Funktionelle Analyse von Cryptochrom 3 aus Arabidopsis thaliana

Cryptochromes are blue/UV-A light photoreceptors, which are closely related to photolyases but do not show DNA repair activity. Apart from the classical plant cryptochromes cry1 and cry2, a third Cryptochrom was identified in Arabidopsis thaliana. Cryptochrome 3 (cry3) belongs to the subfamily of the DASH cryptochromes and was shown to be localized in the chloroplasts and mitochondria. Although cry3 has been characterized quite well considering protein structure and biochemical properties, the biological function of this protein in the plant remains unknown until today. In this thesis the biological function of cry3 in the plant has been investigated. The localization of cry3 in chloroplasts and mitochondria could be confirmed by immunological studies on organellar fractions of Arabidopsis cell culture and immunolocalization studies using gold labelled secondary antibodies. Therefore any artefacts of the previous localization studies by overexpression and GFP fusion of cry3 can be excluded. At transcript level CRY3 expression is regulated by light. During the early phase of deetiolation cry3 is transiently induced mainly by far-red light. Phytochrome A was identified as the responsible photoreceptor for this regulation of cry3. PIF1 and PIF3 are also involved in this phytochrome dependent process. In adult plants grown in light / dark cycles, CRY3 expression shows a circadian regulation, which is also affected by the photoperiod. For functional analysis of cry3, several seed collections were screened for cry3 mutants and transgenic cry3 lines were produced. For further functional analysis cry3 overexpressor lines, RNAi knock-down lines, a cry3 knock-out transposon line and several T-DNA insertion lines with altered CRY3 expression and truncated cry3 c-term are now available. Two of these mutant lines are affected in seed germination under limiting light conditions. However this phenotype could not be proven beyond any doubt. Apart from this reduced germination rate the analyzed cry3 lines did not show any clear phenotypic differences compared to wild type plants. Cry3 does not seem to affect blue, green or UV light dependent regulation of plastid genes in Arabidopsis. Growth of Arabidopsis plants treated with elevated levels of UV-B light is not affected by cry3, although cry3 has been described to repair UV lesions in vitro in single stranded DNA and in double stranded DNA with loop structures. The results of this thesis clearly disagree with a function for cry3 as a DNA repair enzyme, because the PCR based repair assay described here could not detect any effect of cry3 on the repair activity in chloroplasts, mitochondria or in the nucleus.