Cryptochrom 3 aus Arabidopsis thaliana - Kofaktoren, Lokalisation und Funktion in planta

Cry3 from Arabidopsis thaliana is one of the first identified DASH type cryptochromes (Kleine et al., 2003). However, the biological function of cry3 remained elusive and the question whether cry3 could act as a photoreceptor was unanswered. The majority of available data on cry3 were obtained with heterologously expressed cry3. The effective immune-precipitation of in planta expressed cry3-GFP enabled the fluorimetric characterisation of the bound cofactors as FAD and MTHF, and confirmed the data of recombinant E. coli expressed cry3 (Pokorny et al., 2005; Moldt et al., 2009). Thus the E. coli expressed and HIS-tagged cry3 seems to be a valid surrogate of cry3 expressed in planta. To get further insight into the biological function of cry3, its expression and localisation was examined. The relative transcript level of CRY3 was transiently induced by all tested light qualities with a maximum two hours after the onset of light. The transcript level of CRY3 was regulated by cry1, cry2, UVR8 and phyA. Even under red light phyA was the prominent regulator of cry3 expression. Analysing the cry3 deletion mutant revealed a slight but reproducible effect on inhibition of hypocotyl elongation during deetiolation of Arabidopsis seedlings. The cry3 mutant seedlings showed a weaker response to blue light. Though a complementation of this phenotype failed, the chloroplast proteome was altered in a 2D DIGE analysis comparing the cry3 deletion mutant with the wild type. This result was substantiated by Western blot analyses, even though the differences were only observed in chloroplast extracts but not in whole cell extracts. Thus, in summary the obtained results are in line with a function of cry3 as photoreceptor. While cry3 localisation was believed to be restricted to chloroplast and mitochondria, this study showed that cry3-GFP is also present in the nucleus and especially enriched in the nucleolus. Thus, cry3 is present in all DNA containing cell compartments. This is of special interest due to the ability of cry3 to bind single stranded DNA and RNA, as shown in in vitro studies (Pokorny et al., 2008). In chloroplasts, cry3 was found to be a soluble stroma protein and SEC data indicated an association with a large, soluble complex. However, the identification of cry3 interaction partners using CoIP and subsequent mass spectrometry of the eluates failed to detect a specific candidate. Whereas CoIPs were only used to search for interacting chloroplast proteins, in lines expressing cry3-HA and cry3-GFP, a Y2H library screen was performed with a complete cDNA library including proteins of all cellular compartments. Two specific interaction partners were found: NUP205, a nuclear pore protein and DPE2, a cytosolic protein acting in starch metabolism. Further analyses have to verify the Y2H interactions and show the biological relevance of these findings if any.