Divergent roles for the two PolI-like organelle DNA polymerases of Arabidopsis

Abstract

DNA polymerases play a central role in the process of DNA replication. Yet, the proteins in charge of the replication of plant organelle DNA have not been unambiguously identified. There are however many indications that a family of proteins homologous to bacterial DNA polymerase I (PolI) is implicated in organelle DNA replication. Here, we have isolated mutant lines of the PolIA and PolIB genes of Arabidopsis (Arabidopsis thaliana) to test this hypothesis. We find that mutation of both genes is lethal, thus confirming an essential and redundant role for these two proteins. However, the mutation of a single gene is sufficient to cause a reduction in the levels of DNA in both mitochondria and plastids. We also demonstrate that polIb, but not polIa mutant lines, are hypersensitive to ciprofloxacin, a small molecule that specifically induces DNA double-strand breaks in plant organelles, suggesting a function for PolIB in DNA repair. In agreement with this result, a cross between polIb and a plastid Whirly mutant line yielded plants with high levels of DNA rearrangements and severe growth defects, indicating impairments in plastid DNA repair pathways. Taken together, this work provides further evidences for the involvement of the plant PolI-like genes in organelle DNA replication and suggests an additional role for PolIB in DNA repair.

Figure 1.

Isolation of PolIA and PolIB mutant lines. A, Schematic representation of PolIA and PolIB genes. Exons are denotated by blue boxes, the 3′ untranslated region by a red box, and the 5′ untranslated region by a white box. The pale-blue lines represent introns. The position of the T-DNA insertions is indicated by black triangles. Half-arrows stand for the primers used for the qRT-PCR experiments. B, Histogram presenting the results of the qRT-PCR experiments measuring the level of expression of the PolIA gene inside the polIa mutant plants grown on Murashige and Skoog medium for 14 d. Expression levels are represented as percentages of wild-type (WT) level. The experiments were done in technical and biological triplicates. The error bars represent the sem on the biological triplicates. C, Histogram presenting the results of the qRT-PCR experiments measuring the level of expression of the PolIB gene inside the polIb mutant plants grown on Murashige and Skoog medium for 14 d. Expression levels are represented as percentages of wild-type level. The experiments were done in technical and biological triplicates. The error bars represent the sem on the biological triplicates. D, Representative photographs of 4-week-old Arabidopsis plants for wild-type and PolI mutant lines.

Figure 2.

Organelle DNA is less abundant in seedlings of polIa-1 and polIb-1 mutants. A, Histogram presenting the results of the qPCR analysis of mitochondrial genome copy number relative to nuclear genome copy number for 3-, 4-, and 5-d-old seedlings. The experiments were done in technical and biological triplicates. The error bars represent the sem on the biological triplicate. B, Histogram presenting the results of the qPCR analysis of plastid genome copy number relative to nuclear genome copy number for 3-, 4-, and 5-d-old seedlings. The experiments were done in technical and biological triplicates. The error bars represent the sem on the biological triplicate. WT, Wild type.

Figure 3.

PolIB mutants exhibit an increased sensitivity to ciprofloxacin (CIP) and are impaired in their ability to process DNA DSBs. A, Representative photographs of the different polI mutants grown for 21 d on Murashige and Skoog medium containing 0, 0.25, or 0.5 μm ciprofloxacin. B, Representative photographs of the first true leaves of polIb-1 and polIb-2 plants grown for 21 d on Murashige and Skoog medium containing 0.25 μm ciprofloxacin. C, Histogram representing the percentage of plants with white/variegated first true leaves after 21 d of growth on medium with different concentrations of ciprofloxacin. Error bars represent the sd of the counts made on different plates. D, Histogram representing the percentage of plants without any first true leaves after 21 d of growth on medium with different concentrations of ciprofloxacin. Error bars represent the sd of the counts made on different plates. E, Histogram presenting the results of the PCR experiments to evaluate the abundance of DNA lesions upon ciprofloxacin treatment in ptDNA. DNA lesions are significantly more abundant for the polIb-1 mutant when treated with both 0.25 and 0.5 μm ciprofloxacin. Error bars represent the sem of three separate experiments where wild-type signal at 0 μm ciprofloxacin is expressed as 100 for each repetition. One asterisk indicates a significant difference with a P < 0.05 in the Student’s t test and two asterisks, a difference with a P < 0.01. F, Histogram presenting the results of the PCR experiments to evaluate the abundance of DNA lesions in mitochondrial DNA upon treatment with ciprofloxacin. DNA lesions are significantly more abundant for the polIb-1 mutant when treated with 0.5 μm ciprofloxacin. Error bars represent the sem of three separate experiments where wild-type signal at 0 μm ciprofloxacin is expressed as 100 for each repetition. Asterisk indicates a significant difference with a P < 0.05 in the Student’s t test. WT, Wild type.

Figure 4.

A polIb-1/why1why3 triple mutant yields a pale-green dwarf phenotype and gives rise to abundant DNA rearrangements in plastids. A, Representative photographs of 4-week-old plants for the genotypes used in the cross and the triple-mutant progeny compared to the wild type (WT). B, Representative PCR reactions to evaluate the abundance of DNA rearrangements dependant on microhomology in plastids of the plants presented in A. The plants were grown for 21 d on Murashige and Skoog medium containing 0 or 0.25 μm ciprofloxacin. Two DNA samples were tested for each condition. PCR reactions were done using the primers indicated on the right of the gels. C, Schematic representation of DNA rearrangements evaluated by the PCR approach. Inward-facing primers placed far apart will yield a PCR product only if a deletion has occurred in the region that separates the primers. Outward-facing primers will yield a PCR product only if the template DNA is duplicated or circularized. Primers are represented by black half-arrows. D, Schematic representation of the DNA rearrangements amplified by PCR. The top section shows the position of the primers used to test the plastid genome of Arabidopsis. The bottom section gives a graphical representation of the rearranged DNA molecules. The sequences in the white boxes are the microhomologous sequences that were used to prime the MMBIR process. The original position of the microhomologous sequences in wild-type ptDNA is indicated.