Alan C. Christensen
Date of this Version
Ericsson SR. 2021. Mitochondrial Genome Structure and Double Strand Break Repair in Arabidopsis Thaliana. Master's diss., School of Biological Sciences, University of Nebraska-Lincoln
The mitochondrial genomes of plants are known for their variability in size and arrangement, yet low mutation rates in coding sequences. These opposing characteristics have been suggested to be due to the stability of their DNA maintenance processes and the usage of a double strand break repair (DSBR) system. The components of this repair complex remain poorly understood and only a few of the involved enzymes have been identified, making direct analysis difficult. The flowering plant species Arabidopsis thaliana has been used as a model organism for genome modification and subsequent phenotypic and genotypic impacts. The aims of this research were to determine the effectiveness of restriction enzymes inserted into a plant cell to cause mitochondrial double strand breaks (DSBs), to see how these breaks impact recombination at repeat sequences undergoing DSBR, and to determine the influence of stress responses on the functionality of the DSBR system. To this end, a wild type Arabidopsis was inoculated with the horizontal gene transfer bacteria Agrobacterium tumefaciens containing a designed gene cassette. This cassette included the sequence for the PvuII restriction enzyme to induce DSBs, an ethanol inducible promoter for the PvuII sequence, and a mitochondrial-targeting peptide (from the AOX1A gene) to ensure transport into the mitochondria. The transgenic PvuII plants were subjected to various stress conditions, including reduced light and cold stress, and the effect of these stresses on recombination rates following ethanol induction of the transgenic restriction enzyme were compared. Recombination was investigated using the L repeat sequence in the Arabidopsis mitochondrial genome and qPCR analysis of the L1 and L2 repeat locations. The experimental groups for reduced light and cold stress, with and without ethanol induction, showed increased recombination after induction of PvuII, and altered recombination accumulation after stress was also applied. This shows that DSBs can be successfully induced in plant mitochondria through inserted restriction enzymes and that this results in higher recombinant sequence accumulation during DSBR, along with stresses changing the functionality of this repair.
Advisor: Alan Christensen