Functional analysis of chloroplast genes by chloroplast transformation

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This research purpose is to define the function of hypothetical chloroplast reading frames (ycf) and to understand the mechanism of regulation of chloroplast gene expression. The novel findings obtained are as follows.1. Two targeted ycf5-disruptants were obtained. These disruptants were uncapable of electron-fow from photosystem II to photosystem I.This indicates that ycf5 is involved in electron transport in photosynthesis.2. The gene encoding a small stable RNA (tmRNA) was isolated and characterized in the unicellular cyanobacterium, Synechococcus PCC6301. A tmRNA gene is not present in higher plant chloroplast DNA, suggesting that a ancestral chloroplast tmRNA gene lost during evolution.3. By immunoprecipitation, gel filtration, and western blot analysis, we demonstrated that tobacco chloroplast ribonucleoproteins (cpRNPs) are abundant stromal proteins that exist as complexes with ribosome-free mRNAs. In addition, an in vitro mRNA degradation assay revealed that the cpRNPs act as stabilizing factors for nonribosome-bound mRNAs in the stroma.4. We isolated and characterized the three genes, NsRpoT-A, NsRpoT-B and NsRpoT-C, encoding phage-type single subunit RNA polymerase from Nicotiana sylvestris. The gene product of NsRpoT-B was demonstrated to be imported into mitochondria and plastids. This provides a new insight of evolution of plant organelles.