Aerobic organisms, from bacteria to plants and animals, have evolved sensing, signaling, and protective mechanisms to cope with various reactive oxygen species (ROS) that are inevitably generated both intra- and extracellularly. Elucidation of these mechanisms is a problem of broad biological significance. Although cellular responses to ROS such as hydrogen peroxide and superoxide have been extensively studied, relatively little is known about biological responses to singlet oxygen, an especially toxic type of ROS that causes oxidative damage to numerous critical cellular components, such as unsaturated lipids, proteins, and DNA. Singlet oxygen is commonly generated by photosensitization reactions that are the basis for the photooxidative tissue damage observed in humans suffering from porphyria and possibly age-related macular degeneration. On the other hand, photosensitization has been exploited in strategies for photodynamic therapy of certain human cancers. This proposal aims to address the relative deficiency of knowledge about singlet oxygen signaling by using molecular genetic and genomic approaches to dissect the singlet oxygen acclimation response in a unicelluar green alga, Chlamydomonas reinhardtii, a model photosynthetic eukaryote. Like other oxygenic phototrophs, Chlamydomonas must cope with high endogenous concentrations of both oxygen and chlorophyll, a potent photosensitizer and may therefore have evolved particularly robust mechanisms for perceiving and responding to singlet oxygen.
The specific aims of this proposal are (1) to perform physiological and biochemical characterization of the singlet oxygen acclimation response in Chlamydomonas, (2) to use whole-genome microarray analysis to identify the set of genes whose expression changes during acclimation to singlet oxygen, (3) to use reverse genetics approaches to test the necessity of specific candidate genes in singlet oxygen acclimation, and (4) to perform a forward genetic dissection of singlet oxygen acclimation, including (5) the isolation of a gene that is defective in an already identified acclimation mutant, called sos1. This investigation will help illuminate studies of singlet oxygen biology in other organisms and provide fundamental knowledge about how eukaryotic cells sense and respond to singlet oxygen.
