|dc.description.abstract||In mature chloroplasts, the DNA (cpDNA) is surrounded by a potentially genotoxic environment that would make the mitochondrial DNA milieu look like a “nadree” (picnic). And yet, the slower evolution of cpDNA compared to other cellular genomes suggests that this organelle must have efficient mechanisms for repairing DNA. Unfortunately, those mechanisms have been barely noted, much less studied. This dissertation describes a novel approach that was developed to study how chloroplasts of Arabidopsis repair the most severe form of DNA damage, a double-strand break (described in Chapter 2). The success with this approach also prompted the development of a new method for site-specific modification of tobacco cpDNA that is described in Chapter 3.
To study the consequences and repair of a break in the circular plastid genome, we developed an inducible system based on a psbA-intron endonuclease from Chlamydomonas (I-CreII) that specifically cleaves the psbA gene of Arabidopsis. The protein was targeted to the chloroplast using the rbcS1 transit peptide, and activation of the nuclear gene was made dependent on an exogenous inducer (β-estradiol). In Chlamydomonas, I-CreII cleavage at psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20-60 bp) that are abundant in that genome. By comparison, Arabidopsis cpDNA is very repeat-poor. Nonetheless, phenotypically strong and weak transgenic lines were obtained, and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of psbA, but not cpDNA as a whole, in the strongly-expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site that were indicative of repair using microhomology (6-12 bp perfect repeats, or 10-16 bp with mismatches) or no homology. The results provide evidence of alternative repair pathways in the Arabidopsis chloroplast that resemble the nuclear microhomology-mediated and nonhomologous end-joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, plus other considerations, the data suggests that an evolutionary relationship may exist between the repeat structure of cpDNA and the organelle’s ability to repair broken chromosomes.
Taking advantage of the inducible I-CreII system, I developed a method to delete defined regions of cpDNA in tobacco, which was named DREEM (for direct repeat and endonuclease mediated). Chloroplast transformation was used to introduce an I-CreII cleavage site adjacent to an aadA:gfp marker and flanked by a direct repeat of 84 bp. When chloroplast-targeted I-CreII was induced with β-estradiol during germination, complete loss of the aadA:gfp marker occurred by SSA-type repair involving the 84-bp direct repeat. I obtained additional evidence for DREEM effectiveness by deleting 3.5 kb of native cpDNA that included part of the large ycf1 gene. DREEM can be used for other modifications besides gene deletions, partly because it is seamless and leaves no trace of introduced DNA. Since expression of the endonuclease is controlled by steroid application (and concentration), and the deleted cpDNA is probably destroyed during the SSA process, this inducible gene-ablation technique could enable the study of essential chloroplast genes in vivo.||en