@article{CleggWackerSpijkerman2021, author = {Clegg, Mark R. and Wacker, Alexander and Spijkerman, Elly}, title = {Phenotypic Diversity and Plasticity of Photoresponse Across an Environmentally Contrasting Family of Phytoflagellates}, series = {Frontiers in plant science : FPLS}, journal = {Frontiers in plant science : FPLS}, number = {12}, publisher = {Frontiers Media}, address = {Lausanne}, issn = {1664-462X}, doi = {10.3389/fpls.2021.707541}, year = {2021}, abstract = {Organisms often employ ecophysiological strategies to exploit environmental conditions and ensure bio-energetic success. However, the many complexities involved in the differential expression and flexibility of these strategies are rarely fully understood. Therefore, for the first time, using a three-part cross-disciplinary laboratory experimental analysis, we investigated the diversity and plasticity of photoresponsive traits employed by one family of environmentally contrasting, ecologically important phytoflagellates. The results demonstrated an extensive inter-species phenotypic diversity of behavioural, physiological, and compositional photoresponse across the Chlamydomonadaceae, and a multifaceted intra-species phenotypic plasticity, involving a broad range of beneficial photoacclimation strategies, often attributable to environmental predisposition and phylogenetic differentiation. Deceptively diverse and sophisticated strong (population and individual cell) behavioural photoresponses were observed, with divergence from a general preference for low light (and flexibility) dictated by intra-familial differences in typical habitat (salinity and trophy) and phylogeny. Notably, contrasting lower, narrow, and flexible compared with higher, broad, and stable preferences were observed in freshwater vs. brackish and marine species. Complex diversity and plasticity in physiological and compositional photoresponses were also discovered. Metabolic characteristics (such as growth rates, respiratory costs and photosynthetic capacity, efficiency, compensation and saturation points) varied elaborately with species, typical habitat (often varying more in eutrophic species, such as Chlamydomonas reinhardtii), and culture irradiance (adjusting to optimise energy acquisition and suggesting some propensity for low light). Considerable variations in intracellular pigment and biochemical composition were also recorded. Photosynthetic and accessory pigments (such as chlorophyll a, xanthophyll-cycle components, chlorophyll a:b and chlorophyll a:carotenoid ratios, fatty acid content and saturation ratios) varied with phylogeny and typical habitat (to attune photosystem ratios in different trophic conditions and to optimise shade adaptation, photoprotection, and thylakoid architecture, particularly in freshwater environments), and changed with irradiance (as reaction and harvesting centres adjusted to modulate absorption and quantum yield). The complex, concomitant nature of the results also advocated an integrative approach in future investigations. Overall, these nuanced, diverse, and flexible photoresponsive traits will greatly contribute to the functional ecology of these organisms, addressing environmental heterogeneity and potentially shaping individual fitness, spatial and temporal distribution, prevalence, and ecosystem dynamics.}, language = {en} } @misc{CleggWackerSpijkerman2021, author = {Clegg, Mark R. and Wacker, Alexander and Spijkerman, Elly}, title = {Phenotypic Diversity and Plasticity of Photoresponse Across an Environmentally Contrasting Family of Phytoflagellates}, series = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1219}, issn = {1866-8372}, doi = {10.25932/publishup-53617}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-536174}, year = {2021}, abstract = {Organisms often employ ecophysiological strategies to exploit environmental conditions and ensure bio-energetic success. However, the many complexities involved in the differential expression and flexibility of these strategies are rarely fully understood. Therefore, for the first time, using a three-part cross-disciplinary laboratory experimental analysis, we investigated the diversity and plasticity of photoresponsive traits employed by one family of environmentally contrasting, ecologically important phytoflagellates. The results demonstrated an extensive inter-species phenotypic diversity of behavioural, physiological, and compositional photoresponse across the Chlamydomonadaceae, and a multifaceted intra-species phenotypic plasticity, involving a broad range of beneficial photoacclimation strategies, often attributable to environmental predisposition and phylogenetic differentiation. Deceptively diverse and sophisticated strong (population and individual cell) behavioural photoresponses were observed, with divergence from a general preference for low light (and flexibility) dictated by intra-familial differences in typical habitat (salinity and trophy) and phylogeny. Notably, contrasting lower, narrow, and flexible compared with higher, broad, and stable preferences were observed in freshwater vs. brackish and marine species. Complex diversity and plasticity in physiological and compositional photoresponses were also discovered. Metabolic characteristics (such as growth rates, respiratory costs and photosynthetic capacity, efficiency, compensation and saturation points) varied elaborately with species, typical habitat (often varying more in eutrophic species, such as Chlamydomonas reinhardtii), and culture irradiance (adjusting to optimise energy acquisition and suggesting some propensity for low light). Considerable variations in intracellular pigment and biochemical composition were also recorded. Photosynthetic and accessory pigments (such as chlorophyll a, xanthophyll-cycle components, chlorophyll a:b and chlorophyll a:carotenoid ratios, fatty acid content and saturation ratios) varied with phylogeny and typical habitat (to attune photosystem ratios in different trophic conditions and to optimise shade adaptation, photoprotection, and thylakoid architecture, particularly in freshwater environments), and changed with irradiance (as reaction and harvesting centres adjusted to modulate absorption and quantum yield). The complex, concomitant nature of the results also advocated an integrative approach in future investigations. Overall, these nuanced, diverse, and flexible photoresponsive traits will greatly contribute to the functional ecology of these organisms, addressing environmental heterogeneity and potentially shaping individual fitness, spatial and temporal distribution, prevalence, and ecosystem dynamics.}, language = {en} } @article{SpijkermanBehrendFachetal.2018, author = {Spijkerman, Elly and Behrend, Hella and Fach, Bettina and Gaedke, Ursula}, title = {Decreased phosphorus incorporation explains the negative effect of high iron concentrations in the green microalga Chlamydomonas acidophila}, series = {The science of the total environment : an international journal for scientific research into the environment and its relationship with man}, volume = {626}, journal = {The science of the total environment : an international journal for scientific research into the environment and its relationship with man}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0048-9697}, doi = {10.1016/j.scitotenv.2018.01.188}, pages = {1342 -- 1349}, year = {2018}, abstract = {The green microalga Chlamydomonas acidophila is an important primary producer in very acidic lakes (pH 2.0-3.5), characterized by high concentrations of ferric iron (up to 1 g total Fe L-1) and low rates of primary production. It was previously suggested that these high iron concentrations result in high iron accumulation and inhibit photosynthesis in C. acidophila. To test this, the alga was grown in sterilized lake water and in medium with varying total iron concentrations under limiting and sufficient inorganic phosphorus (Pi) supply, because Pi is an important growth limiting nutrient in acidic waters. Photosynthesis and growth of C. acidophila as measured over 5 days were largely unaffected by high total iron concentrations and only decreased if free ionic Fe3+ concentrations exceeded 100 mg Fe3+ L-1. Although C. acidophila was relatively rich in iron (up to 5 mmol Fe: mol C), we found no evidence of iron toxicity. In contrast, a concentration of 260 mg total Fe L-1 (i.e. 15 mg free ionic Fe3+ L-1), which is common in many acidic lakes, reduced Pi-incorporation by 50\% and will result in Pi-limited photosynthesis. The resulting Pi-limitation present at high iron and Pi concentrations was illustrated by elevated maximum Pi-uptake rates. No direct toxic effects of high iron were found, but unfavourable chemical Pi-speciation reduced growth of the acidophile alga.}, language = {en} } @article{DauvilleeChochoisSteupetal.2006, author = {Dauvillee, David and Chochois, Vincent and Steup, Martin and Haebel, Sophie and Eckermann, Nora and Ritte, Gerhard and Ral, Jean-Philippe and Colleoni, Christophe and Hicks, Glenn and Wattebled, Fabrice and Deschamps, Philippe and Lienard, Luc and Cournac, Laurent and Putaux, Jean-Luc and Dupeyre, Danielle and Ball, Steven G.}, title = {Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii}, series = {The plant journal}, volume = {48}, journal = {The plant journal}, number = {2}, publisher = {Blackwell}, address = {Oxford}, issn = {0960-7412}, doi = {10.1111/j.1365-313X.2006.02870.x}, pages = {274 -- 285}, year = {2006}, abstract = {Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.}, language = {en} } @article{LachmannMaberlySpijkerman2017, author = {Lachmann, Sabrina C. and Maberly, Stephen C. and Spijkerman, Elly}, title = {Species-specific influence of P-i-status on inorganic carbon acquisition in microalgae (Chlorophyceae)}, series = {Botany}, volume = {95}, journal = {Botany}, publisher = {NRC Research Press}, address = {Ottawa}, issn = {1916-2790}, doi = {10.1139/cjb-2017-0082}, pages = {943 -- 952}, year = {2017}, abstract = {Inorganic phosphorus (P-i) is often the primary limiting nutrient in freshwater ecosystems. Since P(i-)limitation affects energy transduction, and inorganic carbon (C-i) acquisition can be energy demanding, C(i-)acquisition strategies were compared in four species of green algae grown under P-i-replete and P-i-limited conditions predominantly at low and partly at high CO2. Although P-i-limitation was evident by the 10-fold higher cellular C:P ratio and enhanced phosphatase activity, it only decreased C-i-acquisition to a small extent. Nonetheless, the effects of Pi-limitation on both CO2 and HCO3- acquisition were demonstrated. Decreased CO2 acquisition under conditions of Pi limitation was mainly visible in the maximum uptake rate (V-max) and, for the neutrophile Scenedesmus vacuolatus, in the affinity for CO2 acquisition. Discrimination against C-13 was higher under P-i-limited, high CO2 conditions, compared with P-i-replete, highCO(2) conditions, in Chlamydomonas acidophila and S. vacuolatus. In the pH-drift experiments, HCO3- acquisition was reduced in P-i-limited C. reinhardtii. In general, energy demanding bicarbonate uptake was indicated by the less strong discrimination against (13)Cunder lowCO(2) conditions in the neutrophiles (HCO3- users), separating them from the acidophilic or acidotolerant species (CO2 users). The high variability of the influence of Pi supply among different green algal species is linked to their species-specific C(i-)acquisition strategies.}, language = {en} } @article{LachmannMaberlySpijkerman2016, author = {Lachmann, Sabrina C. and Maberly, Stephen C. and Spijkerman, Elly}, title = {ECOPHYSIOLOGY MATTERS: LINKING INORGANIC CARBON ACQUISITION TO ECOLOGICAL PREFERENCE IN FOUR SPECIES OF MICROALGAE (CHLOROPHYCEAE)}, series = {Journal of phycology}, volume = {52}, journal = {Journal of phycology}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {0022-3646}, doi = {10.1111/jpy.12462}, pages = {1051 -- 1063}, year = {2016}, abstract = {The effect of CO2 supply is likely to play an important role in algal ecology. Since inorganic carbon (C-i) acquisition strategies are very diverse among microalgae and C-i availability varies greatly within and among habitats, we hypothesized that C-i acquisition depends on the pH of their preferred natural environment (adaptation) and that the efficiency of C-i uptake is affected by CO2 availability (acclimation). To test this, four species of green algae originating from different habitats were studied. The pH-drift and C-i uptake kinetic experiments were used to characterize C-i acquisition strategies and their ability to acclimate to high and low CO2 conditions and high and low pH was evaluated. Results from pH drift experiments revealed that the acidophile and acidotolerant Chlamydomonas species were mainly restricted to CO2, whereas the two neutrophiles were efficient bicarbonate users. CO2 compensation points in low CO2-acclimated cultures ranged between 0.6 and 1.4 mu M CO2 and acclimation to different culture pH and CO2 conditions suggested that CO2 concentrating mechanisms were present in most species. High CO2 acclimated cultures adapted rapidly to low CO2 condition during pH-drifts. C-i uptake kinetics at different pH values showed that the affinity for C-i was largely influenced by external pH, being highest under conditions where CO2 dominated the C-i pool. In conclusion, C-i acquisition was highly variable among four species of green algae and linked to growth pH preference, suggesting that there is a connection between C-i acquisition and ecological distribution.}, language = {en} } @article{SpijkermanStojkovicHollandetal.2016, author = {Spijkerman, Elly and Stojkovic, Slobodanka and Holland, Daryl and Lachmann, Sabrina C. and Beardall, John}, title = {Nutrient induced fluorescence transients (NIFTs) provide a rapid measure of P and C (co-)limitation in a green alga}, series = {European journal of phycology}, volume = {51}, journal = {European journal of phycology}, publisher = {Hindawi}, address = {Abingdon}, issn = {0967-0262}, doi = {10.1080/09670262.2015.1095355}, pages = {47 -- 58}, year = {2016}, abstract = {Nutrient Induced Fluorescence Transients (NIFTs) have been shown to be a possible way of testing for the limiting nutrient in algal populations. In this study we tested the hypothesis that NIFTs can be used to detect a (co-)limitation for inorganic phosphorus (Pi) and CO2 in the green alga Chlamydomonas acidophila and that the magnitude of the NIFTs can be related to cellular P:C ratios. We show a co-limitation response for Pi and CO2 via traditional nutrient enrichment experiments in natural phytoplankton populations dominated by C. acidophila. We measured NIFT responses after a Pi- or a CO2-spike in C. acidophila batch cultures at various stages of Pi and inorganic C limitation. Significant NIFTs were observed in response to spikes in both nutrients. The NIFT response to a Pi-spike showed a strong negative correlation with cellular P:C ratio that was pronounced below 3 mmol P: mol C (equivalent to 0.2 pg P cell(-1)). Both cellular P and C content influenced the extent of the Pi-NIFT response. The NIFT response to a CO2-spike correlated to low CO2 culturing conditions and also had a negative correlation with cellular P content. A secondary response within the Pi-NIFT response was related to the CO2 concentration and potentially reflected co-limitation. In conclusion, NIFTs provided a quick and reliable method to detect the growth-limiting nutrient in an extremophile green alga, under Pi-, CO2- and Pi/CO2 (co-)limited growth conditions.}, language = {en} } @phdthesis{Mubeen2018, author = {Mubeen, Umarah}, title = {Regulation of central carbon and nitrogen metabolism by Target of Rapamycin (TOR) kinase in Chlamydomonas reinhardtii}, school = {Universit{\"a}t Potsdam}, pages = {vii, 153}, year = {2018}, abstract = {The highly conserved protein complex containing the Target of Rapamycin (TOR) kinase is known to integrate intra- and extra-cellular stimuli controlling nutrient allocation and cellular growth. This thesis describes three studies aimed to understand how TOR signaling pathway influences carbon and nitrogen metabolism in Chlamydomonas reinhardtii. The first study presents a time-resolved analysis of the molecular and physiological features across the diurnal cycle. The inhibition of TOR leads to 50\% reduction in growth followed by nonlinear delays in the cell cycle progression. The metabolomics analysis showed that the growth repression is mainly driven by differential carbon partitioning between anabolic and catabolic processes. Furthermore, the high accumulation of nitrogen-containing compounds indicated that TOR kinase controls the carbon to nitrogen balance of the cell, which is responsible for biomass accumulation, growth and cell cycle progression. In the second study the cause of the high accumulation of amino acids is explained. For this purpose, the effect of TOR inhibition on Chlamydomonas was examined under different growth regimes using stable 13C- and 15N-isotope labeling. The data clearly showed that an increased nitrogen uptake is induced within minutes after the inhibition of TOR. Interestingly, this increased N-influx is accompanied by increased activities of nitrogen assimilating enzymes. Accordingly, it was concluded that TOR inhibition induces de-novo amino acid synthesis in Chlamydomonas. The recognition of this novel process opened an array of questions regarding potential links between central metabolism and TOR signaling. Therefore a detailed phosphoproteomics study was conducted to identify the potential substrates of TOR pathway regulating central metabolism. Interestingly, some of the key enzymes involved in carbon metabolism as well as amino acid synthesis exhibited significant changes in the phosphosite intensities immediately after TOR inhibition. Altogether, these studies provide a) detailed insights to metabolic response of Chlamydomonas to TOR inhibition, b) identification of a novel process causing rapid upshifts in amino acid levels upon TOR inhibition and c) finally highlight potential targets of TOR signaling regulating changes in central metabolism. Further biochemical and molecular investigations could confirm these observations and advance the understanding of growth signaling in microalgae.}, language = {en} } @article{WinckKwasniewskiWienkoopetal.2011, author = {Winck, Flavia Vischi and Kwasniewski, Miroslaw and Wienkoop, Stefanie and M{\"u}ller-R{\"o}ber, Bernd}, title = {An optimized method for the isolation of nuclei from chlamydomas Reinhardtii (Chlorophyceae)}, series = {Journal of phycology}, volume = {47}, journal = {Journal of phycology}, number = {2}, publisher = {Wiley-Blackwell}, address = {Malden}, issn = {0022-3646}, doi = {10.1111/j.1529-8817.2011.00967.x}, pages = {333 -- 340}, year = {2011}, abstract = {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.}, language = {en} } @article{PiephoArtsWacker2012, author = {Piepho, Maike and Arts, Michael T. and Wacker, Alexander}, title = {Species-specific variation in fatty acid concentrations of four phytoplankton species does phosphorus supply influence the effect of light intensity of temperature?}, series = {Journal of phycology}, volume = {48}, journal = {Journal of phycology}, number = {1}, publisher = {Wiley-Blackwell}, address = {Malden}, issn = {0022-3646}, doi = {10.1111/j.1529-8817.2011.01103.x}, pages = {64 -- 73}, year = {2012}, abstract = {We tested, in the laboratory, the influence of light intensity, temperature, and phosphorus (P) supply on fatty acid (FA) concentrations of four freshwater algae: the green algae Scenedesmus quadricauda (Turpin) Breb. and Chlamydomonas globosa J. Snow, the cryptophyte Cryptomonas ovata Ehrenb., and the diatom Cyclotella meneghiniana Kutz. We investigated the main and interactive effects of two variables on algal FA concentrations (i.e., light intensity and P supply or temperature and P supply). Interactive effects of light intensity and P supply were most pronounced in C. meneghiniana, but were also found in S. quadricauda and C. ovata. Changes in several saturated and unsaturated FA concentrations with light were more distinct in the low-P treatments than in the high-P treatments. Interactive effects of temperature and P supply on various FA concentrations were observed in all four species, but there was no consistent pattern. In lake ecosystems, P limitation often coincides with high light intensities and temperatures in summer. Therefore, it is important to examine how combinations of these environmental conditions affect FA concentrations of primary producers that are important sources of FAs for higher trophic levels.}, language = {en} } @phdthesis{Fleischmann2012, author = {Fleischmann, Tobias}, title = {Deletion plastid{\"a}rer ribosomaler Proteine in Nicotiana tabacum im Kontext reduktiver Genomevolutionund Entwicklung einer Hochdurchsatzplattform zur Analysevon miRNAs in Chlamydomonas reinhardtii}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-60393}, school = {Universit{\"a}t Potsdam}, year = {2012}, abstract = {Im Rahmen des ersten Teils der vorliegenden Doktorarbeit konnten zwei nicht-essentielle (rps15, rpl36) und f{\"u}nf essentielle (rps3, rps16, rpl22, rpl23, rpl32) im Plastom von Nicotiana tabacum kodierte Proteine des plastid{\"a}ren Ribosoms bez{\"u}glich ihrer Essentialit{\"a}t charakterisiert werden. Diese Gene wurden durch gezielte Knockout-Experimente inaktiviert und die resultierenden Effekte untersucht. Die Ergebnisse lassen einen R{\"u}ckschluss auf die Lokalisation der Gene der insgesamt sieben untersuchten ribosomalen Proteine zu, die im Plastom mehrerer parasitischer, Plastiden-besitzender Spezies nicht mehr nachweisbar sind. Im Fall von rps15 k{\"o}nnte tats{\"a}chlich ein Verlust des Genes stattgefunden haben, im Fall der restlichen Gene ist eher mit einem Transfer in den Nukleus zu rechnen (rpl36 ausgenommen). Dies w{\"u}rde bedeuten, dass die Geschwindigkeit der erfolgreichen Etablierung eines Gentransfers in vielen parasitischen Spezies gegen{\"u}ber gr{\"u}nen Pflanzen stark erh{\"o}ht ist. Alle in E. coli nicht-essentiellen Proteine mit Homologen in Plastiden (rps15, rpl33, rpl36) sind auch dort, trotz ~1,5 Milliarden Jahren getrennter Evolution, nicht essentiell. Dieses Ergebnis best{\"a}tigt den schon fr{\"u}her festgestellten hohen Konservierungsgrad der bakteriellen und plastid{\"a}ren Translationsmaschinerien. Die Ph{\"a}notypen der KO-Pflanzen der nicht-essentiellen Gene (rps15, rpl36) weisen auf eine interessante Rolle von S15 w{\"a}hrend der Ribosomenassemblierung hin und im Fall von L36 auf eine wichtige funktionelle Rolle im Plastiden-Ribosomen sowie auf eine Involvierung der Plastidentranslation in der Generierung eines retrograden Signals, welches die Blattform zu beeinflussen im Stande ist. Des Weiteren konnte eine Verbindung der Translationsaktivit{\"a}t mit der Ausbildung von Seitentrieben hergestellt werden, die vermutlich auf ver{\"a}nderte Auxinsynthese im Chloroplast zur{\"u}ckzuf{\"u}hren ist. Aus dem Folgeprojekt, bei dem Doppel-KO-Pflanzen nicht-essentieller ribosomaler Proteine erzeugt wurden, l{\"a}sst sich auf eine relativ große Plastizit{\"a}t der Architektur von Plastidenribosomen schließen. Im zweiten Teil der Arbeit konnte erfolgreich ein Hochdurchsatz-Screeningsystem zur semiquantitativen Analyse von 192 verschiedenen miRNAs aus Chlamydomonas reinhardtii etabliert werden. Es gelang durch die Untersuchung von 23 verschiedenen Wachstums- und Stressbedingungen sowie Entwicklungsstadien mehrere miRNAs zu identifizieren, die eine differenzielle Expression zeigen sowie unter allen untersuchten Bedingungen konstant bleibende miRNAs nachzuweisen. Dadurch konnten mehrere vielversprechende Kandidaten-miRNAs ausgemacht werden, die nun eingehender untersucht werden k{\"o}nnen.}, language = {de} } @phdthesis{Winck2011, author = {Winck, Flavia Vischi}, title = {Nuclear proteomics and transcription factor profiling in Chlamydomonas reinhardtii}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-53909}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {The transcriptional regulation of the cellular mechanisms involves many different components and different levels of control which together contribute to fine tune the response of cells to different environmental stimuli. In some responses, diverse signaling pathways can be controlled simultaneously. One of the most important cellular processes that seem to possess multiple levels of regulation is photosynthesis. A model organism for studying photosynthesis-related processes is the unicellular green algae Chlamydomonas reinhardtii, due to advantages related to culturing, genetic manipulation and availability of genome sequence. In the present study, we were interested in understanding the regulatory mechanisms underlying photosynthesis-related processes. To achieve this goal different molecular approaches were followed. In order to indentify protein transcriptional regulators we optimized a method for isolation of nuclei and performed nuclear proteome analysis using shotgun proteomics. This analysis permitted us to improve the genome annotation previously published and to discover conserved and enriched protein motifs among the nuclear proteins. In another approach, a quantitative RT-PCR platform was established for the analysis of gene expression of predicted transcription factor (TF) and other transcriptional regulator (TR) coding genes by transcript profiling. The gene expression profiles for more than one hundred genes were monitored in time series experiments under conditions of changes in light intensity (200 µE m-2 s-1 to 700 µE m-2 s-1), and changes in concentration of carbon dioxide (5\% CO2 to 0.04\% CO2). The results indicate that many TF and TR genes are regulated in both environmental conditions and groups of co-regulated genes were found. Our findings also suggest that some genes can be common intermediates of light and carbon responsive regulatory pathways. These approaches together gave us new insights about the regulation of photosynthesis and revealed new candidate regulatory genes, helping to decipher the gene regulatory networks in Chlamydomonas. Further experimental studies are necessary to clarify the function of the candidate regulatory genes and to elucidate how cells coordinately regulate the assimilation of carbon and light responses.}, language = {en} } @phdthesis{Boelling2006, author = {B{\"o}lling, Christian}, title = {Comprehensive metabolite analysis in Chlamydomonas reinhardtii : method development and application to the study of environmental and genetic perturbations}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-11329}, school = {Universit{\"a}t Potsdam}, year = {2006}, abstract = {This study introduces a method for multiparallel analysis of small organic compounds in the unicellular green alga Chlamydomonas reinhardtii, one of the premier model organisms in cell biology. The comprehensive study of the changes of metabolite composition, or metabolomics, in response to environmental, genetic or developmental signals is an important complement of other functional genomic techniques in the effort to develop an understanding of how genes, proteins and metabolites are all integrated into a seamless and dynamic network to sustain cellular functions. The sample preparation protocol was optimized to quickly inactivate enzymatic activity, achieve maximum extraction capacity and process large sample quantities. As a result of the rapid sampling, extraction and analysis by gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF) more than 800 analytes from a single sample can be measured, of which over a 100 could be positively identified. As part of the analysis of GC-TOF raw data, aliquot ratio analysis to systematically remove artifact signals and tools for the use of principal component analysis (PCA) on metabolomic datasets are proposed. Cells subjected to nitrogen (N), phosphorus (P), sulfur (S) or iron (Fe) depleted growth conditions develop highly distinctive metabolite profiles with metabolites implicated in many different processes being affected in their concentration during adaptation to nutrient deprivation. Metabolite profiling allowed characterization of both specific and general responses to nutrient deprivation at the metabolite level. Modulation of the substrates for N-assimilation and the oxidative pentose phosphate pathway indicated a priority for maintaining the capability for immediate activation of N assimilation even under conditions of decreased metabolic activity and arrested growth, while the rise in 4-hydroxyproline in S deprived cells could be related to enhanced degradation of proteins of the cell wall. The adaptation to sulfur deficiency was analyzed with greater temporal resolution and responses of wild-type cells were compared with mutant cells deficient in SAC1, an important regulator of the sulfur deficiency response. Whereas concurrent metabolite depletion and accumulation occurs during adaptation to S deprivation in wild-type cells, the sac1 mutant strain is characterized by a massive incapability to sustain many processes that normally lead to transient or permanent accumulation of the levels of certain metabolites or recovery of metabolite levels after initial down-regulation. For most of the steps in arginine biosynthesis in Chlamydomonas mutants have been isolated that are deficient in the respective enzyme activities. Three strains deficient in the activities of N-acetylglutamate-5-phosphate reductase (arg1), N2 acetylornithine-aminotransferase (arg9), and argininosuccinate lyase (arg2), respectively, were analyzed with regard to activation of endogenous arginine biosynthesis after withdrawal of externally supplied arginine. Enzymatic blocks in the arginine biosynthetic pathway could be characterized by precursor accumulation, like the amassment of argininosuccinate in arg2 cells, and depletion of intermediates occurring downstream of the enzymatic block, e.g. N2-acetylornithine, ornithine, and argininosuccinate depletion in arg9 cells. The unexpected finding of substantial levels of the arginine pathway intermediates N-acetylornithine, citrulline, and argininosuccinate downstream the enzymatic block in arg1 cells provided an explanation for the residual growth capacity of these cells in the absence of external arginine sources. The presence of these compounds, together with the unusual accumulation of N-Acetylglutamate, the first intermediate that commits the glutamate backbone to ornithine and arginine biosynthesis, in arg1 cells suggests that alternative pathways, possibly involving the activity of ornithine aminotransferase, may be active when the default reaction sequence to produce ornithine via acetylation of glutamate is disabled.}, language = {en} } @phdthesis{Bissinger2003, author = {Bissinger, Vera}, title = {Factors determining growth and vertical distribution of planktonic algae in extremely acidic mining lakes (pH 2.7)}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-0000695}, school = {Universit{\"a}t Potsdam}, year = {2003}, abstract = {Die vorliegende Dissertation besch{\"a}ftigt sich mit den Faktoren, die das Wachstum und die Vertikalverteilung von Planktonalgen in extrem sauren Tagebaurestseen (TBS; pH 2-3) beeinflussen. Im exemplarisch untersuchten TBS 111 (pH 2.7; Lausitzer Revier) dominiert die Goldalge Ochromonas sp. in oberen und die Gr{\"u}nalge Chlamydomonas sp. in tieferen Wasserschichten, wobei letztere ein ausgepr{\"a}gtes Tiefenchlorophyll-Maximum (DCM) ausbildet. Es wurde ein deutlicher Einfluss von Limitation durch anorganischen Kohlenstoff (IC) auf das phototrophe Wachstum von Chlamydomonas sp. in oberen Wasserschichten nachgewiesen, die mit zunehmender Tiefe von Lichtlimitation abgel{\"o}st wird. Im Vergleich mit Arbeiten aus neutralen Seen zeigte Chlamydomonas sp. erniedrigte maximale Wachstumsraten, einen gesteigerten Kompensationspunkt und erh{\"o}hte Dunkelrespirationsraten, was auf gesteigerte metabolische Kosten unter den extremen physikalisch-chemischen Bedingungen hinweist. Die Photosyntheseleistungen von Chlamydomonas sp. waren in Starklicht-adaptierten Zellen durch IC-Limitation deutlich verringert. Außerdem ergaben die ermittelten minimalen Zellquoten f{\"u}r Phosphor (P) einen erh{\"o}hten P-Bedarf unter IC-Limitation. Anschließend konnte gezeigt werden, dass Chlamydomonas sp. ein mixotropher Organismus ist, der seine Wachstumsraten {\"u}ber die osmotrophe Aufnahme gel{\"o}sten organischen Kohlenstoffs (DOC) erh{\"o}hen kann. Dadurch ist dieser Organismus f{\"a}hig, in tieferen, Licht-limitierten Wasserschichten zu {\"u}berleben, die einen h{\"o}heren DOC-Gehalt aufweisen. Da die Vertikalverteilung der Algen im TBS 111 jedoch weder durch IC-Limitation, P-Verf{\"u}gbarkeit noch die in situ DOC-Konzentrationen abschließend erkl{\"a}rt werden konnte (bottom-up Kontrolle), wurde eine neue Theorie zur Entstehung der Vertikalverteilung gepr{\"u}ft. Grazing der phagotrophen und phototrophen Alge Ochromonas sp. auf der phototrophen Alge Chlamydomonas sp. erwies sich als herausragender Faktor, der {\"u}ber top-down Kontrolle die Abundanz der Beute in h{\"o}heren Wasserschichten beeinflussen kann. Gemeinsam mit der Tatsache, dass Chlamydomonas sp. DOC zur Wachstumssteigerung verwendet, f{\"u}hrt dies zu einer Akkumulation von Chlamydomonas sp. in der Tiefe, ausgepr{\"a}gt als DCM. Daher erscheint grazing als der Hauptfaktor, der die beobachtete Vertikalschichtung der Algen im TBS 111 hervorruft. Die erzielten Ergebnisse liefern grundlegende Informationen, um die Auswirkungen von Strategien zur Neutralisierung der TBS auf das Nahrungsnetz absch{\"a}tzen zu k{\"o}nnen.}, language = {en} }