Mechanisms for maintenance, replication, and repair of the chloroplast genome in plants

Photosynthesis is a complex process that occurs in chloroplasts of higher plants, and requires a large number of proteins to assemble the photosynthetic machinery. Many chloroplast-localized proteins are nuclear-encoded and must be imported into the chloroplasts from the cytoplasm. A considerable number of genes for photosynthesis and other chloroplast functions, including transcription and translation, are encoded in the chloroplast genome (ctDNA), which ranges in size from about 130–160 kbp in most higher plants. CtDNA replication is not linked with the plant cell cycle and the chloroplast genome can be amplified to a very high copy number per cell in rapidly dividing leaf tissue. Later in leaf development and plant growth, the ctDNA levels reduce to very low levels (Oldenburg and Bendich, 2004b). The controls that regulate ctDNA replication initiation, replication, and copy number are not understood. From earlier publications on a number of plant species it appears that ctDNA may replicate by more than one mechanism, including a recombination-dependent replication mechanism (Rowan et al., 2010, this issue; Oldenburg and Bendich, 2004b; Marechal and Brisson, 2010), a double D-loop mechanism (Chiu and Sears, 1992; Kunnimalaiyaan and Nielsen, 1997a, b), and rolling circle replication (Kolodner and Tewari, 1975). In this issue, Rowan et al. (2010) report on the role of chloroplast-targeted RecA (cpRecA) in the maintenance of ctDNA in Arabidopsis. Previously published reports provide evidence that some ctDNA molecules may be recombination intermediates as shown by the presence of branched DNA molecules in some DNA preparations (Oldenburg and Bendich, 2004a, b; Scharff and Koop, 2007). As summarized in a review by Marechal and Brisson (2010), recombination has been shown to be involved in the repair of double-strand breaks and point mutations in ctDNA. It has been known for some time that a plant homologue of bacterial RecA is localized in chloroplasts (Cerutti et al., 1992), but, to date, little is known about the role of DNA recombination in the maintenance of ctDNA. Rowan et al. (2010) show clear evidence that cpRecA is involved in the maintenance of the chloroplast genome copy number in plants, as T-DNA insertions (from the Agrobacterium Ti plasmid) in the nuclear gene encoding this protein led to a reduction in ctDNA copy number in the mutant plants relative to wild-type plants and to a change in the structure of the ctDNA. The levels of detectable single-stranded DNA increased in the mutants, which is compatible with the decreased amount of cpRecA which would normally coat the single-stranded DNA regions and thus block its detection. After a few generations the mutants began to show significant signs of distress and reduced chloroplast function, including variegation and necrosis. These findings represent a significant advance in our understanding of the mechanisms involved in the maintenance of ctDNA integrity. The authors suggest that the role of cpRecA is primarily in DNA repair, as supported by the analysis of wild-type plants that have been treated with ciprofloxacin, which induces double-strand DNA breaks. In these plants, altered ctDNA structures were observed as in the cpRecA plants. Similar experiments with insertions in the DRT 100 homologue, which has only very weak homology to bacterial RecA but can partially complement E. coli recA mutants showed no effect, suggesting that DRT 100 may not be directly involved in the repair of ctDNA. The role of cpRecA in DNA repair is clearly supported by these experiments; it is also possible that cpRecA may be involved in recombination-mediated replication of the chloroplast genome. CpRecA and DRT 100 are not the only RecA homologues localized to chloroplasts. A dual-targeted (to both chloroplasts and mitochondria) RecA (distinguished from the others as RecA2) has been identified in the Arabidopsis nuclear genome (Christensen et al., 2005). T-DNA insertions in this gene lead to non-viable plants (BL Nielsen, JD Cupp, unpublished observations; Shedge et al., 2007), suggesting that RecA2 may be essential for ctDNA and/or mtDNA maintenance and plant development. However, at this point in time there are no data to determine whether the lethal phenotype is due to the disruption of chloroplast or mitochondrial DNA maintenance mechanisms, or both. The RecA2 gene was not included in the current study by Rowan et al. (2010, this issue) but its role in ctDNA replication should be evaluated. The observation that T-DNA insertions in cpRecA were not lethal may be due to functional (at least partial) complementation by RecA2.