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Modes of action of cryptochrome 2 from Arabidopsis thaliana
Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light developmental responses in plants. Arabidopsis thaliana cryptochrome 2 (Atcry2) is the major photoreceptor mediating blue light regulation of flowering induction. Although the biological role of crys in plants is...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2007
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| Zusammenfassung: | Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light developmental responses in plants. Arabidopsis thaliana cryptochrome 2 (Atcry2) is the major photoreceptor mediating blue light regulation of flowering induction. Although the biological role of crys in plants is well-known, the initial photochemistry underlying cryptochrome activation and regulation remain poorly understood. In the present work we addressed several aspects in the early activation events of cry 2.
Many members of the photoreceptor families and components of the light signalling transduction pathway dimerize. Therefore, we studied wheter Atcry2 dimerizes, too. Immunoprecipitation studies of transgenic Arabidopsis extracts expressing both,
cry2-GFP and cry2 revealed that full-lenght Atcry2 is a homodimer in vivo in a
light-independent fashion. The identity of the domains involved in cry2 homodimerization were investigated, and both CNT2 and CCT2 were found as monomers.
Because of the sequence similarity of cry2 with cry1, heterodimerization between cry1 and cry2 was also studied, but no cry1-cry2 heterodimers were found in our experiments.
The in vivo effect of dimerization was investigated using Arabidopsis transgenic lines expression either CCT2-GFP or cry2-GFP in addition of the endogenous cry2.
Cry2-GFP dimers showed phosphorylation and degradation under blue light in the same way as the endogenous cry2, whereas under the same conditions the CCT2-GFP monomers remained stable and unphosphorylated. Moreover, cry2-GFP was able to promote early flowering in plants kept under short day conditions. Whereas under the same conditions CCT2-GFP expressing transgenic Arabidopsis flowered as late as
wild-type.
Crys purified from Escherichia coli contain two chromophores, which were identified as flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF), whereas the presence of MTHF was not found for Atcrys purified from an eukaryotic source as insect cells. The detailed knowledge of which chromophore(s) are attached under natural conditions is important for the interpretation of spectroscopic data of these receptors. Therefore, we overexpressed epitope-tagged cry2 in planta. Specific immuno-precipitation of the tagged cry2 protein allowed purification of sufficient amounts of the photoreceptor to identify its chromophores. Based on fluorescence emission data we found that cry2 binds indeed FAD and MTHF in planta. In addition, energy transfer from MTHF to FAD was observed.
Because of their similarity in aminoacid sequence and structure, photolyases have been taken as a model for crys photocycle. However, crys were shown to undergo a photocycle in which semireduced flavin (FADHo) accumulates upon blue light irradiation in contrast to photolyase that accumulates fully reduced FADH-. Green light irradiation of cry2 causes a change in the equilibrium of flavin oxidation states, and attenuates cry2-controlled responses such as flowering. Here, we provided in vivo evidence for semireduced flavin (FADHo) being the active FAD redox state in Atcry2 by analysis of the expression of flowering genes, linking in vitro with physiological studies.
In order to address further insight into the role of phosphorylation on Atcry2 activity, cry2 protein purification from the plant source following mass spectroscopy was performed. Pitifully, the obtained amounts were too small to allow clear results.
Genes that are specifically blue-light induced in cell cultures were identified by PCR and qRT-PCR that can be used in future studies as reporters for transient studies monitoring cry activity. |
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| Umfang: | 147 Seiten |
| DOI: | 10.17192/z2007.0482 |