Final Report Summary - UVTRANCYCLUM (Molecular characterization of environmental stress responses in the marine diatom Phaeodactylum tricornutum) At the base of our ecosystems, phytoplankton are the main primary producers and among them, diatoms are thought to be responsible for as much as 20 % of global primary productivity (Nelson et al., 1995). Solar UV-B radiations can have a negative impact on the primary producers, reducing the photosynthetic uptake of atmospheric carbon dioxide and consequently affecting ecosystem stability and global biogeochemical cycles (Hader et al. 2011). At a cellular level, UV-B radiations can induce a series of damages that can result in a delay or arrest in the life cycle. Terrestrial plants protect themselves through a massive transcriptome reprogramming under control of a UV-B specific signalling pathway, steering the production of UV-B absorbing compounds and the activation of repair systems (Heijde et al. 2012). Despite the lack of a plant-like UVR8 photoreceptor homolog, diatoms still have the ability to acclimate to UV-B by accumulating UV-B absorbing components, suggesting adaptive responses. Additionally, when exposed to chronic UV-B exposure, diatoms exhibit a decreased growth rate and increased cell volume, which reflects a decoupling between cell division and cell growth. Cell division is known to be under control of environmental factors through the activation of DNA stress checkpoints that sense, react to, and repair DNA damage to sustain viability and adjust growth, if necessary (Cools and De Veylder, 2009). The altered cell cycle kinetics in response to UV-B exposure suggest that diatoms cells use molecular pathways to activate these checkpoints, in order to adjust their cell cycle pace to UV-B induced DNA damage.Because of their peculiar evolutionary history involving sequential endosymbiosis, diatoms were shown to have integrated features from different origins, and thus created a unique metabolic mosaic. Therefore their UV-B adaptation mechanisms and responses might be a chimera of that of different taxa (Bowler et al. 2008). The UVTRANCYCLUM project aimed at defining the genetic basis of systemic and specific signalling pathways in response to UV-B in the diatom Phaeodactylum tricornutum by integrating high throughput methods with in-depth functional characterization methods and computational analyses. For this, the project was divided in 4 main objectives- Objective A: Identification of physiological responses and acclimation capabilities of diatoms to different doses of UV-B- Objective B: Establishment of the UV-B responsive transcriptome- Objective C: Identification of cis-elements and transcription factors that trigger the UV-B transcriptional response- Objective D: Experimental validation of the role of the identified UV-B regulatorsDescription of the work performed and main resultsConsidering the fact that the project has been stopped after 9 months out of the 24 because of a career move by Marc Heijde, some objectives of the project were not addressed as they were planned for later phases of the time period. Objective A: Identification of physiological responses and acclimation capabilities of diatoms to different doses of UV-BIn this part of the project, the objective was to identify the conditions at which UV-B treatment affects the life cycle and the metabolism of the P. tricornutum cell without inducing irreversible damage that might result in a permanent cell cycle arrest. First a suitable UV-B chamber was built with possibility to adjust intensities and use of narrowband UV-B lamps peaking at 312nm, in order to reduce non-specific effects caused by for example UV-A radiation. Subsequently, we identified the optimal growth conditions displaying the best level of synchronization to follow cell cycle progression. At low levels of UV-B diatoms showed no major changes in growth compared to conditions without additional UV-B. Therefore a focus was set on identifying the UV-B dose and time of irradiation needed to induce a temporary arrest of the cell cycle. This experimental setup led to the conclusion that the P. tricornutum cell cycle is reproducibly delayed when an intensity of 1W.m-2 of narrowband UV-B is given for 30 min at 4 hours after the light onset, resulting in a cell cycle delay of 2-3 hours. When using lower intensity, or shortening times of UV-B exposure, no effect on the progress of the cell cycle was observed, indicating that diatoms acclimate quite well to UV-B.Objective B: Establishment of the UV-B responsive transcriptomeThe obtained conditions in the first part formed the biological base for setting up a transcriptome analysis that aimed to unravel the UV-B responses and how these are regulating the cell cycle. From previous work in the laboratory of Pr. Dr. Lieven De Veylder, markers for the different phases of the cell cycle were available. Although we did identify the right irradiance conditions for delaying the cell cycle, molecular markers are necessary to design an optimal time-course for the RNA-Seq experiment. As UV-B is known to damage DNA, mainly by inducing formation of CPD dimers or 6-4 photoproducts, and as the UV-B signaling pathway in Arabidopsis is inducing the expression of photolyases, I decided to focus on the family of photolyases present in P. tricornutum. Based on homology, phylogenetic analysis allowed me to identify four CPD photolyases and three potential cryptochromes. Because of a strong diel regulation identified in preliminary experiments, displaying a peak of expression at the moment where we would treat with UV-B and thus masking UV-B dependent transcriptional activation, we repeated transcription analysis in conditions with constant illumination dampening the diel regulation.Several genes of the CPD photolyase/cryptochrome family displayed a clear response to UV-B, and thus we could identify the potential timings that transcriptome is most affected by UV-B in order to proceed in the establishment of whole transcriptome analyses through RNA-Seq. As the project was stopped after month 9, we did not reach this last stage of things included in Objective BObjective C and Objective D: Identification of cis-elements and transcription factors that trigger the UV-B transcriptional response The Objective C and objective D were planned to be initiated at the end of year one and should have been the main focus of year 2. As the project was stopped at month 9, complete results are not available for these two sections.References- Bowler, C., Allen, A.E. Badger, J.H. Grimwood, J., Jabbari, K., Kuo, A., Maheswari, U., Martens, C., Maumus, F., Otillar, R.P. et al. (2008). The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456, 239-244.- Cools, T., and De Veylder, L. (2009). DNA stress checkpoint control and plant development. Curr Opin Plant Biol 12, 23-28.- Hader, D.P. Helbling, E.W. Williamson, C.E. and Worrest, R.C. (2011). Effects of UV radiation on aquatic ecosystems and interactions with climate change. Photochem Photobiol Sci 10, 242-260.- Heijde, M., and Ulm, R. (2012). UV-B photoreceptor-mediated signalling in plants. Trends in Plant Science 17, 230-237.- Nelson, D.M. Treguer, P., Brzezinski, M.A. Leynaert, A., and Queguiner, B. (1995). Production and Dissolution of Biogenic Silica in the Ocean - Revised Global Estimates, Comparison with Regional Data and Relationship to Biogenic Sedimentation. Global Biogeochem Cy 9, 359-372
