Plastid division in plants is orchestrated by a complex macromolecular apparatus, the components of which are mostly unknown. The functions of a set of predicted and genetically defined Arabidopsis gene products likely to participate in plastid division will be determined. The proteins to be investigated derive from three sources: 1) predicted proteins having sequence or structural homology to known plastid division, bacterial cell division, mitochondrial fission or fusion, or microtubule-based motor proteins; 2) genes defined by the Arabidopsis arc (accumulation and replication of chloroplasts) mutants; and 3) genes encoding proteins with homology to cyanobacterial cell division proteins, to be identified by transposon mutagenesis in Synechococcus PCC 7942. Functional studies will include examination of knockout lines by microscopy for abnormalities in chloroplast number, size, shape, ultrastructure and FtsZ localization pattern, biochemical assays of protein targeting and topology, analysis of in vivo localization using GFP fusion proteins and protein-specific antibodies, and analysis of protein interactions using immunoprecipitation and yeast two-hybrid techniques. The results obtained from functional investigations will be integrated to arrive at a model describing the macromolecular organization and biochemical activity of the chloroplast division complex from which new hypotheses can be derived and tested. This research will be relevant to scientists interested in a variety of biological problems, including organelle fission and dynamics, macromolecular organization, organelle evolution, and plant development. cDNA clones, plasmids, antibodies and other useful reagents generated during these studies will be made available to the broader scientific community. Functional information will be communicated through publication of results and through a website (www.msu.edu/~osteryou/2010.htm) to be updated regularly to reflect data obtained on knockout and arc mutant phenotypes. Database annotations will be revised as new information is obtained. In addition to providing training opportunities to graduate students and postdoctoral scholars, the project is expected to provide research experience to several undergraduate students, including members of underrepresented groups.
