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. 2009 Aug 25;106(34):14693-8.
doi: 10.1073/pnas.0901710106. Epub 2009 Aug 7.

Whirly proteins maintain plastid genome stability in Arabidopsis

Alexandre Maréchal  1 , Jean-Sébastien Parent, Félix Véronneau-Lafortune, Alexandre Joyeux, B Franz Lang, Normand Brisson
Affiliations

Whirly proteins maintain plastid genome stability in Arabidopsis

Alexandre Maréchal et al. Proc Natl Acad Sci U S A. .

Abstract

Maintenance of genome stability is essential for the accurate propagation of genetic information and cell growth and survival. Organisms have therefore developed efficient strategies to prevent DNA lesions and rearrangements. Much of the information concerning these strategies has been obtained through the study of bacterial and nuclear genomes. Comparatively, little is known about how organelle genomes maintain a stable structure. Here, we report that the plastid-localized Whirly ssDNA-binding proteins are required for plastid genome stability in Arabidopsis. We show that a double KO of the genes AtWhy1 and AtWhy3 leads to the appearance of plants with variegated green/white/yellow leaves, symptomatic of nonfunctional chloroplasts. This variegation is maternally inherited, indicating defects in the plastid genome. Indeed, in all variegated lines examined, reorganized regions of plastid DNA are amplified as circular and/or head-tail concatemers. All amplified regions are delimited by short direct repeats of 10-18 bp, strongly suggesting that these regions result from illegitimate recombination between repeated sequences. This type of recombination occurs frequently in plants lacking both Whirlies, to a lesser extent in single KO plants and rarely in WT individuals. Maize mutants for the ZmWhy1 Whirly protein also show an increase in the frequency of illegitimate recombination. We propose a model where Whirlies contribute to plastid genome stability by protecting against illegitimate repeat-mediated recombination.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
AtWhy1 and AtWhy3 are involved in the biogenesis of chloroplasts. (A) Physical maps of the AtWhy1 (AT1G14410) and AtWhy3 (AT2G02740) genes. The position of the T-DNA insertion in the KO1 line is indicated. KO3 is a TILLING line with a mutation that changes a TGG codon to a TGA stop codon in the AtWhy3 gene. An asterisk indicates the position of the mutation. (B) Protein gel blot analysis of simple and double ptWhirlies KO plants. Crude plastid proteins were separated by SDS/PAGE on a 15% polyacrylamide gel. Whirlies were detected by using an anti-AtWhy1/3 antibody. A section of the blot stained with Ponceau red is presented below as a loading control. (C) (Upper) Four-week-old individuals of the indicated genotypes are shown. (Lower) Fluorescence of chlorophyll was visualized by confocal microscopy. (D) Transmission electron microscopy of sections from green (Left) and white (Right) sectors of variegated leaves of KO1/3 plants. (Scale bars: 10 μm in Upper; 2 μm in Lower.) (E) Variegation phenotype varies between independent lines. Four-week-old individuals from the 2 variegated lines Var A (Left) and Var B (Right) are shown.
Fig. 2.
Fig. 2.
Variegated plants contain rearranged amplified ptDNA regions. (A) DNA gel blot (10 μg per lane) of total leaf DNA digested with HindIII and hybridized with the probes indicated below the gels. The probe numbers refer to the nucleotides of the published Arabidopsis chloroplast genome (42). Expected fragments from restriction analysis of Col-0 ptDNA and the size of new fragments observed in variegated lines are presented below the probes. Restriction and gene maps of the reorganized regions in variegated lines are presented in Figs. S4 and S9. A lower exposition of the second panel allowing better visualization of the amplified bands in VarA is presented in Fig. S5. (B) Schematic of the possible arrangements of the reorganized ptDNA in variegated lines. A head–tail dimer and a monomeric circular molecule are represented for Var A (red) and Var B (green). Oligonucleotides used for the PCR amplification of the junctions of reorganized ptDNA are represented by small black arrows. (C) (Lower) PCR amplification of fragments containing the junctions of reorganized ptDNA in Var A and B plants. (Upper) DNA from leaves 4 (variegated) and 8 (nonvariegated) was isolated. The plastidial ycf2 gene was used as a loading control. (D) DNA gel blot analysis showing the arrangement of amplified ptDNA in the Var A line. DNA from the indicated genotypes was digested with the indicated restriction enzymes and separated on an agarose gel. The DNA was hybridized with the probe depicted in E. A 9.2-kb band corresponding to the WT DNA fragment appears in all samples digested with XhoI and PstI restriction enzymes. A band of 9.4 kb expected from digestion of the WT plastid genome with KpnI was found in all genotypes. The asterisks indicate putative circular molecules. (E) Restriction map of the reorganized regions in the Var A line. The red arrow represents the amplified region in Var A. The probe used is represented as a blue line. A portion of ptDNA is represented as a black horizontal line. The restriction sites are indicated by vertical black lines. K = KpnI; P = PstI; X = XhoI. A circular monomer is represented on the right with the expected linear digestion product of this molecule.
Fig. 3.
Fig. 3.
Illegitimate recombination is increased in the absence of Arabidopsis ptWhirlies. For each genotype, PCRs using outward or inward-facing PCR primers were performed on 4 pools of DNA from 4 different plants. Reactions were run on agarose gels containing ethidium bromide. (A–C) Representative PCRs are shown. The oligonucleotides used are indicated above each panel. Individual bands (white numbers) were cut, cloned, and sequenced. Each band represents a unique recombination product (Table S2). The asterisks indicate nonrecombinant products arising from nonspecific hybridization of the 69633F primer at positions 58720–58733 of the plastid genome. (D) The plastidial ycf2 gene was used as a loading control.
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References

    1. Bendich AJ. Circular chloroplast chromosomes: The grand illusion. Plant Cell. 2004;16:1661–1666. - PMC - PubMed
    1. Day A, Madesis P. DNA replication, recombination, and repair in plastids. In: Bock R, editor. Cell and Molecular Biology of Plastids. Vol 19. Berlin: Springer; 2007. pp. 65–119.
    1. Palmer JD. Chloroplast DNA exists in 2 orientations. Nature. 1983;301:92–93.
    1. Staub JM, Maliga P. Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation. Plant Cell. 1992;4:39–45. - PMC - PubMed
    1. Cao J, Combs C, Jagendorf AT. The chloroplast-located homolog of bacterial DNA recombinase. Plant Cell Physiol. 1997;38:1319–1325. - PubMed

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