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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. Cloning and Stress-Induced Expression Analysis of Calmodulin in the Antarctic Alga Chlamydomonas sp. ICE-L.

    PubMed

    He, Ying-Ying; Wang, Yi-Bin; Zheng, Zhou; Liu, Fang-Ming; An, Mei-Ling; He, Xiao-Dong; Qu, Chang-Feng; Li, Lu-Lu; Miao, Jin-Lai

    2017-08-01

    Calmodulin (CaM) is a Ca(2+)-binding protein that plays a role in several Ca(2+) signaling pathways, which dynamically regulates the activities of hundreds of proteins. The ice alga Chlamydomonas sp. ICE-L, which has the ability to adapt to extreme polar conditions, is a crucial primary producer in Antarctic ecosystem. This study hypothesized that Cam helps the ICE-L to adapt to the fluctuating conditions in the polar environment. It first verified the overall length of Cam, through RT-PCR and RACE-PCR, based on partial Cam transcriptome library of ICE-L. Then, the nucleotide and predicted amino acid sequences were, respectively, analyzed by various bioinformatics approaches to gain more insights into the computed physicochemical properties of the CaM. Potential involvements of Cam in responding to certain stimuli (i.e., UVB radiation, high salinity, and temperature) were investigated by differential expression, measuring its transcription levels by means of quantitative RT-PCR. Results showed that CaM was indeed inducible and regulated by high UVB radiation, high salinity, and nonoptimal temperature conditions. Different conditions had different expression tendencies, which provided an important basis for investigating the adaptation mechanism of Cam in ICE-L.

  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.

  4. Molecular cloning and expression analysis of a cytosolic Hsp70 gene from Antarctic ice algae Chlamydomonas sp. ICE-L.

    PubMed

    Liu, Shenghao; Zhang, Pengying; Cong, Bailin; Liu, Chenlin; Lin, Xuezheng; Shen, Jihong; Huang, Xiaohang

    2010-05-01

    A cDNA encoding heat shock protein 70 of Antarctic ice algae Chlamydomonas sp. ICE-L (designated as CiHsp70) was identified by RT-PCR and rapid amplification of cDNA ends approaches. The full-length cDNA of CiHsp70 was 2,232 bp, consisting of a 5'-terminal untranslated region (UTR) of 76 bp, a 3'-terminal UTR of 203 bp with a poly (A) tail, and an open reading frame of 1,953 bp. The CiHsp70 cDNA encoded a polypeptide of 651 amino acids with an ATPase domain of 388 amino acids, the substrate peptide binding domain of 246 amino acids and a C-terminus domain of 17 amino acids. The inducible CiHsp70 cDNA was highly homologous to other plant cytosolic Hsp70 genes and clustered together with green algae and higher plant rather than brown algae, diatom and Cryptophyta. Antarctic ice algae were treated with different stress conditions and messenger RNA (mRNA) expression levels of CiHsp70 were quantified by quantitative RT-PCR. The results showed that both cold and heat shock treatments could stimulate CiHsp70 mRNA expression. Meanwhile, CiHsp70 mRNA expression level increased 2.9-fold in response to UV-B radiation for 6 h, while the expression levels of CiHsp70 were remarkably increased after removing the UV-B radiation and immediately providing additional 6 h visible light. Furthermore, treating with 62 or 93 per thousand NaCl for 2 h, CiHsp70 mRNA expression level increased 3.0- and 2.1-fold, respectively. Together, our observations revealed that CiHsp70 as a molecular chaperone might play an important role in Antarctic ice algae Chlamydomonas sp. ICE-L acclimatizing to polar environment.

  5. Cloning and expression analysis of two different LhcSR genes involved in stress adaptation in an Antarctic microalga, Chlamydomonas sp. ICE-L.

    PubMed

    Mou, Shanli; Zhang, Xiaowen; Ye, Naihao; Dong, Meitao; Liang, Chengwei; Liang, Qiang; Miao, Jinlai; Xu, Dong; Zheng, Zhou

    2012-03-01

    Light-harvesting complexes (LHCs) play essential roles in light capture and photoprotection. Although the functional diversity of individual LHCs in many plants has been well described, knowledge regarding the extent of this family in the majority of green algal groups is still limited. In this study, two different LhcSR genes, LhcSR1 and LhcSR2 from Chlamydomonas sp. ICE-L, were cloned from the total cDNA and characterized in response to high light (HL), low light (LL), UV-B radiation and high salinity. The lower F (v)/F (m) as well as the associated induction of non-photochemical quenching (NPQ), observed under those conditions, indicated that Chlamydomonas sp. ICE-L was under stress. Under HL stress, the expression of LhcSR1 and LhcSR2 increased rapidly from 0.5 h HL and reached a maximum after 3 h. In LL, LhcSR2 expression was up-regulated during the first 0.5 h after which it decreased, while the expression of LhcSR1 decreased gradually from the beginning of the experiment. In addition, the transcript levels of LhcSR1 and LhcSR2 increased under UV-B radiation and high salinity. These results showed that both genes were inducible and up-regulated under stress conditions. A higher NPQ was accompanied by the up-regulated LhcSR genes, suggesting that LhcSR plays a role in thermal energy dissipation. Overall, the results presented here suggest that LhcSR1 and LhcSR2 play a primary role in photoprotection in Chlamydomonas sp. ICE-L under stress conditions and provide an important basis for investigation of the adaptation mechanism of LhcSR in Antarctic green algae.

  6. Analysis of ΔpH and the xanthophyll cycle in NPQ of the Antarctic sea ice alga Chlamydomonas sp. ICE-L.

    PubMed

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

    2013-05-01

    Non-photochemical fluorescence quenching (NPQ) is mainly associated with the transthylakoid proton gradient (ΔpH) and xanthophyll cycle. However, the exact mechanism of NPQ is different in different oxygenic photosynthetic organisms. In this study, several inhibitors were used to study NPQ kinetics in the sea ice alga Chlamydomonas sp. ICE-L and to determine the functions of ΔpH and the xanthophyll cycle in the NPQ process. NH4Cl and nigericin, uncouplers of ΔpH, inhibited NPQ completely and zeaxanthin (Z) was not detected in 1 mM NH4Cl-treated samples. Moreover, Z and NPQ were increased in the samples containing N,N'-dicyclohexyl-carbodiimide (DCCD) under low light conditions. We conclude that ΔpH plays a major role in NPQ, and activation of the xanthophyll cycle is related to ΔpH. In dithiothreitol (DTT)-treated samples, no Z was observed and NPQ decreased. NPQ was completely inhibited when NH4Cl was added suggesting that part of the NPQ process is related to the xanthophyll cycle and the remainder depends on ΔpH. Moreover, lutein and β-carotene were also essential for NPQ. These results indicate that NPQ in the sea ice alga Chlamydomonas sp. ICE-L is mainly dependent on ΔpH which affects the protonation of PSII proteins and de-epoxidation of the xanthophyll cycle, and the transthylakoid proton gradient alone can induce NPQ.

  7. 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.

  8. 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.

  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.

  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.

  11. 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. © 2016 The Authors. Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America.

  12. Growth and lipid content at low temperature of Arctic alga Chlamydomonas sp. KNM0029C.

    PubMed

    Kim, Eun Jae; Jung, Woongsic; Lim, Suyoun; Kim, Sanghee; Han, Se Jong; Choi, Han-Gu

    2016-01-01

    Biodiesel produced from microalgae is a promising source of alternative energy. In winter, however, outdoor mass cultivation for biodiesel production is hampered by poor growth. Here, we report that Arctic Chlamydomonas sp. KNM0029C exhibits optimal growth at 4 °C and reaches densities up to 1.4 × 10(7) cells mL(-1). Lipid body formation in the alga was visualized through BODIPY 505/515 staining and fluorescence microscopy. The fatty acid methyl ester (FAME) production level of KNM0029C was 178.6 mg L(-1) culture and 2.3-fold higher than that of C. reinhardtii CC-125 at 4 °C. Analysis of the FAME content showed a predominance of polyunsaturated fatty acids such as C16:3, C18:2, C18:3, and C20:2. C18:3 fatty acids comprised the largest fraction (20.7%), and the content of polyunsaturated fatty acids (39.6%) was higher than that of saturated fatty acids (6.8%) at 4 °C. These results indicate that Chlamydomonas sp. KNM0029C, as a psychrophilic microalga, might represent a favorable source for biodiesel production in cold environments.

  13. Enzymatic modification by point mutation and functional analysis of an omega-6 fatty acid desaturase from Arctic Chlamydomonas sp.

    PubMed

    Jung, Woongsic; Kim, Eun Jae; Han, Se Jong; Kang, Sung-Ho; Choi, Han-Gu; Kim, Sanghee

    2017-02-07

    Arctic Chlamydomonas sp. is a dominant microalgal strain in cold or frozen freshwater in the Arctic region. The full-length open reading frame of the omega-6 fatty acid desaturase gene (AChFAD6) was obtained from the transcriptomic database of Arctic Chlamydomonas sp. from the KOPRI culture collection of polar micro-organisms. Amino acid sequence analysis indicated the presence of three conserved histidine-rich segments as unique characteristics of omega-6 fatty acid desaturases, and three transmembrane regions transported to plastidic membranes by chloroplast transit peptides in the N-terminal region. The AChFAD6 desaturase activity was examined by expressing wild-type and V254A mutant (Mut-AChFAD6) heterologous recombinant proteins. Quantitative gas chromatography indicated that the concentration of linoleic acids in AChFAD6-transformed cells increased more than 3-fold [6.73 ± 0.13 mg g(-1) dry cell weight (DCW)] compared with cells transformed with vector alone. In contrast, transformation with Mut-AChFAD6 increased the concentration of oleic acid to 9.23 ± 0.18 mg g(-1) DCW, indicating a change in enzymatic activity to mimic that of stearoyl-CoA desaturase. These results demonstrate that AChFAD6 of Arctic Chlamydomonas sp. increases membrane fluidity by enhancing denaturation of C18 fatty acids and facilitates production of large quantities of linoleic fatty acids in prokaryotic expression systems.

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

    DOE PAGES

    Therien, Jesse B.; Zadvornyy, Oleg A.; Posewitz, Matthew C.; ...

    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.

  17. 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.

  18. Proteomic analysis of the response of an acidophilic strain of Chlamydomonas sp. (Chlorophyta) to natural metal-rich water.

    PubMed

    Cid, Cristina; Garcia-Descalzo, Laura; Casado-Lafuente, Victor; Amils, Ricardo; Aguilera, Angeles

    2010-05-01

    A proteomic approach including 2-DE and MALDI-TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas sp. isolated from an extreme acidic environment, Río Tinto (southwest Spain). We have analyzed the soluble proteome obtained from whole cells growing on metal-rich natural acidic water from the river in comparison with the same strain growing in artificial BG-11 media. The most drastic effect was the decrease in the abundance of the ribulose-1,5-biphosphate carboxylase as well as other enzymes related to photosynthesis. However, phytochrome B, phosphoribulokinase, and phosphoglycerate kinase were upregulated when cells were grown in metal-rich acidic water. Besides, increased accumulation of two Hsps, Hsp70 and Hsp90 as well as other stress-related enzymes were also found in the cells growing in natural acidic water. These results suggest that naturally occurring metal-rich water induces a stress response in acidophilic Chlamydomonas forcing algal cells to reorganize their metabolic pathways as an adaptive response to these environmental conditions.

  19. 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

  20. Multiple ice-binding proteins of probable prokaryotic origin in an Antarctic lake alga, Chlamydomonas sp. ICE-MDV (Chlorophyceae).

    PubMed

    Raymond, James A; Morgan-Kiss, Rachael

    2017-08-01

    Ice-associated algae produce ice-binding proteins (IBPs) to prevent freezing damage. The IBPs of the three chlorophytes that have been examined so far share little similarity across species, making it likely that they were acquired by horizontal gene transfer (HGT). To clarify the importance and source of IBPs in chlorophytes, we sequenced the IBP genes of another Antarctic chlorophyte, Chlamydomonas sp. ICE-MDV (Chlamy-ICE). Genomic DNA and total RNA were sequenced and screened for known ice-associated genes. Chlamy-ICE has as many as 50 IBP isoforms, indicating that they have an important role in survival. The IBPs are of the DUF3494 type and have similar exon structures. The DUF3494 sequences are much more closely related to prokaryotic sequences than they are to sequences in other chlorophytes, and the chlorophyte IBP and ribosomal 18S phylogenies are dissimilar. The multiple IBP isoforms found in Chlamy-ICE and other algae may allow the algae to adapt to a greater variety of ice conditions than prokaryotes, which typically have a single IBP gene. The predicted structure of the DUF3494 domain has an ice-binding face with an orderly array of hydrophilic side chains. The results indicate that Chlamy-ICE acquired its IBP genes by HGT in a single event. The acquisitions of IBP genes by this and other species of Antarctic algae by HGT appear to be key evolutionary events that allowed algae to extend their ranges into polar environments. © 2017 Phycological Society of America.

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

    PubMed

    Aguilera, Angeles; Amils, Ricardo

    2005-11-30

    The effects of selected concentrations of Cd on the growth and ultrastructure of three strains of Chlamydomonas sp. isolated from a highly acidic river, Río Tinto (SW Spain) were examined. The river is characterized by its extreme physico-chemical conditions in terms of low pH, mean 2.2 and high concentrations of heavy metals. Growth, Cd accumulation, chlorophyll a, influence of Fe in Cd toxicity and ultrastructural localization were determined. The strains were cultured in both, artificial chemically defined media as well as in natural water from the river. Since iron is the main component of the river water, the effect of different concentrations of this element in relation with Cd toxicity was also analysed. The three strains analysed showed comparable growth and ultrastructural changes. Cd concentration corresponding to 50% growth inhibition (EC50) was 0.2 mM when cells were grown in artificial media. When cells were grown in natural water, no significant differences were found between the controls and the Cd supplemented media even at the highest concentration of 0.8 mM. At an inhibitory level of 0.1 mM of Cd, increasing the concentration of iron up to 90 or 180 mM resulted in a dramatic recovery in algal growth rates in artificial media, reaching normal growth curves. The accumulation of Cd depended on dose and time in the artificial media. The maximal accumulation of Cd was reached after 3 days for all Cd doses, and remained almost unchanged in the subsequent period of time. Chlorophyll a amount depended on dose but not on time in the artificial growth media. At the ultrastructural level, an increase in the periplasmalemmal space was observed due to the presence of a large number of vacuoles, together with a decrease in the relative volume of the nucleus when the cells were incubated in the presence of Cd. Pyrenoid and starch granules were observed and accumulation of spherical electron-dense bodies were also detected. X-ray spectra of these bodies for

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

    PubMed

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

    2014-01-01

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

  3. Biosorption of copper and zinc by immobilised and free algal biomass, and the effects of metal biosorption on the growth and cellular structure of Chlorella sp. and Chlamydomonas sp. isolated from rivers in Penang, Malaysia.

    PubMed

    Maznah, W O Wan; Al-Fawwaz, A T; Surif, Misni

    2012-01-01

    In this study, the biosorption of copper and zinc ions by Chlorella sp. and Chlamydomonas sp. isolated from local environments in Malaysia was investigated in a batch system and by microscopic analyses. Under optimal biosorption conditions, the biosorption capacity of Chlorella sp. for copper and zinc ions was 33.4 and 28.5 mg/g, respectively, after 6 hr of biosorption in an immobilised system. Batch experiments showed that the biosorption capacity of algal biomass immobilised in the form of sodium alginate beads was higher than that of the free biomass. Scanning electron microscopy and energy-dispersive X-ray spectroscopy analyses revealed that copper and zinc were mainly sorbed at the cell surface during biosorption. Exposure to 5 mg/L of copper and zinc affected both the chlorophyll content and cell count of the algal cells after the first 12 hr of contact time.

  4. 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.

  5. 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.

  6. Dynamic metabolic profiling together with transcription analysis reveals salinity-induced starch-to-lipid biosynthesis in alga Chlamydomonas sp. JSC4

    PubMed Central

    Ho, Shih-Hsin; Nakanishi, Akihito; Kato, Yuichi; Yamasaki, Hiroaki; Chang, Jo-Shu; Misawa, Naomi; Hirose, Yuu; Minagawa, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

    2017-01-01

    Biodiesel production using microalgae would play a pivotal role in satisfying future global energy demands. Understanding of lipid metabolism in microalgae is important to isolate oleaginous strain capable of overproducing lipids. It has been reported that reducing starch biosynthesis can enhance lipid accumulation. However, the metabolic mechanism controlling carbon partitioning from starch to lipids in microalgae remains unclear, thus complicating the genetic engineering of algal strains. We here used “dynamic” metabolic profiling and essential transcription analysis of the oleaginous green alga Chlamydomonas sp. JSC4 for the first time to demonstrate the switching mechanisms from starch to lipid synthesis using salinity as a regulator, and identified the metabolic rate-limiting step for enhancing lipid accumulation (e.g., pyruvate-to-acetyl-CoA). These results, showing salinity-induced starch-to-lipid biosynthesis, will help increase our understanding of dynamic carbon partitioning in oleaginous microalgae. Moreover, we successfully determined the changes of several key lipid-synthesis-related genes (e.g., acetyl-CoA carboxylase, pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthetase and pyruvate ferredoxin oxidoreductase) and starch-degradation related genes (e.g., starch phosphorylases), which could provide a breakthrough in the marine microalgal production of biodiesel. PMID:28374798

  7. Dynamic metabolic profiling together with transcription analysis reveals salinity-induced starch-to-lipid biosynthesis in alga Chlamydomonas sp. JSC4.

    PubMed

    Ho, Shih-Hsin; Nakanishi, Akihito; Kato, Yuichi; Yamasaki, Hiroaki; Chang, Jo-Shu; Misawa, Naomi; Hirose, Yuu; Minagawa, Jun; Hasunuma, Tomohisa; Kondo, Akihiko

    2017-04-04

    Biodiesel production using microalgae would play a pivotal role in satisfying future global energy demands. Understanding of lipid metabolism in microalgae is important to isolate oleaginous strain capable of overproducing lipids. It has been reported that reducing starch biosynthesis can enhance lipid accumulation. However, the metabolic mechanism controlling carbon partitioning from starch to lipids in microalgae remains unclear, thus complicating the genetic engineering of algal strains. We here used "dynamic" metabolic profiling and essential transcription analysis of the oleaginous green alga Chlamydomonas sp. JSC4 for the first time to demonstrate the switching mechanisms from starch to lipid synthesis using salinity as a regulator, and identified the metabolic rate-limiting step for enhancing lipid accumulation (e.g., pyruvate-to-acetyl-CoA). These results, showing salinity-induced starch-to-lipid biosynthesis, will help increase our understanding of dynamic carbon partitioning in oleaginous microalgae. Moreover, we successfully determined the changes of several key lipid-synthesis-related genes (e.g., acetyl-CoA carboxylase, pyruvate decarboxylase, acetaldehyde dehydrogenase, acetyl-CoA synthetase and pyruvate ferredoxin oxidoreductase) and starch-degradation related genes (e.g., starch phosphorylases), which could provide a breakthrough in the marine microalgal production of biodiesel.

  8. Molecular mechanisms of the resistance to hydrogen peroxide of enzymes involved in the calvin cycle from halotolerant Chlamydomonas sp. W80.

    PubMed

    Tamoi, M; Kanaboshi, H; Miyasaka, H; Shigeoka, S

    2001-06-15

    cDNA clones encoding NADP(+)-glyceraldehyde-3-phosphate dehydrogenase (NADP(+)-GAPDH) and sedoheptulose-1,7-bisphosphatase (SBPase) were isolated and characterized from halotolerant Chlamydomonas sp. W80 (C. W80) cells. The cDNA clone for NADP(+)-GAPDH encoded 369 amino acid residues, preceded by the chloroplast transit peptide (37 amino acid residues). The cDNA clone for SBPase encoded 351 amino acids with the chloroplast transit peptide. The activities of NADP(+)-GAPDH and SBPase from C. W80 cells were resistant to H(2)O(2) up to 1 mM, as distinct from spinach chloroplastic thiol-modulated enzymes. The illumination to the dark-adapted cells and dithiothreitol treatment to the crude homogenate had little effect on the activities of NADP(+)-GAPDH and SBPase in C. W80. Modeling of the tertiary structures of NADP(+)-GAPDH and SBPase suggests that resistance of the enzymes to H(2)O(2) in C. W80 is due to the different conformational structures in the vicinity of the Cys residues of the chloroplastic enzymes between higher plant and C. W80 cells. Copyright 2001 Academic Press.

  9. Phycoremediation of landfill leachate with the chlorophyte Chlamydomonas sp. SW15aRL and evaluation of toxicity pre and post treatment.

    PubMed

    Paskuliakova, Andrea; McGowan, Ted; Tonry, Steve; Touzet, Nicolas

    2017-09-15

    Landfill leachate treatment is an ongoing challenge in the wastewater management of existing sanitary landfill sites due to the complex nature of leachates and their heavy pollutant load. There is a continuous interest in treatment biotechnologies with expected added benefits for resource recovery; microalgal bioremediation is seen as promising in this regard. Toxicity reduction of landfill leachate subsequent to phycoremediation was investigated in this study. The treatment eventuated from the growth of the ammonia tolerant microalgal strain Chlamydomonas sp. SW15aRL using a N:P ratio adjustment in diluted leachate for facilitating the process. Toxicity tests ranging over a number of trophic levels were applied, including bacterial-yeast (MARA), protistean (microalgae growth inhibition test), crustacean (daphnia, rotifer) and higher plant (monocot, dicot) assays. Ammonia nitrogen in the diluted landfill leachate containing up to 158mgl(-1) NH4(+)-N (60% dilution of the original) was reduced by 83% during the microalgal treatment. Testing prior to remediation indicated the highest toxicity in the crustacean assays Daphnia magna and Brachionus calyciflorus with EC50s at 24h of ~ 35% and 40% leachate dilution, respectively. A major reduction in toxicity was achieved with both bioassays post microalgal treatment with effects well below the EC20s. The microalgae inhibition test on the other hand indicated increased stimulation of growth after treatment as a result of toxicity reduction but also the presence of residual nutrients. Several concurrent processes of both biotic and abiotic natures contributed to pollutant reduction during the treatment. Modifying phosphate dosage especially seems to require further attention. As a by-product of the remediation process, up to 1.2gl(-1) of microalgal biomass was obtained with ~ 18% DW lipid content. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. CHLAMYDOMONAS FLAGELLA

    PubMed Central

    Witman, G. B.; Carlson, K.; Rosenbaum, Joel L.

    1972-01-01

    Quantitative ultrastructural analysis and quantitative gel electrophoresis of preparations of selectively solubilized Chlamydomonas outer doublets indicated that tubulins 1 and 2 were present in both the A tubule and the B tubule, and that only tubulin 1 was present in the three protofilaments which form the wall ("partition") between the lumens of the A and B tubules. The data suggested that the remaining protofilaments of the outer doublet were grouped together in pairs containing the same type of tubulin, pairs containing tubulin 1 alternating with pairs containing tubulin 2. These findings were used to construct models for the arrangement of the two tubulins in the outer doublet. Further analysis by isoelectric focusing resolved tubulins 1 and 2 into at least five bands. PMID:5044758

  11. CHLAMYDOMONAS FLAGELLA

    PubMed Central

    Witman, G. B.; Carlson, K.; Berliner, J.; Rosenbaum, Joel L.

    1972-01-01

    Methods were developed for the isolation of Chlamydomonas flagella and for their fractionation into membrane, mastigoneme, "matrix," and axoneme components. Each component was studied by electron microscopy and acrylamide gel electrophoresis. Purified membranes retained their tripartite ultrastructure and were shown to contain one high molecular weight protein band on electrophoresis in sodium dodecyl sulfate (SDS)-urea gels. Isolated mastigonemes (hairlike structures which extend laterally from the flagellar membrane in situ) were of uniform size and were constructed of ellipsoidal subunits joined end to end. Electrophoretic analysis of mastigonemes indicated that they contained a single glycoprotein of ∼ 170,000 daltons The matrix fraction contained a number of proteins (particularly those of the amorphous material surrounding the microtubules), which became solubilized during membrane removal. Isolated axonemes retained the intact "9 + 2" microtubular structure and could be subfractionated by treatment with heat or detergent. Increasing concentrations of detergent solubilized axonemal microtubules in the following order: one of the two central tubules; the remaining central tubule and the outer wall of the B tubule; the remaining portions of the B tubule; the outer wall of the A tubule; the remainder of the A tubule with the exception of a ribbon of three protofilaments. These three protofilaments appeared to be the "partition" between the lumen of the A and B tubule. Electrophoretic analysis of isolated outer doublets of 9 + 2 flagella of wild-type cells and of "9 + 0" flagella of paralyzed mutants indicated that the outer doublets and central tubules were composed of two microtubule proteins (tubulins 1 and 2) Tubulins 1 and 2 were shown to have apparent molecular weights of 56,000 and 53,000 respectively PMID:4558009

  12. Metabolic and gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C-169.

    PubMed

    Msanne, Joseph; Xu, Di; Konda, Anji Reddy; Casas-Mollano, J Armando; Awada, Tala; Cahoon, Edgar B; Cerutti, Heriberto

    2012-03-01

    Microalgae are emerging as suitable feedstocks for renewable biofuel production. Characterizing the metabolic pathways involved in the biosynthesis of energy-rich compounds, such as lipids and carbohydrates, and the environmental factors influencing their accumulation is necessary to realize the full potential of these organisms as energy resources. The model green alga Chlamydomonas reinhardtii accumulates significant amounts of triacylglycerols (TAGs) under nitrogen starvation or salt stress in medium containing acetate. However, since cultivation of microalgae for biofuel production may need to rely on sunlight as the main source of energy for biomass synthesis, metabolic and gene expression changes occurring in Chlamydomonas and Coccomyxa subjected to nitrogen deprivation were examined under strictly photoautotrophic conditions. Interestingly, nutrient depletion triggered a similar pattern of early synthesis of starch followed by substantial TAG accumulation in both of these fairly divergent green microalgae. A marked decrease in chlorophyll and protein contents was also observed, including reduction in ribosomal polypeptides and some key enzymes for CO₂ assimilation like ribulose-1,5-bisphosphate carboxylase/oxygenase. These results suggest that turnover of nitrogen-rich compounds such as proteins may provide carbon/energy for TAG biosynthesis in the nutrient deprived cells. In Chlamydomonas, several genes coding for diacylglycerol:acyl-CoA acyltransferases, catalyzing the acylation of diacylglycerol to TAG, displayed increased transcript abundance under nitrogen depletion but, counterintuitively, genes encoding enzymes for de novo fatty acid synthesis, such as 3-ketoacyl-ACP synthase I, were down-regulated. Understanding the interdependence of these anabolic and catabolic processes and their regulation may allow the engineering of algal strains with improved capacity to convert their biomass into useful biofuel precursors.

  13. Isolation of Chlamydomonas Flagella

    PubMed Central

    Craige, Branch; Brown, Jason M.; Witman, George B.

    2014-01-01

    A simple, scalable, and fast procedure for the isolation of Chlamydomonas flagella is described. Chlamydomonas can be synchronously deflagellated by treatment with chemicals, pH shock, or mechanical shear. The Basic Protocol describes the procedure for flagellar isolation using dibucaine to induce flagellar abscission; we also describe the pH shock method as an Alternate Protocol when flagellar regeneration is desirable. Sub-fractionation of the isolated flagella into axonemes and the membrane + matrix fraction is described in a Support Protocol. PMID:23728744

  14. The Chlamydomonas cell cycle.

    PubMed

    Cross, Frederick R; Umen, James G

    2015-05-01

    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 has 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 division, probably connected to the interplay between diurnal cycles that drive photosynthetic cell growth and 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 with 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. © 2015 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

  15. Sex determination in Chlamydomonas.

    PubMed

    Goodenough, Ursula; Lin, Huawen; Lee, Jae-Hyeok

    2007-06-01

    The sex-determination system of the unicellular green alga, Chlamydomonas reinhardtii, is governed by genes in the mating-type (MT) locus and entails additional genes located in autosomes. Gene expression is initiated by nitrogen starvation, and cells differentiate into plus or minus gametes within 6h. Reviewed is our current understanding of gametic differentiation and fertilization, initiation of zygote development, and the uniparental inheritance of organelle genomes.

  16. Cytoduction in Chlamydomonas reinhardtii.

    PubMed Central

    Matagne, R F; Remacle, C; Dinant, M

    1991-01-01

    After conjugation between Chlamydomonas gametes of opposite mating type, a transient dikaryon is formed. The two nuclei fuse within 4-6 hr after mating. The young diploid zygote differentiates into dormant zygospore competent to complete meiosis, or more rarely (2-10% of cases) it undergoes mitosis to produce a stable diploid progeny. We here bring genetical, biochemical, and cytological evidence that among the mitotic zygotes, a large proportion of them undergo cytokinesis without fusion of the nuclei-a process that has been termed "cytoduction." By using appropriate genetic markers, haploid cytoductants that possess the nuclear genotype of one parent and the chloroplast marker of the other parent can easily be isolated. Genetical analysis and hybridization experiments moreover show that many haploid cytoductants transmit the chloroplast DNA molecules of both parents and that, as in diploids, these DNA copies occasionally recombine. This process of cytoduction extends the life cycle of Chlamydomonas and provides new tools for its genetic analysis. Images PMID:1871143

  17. The Peroxiredoxin and Glutathione Peroxidase Families in Chlamydomonas reinhardtii

    PubMed Central

    Dayer, Régine; Fischer, Beat B.; Eggen, Rik I. L.; Lemaire, Stéphane D.

    2008-01-01

    Thiol/selenol peroxidases are ubiquitous nonheme peroxidases. They are divided into two major subfamilies: peroxiredoxins (PRXs) and glutathione peroxidases (GPXs). PRXs are present in diverse subcellular compartments and divided into four types: 2-cys PRX, 1-cys PRX, PRX-Q, and type II PRX (PRXII). In mammals, most GPXs are selenoenzymes containing a highly reactive selenocysteine in their active site while yeast and land plants are devoid of selenoproteins but contain nonselenium GPXs. The presence of a chloroplastic 2-cys PRX, a nonselenium GPX, and two selenium-dependent GPXs has been reported in the unicellular green alga Chlamydomonas reinhardtii. The availability of the Chlamydomonas genome sequence offers the opportunity to complete our knowledge on thiol/selenol peroxidases in this organism. In this article, Chlamydomonas PRX and GPX families are presented and compared to their counterparts in Arabidopsis, human, yeast, and Synechocystis sp. A summary of the current knowledge on each family of peroxidases, especially in photosynthetic organisms, phylogenetic analyses, and investigations of the putative subcellular localization of each protein and its relative expression level, on the basis of EST data, are presented. We show that Chlamydomonas PRX and GPX families share some similarities with other photosynthetic organisms but also with human cells. The data are discussed in view of recent results suggesting that these enzymes are important scavengers of reactive oxygen species (ROS) and reactive nitrogen species (RNS) but also play a role in ROS signaling. PMID:18493039

  18. Diversity in photosynthetic electron transport under [CO2]-limitation: the cyanobacterium Synechococcus sp. PCC 7002 and green alga Chlamydomonas reinhardtii drive an O2-dependent alternative electron flow and non-photochemical quenching of chlorophyll fluorescence during CO2-limited photosynthesis.

    PubMed

    Shimakawa, Ginga; Akimoto, Seiji; Ueno, Yoshifumi; Wada, Ayumi; Shaku, Keiichiro; Takahashi, Yuichiro; Miyake, Chikahiro

    2016-12-01

    Some cyanobacteria, but not all, experience an induction of alternative electron flow (AEF) during CO2-limited photosynthesis. For example, Synechocystis sp. PCC 6803 (S. 6803) exhibits AEF, but Synechococcus elongatus sp. PCC 7942 does not. This difference is due to the presence of flavodiiron 2 and 4 proteins (FLV2/4) in S. 6803, which catalyze electron donation to O2. In this study, we observed a low-[CO2] induced AEF in the marine cyanobacterium Synechococcus sp. PCC 7002 that lacks FLV2/4. The AEF shows high affinity for O2, compared with AEF mediated by FLV2/4 in S. 6803, and can proceed under extreme low [O2] (about a few µM O2). Further, the transition from CO2-saturated to CO2-limited photosynthesis leads a preferential excitation of PSI to PSII and increased non-photochemical quenching of chlorophyll fluorescence. We found that the model green alga Chlamydomonas reinhardtii also has an O2-dependent AEF showing the same affinity for O2 as that in S. 7002. These data represent the diverse molecular mechanisms to drive AEF in cyanobacteria and green algae. In this paper, we further discuss the diversity, the evolution, and the physiological function of strategy to CO2-limitation in cyanobacterial and green algal photosynthesis.

  19. 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)

  20. 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)

  1. 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.

  2. Isodityrosine cross-linking mediates insolubilization of cell walls in Chlamydomonas.

    PubMed Central

    Waffenschmidt, S; Woessner, J P; Beer, K; Goodenough, U W

    1993-01-01

    Enzymatic removal of the cell wall induces vegetative Chlamydomonas reinhardtii cells to transcribe wall genes and synthesize new hydroxyproline-rich glycoproteins (HRGPs) related to the extensins found in higher plant cell walls. A cDNA expression library made from such induced cells was screened with antibodies to an oligopeptide containing the (SP)x repetitive domains found in Chlamydomonas wall proteins. One of the selected cDNAs encodes an (SP)x-rich polypeptide that also displays a repeated YGG motif. Ascorbate, a peroxidase inhibitor, and tyrosine derivatives were shown to inhibit insolubilization of both the vegetative and zygotic cell walls of Chlamydomonas, suggesting that oxidative cross-linking of tyrosines is occurring. Moreover, insolubilization of both walls was concomitant with a burst in H2O2 production and in extracellular peroxidase activity. Finally, both isodityrosine and dityrosine were found in hydrolysates of the insolubilized vegetative wall layer. We propose that the formation of tyrosine cross-links is essential to Chlamydomonas HRGP insolubilization. PMID:7689882

  3. Isodityrosine cross-linking mediates insolubilization of cell walls in Chlamydomonas.

    PubMed

    Waffenschmidt, S; Woessner, J P; Beer, K; Goodenough, U W

    1993-07-01

    Enzymatic removal of the cell wall induces vegetative Chlamydomonas reinhardtii cells to transcribe wall genes and synthesize new hydroxyproline-rich glycoproteins (HRGPs) related to the extensins found in higher plant cell walls. A cDNA expression library made from such induced cells was screened with antibodies to an oligopeptide containing the (SP)x repetitive domains found in Chlamydomonas wall proteins. One of the selected cDNAs encodes an (SP)x-rich polypeptide that also displays a repeated YGG motif. Ascorbate, a peroxidase inhibitor, and tyrosine derivatives were shown to inhibit insolubilization of both the vegetative and zygotic cell walls of Chlamydomonas, suggesting that oxidative cross-linking of tyrosines is occurring. Moreover, insolubilization of both walls was concomitant with a burst in H2O2 production and in extracellular peroxidase activity. Finally, both isodityrosine and dityrosine were found in hydrolysates of the insolubilized vegetative wall layer. We propose that the formation of tyrosine cross-links is essential to Chlamydomonas HRGP insolubilization.

  4. 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)

  5. Select Acetophenones Modulate Flagellar Motility in Chlamydomonas

    PubMed Central

    Evans, Shakila K.; Pearce, Austin A.; Ibezim, Prudence K.; Primm, Todd P.; Gaillard, Anne R.

    2009-01-01

    Acetophenones were screened for activity against positive phototaxis of Chlamydomonas cells, a process that requires coordinated flagellar motility. The structure-activity relationships of a series of acetophenones are reported, including acetophenones that affect flagellar motility and cell viability. Notably, 4-methoxyacetophenone, 3,4-dimethoxyacetophenone, and 4-hydroxyacetophenone induced negative phototaxis in Chlamydomonas, suggesting interference with activity of flagellar proteins and control of flagellar dominance. PMID:20659114

  6. Temperature-sensitive rubisco mutant of Chlamydomonas. [Chlamydomonas reinhardtii

    SciTech Connect

    Chen, Z.; Spreitzer, R.J.; Chastain, C.J.

    1987-04-01

    The Chlamydomonas reinhardtii mutant 68-4PP is a temperature-sensitive mutant that lacks photosynthetic ability at 35/sup 0/C, but is able to grow photosynthetically at 25/sup 0/C. Genetic analysis indicated that 68-4PP is a chloroplast mutant that is allelic with known Rubisco large-subunit structural-gene mutants, implying that 68-4PP also resulted from a mutation in the large-subunit gene. The 68-4PP mutant has about 35% of the wild-type level of Rubisco holoenzyme and carboxylase activity when grown at 25/sup 0/C, but it has less than 10% of normal holoenzyme and carboxylase activity when grown at 35/sup 0/C. However, (/sup 35/S)-sulfate pulse labeling showed that Rubisco subunits were synthesized at normal rates at both temperatures. More significantly, the ratio of carboxylase activity in the absence and presence of oxygen at a limiting CO/sub 2/ concentration (6.6 ..mu..M) was about 2.2 for the mutant enzyme, as compared to about 3.0 for the wild-type enzyme. The decreased ratio of the mutant enzyme is maternally inherited, indicating that this reduced oxygen sensitivity results from a mutation in chloroplast DNA. The authors have recently cloned the 68-4PP Rubisco large-subunit gene, and DNA sequencing is in progress.

  7. 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.

  8. 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

  9. Light-Absorbing Carbon in Cloud Residual Nuclei During ICE-L: Combining the Single Particle Soot Photometer and the Counterflow Virtual Impactor

    NASA Astrophysics Data System (ADS)

    Subramanian, R.; Kok, G. L.; Baumgardner, D.; Twohy, C.

    2008-12-01

    The single particle soot photometer (SP2) measures strongly-light absorbing (black) carbon (LAC) using laser incandescence. During the Ice in Clouds Experiment (ICE-L) conducted over Colorado and Wyoming in November/December 2007, the SP2 was operated downstream of a counterflow virtual impactor (CVI) onboard the NCAR C-130 aircraft, when the plane passed through a cloud. The CVI collects cloud droplets and ice crystals larger than 8 μm and evaporates the water content, so that residual nuclei are sampled. The CVI also concentrates the incoming air-stream by as much as a factor of 30 or more. The combination enables measurements of LAC much lower than 1 ng/m3. Results indicate that compared to aerosol in the surrounding air mass, LAC concentrations (per unit volume air) were generally lower in cloud. On November 16, two wave clouds were sampled near Riverton and Wheatland, WY at altitudes between 6-8 km above sea level. LAC mass concentrations upwind of the clouds averaged 5.6 and 4 ng/m3, while in- cloud averages were 0.6 and 0.3 ng/m3 respectively. Average number scavenging ratios of LAC- containing particles measured by the SP2 were 17% and 14% for the two mixed liquid/ice cloud events. In- cloud LAC mass normalized to cloud water content (CWC) was 19 ng/g-CWC in the Riverton cloud, and lower over Wheaton. Multiple passes at different altitudes through the cloud nearer Wheaton did not show a dependence of LAC/CWC on altitude. In a wave cloud over the Wind River Range on November 29, ice-only portions showed LAC/CWC about a factor-of-4 lower than smaller mixed-phase regions of the cloud. Data on LAC measurements in upslope conditions will also be presented.

  10. The Chlamydomonas genome project: a decade on

    PubMed Central

    Blaby, Ian K.; Blaby-Haas, Crysten; Tourasse, Nicolas; Hom, Erik F. Y.; Lopez, David; Aksoy, Munevver; Grossman, Arthur; Umen, James; Dutcher, Susan; Porter, Mary; King, Stephen; Witman, George; Stanke, Mario; Harris, Elizabeth H.; Goodstein, David; Grimwood, Jane; Schmutz, Jeremy; Vallon, Olivier; Merchant, Sabeeha S.; Prochnik, Simon

    2014-01-01

    The green alga Chlamydomonas reinhardtii is a popular unicellular organism for studying photosynthesis, cilia biogenesis and micronutrient homeostasis. Ten years since its genome project was initiated, an iterative process of improvements to the genome and gene predictions has propelled this organism to the forefront of the “omics” era. Housed at Phytozome, the Joint Genome Institute’s (JGI) plant genomics portal, the most up-to-date genomic data include a genome arranged on chromosomes and high-quality gene models with alternative splice forms supported by an abundance of RNA-Seq data. Here, we present the past, present and future of Chlamydomonas genomics. Specifically, we detail progress on genome assembly and gene model refinement, discuss resources for gene annotations, functional predictions and locus ID mapping between versions and, importantly, outline a standardized framework for naming genes. PMID:24950814

  11. A steering mechanism for phototaxis in Chlamydomonas.

    PubMed

    Bennett, Rachel R; Golestanian, Ramin

    2015-03-06

    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. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  12. 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.

  13. 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

  14. 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

  15. Efficient H2 production via Chlamydomonas reinhardtii.

    PubMed

    Esquível, Maria G; Amaro, Helena M; Pinto, Teresa S; Fevereiro, Pedro S; Malcata, F Xavier

    2011-12-01

    Molecular hydrogen (H(2)) obtained from biological sources provides an alternative to bulk chemical processes that is moving towards large-scale, economical generation of clean fuel for automotive engines. This opinion article examines recent improvements in H(2) production by wild and mutant strains of Chlamydomonas reinhardtii - the green microalga currently considered the best eukaryotic H(2) producer. Here, we review various aspects of genetic and metabolic engineering of C. reinhardtii, as well as of process engineering. Additionally, we lay out possible scenarios that would lead to more efficient research approaches in the near future, as part of a consistent strategy for sustainable biohydrogen supply.

  16. [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.

  17. Paternal inheritance of mitochondria in Chlamydomonas.

    PubMed

    Nakamura, Soichi

    2010-03-01

    To analyze mitochondrial DNA (mtDNA)inheritance, differences in mtDNA between Chlamydomonas reinhardtii and Chlamydomonas smithii, respiration deficiency and antibiotic resistance were used to distinguish mtDNA origins. The analyses indicated paternal inheritance. However, these experiments raised questions regarding whether paternal inheritance occurred normally.Mitochondrial nucleoids were observed in living zygotes from mating until 3 days after mating and then until progeny formation. However, selective disappearance of nucleoids was not observed. Subsequently, experimental serial backcrosses between the two strains demonstrated strict paternal inheritance. The fate of mt+ and mt- mtDNA was followed using the differences in mtDNA between the two strains. The slow elimination of mt+ mtDNA through zygote maturation in darkness was observed, and later the disappearance of mt+ mtDNA was observed at the beginning of meiosis. To explain the different fates of mtDNA, methylation status was investigated; however, no methylation was detected. Variously constructed diploid cells showed biparental inheritance. Thus, when the mating process occurs normally, paternal inheritance occurs. Mutations disrupting mtDNA inheritance have not yet been isolated. Mutations that disrupt maternal inheritance of chloroplast DNA (cpDNA) do not disrupt inheritance of mtDNA. The genes responsible for mtDNA inheritance are different from those of chloroplasts.

  18. The Chlamydomonas zygospore: mutant strains of Chlamydomonas monoica blocked in zygospore morphogenesis comprise 46 complementation groups.

    PubMed Central

    VanWinkle-Swift, K; Baron, K; McNamara, A; Minke, P; Burrascano, C; Maddock, J

    1998-01-01

    Chlamydomonas monoica undergoes homothallic sexual reproduction in response to nitrogen starvation. Mating pairs are established in clonal culture via flagellar agglutination and fuse by way of activated mating structures to form the quadriflagellate zygote. The zygote further matures into a dormant diploid zygospore through a series of events that we collectively refer to as zygosporulation. Mutants that arrest development prior to the completion of zygosporulation have been obtained through the use of a variety of mutagens, including ultraviolet irradiation, 5-fluorodeoxyuridine, ethyl methanesulfonate, and methyl methanesulfonate. Complementation analysis indicates that the present mutant collection includes alleles affecting 46 distinct zygote-specific functions. The frequency with which alleles at previously defined loci have been recovered in the most recent mutant searches suggests that as many as 30 additional zygote-specific loci may still remain to be identified. Nevertheless, the present collection should provide a powerful base for ultrastructural, biochemical, and molecular analysis of zygospore morphogenesis and dormancy in Chlamydomonas. PMID:9475727

  19. 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

  20. A brief introduction to the model microswimmer Chlamydomonas reinhardtii

    NASA Astrophysics Data System (ADS)

    Jeanneret, Raphaël; Contino, Matteo; Polin, Marco

    2016-11-01

    The unicellular biflagellate green alga Chlamydomonas reinhardtii has been an important model system in biology for decades, and in recent years it has started to attract growing attention also within the biophysics community. Here we provide a concise review of some of the aspects of Chlamydomonas biology and biophysics most immediately relevant to physicists that might be interested in starting to work with this versatile microorganism.

  1. 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.

  2. 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.

  3. Structure of GUN4 from Chlamydomonas reinhardtii.

    PubMed

    Tarahi Tabrizi, Shabnam; Langley, David B; Harrop, Stephen J; Duff, Anthony P; Willows, Robert D

    2015-08-01

    The genomes uncoupled 4 (GUN4) protein stimulates chlorophyll biosynthesis by increasing the activity of Mg-chelatase, the enzyme that inserts magnesium into protoporphyrin IX (PPIX) in the chlorophyll biosynthesis pathway. One of the roles of GUN4 is in binding PPIX and Mg-PPIX. In eukaryotes, GUN4 also participates in plastid-to-nucleus signalling, although the mechanism for this is unclear. Here, the first crystal structure of a eukaryotic GUN4, from Chlamydomonas reinhardtii, is presented. The structure is in broad agreement with those of previously solved cyanobacterial structures. Most interestingly, conformational divergence is restricted to several loops which cover the porphyrin-binding cleft. The conformational dynamics suggested by this ensemble of structures lend support to the understanding of how GUN4 binds PPIX or Mg-PPIX.

  4. Inorganic Carbon Uptake by Chlamydomonas reinhardtii1

    PubMed Central

    Moroney, James V.; Tolbert, N. Edward

    1985-01-01

    The rates of CO2-dependent O2 evolution by Chlamydomonas reinhardtii, grown with either air levels of CO2 or air with 5% CO2, were measured at varying external pH. Over a pH range of 4.5 to 8.5, the external concentration of CO2 required for half-maximal rates of photosynthesis was constant, averaging 25 micromolar for cells grown with 5% CO2. This is consistent with the hypothesis that these cells take up CO2 but not HCO3− from the medium and that their CO2 requirement for photosynthesis reflects the Km(CO2) of ribulose bisphosphate carboxylase. Over a pH range of 4.5 to 9.5, cells grown with air required an external CO2 concentration of only 0.4 to 3 micromolar for half-maximal rates of photosynthesis, consistent with a mechanism to accumulate external inorganic carbon in these cells. Air-grown cells can utilize external inorganic carbon efficiently even at pH 4.5 where the HCO3− concentration is very low (40 nanomolar). However, at high external pH, where HCO3− predominates, these cells cannot accumulate inorganic carbon as efficiently and require higher concentrations of NaHCO3 to maintain their photosynthetic activity. These results imply that, at the plasma membrane, CO2 is the permeant inorganic carbon species in air-grown cells as well as in cells grown on 5% CO2. If active HCO3− accumulation is a step in CO2 concentration by air-grown Chlamydomonas, it probably takes place in internal compartments of the cell and not at the plasmalemma. PMID:16664038

  5. Photoperiodic control of germination in the unicell Chlamydomonas

    NASA Astrophysics Data System (ADS)

    Suzuki, Lena; Johnson, Carl Hirschie

    2002-03-01

    Photoperiodic time measurement is a well-documented adaptation of multicellular plants and animals to seasonal changes in the environment, but it is unclear whether unicellular organisms can exhibit bona fide photoperiodic responses. We demonstrate that the occurrence of zygospore germination of the unicellular alga Chlamydomonas is a genuine photoperiodic response. Germination efficiency is enhanced in long days as compared with short days. While the total amount of light exposure influences the efficiency of germination, the photoperiod is a significant cue for germination. The developmental stage that senses the photoperiod is just prior to mating and during the first days of zygospore development, so there may be a critical window of zygospore maturation that is regulated by photoperiod. Because zygospores are resistant to freezing injury, whereas vegetative cells are not, it is likely that the suppression of germination by short days is an adaptive response for overwintering of Chlamydomonas. Therefore, Chlamydomonas is a single-celled organism that is capable of photoperiodic responses.

  6. Triacylglycerol mobilization is suppressed by brefeldin A in Chlamydomonas reinhardtii

    PubMed Central

    Kato, Naohiro; Dong, Trung; Bailey, Michael; Lum, Tony; Ingram, Drury

    2013-01-01

    Brefeldin A suppresses vesicle trafficking by inhibiting exchange of GDP for GTP in ADP-ribosylation factor. We report that brefeldin A suppresses mobilization of triacylglycerols in Chlamydomonas reinhardtii, a model organism of green microalgae. Analyses revealed that brefeldin A causes Chlamydomonas to form lipid droplets in which triacylglycerols accumulate in a dose-dependent manner. Pulse labeling experiment using fluorescent fatty acids suggested that brefeldin A inhibits the cells from degrading fatty acids. The experiment also revealed that the cells transiently form novel compartments that accumulate exogenously added fatty acids in the cytoplasm, designated fatty acid-induced microbodies (FAIMs). Brefeldin A up-regulates the formation of FAIMs, whereas nitrogen deprivation that up-regulates triacylglycerol synthesis in Chlamydomonas does not cause the cells to form FAIMs. These results underscore the role of the vesicle trafficking machinery in triacylglycerol metabolism in green microalgae. PMID:23872273

  7. Inorganic Carbon Accumulation by Chlamydomonas reinhardtii1

    PubMed Central

    Manuel, Livingston J.; Moroney, James V.

    1988-01-01

    When the unicellular green alga Chlamydomonas reinhardtii is placed under low CO2 conditions it adapts by making an inorganic carbon accumulating mechanism. Algal cells were labeled with 35SO4−2 during this adaptation period and labeled proteins specific for this low CO2 adaptation were identified. Four major proteins were preferentially synthesized under low CO2 conditions and had Mr of 46, 44, 37, and 20 kilodaltons. The 37 kilodalton protein is most likely the periplasmic carbonic anhydrase previously identified as being part of the inorganic carbon accumulation mechanism of C. reinhardtii. The other three proteins have not been identified. The 46 and the 44 kilodalton proteins were not synthesized by a mutant algal strain, pmp-1, which cannot grow at low CO2 concentrations. This strain does make the 37 and 20 kilodalton proteins, however. These data suggest that at least two or three proteins in addition to the periplasmic carbonic anhydrase are part of the inorganic carbon accumulation mechanism in C. reinhardtii. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:16666333

  8. Eyespot-assembly mutants in Chlamydomonas reinhardtii.

    PubMed Central

    Lamb, M R; Dutcher, S K; Worley, C K; Dieckmann, C L

    1999-01-01

    Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell. PMID:10511552

  9. Analysis of flagellar phosphoproteins from Chlamydomonas reinhardtii.

    PubMed

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2009-07-01

    Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.

  10. 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.

  11. Drosophila roadblock and Chlamydomonas Lc7

    PubMed Central

    Bowman, Aaron B.; Patel-King, Ramila S.; Benashski, Sharon E.; McCaffery, J. Michael; Goldstein, Lawrence S.B.; King, Stephen M.

    1999-01-01

    Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97–amino acid polypeptide that is 57% identical (70% similar) to the 105–amino acid Chlamydomonas outer arm dynein–associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions. PMID:10402468

  12. Radial spoke proteins of Chlamydomonas flagella

    PubMed Central

    Yang, Pinfen; Diener, Dennis R.; Yang, Chun; Kohno, Takahiro; Pazour, Gregory J.; Dienes, Jennifer M.; Agrin, Nathan S.; King, Stephen M.; Sale, Winfield S.; Kamiya, Ritsu; Rosenbaum, Joel L.; Witman, George B.

    2007-01-01

    Summary The radial spoke is a ubiquitous component of ‘9+2’ cilia and flagella, and plays an essential role in the control of dynein arm activity by relaying signals from the central pair of microtubules to the arms. The Chlamydomonas reinhardtii radial spoke contains at least 23 proteins, only 8 of which have been characterized at the molecular level. Here, we use mass spectrometry to identify 10 additional radial spoke proteins. Many of the newly identified proteins in the spoke stalk are predicted to contain domains associated with signal transduction, including Ca2+-, AKAP- and nucleotide-binding domains. This suggests that the spoke stalk is both a scaffold for signaling molecules and itself a transducer of signals. Moreover, in addition to the recently described HSP40 family member, a second spoke stalk protein is predicted to be a molecular chaperone, implying that there is a sophisticated mechanism for the assembly of this large complex. Among the 18 spoke proteins identified to date, at least 12 have apparent homologs in humans, indicating that the radial spoke has been conserved throughout evolution. The human genes encoding these proteins are candidates for causing primary ciliary dyskinesia, a severe inherited disease involving missing or defective axonemal structures, including the radial spokes. PMID:16507594

  13. 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

  14. NUTRITIONAL CONTROL OF SEXUALITY IN CHLAMYDOMONAS REINHARDI

    PubMed Central

    Sager, Ruth; Granick, S.

    1954-01-01

    1. Cells of Chlamydomonas reinhardi grown in the light or dark on standard medium require an additional exposure to light in the absence of a nitrogen source, in order to become sexually active. As the culture ages, the light requirement decreases. 2. This light requirement is a function of nitrogen depletion, as shown by the observation that cells from cultures grown to maturity on a low nitrogen medium in the light or in the dark, have no additional light requirement for zygote formation. The withholding of no other component of the medium has this effect. 3. In cells requiring light for zygote formation, the light can be supplied before the mating types are mixed, indicating that light is required, not for mating per se, but for the conversion of vegetative cells to gametes. 4. Gametes can be dedifferentiated to the vegetative state by any nitrogen compound which the cells can use for growth; and by further exposure to light in the absence of a nitrogen source, these vegetative cells can again become gametic. 5. Cells grown at different nitrogen levels become gametic at widely different cell concentrations of nitrogen and carbon and C/N ratios. 6. It is postulated that the role of light in gametic differentiation is indirect, providing by photosynthesis, energy for the mating process and carbohydrates to tie up excess nitrogenous reserves; and that the concentration of some particular nitrogen fraction or compound determines whether or not gametic differentiation is initiated. PMID:13174779

  15. Recording and analyzing IFT in Chlamydomonas flagella.

    PubMed

    Dentler, William; Vanderwaal, Kristyn; Porter, Mary E

    2009-01-01

    The transport of materials to and from the cell body and tips of eukaryotic flagella and cilia is carried out by a process called intraflagellar transport, or IFT. This process is essential for the assembly and maintenance of cilia and flagella: in the absence of IFT, cilia cannot assemble and, if IFT is arrested in ciliated cells, the cilia disassemble. The major IFT complex proteins and the major motor proteins, kinesin-2 and osm-3 (which transport particles from the cell body to ciliary tips) and cytoplasmic dynein 1b (which transports particles from ciliary tips to the cell body) have been identified. However, we have little understanding of the structure of the IFT particles, the cargo that these particles carry, how cargo is loaded and unloaded from the particles, or how the motor proteins are regulated. The focus of this chapter is to provide methods to observe and quantify the movements of IFT particles in Chlamydomonas flagella. IFT movements can be visualized in paralyzed or partially arrested flagella using either differential interference contrast (IFT) microscopy or, in cells with fluorescently tagged IFT components, with fluorescence microscopy. Methods for recording IFT movements and analyzing movements using kymograms are described.

  16. Genetic tools and techniques for Chlamydomonas reinhardtii.

    PubMed

    Mussgnug, Jan H

    2015-07-01

    The development of tools has always been a major driving force for the advancement of science. Optical microscopes were the first instruments that allowed discovery and descriptive studies of the subcellular features of microorganisms. Although optical and electron microscopes remained at the forefront of microbiological research tools since their inventions, the advent of molecular genetics brought about questions which had to be addressed with new "genetic tools". The unicellular green microalgal genus Chlamydomonas, especially the most prominent species C. reinhardtii, has become a frequently used model organism for many diverse fields of research and molecular genetic analyses of C. reinhardtii, as well as the available genetic tools and techniques, have become increasingly sophisticated throughout the last decades. The aim of this review is to provide an overview of the molecular key features of C. reinhardtii and summarize the progress related to the development of tools and techniques for genetic engineering of this organism, from pioneering DNA transformation experiments to state-of-the-art techniques for targeted nuclear genome editing and high-throughput screening approaches.

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

    PubMed

    Ramos-Martinez, E M; Fimognari, L; Sakuragi, Y

    2017-02-16

    Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability, and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. In order to increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n, wherein n=10 or 20]. The yields of the (SP)n-fused Venus were higher than Venus without the glycomodule by up to 12 folds, with the maximum yield of 15 mg L(-1) . Moreover, the presence of the glycomodules confererred an enhanced proteolytic protein stability. The Venus-(SP)n proteins were shown to be glycosylated, and a treatment of the cells with Brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins. This article is protected by copyright. All rights reserved.

  18. Ammonia Exchange and Photorespiration in Chlamydomonas

    PubMed Central

    Peltier, Gilles; Thibault, Pierre

    1983-01-01

    Two hours after the addition of l-methionine-dl-sulfoximine to the cell suspension, glutamine synthetase activity was inhibited by more than 90% in air-grown Chlamydomonas reinhardii. Cells continued to take up NH3 from the medium provided that the concentration of dissolved CO2 was high (equilibrated with 4% CO2 in air). This NH3 uptake, about 30% of the control, is discussed in terms of glutamate dehydrogenase activity. Without CO2, or with a low CO2 level, a NH3 excretion was observed, the rate of which depended on the actual concentration of the dissolved CO2. Experiments with 15NH3 demonstrated that no NH3 uptake was masked by this excretion and inversely that no excretion occurred during the uptake in the conditions where it took place. Furthermore, the NH3 excretion observed in the absence of CO2 increased when O2 concentration rose to 15% and was inhibited when 10 millimolar isonicotinic acid hydrazide was supplied to the algal suspension. Thus, NH3 excretion in the presence of l-methionine-dl-sulfoximine seems to be related to a photorespiratory process inasmuch as it presents the same properties with regard to the O2 and the isonicotinic acid hydrazide effects. These results favor the hypothesis that NH3 produced in the medium originates from the glycine to serine reaction. On the other hand, partial inhibition (50%) of photosynthesis by l-methionine-dl-sulfoximine was attributed to uncoupling between electron transfer and photophosphorylation due to NH3 accumulation into the cell. PMID:16662924

  19. 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

  20. TETX: a novel nuclear selection marker for Chlamydomonas reinhardtii transformation.

    PubMed

    Garcia-Echauri, Sergio A; Cardineau, Guy A

    2015-01-01

    Transformation of microalgae to obtain recombinant proteins, lipids or metabolites of economic value is of growing interest due to low costs associated with culture growth and scaling up. At present there are only three stable nuclear selection markers for the transformation of Chlamydomonas reinhardtii, which is the most commonly transformed microalgae, specifically: the aminoglycoside phosphotransferaseses aph7and aphVIII and the phleomycin resistance ble gene. As several microalgae are resistant to some of the antibiotics associated with the mentioned resistance genes, we have developed another alternative, tetX, a NADP-requiring Oxidoreductase that hydroxylates tetracycline substrates. We provide evidence that tetX can be used to obtain nuclear transformants of Chlamydomonas reinhardtii. We obtained nuclear transformants harbouring the tetX gene under the control of beta 2 tubulin or HSP70ARBCS2 promoters at an efficiency of transformation of 3.28 and 6.18 colony forming units/μg DNA respectively. This is the first report of a eukaryotic cell transformed using tetracycline as a selectable marker. We developed a protocol for the nuclear transformation of Chlamydomonas reinhardtii using tetX as a selectable marker that confers stable resistance to tetracycline up to 100 μg/mL. We believe tetX can be used to transform Chlamydomonas reinhardtii chloroplasts, related microalgae and other aerobic organisms sensitive to any tetracycline antibiotic.

  1. Separate Origins of Ice-Binding Proteins in Antarctic Chlamydomonas Species

    PubMed Central

    Raymond, James A.; Morgan-Kiss, Rachael

    2013-01-01

    The green alga Chlamydomonas raudensis is an important primary producer in a number of ice-covered lakes and ponds in Antarctica. A C. raudensis isolate (UWO241) from Lake Bonney in the McMurdo Dry Valleys, like many other Antarctic algae, was found to secrete ice-binding proteins (IBPs), which appear to be essential for survival in icy environments. The IBPs of several Antarctic algae (diatoms, a prymesiophyte, and a prasinophyte) are similar to each other (here designated as type I IBPs) and have been proposed to have bacterial origins. Other IBPs (type II IBPs) that bear no resemblance to type I IBPs, have been found in the Antarctic Chlamydomonas sp. CCMP681, a putative snow alga, raising the possibility that chlamydomonad IBPs developed separately from the IBPs of other algae. To test this idea, we obtained the IBP sequences of C. raudensis UWO241 by sequencing the transcriptome. A large number of transcripts revealed no sequences resembling type II IBPs. Instead, many isoforms resembling type I IBPs were found, and these most closely matched a hypothetical protein from the bacterium Stigmatella aurantiaca. The sequences were confirmed to encode IBPs by the activity of a recombinant protein and by the matching of predicted and observed isoelectric points and molecular weights. Furthermore, a mesophilic sister species, C. raudensis SAG49.72, showed no ice-binding activity or PCR products from UWO241 IBP primers. These results confirm that algal IBPs are required for survival in icy habitats and demonstrate that they have diverse origins that are unrelated to the taxonomic positions of the algae. Last, we show that the C. raudensis UWO241 IBPs can change the structure of ice in a way that could increase the survivability of cells trapped in the ice. PMID:23536869

  2. Separate origins of ice-binding proteins in antarctic chlamydomonas species.

    PubMed

    Raymond, James A; Morgan-Kiss, Rachael

    2013-01-01

    The green alga Chlamydomonas raudensis is an important primary producer in a number of ice-covered lakes and ponds in Antarctica. A C. raudensis isolate (UWO241) from Lake Bonney in the McMurdo Dry Valleys, like many other Antarctic algae, was found to secrete ice-binding proteins (IBPs), which appear to be essential for survival in icy environments. The IBPs of several Antarctic algae (diatoms, a prymesiophyte, and a prasinophyte) are similar to each other (here designated as type I IBPs) and have been proposed to have bacterial origins. Other IBPs (type II IBPs) that bear no resemblance to type I IBPs, have been found in the Antarctic Chlamydomonas sp. CCMP681, a putative snow alga, raising the possibility that chlamydomonad IBPs developed separately from the IBPs of other algae. To test this idea, we obtained the IBP sequences of C. raudensis UWO241 by sequencing the transcriptome. A large number of transcripts revealed no sequences resembling type II IBPs. Instead, many isoforms resembling type I IBPs were found, and these most closely matched a hypothetical protein from the bacterium Stigmatella aurantiaca. The sequences were confirmed to encode IBPs by the activity of a recombinant protein and by the matching of predicted and observed isoelectric points and molecular weights. Furthermore, a mesophilic sister species, C. raudensis SAG49.72, showed no ice-binding activity or PCR products from UWO241 IBP primers. These results confirm that algal IBPs are required for survival in icy habitats and demonstrate that they have diverse origins that are unrelated to the taxonomic positions of the algae. Last, we show that the C. raudensis UWO241 IBPs can change the structure of ice in a way that could increase the survivability of cells trapped in the ice.

  3. Flagellar central pair assembly in Chlamydomonas reinhardtii

    PubMed Central

    2013-01-01

    Background Most motile cilia and flagella have nine outer doublet and two central pair (CP) microtubules. Outer doublet microtubules are continuous with the triplet microtubules of the basal body, are templated by the basal body microtubules, and grow by addition of new subunits to their distal (“plus”) ends. In contrast, CP microtubules are not continuous with basal body microtubules, raising the question of how these microtubules are assembled and how their polarity is established. Methods CP assembly in Chlamydomonas reinhardtii was analyzed by electron microscopy and wide-field and super-resolution immunofluorescence microscopy. To analyze CP assembly independently from flagellar assembly, the CP-deficient katanin mutants pf15 or pf19 were mated to wild-type cells. HA-tagged tubulin and the CP-specific protein hydin were used as markers to analyze de novo CP assembly inside the formerly mutant flagella. Results In regenerating flagella, the CP and its projections assemble near the transition zone soon after the onset of outer doublet elongation. During de novo CP assembly in full-length flagella, the nascent CP was first apparent in a subdistal region of the flagellum. The developing CP replaces a fibrous core that fills the axonemal lumen of CP-deficient flagella. The fibrous core contains proteins normally associated with the C1 CP microtubule and proteins involved in intraflagellar transport (IFT). In flagella of the radial spoke-deficient mutant pf14, two pairs of CPs are frequently present with identical correct polarities. Conclusions The temporal separation of flagellar and CP assembly in dikaryons formed by mating CP-deficient gametes to wild-type gametes revealed that the formation of the CP does not require proximity to the basal body or transition zone, or to the flagellar tip. The observations on pf14 provide further support that the CP self-assembles without a template and eliminate the possibility that CP polarity is established by interaction

  4. 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

  5. Strategies to facilitate transgene expression in Chlamydomonas reinhardtii.

    PubMed

    Eichler-Stahlberg, Alke; Weisheit, Wolfram; Ruecker, Ovidiu; Heitzer, Markus

    2009-03-01

    The unicellular green alga Chlamydomonas reinhardtii has been identified as a promising organism for the production of recombinant proteins. While during the last years important improvements have been developed for the production of proteins within the chloroplast, the expression levels of transgenes from the nuclear genome were too low to be of biotechnological importance. In this study, we integrated endogenous intronic sequences into the expression cassette to enhance the expression of transgenes in the nucleus. The insertion of one or more copies of intron sequences from the Chlamydomonas RBCS2 gene resulted in increased expression levels of a Renilla-luciferase gene used as a reporter. Although any of the three RBCS2 introns alone had a positive effect on expression, their integration in their physiological number and order created an over-proportional stimulating effect observed in all transformants. The secretion of the luciferase protein into the medium was achieved by using the export sequence of the Chlamydomonas ARS2 gene in a cell wall deficient strain and Renilla-luciferase could be successfully concentrated with the help of attached C-terminal protein tags. Similarly, a codon adapted gene variant for human erythropoietin (crEpo) was expressed as a protein of commercial relevance. Extracellular erythropoietin produced in Chlamydomonas showed a molecular mass of 33 kDa probably resulting from post-translational modifications. Both, the increased expression levels of transgenes by integration of introns and the isolation of recombinant proteins from the culture medium are important steps towards an extended biotechnological use of this alga.

  6. 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.

  7. Homogentisate phytyltransferase from the unicellular green alga Chlamydomonas reinhardtii.

    PubMed

    Gálvez-Valdivieso, Gregorio; Cardeñosa, Rosa; Pineda, Manuel; Aguilar, Miguel

    2015-09-01

    Homogentisate phytyltransferase (HPT) (EC 2.5.1.-) catalyzes the first committed step of tocopherol biosynthesis in all photosynthetic organisms. This paper presents the molecular characterization and expression analysis of HPT1 gene, and a study on the accumulation of tocopherols under different environmental conditions in the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HPT1 protein conserves all the prenylphosphate- and divalent cation-binding sites that are found in polyprenyltransferases and all the amino acids that are essential for its catalytic activity. Its hydrophobicity profile confirms that HPT is a membrane-bound protein. Chlamydomonas genomic DNA analysis suggests that HPT is encoded by a single gene, HPT1, whose promoter region contains multiple motifs related to regulation by jasmonate, abscisic acid, low temperature and light, and an ATCTA motif presents in genes involved in tocopherol biosynthesis and some photosynthesis-related genes. Expression analysis revealed that HPT1 is strongly regulated by dark and low-temperature. Under the same treatments, α-tocopherol increased in cultures exposed to darkness or heat, whereas γ-tocopherol did it in low temperature. The regulatory expression pattern of HPT1 and the changes of tocopherol abundance support the idea that different tocopherols play specific functions, and suggest a role for γ-tocopherol in the adaptation to growth under low-temperature.

  8. 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

    2014-09-08

    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.

  9. 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.

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

    PubMed

    Suzuki, T; Ohta, H; Enami, I

    2005-06-01

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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. Isolation and characterization of a Chlamydomonas L-asparaginase.

    PubMed Central

    Paul, J H

    1982-01-01

    An L-asparaginase (EC 3.5.1.1) specific for L-asparagine has been purified from a marine Chlamydomonas species, the first such enzyme to be purified from a microalga. The purified enzyme (mol.wt. 275 000) possessed a Km for asparagine of 1.34 x 10(-4) M and showed limited antitumour activity in an antilymphoma assay in vivo. Properties of the enzyme are contrasted with those of asparaginases from prokaryotic and eukaryotic sources. Images Fig. 1. PMID:6896642

  16. Mutations That Alter the Transmission of Chloroplast Genes in Chlamydomonas

    PubMed Central

    Sager, Ruth; Ramanis, Zenta

    1974-01-01

    Two mutations are described that alter the pattern of inheritance of chloroplast genes in Chlamydomonas. The mutant gene mat-1 linked to the mating type allele mt- greatly increases the frequency of exceptional zygotes, i.e., zygotes that transmit chloroplast genes from the mt- (male) parent. In some crosses, 80-90% of the zygotes are biparental, transmitting chloroplast genes from both parents. The mat-2 mutation, linked to mt+, acts to decrease the frequency of exceptional zygotes below the spontaneous level. The mutant effects are discussed in terms of a DNA modification-restriction system, postulated to regulate the transmission of chloroplast DNA in zygotes. PMID:4531010

  17. [Development of the Chlamydomonas actinochloris culture after microwave irradiation].

    PubMed

    Grigor'eva, O O; Berezovskaia, M A; Datsenko, A I

    2012-01-01

    Effect of the microwave irradiation on the subsequent development of the Chlamydomonas actinochloris culture is studied. The number of cells in the suspension was controlled and photoluminescence measurements were performed for 25 days to estimate the functional state of the cells. The exposure at a dose of 80 J/g is shown to negligibly affect the green alga, whereas the 122 J/g dose led to deterioration of the functional state and, thereafter, to the death of most cells. However, the survivors intensively developed, the culture restored the normal state for 20 days, reached and later even left behind the control sample in development.

  18. 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.

  19. Glycolate Metabolism and Excretion by Chlamydomonas reinhardtii1

    PubMed Central

    Moroney, James V.; Wilson, Barbara J.; Tolbert, N. E.

    1986-01-01

    The flux of glycolate through the C2 pathway in Chlamydomonas reinhardtii was estimated after inhibition of the pathway with aminooxyacetate (AOA) or aminoacetonitrile (AAN) by measurement of the accumulation of glycolate and glycine. Cells grown photoautotrophically in air excreted little glycolate except in the presence of 2 mm AOA when they excreted 5 micromoles glycolate per hour per milligram clorophyll. Cells grown on high CO2 (1-5%) when transferred to air produced three times as much glycolate, with half of the glycolate metabolized and half excreted. The lower amount of glycolate produced by the air-grown cells reflects the presence of a CO2 concentrating mechanism which raises the internal CO2 level and decreases the ribulose-1,5-bisP oxygenase reaction for glycolate production. Despite the presence of the CO2 concentrating mechanism, there was still a significant amount of glycolate produced and metabolized by air-grown Chlamydomonas. The capacity of these cells to metabolize between 5 and 10 micromoles of glycolate per hour per milligram chlorophyll was confirmed by measuring the biphasic uptake of added labeled glycolate. The initial rapid (<10 seconds) phase represented uptake of glycolate; the slow phase represented the metabolism of glycolate. The rates of glycolate metabolism were in agreement with those determined using the C2-cycle inhibitors during CO2 fixation. PMID:16665116

  20. 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

  1. Linkage Group Xix of Chlamydomonas Reinhardtii Has a Linear Map

    PubMed Central

    Holmes, J. A.; Johnson, D. E.; Dutcher, S. K.

    1993-01-01

    Linkage group XIX (or the UNI linkage group) of Chlamydomonas reinhardtii has been reported to show a circular meiotic recombination map. A circular map predicts the existence of strong chiasma and chromatid interference, which would lead to an excess number of two-strand double crossovers during meiosis. We have tested this prediction in multipoint crosses. Our results are consistent with a linear linkage group that shows positive chiasma interference and no chromatid interference. Chiasma interference occurs both within arms and across the centromere. Of the original loci that contributed to the circular map, we find that two map to other linkage groups and a third cannot be retested because the mutant strain that defined it has been lost. A second reported unusual property for linkage group XIX was the increase in meiotic recombination with increases in temperature during a period that precedes the onset of meiosis. Although we observed changes in recombination frequencies in some intervals on linkage group XIX in crosses to CC-1952, and in strains heterozygous for the mutation ger1 at 16°, we also show that our strains do not exhibit the previously observed patterns of temperature-sensitive recombination for two different pairs of loci on linkage group XIX. We conclude that linkage group XIX has a linear genetic map that is not significantly different from other Chlamydomonas linkage groups. PMID:8462847

  2. 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

  3. 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

  4. Sulphur responsiveness of the Chlamydomonas reinhardtii LHCBM9 promoter.

    PubMed

    Sawyer, Anne L; Hankamer, Ben D; Ross, Ian L

    2015-05-01

    A 44-base-pair region in the Chlamydomonas reinhardtii LHCBM9 promoter is essential for sulphur responsiveness. The photosynthetic light-harvesting complex (LHC) proteins play essential roles both in light capture, the first step of photosynthesis, and in photoprotective mechanisms. In contrast to the other LHC proteins and the majority of photosynthesis proteins, the Chlamydomonas reinhardtii photosystem II-associated LHC protein, LHCBM9, was recently reported to be up-regulated under sulphur deprivation conditions, which also induce hydrogen production. Here, we examined the sulphur responsiveness of the LHCBM9 gene at the transcriptional level, through promoter deletion analysis. The LHCBM9 promoter was found to be responsive to sulphur deprivation, with a 44-base-pair region between nucleotide positions -136 and -180 relative to the translation start site identified as essential for this response. Anaerobiosis was found to enhance promoter activity under sulphur deprivation conditions, however, alone was unable to induce promoter activity. The study of LHCBM9 is of biological and biotechnological importance, as its expression is linked to photobiological hydrogen production, theoretically the most efficient process for biofuel production, while the simplicity of using an S-deprivation trigger enables the development of a novel C. reinhardtii-inducible promoter system based on LHCBM9.

  5. Amino Acid Sequence of a Novel Calmodulin from the Unicellular Alga Chlamydomonas1

    PubMed Central

    Lukas, Thomas J.; Wiggins, Michael E.; Watterson, D. Martin

    1985-01-01

    An amino acid sequence for a Chlamydomonas calmodulin has been elucidated with emphasis on the characterization of differences that are unique to Chlamydomonas and Dictyostelium calmodulin. While the concentration of calmodulin required for half-maximal activation of plant NAD kinase varies among vertebrate, higher plant, algal, and slime mold calmodulins, only calmodulins from the unicellular alga Chlamydomonas and the slime mold Dictyostelium show increased maximal activation of NAD kinase (Roberts, Burgess, Watterson 1984 Plant Physiol 75: 796-798; Marshak, Clarke, Roberts, Watterson 1984 Biochemistry 23: 2891-2899). The same preparations of calmodulin do not show major differences in phosphodiesterase or myosin light chain kinase activator activity. We report here that a Chlamydomonas calmodulin has four primary structural features similar to Dictyostelium that are not found in other calmodulins characterized to date: an altered carboxy terminus including a novel 11-residue extension for Chlamydomonas calmodulin, unique residues at positions 81 and 118, and an unmethylated lysine at position 115. The only amino acid sequence identity unique to Chlamydomonas and Dictyostelium calmodulin is the presence of a lysine at position 115 instead of a trimethyllysine. These studies indicate that the methylation state of lysine 115 may be important in the maximal NAD kinase activator activity of calmodulin and support the concept that calmodulin has multiple functional domains in addition to multiple structural domains. PMID:16664269

  6. Rapid Triacylglycerol Turnover in Chlamydomonas reinhardtii Requires a Lipase with Broad Substrate Specificity

    PubMed Central

    Li, Xiaobo; Benning, Christoph

    2012-01-01

    When deprived of nitrogen (N), the photosynthetic microalga Chlamydomonas reinhardtii accumulates large quantities of triacylglycerols (TAGs), making it a promising source of biofuel. Prominent transcriptional changes associated with the conditions leading to TAG accumulation have been found, suggesting that the key enzymes for TAG metabolism might be among those that fluctuate in their expression during TAG synthesis and breakdown. Using a Saccharomyces cerevisiae lipase null mutant strain for functional complementation, we identified the CrLIP1 gene from Chlamydomonas based on its ability to suppress the lipase deficiency-related phenotypes of the yeast mutant. In Chlamydomonas, an inverse correlation was found between the CrLIP1 transcript level and TAG abundance when Chlamydomonas cultures were reversibly deprived of N. The CrLIP1 protein expressed and purified from Escherichia coli exhibited lipolytic activity against diacylglycerol (DAG) and polar lipids. The lipase domain of CrLIP1 is most similar to two human DAG lipases, DAGLα and DAGLβ. The involvement of CrLIP1 in Chlamydomonas TAG hydrolysis was corroborated by reducing the abundance of the CrLIP1 transcript with an artificial micro-RNA, which resulted in an apparent delay in TAG lipolysis when N was resupplied. Together, these data suggest that CrLIP1 facilitates TAG turnover in Chlamydomonas primarily by degrading the DAG presumably generated from TAG hydrolysis. PMID:23042128

  7. 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. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  8. Rapid triacylglycerol turnover in Chlamydomonas reinhardtii requires a lipase with broad substrate specificity.

    PubMed

    Li, Xiaobo; Benning, Christoph; Kuo, Min-Hao

    2012-12-01

    When deprived of nitrogen (N), the photosynthetic microalga Chlamydomonas reinhardtii accumulates large quantities of triacylglycerols (TAGs), making it a promising source of biofuel. Prominent transcriptional changes associated with the conditions leading to TAG accumulation have been found, suggesting that the key enzymes for TAG metabolism might be among those that fluctuate in their expression during TAG synthesis and breakdown. Using a Saccharomyces cerevisiae lipase null mutant strain for functional complementation, we identified the CrLIP1 gene from Chlamydomonas based on its ability to suppress the lipase deficiency-related phenotypes of the yeast mutant. In Chlamydomonas, an inverse correlation was found between the CrLIP1 transcript level and TAG abundance when Chlamydomonas cultures were reversibly deprived of N. The CrLIP1 protein expressed and purified from Escherichia coli exhibited lipolytic activity against diacylglycerol (DAG) and polar lipids. The lipase domain of CrLIP1 is most similar to two human DAG lipases, DAGLα and DAGLβ. The involvement of CrLIP1 in Chlamydomonas TAG hydrolysis was corroborated by reducing the abundance of the CrLIP1 transcript with an artificial micro-RNA, which resulted in an apparent delay in TAG lipolysis when N was resupplied. Together, these data suggest that CrLIP1 facilitates TAG turnover in Chlamydomonas primarily by degrading the DAG presumably generated from TAG hydrolysis.

  9. Function and regulation of the glutathione peroxidase homologous gene GPXH/GPX5 in Chlamydomonas reinhardtii.

    PubMed

    Fischer, Beat B; Dayer, Régine; Schwarzenbach, Yvonne; Lemaire, Stéphane D; Behra, Renata; Liedtke, Anja; Eggen, Rik I L

    2009-12-01

    When exposed to strong sunlight, photosynthetic organisms encounter photooxidative stress by the increased production of reactive oxygen species causing harmful damages to proteins and membranes. Consequently, a fast and specific induction of defense mechanisms is required to protect the organism from cell death. In Chlamydomonas reinhardtii, the glutathione peroxidase homologous gene GPXH/GPX5 was shown to be specifically upregulated by singlet oxygen formed during high light conditions presumably to prevent the accumulation of lipid hydroperoxides and membrane damage. We now showed that the GPXH protein is a thioredoxin-dependent peroxidase catalyzing the reduction of hydrogen peroxide and organic hydroperoxides.Furthermore, the GPXH gene seems to encode a dual-targeted protein, predicted to be localized both in the chloroplast and the cytoplasm, which is active with either plastidic TRXy or cytosolic TRXh1. Putative dual-targeting is achieved by alternative transcription and translation start sites expressed independently from either a TATA-box or an Initiator core promoter. Expression of both transcripts was upregulated by photooxidative stress even though with different strengths. The induction required the presence of the core promoter sequences and multiple upstream regulatory elements including a Sp1-like element and an earlier identified CRE/AP-1 homologous sequence. This element was further characterized by mutation analysis but could not be confirmed to be a consensus CRE or AP1 element. Instead, it rather seems to be another member of the large group of TGAC-transcription factor binding sites found to be involved in the response of different genes to oxidative stress.

  10. New thioredoxin targets in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii.

    PubMed

    Lemaire, Stéphane D; Guillon, Blanche; Le Maréchal, Pierre; Keryer, Eliane; Miginiac-Maslow, Myroslawa; Decottignies, Paulette

    2004-05-11

    Proteomics were used to identify the proteins from the eukaryotic unicellular green alga Chlamydomonas reinhardtii that can be reduced by thioredoxin. These proteins were retained specifically on a thioredoxin affinity column made of a monocysteinic thioredoxin mutant able to form mixed disulfides with its targets. Of a total of 55 identified targets, 29 had been found previously in higher plants or Synechocystis, but 26 were new targets. Biochemical tests were performed on three of them, showing a thioredoxin-dependent activation of isocitrate lyase and isopropylmalate dehydrogenase and a thioredoxin-dependent deactivation of catalase that is redox insensitive in Arabidopsis. In addition, we identified a Ran protein, a previously uncharacterized nuclear target in a photosynthetic organism. The metabolic and evolutionary implications of these findings are discussed.

  11. Nuclear Mutation Increases Streptomycin and Spectinomycin Sensitivity in Chlamydomonas

    PubMed Central

    Lee, Robert W.; Sapp, Jan A.

    1978-01-01

    A spontaneously arising nuclear mutation, ss-1, has been identified in Chlamydomonas reinhardtii that decreases both streptomycin and spectinomycin resistance levels about 10-fold after its introduction into all wild-type, streptomycin-resistant and spectinomycin-resistant strains examined. The mutations for resistance map to nuclear and uniparentally inherited (chloroplast) loci. In contrast, no modification of erythromycin resistance was detected after introducing ss-1 into wild-type strains or into strains carrying nuclear or uniparentally inherited erythromycin-resistance mutations. We suggest that ss-1 affects the small subunit of the chloroplast ribosome because others have shown that streptomycin and spectinomycin resistance in C. reinhardtii are associated with this subunit, whereas erythromycin resistance is associated with the large subunit. ss-1 shows no linkage with the nuclear locus for streptomycin resistance. PMID:148390

  12. Enzymatic pretreatment of Chlamydomonas reinhardtii biomass for ethanol production.

    PubMed

    Choi, Seung Phill; Nguyen, Minh Thu; Sim, Sang Jun

    2010-07-01

    The production of ethanol from feedstock other than agriculture materials has been promoted in recent years. Some microalgae can accumulate a high starch content (about 44% of dry base) via photosynthesis. Algal biomass, Chlamydomonas reinhardtii UTEX 90, was converted into a suitable fermentable feedstock by two commercial hydrolytic enzymes. The results showed that almost all starch was released and converted into glucose without steps for the cell wall disruption. Various conditions in the liquefaction and saccharification processes, such as enzyme concentration, pH, temperature, and residence time, have been investigated to obtain an optimum combination using the orthogonal analysis. As a result, approximately 235 mg of ethanol was produced from 1.0 g of algal biomass by a separate hydrolysis and fermentation (SHF) method. The main advantages of this process include the low cost of chemicals, short residence time, and simple equipment system, all of which promote its large-scale application.

  13. 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.

  14. New thioredoxin targets in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii

    PubMed Central

    Lemaire, Stéphane D.; Guillon, Blanche; Le Maréchal, Pierre; Keryer, Eliane; Miginiac-Maslow, Myroslawa; Decottignies, Paulette

    2004-01-01

    Proteomics were used to identify the proteins from the eukaryotic unicellular green alga Chlamydomonas reinhardtii that can be reduced by thioredoxin. These proteins were retained specifically on a thioredoxin affinity column made of a monocysteinic thioredoxin mutant able to form mixed disulfides with its targets. Of a total of 55 identified targets, 29 had been found previously in higher plants or Synechocystis, but 26 were new targets. Biochemical tests were performed on three of them, showing a thioredoxin-dependent activation of isocitrate lyase and isopropylmalate dehydrogenase and a thioredoxin-dependent deactivation of catalase that is redox insensitive in Arabidopsis. In addition, we identified a Ran protein, a previously uncharacterized nuclear target in a photosynthetic organism. The metabolic and evolutionary implications of these findings are discussed. PMID:15123830

  15. Light affects the structure of Chlamydomonas chloroplast chromosomes.

    PubMed

    Thompson, R J; Mosig, G

    1990-05-11

    We have analyzed changes in the structure of chloroplast chromosomes in response to light in growing Chlamydomonas cells using a crosslinking assay based on the intercalation of HMT (4'-hydroxymethyl-4,5',8-trimethylpsoralen) into DNA. Our results show that the structure of chloroplast chromosomes in at least three widely separated regions is different in light-grown vs. dark-grown cells. Structural changes in chloroplast chromosomes occur within 3 hrs after exposure to light or darkness, respectively. The response to light is not inhibited by atrazine and can be elicited by dim blue light incapable of evolving O2, indicating that it does not require photosynthesis. Inhibition of cytoplasmic protein synthesis with cycloheximide prevents this response to light, indicating that it depends, at least in part, on proteins imported from the cytoplasm.

  16. 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.

  17. Regulation of Photosynthetic Capacity in Chlamydomonas mundana 1

    PubMed Central

    Russell, George K.; Gibbs, Martin

    1966-01-01

    A regulatory system has been described in the obligately phototrophic green alga Chlamydomonas mundana. Cells grown in acetate media are unable to fix carbon dioxide in the light but carry out a photoassimilation of acetate to carbohydrate: cells cultured with carbon dioxide as the sole source of cellular carbon carry out typical green plant photosynthesis. The control appears to take place at the level of the reductive pentose phosphate cycle. The presence of sodium acetate in the medium strongly inhibits formation of ribulose-1.5-diphosphate carboxylase, ribulose-5-phosphate kinase, and one of the 2 fructose-1,6-diphosphate aldolase activities of the cell. Ribose-5-phosphate isomerase is present in higher activity in autotrophic cells. Changes in the levels of triose phosphate dehydrogenase were also noted. The total pigment content of the cell and the photosynthetic electron transport reactions are not altered under different conditions of growth. PMID:16656335

  18. Antiphase synchronization in a flagellar-dominance mutant of Chlamydomonas.

    PubMed

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

    2013-10-11

    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.

  19. 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

  20. 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

  1. Effect of selenate on growth and photosynthesis of Chlamydomonas reinhardtii.

    PubMed

    Geoffroy, Laure; Gilbin, Rodolphe; Simon, Olivier; Floriani, Magali; Adam, Christelle; Pradines, Catherine; Cournac, Laurent; Garnier-Laplace, Jacqueline

    2007-06-15

    Algal communities play a crucial role in aquatic food webs by facilitating the transfer of dissolved inorganic selenium (both an essential trace element and a toxic compound for a wide variety of organisms) to higher trophic levels. The dominant inorganic chemical species of selenium in freshwaters are selenite (SeO(3)(2-)) and selenate (SeO(4)(2-)). At environmental concentrations, selenite is not likely to have direct toxic effects on phytoplankton growth [Morlon, H., Fortin, C., Floriani, M., Adam, C., Garnier-Laplace, J., Boudou, A., 2005a. Toxicity of selenite in the unicellular green alga Chlamydomonas reinharditii: comparison between effects at the population and sub-cellular level. Aquat. Toxicol. 73(1), 65-78]. The effects of selenate, on the other hand, are poorly documented. We studied the effects of selenate on Chlamydomonas reinhardtii growth (a common parameter in phytotoxicity tests). Growth inhibition (96-h IC(50)) was observed at 4.5+/-0.2 microM selenate (p<0.001), an effective concentration which is low compared to environmental concentrations. Growth inhibition at high selenium concentrations may result from impaired photosynthesis. This is why we also studied the effects of selenate on the photosynthetic process (not previously assessed in this species to our knowledge) as well as selenate's effects on cell ultrastructure. The observed ultrastructural damage (chloroplast alterations, loss of appressed domains) confirmed that chloroplasts are important targets in the mechanism of selenium toxicity. Furthermore, the inhibition of photosynthetic electron transport evaluated by chlorophyll fluorescence induction confirmed this hypothesis and demonstrated that selenate disrupts the photosynthetic electron chain. Compared to the classical 'growth inhibition' parameter used in phytotoxicity tests, cell diameter and operational photosynthetic yield were more sensitive and may be convenient tools for selenate toxicity assessment in non-target plants.

  2. 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.

  3. 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.

  4. A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii.

    PubMed

    Pollock, Steve V; Mukherjee, Bratati; Bajsa-Hirschel, Joanna; Machingura, Marylou C; Mukherjee, Ananya; Grossman, Arthur R; Moroney, James V

    2017-01-01

    Random insertional mutagenesis of Chlamydomonas reinhardtii using drug resistance cassettes has contributed to the generation of tens of thousands of transformants in dozens of labs around the world. In many instances these insertional mutants have helped elucidate the genetic basis of various physiological processes in this model organism. Unfortunately, the insertion sites of many interesting mutants are never defined due to experimental difficulties in establishing the location of the inserted cassette in the Chlamydomonas genome. It is fairly common that several months, or even years of work are conducted with no result. Here we describe a robust method to identify the location of the inserted DNA cassette in the Chlamydomonas genome. Insertional mutants were generated using a DNA cassette that confers paromomycin resistance. This protocol identified the cassette insertion site for greater than 80% of the transformants. In the majority of cases the insertion event was found to be simple, without large deletions of flanking genomic DNA. Multiple insertions were observed in less than 10% of recovered transformants. The method is quick, relatively inexpensive and does not require any special equipment beyond an electroporator. The protocol was tailored to ensure that the sequence of the Chlamydomonas genomic DNA flanking the random insertion is consistently obtained in a high proportion of transformants. A detailed protocol is presented to aid in the experimental design and implementation of mutant screens in Chlamydomonas.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. Isolation and in vitro binding of mating type plus fertilization tubules from Chlamydomonas.

    PubMed

    Wilson, Nedra F

    2008-01-01

    During fertilization in Chlamydomonas, adhesion and fusion of gametes occur at the tip of specialized regions of the plasma membrane, known as mating structures. The mating type minus (mt[-]) structure is a slightly raised dome-shaped region located at the apical end of the cell body. In contrast, the activated mating type plus (mt[+]) structure is an actin-filled, microvillouslike organelle. Interestingly, a similar type of "fusion organelle" is conserved across diverse groups. Chlamydomonas provides an ideal model system for studying the process of gametic cell fusion in that it is amenable to genetic manipulations as well as cell and molecular biological approaches. Moreover, the ease of culturing Chlamydomonas combined with the ability to isolate the mt(+) fertilization tubule and the development of in vitro assays for adhesion makes it an ideal system for biochemical studies focused on dissecting the molecular mechanisms that underlie the complex process of gametic cell fusion.

  10. 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

  11. 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.

  12. 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

  13. In vivo imaging of IFT in Chlamydomonas flagella.

    PubMed

    Lechtreck, Karl F

    2013-01-01

    Intraflagellar transport (IFT) is a specialized intracellular transport which is required for the assembly and maintenance of cilia and eukaryotic flagella. IFT protein particles move bidirectionally along the flagella in the space between the flagellar membrane and the axonemal doublets. The particles consist of more than 20 different polypeptides and are transported by kinesin-2 from the cell body to the flagellar tip and by cytoplasmic dynein back to the cell body. Chlamydomonas reinhardtii is unique in that IFT can be visualized by two distinct microscopic approaches: differential interference contrast (DIC) and tracking of fluorescently tagged IFT proteins. In vivo imaging of IFT is critical to determine, for example, the role of individual proteins in the IFT pathway and how flagellar proteins are transported by IFT. Here, the microscopic requirements and the procedures for the imaging of IFT by DIC and by total internal reflection fluorescence microscopy will be described. Kymograms, graphical representations of spatial position over time, provide a convenient way to analyze in vivo recordings of IFT. In the future, multicolor in vivo imaging of IFT and its cargoes will be used to understand how flagella are assembled, maintained, and repaired.

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

    PubMed

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

    2016-01-01

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

  15. Regulation of the Chlamydomonas cell cycle by light and dark

    PubMed Central

    1980-01-01

    By growing cells in alternating periods of light and darkness, we have found that the synchronization of phototrophically grown Chlamydomonas populations is regulated at two specific points in the cell cycle: the primary arrest (A) point, located in early G1, and the transition (T) point, located in mid-G1. At the A point, cell cycle progression becomes light dependent. At the T point, completion of the cycle becomes independent of light. Cells transferred from light to dark at cell cycle position between the two regulatory points enter a reversible resting state in which they remain viable and metabolically active, but do not progress through their cycles. The photosystem II inhibitor dichlorophenyldimethylurea (DCMU) mimics the A point block induced by darkness. This finding indicates that the A point block is mediated by a signal that operates through photosynthetic electron transport. Cells short of the T point will arrest in darkness although they contain considerable carbohydrate reserves. After the T point, a sharp increase occurs in starch degradation and in the endogenous respiration rate, indicating that some internal block to the availability of stored energy reserves has now been released, permitting cell cycle progression. PMID:6767730

  16. Establishing Chlamydomonas reinhardtii as an industrial biotechnology host.

    PubMed

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

    2015-05-01

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

  17. Nitric oxide controls nitrate and ammonium assimilation in Chlamydomonas reinhardtii.

    PubMed

    Sanz-Luque, Emanuel; Ocaña-Calahorro, Francisco; Llamas, Angel; Galvan, Aurora; Fernandez, Emilio

    2013-08-01

    Nitrate and ammonium are major inorganic nitrogen sources for plants and algae. These compounds are assimilated by means of finely regulated processes at transcriptional and post-translational levels. In Chlamydomonas, the expression of several genes involved in high-affinity ammonium (AMT1.1, AMT1.2) and nitrate transport (NRT2.1) as well as nitrate reduction (NIA1) are downregulated by ammonium through a nitric oxide (NO)-dependent mechanism. At the post-translational level, nitrate/nitrite uptake and nitrate reductase (NR) are also inhibited by ammonium, but the mechanisms implicated in this regulation are scarcely known. In this work, the effect of NO on nitrate assimilation and the high-affinity ammonium uptake was addressed. NO inhibited the high-affinity uptake of ammonium and nitrate/nitrite, as well as the NR activity, in a reversible form. In contrast, nitrite reductase and glutamine synthetase activities were not affected. The in vivo and in vitro studies suggested that NR enzyme is inhibited by NO in a mediated process that requires the cell integrity. These data highlight a role of NO in inorganic nitrogen assimilation and suggest that this signalling molecule is an important regulator for the first steps of the pathway.

  18. Modes of flagellar assembly in Chlamydomonas reinhardtii and Trypanosoma brucei

    PubMed Central

    Höög, Johanna L; Lacomble, Sylvain; O’Toole, Eileen T; Hoenger, Andreas; McIntosh, J Richard; Gull, Keith

    2014-01-01

    Defects in flagella growth are related to a number of human diseases. Central to flagellar growth is the organization of microtubules that polymerize from basal bodies to form the axoneme, which consists of hundreds of proteins. Flagella exist in all eukaryotic phyla, but neither the mechanism by which flagella grow nor the conservation of this process in evolution are known. Here, we study how protein complexes assemble onto the growing axoneme tip using (cryo) electron tomography. In Chlamydomonas reinhardtii microtubules and associated proteins are added simultaneously. However, in Trypanosoma brucei, disorganized arrays of microtubules are arranged into the axoneme structure by the later addition of preformed protein complexes. Post assembly, the T. brucei transition zone alters structure and its association with the central pair loosens. We conclude that there are multiple ways to form a flagellum and that species-specific structural knowledge is critical before evaluating flagellar defects. DOI: http://dx.doi.org/10.7554/eLife.01479.001 PMID:24448408

  19. Characterizing the Anaerobic Response of Chlamydomonas reinhardtii by Quantitative Proteomics

    PubMed Central

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

    2010-01-01

    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 ([13C6]arginine/[12C6]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

  20. 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

  1. 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. © 2013 John Wiley & Sons Ltd.

  2. Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise

    PubMed Central

    Wakabayashi, Ken-ichi; King, Stephen M.

    2006-01-01

    Redox-based regulatory systems are essential for many cellular activities. Chlamydomonas reinhardtii exhibits alterations in motile behavior in response to different light conditions (photokinesis). We hypothesized that photokinesis is signaled by variations in cytoplasmic redox poise resulting from changes in chloroplast activity. We found that this effect requires photosystem I, which generates reduced NADPH. We also observed that photokinetic changes in beat frequency and duration of the photophobic response could be obtained by altering oxidative/reductive stress. Analysis of reactivated cell models revealed that this redox poise effect is mediated through the outer dynein arms (ODAs). Although the global redox state of the thioredoxin-related ODA light chains LC3 and LC5 and the redox-sensitive Ca2+-binding subunit of the docking complex DC3 did not change upon light/dark transitions, we did observe significant alterations in their interactions with other flagellar components via mixed disulfides. These data indicate that redox poise directly affects ODAs and suggest that it may act in the control of flagellar motility. PMID:16754958

  3. Retrograde bilin signaling enables Chlamydomonas greening and phototrophic survival.

    PubMed

    Duanmu, Deqiang; Casero, David; Dent, Rachel M; Gallaher, Sean; Yang, Wenqiang; Rockwell, Nathan C; Martin, Shelley S; Pellegrini, Matteo; Niyogi, Krishna K; Merchant, Sabeeha S; Grossman, Arthur R; Lagarias, J Clark

    2013-02-26

    The maintenance of functional chloroplasts in photosynthetic eukaryotes requires real-time coordination of the nuclear and plastid genomes. Tetrapyrroles play a significant role in plastid-to-nucleus retrograde signaling in plants to ensure that nuclear gene expression is attuned to the needs of the chloroplast. Well-known sites of synthesis of chlorophyll for photosynthesis, plant chloroplasts also export heme and heme-derived linear tetrapyrroles (bilins), two critical metabolites respectively required for essential cellular activities and for light sensing by phytochromes. Here we establish that Chlamydomonas reinhardtii, one of many chlorophyte species that lack phytochromes, can synthesize bilins in both plastid and cytosol compartments. Genetic analyses show that both pathways contribute to iron acquisition from extracellular heme, whereas the plastid-localized pathway is essential for light-dependent greening and phototrophic growth. Our discovery of a bilin-dependent nuclear gene network implicates a widespread use of bilins as retrograde signals in oxygenic photosynthetic species. Our studies also suggest that bilins trigger critical metabolic pathways to detoxify molecular oxygen produced by photosynthesis, thereby permitting survival and phototrophic growth during the light period.

  4. Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii▿ †

    PubMed Central

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2009-01-01

    Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum. PMID:19429781

  5. Retrograde bilin signaling enables Chlamydomonas greening and phototrophic survival

    PubMed Central

    Duanmu, Deqiang; Casero, David; Dent, Rachel M.; Gallaher, Sean; Yang, Wenqiang; Rockwell, Nathan C.; Martin, Shelley S.; Pellegrini, Matteo; Niyogi, Krishna K.; Merchant, Sabeeha S.; Grossman, Arthur R.; Lagarias, J. Clark

    2013-01-01

    The maintenance of functional chloroplasts in photosynthetic eukaryotes requires real-time coordination of the nuclear and plastid genomes. Tetrapyrroles play a significant role in plastid-to-nucleus retrograde signaling in plants to ensure that nuclear gene expression is attuned to the needs of the chloroplast. Well-known sites of synthesis of chlorophyll for photosynthesis, plant chloroplasts also export heme and heme-derived linear tetrapyrroles (bilins), two critical metabolites respectively required for essential cellular activities and for light sensing by phytochromes. Here we establish that Chlamydomonas reinhardtii, one of many chlorophyte species that lack phytochromes, can synthesize bilins in both plastid and cytosol compartments. Genetic analyses show that both pathways contribute to iron acquisition from extracellular heme, whereas the plastid-localized pathway is essential for light-dependent greening and phototrophic growth. Our discovery of a bilin-dependent nuclear gene network implicates a widespread use of bilins as retrograde signals in oxygenic photosynthetic species. Our studies also suggest that bilins trigger critical metabolic pathways to detoxify molecular oxygen produced by photosynthesis, thereby permitting survival and phototrophic growth during the light period. PMID:23345435

  6. Methylation of chloroplast DNAs in the life cycle of Chlamydomonas

    PubMed Central

    Royer, Hans-Dieter; Sager, Ruth

    1979-01-01

    Methylation patterns of Chlamydomonas chloroplast DNAs (chlDNAs) were examined in the vegetative, gametic, and zygotic stages of the life cycle. Restriction endo-nuclease fragment patterns produced by EcoRI, BamHI, Hpa II, and Msp I were compared; the last two cleave DNA at the sequence C-C-G-G, but Hpa II is blocked by prior methylation of the internal cytidine whereas Msp I is not. chlDNAs from vegetative cells of both mating types showed no evidence of methylation at C-C-G-G. Gametic mt+ chlDNA was heavily methylated at C-C-G-G, whereas the homologous chlDNA from mt- gametes showed very slight methylation at C-C-G-G. Methylation of additional sites in chlDNA from mt+ gametes but not from mt- gametes was shown by blockage of some EcoRI and BamHI sites that were cleaved in the chlDNA from vegetative cells. chlDNA from 6-hr zygotes was much more methylated than gametic mt+ DNA, as shown by its almost total resistance to cleavage by all four restriction enzymes. These findings support and extend previous evidence that chlDNA of mt+ cells is methylated during gametogenesis and that further methylation occurs after gametic fusion in the young zygotes. Images PMID:16592724

  7. 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

  8. Intraflagellar transport (IFT) during assembly and disassembly of Chlamydomonas flagella.

    PubMed

    Dentler, William

    2005-08-15

    Intraflagellar transport (IFT) of particles along flagellar microtubules is required for the assembly and maintenance of eukaryotic flagella and cilia. In Chlamydomonas, anterograde and retrograde particles viewed by light microscopy average 0.12-microm and 0.06-microm diameter, respectively. Examination of IFT particle structure in growing flagella by electron microscopy revealed similar size aggregates composed of small particles linked to each other and to the membrane and microtubules. To determine the relationship between the number of particles and flagellar length, the rate and frequency of IFT particle movement was measured in nongrowing, growing, and shortening flagella. In all flagella, anterograde and retrograde IFT averaged 1.9 microm/s and 2.7 microm/s, respectively, but retrograde IFT was significantly slower in flagella shorter than 4 mum. The number of flagellar IFT particles was not fixed, but depended on flagellar length. Pauses in IFT particle entry into flagella suggest the presence of a periodic "gate" that permits up to 4 particles/s to enter a flagellum.

  9. Chloroplasts Isolation from Chlamydomonas reinhardtii under Nitrogen Stress

    PubMed Central

    Yang, Miao; Jiang, Jun-Peng; Xie, Xi; Chu, Ya-Dong; Fan, Yan; Cao, Xu-Peng; Xue, Song; Chi, Zhan-You

    2017-01-01

    Triacylglycerols are produced in abundance through chloroplast and endoplasmic reticulum pathways in some microalgae exposed to stress, though the relative contribution of either pathway remains elusive. Characterization of these pathways requires isolation of the organelles. In this study, an efficient and reproducible approach, including homogenous batch cultures of nitrogen-deprived algal cells in photobioreactors, gentle cell disruption using a simple custom-made disruptor with mechanical shear force, optimized differential centrifugation and Percoll density gradient centrifugation, was developed to isolate chloroplasts from Chlamydomonas reinhardtii subjected to nitrogen stress. Using this approach, the maximum limited stress duration was 4 h and the stressed cells exhibited 19 and 32% decreases in intracellular chlorophyll and nitrogen content, respectively. Chloroplasts with 48 – 300 μg chlorophyll were successfully isolated from stressed cells containing 10 mg chlorophyll. These stressed chloroplasts appeared intact, as monitored by ultrastructure observation and a novel quality control method involving the fatty acid biomarkers. This approach can provide sufficient quantities of intact stressed chloroplasts for subcellular biochemical studies in microalgae. PMID:28900438

  10. Gene Expression Profiling of Flagellar Disassembly in Chlamydomonas reinhardtii

    PubMed Central

    Chamberlain, Kara L.; Miller, Steven H.; Keller, Laura R.

    2008-01-01

    Flagella are sensory organelles that interact with the environment through signal transduction and gene expression networks. We used microarray profiling to examine gene regulation associated with flagellar length change in the green alga Chlamydomonas reinhardtii. Microarrays were probed with fluorescently labeled cDNAs synthesized from RNA extracted from cells before and during flagellar assembly or disassembly. Evaluation of the gene expression profiles identified >100 clones showing at least a twofold change in expression during flagellar length changes. Products of these genes are associated not only with flagellar structure and motility but also with other cellular responses, including signal transduction and metabolism. Expression of specific genes from each category was further characterized at higher resolution by using quantitative real-time PCR (qRT–PCR). Analysis and comparison of the gene expression profiles coupled to flagellar assembly and disassembly revealed that each process involves a new and uncharacterized whole-cell response to flagellar length changes. This analysis lays the groundwork for a more comprehensive understanding of the cellular and molecular networks regulating flagellar length changes. PMID:18493036

  11. 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/.

  12. Improving Gene-finding in Chlamydomonas reinhardtii:GreenGenie2

    PubMed Central

    Kwan, Alan L; Li, Linya; Kulp, David C; Dutcher, Susan K; Stormo, Gary D

    2009-01-01

    Background The availability of whole-genome sequences allows for the identification of the entire set of protein coding genes as well as their regulatory regions. This can be accomplished using multiple complementary methods that include ESTs, homology searches and ab initio gene predictions. Previously, the Genie gene-finding algorithm was trained on a small set of Chlamydomonas genes and shown to improve the accuracy of gene prediction in this species compared to other available programs. To improve ab initio gene finding in Chlamydomonas, we assemble a new training set consisting of over 2,300 cDNAs by assembling over 167,000 Chlamydomonas EST entries in GenBank using the EST assembly tool PASA. Results The prediction accuracy of our cDNA-trained gene-finder, GreenGenie2, attains 83% sensitivity and 83% specificity for exons on short-sequence predictions. We predict about 12,000 genes in the version v3 Chlamydomonas genome assembly, most of which (78%) are either identical to or significantly overlap the published catalog of Chlamydomonas genes [1]. 22% of the published catalog is absent from the GreenGenie2 predictions; there is also a fraction (23%) of GreenGenie2 predictions that are absent from the published gene catalog. Randomly chosen gene models were tested by RT-PCR and most support the GreenGenie2 predictions. Conclusion These data suggest that training with EST assemblies is highly effective and that GreenGenie2 is a valuable, complementary tool for predicting genes in Chlamydomonas reinhardtii. PMID:19422688

  13. Analysis of cargo transport by IFT and GFP imaging of IFT in Chlamydomonas.

    PubMed

    Diener, Dennis

    2009-01-01

    Chlamydomonas reinhardtii is the organism in which intraflagellar transport (IFT) was first visualized and in which the composition of IFT particles was originally elucidated. As the universality of IFT among ciliated/flagellated cells was uncovered, the diversity of organisms used to study IFT has grown. Still, because of the ease of isolation of flagella from Chlamydomonas and the battery of temperature-sensitive mutants affecting IFT proteins and motors, this unicellular alga remains the principal model for biochemical studies of IFT motors and cargo; furthermore, the long, exposed flagella of this cell are ideally suited for observing IFT in real time with GFP-tagged components of IFT.

  14. 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.

  15. Amino acid utilization by Chlamydomonas reinhardtii: specific study of histidine.

    PubMed

    Hellio, Claire; Veron, Benoit; Le Gal, Yves

    2004-03-01

    Phytoplankton live in fluctuating environments where many factors such as grazing pressure, sinking, light availability, nutrient uptake and turnover influence the distribution of phytoplankton in time and space. The purpose of this study was to investigate if under conditions of depletion of inorganic nitrogen, as recorded in summer in naturals waters, phytoplanktonic species have the capability of using organic nitrogen sources, including free or combined amino acids, in addition to inorganic nitrogen. The study has focussed on histidine, the degradation of which yielding potentially three nitrogen atoms for each molecule of histidine. Chlamydomonas reinhardtii (CCAP 11/32A) was cultivated axenically with two different sources of nitrogen (histidine and/or ammonium). In the presence of histidine as sole source of nitrogen, cell growth was comparable to that observed with the same concentration of nitrogen in ammonium form. In the presence of both histidine and ammonium, histidine degradation was observed only when the concentration of ammonium was depleted. Under these conditions, the first two enzymes of histidine degradation pathway, histidase (EC 4.3.1.3) and urocanase (EC 4.2.1.49) were produced and were co-ordinately regulated. Histidase activity was also controlled by succinate and glutamate as carbon sources. Histidase was purified 1018-fold and partially characterized. The molecular weight of the native enzyme was estimated to 152.4 kDa corresponding to four subunits of 38.1 kDa. The enzyme did not exhibit classical Michaelis-Menten kinetics but showed a relationship between the rate of catalysis (V) and the concentration of substrate (S), characteristic of negative allosteric behavior. A Hill coefficient of 4 was measured for histidine concentrations higher than 20.5 mM.

  16. Flagellar coordination in Chlamydomonas cells held on micropipettes.

    PubMed

    Rüffer, U; Nultsch, W

    1998-01-01

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

  17. 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

  18. 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

  19. 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.

  20. How the green alga Chlamydomonas reinhardtii keeps time.

    PubMed

    Schulze, Thomas; Prager, Katja; Dathe, Hannes; Kelm, Juliane; Kiessling, Peter; Mittag, Maria

    2010-08-01

    The unicellular green alga Chlamydomonas reinhardtii has two flagella and a primitive visual system, the eyespot apparatus, which allows the cell to phototax. About 40 years ago, it was shown that the circadian clock controls its phototactic movement. Since then, several circadian rhythms such as chemotaxis, cell division, UV sensitivity, adherence to glass, or starch metabolism have been characterized. The availability of its entire genome sequence along with homology studies and the analysis of several sub-proteomes render C. reinhardtii as an excellent eukaryotic model organism to study its circadian clock at different levels of organization. Previous studies point to several potential photoreceptors that may be involved in forwarding light information to entrain its clock. However, experimental data are still missing toward this end. In the past years, several components have been functionally characterized that are likely to be part of the oscillatory machinery of C. reinhardtii since alterations in their expression levels or insertional mutagenesis of the genes resulted in defects in phase, period, or amplitude of at least two independent measured rhythms. These include several RHYTHM OF CHLOROPLAST (ROC) proteins, a CONSTANS protein (CrCO) that is involved in parallel in photoperiodic control, as well as the two subunits of the circadian RNA-binding protein CHLAMY1. The latter is also tightly connected to circadian output processes. Several candidates including a significant number of ROCs, CrCO, and CASEIN KINASE1 whose alterations of expression affect the circadian clock have in parallel severe effects on the release of daughter cells, flagellar formation, and/or movement, indicating that these processes are interconnected in C. reinhardtii. The challenging task for the future will be to get insights into the clock network and to find out how the clock-related factors are functionally connected. In this respect, system biology approaches will certainly

  1. An Animal-Like Cryptochrome Controls the Chlamydomonas Sexual Cycle.

    PubMed

    Zou, Yong; Wenzel, Sandra; Müller, Nico; Prager, Katja; Jung, Elke-Martina; Kothe, Erika; Kottke, Tilman; Mittag, Maria

    2017-07-01

    Cryptochromes are known as flavin-binding blue light receptors in bacteria, fungi, plants, and insects. The animal-like cryptochrome (aCRY) of the green alga Chlamydomonas reinhardtii has extended our view on cryptochromes, because it responds also to other wavelengths of the visible spectrum, including red light. Here, we have investigated if aCRY is involved in the regulation of the sexual life cycle of C. reinhardtii, which is controlled by blue and red light at the steps of gametogenesis along with its restoration and germination. We show that aCRY is differentially expressed not only during the life cycle but also within the cell as part of the soluble and/or membrane-associated protein fraction. Moreover, localization of aCRY within the algal cell body varies between vegetative cells and the different cell types of gametogenesis. aCRY is significantly (early day) or to a small extent (late night) enriched in the nucleus in vegetative cells. In pregametes, gametes and dark-inactivated gametes, aCRY is localized over the cell body. aCRY plays an important role in the sexual life cycle of C. reinhardtii: It controls the germination of the alga, under which the zygote undergoes meiosis, in a positive manner, similar to the regulation by the blue light receptors phototropin and plant cryptochrome (pCRY). However, aCRY acts in combination with pCRY as a negative regulator for mating ability as well as for mating maintenance, opposite to the function of phototropin in these processes. © 2017 American Society of Plant Biologists. All Rights Reserved.

  2. 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

  3. Heavy metal-activated synthesis of peptides in Chlamydomonas reinhardtii

    SciTech Connect

    Howe, G.; Merchant, S. )

    1992-01-01

    In this study, the authors have addressed the capacity of the green alga Chlamydomonas reinhardtii to produce metal-binding peptides in response to stress induced by the heavy metals Cd{sup 2+}, Hg{sup 2+}, and Ag{sup +}. Cells cultured in the presence of sublethal concentrations of Cd{sup 2+} 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 {times} 10{sup 3}. 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 Hg{sup 2+}-treated cells, the principal thiol-containing compound induced by Hg{sup 2+} ion 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{sup +} ions also synthesized a sulfur-containing component with a charge to mass ratio similar to Cd{sup 2+}-induced peptides. But, in contrast to the results obtained using Cd{sup 2+} as an inducer, these molecules did not accumulate to significant levels in Ag{sup +}-treated cells. The presence of physiological concentrations of Cu{sup 2+} in the growth medium blocked the synthesis of the Ag{sup +}-inducible component(s) and rendered cells resistant to the toxic effects of Ag{sup +}, suggesting competition between Cu{sup 2+} and Ag{sup +} ions, possibly at the level of metal uptake.

  4. Remodeling of membrane lipids in iron-starved Chlamydomonas.

    PubMed

    Urzica, Eugen I; Vieler, Astrid; Hong-Hermesdorf, Anne; Page, M Dudley; Casero, David; Gallaher, Sean D; Kropat, Janette; Pellegrini, Matteo; Benning, Christoph; Merchant, Sabeeha S

    2013-10-18

    Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24-48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii.

  5. Remodeling of Membrane Lipids in Iron-starved Chlamydomonas*

    PubMed Central

    Urzica, Eugen I.; Vieler, Astrid; Hong-Hermesdorf, Anne; Page, M. Dudley; Casero, David; Gallaher, Sean D.; Kropat, Janette; Pellegrini, Matteo; Benning, Christoph; Merchant, Sabeeha S.

    2013-01-01

    Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24–48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii. PMID:23983122

  6. Chloroplast Genetics of Chlamydomonas. II. Mapping by Cosegregation Frequency Analysis

    PubMed Central

    Sager, Ruth; Ramanis, Zenta

    1976-01-01

    This paper presents segregation and cosegregation data for a set of 15 chloroplast genes of Chlamydomonas, and uses these data to generate a linear map of the chloroplast genome. The data were derived from pedigree analysis of a total of 1596 zoospore clones resulting from 12 crosses in each of which 4 to 7 pairs of chloroplast alleles were segregating. The crosses are a subset of those previously described (Sager and Ramanis 1976). By means of pedigree analysis, Type II segregations (nonreciprocal conversion-like events) were distinguished from Type III segregations (reciprocal events). The average frequency of Type II segregation was found to be the same for all 15 genes, indicating randomness of this event with respect to map location (Figure 1). Type III segregations occurred with a different and characteristic frequency for each gene, and were interpreted as a measure of the distance of each gene from the postulated centromere-like attachment point. Cosegregations, involving two or more genes, occurred with frequencies characteristic of the particular genes and much lower than expected for the product of single-gene events, indicating strong positive interference. Pairwise cosegregation frequencies provided unambiguous data for the gene order, confirmed by cosegregation runs of three or more genes. Apparent lengths of cosegregation runs, as fractions of the total map, indicate much longer stretches of gene conversion-like events than have been reported for other genetic systems. Comparisons of cosegregation frequencies in cross 20 after 15'', 30'' and 15'' UV irradiation of the mt+ before mating, indicate little if any consistent effect of this irradiation on segregation events. PMID:17248717

  7. Nuclear transformation of Chlamydomonas reinhardtii with silicon carbide fibers

    SciTech Connect

    Dunahay, T.G. )

    1992-01-01

    Efficient nuclear transformation of cell wall-deficient strains of the green alga Chlamydomonas reinhardtii can be accomplished by vortexing the cells in the presence of glass beads and polyethylene glycol (Kindle 1990 PNAS 87:1228). Intact (walled) cells can also be transformed using this protocol, but at very low efficiencies. Two recent reports have described the use of silicon carbide fibers to mediate DNA entry into plant suspension cells (Kaeppler et al. 1990 Plant Cell Rep. 9:414; Asano et al. 1991 Plant Sci. 79:247). The author has found that nuclear transformation efficiencies of walled cells of C. reinhardtii can be increased 3 to 10 fold by vortexing the cells in the presence of silicon carbide fibers and PEG. Using a modification of the glass bead transformation procedure, the wild-type nitrate reductase structural gene was used to complement a NR-deficient mutant of C. reinhardtii, nit-1-305. The transformation efficiency increased with longer vortexing times, although the absolute number of transformants varied between experiments, ranging from 10 to 40 transformants per 10[sup 7] cells. In contrast to vortexing with glass beads, cell viability was very high, with greater than 80% cell survival even after vortexing for 10 minutes. Neither cell death nor transformation efficiency increased when cell wall-deficient mutants (cw15 nit-1-305) were used as compared to intact cells. Experiments are in progress to test the applicability of silicon carbide-mediated transformation to other algal strains for which cell wall mutants or protoplasting procedures are unavailabile.

  8. Flagellar tip activation stimulated by membrane adhesions in Chlamydomonas gametes

    PubMed Central

    1980-01-01

    Membrane adhesions between the flagella of mating-type "plus" and "minus" gametes of Chlamydomonas reinhardi are shown to stimulate a rapid change in the ultrastructure of the flagellar tips, designated as flagellar tip activation (FTA). A dense substance, termed fibrous tip material (FTM), accumulates between the flagellar membrane and the nine single A microtubules of the tip. The A microtubules then elongate, growing into the distal region of the tip, increasing tip length by 30%. This study describes FTA kinetics during normal and mutant matings, presents experiments designed to probe its role in the mating reaction, and offers the following conclusions: (a) FTA is elicited by agents that cross-link flagellar membrane components (including natural sexual agglutinins, antiflagellar antisera, and concanavalin A) but not by flagellar adherence to polylysine-coated films. (b) FTA is reversed by flagellar disadhesion. (c) Gametes can undergo repeated cycles of FTA during successive rounds of adhesion/disadhesion. (d) FTA, flagellar tipping, and sexual signaling are simultaneously blocked by colchicine and by vinblastine, suggesting that tubulinlike molecules, perhaps exposed at the membrane surface, are involved in all three responses. (e) FTA is not blocked by short exposure to chymotrypsin, by cytochalasins B and D, nor by concanavalin A, even though all block cell fusion; the response is therefore autonomous and experimentally dissociable from later stages in the mating reaction. (f) Under no experimental conditions is mating-structure activation observed to occur unless FTA also occurs. This study concludes that FTA is a necessary event in the sexual signaling sequence, and presents a testable working model for its mechanism. PMID:7358792

  9. Compartmentalisation of [FeFe]-hydrogenase maturation in Chlamydomonas reinhardtii.

    PubMed

    Sawyer, Anne; Bai, Yu; Lu, Yinghua; Hemschemeier, Anja; Happe, Thomas

    2017-03-13

    Molecular hydrogen (H2 ) can be produced in green microalgae by [FeFe]-hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA1 contains a catalytic site comprising a classic [4Fe4S] cluster linked to a unique 2Fe sub-cluster. From in vitro studies it appears that the [4Fe4S] cluster is incorporated first by the housekeeping FeS cluster assembly machinery, followed by the 2Fe sub-cluster, whose biosynthesis requires the specific maturases HYDEF and HYDG. To investigate the maturation process in vivo, we expressed HYDA1 from the C. reinhardtii chloroplast and nuclear genomes (with and without a chloroplast transit peptide) in a hydrogenase-deficient mutant strain, and examined the cellular enzymatic hydrogenase activity, as well as in vivo H2 production. The transformants expressing HYDA1 from the chloroplast genome displayed H2 production levels comparable to the wild type, as did the transformants expressing full-length HYDA1 from the nuclear genome. In contrast, cells equipped with cytoplasm-targeted HYDA1 produced inactive enzyme, which could only be activated in vitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA1 FeS cluster can only be built by the chloroplastic FeS cluster assembly machinery. Further, the expression of a bacterial hydrogenase gene, CPI, from the C. reinhardtii chloroplast genome resulted in H2 -producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA1 in vivo and that overexpression of foreign hydrogenases in C. reinhardtii is possible. All chloroplast transformants were stable and no toxic effects were seen from HYDA1 or CPI expression. This article is protected by copyright. All rights reserved.

  10. Inhibition of target of rapamycin signaling by rapamycin in the unicellular green alga Chlamydomonas reinhardtii.

    PubMed

    Crespo, José L; Díaz-Troya, Sandra; Florencio, Francisco J

    2005-12-01

    The macrolide rapamycin specifically binds the 12-kD FK506-binding protein (FKBP12), and this complex potently inhibits the target of rapamycin (TOR) kinase. The identification of TOR in Arabidopsis (Arabidopsis thaliana) revealed that TOR is conserved in photosynthetic eukaryotes. However, research on TOR signaling in plants has been hampered by the natural resistance of plants to rapamycin. Here, we report TOR inactivation by rapamycin treatment in a photosynthetic organism. We identified and characterized TOR and FKBP12 homologs in the unicellular green alga Chlamydomonas reinhardtii. Whereas growth of wild-type Chlamydomonas cells is sensitive to rapamycin, cells lacking FKBP12 are fully resistant to the drug, indicating that this protein mediates rapamycin action to inhibit cell growth. Unlike its plant homolog, Chlamydomonas FKBP12 exhibits high affinity to rapamycin in vivo, which was increased by mutation of conserved residues in the drug-binding pocket. Furthermore, pull-down assays demonstrated that TOR binds FKBP12 in the presence of rapamycin. Finally, rapamycin treatment resulted in a pronounced increase of vacuole size that resembled autophagic-like processes. Thus, our findings suggest that Chlamydomonas cell growth is positively controlled by a conserved TOR kinase and establish this unicellular alga as a useful model system for studying TOR signaling in photosynthetic eukaryotes.

  11. Respiratory-deficient mutants of the unicellular green alga Chlamydomonas: a review.

    PubMed

    Salinas, Thalia; Larosa, Véronique; Cardol, Pierre; Maréchal-Drouard, Laurence; Remacle, Claire

    2014-05-01

    Genetic manipulation of the unicellular green alga Chlamydomonas reinhardtii is straightforward. Nuclear genes can be interrupted by insertional mutagenesis or targeted by RNA interference whereas random or site-directed mutagenesis allows the introduction of mutations in the mitochondrial genome. This, combined with a screen that easily allows discriminating respiratory-deficient mutants, makes Chlamydomonas a model system of choice to study mitochondria biology in photosynthetic organisms. Since the first description of Chlamydomonas respiratory-deficient mutants in 1977 by random mutagenesis, many other mutants affected in mitochondrial components have been characterized. These respiratory-deficient mutants increased our knowledge on function and assembly of the respiratory enzyme complexes. More recently some of these mutants allowed the study of mitochondrial gene expression processes poorly understood in Chlamydomonas. In this review, we update the data concerning the respiratory components with a special focus on the assembly factors identified on other organisms. In addition, we make an inventory of different mitochondrial respiratory mutants that are inactivated either on mitochondrial or nuclear genes.

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

    PubMed Central

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

    2013-01-01

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

  13. 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.

  14. 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.

  15. 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.

  16. Historical perspective on Chlamydomonas as a model for basic research: 1950-1970.

    PubMed

    Goodenough, Ursula

    2015-05-01

    During the period 1950-1970, groundbreaking research on the genetic mapping of Chlamydomonas reinhardtii and the use of mutant strains to analyze photosynthesis was conducted in the laboratory of R. Paul Levine at Harvard University. An account of this era, based in part on interviews with Levine, is presented.

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

    PubMed

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

    2013-01-01

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

  18. New insights into the roles of molecular chaperones in Chlamydomonas and Volvox.

    PubMed

    Nordhues, André; Miller, Stephen M; Mühlhaus, Timo; Schroda, Michael

    2010-01-01

    The unicellular green alga Chlamydomonas reinhardtii has been used as a model organism for many decades, mainly to study photosynthesis and flagella/cilia. Only recently, Chlamydomonas has received much attention because of its ability to produce hydrogen and nonpolar lipids that have promise as biofuels. The best-studied multicellular cousin of Chlamydomonas reinhardtii is Volvox carteri, whose life cycle comprises events that have clear parallels in higher plants and/or animals, making it an excellent system in which to study fundamental developmental processes. Molecular chaperones are proteins that guide other cellular proteins through their life cycle. They assist in de novo folding of nascent chains, mediate assembly and disassembly of protein complexes, facilitate protein transport across membranes, disassemble protein aggregates, fold denatured proteins back to the native state, and transfer unfoldable proteins to proteolytic degradation. Hence, molecular chaperones regulate protein function under all growth conditions and play important roles in many basic cellular and developmental processes. The aim of this chapter is to describe recent advances toward understanding molecular chaperone biology in Chlamydomonas and Volvox.

  19. 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

  20. Identification of Chlamydomonas Central Core Centriolar Proteins Reveals a Role for Human WDR90 in Ciliogenesis.

    PubMed

    Hamel, Virginie; Steib, Emmanuelle; Hamelin, Romain; Armand, Florence; Borgers, Susanne; Flückiger, Isabelle; Busso, Coralie; Olieric, Natacha; Sorzano, Carlos Oscar S; Steinmetz, Michel O; Guichard, Paul; Gönczy, Pierre

    2017-08-21

    Centrioles are evolutionarily conserved macromolecular structures that are fundamental to form cilia, flagella, and centrosomes. Centrioles are 9-fold symmetrical microtubule-based cylindrical barrels comprising three regions that can be clearly distinguished in the Chlamydomonas reinhardtii organelle: an ∼100-nm-long proximal region harboring a cartwheel; an ∼250-nm-long central core region containing a Y-shaped linker; and an ∼150-nm-long distal region ending at the transitional plate. Despite the discovery of many centriolar components, no protein has been localized specifically to the central core region in Chlamydomonas thus far. Here, combining relative quantitative mass spectrometry and super-resolution microscopy on purified Chlamydomonas centrioles, we identified POB15 and POC16 as two proteins of the central core region, the distribution of which correlates with that of tubulin glutamylation. We demonstrated that POB15 is an inner barrel protein within this region. Moreover, we developed an assay to uncover temporal relationships between centriolar proteins during organelle assembly and thus established that POB15 is recruited after the cartwheel protein CrSAS-6 and before tubulin glutamylation takes place. Furthermore, we discovered that two poc16 mutants exhibit flagellar defects, indicating that POC16 is important for flagellum biogenesis. In addition, we discovered that WDR90, the human homolog of POC16, localizes to a region of human centrioles that we propose is analogous to the central core of Chlamydomonas centrioles. Moreover, we demonstrate that WDR90 is required for ciliogenesis, echoing the findings in Chlamydomonas. Overall, our work provides novel insights into the identity and function of centriolar central core components. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. 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. © 2016 American Society of Plant Biologists. All Rights Reserved.

  2. Characterization and differential expression of microRNAs elicited by sulfur deprivation in Chlamydomonas reinhardtii

    PubMed Central

    2012-01-01

    Background microRNAs (miRNAs) have been found to play an essential role in the modulation of numerous biological processes in eukaryotes. Chlamydomonas reinhardtii is an ideal model organism for the study of many metabolic processes including responses to sulfur-deprivation. We used a deep sequencing platform to extensively profile and identify changes in the miRNAs expression that occurred under sulfur-replete and sulfur-deprived conditions. The aim of our research was to characterize the differential expression of Chlamydomonas miRNAs under sulfur-deprived conditions, and subsequently, the target genes of miRNA involved in sulfur-deprivation were further predicted and analyzed. Results By using high-throughput sequencing, we characterized the microRNA transcriptomes under sulphur-replete and sulfur-deprived conditions in Chlamydomonas reinhardtii. We predicted a total of 310 miRNAs which included 85 known miRNAs and 225 novel miRNAs. 13 miRNAs were the specific to the sulfur-deprived conditions. 47 miRNAs showed significantly differential expressions responding to sulfur-deprivation, and most were up-regulated in the small RNA libraries with sulfur-deprivation. Using a web-based integrated system (Web MicroRNAs Designer 3) and combing the former information from a transcriptome of Chlamydomonas reinhardtii, 22 miRNAs and their targets involved in metabolism regulation with sulfur-deprivation were verified. Conclusions Our results indicate that sulfur-deprivation may have a significant influence on small RNA expression patterns, and the differential expressions of miRNAs and interactions between miRNA and its targets might further reveal the molecular mechanism responding to sulfur-deprivation in Chlamydomonas reinhardtii. PMID:22439676

  3. 5' sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts.

    PubMed Central

    Salvador, M L; Klein, U; Bogorad, L

    1993-01-01

    We have found that sequences in the 5' leader of the Chlamydomonas chloroplast rbcL gene, when fused 5' to foreign genes, destabilize transcripts of these chimeric genes in the chloroplast of transgenic Chlamydomonas but that 5' sequences of the rbcL structural gene prevent this destabilization. Transcripts of the chloroplast rbcL gene are about equally abundant at all times in Chlamydomonas reinhardtii growing on an alternating 12-h light/12-h dark cycle. However, Chlamydomonas chloroplast transformants, harboring chimeric genes containing the same rbcL promoter with 63 or 92 bp of the rbcL 5' leader sequence fused upstream of the Escherichia coli uidA (beta-glucuronidase, GUS) gene, accumulated GUS transcripts only in the dark. Transcripts disappeared rapidly upon illumination of the cells. The same phenomenon was exhibited by transcripts of chimeric genes in which the GUS gene coding sequence was replaced by other unrelated genes. The precipitous light-induced drop in GUS transcript abundance was found to be due to an approximately 16-fold increase in the rate of degradation of GUS transcripts in light rather than to a decrease in the rate of transcription of the GUS gene. Transcripts of a chimeric rbcL-GUS construct in which the leader sequence of the rbcL gene was replaced by 103 bp of the leader sequence of the atpB gene were stable in illuminated cells. The destabilizing effect of the rbcL 5' leader sequence was reversed by adding 257 bp of the 5' coding region of the rbcL gene. The results show that chloroplast transcript levels in illuminated Chlamydomonas cells--and perhaps in other cases--can be determined, at least to some extent, by sequences and interactions of sequences transcribed from the 5' ends of genes. Images PMID:8434017

  4. Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii

    PubMed Central

    Boyle, Nanette R; Morgan, John A

    2009-01-01

    Background Photosynthetic organisms convert atmospheric carbon dioxide into numerous metabolites along the pathways to make new biomass. Aquatic photosynthetic organisms, which fix almost half of global inorganic carbon, have great potential: as a carbon dioxide fixation method, for the economical production of chemicals, or as a source for lipids and starch which can then be converted to biofuels. To harness this potential through metabolic engineering and to maximize production, a more thorough understanding of photosynthetic metabolism must first be achieved. A model algal species, C. reinhardtii, was chosen and the metabolic network reconstructed. Intracellular fluxes were then calculated using flux balance analysis (FBA). Results The metabolic network of primary metabolism for a green alga, C. reinhardtii, was reconstructed using genomic and biochemical information. The reconstructed network accounts for the intracellular localization of enzymes to three compartments and includes 484 metabolic reactions and 458 intracellular metabolites. Based on BLAST searches, one newly annotated enzyme (fructose-1,6-bisphosphatase) was added to the Chlamydomonas reinhardtii database. FBA was used to predict metabolic fluxes under three growth conditions, autotrophic, heterotrophic and mixotrophic growth. Biomass yields ranged from 28.9 g per mole C for autotrophic growth to 15 g per mole C for heterotrophic growth. Conclusion The flux balance analysis model of central and intermediary metabolism in C. reinhardtii is the first such model for algae and the first model to include three metabolically active compartments. In addition to providing estimates of intracellular fluxes, metabolic reconstruction and modelling efforts also provide a comprehensive method for annotation of genome databases. As a result of our reconstruction, one new enzyme was annotated in the database and several others were found to be missing; implying new pathways or non-conserved enzymes. The use of

  5. 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.

  6. Chloroplast ribosomal proteins of Chlamydomonas synthesized in the cytoplasm are made as precursors

    PubMed Central

    1984-01-01

    Polyadenylated RNA from Chlamydomonas was translated in a cell-free rabbit reticulocyte system that employed [35S]methionine. Antibodies made to four chloroplast ribosomal proteins synthesized in the cytoplasm and imported into the organelle were used for indirect immunoprecipitation of the labeled translation products, which were subsequently visualized on fluorographs of SDS gels. The cytoplasmically synthesized chloroplast ribosomal proteins were first seen as precursors with apparent molecular weights of 1,000 to 6,000 greater than their respective mature forms. Processing of the ribosomal protein precursors to mature proteins was affected by adding a postribosomal supernatant that had been extracted from cells of Chlamydomonas. In contrast to the chloroplast ribosomal proteins synthesized in the cytoplasm, two such proteins made within the chloroplast were found to be synthesized in mature form in cell-free wheat germ translation systems programmed with nonpolyadenylated RNA. PMID:6202701

  7. 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.

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

    PubMed

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

    2017-06-14

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

  9. BiP links TOR signaling to ER stress in Chlamydomonas.

    PubMed

    Crespo, José L

    2012-02-01

    The highly conserved target of rapamycin (TOR) Ser/Thr kinase promotes protein synthesis under favorable growth conditions in all eukaryotes. Downregulation of TOR signaling in the model unicellular green alga Chlamydomonas reinhardtii has recently revealed a link between control of protein synthesis, endoplasmic reticulum (ER) stress and the reversible modification of the BiP chaperone by phosphorylation. Inhibition of protein synthesis by rapamycin or cycloheximide resulted in the phosphorylation of BiP on threonine residues while ER stress induced by tunicamycin or heat shock caused the fast dephosphorylation of the protein. Regulation of BiP function by phosphorylation/dephosphorylation events was proposed in early studies in mammalian cells although no connection to TOR signaling has been established so far. Here I will discuss about the coordinated regulation of BiP modification by TOR and ER stress signals in Chlamydomonas.

  10. 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

  11. 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.

  12. A Plant Cryptochrome Controls Key Features of the Chlamydomonas Circadian Clock and Its Life Cycle.

    PubMed

    Müller, Nico; Wenzel, Sandra; Zou, Yong; Künzel, Sandra; Sasso, Severin; Weiß, Daniel; Prager, Katja; Grossman, Arthur; Kottke, Tilman; Mittag, Maria

    2017-05-01

    Cryptochromes are flavin-binding proteins that act as blue light receptors in bacteria, fungi, plants, and insects and are components of the circadian oscillator in mammals. Animal and plant cryptochromes are evolutionarily divergent, although the unicellular alga Chlamydomonas reinhardtii (Chlamydomonas throughout) has both an animal-like cryptochrome and a plant cryptochrome (pCRY; formerly designated CPH1). Here, we show that the pCRY protein accumulates at night as part of a complex. Functional characterization of pCRY was performed based on an insertional mutant that expresses only 11% of the wild-type pCRY level. The pcry mutant is defective for central properties of the circadian clock. In the mutant, the period is lengthened significantly, ultimately resulting in arrhythmicity, while blue light-based phase shifts show large deviations from what is observed in wild-type cells. We also show that pCRY is involved in gametogenesis in Chlamydomonas pCRY is down-regulated in pregametes and gametes, and in the pcry mutant, there is altered transcript accumulation under blue light of the strictly light-dependent, gamete-specific gene GAS28 pCRY acts as a negative regulator for the induction of mating ability in the light and for the loss of mating ability in the dark. Moreover, pCRY is necessary for light-dependent germination, during which the zygote undergoes meiosis that gives rise to four vegetative cells. In sum, our data demonstrate that pCRY is a key blue light receptor in Chlamydomonas that is involved in both circadian timing and life cycle progression. © 2017 American Society of Plant Biologists. All Rights Reserved.

  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. Inorganic carbon limitation and mixotrophic growth in Chlamydomonas from an acidic mining lake.

    PubMed

    Tittel, Jörg; Bissinger, Vera; Gaedke, Ursula; Kamjunke, Norbert

    2005-06-01

    Plankton communities in acidic mining lakes (pH 2.5-3.3) are species-poor because they face extreme environmental conditions, e.g. 150mg l(-1) Fe2+ +Fe3+. We investigated the growth characteristics of the dominant pigmented species, the flagellate Chlamydomonas acidophila, in semi-continuous culture experiments under in situ conditions. The following hypotheses were tested: (1) Low inorganic carbon (IC) concentrations in the epilimnion (e.g. 0.3 mg l(-1)) arising from the low pH limit phototrophic growth (H-1); (2) the additional use of dissolved organic carbon (mixotrophy) leads to higher growth rates under IC-limitation (H-2), and (3) phagotrophy is not relevant (H-3). H-1 was supported as the culture experiments, in situ PAR and IC concentrations indicated that IC potentially limited phototrophic growth in the mixed surface layers. H-2 was also supported: mixotrophic growth always exceeded pure phototrophic growth even when photosynthesis was saturated. Dark growth in filtered lake water illuminated prior to inoculation provided evidence that Chlamydomonas was able to use the natural DOC. The alga did not grow on bacteria, thus confirming H-3. Chlamydomonas exhibited a remarkable resistance to starvation in the dark. The compensation light intensity (ca. 20 micromol photons m(-2) s(-1)) and the maximum phototrophic growth (1.50 d(-1)) fell within the range of algae from non-acidic waters. Overall, Chlamydomonas, a typical r-strategist in circum-neutral systems, showed characteristics of a K-strategist in the stable, acidic lake environment in achieving moderate growth rates and minimizing metabolic losses.

  15. Predicting the Physiological Role of Circadian Metabolic Regulation in the Green Alga Chlamydomonas reinhardtii

    PubMed Central

    Voytsekh, Olga; Mittag, Maria; Schuster, Stefan

    2011-01-01

    Although the number of reconstructed metabolic networks is steadily growing, experimental data integration into these networks is still challenging. Based on elementary flux mode analysis, we combine sequence information with metabolic pathway analysis and include, as a novel aspect, circadian regulation. While minimizing the need of assumptions, we are able to predict changes in the metabolic state and can hypothesise on the physiological role of circadian control in nitrogen metabolism of the green alga Chlamydomonas reinhardtii. PMID:21887226

  16. Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases.

    PubMed

    Sizova, Irina; Greiner, Andre; Awasthi, Mayanka; Kateriya, Suneel; Hegemann, Peter

    2013-03-01

    The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide range of tools for genetic manipulation have been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Our approach includes (i) design of gene-specific ZFNs using available online tools, (ii) evaluation of the designed ZFNs in a Chlamydomonas in situ model system, (iii) optimization of ZFN activity by modification of the nuclease domain, and (iv) application of the most suitable enzymes for mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs to target the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional aminoglycoside 3'-phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin-resistant (Pm-R) clones with expressing nucleases. Of these Pm-R clones, 1% also contained a modified COP3 locus. In cases where cells were co-transformed with a modified COP3 template, the COP3 locus was specifically modified by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas.

  17. Cloning of Flagellar Genes in Chlamydomonas Reinhardtii by DNA Insertional Mutagenesis

    PubMed Central

    Tam, L. W.; Lefebvre, P. A.

    1993-01-01

    Chlamydomonas is a popular genetic model system for studying many cellular processes. In this report, we describe a new approach to isolate Chlamydomonas genes using the cloned nitrate reductase gene (NIT1) as an insertional mutagen. A linearized plasmid containing the NIT1 gene was introduced into nit1 mutant cells by glass-bead transformation. Of 3000 Nit(+) transformants examined, 74 showed motility defects of a wide range of phenotypes, suggesting that DNA transformation is an effective method for mutagenizing cells. For 13 of 15 such motility mutants backcrossed to nit(-) mutant strains, the motility phenotype cosegregated with the Nit(+) phenotype, indicating that the motility defects of these 13 mutants may be caused by integration of the plasmid. Further genetic analysis indicated that three of these mutants contained alleles of previously identified loci: mbo2 (move backward only), pf13 (paralyzed flagella) and vfl1 (variable flagellar number). Three other abnormal-flagellar-number mutants did not map to any previously described loci at which mutations produce similar phenotypes. Genomic sequences flanking the integrated plasmid in the mbo2 and vfl1 mutants were isolated and used as probes to obtain wild-type genomic clones, which complemented the motility defects upon transformation into cells. Our results demonstrate the potential of this new approach for cloning genes identified by mutation in Chlamydomonas. PMID:8244002

  18. 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

  19. Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii.

    PubMed

    Almeida, Carla Malaquias; Pereira, Cláudia; da Costa, Diana Soares; Pereira, Susana; Pissarra, José; Simões, Isaura; Faro, Carlos

    2012-07-01

    Aspartic proteinases have been extensively characterized in land plants but up to now no evidences for their presence in green algae group have yet been reported in literature. Here we report on the identification of the first (and only) typical aspartic proteinase from Chlamydomonas reinhardtii. This enzyme, named chlapsin, was shown to maintain the primary structure organization of typical plant aspartic proteinases but comprising distinct features, such as similar catalytic motifs DTG/DTG resembling those from animal and microbial counterparts, and an unprecedentedly longer plant specific insert domain with an extra segment of 80 amino acids, rich in alanine residues. Our results also demonstrated that chlapsin accumulates in Chlamydomonas chloroplast bringing this new enzyme to a level of uniqueness among typical plant aspartic proteinases. Chlapsin was successfully expressed in Escherichia coli and it displayed the characteristic enzymatic properties of typical aspartic proteinases, like optimum activity at acidic pH and complete inhibition by pepstatin A. Another difference to plant aspartic proteinases emerged as chlapsin was produced in an active form without its putative prosegment domain. Moreover, recombinant chlapsin showed a restricted enzymatic specificity and a proteolytic activity influenced by the presence of redox agents and nucleotides, further differentiating it from typical plant aspartic proteinases and anticipating a more specialized/regulated function for this Chlamydomonas enzyme. Taken together, our results revealed a pattern of complexity for typical plant aspartic proteinases in what concerns sequence features, localization and biochemical properties, raising new questions on the evolution and function of this vast group of plant enzymes.

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

    PubMed

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

    2017-01-01

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

  1. Reduction of PII signaling protein enhances lipid body production in Chlamydomonas reinhardtii.

    PubMed

    Zalutskaya, Zhanneta; Kharatyan, Nina; Forchhammer, Karl; Ermilova, Elena

    2015-11-01

    In all examined organisms that have the PII signal transduction machinery, PII coordinates the central C/N anabolic metabolism. In green algae and land plants, PII is localized in the chloroplast and controls the L-arginine biosynthetic pathway pathway. To elucidate additional functions of PII in the model photosynthetic organism Chlamydomonas reinhardtii (CrPII), we generated and analyzed four strains, in which PII was strongly under-expressed by artificial microRNA (GLB1-amiRNA strains). In response to nitrogen deficiency, Chlamydomonas produces triacylglycerols (TAGs) that are accumulated in lipid bodies (LB). Quantification of LBs by confocal microscopy in four GLB1-amiRNA strains showed that reduced PII levels resulted in over-accumulation of LBs compared to their parental strains. Moreover, knock-down of PII caused also an increase in the total TAG level. We propose that the larger yields of TAG-filled LBs in N-starved GLB1-amiRNA cells can be attributed to the strain's depleted PII level and their inability to properly control acetyl-CoA carboxylase activity (ACCase). Together, our results imply that PII in Chlamydomonas negatively controls TAG accumulation in LBs during acclimation to nitrogen starvation of the alga.

  2. Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii

    PubMed Central

    Onishi, Masayuki; Pringle, John R.

    2016-01-01

    The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins. PMID:27770025

  3. 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.

  4. Inhibition of glycolate and D-lactate metabolism in a Chlamydomonas reinhardtii mutant deficient in mitochondrial respiration

    PubMed Central

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

    1987-01-01

    The possibility that glycolate oxidation in unicellular green algae is linked to mitochondrial electron transport, rather than to peroxisomal metabolism as in higher plants and animals, was studied in a mutant of Chlamydomonas reinhardtii (dk97) deficient in cytochrome oxidase. This mutant had normal rates of dark respiration (40 ± 15 μmol of O2 uptake per hr per mg of chlorophyll) but had only 11% of wild-type levels of cytochrome oxidase activity. Salicylhydroxamic acid (SHAM) reduced the dark respiration rate of dk97 cells by 71%, but cyanide did not significantly inhibit this rate. During photosynthesis in the presence of SHAM, glycolate oxidation was blocked, resulting in glycolate accumulation and excretion by mutant cells but not by wild-type Chlamydomonas. D-Lactate, which accumulated after brief periods of anaerobiosis in Chlamydomonas, was reoxidized by air-grown cells only aerobically in the light, and reoxidation of D-lactate was blocked by SHAM in the dk97 cells. Thus, glycolate and D-lactate dehydrogenase activities are both linked to mitochondrial electron transport in Chlamydomonas. During photosynthetic 14CO2 fixation by dk97 cells in the presence of SHAM, 14C-labeled tricarboxylic acid cycle intermediates accumulated, indicating that, in Chlamydomonas, mitochondrial respiration functions during photosynthesis. PMID:16578800

  5. Between a rock and a hard place: trace element nutrition in Chlamydomonas.

    PubMed

    Merchant, Sabeeha S; Allen, Michael D; Kropat, Janette; Moseley, Jeffrey L; Long, Joanne C; Tottey, Stephen; Terauchi, Aimee M

    2006-07-01

    Photosynthetic organisms are among the earliest life forms on earth and their biochemistry is strictly dependent on a wide range of inorganic nutrients owing to the use of metal cofactor-dependent enzymes in photosynthesis, respiration, inorganic nitrogen and sulfur assimilation. Chlamydomonas reinhardtii is a photosynthetic eukaryotic model organism for the study of trace metal homeostasis. Chlamydomonas spp. are widely distributed and can be found in soil, glaciers, acid mines and sewage ponds, suggesting that the genus has significant capacity for acclimation to micronutrient availability. Analysis of the draft genome indicates that metal homeostasis mechanisms in Chlamydomonas represent a blend of mechanisms operating in animals, plants and microbes. A combination of classical genetics, differential expression and genomic analysis has led to the identification of homologues of components known to operate in fungi and animals (e.g., Fox1, Ftr1, Fre1, Fer1, Ctr1/2) as well as novel molecules involved in copper and iron nutrition (Crr1, Fea1/2). Besides activating iron assimilation pathways, iron-deficient Chlamydomonas cells re-adjust metabolism by reducing light delivery to photosystem I (to avoid photo-oxidative damage resulting from compromised FeS clusters) and by modifying the ferredoxin profile (perhaps to accommodate preferential allocation of reducing equivalents). Up-regulation of a MnSOD isoform may compensate for loss of FeSOD. Ferritin could function to buffer the iron released from programmed degradation of iron-containing enzymes in the chloroplast. Some metabolic adjustments are made in anticipation of deficiency while others occur only with sustained or severe deficiency. Copper-deficient Chlamydomonas cells induce a copper assimilation pathway consisting of a cell surface reductase and a Cu(+) transporter (presumed CTR homologue). There are metabolic adaptations in addition: the synthesis of "back-up" enzymes for plastocyanin in photosynthesis

  6. Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii.

    PubMed

    Hu, Jinlu; Deng, Xuan; Shao, Ning; Wang, Gaohong; Huang, Kaiyao

    2014-09-01

    The unicellular green algae Chlamydomonas reinhardtii is a classic model for the study of flagella/cilia and photosynthesis, and it has recently been exploited for producing biopharmaceuticals and biofuel. Due to the low frequency of homologous recombination, reverse genetic manipulation in Chlamydomonas relies mainly on miRNA- and siRNA-based knockdown methods. However, the difficulty in constructing artificial miRNA vectors, laborious screening of knockdown transformants, and undesired epigenetic silencing of exogenous miRNA constructs limit their application. We have established a one-step procedure to construct an artificial miRNA precursor by annealing eight oligonucleotides of approximately 40 nucleotides. In the final construct, the Gaussia princeps luciferase gene (G-Luc) is positioned between the promoter and the artificial miRNA precursor so that knockdown strains may quickly be screened by visualizing luciferase luminescence using a photon-counting camera. Furthermore, the luciferase activity of transformants correlates with the knockdown level of two test target proteins: the chloroplast protein VIPP1 (vesicle inducing protein in plastids 1) and the flagellar protein CDPK3 (calcium-dependent protein kinase 3). Adding an intron from RBCS2 (ribulose bisphosphate carboxylase/oxygenase small subunit 2) to the miRNA construct enhanced both the luciferase activity and the miRNA knockdown efficiency. A second miRNA vector incorporated the promoter of the nitrate reductase gene to allow inducible expression of the artificial miRNA. These vectors will facilitate application of the artificial miRNA and provide tools for studying the mechanism of epigenetics in Chlamydomonas, and may also be adapted for use in other model organisms.

  7. Engineering the Chloroplast Targeted Malarial Vaccine Antigens in Chlamydomonas Starch Granules

    PubMed Central

    Dauvillée, David; Delhaye, Stéphane; Gruyer, Sébastien; Slomianny, Christian; Moretz, Samuel E.; d'Hulst, Christophe; Long, Carole A.; Ball, Steven G.; Tomavo, Stanislas

    2010-01-01

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

  8. Phosphatase of Chlamydomonas reinhardi: biochemical and cytochemical approach with specific mutants.

    PubMed Central

    Matagne, R F; Loppes, R; Deltour, R

    1976-01-01

    The unicellular alga Chlamydomonas reinhardi produces two constitutive acid phosphatases and three depressible phosphatases (a neutral and two alkaline ones) that can utilize napthyl phosphate as a substrate. Specific mutants depressible phosphatase were used to investigate biochemical properties and the cytochemical localization of these enzymes. The two constitutive phosphatases show similar pH optima (about 5.0) and Km values (2 x 10(-3) to 3.3 x 10(-3) M) but differ in their heat sensitivity and affinity for glycerophosphate. Images PMID:4437

  9. Chlamydomonas reinhardtii: the model of choice to study mitochondria from unicellular photosynthetic organisms.

    PubMed

    Funes, Soledad; Franzén, Lars-Gunnar; González-Halphen, Diego

    2007-01-01

    Chlamydomonas reinhardtii is a model organism to study photosynthesis, cellular division, flagellar biogenesis, and, more recently, mitochondrial function. It has distinct advantages in comparison to higher plants because it is unicellular, haploid, and amenable to tetrad analysis, and its three genomes are subject to specific transformation. It also has the possibility to grow either photoautotrophically or heterotrophically on acetate, making the assembly of the photosynthetic machinery not essential for cell viability. Methods developed allow the isolation of C. reinhardtii mitochondria free of thylakoid contaminants. We review the general procedures used for the biochemical characterization of mitochondria from this green alga.

  10. Extremely low polymerizability of a highly-divergent Chlamydomonas actin (NAP).

    PubMed

    Kato-Minoura, Takako

    2011-09-09

    Novel actin-like protein (NAP) is a highly divergent actin expressed in Chlamydomonas. With its low sequence similarity, it is uncertain whether NAP can polymerize into filaments. Here I assessed it by ectopically expressing enhanced green fluorescent protein-tagged NAP (EGFP-NAP) in cultured cells. EGFP-NAP was excluded from stress fibres but partially co-localized with endogenous actin in the cell periphery. In fluorescence recovery after photobleaching experiment, turnover rate of EGFP-NAP was similar to the estimated diffusion rate of monomeric actin. Therefore, EGFP-NAP likely accumulates by diffusion. These findings suggest that NAP has extremely poor ability to polymerize.

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

    DOE PAGES

    Jurado-Oller, Jose Luis; Dubini, Alexandra; Galvan, Aurora; ...

    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.

  12. 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.

  13. Comparison of the Microtubule Proteins of Neuroblastoma Cells, Brain, and Chlamydomonas Flagella

    PubMed Central

    Olmsted, J. B.; Witman, G. B.; Carlson, K.; Rosenbaum, Joel L.

    1971-01-01

    Intact A microtubules isolated from outer doublet microtubules of Chlamydomonas flagella contain two separable proteins (tubulins) that differ in molecular weight and in amino-acid composition. The microtubule protein isolated from brain or neuroblastoma cells also has two electrophoretically distinct tubulins. Although the two tubulins of brain and neuroblastoma cells are electrophoretically similar to each other, only one of these tubulins migrates with the flagellar tubulins. This is the first evidence that (a) isolated, morphologically intact, single microtubules from flagella contain at least two different tubulins, and (b) at least one of these tubulins differs from tubulins that are isolated from other sources. Images PMID:5289385

  14. Novel glycopolypeptide synthesis induced by gametic cell fusion in Chlamydomonas reinhardtii

    PubMed Central

    1978-01-01

    Within the first hour of zygote maturation, Chlamydomonas reinhardtii cells stop synthesizing certain polypeptides that characterize the vegetative and gametic stages of the life cycle and initiate the synthesis of novel, zygote-specific polypeptides. At least six of these polypeptides are secreted into the medium, and fine-structural studies indicate that they represent components of the cell wall that is synthesized and secreted early in zygote development. We conclude that a new program of protein synthesis, and possibly also gene transcription, is initiated shortly after gametic cells fuse, a program that appears highly suited to cell-differentiation studies. PMID:659511

  15. [Effect of microwaves on Chlamydomonas actinochloris culture in the stationary phase of growth].

    PubMed

    Grigor'eva, O O; Berezovskaia, M A; Datsenko, A I

    2013-01-01

    Effects of the microwave radiation on the culture of Chlamydomonas actinochloris green flagellar alga in the stationary phase of growth are studied. After exposure to radiation at the maximum dose of 125 J/g, the cell functional state worsened but all the studied parameters were restored in 20 days and in the long run found to be even better than the control indices. The data are compared with the similar ones obtained earlier for the lag phase culture. The studied sample is found to be more resistant to the irradiation than the previous one.

  16. Estimation of Chlamydomonas reinhardtii biomass concentration from chord length distribution data.

    PubMed

    Lopez-Exposito, Patricio; Suarez, Angeles Blanco; Negro, Carlos

    A novel method to estimate the concentration of Chlamydomonas reinhardtii biomass was developed. The method employs the chord length distribution information gathered by means of a focused beam reflectance probe immersed in the culture sample and processes the data through a feedforward multilayer perceptron. The multilayer perceptron architecture was systematically optimised through the application of a simulated annealing algorithm. The method developed can predict the concentration of microalgae with acceptable accuracy and, with further development, it could be implemented online to monitor the aggregation status and biomass concentration of microalgal cultures.

  17. 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.

  18. A phenotypic screening platform to identify small molecule modulators of Chlamydomonas reinhardtii growth, motility and photosynthesis

    PubMed Central

    2012-01-01

    Chemical biology, the interfacial discipline of using small molecules as probes to investigate biology, is a powerful approach of developing specific, rapidly acting tools that can be applied across organisms. The single-celled alga Chlamydomonas reinhardtii is an excellent model system because of its photosynthetic ability, cilia-related motility and simple genetics. We report the results of an automated fitness screen of 5,445 small molecules and subsequent assays on motility/phototaxis and photosynthesis. Cheminformatic analysis revealed active core structures and was used to construct a naïve Bayes model that successfully predicts algal bioactive compounds. PMID:23158586

  19. Actinobacteria associated with the marine sponges Cinachyra sp., Petrosia sp., and Ulosa sp. and their culturability.

    PubMed

    Khan, Shams Tabrez; Takagi, Motoki; Shin-ya, Kazuo

    2012-01-01

    Actinobacteria associated with 3 marine sponges, Cinachyra sp., Petrosia sp., and Ulosa sp., were investigated. Analyses of 16S rRNA gene clone libraries revealed that actinobacterial diversity varied greatly and that Ulosa sp. was most diverse, while Cinachyra sp. was least diverse. Culture-based approaches failed to isolate actinobacteria from Petrosia sp. or Ulosa sp., but strains belonging to 10 different genera and 3 novel species were isolated from Cinachyra sp.

  20. Actinobacteria Associated with the Marine Sponges Cinachyra sp., Petrosia sp., and Ulosa sp. and Their Culturability

    PubMed Central

    Khan, Shams Tabrez; Takagi, Motoki; Shin-ya, Kazuo

    2012-01-01

    Actinobacteria associated with 3 marine sponges, Cinachyra sp., Petrosia sp., and Ulosa sp., were investigated. Analyses of 16S rRNA gene clone libraries revealed that actinobacterial diversity varied greatly and that Ulosa sp. was most diverse, while Cinachyra sp. was least diverse. Culture-based approaches failed to isolate actinobacteria from Petrosia sp. or Ulosa sp., but strains belonging to 10 different genera and 3 novel species were isolated from Cinachyra sp. PMID:22214828

  1. Displacement-Weighted Velocity Analysis of Gliding Assays Reveals that Chlamydomonas Axonemal Dynein Preferentially Moves Conspecific Microtubules

    PubMed Central

    Alper, Joshua D.; Tovar, Miguel; Howard, Jonathon

    2013-01-01

    In vitro gliding assays, in which microtubules are observed to glide over surfaces coated with motor proteins, are important tools for studying the biophysics of motility. Gliding assays with axonemal dyneins have the unusual feature that the microtubules exhibit large variations in gliding speed despite measures taken to eliminate unsteadiness. Because axonemal dynein gliding assays are usually done using heterologous proteins, i.e., dynein and tubulin from different organisms, we asked whether the source of tubulin could underlie the unsteadiness. By comparing gliding assays with microtubules polymerized from Chlamydomonas axonemal tubulin with those from porcine brain tubulin, we found that the unsteadiness is present despite matching the source of tubulin to the source of dynein. We developed a novel, to our knowledge, displacement-weighted velocity analysis to quantify both the velocity and the unsteadiness of gliding assays systematically and without introducing bias toward low motility. We found that the quantified unsteadiness is independent of tubulin source. In addition, we found that the short Chlamydomonas microtubules translocate significantly faster than their porcine counterparts. By modeling the effect of length on velocity, we propose that the observed effect may be due to a higher rate of binding of Chlamydomonas axonemal dynein to Chlamydomonas microtubules than to porcine microtubules. PMID:23663842

  2. Functional Characterization of the Chlamydomonas reinhardtii ERG3 Ortholog, a Gene Involved in the Biosynthesis of Ergosterol

    PubMed Central

    Brumfield, Kristy M.; Moroney, James V.; Moore, Thomas S.; Simms, Tiffany A.; Donze, David

    2010-01-01

    Background The predominant sterol in the membranes of the alga Chlamydomonas reinhardtii is ergosterol, which is commonly found in the membranes of fungi, but is rarely found in higher plants. Higher plants and fungi synthesize sterols by different pathways, with plants producing cycloartenol as a precursor to end-product sterols, while non-photosynthesizing organisms like yeast and humans produce lanosterol as a precursor. Analysis of the C. reinhardtii genome sequence reveals that this algae is also likely to synthesize sterols using a pathway resembling the higher plant pathway, indicating that its sterols are synthesized somewhat differently than in fungi. The work presented here seeks to establish experimental evidence to support the annotated molecular function of one of the sterol biosynthetic genes in the Chlamydomonas genome. Methodology/Principal Findings A gene with homology to the yeast sterol C-5 desaturase, ERG3, is present in the Chlamydomonas genome. To test whether the ERG3 ortholog of C. reinhardtii encodes a sterol C-5 desaturase, Saccharomyces cerevisiae ERG3 knockout strains were created and complemented with a plasmid expressing the Chlamydomonas ERG3. Expression of C. reinhardtii ERG3 cDNA in erg3 null yeast was able to restore ergosterol biosynthesis and reverse phenotypes associated with lack of ERG3 function. Conclusions/Significance Complementation of the yeast erg3 null phenotypes strongly suggests that the gene annotated as ERG3 in C. reinhardtii functions as a sterol C-5 desaturase. PMID:20084111

  3. 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

  4. 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.

  5. 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.

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

    PubMed

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

    2015-12-01

    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.

  7. Altered fermentative metabolism in Chlamydomonas reinhardtii mutants lacking pyruvate formate lyase and both pyruvate formate lyase and alcohol dehydrogenase.

    PubMed

    Catalanotti, Claudia; Dubini, Alexandra; Subramanian, Venkataramanan; Yang, Wenqiang; Magneschi, Leonardo; Mus, Florence; Seibert, Michael; Posewitz, Matthew C; Grossman, Arthur 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 H(2) 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 H(2) 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.

  8. Dissecting the heat stress response in Chlamydomonas by pharmaceutical and RNAi approaches reveals conserved and novel aspects.

    PubMed

    Schmollinger, Stefan; Schulz-Raffelt, Miriam; Strenkert, Daniela; Veyel, Daniel; Vallon, Olivier; Schroda, Michael

    2013-11-01

    To study how conserved fundamental concepts of the heat stress response (HSR) are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotolerance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenuation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell's entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP70B-antisense strains.

  9. Control of phobic behavioral responses by rhodopsin-induced photocurrents in Chlamydomonas.

    PubMed Central

    Holland, E M; Harz, H; Uhl, R; Hegemann, P

    1997-01-01

    Both phototactic and photophobic responses of Chlamydomonas are mediated by a visual system comprising a rhodopsin photoreceptor. Suction pipette recordings have revealed that flash stimulation causes calcium currents into the eyespot and the flagella. These photocurrents have been suggested to be the trigger for all behavioral light responses of the cell. But this has never been shown experimentally. Here we describe a detection technique that combines electrical and optical measurements from individual algae held in a suction pipette. Thus it is possible to record photocurrents and flagellar beating simultaneously and establish a direct link between the two. We demonstrate that in Chlamydomonas only the photoreceptor current in conjuction with a fast flagellar current constitutes the trigger for photophobic responses. Within the time of the action-potential-like flagellar current, the flagella switch from forward to backward swimming, which constitutes the beginning of the photoshock reaction. The switch is accompanied by a complex frequency change and beating pattern modulation. The results are interpreted in terms of a general model for phototransduction in green algae (Chlorophyceae). Images FIGURE 3 PMID:9284306

  10. Control of phobic behavioral responses by rhodopsin-induced photocurrents in Chlamydomonas.

    PubMed

    Holland, E M; Harz, H; Uhl, R; Hegemann, P

    1997-09-01

    Both phototactic and photophobic responses of Chlamydomonas are mediated by a visual system comprising a rhodopsin photoreceptor. Suction pipette recordings have revealed that flash stimulation causes calcium currents into the eyespot and the flagella. These photocurrents have been suggested to be the trigger for all behavioral light responses of the cell. But this has never been shown experimentally. Here we describe a detection technique that combines electrical and optical measurements from individual algae held in a suction pipette. Thus it is possible to record photocurrents and flagellar beating simultaneously and establish a direct link between the two. We demonstrate that in Chlamydomonas only the photoreceptor current in conjuction with a fast flagellar current constitutes the trigger for photophobic responses. Within the time of the action-potential-like flagellar current, the flagella switch from forward to backward swimming, which constitutes the beginning of the photoshock reaction. The switch is accompanied by a complex frequency change and beating pattern modulation. The results are interpreted in terms of a general model for phototransduction in green algae (Chlorophyceae).

  11. The role of nitric oxide signalling in response to salt stress in Chlamydomonas reinhardtii.

    PubMed

    Chen, Xiaodong; Tian, Dagang; Kong, Xiangxiang; Chen, Qian; E F, Abd Allah; Hu, Xiangyang; Jia, Aiqun

    2016-09-01

    Nitric oxide signal and GSNOR activity play an essential role for Chlamydomonas reinhardtii response to salt stress. The unicellular alga Chlamydomonas reinhardtii is one of the most important model organisms phylogenetically situated between higher plants and animals. In the present study, we used comparative proteomics and physiological approaches to study the mechanisms underlying the response to salt stress in C. reinhardtii. We identified 74 proteins that accumulated differentially after salt stress, including oxidative enzymes and enzymes associated with nitric oxide (NO) metabolism, cell damage, and cell autophagy processes. A set of antioxidant enzymes, as well as S-nitrosoglutathione reductase (GSNOR) activity, were induced to balance the cellular redox status during short-term salt stress. Enzymes involved in DNA repair and cell autophagy also contribute to adaptation to short-term salt stress. However, under long-term salt stress, antioxidant enzymes and GSNOR were gradually inactivated through protein S-nitrosylation, leading to oxidative damage and a reduction in cell viability. Modulating the protein S-nitrosylation levels by suppressing GSNOR activity or adding thioredoxin affected the plant's adaptation to salt stress, through altering the redox status and DNA damage and autophagy levels. Based on these data, we propose that unicellular algae use multiple strategies to adapt to salt stress, and that, during this process, GSNOR activity and protein S-nitrosylation levels play important roles.

  12. Rubisco small subunits from the unicellular green alga Chlamydomonas complement Rubisco-deficient mutants of Arabidopsis.

    PubMed

    Atkinson, Nicky; Leitão, Nuno; Orr, Douglas J; Meyer, Moritz T; Carmo-Silva, Elizabete; Griffiths, Howard; Smith, Alison M; McCormick, Alistair J

    2017-04-01

    Introducing components of algal carbon concentrating mechanisms (CCMs) into higher plant chloroplasts could increase photosynthetic productivity. A key component is the Rubisco-containing pyrenoid that is needed to minimise CO2 retro-diffusion for CCM operating efficiency. Rubisco in Arabidopsis was re-engineered to incorporate sequence elements that are thought to be essential for recruitment of Rubisco to the pyrenoid, namely the algal Rubisco small subunit (SSU, encoded by rbcS) or only the surface-exposed algal SSU α-helices. Leaves of Arabidopsis rbcs mutants expressing 'pyrenoid-competent' chimeric Arabidopsis SSUs containing the SSU α-helices from Chlamydomonas reinhardtii can form hybrid Rubisco complexes with catalytic properties similar to those of native Rubisco, suggesting that the α-helices are catalytically neutral. The growth and photosynthetic performance of complemented Arabidopsis rbcs mutants producing near wild-type levels of the hybrid Rubisco were similar to those of wild-type controls. Arabidopsis rbcs mutants expressing a Chlamydomonas SSU differed from wild-type plants with respect to Rubisco catalysis, photosynthesis and growth. This confirms a role for the SSU in influencing Rubisco catalytic properties.

  13. High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

    PubMed Central

    Breker, Michal; Lieberman, Kristi; Tulin, Frej; Cross, Frederick R.

    2016-01-01

    Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript. PMID:28060315

  14. 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

    2016-06-13

    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.

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

    PubMed

    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.

  17. 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

  18. 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

  19. 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

  20. Atomic resolution modeling of the ferredoxin:[FeFe] hydrogenase complex from Chlamydomonas reinhardtii.

    PubMed

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

    2007-11-01

    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 H(2) 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 H(2) evolution, and suggests potential mutagenic strategies to modulate electron flow to HydA2.

  1. System response of metabolic networks in Chlamydomonas reinhardtii to total available ammonium.

    PubMed

    Lee, Do Yup; Park, Jeong-Jin; Barupal, Dinesh K; Fiehn, Oliver

    2012-10-01

    Drastic alterations in macronutrients are known to cause large changes in biochemistry and gene expression in the photosynthetic alga Chlamydomonas reinhardtii. However, metabolomic and proteomic responses to subtle reductions in macronutrients have not yet been studied. When ammonium levels were reduced by 25-100% compared with control cultures, ammonium uptake and growth rates were not affected at 25% or 50% nitrogen-reduction for 28 h. However, primary metabolism and enzyme expression showed remarkable changes at acute conditions (4 h and 10 h after ammonium reduction) compared with chronic conditions (18 h and 28 h time points). Responses of 145 identified metabolites were quantified using gas chromatography-time of flight mass spectrometry; 495 proteins (including 187 enzymes) were monitored using liquid chromatography-ion trap mass spectrometry with label-free spectral counting. Stress response and carbon assimilation processes (Calvin cycle, acetate uptake and chlorophyll biosynthesis) were altered first, in addition to increase in enzyme contents for lipid biosynthesis and accumulation of short chain free fatty acids. Nitrogen/carbon balance metabolism was found changed only under chronic conditions, for example in the citric acid cycle and amino acid metabolism. Metabolism in Chlamydomonas readily responds to total available media nitrogen with temporal increases in short-chain free fatty acids and turnover of internal proteins, long before nitrogen resources are depleted.

  2. A Photoperiod-Regulating Gene CONSTANS Is Correlated to Lipid Biosynthesis in Chlamydomonas reinhardtii

    PubMed Central

    Deng, Xiaodong; Fan, Xinzhao; Li, Ping; Fei, Xiaowen

    2015-01-01

    Background. The regulation of lipid biosynthesis is essential in photosynthetic eukaryotic cells. Thus far, no regulatory genes have been reported in the lipid metabolism pathway. Plant CONSTANS (CO) gene regulates blooming by participating in photoperiod and biological clock. Apart from regulating photoperiod, the Chlamydomonas CO gene also regulates starch content. Results. In this study, the results showed that, under HSM-S condition, cells accumulated more lipids at short-day conditions than at long-day conditions. The silencing of the CrCO gene via RNA interference resulted in an increase in lipid content and an increase in triacylglyceride (TAG) level by 24.5%. CrCO RNAi strains accumulated more lipids at short-day conditions than at long-day conditions. The decrease in CrCO expression resulted in the increased expression of TAG biosynthesis-related genes, such as DGAT2, PAP2, and PDAT3, whereas CIS and FBP1 genes showed a decrease in their mRNA when the CrCO expression was suppressed. On the other hand, the overexpression of CrCO resulted in the decrease in lipid content and TAG level. Conclusions. The results of this study revealed a relationship between CrCO gene and lipid metabolism in Chlamydomonas, suggesting that increasing oil by suppressing CrCO expression in microalgae is feasible. PMID:25654119

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

    PubMed

    Plecenikova, Andrea; Slaninova, Miroslava; Riha, Karel

    2014-01-01

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

  4. 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

  5. A Light Harvesting Complex-Like Protein in Maintenance of Photosynthetic Components in Chlamydomonas.

    PubMed

    Zhao, Lei; Cheng, Dongmei; Huang, Xiahe; Chen, Mei; Dall'Osto, Luca; Xing, Jiale; Gao, Liyan; Li, Lingyu; Wang, Yale; Bassi, Roberto; Peng, Lianwei; Wang, Yingchun; Rochaix, Jean-David; Huang, Fang

    2017-08-01

    Using a genetic approach, we have identified and characterized a novel protein, named Msf1 (Maintenance factor for photosystem I), that is required for the maintenance of specific components of the photosynthetic apparatus in the green alga Chlamydomonas reinhardtii Msf1 belongs to the superfamily of light-harvesting complex proteins with three transmembrane domains and consensus chlorophyll-binding sites. Loss of Msf1 leads to reduced accumulation of photosystem I and chlorophyll-binding proteins/complexes. Msf1is a component of a thylakoid complex containing key enzymes of the tetrapyrrole biosynthetic pathway, thus revealing a possible link between Msf1 and chlorophyll biosynthesis. Protein interaction assays and greening experiments demonstrate that Msf1 interacts with Copper target homolog1 (CHL27B) and accumulates concomitantly with chlorophyll in Chlamydomonas, implying that chlorophyll stabilizes Msf1. Contrary to other light-harvesting complex-like genes, the expression of Msf1 is not stimulated by high-light stress, but its protein level increases significantly under heat shock, iron and copper limitation, as well as in stationary cells. Based on these results, we propose that Msf1 is required for the maintenance of photosystem I and specific protein-chlorophyll complexes especially under certain stress conditions. © 2017 American Society of Plant Biologists. All Rights Reserved.

  6. 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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

    2013-10-17

    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.

  12. First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii.

    PubMed

    Taylor, T C; Backlund, A; Bjorhall, K; Spreitzer, R J; Andersson, I

    2001-12-21

    The crystal structure of Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) from the unicellular green alga Chlamydomonas reinhardtii has been determined to 1.4 A resolution. Overall, the structure shows high similarity to the previously determined structures of L8S8 Rubisco enzymes. The largest difference is found in the loop between beta strands A and B of the small subunit (betaA-betaB loop), which is longer by six amino acid residues than the corresponding region in Rubisco from Spinacia. Mutations of residues in the betaA-betaB loop have been shown to affect holoenzyme stability and catalytic properties. The information contained in the Chlamydomonas structure enables a more reliable analysis of the effect of these mutations. No electron density was observed for the last 13 residues of the small subunit, which are assumed to be disordered in the crystal. Because of the high resolution of the data, some posttranslational modifications are unambiguously apparent in the structure. These include cysteine and N-terminal methylations and proline 4-hydroxylations.

  13. Gene Regulatory Networks for the Haploid-to-Diploid Transition of Chlamydomonas reinhardtii1[OPEN

    PubMed Central

    Joo, Sunjoo; Hong, Ran Ha; Kariyawasam, Thamali; Wang, Ming Hsiu; El Akkad, Saif-El-Din; Suzuki, Takamasa

    2017-01-01

    The sexual cycle of the unicellular Chlamydomonas reinhardtii culminates in the formation of diploid zygotes that differentiate into dormant spores that eventually undergo meiosis. Mating between gametes induces rapid cell wall shedding via the enzyme g-lysin; cell fusion is followed by heterodimerization of sex-specific homeobox transcription factors, GSM1 and GSP1, and initiation of zygote-specific gene expression. To investigate the genetic underpinnings of the zygote developmental pathway, we performed comparative transcriptome analysis of both pre- and post-fertilization samples. We identified 253 transcripts specifically enriched in early zygotes, 82% of which were not up-regulated in gsp1 null zygotes. We also found that the GSM1/GSP1 heterodimer negatively regulates the vegetative wall program at the posttranscriptional level, enabling prompt transition from vegetative wall to zygotic wall assembly. Annotation of the g-lysin-induced and early zygote genes reveals distinct vegetative and zygotic wall programs, supported by concerted up-regulation of genes encoding cell wall-modifying enzymes and proteins involved in nucleotide-sugar metabolism. The haploid-to-diploid transition in Chlamydomonas is masterfully controlled by the GSM1/GSP1 heterodimer, translating fertilization and gamete coalescence into a bona fide differentiation program. The fertilization-triggered integration of genes required to make related, but structurally and functionally distinct organelles—the vegetative versus zygote cell wall—presents a likely scenario for the evolution of complex developmental gene regulatory networks. PMID:28710131

  14. A galactoglycerolipid lipase is required for triacylglycerol accumulation and survival following nitrogen deprivation in Chlamydomonas reinhardtii.

    PubMed

    Li, Xiaobo; Moellering, Eric R; Liu, Bensheng; Johnny, Cassandra; Fedewa, Marie; Sears, Barbara B; Kuo, Min-Hao; Benning, Christoph

    2012-11-01

    Following N deprivation, microalgae accumulate triacylglycerols (TAGs). To gain mechanistic insights into this phenomenon, we identified mutants with reduced TAG content following N deprivation in the model alga Chlamydomonas reinhardtii. In one of the mutants, the disruption of a galactoglycerolipid lipase-encoding gene, designated PLASTID GALACTOGLYCEROLIPID DEGRADATION1 (PGD1), was responsible for the primary phenotype: reduced TAG content, altered TAG composition, and reduced galactoglycerolipid turnover. The recombinant PGD1 protein, which was purified from Escherichia coli extracts, hydrolyzed monogalactosyldiacylglycerol into its lyso-lipid derivative. In vivo pulse-chase labeling identified galactoglycerolipid pools as a major source of fatty acids esterified in TAGs following N deprivation. Moreover, the fatty acid flux from plastid lipids to TAG was decreased in the pgd1 mutant. Apparently, de novo-synthesized fatty acids in Chlamydomonas reinhardtii are, at least partially, first incorporated into plastid lipids before they enter TAG synthesis. As a secondary effect, the pgd1 mutant exhibited a loss of viability following N deprivation, which could be avoided by blocking photosynthetic electron transport. Thus, the pgd1 mutant provides evidence for an important biological function of TAG synthesis following N deprivation, namely, relieving a detrimental overreduction of the photosynthetic electron transport chain.

  15. Calcium mediates the cellular response of Chlamydomonas reinhardtii to the emerging aquatic pollutant Triclosan.

    PubMed

    González-Pleiter, Miguel; Rioboo, Carmen; Reguera, María; Abreu, Isidro; Leganés, Francisco; Cid, Ángeles; Fernández-Piñas, Francisca

    2017-05-01

    The present study was aimed at investigating the role of intracellular free calcium, [Ca(2+)]c, in the early cellular response of the green alga Chlamydomonas reinhardtii to the emergent pollutant Triclosan (13.8μM; 24h of exposure). There is a growing concern about the persistence and toxicity of this antimicrobial in aquatic environments, where non-target organisms such as C. reinhardtii, a primary producer of ecological relevance, might be severely impacted. A mechanistic study was undertaken which combined flow cytometry protocols, physiological as well as gene expression analysis. As an early response, Triclosan strongly altered [Ca(2+)]c homeostasis which could be prevented by prechelation with the intracellular calcium chelator BAPTA-AM. Triclosan induced ROS overproduction which ultimately leads to oxidative stress with loss of membrane integrity, membrane depolarization, photosynthesis inhibition and mitochondrial membrane depolarization; within this context, Triclosan also induced an increase in caspase 3/7 activity and altered the expression of metacaspase genes which are indicative of apoptosis. All these adverse outcomes were dependent on [Ca(2+)]c. Interestingly, an interconnection between [Ca(2+)]c alterations and increased ROS formation by Triclosan was found. Taken altogether these results shed light on the mechanisms behind Triclosan toxicity in the green alga Chlamydomonas reinhardtii and demonstrate the role of [Ca(2+)]c in mediating the observed toxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Interaction between Starch Breakdown, Acetate Assimilation, and Photosynthetic Cyclic Electron Flow in Chlamydomonas reinhardtii*

    PubMed Central

    Johnson, Xenie; Alric, Jean

    2012-01-01

    Spectroscopic studies on photosynthetic electron transfer generally are based upon the monitoring of dark to light changes in the electron transfer chain. These studies, which focus on the light reactions of photosynthesis, also indirectly provide information on the redox or metabolic state of the chloroplast in the dark. Here, using the unicellular microalga Chlamydomonas reinhardtii, we study the impact of heterotrophic/mixotrophic acetate feeding on chloroplast carbon metabolism by using the spectrophotometric detection of P700+, the photooxidized primary electron donor of photosystem I. We show that, when photosynthetic linear and cyclic electron flows are blocked (DCMU inhibiting PSII and methylviologen accepting electrons from PSI), the post-illumination reduction kinetics of P700+ directly reflect the dark metabolic production of reductants (mainly NAD(P)H) in the stroma of chloroplasts. Such results can be correlated to other metabolic studies: in the absence of acetate, for example, the P700+ reduction rate matches the rate of starch breakdown reported previously, confirming the chloroplast localization of the upstream steps of the glycolytic pathway in Chlamydomonas. Furthermore, the question of the interplay between photosynthetic and non-photosynthetic carbon metabolism can be addressed. We show that cyclic electron flow around photosystem I is twice as fast in a starchless mutant fed with acetate than it is in the WT, and we relate how changes in the flux of electrons from carbohydrate metabolism modulate the redox poise of the plastoquinone pool in the dark through chlororespiration. PMID:22692199

  17. AN OPTIMIZED METHOD FOR THE ISOLATION OF NUCLEI FROM CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)(1).

    PubMed

    Winck, Flavia Vischi; Kwasniewski, Miroslaw; Wienkoop, Stefanie; Mueller-Roeber, Bernd

    2011-04-01

    The cell nucleus harbors a large number of proteins involved in transcription, RNA processing, chromatin remodeling, nuclear signaling, and ribosome assembly. The nuclear genome of the model alga Chlamydomonas reinhardtii P. A. Dang. was recently sequenced, and many genes encoding nuclear proteins, including transcription factors and transcription regulators, have been identified through computational discovery tools. However, elucidating the specific biological roles of nuclear proteins will require support from biochemical and proteomics data. Cellular preparations with enriched nuclei are important to assist in such analyses. Here, we describe a simple protocol for the isolation of nuclei from Chlamydomonas, based on a commercially available kit. The modifications done in the original protocol mainly include alterations of the differential centrifugation parameters and detergent-based cell lysis. The nuclei-enriched fractions obtained with the optimized protocol show low contamination with mitochondrial and plastid proteins. The protocol can be concluded within only 3 h, and the proteins extracted can be used for gel-based and non-gel-based proteomic approaches. © 2011 Phycological Society of America.

  18. 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

  19. Generation of the heterodimeric precursor GP3 of the Chlamydomonas cell wall.

    PubMed

    Voigt, Jürgen; Kiess, Michael; Getzlaff, Rita; Wöstemeyer, Johannes; Frank, Ronald

    2010-09-01

    The cell wall of the unicellular green alga Chlamydomonas reinhardtii exclusively consists of hydroxyproline-containing glycoproteins. Protein chemical analysis of its polypeptide constituents was hindered by their cross-linking via peroxidase-catalysed intermolecular isodityrosine formation and transaminase-dependent processes. To overcome this problem, we have identified putative soluble precursors using polyclonal antibodies raised against deglycosylation products of the highly purified insoluble wall fraction and analysed their amino acid sequence. The occurrence of the corresponding polypeptide in the insoluble glycoprotein framework was finally probed by epitope mapping of the polyclonal antibodies using overlapping scan peptides which, together, cover the whole amino acid sequence of the putative precursor. As a control, peptide fragments released from the insoluble wall fraction by trypsin treatment were analysed by mass spectroscopy. By this approach, the heterodimeric, chaotrope-soluble glycoprotein GP3 proved to be a constituent of the insoluble extracellular matrix of Chlamydomonas reinhardtii. Furthermore, we have shown that the polypeptide backbones of both GP3 subunits are encoded by the same gene and differ by a C-terminal truncation in the case of GP3A.

  20. 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

  1. The trafficking of bacterial type rhodopsins into the Chlamydomonas eyespot and flagella is IFT mediated.

    PubMed

    Awasthi, Mayanka; Ranjan, Peeyush; Sharma, Komal; Veetil, Sindhu Kandoth; Kateriya, Suneel

    2016-10-03

    The bacterial type rhodopsins are present in all the three domains of life. In contrast to the animal type rhodopsin that performs mainly sensory functions in higher eukaryotes, the bacterial type rhodopsin could function as ion channel, pumps and as sensory proteins. The functioning of rhodopsin in higher eukaryotes requires the transport of rhodopsin from its site of synthesis to the ciliated outer segment of the photoreceptive cells. However, the trafficking of bacterial type rhodopsin from its site of synthesis to the position of action is not characterized. Here we present the first report for the existence of an IFT-interactome mediated trafficking of the bacterial type rhodopsins into eyespot and flagella of the Chlamydomonas. We show that there is a light-dependent, dynamic localization of rhodopsins between flagella and eyespot of Chlamydomonas. The involvement of IFT components in the rhodopsin trafficking was elucidated by the use of conditional IFT mutants. We found that rhodopsin can be co-immunoprecipitated with the components of IFT machinery and with other protein components required for the IFT-cargo complex formation. These findings show that light-regulated localization of rhodopsin is not restricted to animals thereby suggesting that rhodopsin trafficking is an IFT dependent ancient process.

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

    SciTech Connect

    Chang, C. H.; King, P. W.; Ghirardi, M. L.; Kim, K.

    2007-11-01

    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 20kcal/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.

  3. The trafficking of bacterial type rhodopsins into the Chlamydomonas eyespot and flagella is IFT mediated

    PubMed Central

    Awasthi, Mayanka; Ranjan, Peeyush; Sharma, Komal; Veetil, Sindhu Kandoth; Kateriya, Suneel

    2016-01-01

    The bacterial type rhodopsins are present in all the three domains of life. In contrast to the animal type rhodopsin that performs mainly sensory functions in higher eukaryotes, the bacterial type rhodopsin could function as ion channel, pumps and as sensory proteins. The functioning of rhodopsin in higher eukaryotes requires the transport of rhodopsin from its site of synthesis to the ciliated outer segment of the photoreceptive cells. However, the trafficking of bacterial type rhodopsin from its site of synthesis to the position of action is not characterized. Here we present the first report for the existence of an IFT-interactome mediated trafficking of the bacterial type rhodopsins into eyespot and flagella of the Chlamydomonas. We show that there is a light-dependent, dynamic localization of rhodopsins between flagella and eyespot of Chlamydomonas. The involvement of IFT components in the rhodopsin trafficking was elucidated by the use of conditional IFT mutants. We found that rhodopsin can be co-immunoprecipitated with the components of IFT machinery and with other protein components required for the IFT-cargo complex formation. These findings show that light-regulated localization of rhodopsin is not restricted to animals thereby suggesting that rhodopsin trafficking is an IFT dependent ancient process. PMID:27694882

  4. Chlamydomonas reinhardtii PsbS Protein Is Functional and Accumulates Rapidly and Transiently under High Light1

    PubMed Central

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

    2016-01-01

    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

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

    PubMed

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

    2017-05-16

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

  6. Functional photosystem I maintains proper energy balance during nitrogen depletion in Chlamydomonas reinhardtii, promoting triacylglycerol accumulation.

    PubMed

    Gargouri, Mahmoud; Bates, Philip D; Park, Jeong-Jin; Kirchhoff, Helmut; Gang, David R

    2017-01-01

    Nutrient deprivation causes significant stress to the unicellular microalga, Chlamydomonas reinhardtii, which responds by significantly altering its metabolic program. Following N deprivation, the accumulation of starch and triacylglycerols (TAGs) is significantly altered following massive reprogramming of cellular metabolism. One protein that was found to change dramatically and early to this stress was TAB2, a photosystem I (PSI) translation initiation factor, whose transcript and protein levels increased significantly after only 30 min of N deprivation. A detailed physiological and omics-based analysis of an insertional mutant of Chlamydomonas with reduced TAB2 function was conducted to determine what role the functional PSI plays in regulating the cellular response to N deprivation. The tab2 mutant displayed increased acetate assimilation and elevated starch levels during the first 6 h of N deprivation, followed by a shift toward altered amino acid synthesis, reduced TAG content and altered fatty acid profiles. These results suggested a central role for PSI in controlling cellular metabolism and its implication in regulation of lipid/starch partitioning. Time course analyses of the tab2 mutant versus wild type under N-deprived versus N replete conditions revealed changes in the ATP/NADPH ratio and suggested that TAG biosynthesis may be associated with maintaining the redox state of the cell during N deprivation. The loss of ability to accumulate TAG in the tab2 mutant co-occurred with an up-regulation of photo-protective mechanisms, suggesting that the synthesis of TAG in the wild type occurs not only as a temporal energy sink, but also as a protective electron sink. By exploiting the tab2 mutation in the cells of C. reinhardtii cultured under autotrophic, mixotrophic, and heterotrophic conditions during nitrogen replete growth and for the first 8 days of nitrogen deprivation, we showed that TAG accumulation and lipid/starch partitioning are dynamically

  7. Functional photosystem I maintains proper energy balance during nitrogen depletion in Chlamydomonas reinhardtii, promoting triacylglycerol accumulation

    DOE PAGES

    Gargouri, Mahmoud; Bates, Philip D.; Park, Jeong-Jin; ...

    2017-04-13

    Nutrient deprivation causes significant stress to the unicellular microalga, Chlamydomonas reinhardtii, which responds by significantly altering its metabolic program. In following N deprivation, the accumulation of starch and triacylglycerols (TAGs) is significantly altered following massive reprogramming of cellular metabolism. One protein that was found to change dramatically and early to this stress was TAB2, a photosystem I (PSI) translation initiation factor, whose transcript and protein levels increased significantly after only 30 min of N deprivation. A detailed physiological and omics-based analysis of an insertional mutant of Chlamydomonas with reduced TAB2 function was conducted to determine what role the functional PSImore » plays in regulating the cellular response to N deprivation. The tab2 mutant displayed increased acetate assimilation and elevated starch levels during the first 6 h of N deprivation, followed by a shift toward altered amino acid synthesis, reduced TAG content and altered fatty acid profiles. Our results suggested a central role for PSI in controlling cellular metabolism and its implication in regulation of lipid/starch partitioning. Time course analyses of the tab2 mutant versus wild type under N-deprived versus N replete conditions revealed changes in the ATP/NADPH ratio and suggested that TAG biosynthesis may be associated with maintaining the redox state of the cell during N deprivation. The loss of ability to accumulate TAG in the tab2 mutant co-occurred with an up-regulation of photo-protective mechanisms, suggesting that the synthesis of TAG in the wild type occurs not only as a temporal energy sink, but also as a protective electron sink. By exploiting the tab2 mutation in the cells of C. reinhardtii cultured under autotrophic, mixotrophic, and heterotrophic conditions during nitrogen replete growth and for the first 8 days of nitrogen deprivation, we showed that TAG accumulation and lipid/starch partitioning are

  8. 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%.

  9. 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

  10. A Forward Genetic Approach in Chlamydomonas reinhardtii as a Strategy for Exploring Starch Catabolism

    PubMed Central

    Duchêne, Thierry; Cogez, Virginie; Cousin, Charlotte; Peltier, Gilles; Ball, Steven G.; Dauvillée, David

    2013-01-01

    A screen was recently developed to study the mobilization of starch in the unicellular green alga Chlamydomonas reinhardtii. This screen relies on starch synthesis accumulation during nitrogen starvation followed by the supply of nitrogen and the switch to darkness. Hence multiple regulatory networks including those of nutrient starvation, cell cycle control and light to dark transitions are likely to impact the recovery of mutant candidates. In this paper we monitor the specificity of this mutant screen by characterizing the nature of the genes disrupted in the selected mutants. We show that one third of the mutants consisted of strains mutated in genes previously reported to be of paramount importance in starch catabolism such as those encoding β-amylases, the maltose export protein, and branching enzyme I. The other mutants were defective for previously uncharacterized functions some of which are likely to define novel proteins affecting starch mobilization in green algae. PMID:24019981

  11. 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

  12. Evidence for thylakoid membrane fusion during zygote formation in Chlamydomonas reinhardtii

    PubMed Central

    1991-01-01

    To understand whether fusions of thylakoid membranes from the parental chloroplasts occurred during zygote formation in Chlamydomonas reinhardtii, we performed an ultrastructural analysis of the zygotes produced by crossing mutants lacking photosystem I or II protein complexes, in the absence of de novo chloroplast protein synthesis. Thylakoid membranes from each parent could be distinguished on thin sections due to their organization in "supergrana" in mutants lacking photosystem I centers, by freeze-fracturing due to the absence of most of the exoplasmic-face (EF) particles in mutants lacking photosystem II centers, by immunocytochemistry using antibodies directed against photosystem II subunits. We demonstrate that a fusion of the thylakoid membranes occurred during zygote formation approximately 15 h after mating. These fusions allowed a lateral redistribution of the thylakoid membrane proteins. These observations provide the structural basis for the restoration of photosynthetic electron flow in the mature zygote that we observed in fluorescence induction experiments. PMID:1874788

  13. 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

  14. [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.

  15. Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii.

    PubMed

    Lauersen, Kyle J; Baier, Thomas; Wichmann, Julian; Wördenweber, Robin; Mussgnug, Jan H; Hübner, Wolfgang; Huser, Thomas; Kruse, Olaf

    2016-11-01

    The heterologous expression of terpene synthases in microbial hosts has opened numerous possibilities for bioproduction of desirable metabolites. Photosynthetic microbial hosts present a sustainable alternative to traditional fermentative systems, using freely available (sun)light and carbon dioxide as inputs for bio-production. Here, we report the expression of a patchoulol synthase from Pogostemon cablin Benth in the model green microalga Chlamydomonas reinhardtii. The sesquiterpenoid patchoulol was produced from the alga and was used as a marker of sesquiterpenoid production capacity. A novel strategy for gene loading was employed and patchoulol was produced up to 922±242µgg(-1) CDW in six days. We additionally investigated the effect of carbon source on sesquiterpenoid productivity from C. reinhardtii in scale-up batch cultivations. It was determined that up to 1.03mgL(-1) sesquiterpenoid products could be produced in completely photoautotrophic conditions and that the alga exhibited altered sesquiterpenoid production metabolism related to carbon source.

  16. 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

  17. Ethanol stimulates phospholipid turnover and inositol 1,4,5-trisphosphate production in Chlamydomonas eugametos gametes.

    PubMed

    Musgrave, A; Kuin, H; Jongen, M; de Wildt, P; Schuring, F; Klerk, H; van den Ende, H

    1992-02-01

    Alcohols induce mating-structure activation in Chlamydomonas eugametos gametes. From the effect of ethanol on the (32)P-labelling of polyphosphoinositides, we conclude that the synthesis of these lipids is stimulated. Biologically inactive concentrations of ethanol (<6%) had no effect on synthesis, but 6-8% ethanol stimulated synthesis for upto 60 min. The (32)P incorporated into polyphosphoinositides and phosphatidic acid during ethanol treatment was readily chased out when 1 mM unlabelled Na3PO4 was added. Using a binding assay for inositol 1,4,5-trisphosphate, we show that the production of this phospholipid constituent is dramatically increased after ethanol treatment. This effect, coupled to a rise in intracellular calcium concentration, could explain gamete activation. The significance of these results in explaining other ethanol-induced phenomena in algae is discussed.

  18. [Effect of Methylmercury on the Light Dependence Fluorescence Parameters in a Green Alga Chlamydomonas moewusii].

    PubMed

    Protopopov, F F; Matorin, D N; Seifullina, N H; Bratkovskaya, L B; Zayadan, B K

    2015-01-01

    The effect of a dangerous toxic substance, methylmercury, on light dependence curves of chlorophyll fluorescence in Chlamydomonas moewusii was studied. We found low concentration of methylmercury (10(-7) M) to cause a decrease in the relative rate of the non-cyclic electron transport activity of PS 2, a decline in the maximum utilization of light energy (α), and a decline in the saturation light intensity (E(s)). Non-photochemical fluorescence quenching increased after short-term exposure and decreased in the course of prolonged incubation. These parameters were more sensitive to the action of the toxic substance than the widely used parameter F(V)/F(M), which reflects the maximum quantum yield of PS 2. We propose the use of the method of fast measurement of light dependence curves of fluorescence to detect the changes in algal cells at the early stages of exposure to mercury salts.

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

    DOE PAGES

    Hamaji, Takashi; Lopez, David; Pellegrini, Matteo; ...

    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

  20. 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.

  1. 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.

  2. Reduction-oxidation poise regulates the sign of phototaxis in Chlamydomonas reinhardtii.

    PubMed

    Wakabayashi, Ken-ichi; Misawa, Yuka; Mochiji, Shota; Kamiya, Ritsu

    2011-07-05

    In many phototrophic microorganisms and plants, chloroplasts change their positions relative to the incident light to achieve optimal photosynthesis. In the case of motile green algae, cells change their swimming direction by switching between positive and negative phototaxis, i.e., swimming toward or away from the light source, depending on environmental and internal conditions. However, little is known about the molecular signals that determine the phototactic direction. Using the green alga Chlamydomonas reinhardtii, we found that cellular reduction-oxidation (redox) poise plays a key role: Cells always exhibited positive phototaxis after treatment with reactive oxygen species (ROS) and always displayed negative phototaxis after treatment with ROS quenchers. The redox-dependent switching of the sign of phototaxis may contribute in turn to the maintenance of cellular redox homeostasis.

  3. Reduction-oxidation poise regulates the sign of phototaxis in Chlamydomonas reinhardtii

    PubMed Central

    Wakabayashi, Ken-ichi; Misawa, Yuka; Mochiji, Shota; Kamiya, Ritsu

    2011-01-01

    In many phototrophic microorganisms and plants, chloroplasts change their positions relative to the incident light to achieve optimal photosynthesis. In the case of motile green algae, cells change their swimming direction by switching between positive and negative phototaxis, i.e., swimming toward or away from the light source, depending on environmental and internal conditions. However, little is known about the molecular signals that determine the phototactic direction. Using the green alga Chlamydomonas reinhardtii, we found that cellular reduction-oxidation (redox) poise plays a key role: Cells always exhibited positive phototaxis after treatment with reactive oxygen species (ROS) and always displayed negative phototaxis after treatment with ROS quenchers. The redox-dependent switching of the sign of phototaxis may contribute in turn to the maintenance of cellular redox homeostasis. PMID:21690384

  4. Chlamydomonas swims with two "gears" in a eukaryotic version of run-and-tumble locomotion.

    PubMed

    Polin, Marco; Tuval, Idan; Drescher, Knut; Gollub, J P; Goldstein, Raymond E

    2009-07-24

    The coordination of eukaryotic flagella is essential for many of the most basic processes of life (motility, sensing, and development), yet its emergence and regulation and its connection to locomotion are poorly understood. Previous studies show that the unicellular alga Chlamydomonas, widely regarded as an ideal system in which to study flagellar biology, swims forward by the synchronous action of its two flagella. Using high-speed imaging over long intervals, we found a richer behavior: A cell swimming in the dark stochastically switches between synchronous and asynchronous flagellar beating. Three-dimensional tracking shows that these regimes lead, respectively, to nearly straight swimming and to abrupt large reorientations, which yield a eukaryotic version of the "run-and-tumble" motion of peritrichously flagellated bacteria.

  5. Antimycin A effect on the electron transport in chloroplasts of two Chlamydomonas reinhardtii strains.

    PubMed

    Antal, Taras K; Kukarskikh, Galina P; Bulychev, Alexander A; Tyystjärvi, Esa; Krendeleva, Tatyana

    2013-05-01

    The effects of antimycin A on the redox state of plastoquinone and on electron donation to photosystem I (PS I) were studied in sulfur-deprived Chlamydomonas reinhardtii cells of the strains cc406 and 137c. We found that this reagent suppresses cyclic electron flow around PS I in the cc406 strain, whereas this inhibitory effect was completely absent in the 137c strain. In the latter strain, antimycin A induced rapid reduction of plastoquinone in the dark and considerably enhanced the rate of electron donation to P700 (+) in the dark. Importantly, neither myxothiazol, an inhibitor of mitochondrial respiration, FCCP, a protonophore, nor propyl gallate, an inhibitor of the plastid terminal oxidase, induced such a strong effect like antimycin A. The results indicate that in the chloroplast of the 137c strain, antimycin A has a site of action outside of the machinery of cyclic electron flow.

  6. The chloroplast proteome: a survey from the Chlamydomonas reinhardtii perspective with a focus on distinctive features.

    PubMed

    Terashima, Mia; Specht, Michael; Hippler, Michael

    2011-06-01

    The unicellular green alga Chlamydomonas reinhardtii has emerged to be an important model organism for the study of oxygenic eukaryotic photosynthesis as well as other processes occurring in the chloroplast. However, the chloroplast proteome in C. reinhardtii has only recently been comprehensively characterized, made possible by proteomics emerging as an accessible and powerful tool over the last decade. In this review, we introduce a compiled list of 996 experimentally chloroplast-localized proteins for C. reinhardtii, stemming largely from our previous proteomic dataset comparing chloroplasts and mitochondria samples to localize proteins. In order to get a taste of some cellular functions taking place in the C. reinhardtii chloroplast, we will focus this review particularly on metabolic differences between chloroplasts of C. reinhardtii and higher plants. Areas that will be covered are photosynthesis, chlorophyll biosynthesis, carbon metabolism, fermentative metabolism, ferredoxins and ferredoxin-interacting proteins.

  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. Crystallization and preliminary X-ray characterization of full-length Chlamydomonas reinhardtii centrin

    PubMed Central

    Alfaro, Elisa; del Valle Sosa, Liliana; Sanoguet, Zuleika; Pastrana-Ríos, Belinda; Schreiter, Eric R.

    2008-01-01

    Chlamydomonas reinhardtii centrin is a member of the EF-hand calcium-binding superfamily. It is found in the basal body complex and is important for flagellar motility. Like other members of the EF-hand family, centrin interacts with and modulates the function of other proteins in a calcium-dependent manner. To understand how C. reinhardtii centrin interacts with its protein targets, it has been crystallized in the presence of the model peptide melittin and X-ray diffraction data have been collected to 2.2 Å resolution. The crystals are orthorhombic, with unit-cell parameters a = 52.1, b = 114.4, c = 34.8 Å, and are likely to belong to space group P21212. PMID:18453711

  9. The Effect of Gametogenesis Regimes on the Chloroplast Genetic System of CHLAMYDOMONAS REINHARDTII

    PubMed Central

    Sears, Barbara B.; Boynton, John E.; Gillham, Nicholas W.

    1980-01-01

    In Chlamydomonas reinhardtii, gamete differentiation is induced by nitrogen deprivation. While cellular nitrogen content and amount of chloroplast DNA in cells of both mating types are reduced during gametogenesis, the spontaneous transmission of paternal (mt-) chloroplast alleles in crosses is specifically affected by the stringency of the nitrogen starvation regime used for pregrowth and gametogenesis of the mt- parent. In all cases, reciprocal crosses yielded biparental zygospores whose clones contain predominantly cells expressing only the chloroplast alleles from the maternal (mt+) parent. No differences attributable to strain divergence were seen in chloroplast gene inheritance pattern, DNA content, or the relative frequency of transmission of paternal chloroplast alleles to progeny of biparental zygospores. PMID:17249065

  10. Molecular characterization of a zygote wall protein: an extensin-like molecule in Chlamydomonas reinhardtii.

    PubMed Central

    Woessner, J P; Goodenough, U W

    1989-01-01

    The green alga Chlamydomonas reinhardtii elaborates two biochemically and morphologically distinct cell walls during its life cycle: one surrounds the vegetative and gametic cell and the other encompasses the zygote. Hydroxyproline-rich glycoproteins (HRGPs) constitute a major component of both walls. We describe the isolation and characterization of a zygote-specific gene encoding a wall HRGP. The derived amino acid sequence of this algal HRGP is similar to those of higher plant extensins, rich in proline and serine residues and possessing repeating amino acid motifs, notably X(Pro)3 and (Ser-Pro)n. Antiserum against this zygote wall protein detected common epitopes in several other zygote polypeptides, at least one of which is also encoded by a zygote-specific gene. We conclude that there is one set of HRGP wall genes expressed only in zygotes and another set that is specific to vegetative and gametic cells. PMID:2535530

  11. Isolation and genetic analysis of Chlamydomonas reinhardtii strains resistant to cadmium

    SciTech Connect

    Collard, J.M.; Matagne, R.F. )

    1990-07-01

    In Chlamydomonas reinhardtii, cadmium induces reduction of growth, reduction of chlorophyll content, and lethality. The toxicity was higher in a cell wall-deficient strain than in the wild type. By growing the cells on agar medium containing cadmium at concentrations inducing high lethality, stable resistant clones were isolated. The resistance was due to a nuclear mutation (cadA{sup R}) which probably preexisted in the wild-type cell population, as suggested by the fluctuation test. A double mutant (cadA{sup R} cadB{sup R}) was selected on media containing higher concentrations of cadmium. The cadB{sup R} mutation, which is unlinked to cadA{sup R}, determines a resistance intermediate between the CadA{sup R} mutant and the wild-type strain. Both cadA{sup R} and cadB{sup R} mutations are partially dominant.

  12. Brownian Dynamics and Molecular Dynamics Study of the Association between Hydrogenase and Ferredoxin from Chlamydomonas reinhardtii

    PubMed Central

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

    2008-01-01

    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. Lipid droplet synthesis is limited by acetate availability in starchless mutant of Chlamydomonas reinhardtii.

    PubMed

    Ramanan, Rishiram; Kim, Byung-Hyuk; Cho, Dae-Hyun; Ko, So-Ra; Oh, Hee-Mock; Kim, Hee-Sik

    2013-02-14

    Phenotypic and genotypic changes in Chlamydomonas reinhardtii BafJ5, a starchless mutant, with respect to lipid metabolism was studied in different trophic states under nitrogen (N) sufficient and limited conditions. Interestingly, cellular lipid content increased linearly with input acetate concentration with highest lipid content (∼42%) under nitrogen limitation and mixotrophic state. RT-qPCR studies indicate that key fatty acid biosynthesis genes are down-regulated under N limitation but not under mixotrophic state, whereas, ACS2, encoding Acetyl-CoA synthetase, and DGTT4, encoding Diacylglycerol O-acyltransferase, are up-regulated under all conditions. These results collectively indicate that acetate is the limiting factor and central molecule in lipid droplet synthesis. The study also provides further evidence of the presence of a chloroplast pathway for triacylglycerol synthesis in microalgae.

  14. 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

  15. 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

  16. Cellulose degradation and assimilation by the unicellular phototrophic eukaryote Chlamydomonas reinhardtii.

    PubMed

    Blifernez-Klassen, Olga; Klassen, Viktor; Doebbe, Anja; Kersting, Klaudia; Grimm, Philipp; Wobbe, Lutz; Kruse, Olaf

    2012-01-01

    Plants convert sunlight to biomass, which is primarily composed of lignocellulose, the most abundant natural biopolymer and a potential feedstock for fuel and chemical production. Cellulose assimilation has so far only been described for heterotrophic organisms that rely on photosynthetically active primary producers of organic compounds. Among phototrophs, the unicellular green microalga Chlamydomonas reinhardtii is widely known as one of the best established model organisms. It occupies many habitats, including aquatic and soil ecosystems. This ubiquity underscores the versatile metabolic properties of this microorganism. Here we present yet another paradigm of adaptation for C. reinhardtii, highlighting its photoheterotrophic ability to utilize cellulose for growth in the absence of other carbon sources. When grown under CO(2)-limiting conditions in the light, secretion of endo-β-1,4-glucanases by the cell causes digestion of exogenous cellulose, followed by cellobiose uptake and assimilation. Phototrophic microbes like C. reinhardtii may thus serve as biocatalysts for cellulosic biofuel production.

  17. The Chlamydomonas reinhardtii Nar1 Gene Encodes a Chloroplast Membrane Protein Involved in Nitrite Transport

    PubMed Central

    Rexach, Jesus; Fernández, Emilio; Galván, Aurora

    2000-01-01

    A key step for nitrate assimilation in photosynthetic eukaryotes occurs within chloroplasts, where nitrite is reduced to ammonium, which is incorporated into carbon skeletons. The Nar1 gene from Chlamydomonas reinhardtii is clustered with five other genes for nitrate assimilation, all of them regulated by nitrate. Sequence analysis of genomic DNA and cDNA of Nar1 and comparative studies of strains having or lacking Nar1 have been performed. The deduced amino acid sequence indicates that Nar1 encodes a chloroplast membrane protein with substantial identity to putative formate and nitrite transporters in bacteria. Use of antibodies against NAR1 has corroborated its location in the plastidic membrane. Characterization of strains having or lacking this gene suggests that NAR1 is involved in nitrite transport in plastids, which is critical for cell survival under limiting nitrate conditions, and controls the amount of nitrate incorporated by the cells under limiting CO2 conditions. PMID:10948261

  18. Chlamydomonas reinhardtii cells adjust the metabolism to maintain viability in response to atrazine stress.

    PubMed

    Esperanza, Marta; Seoane, Marta; Rioboo, Carmen; Herrero, Concepción; Cid, Ángeles

    2015-08-01

    Chlamydomonas reinhardtii cells were exposed to a sublethal concentration of the widespread herbicide atrazine for 3 and 24h. Physiological parameters related to cellular energy status, such as cellular activity and mitochondrial and cytoplasmic membrane potentials, monitored by flow cytometry, were altered in microalgal cells exposed to 0.25μM of atrazine. Transcriptomic analyses, carried out by RNA-Seq technique, displayed 12 differentially expressed genes between control cultures and atrazine-exposed cultures at both tested times. Many cellular processes were affected, but the most significant changes were observed in genes implicated in amino acid catabolism and respiratory cellular process. Obtained results suggest that photosynthesis inhibition by atrazine leads cells to get energy through a heterotrophic metabolism to maintain their viability.

  19. IFT proteins accumulate during cell division and localize to the cleavage furrow in Chlamydomonas.

    PubMed

    Wood, Christopher R; Wang, Zhaohui; Diener, Dennis; Zones, James Matt; Rosenbaum, Joel; Umen, James G

    2012-01-01

    Intraflagellar transport (IFT) proteins are well established as conserved mediators of flagellum/cilium assembly and disassembly. However, data has begun to accumulate in support of IFT protein involvement in other processes elsewhere in the cell. Here, we used synchronous cultures of Chlamydomonas to investigate the temporal patterns of accumulation and localization of IFT proteins during the cell cycle. Their mRNAs showed periodic expression that peaked during S and M phase (S/M). Unlike most proteins that are synthesized continuously during G1 phase, IFT27 and IFT46 levels were found to increase only during S/M phase. During cell division, IFT27, IFT46, IFT72, and IFT139 re-localized from the flagella and basal bodies to the cleavage furrow. IFT27 was further shown to be associated with membrane vesicles in this region. This localization pattern suggests a role for IFT in cell division.

  20. 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.

  1. 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

    2016-05-11

    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.

  2. 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

  3. 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

  4. Evidence from Chlamydomonas on the photoactivation of rhodopsins without isomerization of their chromophore

    PubMed Central

    Foster, Kenneth W.; Saranak, Jureepan; Krane, Sonja; Johnson, Randy L.; Nakanishi, Koji

    2011-01-01

    SUMMARY Attachment of retinal to opsin forms the chromophore N-retinylidene which isomerizes during photoactivation of rhodopsins. To test whether isomerization is crucial, custom-tailored chromophores lacking the β-ionone ring and any isomerizable bonds were incorporated in vivo into the opsin of a blind mutant of the eukaryote Chlamydomonas reinhardtii. The analogues restored phototaxis with the anticipated action spectra, ruling out the need for isomerization in photoactivation. To further elucidate photoactivation, responses to chromophores formed from naphthalene aldehydes were studied. The resulting action spectral shifts suggest that charge separation within the excited chromophore leads to electric field induced polarization of nearby amino-acid residues and altered hydrogen bonding. This redistribution of charge faciliates the reported multiple bond rotations and protein rearrangements of rhodopsin activation. These results provide new insight into the activation of rhodopsins and related GPCRs. PMID:21700209

  5. Increased photosystem II stability promotes H2 production in sulfur-deprived Chlamydomonas reinhardtii

    PubMed Central

    Volgusheva, Alena; Styring, Stenbjörn; Mamedov, Fikret

    2013-01-01

    Photobiological H2 production is an attractive option for renewable solar fuels. Sulfur-deprived cells of Chlamydomonas reinhardtii have been shown to produce hydrogen with the highest efficiency among photobiological systems. We have investigated the photosynthetic reactions during sulfur deprivation and H2 production in the wild-type and state transition mutant 6 (Stm6) mutant of Chlamydomonas reinhardtii. The incubation period (130 h) was dissected into different phases, and changes in the amount and functional status of photosystem II (PSII) were investigated in vivo by electron paramagnetic resonance spectroscopy and variable fluorescence measurements. In the wild type it was found that the amount of PSII is decreased to 25% of the original level; the electron transport from PSII was completely blocked during the anaerobic phase preceding H2 formation. This block was released during the H2 production phase, indicating that the hydrogenase withdraws electrons from the plastoquinone pool. This partly removes the block in PSII electron transport, thereby permitting electron flow from water oxidation to hydrogenase. In the Stm6 mutant, which has higher respiration and H2 evolution than the wild type, PSII was analogously but much less affected. The addition of the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea revealed that ∼80% of the H2 production was inhibited in both strains. We conclude that (i) at least in the earlier stages, most of the electrons delivered to the hydrogenase originate from water oxidation by PSII, (ii) a faster onset of anaerobiosis preserves PSII from irreversible photoinhibition, and (iii) mutants with enhanced respiratory activity should be considered for better photobiological H2 production. PMID:23589846

  6. 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.

  7. Biochemical and morphological characterization of sulfur-deprived and H2-producing Chlamydomonas reinhardtii (green alga).

    PubMed

    Zhang, Liping; Happe, Thomas; Melis, Anastasios

    2002-02-01

    Sulfur deprivation in green algae causes reversible inhibition of photosynthetic activity. In the absence of S, rates of photosynthetic O2 evolution drop below those of O2 consumption by respiration. As a consequence, sealed cultures of the green alga Chlamydomonas reinhardtii become anaerobic in the light, induce the "Fe-hydrogenase" pathway of electron transport and photosynthetically produce H2 gas. In the course of such H2-gas production cells consume substantial amounts of internal starch and protein. Such catabolic reactions may sustain, directly or in directly, the H2-production process. Profile analysis of selected photosynthetic proteins showed a precipitous decline in the amount of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) as a function of time in S deprivation, a more gradual decline in the level of photosystem (PS) II and PSI proteins, and a change in the composition of the PSII light-harvesting complex (LHC-II). An increase in the level of the enzyme Fe-hydrogenase was noted during the initial stages of S deprivation (0-72 h) followed by a decline in the level of this enzyme during longer (t >72 h) S-deprivation times. Microscopic observations showed distinct morphological changes in C. reinhardtii during S deprivation and H2 production. Ellipsoid-shaped cells (normal photosynthesis) gave way to larger and spherical cell shapes in the initial stages of S deprivation and H2 production, followed by cell mass reductions after longer S-deprivation and H2-production times. It is suggested that, under S-deprivation conditions, electrons derived from a residual PSII H2O-oxidation activity feed into the hydrogenase pathway, thereby contributing to the H2-production process in Chlamydomonas reinhardtii. Interplay between oxygenic photosynthesis, mitochondrial respiration, catabolism of endogenous substrate, and electron transport via the hydrogenase pathway is essential for this light-mediated H2-production process.

  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. © 2011 The Authors

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

    PubMed

    Demurtas, Olivia C; Massa, Silvia; Ferrante, Paola; Venuti, Aldo; Franconi, Rosella; Giuliano, Giovanni

    2013-01-01

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

  10. A Chlamydomonas-Derived Human Papillomavirus 16 E7 Vaccine Induces Specific Tumor Protection

    PubMed Central

    Demurtas, Olivia C.; Massa, Silvia; Ferrante, Paola; Venuti, Aldo; Franconi, Rosella; Giuliano, Giovanni

    2013-01-01

    Background The E7 protein of the Human Papillomavirus (HPV) type 16, being involved in malignant cellular transformation, represents a key antigen for developing therapeutic vaccines against HPV-related lesions and cancers. Recombinant production of this vaccine antigen in an active form and in compliance with good manufacturing practices (GMP) plays a crucial role for developing effective vaccines. E7-based therapeutic vaccines produced in plants have been shown to be active in tumor regression and protection in pre-clinical models. However, some drawbacks of in whole-plant vaccine production encouraged us to explore the production of the E7-based therapeutic vaccine in Chlamydomonas reinhardtii, an organism easy to grow and transform and fully amenable to GMP guidelines. Methodology/Principal Findings An expression cassette encoding E7GGG, a mutated, attenuated form of the E7 oncoprotein, alone or as a fusion with affinity tags (His6 or FLAG), under the control of the C. reinhardtii chloroplast psbD 5′ UTR and the psbA 3′ UTR, was introduced into the C. reinhardtii chloroplast genome by homologous recombination. The protein was mostly soluble and reached 0.12% of total soluble proteins. Affinity purification was optimized and performed for both tagged forms. Induction of specific anti-E7 IgGs and E7-specific T-cell proliferation were detected in C57BL/6 mice vaccinated with total Chlamydomonas extract and with affinity-purified protein. High levels of tumor protection were achieved after challenge with a tumor cell line expressing the E7 protein. Conclusions The C. reinhardtii chloroplast is a suitable expression system for the production of the E7GGG protein, in a soluble, immunogenic form. The production in contained and sterile conditions highlights the potential of microalgae as alternative platforms for the production of vaccines for human uses. PMID:23626690

  11. Rapid detection and quantification of triacylglycerol by HPLC-ELSD in Chlamydomonas reinhardtii and Chlorella strains.

    PubMed

    Kobayashi, Naoko; Noel, Eric A; Barnes, Austin; Rosenberg, Julian; DiRusso, Concetta; Black, Paul; Oyler, George A

    2013-10-01

    Triacylglycerol (TAG) analysis and quantification are commonly performed by first obtaining a purified TAG fraction from a total neutral lipid extract using thin-layer chromatography (TLC), and then analyzing the fatty acid composition of the purified TAG fraction by gas chromatography (GC). This process is time-consuming, labor intensive and is not suitable for analysis of small sample sizes or large numbers. A rapid and efficient method for monitoring oil accumulation in algae using high performance liquid chromatography for separation of all lipid classes combined with detection by evaporative light scattering (HPLC-ELSD) was developed and compared to the conventional TLC/GC method. TAG accumulation in two Chlamydomonas reinhardtii (21 gr and CC503) and three Chlorella strains (UTEX 1230, CS01 and UTEX 2229) grown under conditions of nitrogen depletion was measured. The TAG levels were found to be 3-6 % DW (Chlamydomonas strains) and 7-12 % DW (Chlorella strains) respectively by both HPLC-ELSD and TLC/GC methods. HPLC-ELSD resolved the major lipid classes such as carotenoids, TAG, diacylglycerol (DAG), free fatty acids, phospholipids, and galactolipids in a 15-min run. Quantitation of TAG content was based on comparison to calibration curves of trihexadecanoin (16:0 TAG) and trioctadecadienoin (18:2 TAG) and showed linearity from 0.2 to 10 μg. Algal TAG levels >0.5 μg/g DW were detectable by this method. Furthermore TAG content in Chlorella kessleri UTEX 2229 could be detected. TAG as well as DAG and TAG content were estimated at 1.6 % DW by HPLC-ELSD, while it was undetectable by TLC/GC method.

  12. 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

  13. 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

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

    PubMed Central

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

    2011-01-01

    Summary 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

  15. 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.

  16. Actinobacillus rossii sp. nov., Actinobacillus seminis sp. nov., nom. rev., Pasteurella bettii sp. nov., Pasteurella lymphangitidis sp. nov., Pasteurella mairi sp. nov., and Pasteurella trehalosi sp. nov.

    PubMed

    Sneath, P H; Stevens, M

    1990-04-01

    Evidence from numerical taxonomic analysis and DNA-DNA hybridization supports the proposal of new species in the genera Actinobacillus and Pasteurella. The following new species are proposed: Actinobacillus rossii sp. nov., from the vaginas of postparturient sows; Actinobacillus seminis sp. nov., nom. rev., associated with epididymitis of sheep; Pasteurella bettii sp. nov., associated with human Bartholin gland abscess and finger infections; Pasteurella lymphangitidis sp. nov. (the BLG group), which causes bovine lymphangitis; Pasteurella mairi sp. nov., which causes abortion in sows; and Pasteurella trehalosi sp. nov., formerly biovar T of Pasteurella haemolytica, which causes septicemia in older lambs.

  17. 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.

  18. Defects in the ratio of the dynein isoform, DHC11 in the long-flagella mutants of Chlamydomonas reinhardtii.

    PubMed

    Sequeira, Marilyn P; Sinha, Sapna; Motiwalla, Mustafa J; Rao, Venkatramanan G; D'Souza, Jacinta S

    2017-01-22

    The long-flagella mutants (lf1, lf2, lf3 and lf4) of Chlamydomonas reinhardtii are defective in proteins that are required for the assembly of normal flagella, their phenotype being long flagella. In a previous study, we biophysically characterized these mutants for their waveform patterns, swimming speeds, beat frequencies and correlated these parameters with their flagellar lengths. We found an anomaly in this correlation and set out to explore the underlying molecular significance, if any. The diverse inner dynein isoforms are the flagellar motors that convert the chemical energy of ATP into the mechanical energy of motility; we probed the presence of one of these isoforms (DHC11, which might help in bend initiation) in the lf mutants and compared it with the wild-type. Our studies show that the ratio of DHC11 is defective in the long-flagella mutants of Chlamydomonas reinhardtii.

  19. Temperature effect on production of hydrogen and oxygen by Chlamydomonas cold strain CCMP1619 and wild-type 137c

    SciTech Connect

    Lee, J.W.; Blankinship, S.L.; Greenbaum, E.

    1995-12-31

    Photosynthetic water splitting for hydrogen and oxygen production is a promising biological process that converts sunlight into useful chemical energy. In green algae, this process becomes active when hydrogenase is induced. In this process, water is split into molecular oxygen, protons, and electrons by photosystem II (PSII). The electrons acquired from water splitting are transferred through PSII to photosystem I (PSI). At PSI, these electrons are further energized by the PSI photochemical reaction. The energized electrons emergent from the reducing side of PSI are transferred to hydrogenase via ferredoxin (Fd), and thereby utilized in a hydrogenase-catalyzed reaction, the reduction of protons and production of molecular hydrogen. The protons consumed in the reduction reaction are derived ultimately from water splitting. The net result of this process is cleavage of water to molecular hydrogen and oxygen. Hydrogenase is a key enzyme in the photoproduction of hydrogen. In multicellular algae and higher plants, this enzyme is lost or no longer inducible for photoproduction of hydrogen. This enzyme is, however, inducible for photoevolution of hydrogen in certain microscopic algae such as Chlamydomonas. However, not all species of Chlamydomonas have an inducible enzyme to produce hydrogen in the light. In the work described in this article, a Chlamydomonas cold strain, CCMP1619, was assayed for its potential hydrogenase activity by measuring anaerobically induced production of dark- and light-dependent hydrogen. This cold strain was originally isolated from Lake Bonney (ice-covered), Antarctica, and known to grow at low temperatures. The effect of temperature on hydrogen production by CCMP1619 was compared with the wild-type Chlamydomonas st rain 137c. The results indicated that 137c and CCMP1619 contain inducible hydrogenase, and that temperature had a significant effect on the rates of hydrogenase induction and on the kinetics of hydrogen production.

  20. Annotation of genes involved in glycerolipid biosynthesis in Chlamydomonas reinhardtii: discovery of the betaine lipid synthase BTA1Cr.

    PubMed

    Riekhof, Wayne R; Sears, Barbara B; Benning, Christoph

    2005-02-01

    Lipid metabolism in flowering plants has been intensely studied, and knowledge regarding the identities of genes encoding components of the major fatty acid and membrane lipid biosynthetic pathways is very extensive. We now present an in silico analysis of fatty acid and glycerolipid metabolism in an algal model, enabled by the recent availability of expressed sequence tag and genomic sequences of Chlamydomonas reinhardtii. Genes encoding proteins involved in membrane biogenesis were predicted on the basis of similarity to proteins with confirmed functions and were organized so as to reconstruct the major pathways of glycerolipid synthesis in Chlamydomonas. This analysis accounts for the majority of genes predicted to encode enzymes involved in anabolic reactions of membrane lipid biosynthesis and compares and contrasts these pathways in Chlamydomonas and flowering plants. As an important result of the bioinformatics analysis, we identified and isolated the C. reinhardtii BTA1 (BTA1Cr) gene and analyzed the bifunctional protein that it encodes; we predicted this protein to be sufficient for the synthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), a major membrane component in Chlamydomonas. Heterologous expression of BTA1Cr led to DGTS accumulation in Escherichia coli, which normally lacks this lipid, and allowed in vitro analysis of the enzymatic properties of BTA1Cr. In contrast, in the bacterium Rhodobacter sphaeroides, two separate proteins, BtaARs and BtaBRs, are required for the biosynthesis of DGTS. Site-directed mutagenesis of the active sites of the two domains of BTA1Cr allowed us to study their activities separately, demonstrating directly their functional homology to the bacterial orthologs BtaARs and BtaBRs.

  1. 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.

  2. 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.

  3. 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

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

    PubMed

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

    2015-03-01

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

  5. 3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process.

    PubMed Central

    Stern, D B; Kindle, K L

    1993-01-01

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

  6. 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

  7. 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

  8. Diversification of the Core RNA Interference Machinery in Chlamydomonas reinhardtii and the Role of DCL1 in Transposon Silencing

    PubMed Central

    Casas-Mollano, J. Armando; Rohr, Jennifer; Kim, Eun-Jeong; Balassa, Eniko; van Dijk, Karin; Cerutti, Heriberto

    2008-01-01

    Small RNA-guided gene silencing is an evolutionarily conserved process that operates by a variety of molecular mechanisms. In multicellular eukaryotes, the core components of RNA-mediated silencing have significantly expanded and diversified, resulting in partly distinct pathways for the epigenetic control of gene expression and genomic parasites. In contrast, many unicellular organisms with small nuclear genomes seem to have lost entirely the RNA-silencing machinery or have retained only a basic set of components. We report here that Chlamydomonas reinhardtii, a unicellular eukaryote with a relatively large nuclear genome, has undergone extensive duplication of Dicer and Argonaute polypeptides after the divergence of the green algae and land plant lineages. Chlamydomonas encodes three Dicers and three Argonautes with DICER-LIKE1 (DCL1) and ARGONAUTE1 being more divergent than the other paralogs. Interestingly, DCL1 is uniquely involved in the post-transcriptional silencing of retrotransposons such as TOC1. Moreover, on the basis of the subcellular distribution of TOC1 small RNAs and target transcripts, this pathway most likely operates in the nucleus. However, Chlamydomonas also relies on a DCL1-independent, transcriptional silencing mechanism(s) for the maintenance of transposon repression. Our results suggest that multiple, partly redundant epigenetic processes are involved in preventing transposon mobilization in this green alga. PMID:18493041

  9. 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

  10. Retinal Chromophore Structure and Schiff Base Interactions in Red-Shifted Channelrhodopsin-1 from Chlamydomonas augustae

    PubMed Central

    2015-01-01

    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

  11. Development of phytase-expressing chlamydomonas reinhardtii for monogastric animal nutrition.

    PubMed

    Erpel, Fernanda; Restovic, Franko; Arce-Johnson, Patricio

    2016-03-12

    In plant-derived animal feedstuffs, nearly 80 % of the total phosphorus content is stored as phytate. However, phytate is poorly digested by monogastric animals such as poultry, swine and fish, as they lack the hydrolytic enzyme phytase; hence it is regarded as a nutritionally inactive compound from a phosphate bioavailability point of view. In addition, it also chelates important dietary minerals and essential amino acids. Therefore, dietary supplementation with bioavailable phosphate and exogenous phytases are required to achieve optimal animal growth. In order to simplify the obtaining and application processes, we developed a phytase expressing cell-wall deficient Chlamydomonas reinhardtii strain. In this work, we developed a transgenic microalgae expressing a fungal phytase to be used as a food supplement for monogastric animals. A codon optimized Aspergillus niger PhyA E228K phytase (mE228K) with improved performance at pH 3.5 was transformed into the plastid genome of Chlamydomonas reinhardtii in order to achieve optimal expression. We engineered a plastid-specific construction harboring the mE228K gene, which allowed us to obtain high expression level lines with measurable in vitro phytase activity. Both wild-type and cell-wall deficient strains were selected, as the latter is a suitable model for animal digestion. The enzymatic activity of the mE228K expressing lines were approximately 5 phytase units per gram of dry biomass at pH 3.5 and 37 °C, similar to physiological conditions and economically competitive for use in commercial activities. A reference basis for the future biotechnological application of microalgae is provided in this work. A cell-wall deficient transgenic microalgae with phytase activity at gastrointestinal pH and temperature and suitable for pellet formation was developed. Moreover, the associated microalgae biomass costs of this strain would be between US$5 and US$60 per ton of feedstuff, similar to the US$2 per ton of feedstuffs

  12. 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.

  13. 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.

  14. The TOR Signaling Network in the Model Unicellular Green Alga Chlamydomonas reinhardtii

    PubMed Central

    Pérez-Pérez, María Esther; Crespo, José L.

    2017-01-01

    Cell growth is tightly coupled to nutrient availability. The target of rapamycin (TOR) kinase transmits nutritional and environmental cues to the cellular growth machinery. TOR functions in two distinct multiprotein complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). While the structure and functions of TORC1 are highly conserved in all eukaryotes, including algae and plants, TORC2 core proteins seem to be missing in photosynthetic organisms. TORC1 controls cell growth by promoting anabolic processes, including protein synthesis and ribosome biogenesis, and inhibiting catabolic processes such as autophagy. Recent studies identified rapamycin-sensitive TORC1 signaling regulating cell growth, autophagy, lipid metabolism, and central metabolic pathways in the model unicellular green alga Chlamydomonas reinhardtii. The central role that microalgae play in global biomass production, together with the high biotechnological potential of these organisms in biofuel production, has drawn attention to the study of proteins that regulate cell growth such as the TOR kinase. In this review we discuss the recent progress on TOR signaling in algae. PMID:28704927

  15. Sulfur availability and the SAC1 gene control adenosine triphosphate sulfurylase gene expression in Chlamydomonas reinhardtii.

    PubMed Central

    Yildiz, F H; Davies, J P; Grossman, A

    1996-01-01

    A Chlamydomonas reinhardtii adenosine triphosphate (ATP) sulfurylase cDNA clone (pATS1) was selected by complementing a mutation in the ATP sulfurylase gene (cysD) of Escherichia coli. E. coli cysD strains harboring pATS1 grow on medium containing sulfate as the sole sulfur source and exhibit ATP sulfurylase activity. The amino acid sequence of the C. reinhardtii ATP sulfurylase, derived from the nucleotide sequence of the complementing gene (ATS1), is 25 to 40% identical to that of ATP sulfurylases in other eukaryotic organisms and has a putative transit peptide at its amino terminus. ATP sulfurylase mRNA was present when cells were grown in sulfur-replete medium, but accumulated to higher levels when the cells were exposed to sulfur-limiting conditions. Furthermore, sulfur-stress-induced accumulation of the ATS1 transcript was reduced in a strain defective in SAC1, a gene that is critical for acclimation to sulfur-limited growth. PMID:8883379

  16. 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.

  17. Dynamic Changes in the Transcriptome and Methylome of Chlamydomonas reinhardtii throughout Its Life Cycle.

    PubMed

    Lopez, David; Hamaji, Takashi; Kropat, Janette; De Hoff, Peter; Morselli, Marco; Rubbi, Liudmilla; Fitz-Gibbon, Sorel; Gallaher, Sean D; Merchant, Sabeeha S; Umen, James; Pellegrini, Matteo

    2015-12-01

    The green alga Chlamydomonas reinhardtii undergoes gametogenesis and mating upon nitrogen starvation. While the steps involved in its sexual reproductive cycle have been extensively characterized, the genome-wide transcriptional and epigenetic changes underlying different life cycle stages have yet to be fully described. Here, we performed transcriptome and methylome sequencing to quantify expression and DNA methylation from vegetative and gametic cells of each mating type and from zygotes. We identified 361 gametic genes with mating type-specific expression patterns and 627 genes that are specifically induced in zygotes; furthermore, these sex-related gene sets were enriched for secretory pathway and alga-specific genes. We also examined the C. reinhardtii nuclear methylation map with base-level resolution at different life cycle stages. Despite having low global levels of nuclear methylation, we detected 23 hypermethylated loci in gene-poor, repeat-rich regions. We observed mating type-specific differences in chloroplast DNA methylation levels in plus versus minus mating type gametes followed by chloroplast DNA hypermethylation in zygotes. Lastly, we examined the expression of candidate DNA methyltransferases and found three, DMT1a, DMT1b, and DMT4, that are differentially expressed during the life cycle and are candidate DNA methylases. The expression and methylation data we present provide insight into cell type-specific transcriptional and epigenetic programs during key stages of the C. reinhardtii life cycle.

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

    PubMed

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

    2017-03-01

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

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

    PubMed Central

    Batyrova, Khorcheska; Hallenbeck, Patrick C.

    2017-01-01

    Chlamydomonas reinhardtii cy6Nac2.49 is a genetically modified algal strain that activates photosynthesis in a cyclical manner, so that photosynthesis is not active constitutively in the presence of oxygen, but is turned on only in response to a metabolic trigger (anaerobiosis). Here, we further investigated hydrogen production by this strain comparing it with the parental wild-type strain under photoheterotrophic conditions in regular tris-acetate-phosphate (TAP) medium with a 10-h:14-h light/dark regime. Unlike the wild-type, whose level of H2 production remained low during illumination, H2 production in the mutant strain increased gradually with each subsequent light period, and by the final light period was significantly higher than the wild-type. The relatively low Photosystem II (PSII) activity of the mutant culture was shown by low fluorescence yield both in the dark (Fv/Fm) and in the light (δF/Fm’) periods. Measurement of oxygen evolution confirmed the low photosynthetic activity of the mutant cells, which gradually accumulated O2 to a lesser extent than the wild-type, thus allowing the mutant strain to maintain hydrogenase activity over a longer time period and to gradually accumulate H2 during periods of illumination. Therefore, controllable expression of PSII can be used to increase hydrogen production under nutrient replete conditions, thus avoiding many of the limitations associated with nutrient deprivation approaches sometimes used to promote hydrogen production. PMID:28300765

  20. 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.

  1. 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.

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

    DOE PAGES

    Hong-Hermesdorf, Anne; Miethke, Marcus; Gallaher, Sean D.; ...

    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

  3. The URF 5 gene of Chlamydomonas reinhardtii mitochondria: DNA sequence and mode of transcription.

    PubMed

    Boer, P H; Gray, M W

    1986-01-01

    A gene homologous to unassigned reading frame (URF) 5 of the mammalian mitochondrial genome has been identified in the mitochondrial DNA of the unicellular green alga, Chlamydomonas reinhardtii. The algal URF 5 gene is closely flanked by the gene for subunit I of cytochrome oxidase (COI) and by an unidentified gene (ORF x). The URF 5 and ORF x genes are transcribed in the same direction, but opposite to that of the COI gene. Transcript analysis reveals a 1.9-kb mRNA whose major 5' terminus maps to the putative URF 5 initiation codon and whose 3' end abuts the 5' end of the ORF x transcript. Characterization of other C. reinhardtii mitochondrial RNAs suggests a general pattern of abutting transcripts and mature mRNAs having little or no 5' leader sequence. While this is reminiscent of post-transcriptional processing in animal mitochondria, different mechanisms must be employed in the two systems, since tRNA sequences (which appear to function as transcript processing signals in animal mitochondria) do not generally flank protein coding sequences in the C. reinhardtii mitochondrial genome. Nevertheless, characteristic secondary structure motifs do occur within the 3'-terminal regions of C. reinhardtii mitochondrial mRNAs, and their location close to mRNA termini suggests that such motifs may play a role in directing the precise endonucleolytic cleavage of long primary transcripts.

  4. The URF 5 gene of Chlamydomonas reinhardtii mitochondria: DNA sequence and mode of transcription.

    PubMed Central

    Boer, P H; Gray, M W

    1986-01-01

    A gene homologous to unassigned reading frame (URF) 5 of the mammalian mitochondrial genome has been identified in the mitochondrial DNA of the unicellular green alga, Chlamydomonas reinhardtii. The algal URF 5 gene is closely flanked by the gene for subunit I of cytochrome oxidase (COI) and by an unidentified gene (ORF x). The URF 5 and ORF x genes are transcribed in the same direction, but opposite to that of the COI gene. Transcript analysis reveals a 1.9-kb mRNA whose major 5' terminus maps to the putative URF 5 initiation codon and whose 3' end abuts the 5' end of the ORF x transcript. Characterization of other C. reinhardtii mitochondrial RNAs suggests a general pattern of abutting transcripts and mature mRNAs having little or no 5' leader sequence. While this is reminiscent of post-transcriptional processing in animal mitochondria, different mechanisms must be employed in the two systems, since tRNA sequences (which appear to function as transcript processing signals in animal mitochondria) do not generally flank protein coding sequences in the C. reinhardtii mitochondrial genome. Nevertheless, characteristic secondary structure motifs do occur within the 3'-terminal regions of C. reinhardtii mitochondrial mRNAs, and their location close to mRNA termini suggests that such motifs may play a role in directing the precise endonucleolytic cleavage of long primary transcripts. Images Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:3007117

  5. The nature of rhodopsin-triggered photocurrents in Chlamydomonas. II. Influence of monovalent ions.

    PubMed Central

    Nonnengässer, C; Holland, E M; Harz, H; Hegemann, P

    1996-01-01

    Chlamydomonas exhibits a sequence of a photoreceptor current and two flagellar currents upon stimulation with bright green flashes. The currents are thought to be a prerequisite for the well-known photophobic responses. In the preceding paper, we analyzed the kinetics of these currents and their dependence on extracellular divalent ions. Here, we show that the photoreceptor current can be carried by monovalent ions (K+ > NH4+ > Na+), provided that the driving force is high enough. The small residual photoreceptor current observed in the absence of Ca2+ is able to evoke flagellar currents at low extracellular pH. This demonstrates that signal transduction from the rhodopsin to the flagella is not inevitably dependent on extracellular Ca2+. Double-flash experiments exclude a contribution of intra-rhodopsin charge movements to the photoreceptor current signal. Evidence will be provided for the existence of nonlocalized K+ outward currents, which counterbalance the localized Ca2+ influx and repolarize the cell after a light flash. A model is presented that explains the different pathways for direction changes and phobic responses. PMID:8789110

  6. The nature of rhodopsin-triggered photocurrents in Chlamydomonas. II. Influence of monovalent ions.

    PubMed

    Nonnengässer, C; Holland, E M; Harz, H; Hegemann, P

    1996-02-01

    Chlamydomonas exhibits a sequence of a photoreceptor current and two flagellar currents upon stimulation with bright green flashes. The currents are thought to be a prerequisite for the well-known photophobic responses. In the preceding paper, we analyzed the kinetics of these currents and their dependence on extracellular divalent ions. Here, we show that the photoreceptor current can be carried by monovalent ions (K+ > NH4+ > Na+), provided that the driving force is high enough. The small residual photoreceptor current observed in the absence of Ca2+ is able to evoke flagellar currents at low extracellular pH. This demonstrates that signal transduction from the rhodopsin to the flagella is not inevitably dependent on extracellular Ca2+. Double-flash experiments exclude a contribution of intra-rhodopsin charge movements to the photoreceptor current signal. Evidence will be provided for the existence of nonlocalized K+ outward currents, which counterbalance the localized Ca2+ influx and repolarize the cell after a light flash. A model is presented that explains the different pathways for direction changes and phobic responses.

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

    PubMed

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

    2014-10-01

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

  8. The Intracellular Localization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Chlamydomonas reinhardtii1

    PubMed Central

    Borkhsenious, Olga N.; Mason, Catherine B.; Moroney, James V.

    1998-01-01

    The pyrenoid is a proteinaceous structure found in the chloroplast of most unicellular algae. Various studies indicate that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in the pyrenoid, although the fraction of Rubisco localized there remains controversial. Estimates of the amount of Rubisco in the pyrenoid of Chlamydomonas reinhardtii range from 5% to nearly 100%. Using immunolocalization, the amount of Rubisco localized to the pyrenoid or to the chloroplast stroma was estimated for C. reinhardtii cells grown under different conditions. It was observed that the amount of Rubisco in the pyrenoid varied with growth condition; about 40% was in the pyrenoid when the cells were grown under elevated CO2 and about 90% with ambient CO2. In addition, it is likely that pyrenoidal Rubisco is active in CO2 fixation because in vitro activity measurements showed that most of the Rubisco must be active to account for CO2-fixation rates observed in whole cells. These results are consistent with the idea that the pyrenoid is the site of CO2 fixation in C. reinhardtii and other unicellular algae containing CO2-concentrating mechanisms. PMID:9536077

  9. Membrane lipid biosynthesis in Chlamydomonas reinhardtii: expression and characterization of CTP:phosphoethanolamine cytidylyltransferase

    PubMed Central

    2004-01-01

    CTP:phosphoethanolamine cytidylyltransferase (ECT) is considered to be the regulatory enzyme in the CDP-ethanolamine pathway of phosphatidylethanolamine (PE) biosynthesis. The ECT cDNA of Chlamydomonas reinhardtii encodes a protein of 443 amino acid residues, which is longer than the same protein in yeast, rat or human. The translated product of cloned cDNA was expressed as a fusion protein in Escherichia coli, and was shown to have ECT activity. The deduced amino acid sequence has 41% identity with that of human or rat, and 30% with yeast. The ECT protein has a repetitive internal sequence in its N- and C-terminal halves and a signature peptide sequence, RTXGVSTT, typical of the cytidylyltransferase family. The first 70 amino acid residues do not match the N-terminal part of the cytidylyltransferases from other organisms, and we hypothesize that it is a subcellular targeting signal to mitochondria. ECT and organelle marker enzyme assays showed that the total activity of ECT correlates well with that of fumarase, a marker enzyme for mitochondria. Northern blots showed an increase in mRNA abundance during reflagellation, indicating a possibility of transcriptional regulation. A notable change in the enzyme activity in C. reinhardtii cells was observed during the cell cycle, increasing during the dark and then decreasing during the light period, while the mRNA level did not alter, providing evidence for post-translational regulation. PMID:15147238

  10. 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

  11. 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.

  12. The function of LHCBM4/6/8 antenna proteins in Chlamydomonas reinhardtii

    PubMed Central

    Girolomoni, Laura; Ferrante, Paola; Berteotti, Silvia; Giuliano, Giovanni; Ballottari, Matteo

    2017-01-01

    Abstract In eukaryotic autotrophs, photosystems are composed of a core moiety, hosting charge separation and electron transport reactions, and an antenna system, enhancing light harvesting and photoprotection. In Chlamydomonas reinhardtii, the major antenna of PSII is a heterogeneous trimeric complex made up of LHCBM1–LHCBM9 subunits. Despite high similarity, specific functions have been reported for several members including LHCBM1, 2, 7, and 9. In this work, we analyzed the function of LHCBM4 and LHCBM6 gene products in vitro by synthesizing recombinant apoproteins from individual sequences and refolding them with pigments. Additionally, we characterized knock-down strains in vivo for LHCBM4/6/8 genes. We show that LHCBM4/6/8 subunits could be found as a component of PSII supercomplexes with different sizes, although the largest pool was free in the membranes and poorly connected to PSII. Impaired accumulation of LHCBM4/6/8 caused a decreased LHCII content per PSII and a reduction in the amplitude of state 1–state 2 transitions. In addition, the reduction of LHCBM4/6/8 subunits caused a significant reduction of the Non-photochemical quenching activity and in the level of photoprotection. PMID:28007953

  13. Bistable retinal schiff base photodynamics of histidine kinase rhodopsin HKR1 from Chlamydomonas reinhardtii.

    PubMed

    Penzkofer, Alfons; Luck, Meike; Mathes, Tilo; Hegemann, Peter

    2014-01-01

    The photodynamics of the recombinant rhodopsin fragment of the histidine kinase rhodopsin HKR1 from Chlamydomonas reinhardtii was studied by absorption and fluorescence spectroscopy. The retinal cofactor of HKR1 exists in two Schiff base forms RetA and RetB. RetA is the deprotonated 13-cis-retinal Schiff base (RSB) absorbing in the UVA spectral region. RetB is the protonated all-trans RSB absorbing in the blue spectral region. Blue light exposure converts RetB fully to RetA. UVA light exposure converts RetA to RetB and RetB to RetA giving a mixture determined by their absorption cross sections and their conversion efficiencies. The quantum efficiencies of conversion of RetA to RetB and RetB to RetA were determined to be 0.096 ± 0.005 and 0.405 ± 0.01 respectively. In the dark thermal equilibration between RetA and RetB with dominant RetA content occurred with a time constant of about 3 days at room temperature. The fluorescence emission behavior of RetA and RetB was studied, and fluorescence quantum yields of ϕ(F) (RetA) = 0.00117 and ϕ(F) (RetB) = 9.4 × 10(-5) were determined. Reaction coordinate schemes of the photodynamics are developed. © 2014 The American Society of Photobiology.

  14. The two parallel photocycles of the Chlamydomonas sensory photoreceptor histidine kinase rhodopsin 1.

    PubMed

    Luck, Meike; Hegemann, Peter

    2017-10-01

    Histidine kinase rhodopsins (HKRs) belong to a class of unexplored sensory photoreceptors that share a similar modular architecture. The light sensing rhodopsin domain is covalently linked to signal-transducing modules and in some cases to a C-terminal guanylyl-cyclase effector. In spite of their wide distribution in unicellular organisms, very little is known about their physiological role and mechanistic functioning. We investigated the photochemical properties of the recombinant rhodopsin-fragment of Cr-HKR1 originating from Chlamydomonas reinhardtii. Our spectroscopic studies revealed an unusual thermal stability of the photoproducts with the deprotonated retinal Schiff base (RSB). Upon UV-irradiation these Rh-UV states with maximal absorbance in the UVA-region (Rh-UV) photochemically convert to stable blue light absorbing rhodopsin (Rh-Bl) with protonated chromophore. The heterogeneity of the sample is based on two parallel photocycles with the chromophore in C15=N-syn- or -anti-configuration. This report represents an attempt to decipher the underlying reaction schemes and interconversions of the two coexisting photocycles. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. 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

  16. Genome-wide characterization of genetic variation in the unicellular, green alga Chlamydomonas reinhardtii.

    PubMed

    Jang, Hyosik; Ehrenreich, Ian M

    2012-01-01

    Chlamydomonas reinhardtii is a model system for studying cilia, photosynthesis, and other core features of eukaryotes, and is also an emerging source of biofuels. Despite its importance to basic and applied biological research, the level and pattern of genetic variation in this haploid green alga has yet to be characterized on a genome-wide scale. To improve understanding of C. reinhardtii's genetic variability, we generated low coverage whole genome resequencing data for nearly all of the available isolates of this species, which were sampled from a number of sites in North America over the past ∼70 years. Based on the analysis of more than 62,000 single nucleotide polymorphisms, we identified two groups of isolates that represent geographical subpopulations of the species. We also found that measurements of genetic diversity were highly variable throughout the genome, in part due to technical factors. We studied the level and pattern of linkage disequilibrium (LD), and observed one chromosome that exhibits elevated LD. Furthermore, we detected widespread evidence of recombination across the genome, which implies that outcrossing occurs in natural populations of this species. In summary, our study provides multiple insights into the sequence diversity of C. reinhardtii that will be useful to future studies of natural genetic variation in this organism.

  17. The circadian clock of the unicellular eukaryotic model organism Chlamydomonas reinhardtii.

    PubMed

    Mittag, Maria; Wagner, Volker

    2003-05-01

    The green unicellular alga Chlamydomonas reinhardtii, also called 'green yeast', emerged in the past years as a model organism for specific scientific questions such as chloroplast biogenesis and function, the composition of the flagella including its basal apparatus, or the mechanism of the circadian clock. Sequencing of its chloroplast and mitochondrial genomes have already been completed and a first draft of its nuclear genome has also been released recently. In C. reinhardtii several circadian rhythms are physiologically well characterized, and one of them has even been shown to operate in outer space. Circadian expression patterns of nuclear and plastid genes have been studied. The mode of regulation of these genes occurs at the transcriptional level, although there is also evidence for posttranscriptional control. A clock-controlled, phylogenetically conserved RNA-binding protein was characterized in this alga, which interacts with several mRNAs that all contain a common cis-acting motif. Its function within the circadian system is currently under investigation. This review summarizes the current state of the knowledge about the circadian system in C. reinhardtii and points out its potential for future studies.

  18. Glutathionylation in the photosynthetic model organism Chlamydomonas reinhardtii: a proteomic survey.

    PubMed

    Zaffagnini, Mirko; Bedhomme, Mariette; Groni, Hayam; Marchand, Christophe H; Puppo, Carine; Gontero, Brigitte; Cassier-Chauvat, Corinne; Decottignies, Paulette; Lemaire, Stéphane D

    2012-02-01

    Protein glutathionylation is a redox post-translational modification occurring under oxidative stress conditions and playing a major role in cell regulation and signaling. This modification has been mainly studied in nonphotosynthetic organisms, whereas much less is known in photosynthetic organisms despite their important exposure to oxidative stress caused by changes in environmental conditions. We report a large scale proteomic analysis using biotinylated glutathione and streptavidin affinity chromatography that allowed identification of 225 glutathionylated proteins in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. Moreover, 56 sites of glutathionylation were also identified after peptide affinity purification and tandem mass spectrometry. The targets identified belong to a wide range of biological processes and pathways, among which the Calvin-Benson cycle appears to be a major target. The glutathionylation of four enzymes of this cycle, phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, ribose-5-phosphate isomerase, and phosphoglycerate kinase was confirmed by Western blot and activity measurements. The results suggest that glutathionylation could constitute a major mechanism of regulation of the Calvin-Benson cycle under oxidative stress conditions.

  19. 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

  20. Molecular and Structural Changes in Chlamydomonas under Limiting CO2 (A Possible Mitochondrial Role in Adaptation).

    PubMed Central

    Geraghty, A. M.; Spalding, M. H.

    1996-01-01

    When Chlamydomonas reinhardtii cells are transferred to limiting CO2, one response is the induction of a CO2-concentrating mechanism (CCM) with components that remain to be identified. Characterization of membrane-associated proteins induced by this transfer revealed that synthesis of the 21-kD protein (LIP-21) was regulated at the level of translatable message abundance and correlated well with the induction of CCM activity. Phase partitioning of LIP-21 and the previously characterized LIP-36 showed that both appeared to be peripherally associated with membranes, which limits their potential to function as transporters of inorganic carbon. Ultrastructural changes that occur when cells are transferred to limiting CO2 were also examined to help form a model for the CCM or other aspects of adaptation to limiting CO2. Changes were observed in vacuolization, starch distribution, and mitochondrial location. The mitochondria relocated from within the cup of the chloroplast to between the chloroplast envelope and the plasma membrane. In addition, immunogold labeling demonstrated that LIP-21 was localized specifically to the peripheral mitochondria. These data suggest that mitochondria, although not previously incorporated into models for the CCM, may play an important role in the cell's adaptation to limiting CO2. PMID:12226366

  1. Evolution of salt tolerance in a laboratory reared population of Chlamydomonas reinhardtii.

    PubMed

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

    2014-06-01

    Understanding the genetic underpinnings of adaptive traits in microalgae is important for the study of evolution and for applied uses. We used long-term selection under a regime of serial transfers with haploid populations of the green alga Chlamydomonas reinhardtii raised in liquid TAP medium containing 200 mM NaCl. After 1255 generations, evolved salt (ES) populations could grow as rapidly in high salt medium as progenitor cells (progenitor light [PL]). Transcriptome data were analysed to elucidate the basis of salt tolerance in ES cells when compared with PL cells and to cells incubated for 48 h in high salt medium (progenitor salt [PS], the short-term acclimation response). These data demonstrate that evolved and short-term acclimation responses to salt stress differ fundamentally from each other. Progenitor salt cells exhibit well-known responses to salt stress such as reduction in photosynthesis, upregulation of glycerophospholipid signaling, and upregulation of the transcription and translation machinery. In contrast, ES cells show downregulation of genes involved in the stress response and in transcription/translation. Our results suggest that gene-rich mixotrophic lineages such as C. reinhardtii may be able to adapt rapidly to abiotic stress engendered either by a rapidly changing climate or physical vicariance events that isolate populations in stressful environments.

  2. Ecotoxicological effects of perfluorooctanoic acid on freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus obliquus.

    PubMed

    Hu, Changwei; Luo, Qi; Huang, Qingguo

    2014-05-01

    As a persistent bioaccumulative compound, perfluorooctanoic acid (PFOA) is found in various ecosystems and receives growing attention. The acute toxicity of PFOA was tested on 2 freshwater microalgae, Chlamydomonas reinhardtii and Scenedesmus obliquus. The 96-h concentration for 50% of maximal effect (EC50) values were measured, physiological responses of the algae were investigated, and uptake of PFOA by the algae was quantified. The EC50 values for C. reinhardtii and S. obliquus were 51.9 ± 1.0 mg/L and 44.0 ± 1.5 mg/L PFOA, respectively. After 8-d exposure to PFOA ranging from 10 mg/L to 40 mg/L, the growth of C. reinhardtii was significantly inhibited, whereas that of S. obliquus was only slightly suppressed. Increases in malonaldehyde and proline levels were observed in the 2 algae when exposed to PFOA at certain concentrations, for instance, 20 mg/L and 40 mg/L, which is indicative of the trigger of a defensive mechanism. The percentage of PFOA that was adsorbed by the algae after 8-d exposure at a dosage between 5 mg/L and 20 mg/L ranged from 5.5% to 7.5%, and the uptake of PFOA by the algae exceeded 10%. © 2014 SETAC.

  3. High light induced changes in organization, protein profile and function of photosynthetic machinery in Chlamydomonas reinhardtii.

    PubMed

    Nama, Srilatha; Madireddi, Sai Kiran; Devadasu, Elsin Raju; Subramanyam, Rajagopal

    2015-11-01

    The green alga Chlamydomonas (C.) reinhardtii is used as a model organism to understand the efficiency of photosynthesis along with the organization and protein profile of photosynthetic apparatus under various intensities of high light exposure for 1h. Chlorophyll (Chl) a fluorescence induction, OJIPSMT transient was decreased with increase in light intensity indicating the reduction in photochemical efficiency. Further, circular dichroism studies of isolated thylakoids from high light exposed cells showed considerable change in the pigment-pigment interactions and pigment-proteins interactions. Furthermore, the organization of supercomplexes from thylakoids is studied, in which, one of the hetero-trimer of light harvesting complex (LHC) II is affected significantly in comparison to other complexes of LHC's monomers. Also, other supercomplexes, photosystem (PS)II reaction center dimer and PSI complexes are reduced. Additionally, immunoblot analysis of thylakoid proteins revealed that PSII core proteins D1 and D2 were significantly decreased during high light treatment. Similarly, the PSI core proteins PsaC, PsaD and PsaG were drastically changed. Further, the LHC antenna proteins of PSI and PSII were differentially affected. From our results it is clear that LHCs are damaged significantly, consequently the excitation energy is not efficiently transferred to the reaction center. Thus, the photochemical energy transfer from PSII to PSI is reduced. The inference of the study deciphers the structural and functional changes driven by light may therefore provide plants/alga to regulate the light harvesting capacity in excess light conditions.

  4. Plastid terminal oxidase 2 (PTOX2) is the major oxidase involved in chlororespiration in Chlamydomonas

    PubMed Central

    Houille-Vernes, Laura; Rappaport, Fabrice; Wollman, Francis-André; Alric, Jean; Johnson, Xenie

    2011-01-01

    By homology with the unique plastid terminal oxidase (PTOX) found in plants, two genes encoding oxidases have been found in the Chlamydomonas genome, PTOX1 and PTOX2. Here we report the identification of a knockout mutant of PTOX2. Its molecular and functional characterization demonstrates that it encodes the oxidase most predominantly involved in chlororespiration in this algal species. In this mutant, the plastoquinone pool is constitutively reduced under dark-aerobic conditions, resulting in the mobile light-harvesting complexes being mainly, but reversibly, associated with photosystem I. Accordingly, the ptox2 mutant shows lower fitness than wild type when grown under phototrophic conditions. Single and double mutants devoid of the cytochrome b6f complex and PTOX2 were used to measure the oxidation rates of plastoquinols via PTOX1 and PTOX2. Those lacking both the cytochrome b6f complex and PTOX2 were more sensitive to light than the single mutants lacking either the cytochrome b6f complex or PTOX2, which discloses the role of PTOX2 under extreme conditions where the plastoquinone pool is overreduced. A model for chlororespiration is proposed to relate the electron flow rate through these alternative pathways and the redox state of plastoquinones in the dark. This model suggests that, in green algae and plants, the redox poise results from the balanced accumulation of PTOX and NADPH dehydrogenase. PMID:22143777

  5. A chlorophyll-protein complex lacking in photosystem I mutants of Chlamydomonas reinhardtii

    PubMed Central

    Chua, N. H.; Matlin, K.; Bennoun, P.

    1975-01-01

    Sodium dodecyl sulfate gel electrophoresis of unheated, detergent-solubilized thylakoid membranes of Chlamydomonas reinhardtii gives two chlorophyll-protein complexes. Chlorophyll-protein complex I (CP I) is the blue-green in color and can be dissociated by heat into "free" chlorophyll and a constituent polypeptide (polypeptide 2; mol wt 66,000). Similar experiments with spinach and Chinese cabbage show that the higher plant CP I contains an equivalent polypeptide but of slightly lower molecular weight (64,000). Both polypeptide 2 and its counterpart in spinach are soluble in a 2:1 (vol/vol) mixture of chloroform-methanol. Chemical analysis reveals that C. reinhardtii CP I has a chlorophyll a to b weight ratio of about 5 and that it contains approximately 5% of the total chlorophyll and 8-9% of the total protein of the thylakoid membranes. Thus, it can be calculated that each constituent polypeptide chain is associated with eight to nine chlorophyll molecules. Attempts to measure the molecular weight of CP I by calibrated SDS gels were unsuccessul since the complex migrates anomalously in such gels. Two Mendelian mutants of C. reinhardtii, F1 and F14, which lack P700 but have normal photosystem I activity, do not contain CP I or the 66,000-dalton polypeptide in their thylakoid membranes. Our results suggest that CP I is essential for photosystem I reaction center activity and that P700 may be associated with the 66,000-dalton polypeptide. PMID:1194353

  6. Acetic acid-induced programmed cell death and release of volatile organic compounds in Chlamydomonas reinhardtii.

    PubMed

    Zuo, Zhaojiang; Zhu, Yerong; Bai, Yanling; Wang, Yong

    2012-02-01

    Acetic acid widely spreads in atmosphere, aquatic ecosystems containing residues and anoxic soil. It can inhibit aquatic plant germination and growth, and even cause programmed cell death (PCD) of yeast. In the present study, biochemical and physiological responses of the model unicellular green algae Chlamydomonas reinhardtii were examined after acetic acid stress. H(2)O(2) burst was found in C. reinhardtii after acetic acid stress at pH 5.0 for 10 min. The photosynthetic pigments were degraded, gross photosynthesis and respiration were disappeared gradually, and DNA fragmentation was also detected. Those results indicated that C. reinhardtii cells underwent a PCD but not a necrotic, accidental cell death event. It was noticed that C. reinhardtii cells in PCD released abundant volatile organic compounds (VOCs) upon acetic acid stress. Therefore, we analyzed the VOCs and tested their effects on other normal cells. The treatment of C. reinhardtii cultures with VOCs reduced the cell density and increased antioxidant enzyme activity. Therefore, a function of VOCs as infochemicals involved in cell-to-cell communication at the conditions of applied stress is suggested.

  7. Spontaneous mutation accumulation in multiple strains of the green alga, Chlamydomonas reinhardtii.

    PubMed

    Morgan, Andrew D; Ness, Rob W; Keightley, Peter D; Colegrave, Nick

    2014-09-01

    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.

  8. Two equilibration pools of chlorophylls in the Photosystem I core antenna of Chlamydomonas reinhardtii.

    PubMed

    Gibasiewicz, Krzysztof; Ramesh, V M; Lin, Su; Redding, Kevin; Woodbury, Neal W; Webber, Andrew N

    2007-04-01

    Femtosecond transient absorption spectroscopy was applied for a comparative study of excitation decay in several different Photosystem I (PSI) core preparations from the green alga Chlamydomonas reinhardtii. For PSI cores with a fully interconnected network of chlorophylls, the excitation energy was equilibrated over a pool of chlorophylls absorbing at approximately 683 nm, independent of excitation wavelength [Gibasiewicz et al. J Phys Chem B 105:11498-11506, 2001; J Phys Chem B 106:6322-6330, 2002]. In preparations with impaired connectivity between chlorophylls, we have found that the spectrum of chlorophylls connected to the reaction center (i.e., with approximately 20 ps decay time) over which the excitation is equilibrated becomes excitation-wavelength-dependent. Excitation at 670 nm is finally equilibrated over chlorophylls absorbing at approximately 675 nm, whereas excitation at 695 nm or 700 nm is equilibrated over chlorophylls absorbing at approximately 683 nm. This indicates that in the vicinity of the reaction center there are two spectrally different and spatially separated pools of chlorophylls that are equally capable of effective excitation energy transfer to the reaction center. We propose that they are related to the two groups of central PSI core chlorophylls lying on the opposite sides of reaction center.

  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.

  10. Ciprofloxacin toxicity and its co-metabolic removal by a freshwater microalga Chlamydomonas mexicana.

    PubMed

    Xiong, Jiu-Qiang; Kurade, Mayur B; Kim, Jung Rae; Roh, Hyun-Seog; Jeon, Byong-Hun

    2017-02-05

    This study evaluated the toxicity and cellular stresses of ciprofloxacin (CIP) and its co-metabolic removal in a freshwater microalga Chlamydomonas mexicana. The toxicological effects of CIP on C. mexicana were assessed by studying the growth and biochemical characteristics of the microalga including total chlorophyll, carotenoid content, malondialdehyde (MDA) and superoxide dismutase (SOD) activity. The calculated effective concentration (EC50) of CIP on C. mexicana was 65±4mgL(-1) at 96h. The growth of C. mexicana was significantly inhibited at increased concentrations of CIP, showing 36±1, 75±3. and 88±3% inhibition at 40, 60 and 100mgL(-1) CIP, respectively, compared to the control after 11days of cultivation. The total chlorophyll, carotenoid, MDA and SOD activity were significantly increased as a result of relatively high concentrations of CIP stress. C. mexicana showed 13±1% removal of CIP (2mgL(-1)) after 11days of cultivation; however, the addition of an electron donor (sodium acetate, 4gL(-1)) highly enhanced the removal of CIP (2mgL(-1)) by>3-fold after 11days. Kinetic studies showed that removal of CIP followed a first-order model (R(2) 0.94-0.97) with the apparent rate constants (k) ranging from 0.0121 to 0.079 d(-1).

  11. Toxicity of atrazine and its bioaccumulation and biodegradation in a green microalga, Chlamydomonas mexicana.

    PubMed

    Kabra, Akhil N; Ji, Min-Kyu; Choi, Jaewon; Kim, Jung Rae; Govindwar, Sanjay P; Jeon, Byong-Hun

    2014-11-01

    This study evaluated the toxicity of herbicide atrazine, along with its bioaccumulation and biodegradation in the green microalga Chlamydomonas mexicana. At low concentration (10 μg L(-1)), atrazine had no profound effect on the microalga, while higher concentrations (25, 50, and 100 μg L(-1)) imposed toxicity, leading to inhibition of cell growth and chlorophyll a accumulation by 22 %, 33 %, and 36 %, and 13 %, 24 %, and 27 %, respectively. Atrazine 96-h EC50 for C. mexicana was estimated to be 33 μg L(-1). Microalga showed a capability to accumulate atrazine in the cell and to biodegrade the cell-accumulated atrazine resulting in 14-36 % atrazine degradation at 10-100 μg L(-1). Increasing atrazine concentration decreased the total fatty acids (from 102 to 75 mg g(-1)) and increased the unsaturated fatty acid content in the microalga. Carbohydrate content increased gradually with the increase in atrazine concentration up to 15 %. This study shows that C. mexicana has the capability to degrade atrazine and can be employed for the remediation of atrazine-contaminated streams.

  12. Impaired Mitochondrial Transcription Termination Disrupts the Stromal Redox Poise in Chlamydomonas1[OPEN

    PubMed Central

    Uhmeyer, Andreas

    2017-01-01

    In photosynthetic eukaryotes, the metabolite exchange between chloroplast and mitochondria ensures efficient photosynthesis under saturating light conditions. The Chlamydomonas reinhardtii mutant stm6 is devoid of the mitochondrial transcription termination factor MOC1 and aberrantly expresses the mitochondrial genome, resulting in enhanced photosynthetic hydrogen production and diminished light tolerance. We analyzed the modulation of mitochondrial and chlororespiration during the acclimation of stm6 and the MOC1-complemented strain to excess light. Although light stress stimulated mitochondrial respiration via the energy-conserving cytochrome c pathway in both strains, the mutant was unable to fine-tune the expression and activity of oxidative phosphorylation complex I in excess light, which was accompanied by an increased mitochondrial respiration via the alternative oxidase pathway. Furthermore, stm6 failed to fully activate chlororespiration and cyclic electron flow due to a more oxidized state of the chloroplast stroma, which is caused by an increased mitochondrial electron sink capacity. Increased susceptibility to photoinhibition of PSII in stm6 demonstrates that the MOC1-dependent modulation of mitochondrial respiration helps control the stromal redox poise as a crucial part of high-light acclimation in C. reinhardtii. PMID:28500267

  13. Cytoplasmic microtubules containing acetylated alpha-tubulin in Chlamydomonas reinhardtii: spatial arrangement and properties

    PubMed Central

    1986-01-01

    A monoclonal antibody, 6-11B-1, specific for acetylated alpha-tubulin (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) was used to study the distribution of this molecule in interphase cells of Chlamydomonas reinhardtii. Double-label immunofluorescence was performed using 6-11B-1, and 3A5, an antibody specific for all alpha- tubulin isoforms. It was found that acetylated alpha-tubulin is not restricted to the axonemes, but is also present in basal bodies and in a subset of cytoplasmic microtubules that radiate from the basal bodies just beneath the plasma membrane. Immunoblotting experiments of basal body polypeptide components using 6-11B-1 as a probe confirmed that basal bodies contain acetylated alpha-tubulin. In the cell body, 6-11B- 1 stained an average of 2.2 microtubules/cell, while 3A5 stained an average of 6.5 microtubules. Although exposure to 0 degrees C depolymerized both types of cytoplasmic microtubules, exposure to various concentrations of colchicine or nocodazole showed that the acetylated microtubules are much more resistant to drug-induced depolymerization than nonacetylated microtubules. Axonemes and basal bodies are already known to be colchicine-resistant. All acetylated microtubules appear, therefore, to be more drug-resistant than nonacetylated microtubules. The acetylation of alpha-tubulin may be part of a mechanism that stabilizes microtubules. PMID:3722261

  14. Cloning and characterization of d-threonine aldolase from the green alga Chlamydomonas reinhardtii.

    PubMed

    Hirato, Yuki; Tokuhisa, Mayumi; Tanigawa, Minoru; Ashida, Hiroyuki; Tanaka, Hiroyuki; Nishimura, Katsushi

    2017-03-01

    d-Threonine aldolase (DTA) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent interconversion of d-threonine and glycine plus acetaldehyde. The enzyme is a powerful tool for the stereospecific synthesis of various β-hydroxy amino acids in synthetic organic chemistry. In this study, DTA from the green alga Chlamydomonas reinhardtii was discovered and characterized, representing the first report to describe the existence of eukaryotic DTA. DTA was overexpressed in recombinant Escherichia coli BL21 (DE3) cells; the specific activity of the enzyme in the cell-free extract was 0.8 U/mg. The recombinant enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose, and Mono Q column chromatographies (purified enzyme 7.0 U/mg). For the cleavage reaction, the optimal temperature and pH were 70 °C and pH 8.4, respectively. The enzyme demonstrated 90% of residual activity at 50 °C for 1 h. The enzyme catalyzed the synthesis of d- and d-allo threonine from a mixture of glycine and acetaldehyde (the diastereomer excess of d-threonine was 18%). DTA was activated by several divalent metal ions, including manganese, and was inhibited by PLP enzyme inhibitors and metalloenzyme inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Carbon Supply and Photoacclimation Cross Talk in the Green Alga Chlamydomonas reinhardtii.

    PubMed

    Polukhina, Iryna; Fristedt, Rikard; Dinc, Emine; Cardol, Pierre; Croce, Roberta

    2016-11-01

    Photosynthetic organisms are exposed to drastic changes in light conditions, which can affect their photosynthetic efficiency and induce photodamage. To face these changes, they have developed a series of acclimation mechanisms. In this work, we have studied the acclimation strategies of Chlamydomonas reinhardtii, a model green alga that can grow using various carbon sources and is thus an excellent system in which to study photosynthesis. Like other photosynthetic algae, it has evolved inducible mechanisms to adapt to conditions where carbon supply is limiting. We have analyzed how the carbon availability influences the composition and organization of the photosynthetic apparatus and the capacity of the cells to acclimate to different light conditions. Using electron microscopy, biochemical, and fluorescence measurements, we show that differences in CO2 availability not only have a strong effect on the induction of the carbon-concentrating mechanisms but also change the acclimation strategy of the cells to light. For example, while cells in limiting CO2 maintain a large antenna even in high light and switch on energy-dissipative mechanisms, cells in high CO2 reduce the amount of pigments per cell and the antenna size. Our results show the high plasticity of the photosynthetic apparatus of C. reinhardtii This alga is able to use various photoacclimation strategies, and the choice of which to activate strongly depends on the carbon availability. © 2016 American Society of Plant Biologists. All Rights Reserved.

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

    PubMed

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

    2016-11-01

    Pharmaceuticals reach terrestrial environments through the application of treated wastewaters and biosolids to agricultural soils. We have investigated the toxicity of 15 common pharmaceuticals, classified as nonsteroidal anti-inflammatory drugs (NSAIDs), blood lipid-lowering agents, β-blockers and antibiotics, in two photosynthetic organisms. Twelve pharmaceuticals caused inhibitory effects on the radicle and hypocotyl elongation of Lactuca sativa seeds. The EC50 values obtained were in the range of 170-5656 mg L(-1) in the case of the radicle and 188-4558 mg L(-1) for the hypocotyl. Propranolol was the most toxic drug for both root and hypocotyl elongation, followed by the NSAIDs, then gemfibrozil and tetracycline. Other effects, such as root necrosis, inhibition of root growth and curly hairs, were detected. However, even at the highest concentrations tested (3000 mg L(-1)), seed germination was not affected. NSAIDs decreased the photosynthetic yield of Chlamydomonas reinhardtii, but only salicylic acid showed EC50 values below 1000 mg L(-1). The first effects detected at low concentrations, together with the concentrations found in environmental samples, indicate that the use of biosolids and wastewaters containing pharmaceuticals should be regulated and their compositions assessed in order to prevent medium- and long-term impacts on agricultural soils and crops.

  17. UV-B photoreceptor-mediated protection of the photosynthetic machinery in Chlamydomonas reinhardtii

    PubMed Central

    Allorent, Guillaume; Lefebvre-Legendre, Linnka; Chappuis, Richard; Kuntz, Marcel; Truong, Thuy B.; Niyogi, Krishna K.; Goldschmidt-Clermont, Michel

    2016-01-01

    Life on earth is dependent on the photosynthetic conversion of light energy into chemical energy. However, absorption of excess sunlight can damage the photosynthetic machinery and limit photosynthetic activity, thereby affecting growth and productivity. Photosynthetic light harvesting can be down-regulated by nonphotochemical quenching (NPQ). A major component of NPQ is qE (energy-dependent nonphotochemical quenching), which allows dissipation of light energy as heat. Photodamage peaks in the UV-B part of the spectrum, but whether and how UV-B induces qE are unknown. Plants are responsive to UV-B via the UVR8 photoreceptor. Here, we report in the green alga Chlamydomonas reinhardtii that UVR8 induces accumulation of specific members of the light-harvesting complex (LHC) superfamily that contribute to qE, in particular LHC Stress-Related 1 (LHCSR1) and Photosystem II Subunit S (PSBS). The capacity for qE is strongly induced by UV-B, although the patterns of qE-related proteins accumulating in response to UV-B or to high light are clearly different. The competence for qE induced by acclimation to UV-B markedly contributes to photoprotection upon subsequent exposure to high light. Our study reveals an anterograde link between photoreceptor-mediated signaling in the nucleocytosolic compartment and the photoprotective regulation of photosynthetic activity in the chloroplast. PMID:27930292

  18. Relationships between surface-bound and internalized copper and cadmium and toxicity in Chlamydomonas reinhardtii.

    PubMed

    Stoiber, Tasha L; Shafer, Martin M; Armstrong, David E

    2012-02-01

    In the present study, the adsorption and uptake of copper (Cu) and cadmium (Cd) in Chlamydomonas reinhardtii were examined to establish fundamental toxicity relationships to glutathione and cell-growth endpoints. Establishing these fundamental relationships of metal accumulation and toxicity metrics is necessary to subsequently implement an algal biotic ligand model. The glutathione response was similar to the response measured from growth endpoints for both internal and adsorbed Cu, indicating that glutathione may be a useful biomarker of toxicity. The glutathione response with Cd contrasted markedly with that observed with Cu and was therefore observed to be a metal-specific biomarker. The density of sites binding metals and the related stability constants for the algal cell surface were also determined. Short exposures to metals (2 h) were conducted, and we determined 6.0 × 10(-6) mol/g sites binding Cu and 2.0 × 10(-6) mol/g sites binding Cd and conditional stability constants as log K' = 7.2 and log K' = 6.7 for Cu and Cd, respectively. Experiments were also conducted to determine the effect on toxicity endpoints of varying nitrate concentrations and different humic acids (HA) in the exposure media. Varying nitrate concentrations did not have an effect on cell growth over 24 h. The surface-adsorbed Cu measurements from the experiments with HA depended on the type and concentration of HA. Copyright © 2011 SETAC.

  19. Crystallization and preliminary X-ray characterization of full-length Chlamydomonas reinhardtii centrin

    SciTech Connect

    Alfaro, Elisa; Valle Sosa, Liliana del; Sanoguet, Zuleika; Pastrana-Ríos, Belinda; Schreiter, Eric R.

    2008-05-01

    C. reinhardtii centrin, an EF-hand calcium-binding protein localized to the microtubule-organizing center of eukaryotic organisms, has been crystallized in the presence of the model peptide melittin. X-ray diffraction data were collected to 2.2 Å resolution. Chlamydomonas reinhardtii centrin is a member of the EF-hand calcium-binding superfamily. It is found in the basal body complex and is important for flagellar motility. Like other members of the EF-hand family, centrin interacts with and modulates the function of other proteins in a calcium-dependent manner. To understand how C. reinhardtii centrin interacts with its protein targets, it has been crystallized in the presence of the model peptide melittin and X-ray diffraction data have been collected to 2.2 Å resolution. The crystals are orthorhombic, with unit-cell parameters a = 52.1, b = 114.4, c = 34.8 Å, and are likely to belong to space group P2{sub 1}2{sub 1}2.

  20. A Spectroscopic Analysis of a High Fluorescent Mutant of Chlamydomonas Reinhardi

    PubMed Central

    Epel, B. L.; Butler, W. L.

    1972-01-01

    Chloroplast fragments of a high fluorescent mutant of Chlamydomonas reinhardi, hfd 91, were compared against those of Acl+, a low chlorophyll variant of the wild type. The chloroplast fragments of the mutant which have a high invariant fluorescence yield lacked photochemical activities associated with photosystem II (PSII) but retained normal photosystem I (PSI) activities. The mutant fragments also lacked the low temperature (-196°C) light-induced absorbance changes due to the photoreduction of C-550 and the photooxidation of cytochrome (cyt) b-559 which are PSII-mediated reactions. A fourth-derivative analysis of the absolute spectra of the chloroplast fragments at different stages of reduction (obtained with ferricyanide, ascorbate, and dithionite) showed both the oxidized and reduced forms of C-550 and the reduced forms of cyt c-553, b-559, and b-564 in wild-type fragments. The mutant fragments lacked C-550 and an ascorbate-reducible cyt b-559 but contained cyt c-553, a dithionite-reducible cyt b-559, and cyt b-564. PMID:5037344

  1. Fitness effects of new mutations in Chlamydomonas reinhardtii across two stress gradients.

    PubMed

    Kraemer, S A; Morgan, A D; Ness, R W; Keightley, P D; Colegrave, N

    2016-03-01

    Most spontaneous mutations affecting fitness are likely to be deleterious, but the strength of selection acting on them might be impacted by environmental stress. Such stress-dependent selection could expose hidden genetic variation, which in turn might increase the adaptive potential of stressed populations. On the other hand, this variation might represent a genetic load and thus lead to population extinction under stress. Previous studies to determine the link between stress and mutational effects on fitness, however, have produced inconsistent results. Here, we determined the net change in fitness in 29 genotypes of the green algae Chlamydomonas reinhardtii that accumulated mutations in the near absence of selection for approximately 1000 generations across two stress gradients, increasing NaCl and decreasing phosphate. We found mutational effects to be magnified under extremely stressful conditions, but such effects were specific both to the type of stress and to the genetic background. The detection of stress-dependent fitness effects of mutations depended on accurately scaling relative fitness measures by generation times, thus offering an explanation for the inconsistencies among previous studies.

  2. 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

  3. 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

  4. Anomalies in the motion dynamics of long-flagella mutants of Chlamydomonas reinhardtii.

    PubMed

    Khona, Dolly K; Rao, Venkatramanan G; Motiwalla, Mustafa J; Varma, P C Sreekrishna; Kashyap, Anisha R; Das, Koyel; Shirolikar, Seema M; Borde, Lalit; Dharmadhikari, Jayashree A; Dharmadhikari, Aditya K; Mukhopadhyay, Siuli; Mathur, Deepak; D'Souza, Jacinta S

    2013-01-01

    Chlamydomonas reinhardtii has long been used as a model organism in studies of cell motility and flagellar dynamics. The motility of the well-conserved '9+2' axoneme in its flagella remains a subject of immense curiosity. Using high-speed videography and morphological analyses, we have characterized long-flagella mutants (lf1, lf2-1, lf2-5, lf3-2, and lf4) of C. reinhardtii for biophysical parameters such as swimming velocities, waveforms, beat frequencies, and swimming trajectories. These mutants are aberrant in proteins involved in the regulation of flagellar length and bring about a phenotypic increase in this length. Our results reveal that the flagellar beat frequency and swimming velocity are negatively correlated with the length of the flagella. When compared to the wild-type, any increase in the flagellar length reduces both the swimming velocities (by 26-57%) and beat frequencies (by 8-16%). We demonstrate that with no apparent aberrations/ultrastructural deformities in the mutant axonemes, it is this increased length that has a critical role to play in the motion dynamics of C. reinhardtii cells, and, provided there are no significant changes in their flagellar proteome, any increase in this length compromises the swimming velocity either by reduction of the beat frequency or by an alteration in the waveform of the flagella.

  5. 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.

  6. The conserved ciliary protein Bug22 controls planar beating of Chlamydomonas flagella.

    PubMed

    Meng, Dan; Cao, Muqing; Oda, Toshiyuki; Pan, Junmin

    2014-01-15

    Eukaryotic flagella and cilia can exhibit planar and non-planar beating, and the mechanism controlling these beating patterns is not well understood. Chlamydomonas reinhardtii flagella beat in approximately the same plane with either an asymmetric ciliary-type or symmetric flagellar-type waveform. Each B-tubule of the number 1, 5 and 6 doublets of the flagellar axoneme possesses a beak-like structure. The number 5 and 6 beak structures are implicated in conversion of ciliary motion into flagellar motion. Here, we show that in a null mutant of Bug22, the asymmetric ciliary waveform is converted into a three-dimensional (non-planar) symmetric flagellar waveform. Bug22 is localized to approximately the proximal half to two-thirds of the flagellum, similar to localization of beak-like structures. However, as shown by immunogold labeling, Bug22 associates with axonemal microtubules without apparent preference for any particular doublets. Interestingly, bug22 mutants lack all beak-like structures. We propose that one function of Bug22 is to regulate the anchoring of the beak-like structures to the doublet microtubules and confine flagellar beating to a plane.

  7. The awesome power of dikaryons for studying flagella and basal bodies in Chlamydomonas reinhardtii.

    PubMed

    Dutcher, Susan K

    2014-02-01

    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.

  8. CDKL5 regulates flagellar length and localizes to the base of the flagella in Chlamydomonas.

    PubMed

    Tam, Lai-Wa; Ranum, Paul T; Lefebvre, Paul A

    2013-03-01

    The length of Chlamydomonas flagella is tightly regulated. Mutations in four genes-LF1, LF2, LF3, and LF4-cause cells to assemble flagella up to three times wild-type length. LF2 and LF4 encode protein kinases. Here we describe a new gene, LF5, in which null mutations cause cells to assemble flagella of excess length. The LF5 gene encodes a protein kinase very similar in sequence to the protein kinase CDKL5. In humans, mutations in this kinase cause a severe form of juvenile epilepsy. The LF5 protein localizes to a unique location: the proximal 1 μm of the flagella. The proximal localization of the LF5 protein is lost when genes that make up the proteins in the cytoplasmic length regulatory complex (LRC)-LF1, LF2, and LF3-are mutated. In these mutants LF5p becomes localized either at the distal tip of the flagella or along the flagellar length, indicating that length regulation involves, at least in part, control of LF5p localization by the LRC.

  9. Characterization of the EYE2 gene required for eyespot assembly in Chlamydomonas reinhardtii.

    PubMed Central

    Roberts, D G; Lamb, M R; Dieckmann, C L

    2001-01-01

    The unicellular biflagellate green alga Chlamydomonas reinhardtii can perceive light and respond by altering its swimming behavior. The eyespot is a specialized structure for sensing light, which is assembled de novo at every cell division from components located in two different cellular compartments. Photoreceptors and associated signal transduction components are localized in a discrete patch of the plasma membrane. This patch is tightly packed against an underlying sandwich of chloroplast membranes and carotenoid-filled lipid granules, which aids the cell in distinguishing light direction. In a prior screen for mutant strains with eyespot defects, the EYE2 locus was defined by the single eye2-1 allele. The mutant strain has no eyespot by light microscopy and has no organized carotenoid granule layers as judged by electron microscopy. Here we demonstrate that the eye2-1 mutant is capable of responding to light, although the strain is far less sensitive than wild type to low light intensities and orients imprecisely. Therefore, pigment granule layer assembly in the chloroplast is not required for photoreceptor localization in the plasma membrane. A plasmid-insertion mutagenesis screen yielded the eye2-2 allele, which allowed the isolation and characterization of the EYE2 gene. The EYE2 protein is a member of the thioredoxin superfamily. Site-directed mutagenesis of the active site cysteines demonstrated that EYE2 function in eyespot assembly is redox independent, similar to the auxiliary functions of other thioredoxin family members in protein folding and complex assembly. PMID:11454753

  10. 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

  11. 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

  12. Production of biodiesel from microalgae Chlamydomonas polypyrenoideum grown on dairy industry wastewater.

    PubMed

    Kothari, Richa; Prasad, Ravindra; Kumar, Virendra; Singh, D P

    2013-09-01

    This study involves a process of phyco-remediation of dairy industry wastewater by algal strain Chlamydomonas polypyrenoideum. The results of selected algal strain indicated that dairy industry wastewater was good nutrient supplement for algal growth in comparable with BG-11 growth medium. Alga grown on dairy industry wastewater reduced the pollution load of nitrate (90%), nitrite (74%), phosphate (70%), chloride (61%), fluoride (58%), and ammonia (90%) on 10th day of its growth as compared to that of uninoculated wastewater. The lipid content of algal biomass grown on dairy wastewater on 10th day (1.6g) and 15th day (1.2 g) of batch experiment was found to be higher than the lipid content of algal biomass grown in BG-11 growth medium on 10th day (1.27 g) and 15th day (1.0 g) of batch experiment. The results on FTIR analysis of the extracted bio-oil through transesterification reaction was comparable with bio-oil obtained from other sources.

  13. Fitness change in relation to mutation number in spontaneous mutation accumulation lines of Chlamydomonas reinhardtii.

    PubMed

    Kraemer, Susanne; Böndel, Katharina B; Ness, Robert W; Keightley, Peter D; Colegrave, Nick

    2017-09-08

    Although all genetic variation ultimately stems from mutations, their properties are difficult to study directly. Here, we used multiple mutation accumulation (MA) lines derived from five genetic backgrounds of the green algae Chlamydomonas reinhardtii that have been previously subjected to whole genome sequencing to investigate the relationship between the number of spontaneous mutations and change in fitness from a non-evolved ancestor. MA lines were on average less fit than their ancestors and we detected a significantly negative correlation between the change in fitness and the total number of accumulated mutations in the genome. Likewise, the number of mutations located within coding regions significantly and negatively impacted MA line fitness. We used the fitness data to parameterize a maximum likelihood model to estimate discrete categories of mutational effects, and found that models containing one to two mutational effect categories (one neutral and one deleterious category) fitted the data best. However, the best-fitting mutational effects models were highly dependent on the genetic background of the ancestral strain. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  14. Purification, characterization, and complete amino acid sequence of a thioredoxin from a green alga, Chlamydomonas reinhardtii.

    PubMed

    Decottignies, P; Schmitter, J M; Jacquot, J P; Dutka, S; Picaud, A; Gadal, P

    1990-07-01

    Two thioredoxins (named Ch1 and Ch2 in reference to their elution pattern on an anion-exchange column) have been purified to homogeneity from the green alga, Chlamydomonas reinhardtii. In this paper, we described the properties and the sequence of the most abundant form, Ch2. Its activity in various enzymatic assays has been compared with those of Escherichia coli and spinach thioredoxins. C. reinhardtii thioredoxin Ch2 can serve as a substrate for E. coli thioredoxin reductase with a lower efficiency when compared to the homologous system. In the presence of dithiothreitol (DTT), the protein is able to catalyze the reduction of porcine insulin. Thioredoxin Ch2 is as efficient as its spinach counterpart in the DTT or light activation of corn NADP-malate dehydrogenase, but it only activates spinach fructose-1, 6-bisphosphatase at very high concentrations. The complete primary structure of the C. reinhardtii thioredoxin Ch2 was determined by automated Edman degradation of the intact protein and of peptides derived from trypsin, chymotrypsin, clostripain, and SV8 protease digestions. It consists of a polypeptide of 106 amino acids (MW 11,808) and contains the well-conserved active site sequence Trp-Cys-Gly-Pro-Cys. The sequence of the algal thioredoxin Ch2 has been compared to that of thioredoxins from other sources and has the greatest similarity (67%) with the thioredoxin from Anabaena 7119.

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

    SciTech Connect

    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.

    2015-05-28

    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. In conclusion, 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.

  16. Effects of chromium on photosynthetic and photoreceptive apparatus of the alga Chlamydomonas reinhardtii.

    PubMed

    Rodríguez, M Cecilia; Barsanti, Laura; Passarelli, Vincenzo; Evangelista, Valter; Conforti, Visitacion; Gualtieri, Paolo

    2007-10-01

    Chromium is a highly toxic non-essential metal for microorganisms and plants. Due to its widespread industrial use, chromium (Cr) has become a serious pollutant in diverse environmental settings. The presence of Cr leads to the selection of algal populations able to tolerate high levels of Cr compounds. The diverse Cr-resistance mechanisms displayed by microorganisms include biosorption, diminished accumulation, precipitation, reduction of Cr(6+) to Cr(3+), and chromate efflux. In this paper we describe the effects of Cr(6+) (the more toxic species) on the photosynthetic and photoreceptive apparatus of the fresh water unicellular alga Chlamydomonas reinhardtii. We measured the effect of the heavy metal by means of in vivo absorption microspectroscopy of both the thylakoid compartments and the eyespot. The decomposition of the overall absorption spectra in pigment constituents indicates that Cr(6+) induced a complete pheophinitization of the chrorophylls and a modification of the carotenoids present in the eyespot only when its concentration is equal or greater than 10 microM. Due to this low tolerance level, C. reinhardtii could be used as indicator of Cr pollution, but it is not feasible for bioremediation purposes.

  17. Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. IV. Purification and Properties of Plastocyanin.

    PubMed

    Gorman, D S; Levine, R P

    1966-12-01

    The copper protein plastocyanin has been found to be an essential component of the photosynthetic electron transport chain of Chlamydomonas reinhardi, and in this paper we describe a method for its isolation and purification from the wild-type strain. In addition, we describe some of its properties and compare them with those reported for spinach plastocyanin.The plastocyanin was extracted from acetone powders prepared from intact cells, and it was purified by ion exchange chromatography on DEAE cellulose and gel filtration on Sephadex G-75. The yield of the purified protein ranged from plastocyanin equivalent to 2.0 to 2.5 mug atoms copper per 1000 mumoles chlorophyll. In general the absorption spectrum of plastocyanin from C. reinhardi resembled that of the plastocyanin from spinach. Some spectral differences were found in the ultraviolet region where, in contrast to spinach plastocyanin, that of C. reinhardi had a greater absorbance (relative to peaks in the visible) and less evidence for phenylalanine fine structure. The normal oxidation-reduction potential of C. reinhardi plastocyanin was found to be + 0.37 volts, the same as reported for spinach plastocyanin. The molecular weight of C. reinhardi plastocyanin has been estimated to be 13,000 +/- 2000. In contrast, the value for spinach plastocyanin has been found to be 21,000.

  18. The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics

    PubMed Central

    Rasala, Beth A

    2011-01-01

    Microalgae are a diverse group of eukaryotic photosynthetic microorganisms. While microalgae play a crucial role in global carbon fixation and oxygen evolution, these organisms have recently gained much attention for their potential role in biotechnological and industrial applications, such as the production of biofuels. We investigated the potential of the microalga Chlamydomonas reinhardtii to be a platform for the production of human therapeutic proteins. C. reinhardtii is a unicellular freshwater green alga that has served as a popular model alga for physiological, molecular, biochemical and genetic studies. As such, the molecular toolkit for this microorganism is highly developed, including well-established methods for genetic transformation and recombinant gene expression. We transformed the chloroplast genome of C. reinhardtii with seven unrelated genes encoding for current or potential human therapeutic proteins and found that four of these genes supported protein accumulation to levels that are sufficient for commercial production. Furthermore, the algal-produced proteins were bioactive. Thus, the microalga C. reinhardtii has the potential to be a robust platform for human therapeutic protein production. PMID:21636988

  19. The TOR Signaling Network in the Model Unicellular Green Alga Chlamydomonas reinhardtii.

    PubMed

    Pérez-Pérez, María Esther; Couso, Inmaculada; Crespo, José L

    2017-07-12

    Cell growth is tightly coupled to nutrient availability. The target of rapamycin (TOR) kinase transmits nutritional and environmental cues to the cellular growth machinery. TOR functions in two distinct multiprotein complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). While the structure and functions of TORC1 are highly conserved in all eukaryotes, including algae and plants, TORC2 core proteins seem to be missing in photosynthetic organisms. TORC1 controls cell growth by promoting anabolic processes, including protein synthesis and ribosome biogenesis, and inhibiting catabolic processes such as autophagy. Recent studies identified rapamycin-sensitive TORC1 signaling regulating cell growth, autophagy, lipid metabolism, and central metabolic pathways in the model unicellular green alga Chlamydomonas reinhardtii. The central role that microalgae play in global biomass production, together with the high biotechnological potential of these organisms in biofuel production, has drawn attention to the study of proteins that regulate cell growth such as the TOR kinase. In this review we discuss the recent progress on TOR signaling in algae.

  20. Stickiness to Glass: Circadian Changes in the Cell Surface of Chlamydomonas reinhardi.

    PubMed

    Straley, S C; Bruce, V G

    1979-06-01

    Conditions were found in which Chlamydomonas reinhardi exhibits a circadian alteration of its cell surface, measured as ability to stick to glass. Under these same conditions the cells also show circadian rhythms of cell division and release of daughter cells. The three rhythmic phenomena were shown to have typical properties of rhythms controlled by the biological clock. The rhythm of stickiness was used to demonstrate that in a mixed culture containing two cell populations with natural periods differing by 2 to 3 hours, the cells did not mutally entrain each other and that this rhythm could be successfully applied in an enrichment procedure for mutants of the biological clock. Stickiness was shown to be independent of growth and motility of the cells and unaffected by red or far red illimination. Minimally sticking cells did not affect the sticking of maximally sticking cells in a mixed culture; nor was there a progressive increase in stickiness shown at the minimum from one cycle to the next in a pure culture. These results indicate that sticking probably is not mediated by long lived adhesive material or enzymes excreted into the medium. Several tests of the sensitivity of stickiness to replacement of the growth medium by distilled water or water containing various compounds suggest that ions might play an important role in the sticking reaction.

  1. Transcriptome for Photobiological Hydrogen Production Induced by Sulfur Deprivation in the Green Alga Chlamydomonas reinhardtii▿ †

    PubMed Central

    Nguyen, Anh Vu; Thomas-Hall, Skye R.; Malnoë, Alizée; Timmins, Matthew; Mussgnug, Jan H.; Rupprecht, Jens; Kruse, Olaf; Hankamer, Ben; Schenk, Peer M.

    2008-01-01

    Photobiological hydrogen production using microalgae is being developed into a promising clean fuel stream for the future. In this study, microarray analyses were used to obtain global expression profiles of mRNA abundance in the green alga Chlamydomonas reinhardtii at different time points before the onset and during the course of sulfur-depleted hydrogen production. These studies were followed by real-time quantitative reverse transcription-PCR and protein analyses. The present work provides new insights into photosynthesis, sulfur acquisition strategies, and carbon metabolism-related gene expression during sulfur-induced hydrogen production. A general trend toward repression of transcripts encoding photosynthetic genes was observed. In contrast to all other LHCBM genes, the abundance of the LHCBM9 transcript (encoding a major light-harvesting polypeptide) and its protein was strongly elevated throughout the experiment. This suggests a major remodeling of the photosystem II light-harvesting complex as well as an important function of LHCBM9 under sulfur starvation and photobiological hydrogen production. This paper presents the first global transcriptional analysis of C. reinhardtii before, during, and after photobiological hydrogen production under sulfur deprivation. PMID:18708561

  2. Method to assemble and integrate biochemical pathways into the chloroplast genome of Chlamydomonas reinhardtii.

    PubMed

    Noor-Mohammadi, Samaneh; Pourmir, Azadeh; Johannes, Tyler W

    2012-11-01

    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. 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 genome of the microalgal species Chlamydomonas 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. 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. This new method represents a useful genetic tool in the construction and integration of complex biochemical pathways into the chloroplast genome of microalgae and should aid current efforts to engineer algae for biofuels production and other desirable natural products.

  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. 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.

  5. The function of LHCBM4/6/8 antenna proteins in Chlamydomonas reinhardtii.

    PubMed

    Girolomoni, Laura; Ferrante, Paola; Berteotti, Silvia; Giuliano, Giovanni; Bassi, Roberto; Ballottari, Matteo

    2017-01-01

    In eukaryotic autotrophs, photosystems are composed of a core moiety, hosting charge separation and electron transport reactions, and an antenna system, enhancing light harvesting and photoprotection. In Chlamydomonas reinhardtii, the major antenna of PSII is a heterogeneous trimeric complex made up of LHCBM1-LHCBM9 subunits. Despite high similarity, specific functions have been reported for several members including LHCBM1, 2, 7, and 9. In this work, we analyzed the function of LHCBM4 and LHCBM6 gene products in vitro by synthesizing recombinant apoproteins from individual sequences and refolding them with pigments. Additionally, we characterized knock-down strains in vivo for LHCBM4/6/8 genes. We show that LHCBM4/6/8 subunits could be found as a component of PSII supercomplexes with different sizes, although the largest pool was free in the membranes and poorly connected to PSII. Impaired accumulation of LHCBM4/6/8 caused a decreased LHCII content per PSII and a reduction in the amplitude of state 1-state 2 transitions. In addition, the reduction of LHCBM4/6/8 subunits caused a significant reduction of the Non-photochemical quenching activity and in the level of photoprotection. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  6. Linear systems analysis of the ciliary steering behavior associated with negative-phototaxis in Chlamydomonas reinhardtii.

    PubMed

    Josef, Keith; Saranak, Jureepan; Foster, Kenneth W

    2006-12-01

    In response to light stimulation Chlamydomonas reinhardtii changes the beating frequency, beating pattern, and beating synchrony of the trans and cis cilia to steer the freely-swimming cell relative to light sources. To understand the cell steering behavior the impulse responses of the beating frequency and stroke velocity of each cilium have been obtained with high temporal resolution on cells held with a micropipette. Interestingly the response of each cilium is quite different. The trans cilium responds with less delay than the cis cilium for both beating frequency and stroke velocity. For light stimulation at 2 Hz, the critical cell-rotation frequency, both responses of the trans and cis cilia are about 180 degrees out of phase. The trans-cilium beating frequency response peaks at a stimulus frequency of 5-6 Hz, higher than the cis at 1-2 Hz. The stroke velocities of the trans and cis cilia have the same stimulus-frequency response (2 Hz), but the trans cilium has a shorter delay than the cis. The times to maximum response are much shorter than the time for a rotation of the cell. The use of two different approaches that enable the trans cilium to respond ahead of the cis for both the beating frequency and stroke velocity responses suggests the importance of both responses to phototaxis. Internal cell processing responsible for the time course of the responses is proposed. Copyright 2006 Wiley-Liss, Inc.

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

    PubMed Central

    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

    2016-01-01

    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. 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.

  9. Flow cytometric analysis to evaluate physiological alterations in herbicide-exposed Chlamydomonas moewusii cells.

    PubMed

    Prado, Raquel; Rioboo, Carmen; Herrero, Concepción; Suárez-Bregua, Paula; Cid, Angeles

    2012-03-01

    Investigation of herbicide toxicology in non-target aquatic primary producers such as microalgae is of great importance from an ecological point of view. In order to study the toxicity of the widely used herbicide paraquat on freshwater green microalga Chlamydomonas moewusii, physiological changes associated with 96 h-exposures to this pollutant were monitored using flow cytometry (FCM) technique. Intracellular reactive oxygen species concentration, cytoplasmic membrane potential, metabolic activity and cell protein content were monitored to evaluate the toxicological impact of paraquat on algal physiology. Results showed that herbicide paraquat induced oxidative stress in C. moewusii cells, as it indicated the increase of both superoxide anion and hydrogen peroxide levels observed in non-chlorotic cells of cultures exposed to increasing herbicide concentrations. Furthermore, a progressive increase in the percentage of depolarised cells and a decrease in the metabolic activity level were observed in response to paraquat when non-chlorotic cells were analysed. Chlorotic cells were probably non-viable cells, based on the cytoplasmic membrane depolarisation, its metabolically non-active state and its drastically reduced protein content. In view of the obtained results, we have concluded that a range of significant physiological alterations, detected by flow cytometry, occur when C. moewusii, an ubiquitous microalga in freshwater environments, is challenged with environmentally relevant concentrations of the herbicide paraquat.

  10. 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.

  11. Application of phosphoproteomics to find targets of casein kinase 1 in the flagellum of chlamydomonas.

    PubMed

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2012-01-01

    The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum.

  12. Application of Phosphoproteomics to Find Targets of Casein Kinase 1 in the Flagellum of Chlamydomonas

    PubMed Central

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2012-01-01

    The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum. PMID:23316220

  13. Proteomic Analysis of a Fraction with Intact Eyespots of Chlamydomonas reinhardtii and Assignment of Protein Methylation.

    PubMed

    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.

  14. 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

  15. 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.

  16. 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

  17. Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii 1

    PubMed Central

    Galván, Aurora; Cárdenas, Jacobo; Fernández, Emilio

    1992-01-01

    In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities. PMID:16668656

  18. 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.

  19. 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.

  20. 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.

  1. 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

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

    PubMed

    Batyrova, Khorcheska; Hallenbeck, Patrick C

    2017-03-16

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

  3. 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.

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

    PubMed Central

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

    2017-01-01

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

  5. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. The sac Mutants of Chlamydomonas reinhardtii Reveal Transcriptional and Posttranscriptional Control of Cysteine Biosynthesis1

    PubMed Central

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

    2002-01-01

    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

  7. 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

  8. 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.

  9. 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. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  10. Temperature-Sensitive, Photosynthesis-Deficient Mutants of Chlamydomonas reinhardtii1

    PubMed Central

    Spreitzer, Robert J.; Al-Abed, Souhail R.; Huether, Michael J.

    1988-01-01

    Mutants of the unicellular, green alga Chlamydomonas reinhardtii were recovered by screening for the absence of photoautotrophic growth at 35°C. Whereas nonconditional mutants required acetate for growth at both 25 and 35°C, the conditional mutants have normal photoautotrophic growth at 25°C. The conditional mutants consisted of two classes: (a) Temperature-sensitive mutants died under all growth conditions at 35°C, but (b) temperature-sensitive, acetate-requiring mutants were capable of heterotrophic growth at 35°C when supplied with acetate in the dark. The majority of mutants within the latter of these two classes had defects in photosynthetic functions. These defects included altered pigmentation, reduced whole-chain electron-transport activity, reduced ribulosebis-phosphate carboxylase activity, or pleiotropic alterations in a number of these photosynthetic components. Both nuclear and chloroplast mutants were identified, and a correlation between light-sensitive and photosynthesis-deficient phenotypes was observed. PMID:16665986

  11. A small multifunctional pentatricopeptide repeat protein in the chloroplast of Chlamydomonas reinhardtii.

    PubMed

    Jalal, Abdullah; Schwarz, Christian; Schmitz-Linneweber, Christian; Vallon, Olivier; Nickelsen, Jörg; Bohne, Alexandra-Viola

    2015-03-01

    Organellar biogenesis is mainly regulated by nucleus-encoded factors, which act on various steps of gene expression including RNA editing, processing, splicing, stabilization, and translation initiation. Among these regulatory factors, pentatricopeptide repeat (PPR) proteins form the largest family of RNA binding proteins, with hundreds of members in flowering plants. In striking contrast, the genome of the unicellular green alga Chlamydomonas reinhardtii encodes only 14 such proteins. In this study, we analyzed PPR7, the smallest and most highly expressed PPR protein in C. reinhardtii. Green fluorescent protein-based localization and gel-filtration analysis revealed that PPR7 forms a part of a high-molecular-weight ribonucleoprotein complex in the chloroplast stroma. RIP-chip analysis of PPR7-bound RNAs demonstrated that the protein associates with a diverse set of chloroplast transcripts in vivo, i.e. rrnS, psbH, rpoC2, rbcL, atpA, cemA-atpH, tscA, and atpI-psaJ. Furthermore, the investigation of PPR7 RNAi strains revealed that depletion of PPR7 results in a light-sensitive phenotype, accompanied by altered levels of its target RNAs that are compatible with the defects in their maturation or stabilization. PPR7 is thus an unusual type of small multifunctional PPR protein, which interacts, probably in conjunction with other RNA binding proteins, with numerous target RNAs to promote a variety of post-transcriptional events.

  12. Lead (Pb) and copper (Cu) share a common uptake transporter in the unicellular alga Chlamydomonas reinhardtii.

    PubMed

    Sánchez-Marín, Paula; Fortin, Claude; Campbell, Peter G C

    2014-02-01

    The unicellular alga Chlamydomonas reinhardtii has a very high rate of lead (Pb) internalization and is known to be highly sensitive to dissolved Pb. However, the transport pathway that this metal uses to cross cellular membranes in microalgae is still unknown. To identify the Pb(2+) transport pathway in C. reinhartdii, we performed several competition experiments with environmentally relevant concentrations of Pb(2+) (~10 nM) and a variety of divalent cations. Among the essential trace metals tested, cobalt, manganese, nickel and zinc had no effect on Pb internalization. A greater than tenfold increase in the concentrations of the major ions calcium and magnesium led to a slight decrease (~34 %) in short-term Pb internalization by the algae. Copper (Cu) was even more effective: at a Cu concentration 50 times higher than that of Pb, Pb internalization by the algae decreased by 87 %. Pre-exposure of the algae to Cu showed that the effect was not due to a physiological effect of Cu on the algae, but rather to competition for the same transporter. A reciprocal effect of Pb on Cu internalization was also observed. These results suggest that Cu and Pb share a common transport pathway in C. reinhardtii at environmentally relevant metal concentrations.

  13. Starchless Mutants of Chlamydomonas reinhardtii Lack the Small Subunit of a Heterotetrameric ADP-Glucose Pyrophosphorylase

    PubMed Central

    Zabawinski, Christophe; Van Den Koornhuyse, Nathalie; D'Hulst, Christophe; Schlichting, Ralf; Giersch, Christoph; Delrue, Brigitte; Lacroix, Jean-Marie; Preiss, Jack; Ball, Steven

    2001-01-01

    ADP-glucose synthesis through ADP-glucose pyrophosphorylase defines the major rate-controlling step of storage polysaccharide synthesis in both bacteria and plants. We have isolated mutant strains defective in the STA6 locus of the monocellular green alga Chlamydomonas reinhardtii that fail to accumulate starch and lack ADP-glucose pyrophosphorylase activity. We show that this locus encodes a 514-amino-acid polypeptide corresponding to a mature 50-kDa protein with homology to vascular plant ADP-glucose pyrophosphorylase small-subunit sequences. This gene segregates independently from the previously characterized STA1 locus that encodes the large 53-kDa subunit of the same heterotetramer enzyme. Because STA1 locus mutants have retained an AGPase but exhibit lower sensitivity to 3-phosphoglyceric acid activation, we suggest that the small and large subunits of the enzyme define, respectively, the catalytic and regulatory subunits of AGPase in unicellular green algae. We provide preliminary evidence that both the small-subunit mRNA abundance and enzyme activity, and therefore also starch metabolism, may be controlled by the circadian clock. PMID:11208806

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

    PubMed Central

    Tomkins, Joseph L.

    2016-01-01

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

  15. Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii

    PubMed Central

    1994-01-01

    The molecular machinery of deflagellation can be activated in detergent permeabilized Chlamydomonas reinhardtii by the addition of Ca2+ (Sanders, M. A., and J. L. Salisbury, 1989. J. Cell Biol. 108:1751- 1760). This suggests that stimuli which induce deflagellation in living cells cause an increase in the intracellular concentration of Ca2+, but this has never been demonstrated. In this paper we report that the wasp venom peptide, mastoparan, and the permeant organic acid, benzoate, activate two different signalling pathways to trigger deflagellation. We have characterized each pathway with respect to: (a) the requirement for extracellular Ca2+; (b) sensitivity to Ca2+ channel blockers; and (c) 45Ca influx. We also report that a new mutant strain of C. reinhardtii, adf-1, is specifically defective in the acid-activated signalling pathway. Both signalling pathways appear normal in another mutant, fa-1, that is defective in the machinery of deflagellation (Lewin, R. and C. Burrascano. 1983. Experientia. 39:1397-1398; Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751-1760). We conclude that mastoparan induces the release of an intracellular pool of Ca2+ whereas acid induces an influx of extracellular Ca2+ to activate the machinery of deflagellation. PMID:8120101

  16. 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

  17. Resistance to Phosphinothricin (Glufosinate) and Its Utilization as a Nitrogen Source by Chlamydomonas reinhardtii

    PubMed Central

    Franco, A. R.; Lopez-Siles, F. J.; Cardenas, J.

    1996-01-01

    Wild-type strain 21gr of the green alga Chlamydomonas reinhardtii was resistant to the ammonium salt of l-phosphinothricin (PPT, also called glufosinate), an irreversible inhibitor of glutamine synthetase activity and the main active component of the herbicide BASTA (AgrEvo, Frankfurt am Main, Germany). Under the same conditions, however, this strain was highly sensitive to l-methionine-S-sulfoximine, a structural analog of PPT which has been reported to be 5 to 10 times less effective than PPT as an inhibitor in plants. Moreover, this alga was able to grow with PPT as the sole nitrogen source when this compound was provided at low concentrations. This utilization of PPT was dependent upon the addition of acetate and light and did not take place in the presence of ammonium. Resistance was due neither to the presence of N-acetyltransferase or transaminase activity nor to the presence of glutamine synthetase isoforms resistant to PPT. By using l-[methyl-(sup14)C]PPT, we demonstrated that resistance is due to lack of PPT transport into the cells. This strongly suggests that PPT and l-methionine-S-sulfoximine enter the cells through different systems. Growth with PPT is supported by its deamination by an l-amino acid oxidase activity which has been previously described to be located at the periplasm. PMID:16535427

  18. 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

  19. Characterization of the truncated hemoglobin THB1 from protein extracts of Chlamydomonas reinhardtii

    PubMed Central

    Johnson, Eric A.; Lecomte, Juliette T.J.

    2014-01-01

    Truncated hemoglobins (TrHbs) belong to the hemoglobin superfamily, but unlike their distant vertebrate relatives, little is known about their principal physiologic functions.  Several TrHbs have been studied in vitro using engineered recombinant peptides.  These efforts have resulted in a wealth of knowledge about the chemical properties of TrHbs and have generated interesting functional leads. However, questions persist as to how closely these engineered proteins mimic their counterparts within the native cell. In this report, we examined THB1, one of several TrHbs from the model organism Chlamydomonas reinhardtii. The recombinant THB1 (rTHB1) has favorable solubility and stability properties and is an excellent candidate for in vitro characterization. Linking rTHB1 to the in vivo protein is a critical step in understanding the physiologic function of this protein. Using a simplified three-step purification protocol, 3.5-L batches of algal culture were processed to isolate 50–60 μL fractions enriched in THB1. These fractions of C. reinhardtii proteins were then subjected to physical examination. Using gel mobility, optical absorbance and immunoreactivity, THB1 was identified in these enriched fractions and its presence correlated with that of a heme molecule. Mass spectrometry confirmed this cofactor to be a type b heme and revealed that the native protein contains a co-translational modification consistent with amino-terminal acetylation following initial methionine cleavage. PMID:25653846

  20. In vivo interactions between photosynthesis, mitorespiration, and chlororespiration in Chlamydomonas reinhardtii.

    PubMed

    Cournac, Laurent; Latouche, Gwendal; Cerovic, Zoran; Redding, Kevin; Ravenel, Jacques; Peltier, Gilles

    2002-08-01

    Interactions between photosynthesis, mitochondrial respiration (mitorespiration), and chlororespiration have been investigated in the green alga Chlamydomonas reinhardtii using flash illumination and a bare platinum electrode. Depending on the physiological status of algae, flash illumination was found to induce either a fast (t(1/2) approximately 300 ms) or slow (t(1/2) approximately 3 s) transient inhibition of oxygen uptake. Based on the effects of the mitorespiratory inhibitors myxothiazol and salicyl hydroxamic acid (SHAM), and of propyl gallate, an inhibitor of the chlororespiratory oxidase, we conclude that the fast transient is due to the flash-induced inhibition of chlororespiration and that the slow transient is due to the flash-induced inhibition of mitorespiration. By measuring blue-green fluorescence changes, related to the redox status of the pyridine nucleotide pool, and chlorophyll fluorescence, related to the redox status of plastoquinones (PQs) in C. reinhardtii wild type and in a photosystem I-deficient mutant, we show that interactions between photosynthesis and chlororespiration are favored when PQ and pyridine nucleotide pools are reduced, whereas interactions between photosynthesis and mitorespiration are favored at more oxidized states. We conclude that the plastid oxidase, similar to the mitochondrial alternative oxidase, becomes significantly engaged when the PQ pool becomes highly reduced, and thereby prevents its over-reduction.

  1. Genetic structure of the mating-type locus of Chlamydomonas reinhardtii.

    PubMed Central

    Ferris, Patrick J; Armbrust, E Virginia; Goodenough, Ursula W

    2002-01-01

    Portions of the cloned mating-type (MT) loci (mt(+) and mt(-)) of Chlamydomonas reinhardtii, defined as the approximately 1-Mb domains of linkage group VI that are under recombinational suppression, were subjected to Northern analysis to elucidate their coding capacity. The four central rearranged segments of the loci were found to contain both housekeeping genes (expressed during several life-cycle stages) and mating-related genes, while the sequences unique to mt(+) or mt(-) carried genes expressed only in the gametic or zygotic phases of the life cycle. One of these genes, Mtd1, is a candidate participant in gametic cell fusion; two others, Mta1 and Ezy2, are candidate participants in the uniparental inheritance of chloroplast DNA. The identified housekeeping genes include Pdk, encoding pyruvate dehydrogenase kinase, and GdcH, encoding glycine decarboxylase complex subunit H. Unusual genetic configurations include three genes whose sequences overlap, one gene that has inserted into the coding region of another, several genes that have been inactivated by rearrangements in the region, and genes that have undergone tandem duplication. This report extends our original conclusion that the MT locus has incurred high levels of mutational change. PMID:11805055

  2. Global expression profiling of Chlamydomonas reinhardtii exposed to trace levels of free cadmium.

    PubMed

    Simon, Dana F; Descombes, Patrick; Zerges, William; Wilkinson, Kevin J

    2008-08-01

    In the natural environment, cadmium is often found as a trace contaminant. Due to the complexity of Cd speciation and the heterogeneity of natural systems and processes, it is often difficult to determine clear relationships between analytical measurements of Cd and its induced biological response. Measurements of gene induction can be used to identify molecular mechanisms underlying toxicity and to quantify sublethal responses to trace contaminants. In the present paper, genes that could be involved in the tolerance of Cd to green algae were examined using two global transcriptome profiling strategies. Microarray and differential display techniques were used for a global transcriptome analysis of Chlamydomonas reinhardtii exposed to micromolar and lower Cd(2+) concentrations for a short period (2 h). Real-time quantitative polymerase chain reaction analysis confirmed that a small set of 10 genes was differentially expressed in response to trace Cd(2+) exposures ranging from 7.8 nM to 9.0 microM. Since induction was only observed for a few genes, none of which are known to function in a general stress response, it was likely the result of relevant responses to Cd exposure. The identified genes are discussed with respect to their possible involvement in Cd tolerance and to their future use as biomarkers for monitoring Cd bioavailability in natural soils and waters.

  3. Cadmium response and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach.

    PubMed

    Gillet, Sylvie; Decottignies, Paulette; Chardonnet, Solenne; Le Maréchal, Pierre

    2006-09-01

    A proteomic approach including two-dimensional electrophoresis and MALDI-TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas reinhardtii. We first described the partial 2D-picture of soluble proteome obtained from whole cells grown on acetate. Then we studied the effects of the exposure of these cells to 150 muM cadmium (Cd). The most drastic effect was the decrease in abundance of both large and small subunits of the ribulose-1,5-bisphosphate carboxylase/oxygenase, in correlation with several other enzymes involved in photosynthesis, Calvin cycle and chlorophyll biosynthesis. Other down-regulated processes were fatty acid biosynthesis, aminoacid and protein biosynthesis. On the other hand, proteins involved in glutathione synthesis, ATP metabolism, response to oxidative stress and protein folding were up-regulated in the presence of cadmium. In addition, we observed that most of the cadmium-sensitive proteins were also regulated via two major cellular thiol redox systems, thioredoxin and glutaredoxin.

  4. 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. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Phosphoregulation of an Inner Dynein Arm Complex in Chlamydomonas reinhardtii Is Altered in Phototactic Mutant Strains

    PubMed Central

    King, Stephen J.; Dutcher, Susan K.

    1997-01-01

    To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning. PMID:9008712

  6. Chlamydomonas Outer Arm Dynein Alters Conformation in Response to Ca2+

    PubMed Central

    Sakato, Miho; Sakakibara, Hitoshi

    2007-01-01

    We have previously shown that Ca2+ directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the β and γ heavy chains (HCs). The γ HC–associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca2+ with KCa = 3 × 10−5 M in vitro, suggesting it may act as a Ca2+ sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and γ HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the γ heavy chain. In vitro experiments indicate that LC4 undergoes a Ca2+-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca2+. The sedimentation profile of the γ HC subunit changed subtly upon Ca2+ addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated γ subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca2+ addition. We propose that Ca2+-dependent conformational change of LC4 has a direct effect on the stem domain of the γ HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm. PMID:17634291

  7. Direct lipid extraction from wet Chlamydomonas reinhardtii biomass using osmotic shock.

    PubMed

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

    2012-11-01

    High-cost downstream process is a major bottleneck for producing microalgal biodiesel at reasonable price. Conventional lipid extraction process necessitates biomass drying process, which requires substantial amount of energy. In this regard, lipid extraction from wet biomass must be an attractive solution. However, it is almost impossible to recover lipid directly from wet microalgae with current technology. In this study, we conceived osmotic shock treatment as a novel method to extract lipid efficiently. Osmotic shock treatment was applied directly to wet Chlamydomonas reinhardtii biomass with water content >99%, along with both polar and non-polar organic solvents. Our results demonstrated that osmotic shock could increase lipid recovery approximately 2 times. We also investigated whether the presence of cell wall or different cell stages could have any impact on lipid recovery. Cell wall-less mutant stains and senescent cell phase could display significantly increased lipid recovery. Taken together, our results suggested that osmotic shock is a promising technique for wet lipid extraction from microalgal biomass and successfully determined that specific manipulation of biomass in certain cell phase could enhance lipid recovery further.

  8. UV-B photoreceptor-mediated protection of the photosynthetic machinery in Chlamydomonas reinhardtii.

    PubMed

    Allorent, Guillaume; Lefebvre-Legendre, Linnka; Chappuis, Richard; Kuntz, Marcel; Truong, Thuy B; Niyogi, Krishna K; Ulm, Roman; Goldschmidt-Clermont, Michel

    2016-12-20

    Life on earth is dependent on the photosynthetic conversion of light energy into chemical energy. However, absorption of excess sunlight can damage the photosynthetic machinery and limit photosynthetic activity, thereby affecting growth and productivity. Photosynthetic light harvesting can be down-regulated by nonphotochemical quenching (NPQ). A major component of NPQ is qE (energy-dependent nonphotochemical quenching), which allows dissipation of light energy as heat. Photodamage peaks in the UV-B part of the spectrum, but whether and how UV-B induces qE are unknown. Plants are responsive to UV-B via the UVR8 photoreceptor. Here, we report in the green alga Chlamydomonas reinhardtii that UVR8 induces accumulation of specific members of the light-harvesting complex (LHC) superfamily that contribute to qE, in particular LHC Stress-Related 1 (LHCSR1) and Photosystem II Subunit S (PSBS). The capacity for qE is strongly induced by UV-B, although the patterns of qE-related proteins accumulating in response to UV-B or to high light are clearly different. The competence for qE induced by acclimation to UV-B markedly contributes to photoprotection upon subsequent exposure to high light. Our study reveals an anterograde link between photoreceptor-mediated signaling in the nucleocytosolic compartment and the photoprotective regulation of photosynthetic activity in the chloroplast.

  9. The Metabolome of Chlamydomonas reinhardtii following Induction of Anaerobic H2 Production by Sulfur Depletion*

    PubMed Central

    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.

    2009-01-01

    The metabolome of the model species Chlamydomonas reinhardtii has been analyzed during 120 h of sulfur depletion to induce anaerobic hydrogen (H2) 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, H2 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 H2 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

  10. The effect of caffeine on repair in Chlamydomonas reinhardtii. I. Enhancement of recombination repair.

    PubMed

    Rosen, H; Rehn, M M; Johnson, B A

    1980-05-01

    The effect of caffeine on repair was studied in the green alga Chlamydomonas reinhardtii. Treatment of UV-irradiated wild-type (UVS+) cells with a sublethal level of caffeine caused a significant increase in survival compared to untreated UV-irradiated cells. Caffeine did not affect survival in the repair-deficient strain UVSE1, which is deficient in repair of UV-induced damage carried out by enzymes associated with recombination during meiosis. A significant increase in survival in the presence of caffeine was observed in the repair-deficient strain UVSE4 in which recombination during meiosis is not affected. Treatment of zygotes homozygous for UVS+, UVSE1, or UVSE4 with sublethal levels of caffeine caused marked increases in recombination frequency in UVS+ and UVSE4 zygotes and no increase in recombination in UVSE1 zygotes. These results indicate that caffeine increases recombination in normal strains. Increased opportunity for recombination caused by caffeine would not result in increased recombination frequency in the UVSE1 strain, assuming limited-recombination enzyme activity in this strain. The observed increase in survival following UV-irradiation in the presence of caffeine in strains having normal recombination would therefore be associated with a caffeine-induced increase in opportunities for recombination repair.

  11. Adenosine 3',5'-cyclic monophosphate in Chlamydomonas reinhardtii. Influence on flagellar function and regeneration.

    PubMed

    Rubin, R W; Filner, P

    1973-03-01

    Adenosine 3',5'-cyclic monophosphate (cAMP) influences both flagellar function and flagellar regeneration in Chlamydomonas reinhardtii. The methylxanthine, aminophylline, which can cause a tenfold increase in cAMP level in C. reinhardtii, inhibits flagellar movement and flagellar regeneration by wild-type cells, without inhibiting cell multiplication. Caffeine, a closely related inhibitor, also inhibits flagellar movement and regeneration, but it inhibits cell multiplication too. Regeneration by a mutant lacking the central pair of flagellar microtubules was found to be more sensitive than wild type to inhibition by caffeine and to be subject to synergistic inhibition by aminophylline plus dibutyryl cAMP. Regeneration by three out of seven mutants with different flagellar abnormalities was more sensitive than wild type to these inhibitors. We interpret these results to mean that cAMP affects a component of the flagellum directly or indirectly, and that the responsiveness of that component to cAMP is enhanced by mutations which affect the integrity of the flagellum. The component in question could be microtubule protein.

  12. 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.

  13. Integrated quantitative analysis of nitrogen stress response in Chlamydomonas reinhardtii using metabolite and protein profiling.

    PubMed

    Wase, Nishikant; Black, Paul N; Stanley, Bruce A; DiRusso, Concetta C

    2014-03-07

    Nitrogen starvation induces a global stress response in microalgae that results in the accumulation of lipids as a potential source of biofuel. Using GC-MS-based metabolite and iTRAQ-labeled protein profiling, we examined and correlated the metabolic and proteomic response of Chlamydomonas reinhardtii under nitrogen stress. Key amino acids and metabolites involved in nitrogen sparing pathways, methyl group transfer reactions, and energy production were decreased in abundance, whereas certain fatty acids, citric acid, methionine, citramalic acid, triethanolamine, nicotianamine, trehalose, and sorbitol were increased in abundance. Proteins involved in nitrogen assimilation, amino acid metabolism, oxidative phosphorylation, glycolysis, TCA cycle, starch, and lipid metabolism were elevated compared with nonstressed cultures. In contrast, the enzymes of the glyoxylate cycle, one carbon metabolism, pentose phosphate pathway, the Calvin cycle, photosynthetic and light harvesting complex, and ribosomes were reduced. A noteworthy observation was that citrate accumulated during nitrogen stress coordinate with alterations in the enzymes that produce or utilize this metabolite, demonstrating the value of comparing protein and metabolite profiles to understand complex patterns of metabolic flow. Thus, the current study provides unique insight into the global metabolic adjustments leading to lipid storage during N starvation for application toward advanced biofuel production technologies.

  14. RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii.

    PubMed

    Moellering, Eric R; Benning, Christoph

    2010-01-01

    Eukaryotic cells store oils in the chemical form of triacylglycerols in distinct organelles, often called lipid droplets. These dynamic storage compartments have been intensely studied in the context of human health and also in plants as a source of vegetable oils for human consumption and for chemical or biofuel feedstocks. Many microalgae accumulate oils, particularly under conditions limiting to growth, and thus have gained renewed attention as a potentially sustainable feedstock for biofuel production. However, little is currently known at the cellular or molecular levels with regard to oil accumulation in microalgae, and the structural proteins and enzymes involved in the biogenesis, maintenance, and degradation of algal oil storage compartments are not well studied. Focusing on the model green alga Chlamydomonas reinhardtii, the accumulation of triacylglycerols and the formation of lipid droplets during nitrogen deprivation were investigated. Mass spectrometry identified 259 proteins in a lipid droplet-enriched fraction, among them a major protein, tentatively designated major lipid droplet protein (MLDP). This protein is specific to the green algal lineage of photosynthetic organisms. Repression of MLDP gene expression using an RNA interference approach led to increased lipid droplet size, but no change in triacylglycerol content or metabolism was observed.

  15. High-throughput fluorescence-activated cell sorting for lipid hyperaccumulating Chlamydomonas reinhardtii mutants.

    PubMed

    Xie, Bo; Stessman, Dan; Hart, Jason H; Dong, Haili; Wang, Yingjun; Wright, David A; Nikolau, Basil J; Spalding, Martin H; Halverson, Larry J

    2014-09-01

    The genetically tractable microalga Chlamydomonas reinhardtii has many advantages as a model for renewable bioproducts and/or biofuels production. However, one limitation of C. reinhardtii is its relatively low-lipid content compared with some other algal species. To overcome this limitation, we combined ethane methyl sulfonate mutagenesis with fluorescence-activated cell sorting (FACS) of cells stained with the lipophilic stain Nile Red to isolate lipid hyperaccumulating mutants of C. reinhardtii. By manipulating the FACS gates, we sorted mutagenized cells with extremely high Nile Red fluorescence signals that were rarely detected in nonmutagenized populations. This strategy successfully isolated several putative lipid hyperaccumulating mutants exhibiting 23% to 58% (dry weight basis) higher fatty acid contents than their progenitor strains. Significantly, for most mutants, nitrogen starvation was not required to attain high-lipid content nor was there a requirement for a deficiency in starch accumulation. Microscopy of Nile Red stained cells revealed that some mutants exhibit an increase in the number of lipid bodies, which correlated with TLC analysis of triacyglycerol content. Increased lipid content could also arise through increased biomass production. Collectively, our findings highlight the ability to enhance intracellular lipid accumulation in algae using random mutagenesis in conjunction with a robust FACS and lipid yield verification regime. Our lipid hyperaccumulating mutants could serve as a genetic resource for stacking additional desirable traits to further increase lipid production and for identifying genes contributing to lipid hyperaccumulation, without lengthy lipid-induction periods.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. Dichromate effect on energy dissipation of photosystem II and photosystem I in Chlamydomonas reinhardtii.

    PubMed

    Perreault, François; Ait Ali, Nadia; Saison, Cyril; Popovic, Radovan; Juneau, Philippe

    2009-07-17

    In this study, we investigated the energy dissipation processes via photosystem II and photosystem I activity in green alga Chlamydomonas reinhardtii exposed to dichromate inhibitory effect. Quantum yield of photosystem II and also photosystem I were highly decreased by dichromate effect. Such inhibition by dichromate induced strong quenching effect on rapid OJIP fluorescence transients, indicating deterioration of photosystem II electron transport via plastoquinone pool toward photosystem I. The decrease of energy dissipation dependent on electron transport of photosystem II and photosystem I by dichromate effect was associated with strong increase of non-photochemical energy dissipation processes. By showing strong effect of dichromate on acceptor side of photosystem I, we indicated that dichromate inhibitory effect was not associated only with PSII electron transport. Here, we found that energy dissipation via photosystem I was limited by its electron acceptor side. By the analysis of P700 oxido-reduction state with methylviolagen as an exogenous PSI electron transport mediator, we showed that PSI electron transport discrepancy induced by dichromate effect was also caused by inhibitory effect located beyond photosystem I. Therefore, these results demonstrated that dichromate has different sites of inhibition which are associated with photosystem II, photosystem I and electron transport sink beyond photosystems.

  1. Asymmetric properties of the Chlamydomonas reinhardtii cytoskeleton direct rhodopsin photoreceptor localization

    PubMed Central

    Mittelmeier, Telsa M.; Boyd, Joseph S.; Lamb, Mary Rose

    2011-01-01

    The eyespot of the unicellular green alga Chlamydomonas reinhardtii is a photoreceptive organelle required for phototaxis. Relative to the anterior flagella, the eyespot is asymmetrically positioned adjacent to the daughter four-membered rootlet (D4), a unique bundle of acetylated microtubules extending from the daughter basal body toward the posterior of the cell. Here, we detail the relationship between the rhodopsin eyespot photoreceptor Channelrhodopsin 1 (ChR1) and acetylated microtubules. In wild-type cells, ChR1 was observed in an equatorial patch adjacent to D4 near the end of the acetylated microtubules and along the D4 rootlet. In cells with cytoskeletal protein mutations, supernumerary ChR1 patches remained adjacent to acetylated microtubules. In mlt1 (multieyed) mutant cells, supernumerary photoreceptor patches were not restricted to the D4 rootlet, and more anterior eyespots correlated with shorter acetylated microtubule rootlets. The data suggest a model in which photoreceptor localization is dependent on microtubule-based trafficking selective for the D4 rootlet, which is perturbed in mlt1 mutant cells. PMID:21555459

  2. 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

  3. 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

  4. 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

  5. Light activates binding of membrane proteins to chloroplast RNAs in Chlamydomonas reinhardtii.

    PubMed

    Zerges, William; Wang, Shengwu; Rochaix, Jean-David

    2002-10-01

    Several membrane proteins were previously shown to bind to the 5' leader of the chloroplast psbC mRNA in the unicellular eukaryotic alga Chlamydomonas reinhardtii. This study showed that these proteins have affinity for AU-rich RNAs, as determined by competition experiments. In addition, their binding activities are enhanced 13-15-fold by light, and a 46 kDa protein is activated within 1-10 min. This activation could be mediated by the modulation of ADP pools by the light-dependent reactions of photosynthesis and ATP synthase because (1) two inhibitors that block ATP synthesis also prevent this activation and (2) ADP inhibits the RNA-binding activity of this protein in vitro. An inhibitor of Photosystem II diminishes this induction, suggesting that reducing potential generated by the photosynthetic electron transport chain modulates this RNA-binding activity. The RNA-binding activities of two proteins (of 46 and 47 kDa) are inhibited by Mg-protoporphyrin IX methyl ester in vitro suggesting they could be regulated by these intermediates in the chlorophyll biosynthetic pathway.

  6. Control of hydrogen photoproduction by the proton gradient generated by cyclic electron flow in Chlamydomonas reinhardtii.

    PubMed

    Tolleter, Dimitri; Ghysels, Bart; Alric, Jean; Petroutsos, Dimitris; Tolstygina, Irina; Krawietz, Danuta; Happe, Thomas; Auroy, Pascaline; Adriano, Jean-Marc; Beyly, Audrey; Cuiné, Stéphan; Plet, Julie; Reiter, Ilja M; Genty, Bernard; Cournac, Laurent; Hippler, Michael; Peltier, Gilles

    2011-07-01

    Hydrogen photoproduction by eukaryotic microalgae results from a connection between the photosynthetic electron transport chain and a plastidial hydrogenase. Algal H₂ production is a transitory phenomenon under most natural conditions, often viewed as a safety valve protecting the photosynthetic electron transport chain from overreduction. From the colony screening of an insertion mutant library of the unicellular green alga Chlamydomonas reinhardtii based on the analysis of dark-light chlorophyll fluorescence transients, we isolated a mutant impaired in cyclic electron flow around photosystem I (CEF) due to a defect in the Proton Gradient Regulation Like1 (PGRL1) protein. Under aerobiosis, nonphotochemical quenching of fluorescence (NPQ) is strongly decreased in pgrl1. Under anaerobiosis, H₂ photoproduction is strongly enhanced in the pgrl1 mutant, both during short-term and long-term measurements (in conditions of sulfur deprivation). Based on the light dependence of NPQ and hydrogen production, as well as on the enhanced hydrogen production observed in the wild-type strain in the presence of the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, we conclude that the proton gradient generated by CEF provokes a strong inhibition of electron supply to the hydrogenase in the wild-type strain, which is released in the pgrl1 mutant. Regulation of the trans-thylakoidal proton gradient by monitoring pgrl1 expression opens new perspectives toward reprogramming the cellular metabolism of microalgae for enhanced H₂ production.

  7. Copper response regulator1-dependent and -independent responses of the Chlamydomonas reinhardtii transcriptome to dark anoxia.

    PubMed

    Hemschemeier, Anja; Casero, David; Liu, Bensheng; Benning, Christoph; Pellegrini, Matteo; Happe, Thomas; Merchant, Sabeeha S

    2013-09-01

    Anaerobiosis is a stress condition for aerobic organisms and requires extensive acclimation responses. We used RNA-Seq for a whole-genome view of the acclimation of Chlamydomonas reinhardtii to anoxic conditions imposed simultaneously with transfer to the dark. Nearly 1.4 × 10(3) genes were affected by hypoxia. Comparing transcript profiles from early (hypoxic) with those from late (anoxic) time points indicated that cells activate oxidative energy generation pathways before employing fermentation. Probable substrates include amino acids and fatty acids (FAs). Lipid profiling of the C. reinhardtii cells revealed that they degraded FAs but also accumulated triacylglycerols (TAGs). In contrast with N-deprived cells, the TAGs in hypoxic cells were enriched in desaturated FAs, suggesting a distinct pathway for TAG accumulation. To distinguish transcriptional responses dependent on copper response regulator1 (CRR1), which is also involved in hypoxic gene regulation, we compared the transcriptomes of crr1 mutants and complemented strains. In crr1 mutants, ~40 genes were aberrantly regulated, reaffirming the importance of CRR1 for the hypoxic response, but indicating also the contribution of additional signaling strategies to account for the remaining differentially regulated transcripts. Based on transcript patterns and previous results, we conclude that nitric oxide-dependent signaling cascades operate in anoxic C. reinhardtii cells.

  8. 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

  9. Nitrogen-Sparing Mechanisms in Chlamydomonas Affect the Transcriptome, the Proteome, and Photosynthetic Metabolism.

    PubMed

    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

    2014-04-01

    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.

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

    PubMed

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

    2014-05-23

    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.

  11. Kinetic Characterization of Nitrite Uptake and Reduction by Chlamydomonas reinhardtii1

    PubMed Central

    Córdoba, Francisco; Cárdenas, Jacobo; Fernández, Emilio

    1986-01-01

    Kinetics of nitrite uptake and reduction by Chlamydomonas reinhardtii cells growing phototrophically has been studied by means of progress curves and the Michaelis-Menten integrated equation. Both uptake and reduction processes exhibited hyperbolic saturation kinetics, the nitrite uptake system lacking a diffusion component. Nitrite uptake and reduction showed significant differences in Ks for nitrite at pH 7.5 (1.6 versus 20 micromolar, respectively), optimal pH, activation energy values, and sensitivity toward reagents of sulfhydryl groups. Ks values for nitrite uptake were halved in cells subjected to darkness or to nitrogen-starvation. Nitrate inhibited nitrite uptake by a partially competitive mechanism. The same inhibition pattern was found for nitrite uptake by C. reinhardtii mutant 305 cells incapable of nitrate assimilation. The results demonstrate that C. reinhardtii cells take up nitrite via a highly specific carrier, probably energy-dependent, kinetically responsive to environmental changes, distinguishable from the enzymic nitrite reduction and endowed with an active site for nitrite not usable for nitrate transport. PMID:16665164

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. Carbon Supply and Photoacclimation Cross Talk in the Green Alga Chlamydomonas reinhardtii1[OPEN

    PubMed Central

    Fristedt, Rikard; Dinc, Emine

    2016-01-01

    Photosynthetic organisms are exposed to drastic changes in light conditions, which can affect their photosynthetic efficiency and induce photodamage. To face these changes, they have developed a series of acclimation mechanisms. In this work, we have studied the acclimation strategies of Chlamydomonas reinhardtii, a model green alga that can grow using various carbon sources and is thus an excellent system in which to study photosynthesis. Like other photosynthetic algae, it has evolved inducible mechanisms to adapt to conditions where carbon supply is limiting. We have analyzed how the carbon availability influences the composition and organization of the photosynthetic apparatus and the capacity of the cells to acclimate to different light conditions. Using electron microscopy, biochemical, and fluorescence measurements, we show that differences in CO2 availability not only have a strong effect on the induction of the carbon-concentrating mechanisms but also change the acclimation strategy of the cells to light. For example, while cells in limiting CO2 maintain a large antenna even in high light and switch on energy-dissipative mechanisms, cells in high CO2 reduce the amount of pigments per cell and the antenna size. Our results show the high plasticity of the photosynthetic apparatus of C. reinhardtii. This alga is able to use various photoacclimation strategies, and the choice of which to activate strongly depends on the carbon availability. PMID:27637747

  17. Light/electricity conversion by defined cocultures of Chlamydomonas and Geobacter.

    PubMed

    Nishio, Koichi; Hashimoto, Kazuhito; Watanabe, Kazuya

    2013-04-01

    Biological energy-conversion systems are attractive in terms of their self-organizing and self-sustaining properties and are expected to be applied towards environmentally friendly bioenergy processes. Recent studies have demonstrated that sustainable light/electricity-conversion systems, termed microbial solar cells (MSCs), can be constructed using naturally occurring microbial communities. To better understand the energy-conversion mechanisms in microbial communities, the present study attempted to construct model MSCs comprised of defined cocultures of a green alga, Chlamydomonas reinhardtii, and an iron-reducing bacterium, Geobacter sulfurreducens, and examined their metabolism and interactions in MSCs. When MSC bioreactors were inoculated with these microbes and irradiated on a 12-h light/dark cycle, periodic current was generated in the dark with energy-conversion efficiencies of 0.1%. Metabolite analyses revealed that G. sulfurreducens generated current by oxidizing formate that was produced by C. reinhardtii in the dark. These results demonstrate that the light/electricity conversion occurs via syntrophic interactions between phototrophs and electricity-generating bacteria. Based on the results and data in literatures, it is estimated that the excretion of organics by the phototroph was the bottleneck step in the syntrophic light/electricity conversion. We also discuss differences between natural-community and defined-coculture MSCs.

  18. Glutathionylation in the Photosynthetic Model Organism Chlamydomonas reinhardtii: A Proteomic Survey*

    PubMed Central

    Zaffagnini, Mirko; Bedhomme, Mariette; Groni, Hayam; Marchand, Christophe H.; Puppo, Carine; Gontero, Brigitte; Cassier-Chauvat, Corinne; Decottignies, Paulette; Lemaire, Stéphane D.

    2012-01-01

    Protein glutathionylation is a redox post-translational modification occurring under oxidative stress conditions and playing a major role in cell regulation and signaling. This modification has been mainly studied in nonphotosynthetic organisms, whereas much less is known in photosynthetic organisms despite their important exposure to oxidative stress caused by changes in environmental conditions. We report a large scale proteomic analysis using biotinylated glutathione and streptavidin affinity chromatography that allowed identification of 225 glutathionylated proteins in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. Moreover, 56 sites of glutathionylation were also identified after peptide affinity purification and tandem mass spectrometry. The targets identified belong to a wide range of biological processes and pathways, among which the Calvin-Benson cycle appears to be a major target. The glutathionylation of four enzymes of this cycle, phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, ribose-5-phosphate isomerase, and phosphoglycerate kinase was confirmed by Western blot and activity measurements. The results suggest that glutathionylation could constitute a major mechanism of regulation of the Calvin-Benson cycle under oxidative stress conditions. PMID:22122882

  19. Phosphoribulokinase from Chlamydomonas reinhardtii: a Benson-Calvin cycle enzyme enslaved to its cysteine residues.

    PubMed

    Thieulin-Pardo, Gabriel; Remy, Thérèse; Lignon, Sabrina; Lebrun, Régine; Gontero, Brigitte

    2015-04-01

    Phosphoribulokinase (PRK) in the green alga Chlamydomonas reinhardtii is a finely regulated and well-studied enzyme of the Benson-Calvin cycle. PRK can form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small chloroplast protein CP12. This study aimed to determine the molecular determinants on PRK involved in the complex and the mechanism of action of a recently described novel regulation of PRK that involves glutathionylation. A combination of mass spectrometry, mutagenesis and activity analyses showed that Cys16, besides its role as the binding site of ATP, was also the site for S-glutathionylation. Previous kinetic analysis of the C55S mutant showed that in the oxidized inactive form of PRK, this residue formed a disulfide bridge with the Cys16 residue. This is the only bridge reported for PRK in the literature. Our data show for the first time that a disulfide bridge between Cys243 and Cys249 on PRK is required to form the PRK-GAPDH-CP12 complex. These results uncover a new mechanism for the PRK-GAPDH-CP12 formation involving a thiol disulfide exchange reaction with CP12 and identify Cys16 of PRK as a target of glutathionylation acting against oxidative stress. Although Cys16 is the key residue involved in binding ATP and acting as a defense against oxidative damage, the formation of the algal ternary complex requires the formation of another disulfide bridge on PRK involving Cys243 and Cys249.

  20. 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

  1. The Chlamydomonas reinhardtii ODA3 Gene Encodes a Protein of the Outer Dynein Arm Docking Complex

    PubMed Central

    Koutoulis, Anthony; Pazour, Gregory J.; Wilkerson, Curtis G.; Inaba, Kazuo; Sheng, Hong; Takada, Saeko; Witman, George B.

    1997-01-01

    We have used an insertional mutagenesis/ gene tagging technique to generate new Chlamydomonas reinhardtii mutants that are defective in assembly of the outer dynein arm. Among 39 insertional oda mutants characterized, two are alleles of the previously unclon