitrogen starvation-induced lipid accumulation in Chlamydomonas reinhardtii

    SciTech Connect

    Garcia de Lomana, Adrian Lopez; Schäuble, Sascha; Valenzuela, Jacob; Imam, Saheed; Carter, Warren; Bilgin, Damla D.; Yohn, Christopher B.; Turkarslan, Serdar; Reiss, David J.; Orellana, Monica V.; Price, Nathan D.; Baliga, Nitin S.

    2015-12-02

    Algae accumulate lipids to endure different kinds of environmental stresses including macronutrient starvation. Although this response has been extensively studied, an in depth understanding of the transcriptional regulatory network (TRN) that controls the transition into lipid accumulation remains elusive. In this study, we used a systems biology approach to elucidate the transcriptional program that coordinates the nitrogen starvation-induced metabolic readjustments that drive lipid accumulation in Chlamydomonas reinhardtii. We demonstrate that nitrogen starvation triggered differential regulation of 2147 transcripts, which were co-regulated in 215 distinct modules and temporally ordered as 31 transcriptional waves. An early-stage response was triggered within 12 min that initiated growth arrest through activation of key signaling pathways, while simultaneously preparing the intracellular environment for later stages by modulating transport processes and ubiquitin-mediated protein degradation. Subsequently, central metabolism and carbon fixation were remodeled to trigger the accumulation of triacylglycerols. Further analysis revealed that these waves of genome-wide transcriptional events were coordinated by a regulatory program orchestrated by at least 17 transcriptional regulators, many of which had not been previously implicated in this process. We demonstrate that the TRN coordinates transcriptional downregulation of 57 metabolic enzymes across a period of nearly 4 h to drive an increase in lipid content per unit biomass. Notably, this TRN appears to also drive lipid accumulation during sulfur starvation, while phosphorus starvation induces a different regulatory program. The TRN model described here is available as a community-wide web-resource at http://networks.systemsbiology.net/chlamy-portal. In conclusion, in this work, we have uncovered a comprehensive mechanistic model of the TRN controlling the transition from N starvation to lipid accumulation

  3. Transcriptional program for nitrogen starvation-induced lipid accumulation in Chlamydomonas reinhardtii

    DOE PAGESBeta

    Garcia de Lomana, Adrian Lopez; Schäuble, Sascha; Valenzuela, Jacob; Imam, Saheed; Carter, Warren; Bilgin, Damla D.; Yohn, Christopher B.; Turkarslan, Serdar; Reiss, David J.; Orellana, Monica V.; et al

    2015-12-02

    Algae accumulate lipids to endure different kinds of environmental stresses including macronutrient starvation. Although this response has been extensively studied, an in depth understanding of the transcriptional regulatory network (TRN) that controls the transition into lipid accumulation remains elusive. In this study, we used a systems biology approach to elucidate the transcriptional program that coordinates the nitrogen starvation-induced metabolic readjustments that drive lipid accumulation in Chlamydomonas reinhardtii. We demonstrate that nitrogen starvation triggered differential regulation of 2147 transcripts, which were co-regulated in 215 distinct modules and temporally ordered as 31 transcriptional waves. An early-stage response was triggered within 12 minmore » that initiated growth arrest through activation of key signaling pathways, while simultaneously preparing the intracellular environment for later stages by modulating transport processes and ubiquitin-mediated protein degradation. Subsequently, central metabolism and carbon fixation were remodeled to trigger the accumulation of triacylglycerols. Further analysis revealed that these waves of genome-wide transcriptional events were coordinated by a regulatory program orchestrated by at least 17 transcriptional regulators, many of which had not been previously implicated in this process. We demonstrate that the TRN coordinates transcriptional downregulation of 57 metabolic enzymes across a period of nearly 4 h to drive an increase in lipid content per unit biomass. Notably, this TRN appears to also drive lipid accumulation during sulfur starvation, while phosphorus starvation induces a different regulatory program. The TRN model described here is available as a community-wide web-resource at http://networks.systemsbiology.net/chlamy-portal. In conclusion, in this work, we have uncovered a comprehensive mechanistic model of the TRN controlling the transition from N starvation to lipid

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

    SciTech Connect

    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

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

    PubMed Central

    2011-01-01

    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 nanoparticles using C. reinhardtii as

  6. Thioredoxin-dependent redox regulation of chloroplastic phosphoglycerate kinase from Chlamydomonas reinhardtii.

    PubMed

    Morisse, Samuel; Michelet, Laure; Bedhomme, Mariette; Marchand, Christophe H; Calvaresi, Matteo; Trost, Paolo; Fermani, Simona; Zaffagnini, Mirko; Lemaire, Stéphane D

    2014-10-24

    In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (-335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys(227) and Cys(361). Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view. PMID:25202015

  7. Uranium accumulation and toxicity in the green alga Chlamydomonas reinhardtii is modulated by pH.

    PubMed

    Lavoie, Michel; Sabatier, Sébastien; Garnier-Laplace, Jacqueline; Fortin, Claude

    2014-06-01

    The effects of pH on metal uptake and toxicity in aquatic organisms are currently poorly understood and remain an evolving topic in studies about the biotic ligand model (BLM). In the present study, the authors investigated how pH may influence long-term (4 d) uranium (U) accumulation and chronic toxicity in batch cultures of the freshwater green alga Chlamydomonas reinhardtii. The toxicity expressed as a function of the free uranyl ion was much greater at pH 7 (effective concentration, 50% [EC50] = 1.8 × 10(-9)  M UO2 (2+) ) than at pH 5 (EC50 = 1.2 × 10(-7)  M UO2 (2+) ). The net accumulation rate of U in algal cells was much higher at pH 7 than at pH 5 for the same free [UO2 (2+) ], but the cells exposed at pH 5 were also more sensitive to intracellular U than the cells at pH 7 with EC50s of 4.0 × 10(-15) and 7.1 × 10(-13)  mol of internalized U cell(-1) , respectively. The higher cellular sensitivity to U at pH 5 than at pH 7 could be explained partly by the increase in cytosolic U binding to algal soluble proteins or enzymes at pH 5 as observed by subcellular fractionation. To predict U accumulation and toxicity in algae accurately, the important modulating effects of pH on U accumulation and U cellular sensitivity should be considered in the BLM. PMID:24596137

  8. CrGNAT gene regulates excess copper accumulation and tolerance in Chlamydomonas reinhardtii.

    PubMed

    Wang, Ye; Cheng, Zhen Zhen; Chen, Xi; Zheng, Qi; Yang, Zhi Min

    2015-11-01

    Excess copper (Cu) in environment affects the growth and metabolism of plants and green algae. However, the molecular mechanism for regulating plant tolerance to excess Cu is not fully understood. Here, we report a gene CrGNAT enconding an acetyltransferase in Chlamydomonas reinhardtii and identified its role in regulating tolerance to Cu toxicity. Expression of CrGNAT was significantly induced by 75-400μM Cu. The top induction occurred at 100μM. Transgenic algae overexpressing CrGNAT (35S::CrGNAT) in C. reinhardtii showed high tolerance to excess Cu, with improved cell population, chlorophyll accumulation and photosynthesis efficiency, but with low degree of oxidation with regard to reduced hydrogen peroxide, lipid peroxides and non-protein thiol compounds. In contrast, CrGNAT knock-down lines with antisense led to sensitivity to Cu stress. 35S::CrGNAT algae accumulated more Cu and other metals (Zn, Fe, Cu, Mn and Mg) than wild-type, whereas the CrGNAT down-regulated algae (35S::AntiCrGNAT) had moderate levels of Cu and Mn, but no effects on Zn, Fe and Mg accumulation as compared to wild-type. The elevated metal absorption in CrGNAT overexpression algae implies that the metals can be removed from water media. Quantitative RT-PCR analysis revealed that expression of two genes encoding N-lysine histone methyltransferases was repressed in 35S::CrGNAT algae, suggesting that CrGNAT-regulated algal tolerance to Cu toxicity is likely associated with histone methylation and chromatin remodeling. The present work provided an example a basis to develop techniques for environmental restoration of metal-contaminated aquatic ecosystems. PMID:26475193

  9. Chlamydomonas reinhardtii chloroplasts contain a homodimeric pyruvate:ferredoxin oxidoreductase that functions with FDX1.

    PubMed

    van Lis, Robert; Baffert, Carole; Couté, Yohann; Nitschke, Wolfgang; Atteia, Ariane

    2013-01-01

    Eukaryotic algae have long been known to live in anoxic environments, but interest in their anaerobic energy metabolism has only recently gained momentum, largely due to their utility in biofuel production. Chlamydomonas reinhardtii figures remarkably in this respect, because it efficiently produces hydrogen and its genome harbors many genes for anaerobic metabolic routes. Central to anaerobic energy metabolism in many unicellular eukaryotes (protists) is pyruvate:ferredoxin oxidoreductase (PFO), which decarboxylates pyruvate and forms acetyl-coenzyme A with concomitant reduction of low-potential ferredoxins or flavodoxins. Here, we report the biochemical properties of the homodimeric PFO of C. reinhardtii expressed in Escherichia coli. Electron paramagnetic resonance spectroscopy of the recombinant enzyme (Cr-rPFO) showed three distinct [4Fe-4S] iron-sulfur clusters and a thiamine pyrophosphate radical upon reduction by pyruvate. Purified Cr-rPFO exhibits a specific decarboxylase activity of 12 µmol pyruvate min⁻¹ mg⁻¹ protein using benzyl viologen as electron acceptor. Despite the fact that the enzyme is very oxygen sensitive, it localizes to the chloroplast. Among the six known chloroplast ferredoxins (FDX1-FDX6) in C. reinhardtii, FDX1 and FDX2 were the most efficient electron acceptors from Cr-rPFO, with comparable apparent K(m) values of approximately 4 µm. As revealed by immunoblotting, anaerobic conditions that lead to the induction of CrPFO did not increase levels of either FDX1 or FDX2. FDX1, being by far the most abundant ferredoxin, is thus likely the partner of PFO in C. reinhardtii. This finding postulates a direct link between CrPFO and hydrogenase and provides new opportunities to better study and engineer hydrogen production in this protist. PMID:23154536

  10. Time-course global expression profiles of Chlamydomonas reinhardtii during photo-biological H₂ production.

    PubMed

    Nguyen, Anh Vu; Toepel, Joerg; Burgess, Steven; Uhmeyer, Andreas; Blifernez, Olga; Doebbe, Anja; Hankamer, Ben; Nixon, Peter; Wobbe, Lutz; Kruse, Olaf

    2011-01-01

    We used a microarray study in order to compare the time course expression profiles of two Chlamydomonas reinhardtii strains, namely the high H₂ producing mutant stm6glc4 and its parental WT strain during H₂ production induced by sulfur starvation. Major cellular reorganizations in photosynthetic apparatus, sulfur and carbon metabolism upon H₂ production were confirmed as common to both strains. More importantly, our results pointed out factors which lead to the higher H₂ production in the mutant including a higher starch accumulation in the aerobic phase and a lower competition between the H₂ase pathway and alternative electron sinks within the H₂ production phase. Key candidate genes of interest with differential expression pattern include LHCSR3, essential for efficient energy quenching (qE). The reduced LHCSR3 protein expression in mutant stm6glc4 could be closely related to the high-light sensitive phenotype. H₂ measurements carried out with the LHCSR3 knock-out mutant npq4 however clearly demonstrated that a complete loss of this protein has almost no impact on H₂ yields under moderate light conditions. The nuclear gene disrupted in the high H₂ producing mutant stm6glc4 encodes for the mitochondrial transcription termination factor (mTERF) MOC1, whose expression strongly increases during -S-induced H₂ production in WT strains. Studies under phototrophic high-light conditions demonstrated that the presence of functional MOC1 is a prerequisite for proper LHCSR3 expression. Furthermore knock-down of MOC1 in a WT strain was shown to improve the total H₂ yield significantly suggesting that this strategy could be applied to further enhance H₂ production in other strains already displaying a high H₂ production capacity. By combining our array data with previously published metabolomics data we can now explain some of the phenotypic characteristics which lead to an elevated H₂ production in stm6glc4. PMID:22242116

  11. Energy-dissipative supercomplex of photosystem II associated with LHCSR3 in Chlamydomonas reinhardtii.

    PubMed

    Tokutsu, Ryutaro; Minagawa, Jun

    2013-06-11

    Plants and green algae have a low pH-inducible mechanism in photosystem II (PSII) that dissipates excess light energy, measured as the nonphotochemical quenching of chlorophyll fluorescence (qE). Recently, nonphotochemical quenching 4 (npq4), a mutant strain of the green alga Chlamydomonas reinhardtii that is qE-deficient and lacks the light-harvesting complex stress-related protein 3 (LHCSR3), was reported [Peers G, et al. (2009) Nature 462(7272):518-521]. Here, applying a newly established procedure, we isolated the PSII supercomplex and its associated light-harvesting proteins from both WT C. reinhardtii and the npq4 mutant grown in either low light (LL) or high light (HL). LHCSR3 was present in the PSII supercomplex from the HL-grown WT, but not in the supercomplex from the LL-grown WT or mutant. The purified PSII supercomplex containing LHCSR3 exhibited a normal fluorescence lifetime at a neutral pH (7.5) by single-photon counting analysis, but a significantly shorter lifetime at pH 5.5, which mimics the acidified lumen of the thylakoid membranes in HL-exposed chloroplasts. The switch from light-harvesting mode to energy-dissipating mode observed in the LHCSR3-containing PSII supercomplex was sensitive to dicyclohexylcarbodiimide, a protein-modifying agent specific to protonatable amino acid residues. We conclude that the PSII-LHCII-LHCSR3 supercomplex formed in the HL-grown C. reinhardtii cells is capable of energy dissipation on protonation of LHCSR3. PMID:23716695

  12. Hydrogen photoproduction is attenuated by disruption of an isoamylase gene in Chlamydomonas reinhardtii.

    PubMed

    Posewitz, Matthew C; Smolinski, Sharon L; Kanakagiri, Saradadevi; Melis, Anastasios; Seibert, Michael; Ghirardi, Maria L

    2004-08-01

    DNA insertional transformants of Chlamydomonas reinhardtii were screened chemochromically for attenuated H(2) production. One mutant, displaying low H(2) gas photoproduction, has a nonfunctional copy of a gene that shows high homology to the family of isoamylase genes found in several photosynthetic organisms. DNA gel blotting and gene complementation were used to link this isoamylase gene to previously characterized nontagged sta7 mutants. This mutant is therefore denoted sta7-10. In C. reinhardtii, the STA7 isoamylase gene is important for the accumulation of crystalline starch, and the sta7-10 mutant reported here contains <3% of the glucose found in insoluble starch when compared with wild-type control cells. Hydrogen photoproduction rates, induced after several hours of dark, anaerobic treatment, are attenuated in sta7 mutants. RNA gel blot analysis indicates that the mRNA transcripts for both the HydA1 and HydA2 [Fe]-hydrogenase genes are expressed in the sta7-10 mutant at greater than wild-type levels 0.5 h after anaerobic induction. However, after 1.5 h, transcript levels of both HydA1 and HydA2 begin to decline rapidly and reach nearly undetectable levels after 7 h. In wild-type cells, the hydrogenase transcripts accumulate more slowly, reach a plateau after 4 h of anaerobic treatment, and maintain the same level of expression for >7 h under anaerobic incubation. Complementation of mutant cells with genomic DNA corresponding to the STA7 gene restores both the starch accumulation and H(2) production phenotypes. The results indicate that STA7 and starch metabolism play an important role in C. reinhardtii H(2) photoproduction. Moreover, the results indicate that mere anaerobiosis is not sufficient to maintain hydrogenase gene expression without the underlying physiology, an important aspect of which is starch metabolism. PMID:15269330

  13. Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii.

    PubMed

    Goold, Hugh Douglas; Cuiné, Stéphan; Légeret, Bertrand; Liang, Yuanxue; Brugière, Sabine; Auroy, Pascaline; Javot, Hélène; Tardif, Marianne; Jones, Brian; Beisson, Fred; Peltier, Gilles; Li-Beisson, Yonghua

    2016-08-01

    Enriching algal biomass in energy density is an important goal in algal biotechnology. Nitrogen (N) starvation is considered the most potent trigger of oil accumulation in microalgae and has been thoroughly investigated. However, N starvation causes the slow down and eventually the arrest of biomass growth. In this study, we show that exposing a Chlamydomonas reinhardtii culture to saturating light (SL) under a nonlimiting CO2 concentration in turbidostatic photobioreactors induces a sustained accumulation of lipid droplets (LDs) without compromising growth, which results in much higher oil productivity than N starvation. We also show that the polar membrane lipid fraction of SL-induced LDs is rich in plastidial lipids (approximately 70%), in contrast to N starvation-induced LDs, which contain approximately 60% lipids of endoplasmic reticulum origin. Proteomic analysis of LDs isolated from SL-exposed cells identified more than 200 proteins, including known proteins of lipid metabolism, as well as 74 proteins uniquely present in SL-induced LDs. LDs induced by SL and N depletion thus differ in protein and lipid contents. Taken together, lipidomic and proteomic data thus show that a large part of the sustained oil accumulation occurring under SL is likely due to the formation of plastidial LDs. We discuss our data in relation to the different metabolic routes used by microalgae to accumulate oil reserves depending on cultivation conditions. Finally, we propose a model in which oil accumulation is governed by an imbalance between photosynthesis and growth, which can be achieved by impairing growth or by boosting photosynthetic carbon fixation, with the latter resulting in higher oil productivity. PMID:27297678

  14. Mechanistic modeling of sulfur-deprived photosynthesis and hydrogen production in suspensions of Chlamydomonas reinhardtii.

    PubMed

    Williams, C R; Bees, M A

    2014-02-01

    The ability of unicellular green algal species such as Chlamydomonas reinhardtii to produce hydrogen gas via iron-hydrogenase is well known. However, the oxygen-sensitive hydrogenase is closely linked to the photosynthetic chain in such a way that hydrogen and oxygen production need to be separated temporally for sustained photo-production. Under illumination, sulfur-deprivation has been shown to accommodate the production of hydrogen gas by partially-deactivating O2 evolution activity, leading to anaerobiosis in a sealed culture. As these facets are coupled, and the system complex, mathematical approaches potentially are of significant value since they may reveal improved or even optimal schemes for maximizing hydrogen production. Here, a mechanistic model of the system is constructed from consideration of the essential pathways and processes. The role of sulfur in photosynthesis (via PSII) and the storage and catabolism of endogenous substrate, and thus growth and decay of culture density, are explicitly modeled in order to describe and explore the complex interactions that lead to H2 production during sulfur-deprivation. As far as possible, functional forms and parameter values are determined or estimated from experimental data. The model is compared with published experimental studies and, encouragingly, qualitative agreement for trends in hydrogen yield and initiation time are found. It is then employed to probe optimal external sulfur and illumination conditions for hydrogen production, which are found to differ depending on whether a maximum yield of gas or initial production rate is required. The model constitutes a powerful theoretical tool for investigating novel sulfur cycling regimes that may ultimately be used to improve the commercial viability of hydrogen gas production from microorganisms. PMID:24026984

  15. Transcriptional and cellular responses of the green alga Chlamydomonas reinhardtii to perfluoroalkyl phosphonic acids.

    PubMed

    Sanchez, David; Houde, Magali; Douville, Mélanie; De Silva, Amila O; Spencer, Christine; Verreault, Jonathan

    2015-03-01

    Perfluoroalkyl phosphonic acids (PFPAs), a new class of perfluoroalkyl substances used primarily in the industrial sector as surfactants, were recently detected in surface water and wastewater treatment plant effluents. Toxicological effects of PFPAs have as yet not been investigated in aquatic organisms. The objective of the present study was to evaluate the effects of perfluorooctylphosphonic acid (C8-PFPA) and perfluorodecylphosphonic acid (C10-PFPA) exposure (31-250μg/L) on Chlamydomonas reinhardtii using genomic (qRT-PCR), biochemical (reactive oxygen species production (ROS) and lipid peroxidation), and physiological (cellular viability) indicators. After 72h of exposure, no differences were observed in cellular viability for any of the two perfluorochemicals. However, increase in ROS concentrations (36% and 25.6% at 125 and 250μg/L, respectively) and lipid peroxidation (35.5% and 35.7% at 125 and 250μg/L, respectively) was observed following exposure to C10-PFPA. C8-PFPA exposure did not impact ROS production and lipid peroxidation in algae. To get insights into the molecular response and modes of action of PFPA toxicity, qRT-PCR-based assays were performed to analyze the transcription of genes related to antioxidant responses including superoxide dismutase (SOD-1), glutathione peroxidase (GPX), catalase (CAT), glutathione S-transferase (GST), and ascorbate peroxidase (APX I). Genomic analyses revealed that the transcription of CAT and APX I was up-regulated for all the C10-PFPA concentrations. In addition, PFPAs were quantified in St. Lawrence River surface water samples and detected at concentrations ranging from 250 to 850pg/L for C8-PFPA and 380 to 650pg/L for C10-PFPA. This study supports the prevalence of PFPAs in the aquatic environment and suggests potential impacts of PFPA exposure on the antioxidant defensive system in C. reinhardtii. PMID:25621396

  16. Mechanistic modeling of sulfur-deprived photosynthesis and hydrogen production in suspensions of Chlamydomonas reinhardtii

    PubMed Central

    Williams, C R; Bees, MA

    2014-01-01

    The ability of unicellular green algal species such as Chlamydomonas reinhardtii to produce hydrogen gas via iron-hydrogenase is well known. However, the oxygen-sensitive hydrogenase is closely linked to the photosynthetic chain in such a way that hydrogen and oxygen production need to be separated temporally for sustained photo-production. Under illumination, sulfur-deprivation has been shown to accommodate the production of hydrogen gas by partially-deactivating O2 evolution activity, leading to anaerobiosis in a sealed culture. As these facets are coupled, and the system complex, mathematical approaches potentially are of significant value since they may reveal improved or even optimal schemes for maximizing hydrogen production. Here, a mechanistic model of the system is constructed from consideration of the essential pathways and processes. The role of sulfur in photosynthesis (via PSII) and the storage and catabolism of endogenous substrate, and thus growth and decay of culture density, are explicitly modeled in order to describe and explore the complex interactions that lead to H2 production during sulfur-deprivation. As far as possible, functional forms and parameter values are determined or estimated from experimental data. The model is compared with published experimental studies and, encouragingly, qualitative agreement for trends in hydrogen yield and initiation time are found. It is then employed to probe optimal external sulfur and illumination conditions for hydrogen production, which are found to differ depending on whether a maximum yield of gas or initial production rate is required. The model constitutes a powerful theoretical tool for investigating novel sulfur cycling regimes that may ultimately be used to improve the commercial viability of hydrogen gas production from microorganisms. Biotechnol. Bioeng. 2014;111: 320–335. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:24026984

  17. Heavy Metal-Activated Synthesis of Peptides in Chlamydomonas reinhardtii 1

    PubMed Central

    Howe, Gregg; Merchant, Sabeeha

    1992-01-01

    In this study, we have addressed the capacity of the green alga Chlamydomonas reinhardtii to produce metal-binding peptides in response to stress induced by the heavy metals Cd2+, Hg2+, and Ag+. Cells cultured in the presence of sublethal concentrations of Cd2+ synthesized and accumulated oligopeptides consisting solely of glutamic acid, cysteine, and glycine in an average ratio of 3:3:1. Cadmium-induced peptides were isolated in their native form as higher molecular weight peptide-metal complexes with an apparent molecular weight of approximately 6.5 × 103. The isolated complex bound cadmium (as evidenced by absorption spectroscopy) and sequestered (with a stoichiometry of 0.7 moles of cadmium per mole of cysteine) up to 70% of the total cadmium found in extracts of cadmium-treated cells. In Hg2+-treated cells, the principal thiol-containing compound induced by Hg2+ ions was glutathione. It is possible that glutathione functions in plant cells (as it does in animal cells) to detoxify heavy metals. Cells treated with Ag+ ions also synthesized a sulfur-containing component with a charge to mass ratio similar to Cd2+-induced peptides. But, in contrast to the results obtained using Cd2+ as an inducer, these molecules did not accumulate to significant levels in Ag+-treated cells. The presence of physiological concentrations of Cu2+ in the growth medium blocked the synthesis of the Ag+-inducible component(s) and rendered cells resistant to the toxic effects of Ag+, suggesting competition between Cu2+ and Ag+ ions, possibly at the level of metal uptake. ImagesFigure 2Figure 6Figure 7Figure 8Figure 9Figure 11 PMID:16668603

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

    PubMed Central

    Liu, Cuimin; Hartl, F. Ulrich; Hayer-Hartl, Manajit

    2015-01-01

    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. PMID:26305355

  19. Lysis of Chlamydomonas reinhardtii by high-intensity focused ultrasound as a function of exposure time.

    PubMed

    Bigelow, Timothy A; Xu, Jin; Stessman, Dan J; Yao, Linxing; Spalding, Martin H; Wang, Tong

    2014-05-01

    Efficient lysis of microalgae for lipid extraction is an important concern when processing biofuels. Historically, ultrasound frequencies in the range of 10-40 kHz have been utilized for this task. However, greater efficiencies might be achievable if higher frequencies could be used. In our study, we evaluated the potential of using 1.1 MHz ultrasound to lyse microalgae for biofuel production while using Chlamydomonas reinhardtii as a model organism. The ultrasound was generated using a spherically focused transducer with a focal length of 6.34 cm and an active diameter of 6.36 cm driven by 20 cycle sine-wave tone bursts at a pulse repetition frequency of 2 kHz (3.6% duty cycle). The time-average acoustic power output was 26.2 W while the spatial-peak-pulse-average intensity (ISPPA) for each tone burst was 41 kW/cm(2). The peak compressional and rarefactional pressures at the focus were 102 and 17 MPa, respectively. The exposure time was varied for the different cases in the experiments from 5s to 9 min and cell lysis was assessed by quantifying the percentage of protein and chlorophyll release into the supernate as well as the lipid extractability. Free radical generation and lipid oxidation for the different ultrasound exposures were also determined. We found that there was a statistically significant increase in lipid extractability for all of the exposures compared to the control. The longer exposures also completely fragmented the cells releasing almost all of the protein and chlorophyll into the supernate. The cavitation activity did not significantly increase lipid oxidation while there was a minor trend of increased free radical production with increased ultrasound exposure. PMID:24355286

  20. Isolation, purification, and characterization of coupling factor 1 from Chlamydomonas reinhardi.

    PubMed

    Selman-Reimer, S; Merchant, S; Selman, B R

    1981-09-15

    Chloroplast thylakoid particles were prepared from wild-type Chlamydomonas reinhardi by gentle sonication. These particles catalyzed phenazine methosulfate dependent photophosphorylation with rates ranging from 300 to 700 mumol of adenosine 5'-triphosphate (ATP) formed (mg of chlorophyll)-1h-1. Photophosphorylation was not sensitive to tentoxin but was sensitive to an anticoupling factor 1 (CF1) antiserum preparation made against spinach CF1. The C. reinhardi chloroplast CF1 was isolated from thylakoid particles by either chloroform or ethylenediaminetraacetic acid extraction. The former enzyme appeared to be missing the gamma subunit and did not reconstitute with partially resolved thylakoid particles. The latter enzyme reconstituted with partially resolved particles and had a specific activity at 37 degrees C of 2-5 umol of ATP hydrolyzed (mg of protein)-1 min-1. The enzyme utilized both MnATP and MgATP. CaATP was a poor substrate, and SrATP was not hydrolyzed. The enzyme was not activated by heat or proteolysis but was stimulated approximately 2-fold by 50 mM dithiothreitol. Alcohols reversibly stimulated the ATPase activity of the enzyme 5-25-fold. Ethanol, 20%, dramatically lowered the temperature optimum from approximately 75 to approximately 45 degrees C and slightly lowered the pH optimum from 8.5 to 8.2. Ethanol had no effect on the activation energy of the ATPase reaction (17 +/- 1.7 kcal/mol). The kinetics of the ATPase reaction catalyzed by the C. reinhardi enzyme are complex. Both free divalent cations and divalent cation ATP inhibited the activity of the enzyme. The apparent Km for MgTAP (55 uM free Mg2+) was approximately 0.2 mM. PMID:6457633

  1. Systems level analysis of the Chlamydomonas reinhardtii metabolic network reveals variability in evolutionary co-conservation.

    PubMed

    Chaiboonchoe, Amphun; Ghamsari, Lila; Dohai, Bushra; Ng, Patrick; Khraiwesh, Basel; Jaiswal, Ashish; Jijakli, Kenan; Koussa, Joseph; Nelson, David R; Cai, Hong; Yang, Xinping; Chang, Roger L; Papin, Jason; Yu, Haiyuan; Balaji, Santhanam; Salehi-Ashtiani, Kourosh

    2016-07-19

    Metabolic networks, which are mathematical representations of organismal metabolism, are reconstructed to provide computational platforms to guide metabolic engineering experiments and explore fundamental questions on metabolism. Systems level analyses, such as interrogation of phylogenetic relationships within the network, can provide further guidance on the modification of metabolic circuitries. Chlamydomonas reinhardtii, a biofuel relevant green alga that has retained key genes with plant, animal, and protist affinities, serves as an ideal model organism to investigate the interplay between gene function and phylogenetic affinities at multiple organizational levels. Here, using detailed topological and functional analyses, coupled with transcriptomics studies on a metabolic network that we have reconstructed for C. reinhardtii, we show that network connectivity has a significant concordance with the co-conservation of genes; however, a distinction between topological and functional relationships is observable within the network. Dynamic and static modes of co-conservation were defined and observed in a subset of gene-pairs across the network topologically. In contrast, genes with predicted synthetic interactions, or genes involved in coupled reactions, show significant enrichment for both shorter and longer phylogenetic distances. Based on our results, we propose that the metabolic network of C. reinhardtii is assembled with an architecture to minimize phylogenetic profile distances topologically, while it includes an expansion of such distances for functionally interacting genes. This arrangement may increase the robustness of C. reinhardtii's network in dealing with varied environmental challenges that the species may face. The defined evolutionary constraints within the network, which identify important pairings of genes in metabolism, may offer guidance on synthetic biology approaches to optimize the production of desirable metabolites. PMID:27357594

  2. Culture of microalgae Chlamydomonas reinhardtii in wastewater for biomass feedstock production.

    PubMed

    Kong, Qing-xue; Li, Ling; Martinez, Blanca; Chen, Paul; Ruan, Roger

    2010-01-01

    The objective of this research was to develop large-scale technologies to produce oil-rich algal biomass from wastewater. The experiments were conducted using Erlenmeyer flasks and biocoil photobioreactor. Chlamydomonas reinhardtii was grown in artificial media and wastewaters taken from three different stages of the treatment process, namely, influent, effluent, and centrate. Each of wastewaters contained different levels of nutrients. The specific growth rate of C. reinhardtii in different cultures was monitored over a period of 10 days. The biomass yield of microalgae and associated nitrogen and phosphorous removal were evaluated. Effects of CO(2) and pH on the growth were also studied. The level of nutrients greatly influenced algae growth. High levels of nutrients seem to inhibit algae growth in the beginning, but provided sustained growth to a high degree. The studies have shown that the optimal pH for C. reinhardtii is in the range of 7.5. An injection of air and a moderate amount of CO(2) promoted algae growth. However, too much CO(2) inhibited algae growth due to a significant decrease in pH. The experimental results showed that algal dry biomass yield reached a maximum of 2.0 g L(-1) day(-1) in the biocoil. The oil content of microalgae of C. reinhardtii was 25.25% (w/w) in dry biomass weight. In the biocoil, 55.8 mg nitrogen and 17.4 mg phosphorus per liter per day were effectively removed from the centrate wastewater. Ferric chloride was found to be an effective flocculent that helps the algae settle for easy harvest and separation from the culture media. PMID:19507059

  3. Tying Down Loose Ends in the Chlamydomonas Genome: Functional Significance of Abundant Upstream Open Reading Frames

    PubMed Central

    Cross, Frederick R.

    2015-01-01

    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. PMID:26701783

  4. Tying Down Loose Ends in the Chlamydomonas Genome: Functional Significance of Abundant Upstream Open Reading Frames.

    PubMed

    Cross, Frederick R

    2016-02-01

    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. PMID:26701783

  5. Retrograde Intraflagellar Transport Mutants Identify Complex A Proteins With Multiple Genetic Interactions in Chlamydomonas reinhardtii

    PubMed Central

    Iomini, Carlo; Li, Linya; Esparza, Jessica M.; Dutcher, Susan K.

    2009-01-01

    The intraflagellar transport machinery is required for the assembly of cilia. It has been investigated by biochemical, genetic, and computational methods that have identified at least 21 proteins that assemble into two subcomplexes. It has been hypothesized that complex A is required for retrograde transport. Temperature-sensitive mutations in FLA15 and FLA17 show defects in retrograde intraflagellar transport (IFT) in Chlamydomonas. We show that IFT144 and IFT139, two complex A proteins, are encoded by FLA15 and FLA17, respectively. The fla15 allele is a missense mutation in a conserved cysteine and the fla17 allele is an in-frame deletion of three exons. The flagellar assembly defect of each mutant is rescued by the respective transgenes. In fla15 and fla17 mutants, bulges form in the distal one-third of the flagella at the permissive temperature and this phenotype is also rescued by the transgenes. These bulges contain the complex B component IFT74/72, but not α-tubulin or p28, a component of an inner dynein arm, which suggests specificity with respect to the proteins that accumulate in these bulges. IFT144 and IFT139 are likely to interact with each other and other proteins on the basis of three distinct genetic tests: (1) Double mutants display synthetic flagellar assembly defects at the permissive temperature, (2) heterozygous diploid strains exhibit second-site noncomplemention, and (3) transgenes confer two-copy suppression. Since these tests show different levels of phenotypic sensitivity, we propose they illustrate different gradations of gene interaction between complex A proteins themselves and with a complex B protein (IFT172). PMID:19720863

  6. Pyruvate:Ferredoxin Oxidoreductase Is Coupled to Light-independent Hydrogen Production in Chlamydomonas reinhardtii*

    PubMed Central

    Noth, Jens; Krawietz, Danuta; Hemschemeier, Anja; Happe, Thomas

    2013-01-01

    In anaerobiosis, the green alga Chlamydomonas reinhardtii evolves molecular hydrogen (H2) as one of several fermentation products. H2 is generated mostly by the [Fe-Fe]-hydrogenase HYDA1, which uses plant type ferredoxin PETF/FDX1 (PETF) as an electron donor. Dark fermentation of the alga is mainly of the mixed acid type, because formate, ethanol, and acetate are generated by a pyruvate:formate lyase pathway similar to Escherichia coli. However, C. reinhardtii also possesses the pyruvate:ferredoxin oxidoreductase PFR1, which, like pyruvate:formate lyase and HYDA1, is localized in the chloroplast. PFR1 has long been suggested to be responsible for the low but significant H2 accumulation in the dark because the catalytic mechanism of pyruvate:ferredoxin oxidoreductase involves the reduction of ferredoxin. With the aim of proving the biochemical feasibility of the postulated reaction, we have heterologously expressed the PFR1 gene in E. coli. Purified recombinant PFR1 is able to transfer electrons from pyruvate to HYDA1, using the ferredoxins PETF and FDX2 as electron carriers. The high reactivity of PFR1 toward oxaloacetate indicates that in vivo, fermentation might also be coupled to an anaerobically active glyoxylate cycle. Our results suggest that C. reinhardtii employs a clostridial type H2 production pathway in the dark, especially because C. reinhardtii PFR1 was also able to allow H2 evolution in reaction mixtures containing Clostridium acetobutylicum 2[4Fe-4S]-ferredoxin and [Fe-Fe]-hydrogenase HYDA. PMID:23258532

  7. Psychrophily is associated with differential energy partitioning, photosystem stoichiometry and polypeptide phosphorylation in Chlamydomonas raudensis.

    PubMed

    Szyszka, Beth; Ivanov, Alexander G; Hüner, Norman P A

    2007-06-01

    Chlamydomonas raudensis UWO 241 and SAG 49.72 represent the psychrophilic and mesophilic strains of this green algal species. This novel discovery was exploited to assess the role of psychrophily in photoacclimation to growth temperature and growth irradiance. At their optimal growth temperatures of 8 degrees C and 28 degrees C respectively, UWO 241 and SAG 49.72 maintained comparable photostasis, that is energy balance, as measured by PSII excitation pressure. Although UWO 241 exhibited higher excitation pressure, measured as 1-qL, at all growth light intensities, the relative changes in 1-qL were similar to that of SAG 49.72 in response to growth light. In response to suboptimal temperatures and increased growth irradiance, SAG 49.72 favoured energy partitioning of excess excitation energy through inducible, down regulatory processes (Phi(NPQ)) associated with the xanthophyll cycle and antenna quenching, while UWO 241 favoured xanthophyll cycle-independent energy partitioning through constitutive processes involved in energy dissipation (Phi(NO)). In contrast to SAG 49.72, an elevation in growth temperature induced an increase in PSI/PSII stoichiometry in UWO 241. Furthermore, SAG 49.72 showed typical threonine-phosphorylation of LHCII, whereas UWO 241 exhibited phosphorylation of polypeptides of comparable molecular mass to PSI reaction centres but the absence of LHCII phosphorylation. Thus, although both strains maintain an energy balance irrespective of their differences in optimal growth temperatures, the mechanisms used to maintain photostasis were distinct. We conclude that psychrophily in C. raudensis is complex and appears to involve differential energy partitioning, photosystem stoichiometry and polypeptide phosphorylation. PMID:17234152

  8. PLASTICITY OF THE PSYCHROPHILIC GREEN ALGA CHLAMYDOMONAS RAUDENSIS (UWO 241) (CHLOROPHYTA) TO SUPRAOPTIMAL TEMPERATURE STRESS(1).

    PubMed

    Possmayer, Marc; Berardi, Gino; Beall, Benjamin F N; Trick, Charles G; Hüner, Norman P A; Maxwell, Denis P

    2011-10-01

    Chlamydomonas raudensis  H. Ettl (UWO 241) is a psychrophilic green alga endemic to Lake Bonney, Antarctica. The objective of this study was to investigate the response of UWO 241 to incubation at 24°C, a temperature close to optimum for related mesophilic species. Using chl a fluorescence analysis, shifting cells from a growth temperature of 10°C-24°C resulted in a decline in PSII photochemical efficiency with light energy being directed away from photochemistry and toward dissipative pathways. Using the SYTOX Green assay, it was determined that UWO 241 cells die when incubated at 24°C under growth irradiance with a half-time of 34.9 h. The role of light in cell death was minor as cell death occurred in darkness at 24°C with a half-time of 43.7 h. To examine the plasticity of UWO 241 to temperature stress, 10°C-grown cells were shifted to 24°C for 12 h and then returned to 10°C to recover. The 12 h incubation at 24°C, which resulted in <10% cell death, led to declines in both light-saturated rates of photosynthesis and respiration, PSII photochemistry and energy partitioning, and changes to transcript abundances-those associated with the light-harvesting protein of PSII and ferredoxin declining rapidly, whereas transcripts of specific heat-shock proteins (HSPs) increased. Within 24-48 h of being transferred back to 10°C, all parameters returned to levels occurring in 10°C-grown cells. This research shows, for the first time, that 24°C is a temperature that is lethal to UWO 241, and yet this organism displays considerable physiological and molecular plasticity. PMID:27020192

  9. Computational comparison of mediated current generation capacity of Chlamydomonas reinhardtii in photosynthetic and respiratory growth modes.

    PubMed

    Mao, Longfei; Verwoerd, Wynand S

    2014-11-01

    Chlamydomonas reinhardtii possesses many potential advantages to be exploited as a biocatalyst in microbial fuel cells (MFCs) for electricity generation. In the present study, we performed computational studies based on flux balance analysis (FBA) to probe the maximum potential of C. reinhardtii for current output and identify the metabolic mechanisms supporting a high current generation in three different cultivation conditions, i.e., heterotrophic, photoautotrophic and mixotrophic growth. The results showed that flux balance limitations allow the highest current output for C. reinhardtii in the mixotrophic growth mode (2.368 A/gDW), followed by heterotrophic growth (1.141 A/gDW) and photoautotrophic growth the lowest (0.7035 A/gDW). The significantly higher mediated electron transfer (MET) rate in the mixotrophic mode is in complete contrast to previous findings for a photosynthetic cyanobacterium, and was attributed to the fact that for C. reinhardtii the photophosphorylation improved the efficiency of converting the acetate into biomass and NADH production. Overall, the cytosolic NADH-dependent current production was mainly associated with five reactions in both mixotrophic and photoautotrophic nutritional modes, whereas four reactions participated in the heterotrophic mode. The mixotrophic and photoautotrophic metabolisms were alike and shared the same set of reactions for maximizing current production, whereas in the heterotrophic mode, the current production was additionally contributed by the metabolic activities in the two organelles: glyoxysome and chloroplast. In conclusion, C. reinhardtii has a potential to be exploited in MFCs of MET mode to produce a high current output. PMID:24875305

  10. METAL-INDUCED REACTIVE OXYGEN SPECIES PRODUCTION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)(1).

    PubMed

    Szivák, Ilona; Behra, Renata; Sigg, Laura

    2009-04-01

    Toxic effects of metals appear to be partly related to the production of reactive oxygen species (ROS), which can cause oxidative damage to cells. The ability of several redox active metals [Fe(III), Cu(II), Ag(I), Cr(III), Cr(VI)], nonredox active metals [Pb(II), Cd(II), Zn(II)], and the metalloid As(III) and As(V) to produce ROS at environmentally relevant metal concentrations was assessed. Cells of the freshwater alga Chlamydomonas reinhardtii P. A. Dang. were exposed to various metal concentrations for 2.5 h. Intracellular ROS accumulation was detected using an oxidation-sensitive reporter dye, 5-(and-6)-carboxy-2',7'-dihydrodifluorofluorescein diacetate (H2 DFFDA), and changes in the fluorescence signal were quantified by flow cytometry (FCM). In almost all cases, low concentrations of both redox and nonredox active metals enhanced intracellular ROS levels. The hierarchy of maximal ROS induction indicated by the increased number of stained cells compared to the control sample was as follows: Pb(II) > Fe(III) > Cd(II) > Ag(I) > Cu(II) > As(V) > Cr(VI) > Zn(II). As(III) and Cr(III) had no detectable effect. The effective free metal ion concentrations ranged from 10(-6) to 10(-9)  M, except in the case of Fe(III), which was effective at 10(-18)  M. These metal concentrations did not affect algal photosynthesis. Therefore, a slightly enhanced ROS production is a general and early response to elevated, environmentally relevant metal concentrations. PMID:27033821

  11. Cryopreservation of Chlamydomonas reinhardtii: a cause of low viability at high cell density.

    PubMed

    Piasecki, Brian P; Diller, Kenneth R; Brand, Jerry J

    2009-02-01

    Cryopreservation is a practical method for stabilizing the genetic content of living algae over long periods of time. Yet, Chlamydomonas reinhardtii, the algal species most often utilized in studies requiring genetically defined strains, is difficult to cryopreserve with a consistently high post-thaw viability. Work described here demonstrates that C. reinhardtii retains high viability only when cryopreserved at a low cell density. Low viability at high cell density was caused by the release of an injurious substance into the culture medium. Rapid freezing and thawing under non-cryoprotective conditions released large amounts of the injurious substance. Heat denaturation of cells prevented the release of the injurious substance, but heating did not inactivate it after it was released. Even when concentrated, the injurious substance was non-toxic to cells under normal culture conditions. Reduced viability of cells cryopreserved in the presence of the injurious substance could not be attributed to changes in the tonicity of the medium. A mutant strain of C. reinhardtii (cw10) with a greatly diminished cell wall did not release a substance that reduced the post-thaw viability of wild-type or cw10 cryopreserved cells. Cryopreservation of cw10 cells was achieved with approximately the same post-thaw viability irrespective to the cell concentration at the time of freezing. Acid treatment of the injurious substance was able to partially diminish its injurious effect on cells during cryopreservation. We propose that diminished viability of C. reinhardtii cells cryopreserved at high cell densities is caused by the enzymatic release of a cell-wall component. PMID:19041638

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

    PubMed

    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. PMID:24463493

  13. Mastoparan-induced programmed cell death in the unicellular alga Chlamydomonas reinhardtii

    PubMed Central

    Yordanova, Zhenya P.; Woltering, Ernst J.; Kapchina-Toteva, Veneta M.; Iakimova, Elena T.

    2013-01-01

    Background and Aims Under stress-promoting conditions unicellular algae can undergo programmed cell death (PCD) but the mechanisms of algal cellular suicide are still poorly understood. In this work, the involvement of caspase-like proteases, DNA cleavage and the morphological occurrence of cell death in wasp venom mastoparan (MP)-treated Chlamydomonas reinhardtii were studied. Methods Algal cells were exposed to MP and cell death was analysed over time. Specific caspase inhibitors were employed to elucidate the possible role of caspase-like proteases. YVADase activity (presumably a vacuolar processing enzyme) was assayed by using a fluorogenic caspase-1 substrate. DNA breakdown was evaluated by DNA laddering and Comet analysis. Cellular morphology was examined by confocal laser scanning microscopy. Key Results MP-treated C. reinhardtii cells expressed several features of necrosis (protoplast shrinkage) and vacuolar cell death (lytic vesicles, vacuolization, empty cell-walled corpse-containing remains of digested protoplast) sometimes within one single cell and in different individual cells. Nucleus compaction and DNA fragmentation were detected. YVADase activity was rapidly stimulated in response to MP but the early cell death was not inhibited by caspase inhibitors. At later time points, however, the caspase inhibitors were effective in cell-death suppression. Conditioned medium from MP-treated cells offered protection against MP-induced cell death. Conclusions In C. reinhardtii MP triggered PCD of atypical phenotype comprising features of vacuolar and necrotic cell deaths, reminiscent of the modality of hypersensitive response. It was assumed that depending on the physiological state and sensitivity of the cells to MP, the early cell-death phase might be not mediated by caspase-like enzymes, whereas later cell death may involve caspase-like-dependent proteolysis. The findings substantiate the hypothesis that, depending on the mode of induction and sensitivity of

  14. Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase[W

    PubMed Central

    Catalanotti, Claudia; Dubini, Alexandra; Subramanian, Venkataramanan; Yang, Wenqiang; Magneschi, Leonardo; Mus, Florence; Seibert, Michael; Posewitz, Matthew C.; Grossman, Arthur R.

    2012-01-01

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. 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 it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism. PMID:22353371

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

    PubMed

    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. PMID:26747175

  16. Taxonomic revision of Chlamydomonas subg. Amphichloris (Volvocales, Chlorophyceae), with resurrection of the genus Dangeardinia and descriptions of Ixipapillifera gen. nov. and Rhysamphichloris gen. nov.

    PubMed

    Nakada, Takashi; Tomita, Masaru; Wu, Jiunn-Tzong; Nozaki, Hisayoshi

    2016-04-01

    Chlamydomonas (Cd.) is one of the largest but most polyphyletic genera of freshwater unicellular green algae. It consists of 400-600 morphological species and requires taxonomic revision. Toward reclassification, each morphologically defined classical subgenus (or subgroup) should be examined using culture strains. Chlamydomonas subg. Amphichloris is characterized by a central nucleus between two axial pyrenoids, however, the phylogenetic structure of this subgenus has yet to be examined using molecular data. Here, we examined 12 strains including six newly isolated strains, morphologically identified as Chlamydomonas subg. Amphichloris, using 18S rRNA gene phylogeny, light microscopy, and mitochondria fluorescent microscopy. Molecular phylogenetic analyses revealed three independent lineages of the subgenus, separated from the type species of Chlamydomonas, Cd. reinhardtii. These three lineages were further distinguished from each other by light and fluorescent microscopy-in particular by the morphology of the papillae, chloroplast surface, stigmata, and mitochondria-and are here assigned to three genera: Dangeardinia emend., Ixipapillifera gen. nov., and Rhysamphichloris gen. nov. Based on the molecular and morphological data, two to three species were recognized in each genus, including one new species, I. pauromitos. In addition, Cd. deasonii, which was previously assigned to subgroup "Pleiochloris," was included in the genus Ixipapillifera as I. deasonii comb. nov. PMID:27037593

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

    PubMed

    Huang, Li-Fen; Lin, Ji-Yu; Pan, Kui-You; Huang, Chun-Kai; Chu, Ying-Kai

    2015-01-01

    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. PMID:26287179

  18. Replacement of tyrosine D with phenylalanine affects the normal proton transfer pathways for the reduction of P680+ in oxygen-evolving photosystem II particles from Chlamydomonas.

    PubMed

    Jeans, C; Schilstra, M J; Ray, N; Husain, S; Minagawa, J; Nugent, J H A; Klug, D R

    2002-12-31

    We have probed the electrostatics of P680(+) reduction in oxygenic photosynthesis using histidine-tagged and histidine-tagged Y(D)-less Photosystem II cores. We make two main observations: (i) that His-tagged Chlamydomonas cores show kinetics which are essentially identical to those of Photosystem II enriched thylakoid membranes from spinach; (ii) that the microsecond kinetics, previously shown to be proton/hydrogen transfer limited [Schilstra et al. (1998) Biochemistry 37, 3974-3981], are significantly different in Y(D)-less Chlamydomonas particles when compared with both the His-tagged Chlamydomonas particles and the spinach membranes. The oscillatory nature of the kinetics in both Chlamydomonas samples is normal, indicating that S-state cycling is unaffected by either the histidine-tagging or the replacement of tyrosine D with phenylalanine. We propose that the effects on the proton-coupled electron transfers of P680(+) reduction in the absence of Y(D) are likely to be due to pK shifts of residues in a hydrogen-bonded network of amino acids in the vicinity of Y(Z). Tyrosine D is 35 A from Y(Z) and yet has a significant influence on proton-coupled electron transfer events in the vicinity of Y(Z). This finding emphasizes the delicacy of the proton balance that Photosystem II has to achieve during the water splitting process. PMID:12501204

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

    PubMed Central

    Huang, Li-Fen; Lin, Ji-Yu; Pan, Kui-You; Huang, Chun-Kai; Chu, Ying-Kai

    2015-01-01

    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. PMID:26287179

  20. Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates

    PubMed Central

    Ning, Jue; Otto, Thomas D.; Pfander, Claudia; Schwach, Frank; Brochet, Mathieu; Bushell, Ellen; Goulding, David; Sanders, Mandy; Lefebvre, Paul A.; Pei, Jimin; Grishin, Nick V.; Vanderlaan, Gary; Billker, Oliver; Snell, William J.

    2013-01-01

    Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa. PMID:23699412

  1. Actinoplanes couchii sp. nov.

    PubMed

    Kämpfer, Peter; Huber, Birgit; Thummes, Kathrin; Grün-Wollny, Iris; Busse, Hans-Jürgen

    2007-04-01

    A Gram-positive bacterium, strain GW8-1761(T), was isolated from soil close to the Marmore waterfalls, Terni, Italy. 16S rRNA gene sequence similarity studies showed that strain GW8-1761(T) belonged to the genus Actinoplanes, being most closely related to Actinoplanes italicus JCM 3165(T) (98.9 %), A. rectilineatus IFO 13941(T) (98.5 %), A. palleronii JCM 7626(T) (97.8 %), A. utahensis IFO 13244(T) (97.6 %) and A. cyaneus DSM 46137(T) (97.6 %). Strain GW8-1761(T) could be distinguished from any other Actinoplanes species with validly published names by 16S rRNA gene sequence similarity values of less than 97.5 %. Chemotaxonomic data [major menaquinone MK-9(H(4)); major polar lipids diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol, with phosphatidylcholine and aminoglycolipids absent; major fatty acids C(15 : 0), C(16 : 0), C(16 : 0) iso, C(17 : 1)omega8c and summed feature 3 (C(16 : 1)omega7c and/or C(15 : 0) iso 2-OH)] supported the affiliation of strain GW8-1761(T) to the genus Actinoplanes. The results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain GW8-1761(T) from the most closely related species. Strain GW8-1761(T) therefore merits species status, and we propose the name Actinoplanes couchii sp. nov., with the type strain GW8-1761(T) (=DSM 45050(T)=CIP 109316(T)). PMID:17392194

  2. Pseudomonas psychrotolerans sp. nov.

    PubMed

    Hauser, Elke; Kämpfer, Peter; Busse, Hans-Jürgen

    2004-09-01

    Three yellow-pigmented, Gram-negative, rod-shaped, non-spore-forming bacterial strains, C36T, C37 and C39, were isolated in the Medical Clinic for Small Animals and Ungulates at the University for Veterinary Medicine in Vienna, Austria. On the basis of 16S rRNA gene sequence similarity, strain C36T was shown to belong to the genus Pseudomonas; Pseudomonas oleovorans DSM 1045T was the nearest relative (99.5 % sequence similarity). Other Pseudomonas species shared <97 % sequence similarity with strain C36T. The presence of Q-9 as the major ubiquinone, the predominance of putrescine and spermidine in its polyamine patterns and its fatty acid profile [i.e. the predominance of C(16 : 0), summed feature 3 (C(16 : 1)omega7c and/or 2-OH C(15 : 0) iso), C(18 : 1)omega7c and the presence of 3-OH C(10 : 0), 3-OH C(12 : 0) and 2-OH C(12 : 0)] were in agreement with identification of this strain as a member of the genus Pseudomonas. Physiological and biochemical characteristics and the results of genomic fingerprinting clearly differentiated strain C36T from its phylogenetic relative P. oleovorans DSM 1045T. Results from DNA-DNA hybridization showed that strain C36T represents a species that is distinct from P. oleovorans DSM 1045T. These data demonstrate that strain C36T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas psychrotolerans sp. nov. is proposed. The type strain is C36T (= LMG 21977T = DSM 15758T). Additionally, physiological, biochemical, chemotaxonomic and genomic fingerprints indicate that P. oleovorans ATCC 29347 may not be a member of the species P. oleovorans sensu stricto. PMID:15388721

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

    NASA Technical Reports Server (NTRS)

    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.

  4. Getting to the heart of intraflagellar transport using Trypanosoma and Chlamydomonas models: the strength is in their differences

    PubMed Central

    2013-01-01

    Cilia and flagella perform diverse roles in motility and sensory perception, and defects in their construction or their function are responsible for human genetic diseases termed ciliopathies. Cilia and flagella construction relies on intraflagellar transport (IFT), the bi-directional movement of ‘trains’ composed of protein complexes found between axoneme microtubules and the flagellum membrane. Although extensive information about IFT components and their mode of action were discovered in the green algae Chlamydomonas reinhardtii, other model organisms have revealed further insights about IFT. This is the case of Trypanosoma brucei, a flagellated protist responsible for sleeping sickness that is turning out to be an emerging model for studying IFT. In this article, we review different aspects of IFT, based on studies of Chlamydomonas and Trypanosoma. Data available from both models are examined to ask challenging questions about IFT such as the initiation of flagellum construction, the setting-up of IFT and the mode of formation of IFT trains, and their remodeling at the tip as well as their recycling at the base. Another outstanding question is the individual role played by the multiple IFT proteins. The use of different models, bringing their specific biological and experimental advantages, will be invaluable in order to obtain a global understanding of IFT. PMID:24289478

  5. The ubiquitin–proteasome pathway protects Chlamydomonas reinhardtii against selenite toxicity, but is impaired as reactive oxygen species accumulate

    PubMed Central

    Vallentine, Patrick; Hung, Chiu-Yueh; Xie, Jiahua; Van Hoewyk, Doug

    2014-01-01

    The ubiquitin–proteasome pathway (UPP) coordinates a myriad of physiological processes in higher plants, including abiotic stress responses, but it is less well characterized in algal species. In this study, the green alga Chlamydomonas reinhardtii was used to gain insights into the role of the UPP during moderate and severe selenite stress at three different time points. The data indicate that activity of the UPP in response to selenium (Se) stress was both time and dose dependent. Moderate selenite stress increased proteasome activity, protein ubiquitination and the proteasomal removal of malformed selenoproteins. However, severe Se stress caused by prolonged selenite treatment or high selenite concentration decreased proteasome activity, inhibited protein ubiquitination and prevented the proteasomal removal of selenoproteins. The UPP impairment during severe Se stress was associated with the observed accumulation of reactive oxygen species (ROS), including mitochondrial superoxide. Additionally, proteasomal inhibition decreased the concentration of chlorophyll in cultures challenged with Se. Therefore, although the UPP protects Chlamydomonas against Se stress, severe oxidative stress induced by selenite toxicity likely hinders the UPP's capacity to mediate a stress response. The possibility that stress tolerance in plants is dependent upon optimal UPP activity and maintenance is discussed. PMID:25301821

  6. Channelrhodopsin-1 Initiates Phototaxis and Photophobic Responses in Chlamydomonas by Immediate Light-Induced Depolarization[W

    PubMed Central

    Berthold, Peter; Tsunoda, Satoshi P.; Ernst, Oliver P.; Mages, Wolfgang; Gradmann, Dietrich; Hegemann, Peter

    2008-01-01

    Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. In neuroscience, they are used to trigger action potentials by light in neuronal cells, tissues, or living animals. Here, we demonstrate that Chlamydomonas cells with low CHR2 content exhibit photophobic and phototactic responses that strictly depend on the availability of CHR1. Since CHR1 was described as a H+-channel, the ion specificity of CHR1 was reinvestigated in Xenopus laevis oocytes. Our experiments show that, in addition to H+, CHR1 also conducts Na+, K+, and Ca2+. The kinetic selectivity analysis demonstrates that H+ selectivity is not due to specific translocation but due to selective ion binding. Purified recombinant CHR1 consists of two isoforms with different absorption maxima, CHR1505 and CHR1463, that are in pH-dependent equilibrium. Thus, CHR1 is a photochromic and protochromic sensory photoreceptor that functions as a light-activated cation channel mediating phototactic and photophobic responses via depolarizing currents in a wide range of ionic conditions. PMID:18552201

  7. Dehydroascorbate: a possible surveillance molecule of oxidative stress and programmed cell death in the green alga Chlamydomonas reinhardtii.

    PubMed

    Murik, Omer; Elboher, Ahinoam; Kaplan, Aaron

    2014-04-01

    Chlamydomonas reinhardtii tolerates relatively high H2 O2 levels that induce an array of antioxidant activities. However, rather than rendering the cells more resistant to oxidative stress, the cells become far more sensitive to an additional H2 O2 dose. If H2 O2 is provided 1.5-9 h after an initial dose, it induces programmed cell death (PCD) in the wild-type, but not in the dum1 mutant impaired in the mitochondrial respiratory complex III. This mutant does not exhibit a secondary oxidative burst 4-5 h after the inducing H2 O2 , nor does it activate metacaspase-1 after the second H2 O2 treatment. The intracellular dehydroascorbate level, a product of ascorbate peroxidase, increases under conditions leading to PCD. The addition of dehydroascorbate induces PCD in the wild-type and dum1 cultures, but higher levels are required in dum1 cells, where it is metabolized faster. The application of dehydroascorbate induces the expression of metacaspase-2, which is much stronger than the expression of metacaspase-1. The presence or absence of oxidative stress, in addition to the rise in internal dehydroascorbate, may determine which metacaspase is activated during Chlamydomonas PCD. Cell death is strongly affected by the timing of H2 O2 or dehydroascorbate admission to synchronously grown cultures, suggesting that the cell cycle phase may distinguish cells that perish from those that do not. PMID:24345283

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

    SciTech Connect

    Singh, K.K.; Changguo Chen; Gibbs, M. )

    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.

  9. Efficient Heterologous Transformation of Chlamydomonas reinhardtii npq2 Mutant with the Zeaxanthin Epoxidase Gene Isolated and Characterized from Chlorella zofingiensis

    PubMed Central

    Couso, Inmaculada; Cordero, Baldo F.; Vargas, María Ángeles; Rodríguez, Herminia

    2012-01-01

    In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga Chlorella zofingiensis (Czzep) has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm). These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications. PMID:23118714

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

    PubMed Central

    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. PMID:15738400

  11. An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii[OPEN

    PubMed Central

    Gang, Spencer S.; Blum, Sean R.; Ivanova, Nina; Yue, Rebecca; Grossman, Arthur R.

    2016-01-01

    The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids. PMID:26764374

  12. Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are non-oncogene addiction genes in cancer cells

    PubMed Central

    Hedrick, Erik; Cheng, Yating; Jin, Un-Ho; Kim, Kyounghyun; Safe, Stephen

    2016-01-01

    Specificity protein (Sp) transcription factor (TF) Sp1 is overexpressed in multiple tumors and is a negative prognostic factor for patient survival. Sp1 and also Sp3 and Sp4 are highly expressed in cancer cells and in this study, we have used results of RNA interference (RNAi) to show that the three TFs individually play a role in the growth, survival and migration/invasion of breast, kidney, pancreatic, lung and colon cancer cell lines. Moreover, tumor growth in athymic nude mice bearing L3.6pL pancreatic cancer cells as xenografts were significantly decreased in cells depleted for Sp1, Sp3 and Sp4 (combined) or Sp1 alone. Ingenuity Pathway Analysis (IPA) of changes in gene expression in Panc1 pancreatic cancer cells after individual knockdown of Sp1, Sp3 and Sp4 demonstrates that these TFs regulate genes and pathways that correlated with the functional responses observed after knockdown but also some genes and pathways that inversely correlated with the functional responses. However, causal IPA analysis which integrates all pathway-dependent changes in all genes strongly predicted that Sp1-, Sp3- and Sp4-regulated genes were associated with the pro-oncogenic activity. These functional and genomic results coupled with overexpression of Sp transcription factors in tumor vs. non-tumor tissues and decreased Sp1 expression with age indicate that Sp1, Sp3 and Sp4 are non-oncogene addiction (NOA) genes and are attractive drug targets for individual and combined cancer chemotherapies. PMID:26967243

  13. H2 and CO2 Evolution by Anaerobically Adapted Chlamydomonas reinhardtii F-60 1

    PubMed Central

    Bamberger, Elchanan S.; King, Dan; Erbes, David L.; Gibbs, Martin

    1982-01-01

    Using manometric and enzymic techniques, H2 and CO2 evolution in darkness and light has been studied in the green alga Chlamydomonas reinhardtii F-60. F-60 is a mutant strain characterized by an incomplete photosynthetic carbon reduction cycle but an intact electron transport chain. In the dark, starch was broken down, and H2 and CO2 was released. The uncoupler, carbonyl cyanide m-fluorophenylhydrazone with an optimum concentration of 5 to 10 micromolar, increased the rate of CO2 release and starch breakdown but depressed H2 formation. It was suggested that carbonyl cyanide m-fluorophenylhydrazone increased the rate of starch breakdown by making the chloroplast membrane permeable to H+, removing a rate-limiting step, and leading to an altered fermentative pattern. Photoevolution of H2 and CO2, but not starch breakdown, was stimulated by acetate. Maximum stimulation occurred at concentrations from 1 to 10 millimolar. Carbonyl cyanide m-fluorophenylhydrazone stimulated starch breakdown and CO2 and H2 release in the light, but not to the extent of acetate. Inasmuch as the uptake and subsequent metabolism of acetate required ATP, it was suggested that acetate, like carbonyl cyanide m-fluorophenylhydrazone, stimulated H2 photoproduction by removing ATP which limited the sequence of reactions. The contribution of photosystem II to the photoproduction of H2, as judged from the effect of 10 micromolar 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, was at least 80%. CO2 photoevolution increased linearly with time, but H2 photoevolution occurred in two phases: a rapid initial phase followed by a second slower phase. The rate of H2 release increased hyperbolically with light intensity, but the rate of CO2 production tended to level off and decrease with increasing light intensity, up to 145 watts per square meter. It was proposed that a changing CO2 and H2 ratio is the result of interaction between the carbon and hydrogen metabolism and the photosynthetic electron transport chain

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

    NASA Astrophysics Data System (ADS)

    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

  15. Time-Course Global Expression Profiles of Chlamydomonas reinhardtii during Photo-Biological H2 Production

    PubMed Central

    Nguyen, Anh Vu; Toepel, Joerg; Burgess, Steven; Uhmeyer, Andreas; Blifernez, Olga; Doebbe, Anja; Hankamer, Ben; Nixon, Peter; Wobbe, Lutz; Kruse, Olaf

    2011-01-01

    We used a microarray study in order to compare the time course expression profiles of two Chlamydomonas reinhardtii strains, namely the high H2 producing mutant stm6glc4 and its parental WT strain during H2 production induced by sulfur starvation. Major cellular reorganizations in photosynthetic apparatus, sulfur and carbon metabolism upon H2 production were confirmed as common to both strains. More importantly, our results pointed out factors which lead to the higher H2 production in the mutant including a higher starch accumulation in the aerobic phase and a lower competition between the H2ase pathway and alternative electron sinks within the H2 production phase. Key candidate genes of interest with differential expression pattern include LHCSR3, essential for efficient energy quenching (qE). The reduced LHCSR3 protein expression in mutant stm6glc4 could be closely related to the high-light sensitive phenotype. H2 measurements carried out with the LHCSR3 knock-out mutant npq4 however clearly demonstrated that a complete loss of this protein has almost no impact on H2 yields under moderate light conditions. The nuclear gene disrupted in the high H2 producing mutant stm6glc4 encodes for the mitochondrial transcription termination factor (mTERF) MOC1, whose expression strongly increases during –S-induced H2 production in WT strains. Studies under phototrophic high-light conditions demonstrated that the presence of functional MOC1 is a prerequisite for proper LHCSR3 expression. Furthermore knock-down of MOC1 in a WT strain was shown to improve the total H2 yield significantly suggesting that this strategy could be applied to further enhance H2 production in other strains already displaying a high H2 production capacity. By combining our array data with previously published metabolomics data we can now explain some of the phenotypic characteristics which lead to an elevated H2 production in stm6glc4. PMID:22242116

  16. Harvesting microalgae cultures with superabsorbent polymers: desulfurization of Chlamydomonas reinhardtii for hydrogen production.

    PubMed

    Martín del Campo, Julia S; Patiño, Rodrigo

    2013-12-01

    It is presented in this work a new methodology to harvest fresh water microalgae cultures by extracting the culture medium with superabsorbent polymers (SAPs). The microalgae Chlamydomonas reinhardtii were grown in the Sueoka culture medium, harvested with polyacrylic SAPs and re-suspended in the culture medium tris-acetate-potassium without sulfur (TAP-S) to generate hydrogen (H2 ) under anoxic conditions. The H2 production as an alternative fuel is relevant since this gas has high-energy recovery without involving carbon. Before microalgae harvesting, a number of range diameters (1-7 mm) for SAPs spherical particles were tested, and the initial rate (V0 ) and the maximal capacity (Qmax ) were determined for the Sueoka medium absorption. The SAP particles with the diameter range 2.0-2.5 mm performed the best and these were employed for the rest of the experiments. The Sueoka medium has a high salt content and the effect of the ionic strength was also studied for different medium concentrations (0-400%). The SAPs were reused in consecutive absorption/desorption cycles, maintaining their absorption capacity. Although the Sueoka medium reduces the SAPs absorption capacity to 40% compared with deionized water, the use of SAPs was very significant for the desulfurization process of C. reihardtii. The presence of C. reinhardtii at different concentrations does not affect the absorption capacity of the Sueoka culture medium by the SAPs. In order to reduce the time of the process, an increase of the SAPs concentration was tested, being 20 g of SAP per liter of medium, a condition to harvest the microalgae culture in 4 h. There were no evident cell ruptures during the harvesting process and the cells remained alive. Finally, the harvested biomass was re-suspended in TAP-S medium and kept under anaerobic conditions and illumination to produce H2 that was monitored by a PEM fuel cell. The use of SAPs for microalgae harvesting is a feasible non-invasive procedure to obtain

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

    PubMed

    Cheloni, Giulia; Marti, Elodie; Slaveykova, Vera I

    2016-01-01

    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 24h were found under all studied conditions. Concentrations of CuO-NPs below 1mgL(-1) do not attenuate the light penetration in the algal suspensions in comparison with NP-free system. Exposure to a combination of 8μgL(-1) or 0.8mgL(-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 0.8mgL(-1) CuO-NPs and SNL for 4 and 8h. Combination of UVR* and 0.8mgL(-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 CuSO4 confirmed the important role of dissolution as toxicity driving force for lower CuO-NP concentration. However, they failed to clarify the contribution of dissolved Cu on the combined effects at 0.8mgL(-1) CuO-NPs. The results point out the necessity of taking into account the possible interactions between ENPs and changing light conditions when evaluating the potential effects of ENPs to phytoplankton in natural waters. PMID:26655656

  18. Effects of nitrogen and phosphorus on arsenite accumulation, oxidation, and toxicity in Chlamydomonas reinhardtii.

    PubMed

    Wang, Ning-Xin; Huang, Bin; Xu, Shen; Wei, Zhong-Bo; Miao, Ai-Jun; Ji, Rong; Yang, Liu-Yan

    2014-12-01

    We studied arsenite (iAs(III)) accumulation, oxidation, and toxicity in the freshwater green alga Chlamydomonas reinhardtii under nutrient-enriched (+NP), phosphorus-limited (-P), and nitrogen-limited (-N) conditions. The -P alga (55.1 μM) had a Michaelis constant (Kd) for uptake approximately one tenth of the +NP (419 μM) and -N (501 μM) cells, indicating iAs(III) uptake inhibition by extracellular phosphate. This conclusion was supported by the hyperbolic reduction in iAs(III) uptake rate (V) from 9.2 to 0.8 μmol/g-dw/h when the extracellular phosphate concentration went up from 0 to 250 μM. The maximal iAs(III) uptake rate (Vmax) of the -N alga (24.3 μmol/g-dw/h) was twice as much as that of the +NP (12 μmol/g-dw/h) and -P (8.1 μmol/g-dw/h) cells. It implies that more arsenic transporters were synthesized under the -N condition. Once accumulated, iAs(III) was oxidized and a higher proportion of arsenate (iAs(V)) was observed at lower [As]dis or under nutrient-limited conditions. Nevertheless, iAs(III) oxidation mainly occurred outside the cells with the extent of oxidation reciprocal to [As]dis. Based on the logistic modeling of the concentration-response curves in the +NP, -P, and -N toxicity tests, iAs(III) had an [As]dis-based EC50 of 1763, 13.1, and 1208 μM and an intracellular arsenic concentration based EC50 of 35.6, 28.8, and 195 μmol/g-dw, respectively. Higher iAs(III) toxicity to the -P cells occured because of their increased iAs(III) accumulation, whereas the underlying mechanisms why the -N alga was more tolerant need to be further revealed. Overall, both N and P had remarkable effects on the behavior and effects of iAs(III), which cannot be disregarded in the biogeochemical cycling research of arsenic. PMID:25456231

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

    PubMed

    Esperanza, Marta; Cid, Ángeles; Herrero, Concepción; Rioboo, Carmen

    2015-08-01

    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 mitochondrial membrane), genotoxicity (oxidative DNA damage, DNA strand breakage, alterations in nuclear morphology), and cell cycle disturbances (subG1-nuclei, decrease of 4N population) in paraquat-treated cells. Overall, the genotoxicity results indicate that the production of ROS caused by exposure to paraquat induces oxidative DNA damage followed by DNA single- and double-strand breaks and cell cycle alterations, possibly leading to apoptosis

  20. Sustainable Hydrogen Photoproduction by Phosphorus-Deprived Marine Green Microalgae Chlorella sp.

    PubMed Central

    Batyrova, Khorcheska; Gavrisheva, Anastasia; Ivanova, Elena; Liu, Jianguo; Tsygankov, Anatoly

    2015-01-01

    Previously it has been shown that green microalga Chlamydomonas reinhardtii is capable of prolonged H2 photoproduction when deprived of sulfur. In addition to sulfur deprivation (-S), sustained H2 photoproduction in C. reinhardtii cultures can be achieved under phosphorus-deprived (-P) conditions. Similar to sulfur deprivation, phosphorus deprivation limits O2 evolving activity in algal cells and causes other metabolic changes that are favorable for H2 photoproduction. Although significant advances in H2 photoproduction have recently been realized in fresh water microalgae, relatively few studies have focused on H2 production in marine green microalgae. In the present study phosphorus deprivation was applied for hydrogen production in marine green microalgae Chlorella sp., where sulfur deprivation is impossible due to a high concentration of sulfates in the sea water. Since resources of fresh water on earth are limited, the possibility of hydrogen production in seawater is more attractive. In order to achieve H2 photoproduction in P-deprived marine green microalgae Chlorella sp., the dilution approach was applied. Cultures diluted to about 0.5–1.8 mg Chl·L−1 in the beginning of P-deprivation were able to establish anaerobiosis, after the initial growth period, where cells utilize intracellular phosphorus, with subsequent transition to H2 photoproduction stage. It appears that marine microalgae during P-deprivation passed the same stages of adaptation as fresh water microalgae. The presence of inorganic carbon was essential for starch accumulation and subsequent hydrogen production by microalgae. The H2 accumulation was up to 40 mL H2 gas per 1iter of the culture, which is comparable to that obtained in P-deprived C. reinhardtii culture. PMID:25629229

  1. Sustainable hydrogen photoproduction by phosphorus-deprived marine green microalgae Chlorella sp.

    PubMed

    Batyrova, Khorcheska; Gavrisheva, Anastasia; Ivanova, Elena; Liu, Jianguo; Tsygankov, Anatoly

    2015-01-01

    Previously it has been shown that green microalga Chlamydomonas reinhardtii is capable of prolonged H2 photoproduction when deprived of sulfur. In addition to sulfur deprivation (-S), sustained H2 photoproduction in C. reinhardtii cultures can be achieved under phosphorus-deprived (-P) conditions. Similar to sulfur deprivation, phosphorus deprivation limits O2 evolving activity in algal cells and causes other metabolic changes that are favorable for H2 photoproduction. Although significant advances in H2 photoproduction have recently been realized in fresh water microalgae, relatively few studies have focused on H2 production in marine green microalgae. In the present study phosphorus deprivation was applied for hydrogen production in marine green microalgae Chlorella sp., where sulfur deprivation is impossible due to a high concentration of sulfates in the sea water. Since resources of fresh water on earth are limited, the possibility of hydrogen production in seawater is more attractive. In order to achieve H2 photoproduction in P-deprived marine green microalgae Chlorella sp., the dilution approach was applied. Cultures diluted to about 0.5-1.8 mg Chl·L-1 in the beginning of P-deprivation were able to establish anaerobiosis, after the initial growth period, where cells utilize intracellular phosphorus, with subsequent transition to H2 photoproduction stage. It appears that marine microalgae during P-deprivation passed the same stages of adaptation as fresh water microalgae. The presence of inorganic carbon was essential for starch accumulation and subsequent hydrogen production by microalgae. The H2 accumulation was up to 40 mL H2 gas per 1iter of the culture, which is comparable to that obtained in P-deprived C. reinhardtii culture. PMID:25629229

  2. The small domain of cytochrome f from the psychrophile Chlamydomonas raudensis UWO 241 modulates the apparent molecular mass and decreases the accumulation of cytochrome f in the mesophile Chlamydomonas reinhardtii.

    PubMed

    Gudynaite-Savitch, Loreta; Loiselay, Christelle; Savitch, Leonid V; Simmonds, John; Kohalmi, Susanne E; Choquet, Yves; Hüner, Norman P A

    2007-10-01

    Cytochrome f from the psychrophile Chlamydomonas raudensis UWO 241 has a lower thermostability of its c-type heme and an apparent molecular mass that is 7 kDa lower than that of the model mesophilic green alga Chlamydomonas reinhardtii. We combined chloroplast transformation, site-directed mutagensis, and the creation of chimeric fusion constructs to assess the contribution of specific domains and (or) amino acids residues to the structure, stability, and accumulation of cytochrome f, as well as its function in photosynthetic intersystem electron transport. We demonstrate that differences in the amino acid sequence of the small domain and specific charged amino acids in the large domain of cytochrome f alter the physical properties of this protein but do not affect either the thermostability of the c-type heme, the apparent half-life of cytochrome f in the presence of the chloroplastic protein synthesis inhibitor chloramphenicol, or the capacity for photosynthetic intersystem electron transport, measured as e-/P700. However, pulse-labeling with [14C]acetate, combined with immunoblotting, indicated that the negative autoregulation of cytochrome f accumulation observed in mesophilic C. reinhardtii transformed with chimeric constructs from the psychrophile was likely the result of the defective association of the chimeric forms of cytochrome f with the other subunits of the cytochrome b6/f complex native to the C. reinhardtii wild type. These results are discussed in terms of the unique fatty acid composition of the thylakoid membranes of C. raudensis UWO 241 adapted to cold environments. PMID:17901903

  3. Lactobacillus apinorum sp. nov., Lactobacillus mellifer sp. nov., Lactobacillus mellis sp. nov., Lactobacillus melliventris sp. nov., Lactobacillus kimbladii sp. nov., Lactobacillus helsingborgensis sp. nov. and Lactobacillus kullabergensis sp. nov., isolated from the honey stomach of the honeybee Apis mellifera

    PubMed Central

    Alsterfjord, Magnus; Nilson, Bo; Butler, Èile; Vásquez, Alejandra

    2014-01-01

    We previously discovered a symbiotic lactic acid bacterial (LAB) microbiota in the honey stomach of the honeybee Apis mellifera. The microbiota was composed of several phylotypes of Bifidobacterium and Lactobacillus. 16S rRNA gene sequence analyses and phenotypic and genetic characteristics revealed that the phylotypes isolated represent seven novel species. One grouped with Lactobacillus kunkeei and the others belong to the Lactobacillus buchneri and Lactobacillus delbrueckiisubgroups of Lactobacillus. We propose the names Lactobacillus apinorum sp. nov., Lactobacillus mellifer sp. nov., Lactobacillus mellis sp. nov., Lactobacillus melliventris sp. nov., Lactobacillus kimbladii sp. nov., Lactobacillus helsingborgensis sp. nov. and Lactobacillus kullabergensis sp. nov. for these novel species, with the respective type strains being Fhon13NT ( = DSM 26257T = CCUG 63287T), Bin4NT ( = DSM 26254T = CCUG 63291T), Hon2NT ( = DSM 26255T = CCUG 63289T), Hma8NT ( = DSM 26256T = CCUG 63629T), Hma2NT ( = DSM 26263T = CCUG 63633T), Bma5NT ( = DSM 26265T = CCUG 63301T) and Biut2NT ( = DSM 26262T = CCUG 63631T). PMID:24944337

  4. Interplay of posttranslational modifications in Sp1 mediates Sp1 stability during cell cycle progression.

    PubMed

    Wang, Yi-Ting; Yang, Wen-Bin; Chang, Wen-Chang; Hung, Jan-Jong

    2011-11-18

    Although Sp1 is known to undergo posttranslational modifications such as phosphorylation, glycosylation, acetylation, sumoylation, and ubiquitination, little is known about the possible interplay between the different forms of Sp1 that may affect its overall levels. It is also unknown whether changes in the levels of Sp1 influence any biological cell processes. Here, we identified RNF4 as the ubiquitin E3 ligase of Sp1. From in vitro and in vivo experiments, we found that sumoylated Sp1 can recruit RNF4 as a ubiquitin E3 ligase that subjects sumoylated Sp1 to proteasomal degradation. Sp1 mapping revealed two ubiquitination-related domains: a small ubiquitin-like modifier in the N-terminus of Sp1(Lys16) and the C-terminus of Sp1 that directly interacts with RNF4. Interestingly, when Sp1 was phosphorylated at Thr739 by c-Jun NH(2)-terminal kinase 1 during mitosis, this phosphorylated form of Sp1 abolished the Sp1-RNF4 interaction. Our results show that, while sumoylated Sp1 subjects to proteasomal degradation, the phosphorylation that occurs during the cell cycle can protect Sp1 from degradation by repressing the Sp1-RNF4 interaction. Thus, we propose that the interplay between posttranslational modifications of Sp1 plays an important role in cell cycle progression and keeps Sp1 at a critical level for mitosis. PMID:21983342

  5. Pantoea vagans sp. nov., Pantoea eucalypti sp. nov., Pantoea deleyi sp. nov. and Pantoea anthophila sp. nov.

    PubMed

    Brady, Carrie L; Venter, Stephanus N; Cleenwerck, Ilse; Engelbeen, Katrien; Vancanneyt, Marc; Swings, Jean; Coutinho, Teresa A

    2009-09-01

    Bacteria isolated from eucalyptus leaves and shoots showing symptoms of blight and die-back collected in Uganda, Uruguay and Argentina and from maize displaying brown stalk rot symptoms in South Africa were tentatively placed in the genus Pantoea on the basis of phenotypic and biochemical tests. These isolates, together with two strains (LMG 2558 and LMG 2560) previously assigned to Pantoea agglomerans based on protein electrophoregrams but later excluded from this species, were further investigated using molecular techniques. 16S rRNA gene sequencing and multilocus sequence analyses (MLSA) revealed that the strains were phylogenetically closely related to Pantoea agglomerans, Pantoea stewartii and Pantoea ananatis. MLSA and amplified fragment length polymorphism analysis placed the strains into four separate clusters, not containing any of the type strains of species of the genus Pantoea. DNA-DNA hybridization confirmed the classification of the isolates into four novel species, for which the names Pantoea vagans sp. nov. (type strain R-21566T=LMG 24199T=BCC 105T=BD 765T), Pantoea eucalypti sp. nov. (type strain R-25678T=LMG 24197T=BCC 076T=BD 769T), Pantoea deleyi sp. nov. (type strain R-31523T=LMG 24200T=BCC 109T=BD 767T) and Pantoea anthophila sp. nov. (type strain LMG 2558T=BD 871T=NCPPB 1682T) are proposed. PMID:19620357

  6. Argonne's SpEC Module

    SciTech Connect

    Harper, Jason

    2014-05-05

    Jason Harper, an electrical engineer in Argonne National Laboratory's EV-Smart Grid Interoperability Center, discusses his SpEC Module invention that will enable fast charging of electric vehicles in under 15 minutes. The module has been licensed to BTCPower.

  7. The Sp(1)-Kepler problems

    SciTech Connect

    Meng Guowu

    2009-07-15

    Let n{>=}2 be a positive integer. To each irreducible representation {sigma} of Sp(1), an Sp(1)-Kepler problem in dimension (4n-3) is constructed and analyzed. This system is superintegrable, and when n=2 it is equivalent to a generalized MICZ-Kepler problem in dimension of 5. The dynamical symmetry group of this system is O-tilde*(4n) with the Hilbert space of bound states H({sigma}) being the unitary highest weight representation of O*-tilde(4n) with highest weight, (-1,{center_dot}{center_dot}{center_dot},-1,-(1+{sigma})), which occurs at the rightmost nontrivial reduction point in the Enright-Howe-Wallach classification diagram for the unitary highest weight modules. Here {sigma} is the highest weight of {sigma}. Furthermore, it is shown that the correspondence {sigma}{r_reversible}H({sigma}) is the theta-correspondence for dual pair (Sp(1),O*(4n))subset Sp(8n,R)

  8. Argonne's SpEC Module

    ScienceCinema

    Harper, Jason

    2014-06-05

    Jason Harper, an electrical engineer in Argonne National Laboratory's EV-Smart Grid Interoperability Center, discusses his SpEC Module invention that will enable fast charging of electric vehicles in under 15 minutes. The module has been licensed to BTCPower.

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

    SciTech Connect

    Willeford, K.O.; Gibbs, M. )

    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}.

  10. Photo-cycle dynamics of LOV1-His domain of phototropin from Chlamydomonas reinhardtii with roseoflavin monophosphate cofactor

    NASA Astrophysics Data System (ADS)

    Tyagi, A.; Penzkofer, A.; Mathes, T.; Hegemann, P.

    2010-09-01

    The wild-type phototropin protein phot from the green alga Chlamydomonas reinhardtii consists of two N-terminal LOV domains LOV1 and LOV2 with flavin mononucleotide (FMN) cofactor and a C-terminal serine-threonine kinase domain. It controls multiple steps in the sexual lifecycle of the alga. Here the LOV1-His domain of phot with modified cofactor is studied. FMN is replaced by roseoflavin monophosphate (8-dimethylamino-8-demethyl-FMN, RoFMN). The modified LOV1 domain is called RoLOV1. The photo-dynamics consequences of the cofactor change are studied. The absorption, emission, and photo-cyclic behaviour of LOV1-His and RoLOV1-His are compared. A spectroscopic characterisation of the cofactors FMN and RoFMN (roseoflavin) is given.

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

    SciTech Connect

    Greenbaum, E.; Lee, J.W.; Tevault, C.V.

    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.

  12. Site-specific mutagenesis of the D1 subunit of photosystem II in wild-type Chlamydomonas.

    PubMed Central

    Przibilla, E; Heiss, S; Johanningmeier, U; Trebst, A

    1991-01-01

    The structure and functional mode of photosystem II reaction center protein D1 can be studied by analyzing the effects of amino acid substitutions within the binding niche for QB, the second stable electron acceptor of photosystem II, on herbicide binding. Here we report on site-directed mutagenesis of the psbA gene coding for the D1 protein in the unicellular alga Chlamydomonas reinhardtii. The chloroplasts of wild-type cells were transformed using the particle gun. The plasmids introduced carried an in vitro mutated fragment of the psbA gene. We obtained a double mutant with replacements of amino acids 264 and 266 and a triple mutant having an additional substitution in position 259. The sensitivities of both mutants toward several types of herbicides are given and compared with those of a mutant having only a substitution at position 264. PMID:1840907

  13. Early alterations on photosynthesis-related parameters in Chlamydomonas reinhardtii cells exposed to atrazine: A multiple approach study.

    PubMed

    Esperanza, Marta; Seoane, Marta; Rioboo, Carmen; Herrero, Concepción; Cid, Ángeles

    2016-06-01

    Chlamydomonas reinhardtii cells were exposed to a sublethal concentration of the widespread herbicide atrazine for 3h. Physiological cellular parameters, such as chlorophyll a fluorescence and oxidative stress monitored by flow cytometry and pigments levels were altered in microalgal cells exposed to 0.25 μM of atrazine. Furthermore, the effects of this herbicide on C. reinhardtii were explored using "omics" techniques. Transcriptomic analyses, carried out by RNA-Seq technique, displayed 9 differentially expressed genes, related to photosynthesis, between control cultures and atrazine exposed cultures. Proteomic profiles were obtained using iTRAQ tags and MALDI-MS/MS analysis, identifying important changes in the proteome during atrazine stress; 5 proteins related to photosynthesis were downexpressed. The results of these experiments advance the understanding of photosynthetic adjustments that occur during an early herbicide exposure. Inhibition of photosynthesis induced by atrazine toxicity will affect the entire physiological and biochemical states of microalgal cells. PMID:26950638

  14. Fatty acid biosynthesis in eukaryotic photosynthetic microalgae: identification of a microsomal delta 12 desaturase in Chlamydomonas reinhardtii.

    PubMed

    Chi, Xiaoyuan; Zhang, Xiaowen; Guan, Xiangyu; Ding, Ling; Li, Youxun; Wang, Mingqing; Lin, Hanzhi; Qin, Song

    2008-04-01

    Polyunsaturated fatty acids (PUFAs) are important components of infant and adult nutrition because they serve as structural elements of cell membranes. Fatty acid desaturases are responsible for the insertion of double bonds into pre-formed fatty acid chains in reactions that require oxygen and reducing equivalents. In this study, the genome-wide characterization of the fatty acid desaturases from seven eukaryotic photosynthetic microalgae was undertaken according to the conserved histidine-rich motifs and phylogenetic profiles. Analysis of these genomes provided insight into the origin and evolution of the pathway of fatty acid biosynthesis in eukaryotic plants. In addition, the candidate enzyme from Chlamydomonas reinhardtii with the highest similarity to the microsomal delta 12 desaturase of Chlorella vulgaris was isolated, and its function was verified by heterologous expression in yeast (Saccharomyces cerevisiae). PMID:18545969

  15. A sex recognition glycoprotein is encoded by the plus mating-type gene fus1 of Chlamydomonas reinhardtii.

    PubMed Central

    Ferris, P J; Woessner, J P; Goodenough, U W

    1996-01-01

    Sexual fusion between plus and minus gametes of the unicellular green alga Chlamydomonas reinhardtii entails adhesion between plus-specific and minus-specific "fringe" proteins displayed on the plasma membrane of gametic mating structures. We report the identification of the gene (fus1) encoding the plus fringe glycoprotein, which resides in a unique domain of the mating-type plus (mt+) locus, and which was identified by transposon insertions in three fusion-defective mutant strains. Transformation with fus1+ restores fringe and fusion competence to these mutants and to the pseudo-plus mutant imp11 mt-, defective in minus differentiation. The fus1 gene is remarkable in lacking the codon bias found in all other nuclear genes of C. reinhardtii. Images PMID:8856667

  16. Identification of a highly conserved hydroxyproline-rich glycoprotein in the cell walls of Chlamydomonas reinhardtii and two other Volvocales.

    PubMed

    Adair, W S; Appel, H

    1989-10-01

    The unicellular alga Chlamydomonas reinhardtii Dang, has a cell wall made entirely from hydroxyproline-rich glycoproteins (HRGPs). We recently employed a quantiative in vitro reconstitution system (Adair et al. 1987, J. Cell Biol. 105, 2373-2382) to assign outer-wall HRGPs of C. reinhardtii to specific sublayers, and describe the major interactions responsible for their assembly. Some of these interactions appear to involve relatively conserved HRGP domains, as evidenced by interspecific cell-wall reconstitution between C. reinhardtii and two multicellular Volvocales (Volvoxcarteri lyengar and Gonium pectorale Müller). In the present report we provide biochemical and immunological evidence that the outer cell-walls of V. carteri and G. pectorale both contain prominent HRGPs closely related to C. reinhardtii GP2. Identification of conserved GP2 homologues indicates a molecular basis for interspecific reconstitution and provides a useful avenue for characterization of HRGP domains mediating cell-wall formation in these algae. PMID:24201668

  17. Environmental Feedbacks and Engineered Nanoparticles: Mitigation of Silver Nanoparticle Toxicity to Chlamydomonas reinhardtii by Algal-Produced Organic Compounds

    PubMed Central

    Stevenson, Louise M.; Dickson, Helen; Klanjscek, Tin; Keller, Arturo A.; McCauley, Edward; Nisbet, Roger M.

    2013-01-01

    The vast majority of nanotoxicity studies measures the effect of exposure to a toxicant on an organism and ignores the potentially important effects of the organism on the toxicant. We investigated the effect of citrate-coated silver nanoparticles (AgNPs) on populations of the freshwater alga Chlamydomonas reinhardtii at different phases of batch culture growth and show that the AgNPs are most toxic to cultures in the early phases of growth. We offer strong evidence that reduced toxicity occurs because extracellular dissolved organic carbon (DOC) compounds produced by the algal cells themselves mitigate the toxicity of AgNPs. We analyzed this feedback with a dynamic model incorporating algal growth, nanoparticle dissolution, bioaccumulation of silver, DOC production and DOC-mediated inactivation of nanoparticles and ionic silver. Our findings demonstrate how the feedback between aquatic organisms and their environment may impact the toxicity and ecological effects of engineered nanoparticles. PMID:24086348

  18. Effect of tetracycline on the growth and nutrient removal capacity of Chlamydomonas reinhardtii in simulated effluent from wastewater treatment plants.

    PubMed

    Li, Jie; Zheng, Xiaoqian; Liu, Kaichuan; Sun, Shujuan; Li, Xiaochen

    2016-10-01

    The aim of this work was to study the effect of tetracycline, which is on the growth, physiological characteristics, and contaminants removal by Chlamydomonas reinhardtii. The results showed that the biomass and photosynthetic pigment concentration of C. reinhardtii exposed to tetracycline were lower than those of the control, while the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, and the malondialdehyde (MDA) content, were higher than those of the control. Additionally, when the tetracycline concentration reached 0.25mg/L, the removal of total nitrogen (TN), total phosphorus (TP), and chemical oxygen demand (COD) decreased from 80.8 to 55.0%, 100 to 92.5%, and 36.5 to 11.5%, respectively. Thus, tetracycline concentrations of 0-0.25mg/L are expected to have a significant effect on the growth and nutrient removal of C. reinhardtii in recycled water from wastewater treatment plants. PMID:27472492

  19. Continuous hydrogen photoproduction by Chlamydomonas reinhardtii: using a novel two-stage, sulfate-limited chemostat system.

    PubMed

    Fedorov, Alexander S; Kosourov, Sergey; Ghirardi, Maria L; Seibert, Michael

    2005-01-01

    This study demonstrates, for the first time, that it is possible to couple sulfate-limited Chlamydomonas reinhardtii growth to continuous H2 photoproduction for more than 4000 h. A two-stage chemostat system physically separates photosynthetic growth from H2 production, and it incorporates two automated photobioreactors (PhBRs). In the first PhBR, the algal cultures are grown aerobically in chemostat mode under limited sulfate to obtain photosynthetically competent cells. Active cells are then continuously delivered to the second PhBR, where H2 production occurs under anaerobic conditions. The dependence of the H2 production rate on sulfate concentration in the medium, dilution rates in the PhBRs, and incident light intensity is reported. PMID:15917617

  20. Transcriptomic and Physiological Responses of the Green Microalga Chlamydomonas reinhardtii during Short-Term Exposure to Subnanomolar Methylmercury Concentrations.

    PubMed

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

    2016-07-01

    The effects of short-term exposure to subnanomolar methyl-mercury (MeHg) concentrations, representative of contaminated environments, on the microalga Chlamydomonas reinhardtii were assessed using both physiological end points and gene expression analysis. MeHg bioaccumulated and induced significant increase of the photosynthesis efficiency, while the algal growth, oxidative stress, and chlorophyll fluorescence were unaffected. At the molecular level, MeHg significantly dysregulated the expression of genes involved in motility, energy metabolism, lipid metabolism, metal transport, and antioxidant enzymes. Data suggest that the cells were able to cope with subnanomolar MeHg exposure, but this tolerance resulted in a significant cost to the cell energy and reserve metabolism as well as ample changes in the nutrition and motility of C. reinhardtii. The present results allowed gaining new insights on the effects and uptake mechanisms of MeHg at subnanomolar concentrations in aquatic primary producers. PMID:27254783

  1. Blastocladiella emersonii expresses a centrin similar to Chlamydomonas reinhardtii isoform not found in late-diverging fungi.

    PubMed

    Ribichich, Karina Fabiana; Gomes, Suely Lopes

    2005-08-15

    Centrins are members of the calcium-binding EF-hand protein superfamily which can be divided into two subfamilies, probably associated with different functions: one related to Chlamydomonas reinhardtii centrin, CrCenp, and the other, represented by Saccharomyces cerevisiae isoform, ScCdc31p. ESTs encoding the two isoforms (BeCen1 and BeCen3) from the chytridiomycete Blastocladiella emersonii were isolated, and expression of the CrCenp-type centrin, BeCen1, was analyzed throughout the fungus life cycle. Becen1 mRNA levels increase transiently during sporulation and protein levels present a similar pattern. Immunolocalization studies seem to localize BeCen1 at the basal body zone and in the cytoplasm surrounding the nuclear cap, a zoospore organelle. PMID:16051227

  2. Repression of Essential Chloroplast Genes Reveals New Signaling Pathways and Regulatory Feedback Loops in Chlamydomonas[W

    PubMed Central

    Ramundo, Silvia; Rahire, Michèle; Schaad, Olivier; Rochaix, Jean-David

    2013-01-01

    Although reverse genetics has been used to elucidate the function of numerous chloroplast proteins, the characterization of essential plastid genes and their role in chloroplast biogenesis and cell survival has not yet been achieved. Therefore, we developed a robust repressible chloroplast gene expression system in the unicellular alga Chlamydomonas reinhardtii based mainly on a vitamin-repressible riboswitch, and we used this system to study the role of two essential chloroplast genes: ribosomal protein S12 (rps12), encoding a plastid ribosomal protein, and rpoA, encoding the α-subunit of chloroplast bacterial-like RNA polymerase. Repression of either of these two genes leads to the arrest of cell growth, and it induces a response that involves changes in expression of nuclear genes implicated in chloroplast biogenesis, protein turnover, and stress. This response also leads to the overaccumulation of several plastid transcripts and reveals the existence of multiple negative regulatory feedback loops in the chloroplast gene circuitry. PMID:23292734

  3. Apparent lack of an O/sup 6/-methylguanine repair mechanism in the unicellular alga, Chlamydomonas ReinhardtII

    SciTech Connect

    Frost, B.; Small, G.D.

    1986-05-01

    O/sup 6/-Methylguanine (O/sup 6/meG) is the presumed major mutagenic lesion formed by the treatment of DNA with methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The repair of this lesion has been shown to involve a protein which selectively removes the O/sup 6/-methyl group by transferring the group to one of the protein's cysteinyl residues. Several prokaryotic and eukaryotic organisms have this O/sup 6/-meG transferase (O/sup 6/MGT) activity, while other (e.g., yeast) lack any apparent O/sup 6/MGT. In some organisms, the O/sup 6/MGT is inducible in response to sublethal doses of methylating agent. The authors have examined Chlamydomonas for such a repair system. This is the first report of a search for O/sup 6/meG repair in a plant system. O/sup 6/meG repair was examined on three levels: in vivo removal of O/sup 6/meG, inducible repair of O/sup 6/meG, the presence of O/sup 6/MGT activity in cellular extracts. They observed no obvious removal of O/sup 6/meG from cellular DNA at various times (up to 30 hours) after treatment of cells with MNNG. The authors were unable to detect any inducible repair of O/sup 6/meG upon pretreatment of cells with sublethal doses of MNNG. Finally, they observed no apparent O/sup 6/MGT activity in cell-free extracts prepared two different ways following the protocols used in E. coli and in rat liver. These results suggest Chlamydomonas apparently lacks a repair mechanism for O/sup 6/meG.

  4. Posttranslational events leading to the assembly of photosystem II protein complex: a study using photosynthesis mutants from Chlamydomonas reinhardtii.

    PubMed

    de Vitry, C; Olive, J; Drapier, D; Recouvreur, M; Wollman, F A

    1989-09-01

    We studied the assembly of photosystem II (PSII) in several mutants from Chlamydomonas reinhardtii which were unable to synthesize either one PSII core subunit (P6 [43 kD], D1, or D2) or one oxygen-evolving enhancer (OEE1 or OEE2) subunit. Synthesis of the PSII subunits was analyzed on electrophoretograms of cells pulse labeled with [14C]acetate. Their accumulation in thylakoid membranes was studied on immunoblots, their chlorophyll-binding ability on nondenaturating gels, their assembly by detergent fractionation, their stability by pulse-chase experiments and determination of in vitro protease sensitivity, and their localization by immunocytochemistry. In Chlamydomonas, the PSII core subunits P5 (47 kD), D1, and D2 are synthesized in a concerted manner while P6 synthesis is independent. P5 and P6 accumulate independently of each other in the stacked membranes. They bind chlorophyll soon after, or concomitantly with, their synthesis and independently of the presence of the other PSII subunits. Resistance to degradation increases step by step: beginning with assembly of P5, D1, and D2, then with binding of P6, and, finally, with binding of the OEE subunits on two independent high affinity sites (one for OEE1 and another for OEE2 to which OEE3 binds). In the absence of PSII cores, the OEE subunits accumulate independently in the thylakoid lumen and bind loosely to the membranes; OEE1 was found on stacked membranes, but OEE2 was found on either stacked or unstacked membranes depending on whether or not P6 was synthesized. PMID:2670960

  5. High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes.

    PubMed

    Remacle, Claire; Cardol, Pierre; Coosemans, Nadine; Gaisne, Mauricette; Bonnefoy, Nathalie

    2006-03-21

    Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100-250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This Deltand4 mutation causes a misassembly of complex I, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex I. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex I, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex I. PMID:16537419

  6. A C2H2 Zinc Finger Protein FEMU2 Is Required for fox1 Expression in Chlamydomonas reinhardtii

    PubMed Central

    Deng, Xiaodong; Yang, Jinghao; Wu, Xiaoxia; Li, YaJun; Fei, Xiaowen

    2014-01-01

    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/T)TTGG(G/T)(G/T)T) 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. PMID:25485540

  7. A fluorescence-activated cell sorting-based strategy for rapid isolation of high-lipid Chlamydomonas mutants

    PubMed Central

    Terashima, Mia; Freeman, Elizabeth S; Jinkerson, Robert E; Jonikas, Martin C

    2015-01-01

    There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the Chlamydomonas high-lipid sorting (CHiLiS) strategy, which enables enrichment of high-lipid mutants by fluorescence-activated cell sorting (FACS) of pooled mutants stained with the lipid-sensitive dye Nile Red. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high-lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20-fold enrichment of the known high-lipid mutant sta1 from a mixture of sta1 and wild-type cells. We then applied CHiLiS to sort thousands of high-lipid cells from a pool of about 60 000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high-lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole-cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS opens the door to a systems-level understanding of green algal lipid biology by enabling genome-saturating isolation of mutants in key genes. PMID:25267488

  8. Characterization of type 2 diacylglycerol acyltransferases in Chlamydomonas reinhardtii reveals their distinct substrate specificities and functions in triacylglycerol biosynthesis.

    PubMed

    Liu, Jin; Han, Danxiang; Yoon, Kangsup; Hu, Qiang; Li, Yantao

    2016-04-01

    Diacylglycerol acyltransferases (DGATs) catalyze a rate-limiting step of triacylglycerol (TAG) biosynthesis in higher plants and yeast. The genome of the green alga Chlamydomonas reinhardtii has multiple genes encoding type 2 DGATs (DGTTs). Here we present detailed functional and biochemical analyses of Chlamydomonas DGTTs. In vitro enzyme analysis using a radiolabel-free assay revealed distinct substrate specificities of three DGTTs: CrDGTT1 preferred polyunsaturated acyl CoAs, CrDGTT2 preferred monounsaturated acyl CoAs, and CrDGTT3 preferred C16 CoAs. When diacylglycerol was used as the substrate, CrDGTT1 preferred C16 over C18 in the sn-2 position of the glycerol backbone, but CrDGTT2 and CrDGTT3 preferred C18 over C16. In vivo knockdown of CrDGTT1, CrDGTT2 or CrDGTT3 resulted in 20-35% decreases in TAG content and a reduction of specific TAG fatty acids, in agreement with the findings of the in vitro assay and fatty acid feeding test. These results demonstrate that CrDGTT1, CrDGTT2 and CrDGTT3 possess distinct specificities toward acyl CoAs and diacylglycerols, and may work in concert spatially and temporally to synthesize diverse TAG species in C. reinhardtii. CrDGTT1 was shown to prefer prokaryotic lipid substrates and probably resides in both the endoplasmic reticulum and chloroplast envelope, indicating its role in prokaryotic and eukaryotic TAG biosynthesis. Based on these findings, we propose a working model for the role of CrDGTT1 in TAG biosynthesis. This work provides insight into TAG biosynthesis in C. reinhardtii, and paves the way for engineering microalgae for production of biofuels and high-value bioproducts. PMID:26919811

  9. Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant.

    PubMed

    Krishnan, Anagha; Kumaraswamy, G Kenchappa; Vinyard, David J; Gu, Huiya; Ananyev, Gennady; Posewitz, Matthew C; Dismukes, G Charles

    2015-03-01

    Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP-glucose pyrophosphorylase. This mutant is unable to convert glucose-1-phosphate to ADP-glucose, the precursor of starch biosynthesis. During nutrient-replete culturing, sta6 does not re-direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC-MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO2) decrease in sta6 due to attenuated rates of NADPH re-oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system-wide consequences of slower NADPH re-oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high-light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient-replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress. PMID:25645872

  10. 3[prime] end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process

    SciTech Connect

    Stern, D.B.; Kindle, K.L. )

    1993-04-01

    The research studied the 3[prime] end maturation of green algae chloroplast atpB mRNA. Most data on transcription termination and 3[prime] end maturation in chloroplasts have been based on in vitro experiments. Newly developed chloroplast transformation techniques have allowed the use of a green algae, Chlamydomonas reinhardtii, to examine chloroplast mRNA 3[prime] end stability determinants and mRNA processing both in vitro and in vivo. The results of this research showed that Chlamydomonas chloroplast protein extracts contain an endonuclease activity that cleaves a synthetic precursor of atpB mRNA 10 nucleotides downstream on the mature 3[prime] end in vitro. Rapid cleavage by this endonuclease is followed by exonucleolytic removal of 10 nucleotides to yield the mature 3[prime] end.

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

    SciTech Connect

    Singh, K.K.; Chen, C.; Epstein, D.K.; Gibbs, M. )

    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.

  12. Alternative Acetate Production Pathways in Chlamydomonas reinhardtii during Dark Anoxia and the Dominant Role of Chloroplasts in Fermentative Acetate Production[W

    PubMed Central

    Catalanotti, Claudia; D’Adamo, Sarah; Wittkopp, Tyler M.; Ingram-Smith, Cheryl J.; Mackinder, Luke; Miller, Tarryn E.; Heuberger, Adam L.; Peers, Graham; Smith, Kerry S.; Jonikas, Martin C.; Grossman, Arthur R.; Posewitz, Matthew C.

    2014-01-01

    Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes. PMID:25381350

  13. Different B-type methionine sulfoxide reductases in Chlamydomonas may protect the alga against high-light, sulfur-depletion, or oxidative stress.

    PubMed

    Zhao, Lei; Chen, Mei; Cheng, Dongmei; Yang, Haomeng; Sun, Yongle; Zhou, Heyi; Huang, Fang

    2013-11-01

    The genome of unicellular green alga Chlamydomonas reinhardtii contains four genes encoding B-type methionine sulfoxide reductases, MSRB1.1, MSRB1.2, MSRB2.1, and MSRB2.2, with functions largely unknown. To understand the cell defense system mediated by the methionine sulfoxide reductases in Chlamydomonas, we analyzed expression and physiological roles of the MSRBs under different abiotic stress conditions using immunoblotting and quantitative polymerase chain reaction (PCR) analyses. We showed that the MSRB2.2 protein was accumulated in cells treated with high light (1,300 µE/m² per s), whereas MSRB1.1 was accumulated in the cells under 1 mmol/L H₂O₂ treatment or sulfur depletion. We observed that the cells with the MSRB2.2 knockdown and overexpression displayed increased and decreased sensitivity to high light, respectively, based on in situ chlorophyll a fluorescence measures. We also observed that the cells with the MSRB1.1 knockdown and overexpression displayed decreased and increased tolerance to sulfur-depletion and oxidative stresses, respectively, based on growth and H₂-producing performance. The physiological implications revealed from the experimental data highlight the importance of MSRB2.2 and MSRB1.1 in protecting Chlamydomonas cells against adverse conditions such as high-light, sulfur-depletion, and oxidative stresses. PMID:24034412

  14. Effect of Triacontanol on Chlamydomonas: II. Specific Activity of Ribulose-Bisphosphate Carboxylase/Oxygenase, Ribulose-Bisphosphate Concentration, and Characteristics of Photorespiration.

    PubMed

    Houtz, R L; Ries, S K; Tolbert, N E

    1985-10-01

    Increased photosynthetic CO(2) assimilation by Chlamydomonas reinhardtii cells treated with triacontanol (TRIA) was not due to changes in glycolate excretion, CO(2) compensation point, or the sensitivity of photosynthetic CO(2) assimilation to O(2). Kinetic analysis of TRIA-treated cells showed that the increase in photosynthetic CO(2) assimilation was a result of an increase in the apparent V(max) for intact cells. The total activity of ribulose-P(2) carboxylase/oxygenase was higher in cell lysates from TRIA-treated cells. However quantification of this enzyme concentration by binding of [(14)C]carboxyarabinitol-P(2) did not show an increase in TRIA-treated cells. Thus, there was an increase in the specific activity of ribulose-P(2) carboxylase/oxygenase extracted from Chlamydomonas cells treated with TRIA. TRIA alone had no effect on the activity of the enzyme in cell lysates from Chlamydomonas or purified from spinach (Spinacia oleracea L.) leaves.The ribulose-P(2) pool was 50 to 60% higher in cells treated with TRIA that were assayed for photosynthetic CO(2) assimilation at high- and low-CO(2). TRIA also increased ribulose-P(2) levels in the absence of CO(2) in the light with atmospheres of N(2) or N(2) with 21% O(2). PMID:16664415

  15. A NIMA-related kinase, Fa2p, localizes to a novel site in the proximal cilia of Chlamydomonas and mouse kidney cells.

    PubMed

    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. PMID:15371535

  16. SP-100 Reactor Subsystem Development

    NASA Astrophysics Data System (ADS)

    Demuth, Scott F.

    1994-07-01

    The SP-100 reactor subsystem consists of the pressure vessel, vessel internals, and fuel elements. Type A (standard) Nb-1Zr and rhenium materials development efforts related to fabrication of the vessel, vessel internals, and fuel cladding/liner have been completed. Type A and Type C (PWC-11) Nb-1Zr loop fabrication has been successfully demonstrated by prototypic testing with flowing lithium at 1350 K for 1500 hr. Development of UN fuel has been completed, and the performance validated by irradiation testing to the full life (7 yr. full power) burnup of 6 atom %. Neutronic and hydraulic core performance have been validated by engineering mockup critical experiments in the Zero Power Physics Reactor at Argonne National Laboratory, and detailed core hydraulic flow testing with water. Essentially all feasibility issues have been settled for the full life SP-100 reactor subsystem. Remaining SP-100 reactor subsystem development efforts are focused on further reducing mass by the use of Type C (PWC-11) Nb-1Zr rather than Type A, and demonstrating fuel life for beyond full life to perhaps 9 atom % burnup.

  17. Cultivation of Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae in Batch culture using Nile tilapia effluent.

    PubMed

    Guerrero-Cabrera, Luis; Rueda, José A; García-Lozano, Hiram; Navarro, A Karin

    2014-06-01

    Monoraphidium sp., Chlorella sp. and Scenedesmus sp. algae were cultured in three volumes of Tilapia Effluent Medium (TEM) in comparison with the Bold Basal Medium (BBM) (Nichols and Bold, 1965). Specific growth rate (μ'), biomass dry productivity (Q), volumetric productivity (Qv) as well as lipid and protein content were measured. Then, volumetric productivities for both lipids and proteins were calculated (QVL and QVP). In Scenedesmus sp., BBM produced higher μ' and Qv than TEM in 1.5L volume. Chlorella sp. showed a higher QVL for BBM than TEM. Any observed difference in protein or lipid productivities among volumes was in favor of a greater productivity for 1.5L volume. Even when TEM had a larger protein content in Chlorella sp. than BBM, QVP was not different. Current results imply that TEM can be used as an alternative growth medium for algae when using Batch cultures, yet productivity is reduced. PMID:24736090

  18. Whole Genome Sequencing Identifies a Deletion in Protein Phosphatase 2A That Affects Its Stability and Localization in Chlamydomonas reinhardtii

    PubMed Central

    Lin, Huawen; Miller, Michelle L.; Granas, David M.; Dutcher, Susan K.

    2013-01-01

    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. PMID:24086163

  19. Comparison of the structural changes occurring during the primary phototransition of two different channelrhodopsins from Chlamydomonas algae.

    PubMed

    Ogren, John I; Yi, Adrian; Mamaev, Sergey; Li, Hai; Lugtenburg, Johan; DeGrip, Willem J; Spudich, John L; Rothschild, Kenneth J

    2015-01-20

    Channelrhodopsins (ChRs) from green flagellate algae function as light-gated ion channels when expressed heterologously in mammalian cells. Considerable interest has focused on understanding the molecular mechanisms of ChRs to bioengineer their properties for specific optogenetic applications such as elucidating the function of specific neurons in brain circuits. While most studies have used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), in this work low-temperature Fourier transform infrared-difference spectroscopy is applied to study the conformational changes occurring during the primary phototransition of the red-shifted ChR1 from Chlamydomonas augustae (CaChR1). Substitution with isotope-labeled retinals or the retinal analogue A2, site-directed mutagenesis, hydrogen-deuterium exchange, and H2(18)O exchange were used to assign bands to the retinal chromophore, protein, and internal water molecules. The primary phototransition of CaChR1 at 80 K involves, in contrast to that of CrChR2, almost exclusively an all-trans to 13-cis isomerization of the retinal chromophore, as in the primary phototransition of bacteriorhodopsin (BR). In addition, significant differences are found for structural changes of the protein and internal water(s) compared to those of CrChR2, including the response of several Asp/Glu residues to retinal isomerization. A negative amide II band is identified in the retinal ethylenic stretch region of CaChR1, which reflects along with amide I bands alterations in protein backbone structure early in the photocycle. A decrease in the hydrogen bond strength of a weakly hydrogen bonded internal water is detected in both CaChR1 and CrChR2, but the bands are much broader in CrChR2, indicating a more heterogeneous environment. Mutations involving residues Glu169 and Asp299 (homologues of the Asp85 and Asp212 Schiff base counterions, respectively, in BR) lead to the conclusion that Asp299 is protonated during P1 formation and suggest that

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

    SciTech Connect

    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

  1. Manufacturing SP-100 rhenium tubes

    NASA Astrophysics Data System (ADS)

    Sayre, Edwin D.; Ruffo, Thomas J.

    1992-01-01

    A process for producing high quality, thin walled, wrought, rhenium tubing was successfully developed and qualified in the SP-100 fuel fabrication program. Rhenium was selected for the fuel-cladding barrier versus tungsten because of the cold workability and nuclear characteristics of rhenium. Several tube fabricating processes including swaging, drawing, and extruding sintered tube shells and chemical vapor deposition were evaluated before a drawn tube made by forming and electron beam welding rhenium strip was selected as the most cost effective. The process for making the rhenium tubes is discussed in general and the tube, room temperature, tensile properties are compared favorably with the properties reported in the literature.

  2. Two New Species of Cryptococcus sp. and Candida sp. from Wild Flowers in Korea

    PubMed Central

    Min, Jin-Hong; Kang, Min-Gu; Ryu, Jin-Ju; Lee, Hyang-Burm; Kim, Chang-Mu; Kim, Ha-Kun

    2012-01-01

    Among 80 types of yeast isolated from wild flowers in Daejeon, Korea, two species that have not yet been identified by phylogenetic analysis of the internal transcribed spacer-2 (ITS2) genes and 26S rDNA sequences were identified as Candida sp. 44-C-1 and Cryptococcus sp. 9-D-1. Neither of the newly identified species formed ascospores, while Candida sp. 44-C-1 formed pseudomycelium and Cryptococcus sp. 9-D-1 did not. PMID:23323051

  3. Zinc Finger Independent Genome-Wide Binding of Sp2 Potentiates Recruitment of Histone-Fold Protein Nf-y Distinguishing It from Sp1 and Sp3

    PubMed Central

    Finkernagel, Florian; Stiewe, Thorsten; Nist, Andrea; Suske, Guntram

    2015-01-01

    Transcription factors are grouped into families based on sequence similarity within functional domains, particularly DNA-binding domains. The Specificity proteins Sp1, Sp2 and Sp3 are paradigmatic of closely related transcription factors. They share amino-terminal glutamine-rich regions and a conserved carboxy-terminal zinc finger domain that can bind to GC rich motifs in vitro. All three Sp proteins are ubiquitously expressed; yet they carry out unique functions in vivo raising the question of how specificity is achieved. Crucially, it is unknown whether they bind to distinct genomic sites and, if so, how binding site selection is accomplished. In this study, we have examined the genomic binding patterns of Sp1, Sp2 and Sp3 in mouse embryonic fibroblasts by ChIP-seq. Sp1 and Sp3 essentially occupy the same promoters and localize to GC boxes. The genomic binding pattern of Sp2 is different; Sp2 primarily localizes at CCAAT motifs. Consistently, re-expression of Sp2 and Sp3 mutants in corresponding knockout MEFs revealed strikingly different modes of genomic binding site selection. Most significantly, while the zinc fingers dictate genomic binding of Sp3, they are completely dispensable for binding of Sp2. Instead, the glutamine-rich amino-terminal region is sufficient for recruitment of Sp2 to its target promoters in vivo. We have identified the trimeric histone-fold CCAAT box binding transcription factor Nf-y as the major partner for Sp2-chromatin interaction. Nf-y is critical for recruitment of Sp2 to co-occupied regulatory elements. Equally, Sp2 potentiates binding of Nf-y to shared sites indicating the existence of an extensive Sp2-Nf-y interaction network. Our results unveil strikingly different recruitment mechanisms of Sp1/Sp2/Sp3 transcription factor members uncovering an unexpected layer of complexity in their binding to chromatin in vivo. PMID:25793500

  4. PSR1 Is a Global Transcriptional Regulator of Phosphorus Deficiency Responses and Carbon Storage Metabolism in Chlamydomonas reinhardtii.

    PubMed

    Bajhaiya, Amit K; Dean, Andrew P; Zeef, Leo A H; Webster, Rachel E; Pittman, Jon K

    2016-03-01

    Many eukaryotic microalgae modify their metabolism in response to nutrient stresses such as phosphorus (P) starvation, which substantially induces storage metabolite biosynthesis, but the genetic mechanisms regulating this response are poorly understood. Here, we show that P starvation-induced lipid and starch accumulation is inhibited in a Chlamydomonas reinhardtii mutant lacking the transcription factor Pi Starvation Response1 (PSR1). Transcriptomic analysis identified specific metabolism transcripts that are induced by P starvation but misregulated in the psr1 mutant. These include transcripts for starch and triacylglycerol synthesis but also transcripts for photosynthesis-, redox-, and stress signaling-related proteins. To further examine the role of PSR1 in regulating lipid and starch metabolism, PSR1 complementation lines in the psr1 strain and PSR1 overexpression lines in a cell wall-deficient strain were generated. PSR1 expression in the psr1 lines was shown to be functional due to rescue of the psr1 phenotype. PSR1 overexpression lines exhibited increased starch content and number of starch granules per cell, which correlated with a higher expression of specific starch metabolism genes but reduced neutral lipid content. Furthermore, this phenotype was consistent in the presence and absence of acetate. Together, these results identify a key transcriptional regulator in global metabolism and demonstrate transcriptional engineering in microalgae to modulate starch biosynthesis. PMID:26704642

  5. Carbon dioxide and light regulation of promoters controlling the expression of mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii.

    PubMed

    Villand, P; Eriksson, M; Samuelsson, G

    1997-10-01

    Nuclear genes coding for carbonic anhydrase, a major mitochondrial constituent in Chlamydomonas reinhardtii grown under limited CO2, were characterized. Two genes, ca1 and ca2, were found within 7 kb of genomic DNA, organized 'head to head' in a large inverted repeat. The DNA sequences for the two genes were very similar, even in the promoter regions and in introns, indicating that the repeat is a result of a recent duplication. To study gene regulation, elements from the upstream region of ca1 were fused to the arylsulphatase reporter gene. After transformation, the expression of arylsulphatase was regulated similarly to the endogenous ca1/ca2 genes, even when the promoter was trimmed down to 194 nt. Expression could not be detected when 5% CO2 was bubbled into the growth medium, but was induced within hours after transfer to air. The ca1 promoter was not induced in low light, but at intermediate light levels its activity was dependent on the irradiance. O2 concentration had no effect on the promoter activity, indicating that photorespiratory metabolites are not triggering the response. The availability of cells transformed with a CO2-regulated reporter gene should facilitate further studies on the metabolic adaptations that occur in some green algae in response to the external CO2 level. PMID:9355734

  6. Expression of chloroplast protein genes during the cell cycle of Chlamydomonas reinhardtii: evidence for transcriptional and translocational control

    SciTech Connect

    Herrin, D.L.

    1986-01-01

    Chlamydomonas reinhardtii cells, growing synchronously under a repeating 12 h light:12 h dark cycle, were used to investigate the synthesis and regulation of chloroplast proteins. The cells accumulate chlorophyll, the major thylakoid membrane proteins, and ribulose-1,5-bisphosphate carboxylase (RuBPCase) during the light (G1) period of the cell cycle. Pulse-labeling in vivo with (/sup 3/H)arginine, and analysis of the protein synthetic capacity of thylakoid-bound polysomes in vitro, shows that these proteins are synthesized de novo during the light. Specific antibody and cloned DNA probes were obtained and used to estimate translatable and/or steady-state mRNA levels for light-harvesting (LHCII) and reaction center (D-1 and D-2) polypeptides of photosystem II, a light-harvesting polypeptide of photosystem I (LHCI), and the large (LS) and small (SS) subunits of RuBPCase. Levels of mRNA for the nuclear-encoded LHCI, LHCII and SS correlated with the synthesis of these polypeptides in vivo; they were higher in the light period and several-folded lower or absent during the dark period. The results suggest that synthesis of nuclear-encoded chloroplast proteins are regulated primarily by the level of mRNA. In contrast, regulation of chloroplast-encoded genes is achieved by controlling the translation of mRNA that is constitutively present, and by transcriptional mechanisms during light induction.

  7. Biogenesis and fate of the cell-cell adhesion molecule, agglutinin, during gametogenesis and fertilization of Chlamydomonas reinhardtii

    SciTech Connect

    Hunnicutt, G.R.

    1989-01-01

    Fertilization in Chlamydomonas begins with the species-specific recognition and adhesion between gametes of opposite mating types via agglutinin molecules on the flagellar surface. This adhesion generates a cAMP-mediated sexual signal that initiates the subsequent events of call wall release, mating structure activation, and cell fusion. Although flagella of paired gametes remain attached to each other until the zygote forms, the process is dynamic. Engaged agglutinins rapidly become inactivated and turnover, requiring the constant supply of new agglutinins to replace the lost molecules. A population of cell body associated agglutinins has been postulated to the pool of agglutinins recruited during this turnover. Cell body agglutinins, therefore were identified, purified, localized within the cells and compared to flagellar agglutinins. The relationship between these two agglutinin populations was also examined. Cell body agglutinins were biochemically indistinguishable from the flagellar form with respect to their M{sub r}, sedimentation coefficient, and hydrophobicity elution properties. Functionally, however, these molecules were inactive in situ. The calculated surface density of agglutinins in the cell body and flagellar domains was similar and thus could not explain their functional difference, but two domains contiguous and yet distinctive suggested they may be separated by a functional barrier. To test this, a method was developed, using a monoclonal antibody and cycloheximide, that removed the flagellar agglutinins so movement between the domains could be monitored. Mobilization of agglutinins onto the flagella did not occur unless sexual signaling was induced with cAMP and papaverine.

  8. A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii[W

    PubMed Central

    Beel, Benedikt; Prager, Katja; Spexard, Meike; Sasso, Severin; Weiss, Daniel; Müller, Nico; Heinnickel, Mark; Dewez, David; Ikoma, Danielle; Grossman, Arthur R.; Kottke, Tilman; Mittag, Maria

    2012-01-01

    Cryptochromes are flavoproteins that act as sensory blue light receptors in insects, plants, fungi, and bacteria. We have investigated a cryptochrome from the green alga Chlamydomonas reinhardtii with sequence homology to animal cryptochromes and (6-4) photolyases. In response to blue and red light exposure, this animal-like cryptochrome (aCRY) alters the light-dependent expression of various genes encoding proteins involved in chlorophyll and carotenoid biosynthesis, light-harvesting complexes, nitrogen metabolism, cell cycle control, and the circadian clock. Additionally, exposure to yellow but not far-red light leads to comparable increases in the expression of specific genes; this expression is significantly reduced in an acry insertional mutant. These in vivo effects are congruent with in vitro data showing that blue, yellow, and red light, but not far-red light, are absorbed by the neutral radical state of flavin in aCRY. The aCRY neutral radical is formed following blue light absorption of the oxidized flavin. Red illumination leads to conversion to the fully reduced state. Our data suggest that aCRY is a functionally important blue and red light–activated flavoprotein. The broad spectral response implies that the neutral radical state functions as a dark form in aCRY and expands the paradigm of flavoproteins and cryptochromes as blue light sensors to include other light qualities. PMID:22773746

  9. The ultrastructure of a Chlamydomonas reinhardtii mutant strain lacking phytoene synthase resembles that of a colorless alga.

    PubMed

    Inwood, William; Yoshihara, Corinne; Zalpuri, Reena; Kim, Kwang-Seo; Kustu, Sydney

    2008-11-01

    Chlamydomonas reinhardtii strains lacking phytoene synthase, the first enzyme of carotenoid biosynthesis, are white. They lack carotenoid pigments, have very low levels of chlorophyll, and can grow only heterotrophically in the dark. Our electron and fluorescence microscopic studies showed that such a mutant strain (lts1-204) had a proliferated plastid envelope membrane but no stacks of thylakoid membranes within the plastid. It accumulated cytoplasmic compartments that appeared to be autophagous vacuoles filled with membranous material. The lts1 mutants apparently lacked pyrenoid bodies, which normally house ribulose bisphosphate carboxylase-oxygenase (Rubisco), and accumulated many starch granules. Although these mutant strains cannot synthesize the carotenoid and carotenoid-derived pigments present in the phototactic organelle (eyespot), the mutant we examined made a vestigial eyespot that was disorganized and often mislocalized to the posterior end of the cell. The absence of a pyrenoid body, the accumulation of starch, and the disorganization of the eyespot may all result from the absence of thylakoids. The ultrastructure of lts1 mutant strains is similar to but distinct from that of previously described white and yellow mutant strains of C. reinhardtii and is similar to that of naturally colorless algae of the Polytoma group. PMID:19825593

  10. Linking toxicity and adaptive responses across the transcriptome, proteome, and phenotype of Chlamydomonas reinhardtii exposed to silver

    PubMed Central

    Pillai, Smitha; Behra, Renata; Nestler, Holger; Suter, Marc J.-F.; Sigg, Laura; Schirmer, Kristin

    2014-01-01

    Understanding mechanistic and cellular events underlying a toxicological outcome allows the prediction of impact of environmental stressors to organisms living in different habitats. A systems-based approach aids in characterizing molecular events, and thereby the cellular pathways that have been perturbed. However, mapping only adverse outcomes of a toxicant falls short of describing the stress or adaptive response that is mounted to maintain homeostasis on perturbations and may confer resistance to the toxic insult. Silver is a potential threat to aquatic organisms because of the increasing use of silver-based nanomaterials, which release free silver ions. The effects of silver were investigated at the transcriptome, proteome, and cellular levels of Chlamydomonas reinhardtii. The cells instigate a fast transcriptome and proteome response, including perturbations in copper transport system and detoxification mechanisms. Silver causes an initial toxic insult, which leads to a plummeting of ATP and photosynthesis and damage because of oxidative stress. In response, the cells mount a defense response to combat oxidative stress and to eliminate silver via efflux transporters. From the analysis of the perturbations of the cell’s functions, we derived a detailed mechanistic understanding of temporal dynamics of toxicity and adaptive response pathways for C. reinhardtii exposed to silver. PMID:24550482

  11. 3D Ultrastructural Organization of Whole Chlamydomonas reinhardtii Cells Studied by Nanoscale Soft X-Ray Tomography

    PubMed Central

    Hummel, Eric; Guttmann, Peter; Werner, Stephan; Tarek, Basel; Schneider, Gerd; Kunz, Michael; Frangakis, Achilleas S.; Westermann, Benedikt

    2012-01-01

    The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology. PMID:23300909

  12. Isolation and characterization of a mutant of Chlamydomonas reinhardtii deficient in the CO sub 2 concentrating mechanism

    SciTech Connect

    Moroney, J.V.; Manual, L.J. ); Husic, H.D. ); Tolbert, N.E. ); Kitayama, M.; Togasaki, R.K. )

    1989-03-01

    A Chlamydomonas reinhardtii mutant has been isolated that cannot grow photoautotrophically on low CO{sub 2} concentrations but can grow on elevated CO{sub 2}. In a test cross, the high CO{sub 2}-requirement for growth showed a 2:2 segregation. This mutant, designated CIA-5, had a phenotype similar to previously identified mutants that were defective in some aspect of CO{sub 2} accumulation. Unlike previously isolated mutants, CIA-5 did not have detectable levels of the periplasmic carbonic anhydrase, an inducible protein that participates in the acquisition of CO{sub 2} by C. reinhardtii. CIA-5 also did not accumulate inorganic carbon to levels higher than could be accounted for by diffusion. This mutant strain did not synthesize any of the four polypeptides preferentially made by wild type C. reinhardtii when switched from an environment containing elevated CO{sub 2} levels to an environment low in CO{sub 2}. It is concluded that this mutant fails to induce the CO{sub 2} concentrating system and is incapable of adapting to low CO{sub 2} conditions.

  13. Direct Estimate of the Spontaneous Mutation Rate Uncovers the Effects of Drift and Recombination in the Chlamydomonas reinhardtii Plastid Genome.

    PubMed

    Ness, Rob W; Kraemer, Susanne A; Colegrave, Nick; Keightley, Peter D

    2016-03-01

    Plastids perform crucial cellular functions, including photosynthesis, across a wide variety of eukaryotes. Since endosymbiosis, plastids have maintained independent genomes that now display a wide diversity of gene content, genome structure, gene regulation mechanisms, and transmission modes. The evolution of plastid genomes depends on an input of de novo mutation, but our knowledge of mutation in the plastid is limited to indirect inference from patterns of DNA divergence between species. Here, we use a mutation accumulation experiment, where selection acting on mutations is rendered ineffective, combined with whole-plastid genome sequencing to directly characterize de novo mutation in Chlamydomonas reinhardtii. We show that the mutation rates of the plastid and nuclear genomes are similar, but that the base spectra of mutations differ significantly. We integrate our measure of the mutation rate with a population genomic data set of 20 individuals, and show that the plastid genome is subject to substantially stronger genetic drift than the nuclear genome. We also show that high levels of linkage disequilibrium in the plastid genome are not due to restricted recombination, but are instead a consequence of increased genetic drift. One likely explanation for increased drift in the plastid genome is that there are stronger effects of genetic hitchhiking. The presence of recombination in the plastid is consistent with laboratory studies in C. reinhardtii and demonstrates that although the plastid genome is thought to be uniparentally inherited, it recombines in nature at a rate similar to the nuclear genome. PMID:26615203

  14. A Nucleus-Encoded Chloroplast Phosphoprotein Governs Expression of the Photosystem I Subunit PsaC in Chlamydomonas reinhardtii.

    PubMed

    Douchi, Damien; Qu, Yujiao; Longoni, Paolo; Legendre-Lefebvre, Linnka; Johnson, Xenie; Schmitz-Linneweber, Christian; Goldschmidt-Clermont, Michel

    2016-05-01

    The nucleo-cytoplasmic compartment exerts anterograde control on chloroplast gene expression through numerous proteins that intervene at posttranscriptional steps. Here, we show that the maturation of psaC mutant (mac1) of Chlamydomonas reinhardtii is defective in photosystem I and fails to accumulate psaC mRNA. The MAC1 locus encodes a member of the Half-A-Tetratricopeptide (HAT) family of super-helical repeat proteins, some of which are involved in RNA transactions. The Mac1 protein localizes to the chloroplast in the soluble fraction. MAC1 acts through the 5' untranslated region of psaC transcripts and is required for their stability. Small RNAs that map to the 5'end of psaC RNA in the wild type but not in the mac1 mutant are inferred to represent footprints of MAC1-dependent protein binding, and Mac1 expressed in bacteria binds RNA in vitro. A coordinate response to iron deficiency, which leads to dismantling of the photosynthetic electron transfer chain and in particular of photosystem I, also causes a decrease of Mac1. Overexpression of Mac1 leads to a parallel increase in psaC mRNA but not in PsaC protein, suggesting that Mac1 may be limiting for psaC mRNA accumulation but that other processes regulate protein accumulation. Furthermore, Mac 1 is differentially phosphorylated in response to iron availability and to conditions that alter the redox balance of the electron transfer chain. PMID:27113776

  15. Origin of pronounced differences in 77 K fluorescence of the green alga Chlamydomonas reinhardtii in state 1 and 2.

    PubMed

    Ünlü, Caner; Polukhina, Iryna; van Amerongen, Herbert

    2016-04-01

    In response to changes in the reduction state of the plastoquinone pool in its thylakoid membrane, the green alga Chlamydomonas reinhardtti is performing state transitions: remodelling of its thylakoid membrane leads to a redistribution of excitations over photosystems I and II (PSI and PSII). These transitions are accompanied by marked changes in the 77 K fluorescence spectrum, which form the accepted signature of state transitions. The changes are generally thought to reflect a redistribution of light-harvesting complexes (LHCs) over PSII (fluorescing below 700 nm) and PSI (fluorescing above 700 nm). Here we studied the picosecond fluorescence properties of C. reinhardtti over a broad range of wavelengths with very low excitation intensities (0.2 nJ per laser pulse). Cells were directly used for time-resolved fluorescence measurements at 77 K without further treatment, such as medium exchange with glycerol. It is observed that upon going from state 1 (relatively more fluorescence below 700 nm) to state 2 (relatively more fluorescence above 700 nm), a large part of the fluorescence of LHC/PSII becomes substantially quenched in concurrence with LHC detachment from PSII, whereas the absolute amount of PSI fluorescence hardly changes. These results are in agreement with the recent proposal that the amount of LHC moving from PSII to PSI upon going from state 1 to state 2 is rather limited (Unlu et al. Proc Natl Acad Sci USA 111 (9):3460-3465, 2014). PMID:26518693

  16. Opportunistic proteolytic processing of carbonic anhydrase 1 from Chlamydomonas in Arabidopsis reveals a novel route for protein maturation.

    PubMed

    Juvale, Parijat S; Wagner, Ryan L; Spalding, Martin H

    2016-04-01

    Proteolytic processing of secretory proteins to yield an active form generally involves specific proteolytic cleavage of a pre-protein. Multiple specific proteases have been identified that target specific pre-protein processing sites in animals. However, characterization of site-specific proteolysis of plant pre-proteins is still evolving. In this study, we characterized proteolytic processing of Chlamydomonas periplasmic carbonic anhydrase 1 (CAH1) in Arabidopsis. CAH1 pre-protein undergoes extensive post-translational modification in the endomembrane system, including glycosylation, disulfide bond formation and proteolytic removal of a peptide 'spacer' region, resulting in a mature, heterotetrameric enzyme with two large and two small subunits. We generated a series of small-scale and large-scale modifications to the spacer and flanking regions to identify potential protease target motifs. Surprisingly, we found that the endoproteolytic removal of the spacer from the CAH1 pre-protein proceeded via an opportunistic process apparently followed by further maturation via amino and carboxy peptidases. We also discovered that the spacer itself is not required for processing, which appears to be dependent only on the number of amino acids separating two key disulfide-bond-forming cysteines. Our data suggest a novel, opportunistic route for pre-protein processing of CAH1. PMID:26917556

  17. A Transient Receptor Potential Ion Channel in Chlamydomonas Shares Key Features with Sensory Transduction-Associated TRP Channels in Mammals

    PubMed Central

    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. PMID:25595824