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Dal Bosco, Cristina (2006): The role of thylakoid ATP synthase subunit gamma and attempts to transform the organelles of A. thaliana. Dissertation, LMU München: Faculty of Biology
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Abstract

ATP synthase is one of the major photosynthetic complexes that represents one of the smallest molecular motors known in nature. The rotating γ subunit is a key feature of this enzyme. It contains features specific for the chloroplast ATP synthase. In this work the γ subunit has been functionally analyzed in Arabidopsis thaliana. - The nuclear gene atpC1 encoding the γ subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis. In the seedling-lethal dpa1 mutant the absence of detectable amounts of the γ subunit destabilizes the entire ATP synthase complex and consequently photophosphorylation is abolished. However, in vivo protein labelling analysis suggests that assemαβ bly of the ATP synthase and subunits into the thylakoid membrane still occurs in dpa1. Further effects of the mutation include an increased light sensitivity of the plants and an altered photosystem II activity. A high non-photochemical quenching develops with increasing actinic light intensity. It has been shown that a high proton gradient is responsible for most quenching (qE). The photoprotective role of qE was further demonstrated in the double mutant dpa1 x psbS in which PsbS, essential factor for qE, is missing. - The expression of a second gene copy, atpC2, is unaltered in dpa1 and is not sufficient to compensate for the lack of atpC1 expression. The two proteins, AtpC1 and AtpC2, share less similarity than AtpC1 of Arabidopsis with γ subunits of other plant species suggesting that the γ subunits so far isolated in other plant species are AtpC1 orthologs. It has been established that AtpC2 is also imported into the chloroplast. Therefore, it is likely that the chloroplast ATP synthase complexes contain both atpC1 and atpC2 encoded γ subunits. However, the atpC2 gene is expressed more than hundred times at a lower level than atpC1 and array data show the differential and tissue specific expression of the two genes. The function of AtpC2 could not be revealed by inactivating the gene. Overexpression of atpC2 in dpa1 generated viable lines with an ATP synthase complex containing only γ2, although wild type phenotype is not completely restored. The second part of this work regarded the optimization of conditions for plastid transformation in Arabidopsis thaliana. An efficient and fast regeneration system from cotyledon protoplasts was established for Arabidopsis thaliana accessions C24, Columbia, and Wassilewskija. Culture conditions and media compositions were optimized for the development of protoplasts embedded in thin alginate layers. The protocol is reproducible, efficient, extremely fast, and regenerated plants are fertile. Thus, this cotyledon-based system could prove useful for studying plant cell and molecular biology in A. thaliana. - The sul gene appeared to be a potential novel candidate as selectable marker for plastid transformation. However, genetic and molecular studies demonstrated that sul can not be used for this purpose. On the other hand a new function of sul appeared. The gene could be the missing marker for mitochondria transformation in higher plants.