Mitochondrial DNA Repair in an Arabidopsis thaliana Uracil N-Glycosylase Mutant

Abstract

Substitution rates in plant mitochondrial genes are extremely low, indicating strong selective pressure as well as efficient repair. Plant mitochondria possess base excision repair pathways; however, many repair pathways such as nucleotide excision repair and mismatch repair appear to be absent. In the absence of these pathways, many DNA lesions must be repaired by a different mechanism. To test the hypothesis that double-strand break repair (DSBR) is that mechanism, we maintained independent self-crossing lineages of plants deficient in uracil-N-glycosylase (UNG) for 11 generations to determine the repair outcomes when that pathway is missing. Surprisingly, no single nucleotide polymorphisms (SNPs) were fixed in any line in generation 11. The pattern of heteroplasmic SNPs was also unaltered through 11 generations. When the rate of cytosine deamination was increased by mitochondrial expression of the cytosine deaminase APOBEC3G, there was an increase in heteroplasmic SNPs but only in mature leaves. Clearly, DNA maintenance in reproductive meristem mitochondria is very effective in the absence of UNG while mitochondrial genomes in differentiated tissue are maintained through a different mechanism or not at all. Several genes involved in DSBR are upregulated in the absence of UNG, indicating that double-strand break repair is a general system of repair in plant mitochondria. It is important to note that the developmental stage of tissues is critically important for these types of experiments.

Keywords: DNA repair; double-strand break repair; mitochondria; uracil-N-glycosylase.

Figure 1

Quantitative RT-PCR assays of enzymes involved in double-strand break repair (DSBR) in UNG lines relative to wild-type: Fold change in transcript level is shown on the Y-axis. Error bars are standard deviation of three biological replicates. MSH1 and RECA2 are significantly transcriptionally upregulated in UNG lines relative to wild-type (5.60-fold increase and 3.19-fold increase, respectively. Unpaired, 2-tailed student’s t-test, * indicates p < 0.05). OSB1 is nearly significantly upregulated in UNG lines relative to wild-type (3.07-fold increase. Unpaired t-test p = 0.053).

Figure 2

qPCR analysis of intermediate repeat recombination in UNG lines compared to wild-type: Recombination at intermediate repeats is an indicator of increased double-strand breaks in plant mitochondrial genomes. (a) Primer scheme for detecting parental and recombinant repeats: Using different combinations of primers that anneal to the unique sequence flanking the repeats, either parental type (1/1 and 2/2) or recombinant type (1/2 and 2/1) repeats can be amplified. (b) Fold change of intermediate repeats in young leaves of UNG lines relative to wild-type: Error bars are standard deviation of three biological replicates. (c) Fold change of intermediate repeats in mature leaves of UNG lines relative to wild-type: Error bars are standard deviation of three biological replicates. B1/1, B2/2, D2/2, and L2/2 show significant reduction in copy number (unpaired, 2-tailed student’s t-test, * indicates p < 0.05).