Besides the photosynthetically active chloroplasts, a number of differentiated plastid types have been found in various organs of plants. In Lycopersicon esculentum, chloroplasts develop into functionally different chromoplasts during fruit ripening. The chloroplast/chromoplast conversion is accompanied by major ultrastructural changes and loss of the photosynthetic apparatus. This project is focused on the expression of photosynthesis-specific genes, such as the large subunit of the ribulose-1.5-bisphosphate carboxylase (rbcL) and the Mr 32.000 polypeptide (psbA), and on understanding mechanisms involved in the regulation of these genes during chromoplast development. In addition, we will attempt to identify plastid genes which might be expressed only in chromoplasts. Since plastid genomes are of small size, easily cloned in fragments of desired size as recombinant DNAs, and cloned genes can be transcribed in vitro, it is a feasible goal to determine the mechanisms for differential gene expression. The rbcL and psbA loci from L. esculentum chloroplast DNA will be cloned and sequenced with either the chemical cleavage or primed, dideoxy method. Characterization of rbcL and psbA transcription will be based on studies with homologous in vitro transcription systems from both chloroplasts and chromoplasts. These systems, together with modified DNA templates, will be used to precisely define the DNA sequences involved in initiation and regulation of transcription. Finally, we will construct a cDNA library from chromoplast RNA in order to identify and locate chromoplast-specific genes.
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