In vivo Analysis of the Role of FtsZ1 and FtsZ2 Proteins in Chloroplast Division in Arabidopsis thaliana

Chloroplasts divide by a constrictive fission process that is regulated by FtsZ proteins. Given the importance of photosynthesis and chloroplasts in general, it is important to understand the mechanisms and molecular biology of chloroplast division. An FtsZ gene is known to be of prokaryotic origin and to have been transferred from a symbiont's genome to host genome via lateral transfer. Subsequent duplication of the initial FtsZ gene gave rise to the FtsZ1 and FtsZ2 genes and protein families in eukaryotes. These proteins co-localize mid-chloroplast to form the Z-ring. Z-ring assembly initiates chloroplast division, and it serves as a scaffold for other chloroplast division proteins. Little is known, however, about the FtsZ protein subunit turnover within the Z-ring, the effects of accessory proteins on Z-ring turnover assemblies, as well as the in vivo ultrastructure of the Z-ring in plants. To investigate the Arabidopsis thaliana FtsZ subunit turnover rate within the Z-ring, a section of the Z-ring in the chloroplasts of living plants expressing fluorescently tagged FtsZ1 or FtsZ2 proteins was photobleached and the recovery rate was monitored. The results show that the fluorescence recovery half times for the FtsZ1 and FtsZ2 proteins are 117s and 325s, respectively. This is significant as these data mirror their differences in GTP hydrolysis rates.

To elucidate in vivo structure and ultrastructure of the Z-ring, a protocol was established that maintained fluorescence during high pressure freezing, freeze substitution and low temperature embedding. Afterwards, a correlative microscopy approach was employed to visualize and identify fluorescently labeled puncta, circular structures, at the light microscopy level. These puncta were further resolved as mini-rings using optical microscopy eXperimental (OMX) superresolution microscopy. Electron microscopy (EM) analysis imaged mini-rings and filament assemblies comprised of dense subunits. Electron tomography (ET) showed mini-rings composed of protofilaments.