Project Prof. Dr. Claudia Büchel

"Light harvesting proteins in diatoms - functional heterogeneity and regulation"

The major aim of the project is to examine more deeply the effects of the newly identified, unusual cryptochrome CryP in the pennate diatom Phaeodactylum tricornutum. CryP, having DASH like features according to spectroscopic analyses but also grouping close to plant Crys in sequence analyses, was shown to be involved in the regulation of the overall protein levels of different light-harvesting proteins (fucoxanthin chlorophyll proteins, FCPs). Here, we will focus on the influence of CryP on the organisation of light harvesting proteins. Many FCPs are expressed in diatoms and assemble mostly in trimers, which differ in polypeptide composition and also slightly in pigmentation. We want to use our CryP knock-down lines to test biochemically, which FCP trimers are influenced by the action of CryP. Whether this influence is in relation to the circadian clock will be determined on mRNA level, since CryP is related to plant Crys. In addition, the cellular localization of CryP should be clarified. Since the spectroscopic features of CryP resemble CRY-DASH proteins, its possible bi-functionality, i.e. an involvement in DNA repair, will be tested as well. Spectroscopic and structural investigations on the protein together with mRNA quantifications under different light conditions should provide further insight into the mode of action, as well as the identification of possible interaction partners.

Project Prof Dr. Peter Hegemann

"Functional characterization of novel rhodopsins of Chlamydomonas and other algae"

During the current funding period we were able to characterize a number of so far non described rhodopsins of green algae. Most effords we have invested in the characterization of rhodopsin domain that is part of the novel Histidin-Kinase Rhodopsin (HKR), a familiy that we recently discovered in Chlamydomonas. This protein is an UVA-receptor (λmax = 380 nm). After UV-light absorption it is converted into the stable Metaform Rh-Bl (λmax = 490 nm) and the reaction is reversed by blue light. Chromophore dynamics and photochemistry were studied by time reolved UV/Vis and Resonance Raman spectroscopy. In parallel we have characterized a Channelrhodopsin of the halotolerant alga Dunaliella salina, which exclusivly conducts H+, and a light driven proton pump of Chlorella vulgaris. The Chlorella protein is of high interest for optogenetic applications.
During the funding period we are applying for we would like to continue the characterization of the HKRs on a molecular level in vitro and in vivo. We will undertake efforts to functionally express the rhodopsins of HKR2 and HKR4 of Chlamydomonas and the only HKR of Ostreococcus to study spectral properties and photoreactions. We will try to express the full-length proteins and to biochemically characterize the signaling process in-cluding light regulated cyclase activity. We will delete the genes by homologous recombination and characterize the deletion strains by biophysical and physiological methods. As a side project we will continue the characterization of the only rhodopsins of the cryophilic alga Fragilariopsis cylindrus and the green alga Chlorella vulgaris.

Project Prof. Dr. Tilman Kottke

"Light responses of animal-like cryptochromes and aureochromes from microalgae"

Cryptochromes and aureochromes are plant photosensors incorporating a flavin as a chromophore. The mechanism and the role of these receptors in microalgae remain largely unclear. Cryptochromes regulate blue-light-dependent processes such as photo-morphogenesis and circadian rhythm in higher plants. In the green alga Chlamydomonas reinhardtii, however, an additional animal-like cryptochrome (aCRY) is found. aCRY is currently the only sensory flavoprotein, which is activated not only by blue but also by red light.
The structural basis of this unique red-light sensitivity of aCRY needs to be elucidated. We have demonstrated that heterologously expressed aCRY forms a neutral radical of flavin with an absorption spectrum similar to the spectral response in vivo. The neutral radical state was therefore postulated as dark state. The influence of reductants and point mutations on formation and stabilisation of this dark state will be investigated. The course of the photoreaction will be followed by time-resolved spectroscopy. aCRY differs from homologous proteins with a function as core clock component or DNA repair enzyme in a specific C-terminal extension. Infrared difference spectroscopy will be applied to search for light-dependent structural changes in this extension. Further promising candidates for sensory cryptochromes from microalgae will be characterized by spectroscopy.
Aureochromes have only been identified in algae up to now including the diatom Phaeodactylum tricornutum. Aureochrome differs from all other proteins containing LOV (light-, oxygen-, or voltage-sensitive) domains such as phototropin in a reverse arrangement of effector and sensor domains. We have shown that despite the reverse arrangement, the C-terminal LOV-J(alpha) helix unfolds in aureochrome similar to phototropin. We will investigate the progress of signal transfer by the J(alpha) helix or other segments in the full-length AUREO1a using infrared double difference spectroscopy. The DNA binding ability of the putative transcription factor will be studied. Other aureochromes from P. tricornutum will be heterologously expressed and their photochemical properties will be analyzed with respect to their role in vivo. The investigation of the novel pathways of signal transfer in these aureochromes and cryptochromes will significantly broaden our current understanding of photosensors.

Project Prof. Dr. Maria Mittag

"The involvement of cryptochromes in the light- and circadian signaling pathways in Chlamydomonas reinhardtii"

Cryptochromes (CRYs) are flavoproteins that act as sensory blue-light receptors in plants thereby mediating the input of blue light for the entrainment of the circadian clocks. In contrast, mammalian CRYs are components of the circadian oscillatory machinery. In addition, there are DASH-type CRYs, but their biological function is still largely unknown. In the green biflagellate alga Chlamydomonas reinhardtii, a plant (pCRY) and an animal-like CRY (aCRY) exist as well as two DASH-type CRYs. We discovered that aCRY functions as a sensory blue-light receptor as well as a sensory red-light receptor. This is novel for a flavoprotein in general and a CRY in particular. Recent data also indicate that pCRY and aCRY in C. reinhardtii are functionally intertwined. We will focus on the in vivo mechanism of signal transduction of aCRY based on its spectral properties, its potential light-driven DNA repair function and the detailed role of both CRYs within the circadian clock of C. reinhardtii. Moreover, we will investigate the interplay of both CRYs as well as the function of a DASH-type CRY at different levels. The combination of the interdisciplinary scientific expertises within the research group will allow us to gain information about the spectroscopic, molecular biological and structural properties of the different CRYs.

Project Prof. Dr. Peter Kroth and Prof. Dr. Christian Wilhelm

"Biological function of aureochromes in the diatom Phaeodactylum tricornutum"

Diatoms are unicellular algae that can be found in any aquatic habitat. Being mainly photoautotrophs, diatoms use light as a driving force for photosynthesis but also for cellular regulation. In the first funding period of the project we have shown that a blue light receptor is responsible for triggering the metabolic changes which induce high light acclimation. There is some evidence that aureochromes (AUREOs) which are blue light receptors and transcriptions factors possessing a bZIP and a LOV domain may be responsible for this light acclimation reaction. We have generated Aureo mutants which are significantly decreased in AUREO1a on the protein level. These mutants will be studied on the physiological level if the light acclimation reaction is impaired. On the genomic level we want to identify cis-acting elements as well as interaction partners of the AUREO proteins.

Project Prof. Dr. Lars-Oliver Essen

"Studies on the structures and mechanisms of algal photoreceptors"

Light-dependent responses play major roles not only in the morphogenesis of higher plants, but also in the regulation of cellular responses by green and red algae. Several cryptochromes are now demonstrated to mediate blue-light and, in the case of aCRY from Chlamydomonas reinhardtii, even red-light responses in green algae. An important prerequisite for understanding their in vivo photobiology is first the identification of cognate antenna cofactors in algal cryptochromes. Secondly, analysis of their redox-state dependent structures will then allow to correlate their photochemistry and signalling function with structural alterations and delineate interaction sites with putative down-stream regulators. Aureochromes are a novel class of blue-light-dependent transcription factors, which are found only in several algae and differ by their inverted domain organisation from other LOV-regulated signalling proteins. We will analyse their lit state structures by X-ray crystallography when complexed to cognate DNA and compare them with their dark state structures. Here, the final goal is to obtain the molecular basis for intra-molecular signal transfer and transcription activation by aureochromes.