Abstract

This thesis describes my research in the cell and molecular biology of the chloroplast of the green alga Chlamydomonas reinhardtii. Following a general introduction to all relevant topics in Chapter 1, Chapters 2 to 4 present different projects, each with the organization of a publication.
Chapter 2 describes my investigation of the management of oxidized RNA in the chloroplast a semi-autonomous, bacterial-type cell organelle. I show that the large subunit of Rubisco, RBCL, has a “moonlighting” function in controlling the level of oxidized RNA in the chloroplast. I also show in this chapter that a complex of RBCL, correlates with the RBCL moonlighting function, with results of native polyacrylamide gel electrophoresis and size-exclusion chromatography. The identification of this RBCL complex and aggregated form is a step towards understanding how RBCL mitigates RNA oxidation.
Results of immunofluorescence microscopy reveal that oxidized RNA localized in the pyrenoid, a chloroplast micro-compartment where CO2 is assimilated by the Calvin cycle enzyme Rubisco. This finding, together with previous research of chloroplast stress granules, provoked my interest in the potential functions of pyrenoid in RNA metabolism and led me to undertake a proteomic characterization of this microcompartment. Chapter 3 reports a partial pyrenoid proteome. I optimized the subcellular fractionation methods to obtain pyrenoid-enriched fractions and devised a means of identifying contaminant proteins, by preparing equivalent fractions from mutants that lack a pyrenoid. Results of bioinformatic analyses of this pyrenoid proteome further substantiate its role in RNA metabolism, and also confirm its long-suspected role in starch metabolism. The results also suggest additional unexpected possible functions in translation, lipid metabolism, chlorophyll biosynthesis and stress responses.
Chapter 4 describes results that further support the role of the pyrenoid in chlorophyll synthesis and reveal other new and unexpected findings. The localization of chlorophyll synthesis could closely relate to the assembly of photosystem or light harvesting complex proteins. These results also give hints regarding the location(s) of thylakoid biogenesis. My work reveals a novel function of the photosynthesis protein RBCL in the control of oxidized RNA and the important potential functions of the pyrenoid, in RNA metabolism and chlorophyll biosynthesis.