261 research outputs found

    Induction of alternative pathway respiration by nitrate in Chlamydomonas

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    Abstract Besides the ubiquitous cytochrome pathway of mitochondrial respiration, the green algae Chlamydomonas reinhardtii possesses an alternative pathway of respiration, which is comprised of a single protein, alternative oxidase (AOX). AOX is induced when C. reinhardtii cells are shifted from a growth medium containing ammonium as the nitrogen source to one with nitrate. The primary aim of this thesis was to understand the metabolic connections between nitrate assimilation and the induction of the alternative pathway. That these two metabolic processes are closely linked is supported by the fact that a gene encoding AOX (AOX1) is clustered with the genes required for nitrate assimilation (NAR genes). To investigate if the clustering of AOX1 with NAR genes occurs in other green algae, publicly available genome databases were searched. It was found that while the clustering of NAR genes is widespread in many lineages of green algae, the presence of AOX1 within a NAR cluster is a characteristic of only a single group of green algae, the chlorophytes. Interestingly, it was found that lineages that lack NAR gene clustering seem to have a preference for importing amino acids instead of nitrate as their dominant source of nitrogen. The role of AOX in nitrate assimilation was investigated by constitutively blocking AOX1 expression. AOX knockdown cells displayed a slightly decreased rate of growth and distinctly different photosynthetic electron transport characteristics. Whole-cell metabolite analysis showed that the lack of AOX in the mitochondrion in knockdown cells suppressed the stimulatory effect of nitrate on oxidative pentose phosphate pathway (oxPPP) activity within the chloroplast. Interestingly, this regulation also occurred in the opposite direction. In wild-type cells, the biochemical inhibition of oxPPP by glycerol in the chloroplast decreased the accumulation of AOX in the mitochondrion. Overall, the data suggests that the induction of AOX by nitrate relieves respiratory electron transport from adenylate control. During nitrate assimilation increased demand by the chloroplast for ATP would decrease the transfer of ADP and Pi to the mitochondria restricting the rate of the cytochrome pathway. Up regulation of the alternative pathway would allow for continued upstream respiratory carbon flow under limiting adenylate conditions. Keywords Alternative oxidase, nitrate, Chlamydomonas, respiration, oxidative pentose phosphate pathwa

    A combined modelling and experimental characterisation of Chlamydomonas reinhardtii under monochromatic LED illumination

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    Industrial biotechnology is currently synonymous with heterotrophic processes that rely on bacterial, yeast, insect or mammalian cells to biosynthesise products of interest. Microalgae are of substantial biotechnological interest due their polyphyletic nature which grants them access to a wide array of high-value metabolites and their ability to grow under a variety of trophic strategies, including phototrophy. Despite significant process development and optimisation efforts, the full potential of these photosynthetic organisms has yet to be realised. One of the most impactful process parameters when cultivating microalgae is light. It is essential for phototrophic growth and remains highly influential on mixotrophic growth. Indoor cultivations relying on artificial light allow full control of illumination conditions. The advent of LED lights has lowered the costs and improved the flexibility of such installations. Specifically, the spectral composition of LED lights can be accurately and dynamically tailored to the needs of the culture. Spectral composition is known to exert regulatory control over the cell cycle and can affect the cell’s biochemical make up. The effects of illumination strategy on the model microalgae Chlamydomonas reinhardtii were characterised at three different levels (a) growth kinetics, (b) biochemical composition and, (c) transcriptional activity at key carbon nodes. To obtain the transcriptional data, RNA extraction protocols were compared and optimised. Additionally, a suite of candidate reference genes was validated to ensure accurate gene expression normalisation was possible in reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) studies. The growth kinetics and biochemical composition data obtained served as inputs for a previously published genome scale metabolic model. An algorithm was developed to approximate the default biomass composition in the model to experimental data in an effort to increase the fidelity of the simulations. The flux distributions obtained thereafter helped to describe the distinct metabolic fingerprints created under different trophic and illumination strategies

    Reverse genetics of PsaA and PsaB to dissect their function in binding and electron transfer from plastocyanin or cytochrome c6 to the core of photosystem 1

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    Um die Bindungsstelle der Elektronendonoren Plastocyanin (Pc) und Cytochrom c6 (Cyt c6) am Photosystem 1 zu spezifizieren wurden mittels gerichteter Mutagenese die beide Kernuntereinheiten von PS1, PSaA und PSaB, in Chlamydomonas reinhardtii verändert. Die Interaktion zwischen Pc oder Cyt c6 und PS1 aus den erzeugten Mutanten wurden in vitro mittels Cross-linking, NADP+-Photoreduktion und Laserblitz-induzierter Absorbtionskinetik untersucht. Weiterhin wurden die Effekte von Lichtstress auf die erzeugten PS1 Donorseitenmutanten mit Mutanten an der Acceptorseite mittels verschiedener in vivo und in vitro Methoden verglichen

    Applied Photosynthesis

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    Using the energy from sunlight, photosynthesis usually converts carbon dioxide into organic compounds, which are important for all living creatures. Photosynthesis is one of the most important reactions on Earth, and it is a scientific field that is intrinsically interdisciplinary, and many research groups have considered photosynthesis. The aim of this book is to provide new progresses on applied aspects of photosynthesis, and different research groups collected their voluble results from study of this interesting process. All sections have been written by experts in their fields, and book chapters present different and new subjects on photosynthesis

    Evaluation of the plant biostimulant effects of selected eukaryotic green microalgae

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    Microalgae are ubiquitous photosynthetic microorganisms found in nature. They have been reported to synthesize many bioactive chemicals that stimulate plant development. Our experiments aimed to examine the effects of three selected strains of eukaryotic green microalgae on plant growth. Two Chlorella species (Mosonmagyaróvár Algal Culture Collection (MACC)-360 and MACC-38) and a Chlamydomonas reinhardtii strain (cc124) were examined in Medicago truncatula, A17 ecotype, in the first portion of our investigation. First, using growth curves and microscopy, the growth patterns, cell size, and morphology of the microalgal strains were determined. In addition, their ability to synthesize auxins was evaluated. In the greenhouse, M. truncatula was grown in pots containing a mixture of vermiculite and soil (1:3) with a clay layer at the bottom. Living algae cells were applied to the plants using the soil drench method. The plants' physiological reactions to adding algal biomass were comprehensively studied. Microalgae substantially boosted the plant's stem length, leaf size, fresh weight, number of flowers, and pigment content. For most of the investigated factors, there was a strain-specific effect. Overall, the application of Chlorella sp. MACC-360 resulted in more robust plants with greater fresh biomass, larger leaves, and more flowers/pods than the control, which received the same total nutrients. In the second phase of the investigation, the biostimulant effects of Chlorella sp. MACC-360 and C. reinhardtii cc124 on Solanum lycopersicum (tomato) were studied. This study's first purpose was to determine whether the two strains had biostimulant effects on tomato plants. The significance of application mode and timing (plant age) was also studied. Thirdly, the strain-specific effects of the two algal strains were evaluated. Finally, transcriptome and metagenomic analyses were conducted to identify the molecular effect of algae and the microbial community of the rhizosphere. Tomatoes were grown in pots with a clay layer at the bottom and a mixture of soil and vermiculate (2:1). In two sets of trials, living algae and algal extract and living algae and spent media plus extracts were applied to the soil and plant leaves, respectively. In the first group, the culture suspension (treatment A) was centrifuged, the algae pellet was re-suspended in water to make (Treatment B), and this was applied weekly to the soil, while the algae extract (cell disrupted algae suspension – Treatment C) was sprayed on the leaves bi-weekly. Analyses were conducted on the blooming process, plant morphology, fruit characteristics, and pigment content. In the second set of tests, culture suspension (A) was administered weekly to the soil, and C was sprayed bi-weekly on the leaves. The kinetics of flowering, reproductive capability, and photosynthetic characteristics were investigated. Both algal strains enhanced the leaf pigments, fruit weight, and fruit diameter. The age of the plant at the onset of treatment was a significant determinant of the outcome; treatments initiated later (week 5) produced superior results than those initiated at a juvenile level (week 1). Chlorella sp. MACC-360 stimulated early blooming and fruit development, whereas C. reinhardtii cc124 greatly slowed these processes. Chlorella facilitated the transformation of light energy into chemical energy, whereas Chlamydomonas boosted the protection of photosynthetic parameters. Both strains increased leaf thickness and leaf temperature differential. Both algal strains enhanced crucial agronomically useful tomato processes. The upregulation of genes involved in systemic resistance demonstrated that microalgae readied plants to respond to abiotic stress and pathogen attacks, as evidenced by the transcription data. According to soil metagenomics research results, algae influenced the construction of the tomato rhizosphere microbiome. In soils saturated with microalgae, the number of Ascomycota fungus, Streptomyces, Brevundimonas, and other helpful bacteria that provided plant nutrition and defense against dangerous microbes increased

    Transcriptional landscapes of lipid producing microalgae

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    Microalgae are unicellular microscopic and photosynthetic organisms. They are found all over the planet and in all sorts of environments. Their role has been and is still very important for the planet, most notably that they are currently accounted for producing half of the atmospheric oxygen. While they used to be studied because of their capabilities to depollute water, the interests have shifted towards oleaginous microalgae and their high level of fatty-acids accumulation. Fatty-acids such as Triacylglycerides (TAG) are of particular interest for their easy chemical treatment to produce clean biodiesel. Even if microalgae have higher energy conversion efficiency than plants, do not need arable lands to grow on, and do not compete with feed production, the optimal conditions of production are still too costly to compete with fossil fuel pricing. To decrease production costs, growth conditions and the physiological efficiency of the microalgae needs to be optimized. This requires a deep understanding of the microalgae phenotype in the relevant growth conditions. A phenotype represents a set of behavioral traits of an organism In this thesis, the internal phenotype, the transcriptional landscape of two oleaginous microalgae species was studied using different growth conditions. The RNA content was chosen because it provides a dynamic system-wide view, can be done in high-throughput and, as proxy, can inform about the cellular and metabolic activities in response to a changing environment. To analyze the transcriptome, it is necessary to know the functions of the transcripts. In contrast to model organisms like human or Arabidopsis thaliana whose genomes have been deeply annotated and studied, microalgae are far from that state. For both organisms studied in this thesis, Neochloris oleoabundans and Tetradesmus obliquus, it was necessary to annotate the genes and transcripts since it was never done before. To functionally annotate a gene, most methods rely on sequence similarities to identify the closest gene in known organisms. Green algae are a difficult case due to the large genetic distance between them and the greater distance to reference organisms from land plants. Chapter 2 treats about the particular problem of protein annotation in microalgae. I analyzed the general state of data availability in microalgae, and we discussed about several annotation methods that are better suited than sequence similarity and discussed the limitations of using domain-based recognition methods. Besides, we also discussed the identification of the protein localization within the different subcellular compartment in microalgae. Finally, we stressed the importance of a large-scale wet-lab efforts for a few selected microalgae in order to provide a solid foundation for computation based methods. To obtain more insight into the metabolism of Ettlia oleoabundans in Chapter 3 , a constraint-based metabolic model of Ettlia oleoabundans was built around the central carbon metabolism. This model was built based on the knowledge of central metabolism of algae at that moment and was cross checked with the de novo assembled annotated transcriptome. Experiments in controlled turbidostat were conducted in different combinations of light intensity and nitrogen supply. The measurements from the experimental conditions were used as constraints on the inputs and outputs of the model, effectively allowing us to estimate the metabolic flux distributions. In addition, RNA samples from the different experimental conditions were sequenced and analyzed. These data were used to validate the model structure as stated before, correlate expression levels with flux distributions and get a better understanding of the effect of light and nutrient conditions on algal physiology. The metabolic model calculates a maximum TAG yield of N. oleoabundans on light of 1.06 g (mol photons)-1, more than 3 times the current experimental yield under optimal conditions. The model also shows that TAG yield on light can be more efficiently improved by optimizing photosynthetic conversion than by blocking competing pathways. Geranylgeranyl diphosphate reductase was identified as a potential regulator for photosynthetic capability that complements the fine-tuning of chlorophyll levels from synthesis and degradation. Finally, we identified some key reactions that could be targeted to improve TAG yield, by not only specifically increasing the flux within the lipids and TAG pathways, but also potentially redirect carbons from other pathways. Water is a precious resource, and using fresh drinkable water to grow plants or microalgae could be considered non sustainable. However, salt-water is abundantly available and would be cheaper to supply. Therefore it is important to understand how algae deal with a salt water environment under growth and production conditions. This allowed us in Chapter 4 to study how algae deal with high salinity conditions under nitrogen replete (growth) and nitrogen deplete (TAG accumulation) conditions using a transcriptomics approach. The oleaginous microalgae, Ettlia oleoabundans (formally known as Neochloris oleoabundans) was chosen as a model algae, since it can accumulate large amounts of TAG and can grow in both fresh and salt-water. For this algae experiments were done in fresh water and salt water in combination with nitrogen replete and nitrogen deplete conditions. In addition to the transcriptome, we analyzed the biomass composition including TAG and starch accumulation and used the data to look into different salt resistance mechanisms. We found that Proline and the ascorbate-glutathione cycle seem to be of importance for successful osmoregulation in N. oleoabundans. Genes involved in Proline biosynthesis were found to be upregulated in salt water, which is supported by Nuclear magnetic resonance (NMR) spectroscopy. Oil accumulation is increased under nitrogen-deplete conditions in a comparable way in both fresh and salt water. The mechanism behind the biosynthesis of compatible osmolytes can be used to improve N. oleoabundans and other industrially relevant microalgal strains to create a more robust and sustainable production platform for microalgae derived products in the future. Although the TAG content that can be reached in Ettlia oleoabundans is high, the volumetric TAG productivity in Tetradesmus obliquus was evaluated to be clearly higher, while reaching the same TAG content. This was mainly due to the ability of T. obliquus to maintain photosynthetic efficiency for a longer time longer during the nitrogen depletion phase. Therefore, it was decided to switch to T. obliquus as a model organism. To obtain an idea of the capabilities of T. obliquus and to make transcriptome experiments easier to analyze, the genome of T. obliquus was sequenced. In Chapter 5 , the sequencing of the genome of T. obliquus is presented. The assembly approach was unconventional by combining two different methods and was able to combine the higher coverage from one method with the precision of the other method. In this way, the coverage and the accuracy of the assembly was maximized. Production using microalgae will in many cases occur outdoors using sunlight. Consequently the algae will be exposed to the naturally occurring day night cycle. To better understand the effect of these day night cycles, in Chapters 6 and 7, the transcriptional response of algae to diurnal cycles was studied under nitrogen replete conditions and nitrogen limiting conditions for the wild type and a mutant that can not synthesize starch. In Chapter 6 , hourly samples of RNA of Tetradesmus obliquus UTEX 393 were taken from a turbidostat culture operated under nitrogen replete conditions over a diurnal cycle of 16 hour light and 8 hours dark, to obtain more insight in in the transcriptional response towards diurnal cycles. In addition, to understand the effects of a lack of starch, the major transient energy storage, we sequenced samples of the starchless mutant slm1 that were collected every three hours under the same conditions. At the same time, samples were collected and measurements of the biochemical composition of biomass and the specific light absorption rate were performed. These data are presented in a previously published article [24]⁠. The genome features were annotated using more than 38 RNA-seq samples from this study, using a specially developed extension of the FAIR principle based framework called SAPP. The work done to extend this framework for transcriptome analysis is described in the discussion Chapter 8 . We described the succession of metabolic events that occurred during the diurnal cycle, which are in agreement with the biochemical measurements. Comparing the wild-type with the starchless mutant slm1, we found a few temporal shifts in expression that reflect transcriptional adaptation to the lack of a transient energy storage compound during the dark period. Our study provides new perspectives on the role of starch and the adaptation to LD cycles of oleaginous microalgae. In Chapter 7 a similar experimental approach was taken, where samples were taken for biochemical and transcriptome analysis every 3 hours from a turbidostat culture operated at the same diurnal cycle of 16 hours light and 8 hours dark, but this time in nitrogen limiting conditions, resulting in TAG accumulation. Again this was done for the wild type and the starchless mutant. The transcriptional landscape and biochemical data are compared to the nitrogen replete condition in Chapter 6 , to evaluate the effect of nitrogen limitation in general and study how the lack of starch is affecting TAG accumulation under nitrogen limiting conditions. We observed that the cycling diurnal effect is greatly reduced in comparison to nitrogen replete condition. The wild-type accumulated more starch than in nitrogen replete condition (Chapter 6 ), and small oscillation was observed, indicating that it is being used as transient energy storage during the dark period. While the biochemical analysis did not reveal any oscillation in total lipids content in either strain, slm1 over-expresses transcripts associated to TAG and lipid degradation during the dark period. Besides, while slm1 accumulated more TAG than the wild-type, its conversion efficiency was only half of the wild-type. It appears also that the organism recycles more proteins during the dark period to supply nitrogen for the strong increase in amino acid synthesis right after light is turned on. In Chapter 8 , the results of this thesis are discussed. The interest in analyzing the transcriptome of microalgae is explained. Then, the annotation methods are explained to show the large improvements between them, and could it still be improved. Basic numbers of the later annotation results are compared to the current status in UniProtKB. Particular points from the transcriptomics data are discussed, notably the expression of the mutant gene responsible for slm1 phenotype, and the interests from using single-cell technologies. Suggestions are then made to improve the experimental conditions and the photobioreactors setups. The efforts made in the thesis to generate and store the data according to the FAIR principles are explained. Finally, using the knowledge acquired during this thesis, suggestions are made to improve the growth conditions and to improve TAG production with divers metabolic engineering strategies. The work of this thesis contributes for the future of sustainable production of biofuels, which ultimately will help alleviating human society’s dependence on fossil fuels.</p

    Mutations that Affect the Bidirectional Electron Transfer in Photosystem I

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    abstract: Photosystem I (PSI) is a multi-subunit, pigment-protein complex that catalyzes light-driven electron transfer (ET) in its bi-branched reaction center (RC). Recently it was suggested that the initial charge separation (CS) event can take place independently within each ec2/ec3 chlorophyll pair. In order to improve our understanding of this phenomenon, we have generated new mutations in the PsaA and PsaB subunits near the electron transfer cofactor 2 (ec2 chlorophyll). PsaA-Asn604 accepts a hydrogen bond from the water molecule that is the axial ligand of ec2B and the case is similar for PsaB-Asn591 and ec2A. The second set of targeted sites was PsaA-Ala684 and PsaB-Ala664, whose methyl groups are present near ec2A and ec2B, respectively. We generated a number of mutants by targeting the selected protein residues. These mutations were expected to alter the energetics of the primary charge separation event. The PsaA-A684N mutants exhibited increased ET on the B-branch as compared to the A-branch in both in vivo and in vitro conditions. The transient electron paramagnetic resonance (EPR) spectroscopy revealed the formation of increased B-side radical pair (RP) at ambient and cryogenic temperatures. The ultrafast transient absorption spectroscopy and fluorescence decay measurement of the PsaA-A684N and PsaB-A664N showed a slight deceleration of energy trapping. Thus making mutations near ec2 on each branch resulted into modulation of the charge separation process. In the second set of mutants, where ec2 cofactor was target by substitution of PsaA-Asn604 or PsaB-Asn591 to other amino acids, a drop in energy trapping was observed. The quantum yield of CS decreases in Asn to Leu and His mutants on the respective branch. The P700 triplet state was not observed at room and cryogenic temperature for these mutants, nor was a rapid decay of P700+ in the nanosecond timescale, indicating that the mutations do not cause a blockage of electron transfer from the ec3 Chl. Time-resolved fluorescence results showed a decrease in the lifetime of the energy trapping. We interpret this decrease in lifetime as a new channel of excitation energy decay, in which the untrapped energy dissipates as heat through a fast internal conversion process. Thus, a variety of spectroscopic measurements of PSI with point mutations near the ec2 cofactor further support that the ec2 cofactor is involved in energy trapping process.Dissertation/ThesisDoctoral Dissertation Biochemistry 201
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