The role of homologous recombination in the production of debilitating mitochondrial DNA rearrangements in plants

Recombination plays a significant role causing mitochondrial disorders such as cytoplasmic male sterility as well as in the maintenance and expression of plant mitochondrial genomes. Despite this knowledge, the proteins involved and the mechanism by which recombination occurs in plant mitochondria remains uninvestigated. In bacteria the best studied mechanism of homologous recombination requires the RecA protein. These studies addressed whether the E. coli RecA protein could play a role in generating rearrangements that cause mitochondrial disorders. Over-expression of the E. coli RecA in plant mitochondria was anticipated to increase the number of mitochondrial DNA (mtDNA) rearrangements. An increase in mtDNA rearrangements was expected to result in the appearance of one or a combination of the following phenotypes: variegation, male sterility, improper leaf expansion and overall diminished growth or yield.; To test the hypothesis, constructs were created in which the E. coli RecA gene and its dominant negative derivative were ligated behind a mitochondrial targeting sequence and the CaMV promoter. Agrobacterium-mediated transformation was used to integrate these genes into the nucleus of Arabidopsis thaliana (Columbia and chm lines) as well as Nicotiana tabacum. These studies showed that the over-expression of E. coli RecA in Arabidopsis did not result in phenotypic manifestation of plant mitochondrial syndromes. In contrast phenotypic characteristics of mitochondrial syndromes were apparent in Nicotiana, where multiple homoetic-like changes were seen during floral development. These changes were expressed as a mosaic on each plant, with normal flowers appearing next to those with developmental abnormalities. Once these abnormalities were induced, their expression and transmission was independent of the presence of the E. coli RecA transgene. T1 plants showed additional characteristics of plant mitochondrial dysfunction including: infertility, dwarfism, changes in leaf expansion and variegation. To assess whether these abnormalities were maternally inherited, a back-cross population was generated. Maternal inheritance was confirmed because all the progeny exhibited some level of floral abnormality as well as infertility. Additional support for the mitochondrial basis of these changes came in the identification of a mtDNA RFLP in nine of the backcross plants.; The production of the plants provides a new resource in which to study the roles of mitochondrial segregation and threshold effects of a mixed population of mtDNA molecules. The transgenic plants are phenotypically novel compared to other plant mitochondrial mutants in that the manifestations of mitochondrial disorders are not uniformly presented. The appearance of floral abnormalities as a mosaic, in conjunction with the number and variability of the floral phenotypes, suggest that active segregation is occurring in the plants, and proper development of floral organs is responsive to thresholds of functional mitochondria.