Reverse genetics in higher plant chloroplasts
Bock, R., Ruf, S. and Drescher, A.
Institut für Biologie III, Universität Freiburg, Schänzlestraße 1, D-79104 Freiburg, Germany
The complete sequence analysis of the tobacco chloroplast genome more than ten years ago marks a milestone in plastid genetics and has had a profound influence on our understanding of the structure and function of plant organellar genomes. Detailed computer analyses of the sequence data allowed the identification of numerous regions potentially encoding novel proteins. In the following years, most of these open reading frames could be assigned to functional gene products involved in either genetic system functions or in photosynthesis. However, there are about ten conserved reading frames left the function of which is still elusive.
The recent development of a transformation technology for tobacco chloroplasts has enabled us to address functional aspects of plastid open reading frames by reverse genetics. By deletional or insertional mutagenesis we can create null alleles for any plastid genome-encoded open reading frame to reveal the phenotype of plants deficient for the respective gene product. We generated such mutants for some of the open reading frames encoded in the tobacco plastome and characterized the mutant phenotypes by employing physiological, biochemical and molecular biological techniques.
This approach resulted in the identification of novel plastid-encoded photosynthesis genes but also led to the discovery of genes that are essential for plant viability even under heterotrophic culture conditions. Such chloroplast transformants remain heteoplasmic and contain a mixture of wild type and transformed genomes. Typically, we observe a balanced selection in the presence of the antibiotic (i. e. maintenance of a constant ratio of wild type vs. transformed genome copies). Under non-selective conditions, however, this heteroplasmy is unstable and eventually results in sorting out towards the wild type.
Svab, Z. and Maliga, P. (1993). High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc. Natl. Acad. Sci. USA, 90, 913-917.
Ruf, S., Kössel, H. and Bock, R. (1997). Targeted inactivation of a tobacco intron-containing open reading frame reveals a novel chloroplast-encoded photosystem I-related gene. J. Cell Biol., 139, 95-102.
| LOCATION |
DATE |
TIME |
| Lecture Hall I |
Wednesday, April 8 |
02:00 pm |