Regulation of the Calvin cycle in the diatom Phaeodactylum tricornutum

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SACHSE, Matthias, 2013. Regulation of the Calvin cycle in the diatom Phaeodactylum tricornutum [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Sachse2013Regul-26054, title={Regulation of the Calvin cycle in the diatom Phaeodactylum tricornutum}, year={2013}, author={Sachse, Matthias}, address={Konstanz}, school={Universität Konstanz} }

eng Sachse, Matthias Regulation of the Calvin cycle in the diatom Phaeodactylum tricornutum 2013 2014-01-24T11:02:15Z Phaeodactylum tricornutum is an important model organism for diatoms, one of the prevalent phytoplankton on earth. Diatoms are unicellular, eukaryotic and photoautotrophic microalgae, which contribute greatly to the global carbon cycle efficiently transforming CO2 into carbon compounds via the Calvin cycle pathway. There is strong evidence that the regulation of the Calvin cycle in diatoms is different to what is known for higher plants and green algae and that especially blue light receptors of the aureochrome (AUREO) type may play an important role in the cellular responses for photosynthesis and carbon fixation.<br /><br />Thus, in this doctoral thesis the regulatory mechanisms involved in the Calvin cycle control were investigated and secondly the novel blue light receptors called aureochromes were characterised. To this end a comprehensive expression analysis of the putative pacemaker enzymes of the Calvin cycle, as deduced from the higher plant enzyme redox regulation, and plastidic thioredoxins was performed. The expression of these enzymes, namely the plastidic glyceraldehyde-3-phosphate dehydrogenase (GAP C1), the phosphoribulokinase (PRK), the Ribulose-1,5-bisposhphate carboxylase/oxygenase (RuBisCO) and the plastdic fructose-1,6-bisphosphatases (FBP) was monitored at transcript and protein levels over time in two conditions, once a simulated day night cycle and once after transfer into complete darkness. This setup allows the differentiation between circadian and light dependent effects on the transcript levels. In the context of this expression analysis a set of endogenous reference genes for diatoms stably expressed independent of time and light was identified, namely the genes for hypoxanthine-guanine phosphoribosyltransferase (HPRT), ribosomal protein S1 (RPS)and TATA-box binding protein (TBP). The expression analysis revealed concerted expression patterns for most investigated Calvin cycle genes and thioredoxins respectively. While most transcriptional relative changes were weak to moderate, the genes for the GAP C1 and PRK were drastically regulated at the transcript level in P. tricornutum during the day. Interestingly both enzymes are usually posttranslationally co-regulated by redox interactions with the CP12 protein in the green lineage, a mechanism apparently missing in most diatoms due to missing genes for CP12. This could indicate that this missing redox regulation may be compensated in part by the strong transcriptional regulation of these genes. The analysis of the corresponding protein levels were in accordance to the transcript level expectations in case of PRK but different in case of GAP C1. The protein data on GAP C1 is inconsistent but it likely indicates a high protein level at all times and thus a light independent functionality for GAP C1. FBPs are the only known Calvin cycle enzyme to be putatively redox regulated in diatoms. In preparation for future research and analysis of this regulation an enzymatic assay for FBP activity was established and intein overexpression constructs for two FBP isoforms were generated.<br /><br />In this thesis first indications for an involvement of the blue light receptor aureochromes in the Calvin cycle regulation are described: the screening for homologous regions to the DNA recognition site described for AUREO1 of Vaucheria frigida revealed potential gene regulatory regions in the genes of GAP C1 and PRK. For the characterisation of AUREOs in P. tricornutum peptide antibodies against AUREO1a, 1b and 2 were designed and generated. The antiserum against AUREO1a was thoroughly tested and verified. AUREOs are putatively acting as blue light sensing transcription factors, due to their blue light sensing LOV (light, oxygen, voltage) and DNA binding bZIP (basic leucine zipper) domains. In this thesis the nuclear localisation for three AUREOs was verified by the expression of the respective full length GFP fusion proteins in P. tricornutum and their analysis via laser scanning microscopy, which supports functionality as transcription factors. An RNA interference (RNAi) silencing construct for the AUREO1a gene was generated and transformed into P. tricornutum. For two strains a clear down regulation of AUREO1a could be demonstrated and these strains were thoroughly physiologically investigated. Interestingly against expectations the AUREO1a knockdown not only affected the light dependent physiological response of blue light acclimated cells but also of cells acclimated to red light. Under both conditions the mutant strains exhibited a hyper response to light reacting similar to acclimating to light stress: a decreased Chlorophyll a content, the maximum photosynthesis rates were increased, as was their potential for non-photochemical quenching (NPQ). This supports a photoacclimation inhibiting function of AUREO1a, which is released by blue light, and putative interaction with red light sensing pathways. Sachse, Matthias terms-of-use

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