Como as plantas protegem os seus genes: acaso, necessidade ou design inteligente?

Whirly proteins maintain plastid genome stability in Arabidopsis

Alexandre Maréchal,1, Jean-Sébastien Parent,1, Félix Véronneau-Lafortune, Alexandre Joyeux, B. Franz Lang and Normand Brisson,2

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Department of Biochemistry, Université de Montréal, P.O. Box 6128, Station Centre-ville, Montréal, QC, Canada H3C 3J7

Edited by Patricia C. Zambryski, University of California, Berkeley, CA, and approved June 19, 2009

↵1A.M. and J.-S.P. contributed equally to this work. (received for review February 13, 2009)

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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.

genome maintenance microhomology recombination

Plastids play diverse and essential roles in plants. Despite this central importance, surprisingly little is known about even the most basic aspects of the plastid genome structure, maintenance, and propagation. For example, while the textbook depiction of plastid DNA (ptDNA) is that of a genome-sized circular DNA molecule, recent evidence suggests instead that most of the ptDNA is organized in concatenated, branched linear forms with T4 phage-like features (1). This change of perception of plastid genome architecture requires a re-evaluation of the current rolling-circle model for plastid genome replication. It is now considered that a recombination-dependent replication process is responsible for the branched, multigenomic structures present in plastids (1). Recombination is also expected to play a crucial role in plastid genome maintenance. Indeed, because of its exposure to radiation and reactive oxygen species, the plastid genome is expected to accumulate mutations at a high rate. This situation stresses the importance of efficient DNA replication, recombination, and repair (DNA-RRR) pathways in these organelles (2). However, to date the mechanisms and enzymes involved in these pathways remain poorly characterized.

Evidence for recombination in plastid genomes abounds in the literature (2). An example is the recombination between the large inverted repeat sequences present in many plastid genomes (3). This flip-flop recombination is responsible for the 2 isomers of ptDNA, which differ only with respect to the orientation of the single-copy regions. More direct evidence of recombination comes from plastid transformation experiments, which demonstrate that foreign DNA is integrated into ptDNA by homologous recombination (4).

Homologues of bacterial genes involved in DNA-RRR are present in the nuclear genome of plants, and some of their encoded proteins are targeted to plastids. These include the recA homologs RECA1 (5) and RECA2, whose disruption is lethal in Arabidopsis (6), a Rec Q-like DNA helicase from rice (7), and genes for a gyrase A-like and 2 gyrase B-like subunits in Arabidopsis (8). Recently, 2 homologs of OSB1, a ssDNA-binding protein (SSB) that regulates recombination in mitochondria (9), were shown to localize to plastids. However, no role has yet been ascribed to these proteins. Finally, homologs of replication protein A (RPA), another ssDNA-binding protein family that plays an essential role in mammalian DNA-RRR, have recently been identified in plants. One member of this family is targeted to plastids (10).

Similar to many DNA-RRR proteins, Whirlies form a small family of ssDNA-binding proteins (11). They are involved in a variety of phenomena, ranging from pathogen defense (12) to telomeric homeostasis (13). In Arabidopsis, 3 Whirly genes are present and their proteins localize to organelles; AtWhy1 and AtWhy3 are targeted to plastids and AtWhy2 is targeted to mitochondria (14, 15).

Recent evidence indicates that Whirlies bind organelle DNA without apparent sequence specificity in vivo. In Arabidopsis, AtWhy2 binds to many regions of the mitochondrial genome with no obvious sequence consensus (15). Similarly, in maize, the plastid-localized ZmWhy1 interacts with DNA from throughout the plastid genome (16). Knockdown mutations of ZmWhy1 lead to ivory or pale green plants, indicating that this Whirly is involved in chloroplast biogenesis. This phenotype was attributed to a defect in the maturation of the atpF and 23S ribosomal RNAs, but the participation of ZmWhy1 in DNA recombination or repair has not been ruled out.

To better understand the role of plastid-targeted Whirlies (ptWhirlies), we characterized an Arabidopsis double KO line of the AtWhy1 and AtWhy3 genes (KO1/3). Variegation patterns, which appear on leaves in ≈4.6% of the progeny, correlated with the selective rearrangement and amplification of large regions of the plastid genome. We show that the rearrangements are produced by illegitimate recombination at short direct repeats that border the amplified regions in intact ptDNA. We suggest that AtWhy1 and AtWhy3 function as antirecombination proteins, contributing to safeguard plastid genome integrity.

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