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Lezhneva, Lina (2005): Identification of novel nuclear factors required for chloroplast gene expression and photosystem I assembly. Dissertation, LMU München: Faculty of Biology
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Abstract

During evolution of photoautotrophic eukaryotes, the nucleus has gained a dominant role in the coordination of the integrated genetic system of the cell consisting of three specifically coevolved genetic compartments. The photosynthetic machinery is encoded by the chloroplast and nuclear genomes. Therefore, biosynthesis and assembly of stochiometric amounts of subunits as well as association of the proteins with corresponding cofactors need to be managed and precisely regulated. To identify novel nuclear-encoded factors involved in the regulation of chloroplast gene expression at different levels, 12 nuclear mutants with high chlorophyll fluorescence (hcf) phenotypes denoting quite diverse defects in the photosynthetic apparatus were selected. Three of them, hcf145, hcf109 and hcf101, were analysed and the affected genes were characterized in more detail. Spectroscopic, fluorimetric and immunological studies have revealed that hcf145 and hcf101 were predominantly affected in photosystem I (PSI), while hcf109 had pleiotropic deficiencies. Remarkably, the dramatic reduction of PSI core complex accumulation in hcf145 was not accompanied by corresponding deficiencies of the outer light-harvesting antenna complex. A comparison of stationary transcript levels with rates of transcription, as estimated by Northern and chloroplast run-on transcription analysis, revealed that the hcf145 mutant is primarily and specifically characterised by a reduced stability of tricistronic chloroplast psaA-psaB-rps14 transcripts. The corresponding operon encodes the two large PSI polypeptides PsaA and PsaB, which form the heterodimeric PSI reaction centre, and the ribosomal protein S14. Chloroplast translation inhibition experiments excluded translational defects as the primary cause of impaired mRNA stability. Defined intervals of the tricistronic transcript were quantified by real-time RT-PCR which established that the psaA region is less stable than the rps14 region in hcf145. Therefore, although up to date, no 5'-3' exoribonucleases have been found in eubacteria (including the ancestors of plants), factor HCF145 appears to be required for the protection of the psaA-psaB-rps14 mRNA against progressive ribonucleolytic degradation starting at the 5' end. In the hcf109 mutant, exclusively plastid transcripts containing UGA stop codons are unstable. The affected gene encodes the first described chloroplast peptide chain release factor AtprfB. Its full-length cDNA, introduced into hcf109 via Agrobacterium-mediated transformation, could functionally complement the mutant. Homology of AtprfB to eubacterial release factors indicates that processes of translational termination in chloroplasts resemble those in eubacteria. The mutant phenotype revealed that translation of all plastid mRNAs containing UGA stop codons is exclusively terminated by AtprfB. However, besides its peptide chain release function, AtprfB appears to acquire yet unknown roles in regulating the stability and translation of the chloroplast mRNAs containing UGA stop codons. These additional regulatory functions could reflect evolutionary constraints which keep the number of plastid TGA stop codons high in vascular plant organelles in contrast to those of algae, mosses and ferns. In contrast to hcf145, steady-state levels and translation of photosynthetic transcripts are not altered in the PSI mutant hcf101. Separation of thylakoid membrane complexes by sucrose gradient centrifugation has uncovered that, similar to hcf145, accumulation of the outer antenna of PSI is not changed in hcf101. Therefore, hcf101 is affected in the assembly of the PSI core complexes. Expression of the HCF101 full-length cDNA in the hcf101 genetic background functionally complemented the mutant. The HCF101 protein encodes a very ancient and universally conserved protein of P-loop ATPases. HCF101 is plastid-localised and represents the first described factor essentially required for the assembly of PSI and other [4Fe-4S]-containing protein complexes in the chloroplast. Relatives of HCF101 are divided into four classes present in all organisms and in all cellular compartments. The antiquity of HCF101 points to the importance of Fe-S cluster biogenesis during the earliest phases of cell evolution. The ubiquity of HCF101 indicates that it is essential for all free-living cells.