| Eukaryotic cells are highly compartmentalized. Many processes and metabolic pathways occur within specific organelles such as the nucleus, endoplasmic reticulum, Golgi apparatus, mitochondria, and peroxisomes. These compartments themselves are highly compartmentalized. In plants and algae, chloroplasts carry out photosynthesis, the biosynthesis of lipids, pigments, cofactors, amino acids, and function in the assimilation of S, P, and N. However, chloroplast cell biology has lagged behind that of other organelles. Classic models for the spatial organization of these processes are contradictory and based on EM and subcellular fractionation with no use of modern fluorescence microscopy. Here, chloroplast processes are revealed as highly compartmentalized within the chloroplast of Chlamydomonas reinhardtii with the first extensive use of fluorescence in situ hybridization, immunofluorescence staining and confocal microscopy. In Chapter 2, I propose that the processes underlying the biogenesis of thylakoids are compartmentalized in the chloroplast of C. reinhardtii. Chapter 4 describes the discovery of chloroplast stress granules, a compartment that manages mRNAs during oxidative stress. Chapter 3 assigned known targeting mechanisms to specific chloroplast proteins while identifying new examples of localized translation. Because the pyrenoid, a largely unexplored chloroplast compartment in most algae, plays a central role in many of my findings, and little is known about this compartment, I isolated it and characterized its proteome by mass spectrometry. The pyrenoid proteome consisted of proteins with roles in starch metabolism, CO2 assimilation and nitrite reduction. Taken together, these findings reveal that the Chlamydomonas chloroplast is more highly compartmentalized and complex than has been appreciated and that it can be used as a model system to study fundamental cell biological questions. This work has advanced our understanding of the cell biology of chloroplasts and provides a new conceptual framework for research into chloroplast biogenesis. |
