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Joris J Snellenburg, Lucyna M Wlodarczyk, Jan P Dekker, Rienk van Grondelle, Ivo H M van Stokkum
The regulatory mechanism of state transitions was studied in Chlamydomonas reinhardtii (C.r.) wild type (WT) as well as mutant strains deficient in the photosystem I (PSI) or the photosystem II (PSII) core. Time-resolved fluorescence measurements were obtained on instantly frozen cells incubated beforehand in the dark in aerobic or anaerobic conditions which leads to state 1 (S1) or state 2 (S2). WT data contains information on the light-harvesting complex (LHC) connected to PSI and PSII. The mutants' data contain information on either LHCII-LHCI-PSI or LHCII-PSII, plus information on LHC antennas devoid of a PS core...
October 21, 2016: Biochimica et Biophysica Acta
Clare F Megarity, Julian Esselborn, Suzannah V Hexter, Florian Wittkamp, Ulf-Peter Apfel, Thomas Happe, Fraser A Armstrong
Protein film electrochemistry (PFE) has been used to study the assembly of the complex 6Fe active site of [FeFe]-hydrogenases (known as the H-cluster) from its precursors the [4Fe 4S] domain that is already coordinated within the host and the 2Fe domain that is presented as a synthetic water-soluble complex stabilized by an additional CO. Not only does PFE allow control of redox states via the electrode potential, but the immobilized state of the enzyme facilitates control of extremely low concentrations of the 2Fe complex...
October 24, 2016: Journal of the American Chemical Society
Masayuki Onishi, John R Pringle
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...
October 21, 2016: G3: Genes—Genomes—Genetics
Jingyi Zhu, Tilo Mathes, Yusaku Hontani, Maxime T A Alexandre, Kee Chua Toh, Peter Hegemann, John T M Kennis
The two Light, Oxygen, and Voltage domains of phototropin are blue-light photoreceptor domains which control various functions in plants and green algae. The key step of the light-driven reaction is the formation of a photoadduct between its FMN chromophore and a conserved cysteine, where the canonical reaction proceeds through the FMN triplet state. Here, complete photoreaction mapping of CrLOV2 from Chlamydomonas reinhardtii phototropin and AsLOV2 from Avena sativa phototropin-1 was realized by ultrafast broadband spectroscopy from femtoseconds to microseconds...
October 21, 2016: Journal of Physical Chemistry Letters
Sari Dewi Kurniasih, Tomohito Yamasaki, Fantao Kong, Sigeru Okada, Dwiyantari Widyaningrum, Takeshi Ohama
In this investigation, we succeeded to generate Chlamydomonas mutants that bear dramatically enhanced ability for transgene expression. To yield these mutants, we utilized DNA methyltransferase deficient strain. These mutants must be useful as a plant cell factory. Chlamydomonas reinhardtii (hereafter Chlamydomonas) is a green freshwater microalga. It is a promising cell factory for the production of recombinant proteins because it rapidly grows in simple salt-based media. However, expression of transgenes integrated into the nuclear genome of Chlamydomonas is very poor, probably because of severe transcriptional silencing irrespective of the genomic position...
October 19, 2016: Plant Molecular Biology
Jooyeon Jeong, Kwangryul Baek, Henning Kirst, Anastasios Melis, EonSeon Jin
The Chlamydomonas reinhardtii truncated light-harvesting antenna 4 (tla4) DNA transposon mutant has a pale green phenotype, a lower chlorophyll (Chl) per cell and a higher Chl a/b ratio in comparison with the wild type. It required a higher light intensity for the saturation of photosynthesis and displayed a greater per chlorophyll light-saturated rate of oxygen evolution than the wild type. The Chl antenna size of the photosystems in the tla4 mutant was only about 65% of that measured in the wild type. Molecular genetic analysis revealed that a single plasmid DNA insertion disrupted two genes on chromosome 11 of the mutant...
October 17, 2016: Biochimica et Biophysica Acta
Masaki Odahara, Yusuke Kobayashi, Toshiharu Shikanai, Yoshiki Nishimura
The chloroplast (cp) genome is organized as nucleoids that are dispersed throughout the cp stroma. Previously, a cp homolog of bacterial recombinase RecA (cpRECA) was shown to be involved in maintenance of cp genome integrity by repairing damaged cpDNA and by suppressing aberrant recombination between short dispersed repeats (SDRs) in the moss Physcomitrella patens. Here, overexpression and knockdown analysis of cpRECA in the green alga Chlamydomonas reinhardtii revealed that cpRECA was involved in cp nucleoid dynamics as well as having a role in maintaining cp genome integrity...
October 17, 2016: Plant Physiology
Maria Esther Perez-Perez, Stéphane D Lemaire, Jose L Crespo
Autophagy is a major catabolic pathway by which eukaryotic cells deliver unnecessary or damaged cytoplasmic material to the vacuole for its degradation and recycling in order to maintain cellular homeostasis. Control of autophagy has been associated with the production of reactive oxygen species in several organisms including plants and algae but the precise regulatory molecular mechanisms remain unclear. Here, we show that the ATG4 protease, an essential protein for autophagosome biogenesis, plays a central role for the redox regulation of autophagy in the model green alga Chlamydomonas reinhardtii...
October 17, 2016: Plant Physiology
Sandrine Bujaldon, Natsumi Kodama, Fabrice Rappaport, Rajagopal Subramanyam, Catherine de Vitry, Yuichiro Takahashi, Francis-André Wollman
The green alga Chlamydomonas reinhardtii contains several light-harvesting chlorophyll a/b complexes (LHC); four major LHCIIs, two minor LHCIIs and nine LHCIs. We characterized three chlorophyll b-less mutants to assess the effect of chlorophyll b deficiency on the function, assembly and stability of these chlorophyll a/b-binding proteins. We identified point mutations in two mutants that inactivate the CAO gene responsible for chlorophyll a to chlorophyll b conversion. All LHCIIs accumulated to wild type levels in a CAO mutant but their light-harvesting function for photosystem-II was impaired...
October 11, 2016: Molecular Plant
Kenichi Yamaguchi
Plastid ribosomes are responsible for a large part of the protein synthesis in plant leaves, green algal cells, and the vast majority in the thalli of red algae. Plastid translation is necessary not only for photosynthesis but also for development/differentiation of plants and algae. While some isolated plastid ribosomes from a few green lineages have been characterized by biochemical and proteomic approaches, in-depth proteomics including analyses of posttranslational modifications and processing, comparative proteomics of plastid ribosomes isolated from the cells grown under different conditions, and those from different taxa are still to be carried out...
2017: Methods in Molecular Biology
Fei Wang, Yafei Qi, Alizée Malnoë, Yves Choquet, Francis-André Wollman, Catherine de Vitry
In Chlamydomonas reinhardtii, the major protease involved in the maintenance of the photosynthetic machinery in thylakoid membranes - the FtsH protease - forms mostly large hetero-oligomers (∼1 MDa) comprising FtsH1 and FtsH2 subunits, whatever the light intensity for growth. Upon high light exposure, the FtsH subunits display a shorter half-life, which is counterbalanced by an increase in FTSH1/2 mRNA levels, resulting in a modest upregulation of FtsH1/2 proteins. Furthermore, we show that high light increases the protease activity through a hitherto unnoticed redox-controlled reduction of intermolecular disulfide bridges...
October 1, 2016: Molecular Plant
Scott M Plummer, Mark A Plummer, Patricia A Merkel, Moira Hagen, Jennifer F Biddle, Lisa A Waidner
Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe-Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production...
November 2016: Enzyme and Microbial Technology
Takashi Hirashima, Naoyuki Tajima, Naoki Sato
Phosphatidylcholine (PC) is one of the essential phospholipids for most eukaryotes. Although the model green alga Chlamydomonas reinhardtii lacks PC, four species containing PC were found in the genus Chlamydomonas Here, we report the draft genome sequences of the four species of Chlamydomonas containing PC.
September 29, 2016: Genome Announcements
Roshni J Best, Jan J Lyczakowski, Sara Abalde-Cela, Ziyi Yu, Chris Abell, Alison G Smith
Microalgae and cyanobacteria are promising organisms for sustainable biofuel production, but several challenges remain to make this economically viable, including identification of optimized strains with high biomass productivity. Here we report on a novel methodology for the label-free screening and sorting of cyanobacteria and microalgae in a microdroplet platform. We show for the first time that chlorophyll fluorescence can be used to measure differences in biomass between populations of picoliter microdroplets containing different species of cyanobacteria, Synechocystis PCC 6803 and Synechococcus PCC 7002, which exhibit different growth dynamics in bulk culture...
October 21, 2016: Analytical Chemistry
Catherine E Seed, Joseph L Tomkins
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...
2016: PloS One
Daniel Eugenio Gaytán Luna, Ana Erika Ochoa Alfaro, Alejandro Rocha-Uribe, Ana Silvia Pérez-Martínez, Ángel Gabriel Alpuche-Solís, Ruth Elena Soria-Guerra
Microalgae have the potential to accumulate triacylglycerols (TAGs) under different light spectra. In this work Chlamydomonas reinhardtii was grown under white (400-700 nm), red (650 nm) and green (550 nm) lights. According to our results, red light (650nm) has a positive effect in the microalgae growth and chlorophyll concentration. Regarding the lipid content, the control culture (white light-illuminated) reached a 4.4% of dry cell weight (dcw) while the culture grown at 550nm showed an increase in the lipids accumulation of 1...
September 27, 2016: Biotechnology Progress
Emily G Werth, Evan W McConnell, T S Karim Gilbert, Inmaculada Couso Lianez, Carlos A Perez, Cherrel K Manley, Lee M Graves, James G Umen, Leslie M Hicks
Identifying dynamic protein phosphorylation events is critical for understanding kinase/phosphatase regulated signaling pathways. To date, protein phosphorylation and kinase expression have been examined independently in photosynthetic organisms. Presented here is a method to study the global kinome and phosphoproteome in tandem in the model photosynthetic organism, the alga Chlamydomonas reinhardtii (Chlamydomonas), using mass spectrometry-based label-free proteomics. A dual enrichment strategy targets intact protein kinases via capture on immobilized multiplexed inhibitor beads (MIBs) with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment...
September 27, 2016: Plant Journal: for Cell and Molecular Biology
Hui Li, Yuting Wang, Meirong Chen, Peng Xiao, Changxing Hu, Zhiyong Zeng, Chaogang Wang, Jiangxin Wang, Zhangli Hu
Microalgae are regarded as the most promising biofuel candidates and extensive metabolic engineering were conducted but very few improvements were achieved. Long non-coding RNA (lncRNA) investigation and manipulation may provide new insights for this issue. LncRNAs refer to transcripts that are longer than 200 nucleotides, do not encode proteins but play important roles in eukaryotic gene regulation. However, no information of potential lncRNAs has been reported in eukaryotic alga. Recently, we performed RNA sequencing in Chlamydomonas reinhardtii, and obtained totally 3,574 putative lncRNAs...
2016: Scientific Reports
Olga Reifschneider, Christina Marx, Jessica Jacobs, Laxmikanth Kollipara, Albert Sickmann, Dirk Wolters, Ulrich Kück
In the chloroplast of the green alga Chlamydomonas reinhardtii, two discontinuous group II introns, psaA-i1 and psaA-i2, splice in trans and thus their excision process resembles the nuclear spliceosomal splicing pathway. Here, we address the question whether fragmentation of trans-acting RNAs is accompanied by the formation of a chloroplast spliceosome-like machinery. Using a combination of liquid chromatography-mass spectrometry (LC-MS), size exclusion chromatography (SEC), and quantitative RT-PCR, we provide the first characterization of a high molecular weight ribonucleoprotein (RNP) apparatus participating in psaA mRNA splicing...
September 19, 2016: Journal of Biological Chemistry
Kai Sun, Fuxing Kang, Michael Gatheru Waigi, Yanzheng Gao, Qingguo Huang
Triclosan (TCS) is a broad-spectrum antimicrobial agent that is found extensively in natural aquatic environments. Enzyme-catalyzed oxidative coupling reactions (ECOCRs) can be used to remove TCS in aqueous solution, but there is limited information available to indicate how metal cations (MCs) and natural organic matter (NOM) influence the environmental fate of TCS during laccase-mediated ECOCRs. In this study, we demonstrated that the naturally occurring laccase from Pleurotus ostreatus was effective in removing TCS during ECOCRs, and the oligomerization of TCS was identified as the dominant reaction pathway by high-resolution mass spectrometry (HRMS)...
September 15, 2016: Environmental Pollution
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