| Reactive oxygen species (ROS) are harmful forms of oxygen and its derivatives, generated especially in cells of organisms that perform oxygenic photosynthesis. Plants and algae have evolved several ways to protect themselves from oxidative damage such as generation of antioxidants and related enzymes. Some antioxidants seem to have overlapping functions, which might act as a backup mechanism in conditions where one antioxidant cannot fully perform its function. It is likely that different antioxidants do not perform exactly the same role but rather have specific roles in protecting cells at a particular site in a particular condition.;I examined the role of tocopherols and their protective role against oxidative damage using the unicellular green alga Chlamydomonas reinhardtii as a model organism. I characterized the vte3 mutant, which lacks alpha-tocopherol, but instead accumulates beta-tocopherol. The mutant does not show an observable phenotype. However, the vte3 phenotypes emerged when studied in a background that lacks two carotenoids, lutein and zeaxanthin. My research showed that beta-tocopherol is better than alpha-tocopherol at protecting the carotenoid-deficient cells against lipid peroxidation.;Unlike the vte3 mutant in Arabidopsis, which also affects plastoquinone biosynthesis, the Chlamydomonas vte3 mutant only affects tocopherol biosynthesis. I used this mutant as a background strain for RNAi targeting a candidate gene for plastoquinone synthesis in Chlamydomonas. I identified one strain that has low plastoquinone level. The mRNA level of the target gene still needs to be examined to confirm that the phenotype is due to the RNAi construct. In addition to studying a lipid-soluble antioxidant, tocopherol, I also attempted to investigate the role of a water-soluble antioxidant, glutathione. I selected for mutants that are resistant to the N-nitroso compound N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG). I isolated three mutants but they are not glutathione-deficient as expected. They showed resistance to tert-butyl hydroperoxide and methyl viologen, and sensitivity to rose bengal, hydrogen peroxide, and metronidazole. Using map-based cloning, I identified a number of interesting candidate genes, many of which are related to oxidative stress response. |
