John D. Cupp

The Arabidopsis At1g30680 gene encodes a homologue to the phage T7 gp4 protein that has both DNA primase and DNA helicase activities

BackgroundThe Arabidopsis thaliana genome encodes a homologue of the full-length bacteriophage T7 gp4 protein, which is also homologous to the eukaryotic Twinkle protein. While the phage protein has both DNA primase and DNA helicase activities, in animal cells Twinkle is localized to mitochondria and has only DNA helicase activity due to sequence changes in the DNA primase domain. However, Arabidopsis and other plant Twinkle homologues retain sequence homology for both functional domains of the phage protein. The Arabidopsis Twinkle homologue has been shown by others to be dual targeted to mitochondria and chloroplasts.ResultsTo determine the functional activity of the Arabidopsis protein we obtained the gene for the full-length Arabidopsis protein and expressed it in bacteria. The purified protein was shown to have both DNA primase and DNA helicase activities. Western blot and qRT-PCR analysis indicated that the Arabidopsis gene is expressed most abundantly in young leaves and shoot apex tissue, as expected if this protein plays a role in organelle DNA replication. This expression is closely correlated with the expression of organelle-localized DNA polymerase in the same tissues. Homologues from other plant species show close similarity by phylogenetic analysis.ConclusionsThe results presented here indicate that the Arabidopsis phage T7 gp4/Twinkle homologue has both DNA primase and DNA helicase activities and may provide these functions for organelle DNA replication.

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.

Minireview: DNA replication in plant mitochondria.

Higher plant mitochondrial genomes exhibit much greater structural complexity compared to most other organisms. Unlike well-characterized metazoan mitochondrial DNA (mtDNA) replication, an understanding of the mechanism(s) and proteins involved in plant mtDNA replication remains unclear. Several plant mtDNA replication proteins, including DNA polymerases, DNA primase/helicase, and accessory proteins have been identified. Mitochondrial dynamics, genome structure, and the complexity of dual-targeted and dual-function proteins that provide at least partial redundancy suggest that plants have a unique model for maintaining and replicating mtDNA when compared to the replication mechanism utilized by most metazoan organisms.

Arabidopsis thaliana organellar DNA polymerase IB mutants exhibit reduced mtDNA levels with a decrease in mitochondrial area density

Plant organelle genomes are complex and the mechanisms for their replication and maintenance remain unclear. Arabidopsis thaliana has two DNA polymerase genes, DNA polymerase IA (polIA) and polIB, that are dual targeted to mitochondria and chloroplasts and are differentially expressed in primary plant tissues. PolIB gene expression occurs at higher levels in tissues not primary for photosynthesis. Arabidopsis T-DNA polIB mutants have a 30% reduction in relative mitochondrial DNA (mtDNA) levels, but also exhibit a 70% increase in polIA gene expression. The polIB mutant shows an increase in mitochondrial numbers but a significant decrease in mitochondrial area density within the hypocotyl epidermis, shoot apex and root tips. Chloroplast numbers are not significantly different in mesophyll protoplasts. These mutants do not have a significant difference in total dark mitorespiration levels but exhibit a difference in light respiration levels and photosynthesis capacity. Organelle-encoded genes for components of respiration and photosynthesis are upregulated in polIB mutants. The mutants exhibited slow growth in conjunction with a decreased rate of cell expansion and other secondary phenotypic effects. Evidence suggests that early plastid development and DNA levels are directly affected by a polIB mutation but are resolved to wild-type levels over time. However, mitochondria numbers and DNA levels never reach wild-type levels in the polIB mutant. We propose that both polIA and polIB are required for mtDNA replication. The results suggest that polIB mutants undergo an adjustment in cell homeostasis, enabling them to maintain functional mitochondria at the cost of normal cell expansion and plant growth.