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1.
Bioresour Technol ; 385: 129391, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37364649

ABSTRACT

Microalgae are promising sources of valuable bioproducts such as biofuels, food, and nutraceuticals. However, harvesting microalgae is challenging due to their small size and low biomass concentrations. To address this challenge, bio-flocculation of starchless mutants of Chlamydomonas reinhardtii (sta6/sta7) was investigated with Mortierella alpina, an oleaginous fungus with high concentrations of arachidonic acid (ARA). Triacylglycerides (TAG) reached 85 % of total lipids in sta6 and sta7 through a nitrogen regime. Scanning electron microscopy determined cell-wall attachment and extra polymeric substances (EPS) to be responsible for flocculation. An algal-fungal biomass ratio around 1:1 (three membranes) was optimal for bio-flocculation (80-85 % flocculation efficiency in 24 h). Nitrogen-deprived sta6/sta7 were flocculated with strains of M. alpina (NVP17b, NVP47, and NVP153) with aggregates exhibiting fatty acid profiles similar to C. reinhardtii, with ARA (3-10 % of total fatty acids). This study showcases M. alpina as a strong bio-flocculation candidate for microalgae and advances a mechanistic understanding of algal-fungal interaction.


Subject(s)
Chlorophyta , Mortierella , Flocculation , Fatty Acids , Arachidonic Acid , Mortierella/genetics , Nitrogen
2.
Elife ; 102021 12 22.
Article in English | MEDLINE | ID: mdl-34936552

ABSTRACT

In algae, it is well established that the pyrenoid, a component of the carbon-concentrating mechanism (CCM), is essential for efficient photosynthesis at low CO2. However, the signal that triggers the formation of the pyrenoid has remained elusive. Here, we show that, in Chlamydomonas reinhardtii, the pyrenoid is strongly induced by hyperoxia, even at high CO2 or bicarbonate levels. These results suggest that the pyrenoid can be induced by a common product of photosynthesis specific to low CO2 or hyperoxia. Consistent with this view, the photorespiratory by-product, H2O2, induced the pyrenoid, suggesting that it acts as a signal. Finally, we show evidence for linkages between genetic variations in hyperoxia tolerance, H2O2 signaling, and pyrenoid morphologies.


Subject(s)
Chlamydomonas/physiology , Hydrogen Peroxide/metabolism , Photosynthesis , Signal Transduction , Anaerobiosis
3.
Molecules ; 26(12)2021 Jun 11.
Article in English | MEDLINE | ID: mdl-34208082

ABSTRACT

Cannabidiol (CBD) is a hydrophobic non-psychoactive compound with therapeutic characteristics. Animal and human studies have shown its poor oral bioavailability in vivo, and the impact of consuming lipid-soluble CBD with and without food on gut bioaccessibility has not been explored. The purpose of this research was to study the bioaccessibility of CBD after a three-phase upper digestion experiment with and without food, and to test lipase activity with different substrate concentrations. Our results showed that lipase enzyme activity and fatty acid absorption increased in the presence of bile salts, which may also contribute to an increase in CBD bioaccessibility. The food matrix used was a mixture of olive oil and baby food. Overall, the fed-state digestion revealed significantly higher micellarization efficiency for CBD (14.15 ± 0.6% for 10 mg and 22.67 ± 2.1% for 100 mg CBD ingested) than the fasted state digestion of CBD (0.65 ± 0.7% for 10 mg and 0.14 ± 0.1% for 100 mg CBD ingested). The increase in bioaccessibility of CBD with food could be explained by the fact that micelle formation from hydrolyzed lipids aid in bioaccessibility of hydrophobic molecules. In conclusion, the bioaccessibility of CBD depends on the food matrix and the presence of lipase and bile salts.


Subject(s)
Bile Acids and Salts/metabolism , Cannabidiol/pharmacokinetics , Food , Lipase/metabolism , Biological Availability , Cannabidiol/pharmacology , Digestion , Food-Drug Interactions , Humans , In Vitro Techniques , Lipid Metabolism , Micelles
4.
Plant Cell ; 30(2): 447-465, 2018 02.
Article in English | MEDLINE | ID: mdl-29437989

ABSTRACT

Photosynthesis occurs in the thylakoid membrane, where the predominant lipid is monogalactosyldiacylglycerol (MGDG). As environmental conditions change, photosynthetic membranes have to adjust. In this study, we used a loss-of-function Chlamydomonas reinhardtii mutant deficient in the MGDG-specific lipase PGD1 (PLASTID GALACTOGLYCEROLIPID DEGRADATION1) to investigate the link between MGDG turnover, chloroplast ultrastructure, and the production of reactive oxygen species (ROS) in response to different adverse environmental conditions. The pgd1 mutant showed altered MGDG abundance and acyl composition and altered abundance of photosynthesis complexes, with an increased PSII/PSI ratio. Transmission electron microscopy showed hyperstacking of the thylakoid grana in the pgd1 mutant. The mutant also exhibited increased ROS production during N deprivation and high light exposure. Supplementation with bicarbonate or treatment with the photosynthetic electron transport blocker DCMU protected the cells against oxidative stress in the light and reverted chlorosis of pgd1 cells during N deprivation. Furthermore, exposure to stress conditions such as cold and high osmolarity induced the expression of PGD1, and loss of PGD1 in the mutant led to increased ROS production and inhibited cell growth. These findings suggest that PGD1 plays essential roles in maintaining appropriate thylakoid membrane composition and structure, thereby affecting growth and stress tolerance when cells are challenged under adverse conditions.


Subject(s)
Algal Proteins/metabolism , Chlamydomonas reinhardtii/enzymology , Galactolipids/metabolism , Lipase/metabolism , Thylakoids/metabolism , Algal Proteins/genetics , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/physiology , Chloroplasts/metabolism , Electron Transport , Environment , Lipase/genetics , Photosynthesis , Stress, Physiological
5.
Plant J ; 92(4): 744-756, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28865165

ABSTRACT

Photosynthetic organisms rapidly adjust the capture, transfer and utilization of light energy to optimize the efficiency of photosynthesis and avoid photodamage. These adjustments involve fine-tuning of expression levels and mutual interactions among electron/proton transfer components and their associated light-harvesting antenna. Detailed studies of these interactions and their dynamics have been hindered by the low throughput and resolution of currently available research tools, which involve laborious isolation, separation and characterization steps. To address these issues, we developed an approach that measured multiple spectroscopic properties of thylakoid preparations directly in native polyacrylamide gel electrophoresis gels, enabling unprecedented resolution of photosynthetic complexes, both in terms of the spectroscopic and functional details, as well as the ability to distinguish separate complexes and thus test their functional connections. As a demonstration, we explore the thylakoid membrane components of Chlamydomonas reinhardtii acclimated to high and low light, using a combination of room temperature absorption and 77K fluorescence emission to generate a multi-dimensional molecular and spectroscopic map of the photosynthetic apparatus. We show that low-light-acclimated cells accumulate a photosystem I-containing megacomplex that is absent in high-light-acclimated cells and contains distinct LhcII proteins that can be distinguished based on their spectral signatures.


Subject(s)
Chlamydomonas reinhardtii/metabolism , Photosystem I Protein Complex/metabolism , Tandem Mass Spectrometry/methods , Thylakoids/metabolism , Acclimatization , Chromatography, Liquid/instrumentation , Chromatography, Liquid/methods , Native Polyacrylamide Gel Electrophoresis , Photosynthesis , Tandem Mass Spectrometry/instrumentation
6.
Plant Physiol ; 167(2): 558-73, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25489023

ABSTRACT

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


Subject(s)
Chlamydomonas reinhardtii/physiology , Nitrogen/deficiency , Photosynthesis , Carbon Cycle , Carbon Isotopes , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/ultrastructure , Chlorophyll/metabolism , Down-Regulation/genetics , Energy Metabolism , Fluorescence , Gene Expression Regulation, Plant , Lipids/analysis , Oxygen/metabolism , Photosystem I Protein Complex/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Proton-Motive Force , RNA, Messenger/genetics , RNA, Messenger/metabolism , Starch/biosynthesis , Thylakoids/metabolism , Thylakoids/ultrastructure
7.
Photosynth Res ; 117(1-3): 449-59, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24113925

ABSTRACT

The chloroplast must rapidly and precisely adjust photosynthetic ATP and NADPH output to meet changing metabolic demands imposed by fluctuating environmental conditions. Cyclic electron flow (CEF) around photosystem I is thought to contribute to this adjustment by providing ATP in excess of that supplied by linear electron low, balancing chloroplast energy budget when relative demand for ATP is high. We assessed the kinetics and energy production of CEF activation in Chlamydomonas reinhardtii under rapid changes of organic and inorganic carbon availability. Comparisons of transient electric field and chlorophyll fluorescence measurements indicated CEF was activated under conditions where ATP demand is expected to be high, consistent with a role in balancing the cellular ATP/NADPH budget under fluctuating environmental or metabolic conditions. CEF activation was not correlated with antenna state transitions, both in wild-type and the state transition mutant stt7-9, suggesting that CEF is rapidly regulated by allosteric or redox modulators. Comparing the CEF under ambient and high CO2 conditions suggests an increase in required energy output of approximately 1ATP/CO2 fixed, nearly sufficient to power proposed mechanistic models for the carbon-concentrating mechanism. Additionally, we see three-fold higher CEF rates in cells under steady-state conditions than cells under similar conditions with inhibited photosystem II, and up to five times higher in cells with severe depletion of inorganic carbon, implying that CEF has larger energetic capacity than predicted from some previous work.


Subject(s)
Chlamydomonas reinhardtii/drug effects , Chlamydomonas reinhardtii/metabolism , Carbon/pharmacology , Electron Transport/drug effects , Environment , Inorganic Chemicals/pharmacology , Kinetics , Organic Chemicals/pharmacology , Oxidation-Reduction/drug effects , Photosynthesis/drug effects , Photosystem II Protein Complex/metabolism
8.
J Biol Chem ; 287(15): 11689-703, 2012 Apr 06.
Article in English | MEDLINE | ID: mdl-22170070

ABSTRACT

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


Subject(s)
Chlamydomonas reinhardtii/metabolism , Flagella/metabolism , Plant Proteins/metabolism , Amino Acid Sequence , Biological Transport , Chlamydomonas reinhardtii/genetics , Escherichia coli , Flagella/chemistry , Gene Knockout Techniques , Immunoprecipitation , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Phylogeny , Plant Proteins/chemistry , Plant Proteins/genetics , Protein Binding , Protein Interaction Domains and Motifs , Protein Stability , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Two-Hybrid System Techniques
9.
Bioresour Technol ; 103(1): 484-8, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22023968

ABSTRACT

A two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production was studied, wherein high density heterotrophic cultures of Chlorellasorokiniana serve as seed for subsequent phototrophic growth. The data showed growth rate, cell density and productivity of heterotrophic C.sorokiniana were 3.0, 3.3 and 7.4 times higher than phototrophic counterpart, respectively. Hetero- and phototrophic algal seeds had similar biomass/lipid production and fatty acid profile when inoculated into phototrophic culture system. To expand the application, food waste and wastewater were tested as feedstock for heterotrophic growth, and supported cell growth successfully. These results demonstrated the advantages of using heterotrophic algae cells as seeds for open algae culture system. Additionally, high inoculation rate of heterotrophic algal seed can be utilized as an effective method for contamination control. This two-stage heterotrophic phototrophic process is promising to provide a more efficient way for large scale production of algal biomass and biofuels.


Subject(s)
Biomass , Cell Culture Techniques/methods , Chlorella/growth & development , Heterotrophic Processes , Lipids/biosynthesis , Phototrophic Processes , Cell Count , Chlamydomonas/cytology , Chlamydomonas/growth & development , Chlorella/cytology , Escherichia coli/growth & development , Escherichia coli/metabolism , Fatty Acids/metabolism , Food , Waste Disposal, Fluid , Waste Products
10.
PLoS One ; 5(9)2010 Sep 30.
Article in English | MEDLINE | ID: mdl-20927350

ABSTRACT

Previous observations have demonstrated that embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination promotes transgenerational adult onset disease such as male infertility, kidney disease, prostate disease, immune abnormalities and tumor development. The current study investigates genome-wide promoter DNA methylation alterations in the sperm of F3 generation rats whose F0 generation mother was exposed to vinclozolin. A methylated DNA immunoprecipitation with methyl-cytosine antibody followed by a promoter tilling microarray (MeDIP-Chip) procedure was used to identify 52 different regions with statistically significant altered methylation in the sperm promoter epigenome. Mass spectrometry bisulfite analysis was used to map the CpG DNA methylation and 16 differential DNA methylation regions were confirmed, while the remainder could not be analyzed due to bisulfite technical limitations. Analysis of these validated regions identified a consensus DNA sequence (motif) that associated with 75% of the promoters. Interestingly, only 16.8% of a random set of 125 promoters contained this motif. One candidate promoter (Fam111a) was found to be due to a copy number variation (CNV) and not a methylation change, suggesting initial alterations in the germline epigenome may promote genetic abnormalities such as induced CNV in later generations. This study identifies differential DNA methylation sites in promoter regions three generations after the initial exposure and identifies common genome features present in these regions. In addition to primary epimutations, a potential indirect genetic abnormality was identified, and both are postulated to be involved in the epigenetic transgenerational inheritance observed. This study confirms that an environmental agent has the ability to induce epigenetic transgenerational changes in the sperm epigenome.


Subject(s)
Epigenesis, Genetic , Genome/drug effects , Oxazoles/pharmacology , Promoter Regions, Genetic/drug effects , Rats/genetics , Spermatozoa/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA Copy Number Variations/drug effects , DNA Methylation/drug effects , Epigenesis, Genetic/drug effects , Female , Male , Molecular Sequence Data , Pedigree , Rats/metabolism , Rats, Sprague-Dawley , Spermatozoa/drug effects
11.
J Biol Chem ; 285(28): 21508-18, 2010 Jul 09.
Article in English | MEDLINE | ID: mdl-20435895

ABSTRACT

Intraflagellar transport (IFT) particles of Chlamydomonas reinhardtii contain two distinct protein complexes, A and B, composed of at least 6 and 15 protein subunits, respectively. As isolated from C. reinhardtii flagella, IFT complex B can be further reduced to a approximately 500-kDa core that contains IFT88, 2x IFT81, 2x IFT74/72, IFT52, IFT46, IFT27, IFT25, and IFT22. In this study, yeast-based two-hybrid analysis was combined with bacterial coexpression to show that three of the core B subunits, IFT88, IFT52, and IFT46, interact directly with each other and, together, are capable of forming a ternary complex. Chemical cross-linking results support the IFT52-IFT88 interaction and provide additional evidence of an association between IFT27 and IFT81. With previous studies showing that IFT81 and IFT74/72 interact to form a (IFT81)(2)(IFT74/72)(2) heterotetramer and that IFT27 and IFT25 form a heterodimer, the architecture of complex B is revealing itself. Last, electroporation of recombinant IFT46 was used to rescue flagellar assembly of a newly identified ift46 mutant and to monitor in vivo localization and movement of the IFT particles.


Subject(s)
Algal Proteins/metabolism , Carrier Proteins/metabolism , Chlamydomonas reinhardtii/metabolism , Flagella/metabolism , Gene Expression Regulation , Protozoan Proteins/metabolism , Biological Transport , Chlamydomonas reinhardtii/genetics , Cross-Linking Reagents/chemistry , Electroporation , Models, Biological , Mutagenesis , Phenotype , Plant Proteins , Plasmids/metabolism , Protein Binding , Recombinant Proteins/chemistry , Two-Hybrid System Techniques
12.
J Cell Biol ; 183(2): 313-22, 2008 Oct 20.
Article in English | MEDLINE | ID: mdl-18852297

ABSTRACT

Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.


Subject(s)
Axoneme/metabolism , Dyneins/metabolism , Flagella/metabolism , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , Axoneme/ultrastructure , Biological Transport , Chlamydomonas reinhardtii/cytology , Chlamydomonas reinhardtii/metabolism , Chlamydomonas reinhardtii/ultrastructure , Cytoplasm/metabolism , Humans , Mice , Molecular Sequence Data , Protein Binding , Protein Transport , Protozoan Proteins/chemistry , Two-Hybrid System Techniques
13.
J Biol Chem ; 280(30): 27688-96, 2005 Jul 29.
Article in English | MEDLINE | ID: mdl-15955805

ABSTRACT

Required for the assembly and maintenance of eukaryotic cilia and flagella, intraflagellar transport (IFT) consists of the bidirectional movement of large protein particles between the base and the distal tip of the organelle. Anterograde movement of particles away from the cell body is mediated by kinesin-2, whereas retrograde movement away from the flagellar tip is powered by cytoplasmic dynein 1b/2. IFT particles contain multiple copies of two distinct protein complexes, A and B, which contain at least 6 and 11 protein subunits, respectively. In this study, we have used increased ionic strength to remove four peripheral subunits from the IFT complex B of Chlamydomonas reinhardtii, revealing a 500-kDa core that contains IFT88, IFT81, IFT74/72, IFT52, IFT46, and IFT27. This result demonstrates that the complex B subunits, IFT172, IFT80, IFT57, and IFT20 are not required for the core subunits to stay associated. Chemical cross-linking of the complex B core resulted in multiple IFT81-74/72 products. Yeast-based two-hybrid and three-hybrid analyses were then used to show that IFT81 and IFT74/72 directly interact to form a higher order oligomer consistent with a tetrameric complex. Similar analysis of the vertebrate IFT81 and IFT74/72 homologues revealed that this interaction has been evolutionarily conserved. We hypothesize that these proteins form a tetrameric complex, (IFT81)2(IFT74/72)2, which serves as a scaffold for the formation of the intact IFT complex B.


Subject(s)
Carrier Proteins/physiology , Flagella/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/physiology , Animals , Blotting, Northern , Carrier Proteins/metabolism , Chlamydomonas reinhardtii/metabolism , Cloning, Molecular , Cross-Linking Reagents/pharmacology , Cytoplasm/metabolism , DNA, Complementary/metabolism , Hydrogen-Ion Concentration , Membrane Proteins/metabolism , Models, Biological , Models, Genetic , Protein Binding , Protein Structure, Tertiary , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Temperature , Trypsin/pharmacology , Two-Hybrid System Techniques
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