Signalling by a plant-like cryptochrome in diatoms

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Cryptochromes are photoreceptors found in all phyla. Whereas animal and plant cryptochromes have been studied for long, plant-like cryptochromes were only discovered recently. They can be found in stramenopiles like diatoms, but also in chlorophytes and some animal groups, including teleosts and annelids. CryP of the diatom Phaeodactylum tricornutum belongs to this group and was shown by us to act as a blue-light photoreceptor. As common, FAD is bound, which is accompanied by an antenna chromophore (methenyltetrahydrofolate). When overexpressing CryP in E. coli, FAD is in its semireduced radical state (FADH•), showing absorbance peaks in the yellow and red range of the spectrum. The FADH• bound to CryP is unusually stable. It can be reversibly reduced to the fully reduced FADH2, but oxidation to the fully oxidised state is extremely slow. This led to the hypothesis that FADH• is the dark state of CryP. Genes for the usual protein interaction partners of plant cryptochromes are missing in diatoms. Thus, the signal transduction pathway is different and has to be examined from scratch. We were able to identify protein interaction partners, a putative transcription factor BolA and a protein of unknown function only present in diatoms and close relatives, ID42612. Those experiments were done in vitro and nothing is known so far about the in vivo interaction and the localisation of the proteins inside the diatom cells. In addition, preliminary experiments have shown that CryP is degraded in a blue-light dependent manner. To elucidate some crucial steps in the signal transduction of CryP we want to tackle three questions: (i) the dark state of CryP in vivo should be proven by quantifying the expression levels of CryP-induced genes under blue light, red light and yellow light in comparison to darkness in WT and knock-out mutants of CryP. If FADH• were the dark state, signalling should be inducible in WT by yellow and red light as well, but not in the mutants. (ii) the interaction of CryP and ID42612 and BolA should be proven in vivo as well, and the localisation of the proteins determined. This will be done using biFC in confocal microscopy, i.e. by coupling parts of GFP to CryP and BolA or ID42612, respectively, that reconstitute the fluorophore when coming close enough. (iii) degradation of CryP ends signalling and a putative function of ID4612 might be degradation of CryP either directly or by recruiting proteases, or protection from degradation. This will be examined using CryP-His overexpressed in E. coli incubated with whole cell extract of P. tricornutum under different light colours and in darkness. The degradation can then be followed by immunoblots and inhibitor studies will provide information on the kind of proteases involved. In summary the project will provide first insights into the peculiar signalling pathway of a plant-like cryptochrome.