Abstract

Reactive oxygen species (ROS) are known to oxidize macromolecules, causing them to be non-functional or harmful. When adverse conditions cause the production of ROS to exceed the capacities of antioxidant responses, oxidative stress arises. During stress, RNA granules called chloroplast stress granules form in the chloroplast of Chlamydomonas reinhardtii. It is hypothesized that the large subunit of Rubisco, RbcL, has a moonlighting function in oxidative stress tolerance as it localizes to chloroplast stress granules without the small subunit (RbcS), and has an RNA-binding activity that is activated under oxidizing conditions (Uniacke & Zerges, 2008; Yosef et al., 2004). This thesis analyzes multiple RbcL mutants to 1) identify the supramolecular state of RbcL that accumulates during hydrogen peroxide-induced stress tolerance, and 2) characterize whether certain regions of the protein are required for the moonlighting function. My results reveal that it is specifically the RbcL dimer, as well as select mutations in the RNA-binding domain and cysteines, that are involved in this alternate function. I further examine if a correlation exists between enhanced stress tolerance and a Triton X-100 insoluble pool of RbcL. This RbcL subpool was found in four of seven mutants with elevated stress tolerance, partially supporting this hypothesis. I also investigate where this non-Rubisco form of RbcL localizes in the chloroplast. Immunofluorescence microscopy results show RbcL in punctate bodies in mutants that have both excess Triton X-100 insoluble RbcL and elevated stress tolerance. This raises the possibility that RbcL in punctate structures carries out the moonlighting function.