PROJECT SUMMARY Mitochondrial mutations occur at a very high rate in humans and are a major cause of inherited and age- related diseases. Although elevated mutation rates have long been considered a byproduct of the intense metabolic activity that occurs within mitochondria, recent evidence has called this view into question, creating enormous uncertainty in the field about the causes of mitochondrial mutations. In contrast to humans, some eukaryotes exhibit extremely low rates of mutation in their mitochondrial DNA. Answering the question of how some organisms are able to maintain low mitochondrial mutation rates has the potential to inform our understanding of what causes them to be so high in humans. Remarkably, however, little effort has been made to address this fundamental question of eukaryotic genetics. The proposed research will focus on flowering plants as a model for understanding the mechanisms responsible for variation in mitochondrial mutation rate. Rates of mitochondrial (and plastid) DNA substitutions in plants are generally lower than in plant nuclear genomes and orders of magnitude lower than in animal mitochondria. However, plants also exhibit extreme fluctuations in rates of mitochondrial sequence evolution even among closely related species. Progress in understanding the mechanisms responsible for the extremely low rates in most plant species has been impeded by the inherent technical difficulties in studying rare mutation events. The advent of new methodologies that leverage deep sequencing and quantitative PCR technologies to directly measure rare mutations and quantify rates of DNA damage presents an exciting opportunity to overcome these historical barriers. The proposed research will apply these methodologies to both wild-type and mutant backgrounds in the model angiosperm Arabidopsis thaliana to test a suite of alternative hypotheses, relating to the fidelity of DNA polymerases, the efficacy of recombinational repair mechanisms, the effects of biased gene conversion, and exposure/susceptibility to DNA damage in plant organelles. Analyses will be conducted on both vegetative and meristematic tissues to distinguish mutations that simply accumulate in plant tissues from those that are actually transmitted to offspring. The research will also be extended to related species of flowering plants in which there has been a recent and massive acceleration in rates of mitochondrial sequence evolution. These investigations will elucidate the mechanisms responsible for variation in mitochondrial mutation rates across eukaryotes and inform ongoing debates about the role of oxidative damage as a mutagenic force in human mitochondria.