Intellectual merit: Chloroplasts are the subcellular compartments in plants and algae that harness the energy of sunlight and convert that energy into chemical energy in the form of sugar (glucose) via the process of photosynthesis. Evolutionarily, they are derived from endosymbiotic photosynthetic bacteria; they are structurally highly complex, with two enveloping membranes and an internal set of membranes termed 'thylakoids' where the photosynthetic enzymes reside. When plant and algal cells grow and divide, it is critically important that each daughter cell receive its share of chloroplasts. The division of chloroplasts in eukaryotic cells is coordinated by a topologically complex macromolecular machine that drives the constriction and fission of the two envelope membranes surrounding the organelle. The long-term goal of Dr. Osteryoung's laboratory is to elucidate the molecular pathway and biochemical mechanisms that orchestrate chloroplast division in plant cells. The studies that will be carried out with support from this award will focus on two chloroplast division proteins closely related to the key bacterial cell division protein FtsZ. FtsZ is a tubulin-like GTPase and essential ring-forming component of the bacterial cell division apparatus, and is usually encoded by a single gene in prokaryotes. Dr. Osteryoung's previous work has established that two distinct forms of FtsZ in plants, FtsZ1 and FtsZ2, are core components of the chloroplast division machinery, and that they co-localize to a dynamic mid-chloroplast ring that functions throughout division of the organelle. FtsZ1 and FtsZ2 are tightly colocalized in vivo, copurify in a soluble complex and can be co-immunoprecipitated, and interact directly in yeast two-hybrid assays. In addition, Dr. Osteryoung has demonstrated that, in the model plant Arabidopsis, FtsZ1 and FtsZ2 are maintained at a constant molar ratio of 1:2 throughout the plant's growth and development and that alterations in FtsZ1 or FtsZ2 levels in Arabidopsis confer dose-dependent defects in chloroplast division. Dr. Osteryoung will now build on these findings to investigate the biochemical relationship between FtsZ1 and FtsZ2. Specifically, this award will support the following aims: test the importance of FtsZ1:FtsZ2 stoichiometry and total FtsZ abundance for chloroplast division in Arabidopsis via genetic manipulation of their in vivo levels and ratios; analyze the in vitro GTPase activities of recombinant FtsZ1 and FtsZ2 separately and in combination, and investigate their polymer-assembly properties using recently developed fluorescence assays; and investigate the functional significance structural features that distinguish FtsZ1 and FtsZ2. These studies will lay the groundwork for the development of models describing the structural organization and dynamic properties underlying the activity of the chloroplast FtsZ ring during division of the organelle.
Broader impacts: Chloroplast division shares some similarities with the functionally and evolutionarily related processes of mitochondrial and bacterial cell division. Therefore, this research will deepen understanding of these fundamental aspects of eukaryotic and prokaryotic cell biology. The project will provide graduate student training in structural and kinetic analysis of protein complexes, and provide additional training opportunities for graduate students, postdoctoral scholars, and undergraduates.
