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b'Plastid chromosomes from the variety of plant species contain several conserved open reading\\nframes of unknown function, which most probably represent functional genes. The primary\\naim of this thesis was the analysis of the role of two such ORFs, designated ycfs or\\nhypothetical chloroplast reading frames, namely ycf9 (ORF62) and ycf10 (ORF229, cemA).\\nBoth were analyzed in Nicotiana tabacum (tobacco) via their inactivation using biolistic\\nplastid transformation. A new experimental protocol, based on pulsed-field gel\\nelectrophoresis (PFGE), was established to reliably assess the homoplastomic state of\\ntransformed plants.\\n1. Functional analysis of the ycf9 gene product: The inactivation of ycf9 in N. tabacum as\\nwell as in Chlamydomonas reinhardtii yielded a homoplastomic mutant phenotype after\\nseveral rounds of regeneration under selective pressure. The mutant plants grew\\nphotoautotrophically, but displayed two clear phenotypes, a light-sensitive one, increasing\\nwith the light intensity, and a dwarf phenotype under low-light combined with\\ntemperatures below 20\\xb0C. The ycf9 gene product was exclusively located in PSII core\\ncomplexes. This localization was based on the isolation of protein complexes released\\nfrom thylakoids by controlled, partial lysis, followed by sucrose density gradient\\ncentrifugation or 2D gel electrophoresis. This finding revised data of the literature.\\nBiochemical analysis indicated an involvement of the protein in the interaction of the light\\nharvesting antenna II complex (LHCII) with PSII cores. In particular, PSII-LHCII\\nsupercomplexes could no longer be isolated from transplastomic tobacco plants.\\nFurthermore, the minor chlorophyll a/b-binding proteins CP26, and to a lesser extent\\nCP29, were substantially reduced under most growth conditions analyzed, in both,\\ntobacco and photoautotrophically grown Chlamydomonas mutants (Swiatek et al. 2001).\\nThe gene was therefore renamed psbZ. The \\u2206psbZ-related alterations in the\\nsupramolecular organization of PSII complexes were accompanied by considerable\\nmodification in (i) the phosphorylation pattern of PSII subunits, (ii) the rate of deepoxydation\\nof xanthophylls, and (iii) the kinetics and amplitude of non-photochemical\\nquenching. The proposed position of PsbZ in close proximity to CP43 enables the protein\\nto interact with PSII cores to elicit an adaptation process in response to excess light\\nexcitation. The molecular mechanism underlying this energy dissipation process remains\\nto be investigated. 2. Functional analysis of the ycf10 gene product: Biolistic plastid transformation was also\\nused to inactivate the ycf10 reading frame in tobacco. After several rounds of regeneration\\nunder selective pressure, homoplastomic plants were obtained. Northern analysis\\nuncovered co-transcription of ycf10 within the psaI-ycf4-ycf10-petA gene cluster, with at\\nleast two promotor regions upstream of the psaI gene. The mutant plants grew\\nphotoautotrophically and developed dark green leaves with numerous pale green to white\\nregions, the latter devoid of photosynthetic activity. The loss of ycf10 did not affect\\nphotosynthetic activity, as indicated by unaltered chlorophyll fluorescence. The tobacco\\nycf10 gene product was localized in the chloroplast inner envelope membrane. Neither\\nprotein composition of stroma or thylakoid fractions, nor the stability of the\\nphotosynthetic protein complexes were affected in the mutant plants. In contrast, CO2-\\ndependent oxygen evolution was strongly reduced, with a maximum rate of Ci-dependent\\nphotosynthesis being approximately 50% lower than in wild-type plants. Two\\nexplanations can account for the observed phenomenon: (i) de-regulation of carbonconcentrating\\nmechanisms in transformed cells, or (ii) an indirect effect on CO2-uptake in\\n\\u2206ycf10 plants.\\n3. Pulsed-field gel electrophoresis is an ideal tool to verify the homoplastomic state of\\ntransformed plants: To enhance the sensitivity of detection of heteroplastomic states, and\\nto distinguish between plastome-located wild-type segments in transplastomic material\\nand promiscuous DNA, a new approach was developed. Customary Southern and PCR\\ntechniques are not sensitive enough or not discriminating the latter alternatives,\\nrespectively. Pulsed-field gel electrophoresis allows to isolate virtually contamination-free\\nplastid DNA. Plastid DNA isolated this way lacked traces of nuclear and mitochondrial\\nDNA at a detection level of 50 DNA molecules. This excludes that gene-specific PCR\\namplification products originate from promiscuous nuclear or mitochondrial gene copies.\\nTherefore, PFGE appears to be an ideal tool to investigate the homoplastomic state of\\ntransformed plants, especially when combined with radiolabeled probes and Southern\\ntechniques.'