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Recombination and the maintenance
of plant organelle genome stability
Author for correspondence:
Normand Brisson
Tel: +1 514 343 6984
Email: [email protected]
Alexandre Maréchal and Normand Brisson
Department of Biochemistry, Université de Montréal, PO Box 6128, Station Centre-ville, Montréal,
QC H3C 3J7, Canada
Received: 30 October 2009
Accepted: 24 December 2009
Contents
IV. Conclusion and perspectives
312
Summary
299
I.
Introduction
299
Acknowledgements
313
II.
Roles of recombination in organelle genome stability
301
References
313
III.
Recombination surveillance machinery in plant organelles 304
Summary
New Phytologist (2010) 186: 299–317
doi: 10.1111/j.1469-8137.2010.03195.x
Key words: DNA repair, genome stability,
microhomology, mitochondria, organelle,
plastid, recombination, replication.
Like their nuclear counterpart, the plastid and mitochondrial genomes of plants
have to be faithfully replicated and repaired to ensure the normal functioning of
the plant. Inability to maintain organelle genome stability results in plastid and ⁄ or
mitochondrial defects, which can lead to potentially detrimental phenotypes. Fortunately, plant organelles have developed multiple strategies to maintain the integrity of their genetic material. Of particular importance among these processes is
the extensive use of DNA recombination. In fact, recombination has been implicated in both the replication and the repair of organelle genomes. Revealingly,
deregulation of recombination in organelles results in genomic instability, often
accompanied by adverse consequences for plant fitness. The recent identification
of four families of proteins that prevent aberrant recombination of organelle DNA
sheds much needed mechanistic light on this important process. What comes out
of these investigations is a partial portrait of the recombination surveillance
machinery in which plants have co-opted some proteins of prokaryotic origin but
have also evolved whole new factors to keep their organelle genomes intact. These
new features presumably optimized the protection of plastid and mitochondrial
genomes against the particular genotoxic stresses they face.
I. Introduction
Maintenance of genome stability is an absolute requirement
for cell growth and proliferation. Failure to maintain such
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stability results in the accumulation of mutations and genomic rearrangements that can quickly become deleterious.
For example, genomic instability characterizes the majority
of human cancers (Halazonetis et al., 2008). Thus, at the
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single organism level, it seems preferable to keep the genome absolutely intact. Nevertheless, a certain level of instability is crucial for the evolution of species as the reshaping
of genomes generates genetic diversity. Eliminating the
mechanisms that produce this diversity would also destroy
the resilience and adaptability of species to changing environmental conditions.
Consequently, all organisms have systems that maintain
the stability of their genome to a degree that prevents excessive mutations but does not preclude evolution. Of prime
importance for the generally faithful propagation of genomes are proteins implicated in DNA replication, recombination and repair (RRR) (see Glossary, Box 1). Genetic
studies have shown that mutation of these proteins in prokaryotes and eukaryotes often results in increased genomic
instability (Aguilera & Gomez-Gonzalez, 2008). Most of
the research effort so far has been directed towards unravelling the systems that regulate genome stability in bacterial
and nuclear genomes, and thus little is known about how
organelle genomes are preserved.
Plant cells contain two types of endosymbiotic organelles
and must therefore maintain the stability of three separate
genomes located in the nucleus, mitochondria and plastids.
In this regard, plants provide a unique opportunity to study
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the strategies used by organelles to protect their genomes.
The organelle genomes of flowering plants do not encode
any of the RRR proteins required for the maintenance of
DNA (Bock, 2007; O’Brien et al., 2009). Consequently,
organelles have to import these factors to ensure that their
genetic material remains functional and is accurately transmitted. Until recently, none of these factors was known,
but a series of reports have now identified several families of
nuclear-encoded proteins implicated in the maintenance of
plant organelle genome stability (Abdelnoor et al., 2003;
Zaegel et al., 2006; Shedge et al., 2007; Maréchal et al.,
2009). A common theme among all these protein families is
their involvement in the suppression of recombination
between repeated DNA sequences. Indeed, elimination or
mutation of these proteins results in large-scale rearrangements of organelle genomes that eventually perturb the
function of plastids and ⁄ or mitochondria. This in turn is
often accompanied by a variety of striking phenotypes,
ranging from variegation to cytoplasmic male sterility
(CMS), underlining the importance of the small organelle
genomes for plant development and function.
In this article, we summarize current knowledge concerning the relationship between the regulation of recombination and the maintenance of genome stability in plant
Box 1 Glossary
Chromatids: each of the two copies of DNA that constitute a replicated chromosome.
Crossing-over: reciprocal exchange of distal sections of two homologous DNA molecules that accompanies gene
conversion of the region proximal to the double-stranded break.
Cytoplasmic male sterility (CMS): a maternally inherited trait that renders plants unable to produce viable pollen. CMS
is frequently associated with rearrangements of the mitochondrial genome.
Gene conversion: nonreciprocal transfer of information from one DNA sequence to another. The damaged recipient
sequence is converted to its intact donor homologue. Gene conversion events can be associated with crossing-over or not.
Head-tail concatemers: DNA concatemers contain multiple successive copies of the same sequence. In a head-tail concatemer, one end of a copy (the head) is linked to the opposite end of the following copy (the tail).
Holliday junction: cross-shaped mobile junction between four different strands of DNA.
Homologous recombination (HR): conservative repair process based on the exchange of genetic information between
two quasi-identical DNA sequences.
Homeologous: homeologous DNA sequences are only partially homologous.
Hypomorphic mutation: Type of mutation where the modified gene product has a reduced activity.
Loss of heterozygosity (LOH): loss of an allelic difference between two homologous chromosomes. Following LOH, the
cell becomes homozygous for a given allele or series of alleles.
Microhomology: region of homology shorter than 30 bp.
Nonchromosomal stripe (NCS): maternally inherited phenotype in which maize (Zea mays) plants exhibit poor growth,
aberrant morphology and leaf striping as a result of chloroplast defects.
Recombination: formation of new alleles or combination of genes through any mechanism.
Substoichiometric shifting (SSS): rapid changes in the stoichiometry of rearranged DNA molecules present in plant
mitochondrial genomes. These molecules can be either dramatically amplified or suppressed.
Unequal crossing-over (UCO): crossing-over between improperly aligned chromosomes. UCO results in nonreciprocal
exchange of genetic material and generates chromosomes of different lengths.
Variegation: the presence of differently coloured sectors on various parts of the plant, usually the leaves or the stems.
Variegation is often caused by a perturbation of chlorophyll biosynthesis by chloroplasts.
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organelles. We discuss the roles of recombination-based
mechanisms in the replication, repair and evolution of plant
organelle DNA and also present the different protein families that modulate recombination in plastid and mitochondria genomes. This review shows that, while the basic
recombination mechanisms are broadly conserved among
nuclear, bacterial and organelle genomes, the recombination
surveillance machinery of plant organelles comprises newly
evolved protein families specific to plants working side by
side with conserved factors of prokaryotic origin. Studying
the interplay between these different proteins will surely
yield surprising new insights into the maintenance of genome stability.
II. Roles of recombination in organelle genome
stability
1. Homology-dependent DNA repair pathways
The capacity of cells to repair damage incurred by their
DNA is a significant aspect of genome stability. In all
organisms, lesions can be fixed by either conservative or
error-prone repair pathways. The former accurately repairs
damaged DNA while the latter can result in slight to substantial modifications of the genome (see Kimura & Sakaguchi (2006) for a comprehensive review of these processes
in plants).
Homologous recombination (HR) is an important and
evolutionarily conserved error-free homology-dependent
DNA repair process used to eliminate potentially harmful
lesions, particularly double-stranded breaks (DSBs). This
type of lesion can result from the exposure of cells to a variety of genotoxic stresses including ionizing radiation, chemical agents and endonucleolytic digestion. DSBs are
traditionally envisioned as double-ended breaks arising
from a fracture in the duplex. However, recent research has
shown that a significant portion of these lesions are actually
single-ended DSBs produced when replication forks
encounter single-stranded breaks in DNA, or as a consequence of replication fork stalling ⁄ collapse at particular base
modifications or DNA sequences (Wyman & Kanaar,
2006). Homology-directed repair of these replication fork
breakdowns and double-ended DSBs is critical for cell viability and relies on the exchange of genetic information
between two DNA molecules that share a substantial length
of quasi-identical sequence (Cox et al., 2000) (at least 50 to
100 bp of homology is required; Singer et al., 1982; Watt
et al., 1985). During this process, the damaged recipient
molecule obtains information to guide its repair from an
intact donor duplex.
Most of what is known about HR is derived from observations made in bacteria and yeast models. Still, studies
using more complex organisms have demonstrated that the
fundamental mechanisms underlying this type of recombi-
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nation are highly conserved (Pâques & Haber, 1999; Persky
& Lovett, 2008). Multiple different mechanisms of HR
that promote accurate DNA repair have been inferred from
experimental results using a variety of reporter systems to
monitor recombination (Fig. 1). The double-stranded
break repair (DSBR) model, also known as the double
Holliday junction model, can fix DNA by gene conversion
that can be coupled with crossing-over (Szostak et al.,
1983). Crossing-over during meiosis is an important source
of genetic diversity and is well explained by the DSBR
model although some species actually use a single Holliday
junction to carry out meiotic crossing-over (Cromie et al.,
2006). However, when crossing-over occurs between
homologous chromosomes in somatic cells, it can lead to
loss of heterozygosity if chromatids carrying the same alleles
segregate together during mitosis, and this can have devastating consequences (Stark & Jasin, 2003). Moreover, if
crossing-over occurs between nonallelic repeated sequences
(unequal crossing-over), it can duplicate or delete the DNA
located between the repeats, thereby producing genome
rearrangements and effectively promoting genomic instability. Accordingly, in all organisms tested, mitotic recombination is not frequently associated with crossing-over. To
explain this bias, variants of another model called synthesisdependent strand annealing (SDSA) were postulated. In this
type of HR, gene conversion occurs without any crossingover (Nasmyth, 1982; Thaler & Stahl, 1988). Finally,
because the late stages of DSBR and SDSA rely on the capture of the second end of the DSB, these models cannot
readily explain the repair of single-ended DSBs (Fig. 1a).
Fortunately, these frequently occurring lesions can be fixed
by the break-induced replication (BIR) pathway of HR, also
known as recombination-dependent replication (RDR). In
contrast to the other two HR mechanisms, in which DNA
polymerization occurs only on leading strands, BIR ⁄ RDR
requires the formation of a full replication fork capable of
both leading and lagging strand synthesis (Llorente et al.,
2008). This HR pathway thus results in the repair of broken
replication forks through gene conversion associated with
nonreciprocal crossing-over (Fig. 1b). All of the different
pathways of HR apparently coexist in a sort of hierarchical
competition, which provides organisms with a flexible
homology-directed DNA repair machinery that confers
resistance to a wide variety of genotoxic stresses and maximizes genomic stability.
2. Homology-dependent DNA repair in plant
organelles
Although knowledge concerning homology-dependent
DNA repair has been mainly acquired through the study of
nuclear and prokaryotic genomes, there is now ample evidence for an active system of recombination in both plant
organelles and the mitochondria of several other taxa
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(a)
(b)
Fig. 1 Mechanisms of homologous recombination. Solid lines correspond to single strands of DNA, small arrowheads represent elongating ⁄
invading 3¢-ends and dotted lines represent new DNA synthesis. Solid pointed arrowheads indicate single-stranded breaks. The damaged recipient duplex is coloured in blue whereas the intact donor duplex is shown in red. (a) Double-ended double-stranded breaks (DSBs) can be fixed
by double-stranded break repair (DSBR) or synthesis-dependent strand annealing (SDSA) pathways. During DSBR, both 5¢-ends of the DSB on
the damaged recipient duplex are first resected to yield 3¢ single-stranded overhangs. A free 3¢-end can then invade a donor duplex at a homologous sequence, forming a D-loop. DNA synthesis can now proceed from the 3¢-end past the position of the original DSB. The displaced donor
strand can then anneal to the other side of the break on the recipient DNA molecule. DNA polymerization and ligation of the free ends yield
two Holliday junctions that can be resolved in a variety of ways by cutting either the crossed (open arrowheads) or noncrossed strands (black
arrowheads). If both junctions are resolved in the same orientation, gene conversion without crossing-over is achieved. Alternatively, if the
junctions are resolved in opposite orientation, gene conversion is accompanied by reciprocal crossing-over. SDSA follows the same steps as
DSBR until the extension of the invading 3¢-end. Once the extension has gone past the original break site, the invading end dissociates from
the donor duplex and re-associates with the recipient DNA by complementary base pairing. DNA synthesis can now fill in the gaps and ligation
always yields a gene conversion event without crossing-over. (b) Single-ended DSBs produced by the demise of replication forks can be successfully repaired by the break-induced replication ⁄ recombination-dependent replication pathway (BIR ⁄ RDR). In the illustrated case, a singlestranded break on the lagging strand template leads to replication fork collapse. Then, as in DSBR and SDSA, resection of the 5¢-end on the
broken DNA frees a 3¢-OH single-stranded overhang which can invade a homologous donor duplex. DNA synthesis starts and the extended
3¢-end is unable to find a complementary second end. To synthesize the missing strand, a replication fork is established and polymerization proceeds to the end of the donor molecule. This results in gene conversion accompanied by nonreciprocal crossing-over. This figure is modified
from Hastings et al. (2009b).
(reviewed in Barr et al., 2005; Kmiec et al., 2006). In the
plastids of most flowering plants, a frequent and easily
observable flip-flop recombination event occurs between
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the large inverted repeats and gives rise to two equimolar
plastome isoforms which differ in the orientation of the two
single copy regions (Palmer, 1983). This provides evidence
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for a DSBR-like HR mechanism in plastids as reciprocal
crossing-over is required for the orientation change of the
single-copy regions in genome-sized circular DNA molecules. However, it is now recognized that most plastid
DNA (ptDNA) exists as linear ⁄ concatemeric ⁄ highly
branched complex molecules and these inversions were reinterpreted as resulting from a BIR-like, recombinationdependent replication mechanism between different linear
copies of the plastome (Bendich, 2004; Oldenburg &
Bendich, 2004) (see Section II.3 and Fig. 2). Additional
and abundant proof for the presence of an efficient recombination apparatus in plastids comes from the production
of transplastomic plants (Boynton et al., 1988; Svab et al.,
1990; Koop et al., 2007). Integration of plasmid-borne foreign DNA within the plastid genome occurs by gene conversion with or without crossing-over, consistent with
DSBR, and requires a substantial length of homology on
both sides of the sequence to be integrated (‡ c. 150 bp)
(Day & Madesis, 2007). Similarly, excision of intervening
sequences between short DNA repeats introduced in the
plastid genome constitutes a viable strategy to remove antibiotic resistance marker genes or even endogenous genes
from transformed plastid genomes and also argues in favour
of an active DSBR pathway in plastids (Fischer et al., 1996;
Iamtham & Day, 2000; Klaus et al., 2004; Kode et al.,
2006).
The mending of DNA damage through homology-directed DNA repair has been demonstrated in plastids. Induced
DNA breaks in the plastid genome of the green alga
Chlamydomonas reinhardtii using the homing endonuclease
I-CreI were fixed by HR (Durrenberger et al., 1996). A
more recent study suggests that the chloroplast of
Chlamydomonas can use both the DSBR and SDSA pathways to fix double-stranded lesions (Odom et al., 2008).
Recombination can also act as a repair mechanism in flowering plants as it was shown to be highly efficient at fixing
point mutations in tobacco (Nicotiana tabacum) plastids
(Khakhlova & Bock, 2006).
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Recombination is also an active process in mitochondria
and is responsible for the highly dynamic character of plant
mitochondrial genomes (Mackenzie, 2007). Indeed, the
mitochondrial genome of angiosperms (flowering plants)
generally contains many fairly large repeated sequences
(> 1 kbp) which can frequently recombine intra- or intermolecularly. These multiple HR events result in a multipartite highly redundant organization of the mitochondrial
genome where subgenomes coexist in approximately equal
stoichiometry (Palmer & Shields, 1984; Fauron et al., 1995).
Homologous recombination also occurs sporadically between
shorter repeats (> 100 bp, < 1 kbp) and produces rearranged
DNA molecules called sublimons (substoichiometric
genomes) that are generally present in low copy numbers in
wild-type (WT) mitochondria (Small et al., 1987). These
recombined molecules have been proposed to constitute
important intermediates during the evolution of plant mitochondrial genomes (Small et al., 1989; Shedge et al., 2007).
Finally, a low frequency of illegitimate recombination
between regions of microhomology has also been reported
in both organelle genomes (Ogihara et al., 1988; Kanno
et al., 1993; Hartmann et al., 1994; Moeykens et al., 1995;
Maréchal et al., 2009). Thus, recombination definitely happens in plastids and mitochondria and appears to be limited
to large repeats (> 100–200 bp) in normal genomes or to
rare events involving microhomologous repeats that are easily observable only on an evolutionary timescale.
3. Recombination-dependent replication of plant
organelle genomes
In addition to its roles in the repair of a wide variety of
DNA lesions and in the dynamics and evolution of organelle genomes, recombination also appears to be implicated
in the replication of organelle DNA. Studying the topology
of a given DNA molecule provides insights into its replication mechanism. Despite a widely held assumption, plant
organelle DNA is not present exclusively as genome-sized
Fig. 2 Recombination-dependent replication produces both isomers of the plastid genome. A free 3¢-OH end of one copy of the plastid
genome invades a homologous head-tail concatemeric duplex inside an inverted repeat (IR) and initiates replication. (1) If BIR ⁄ RDR initiates at
one repeat, the final product is a head-tail concatemer with both single copy regions present in the same orientation. (2) If initiation occurs at
the other IR, the final product yields a head-tail concatemer where one pair of successive small and large single copies is arranged in opposite
orientations. In maize, the use of pulsed field gel electrophoresis, which was not employed in the original study (Palmer, 1983) to examine the
arrangement of chloroplast DNA, demonstrates that the previously observed inversion isomers are present only in the context of head-tail
concatemers in vivo. This invalidates the flip-flop recombination model that used genome-sized circular DNA to produce the ptDNA isomers
(Oldenburg & Bendich, 2004). The pointed arrows with dotted lines indicate the progression of replication forks. The short red and green
blocky arrows represent the inverted repeats. The large and small single copy regions (LSC and SSC) are depicted as long blue and short black
thin arrows, respectively.
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circles in vivo. Restriction mapping data arising from plastid
and plant mitochondrial genomes can indeed be interpreted
as circular molecules but the actual structure of these genomes can only be confidently assigned after direct observation of DNA. Early electron microscopy observations of
purified organelle DNA showed abundant linear DNA molecules but these were dismissed as probable degradation
products of larger circular molecules. In both plastids and
mitochondria, electron microscopy also revealed the presence of circular DNA molecules harbouring tails (r-like
structures), suggesting that rolling-circle replication is
involved in the maintenance of both organelle genomes
(Kolodner & Tewari, 1975; Backert & Borner, 2000).
Concomitant observation of displacement loops (D-loops)
in purified ptDNA led to the proposal of a dual origindependent h to rolling-circle replication model for the plastid genome (Kolodner & Tewari, 1975). However, these
models strongly relied on the postulate that most organelle
DNA exists as genome-sized circles.
With the advent of new techniques which prevent potential shearing, for example pulsed-field gel electrophoresis, it
became possible to observe intact organelle DNA (reviewed
in Bendich, 1993, 2004; Day & Madesis, 2007). It is now
well established that plant organelle DNA is constituted by
a mixture of monomers and head-tail concatemers of circular and linear molecules together with highly complex
branched structures (Deng et al., 1989; Backert et al.,
1996; Oldenburg & Bendich, 1996, 2001, 2004; Backert
& Borner, 2000; Lilly et al., 2001). These observations
allowed the proposal of an RDR mechanism similar to that
found in bacteriophage T4 and in bacteria to explain the
complex arrangement of DNA in plant organelles (Luder &
Mosig, 1982; Asai et al., 1994; Oldenburg & Bendich,
1996, 2001, 2004; Backert & Borner, 2000). In this model,
a free 3¢-OH single-stranded DNA overhang from one copy
of the genome can invade another copy at a homologous
site (Fig. 1b; RDR starts at the D-loop formation step).
This primes DNA synthesis on the leading strand and a full
replication fork is eventually established. Invasion of a linear
DNA molecule by another one produces a branched structure, while invasion of a circular molecule produces a tailed
circle containing a bubble. BIR ⁄ RDR can also conveniently
explain flip-flop recombination if the invasion step is mediated by the inverted repeats of the plastid genome. In this
case, RDR results in gene conversion accompanied by nonreciprocal crossing-over, thereby generating the inversion of
the single copy regions (Fig. 2). Thus, RDR constitutes the
replication model that best accounts for the structural complexity of DNA in plant organelles (Oldenburg & Bendich,
1996, 2001, 2004; Backert & Borner, 2000; Manchekar
et al., 2006). At the present time, the relationship between
RDR and the previously proposed replication models as
well as the relative importance of each mechanism in the
actual replication of organelle DNA remain unclear.
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III. Recombination surveillance machinery in
plant organelles
1. Perturbations of the recombination surveillance
systems
Compared with plastid genomes, plant mitochondrial
genomes are typically rich in fairly large repeated sequences
(> 50 bp), and strict control of HR is required to maintain
genome stability in this organelle (Table 1). Indeed, because
of these numerous repeats, runaway recombination of mitochondrial DNA (mtDNA) is relatively common and can lead
to vast rearrangements of the genome with ensuing changes
in the gene expression pattern of mitochondria (Sakamoto
et al., 1996; Arrieta-Montiel et al., 2009; and reviewed in
Mackenzie, 2007). These modifications can have profound
adverse effects on the functionality of mitochondria.
One of the most studied consequences of mitochondrial
dysfunction caused by genomic instability is CMS, an agronomically important trait that facilitates the large-scale production of high-yielding hybrid plants. It is now clear that
mitochondrial genomes are the major genetic determinants
of this maternally transmitted phenotype. Importantly,
recombination-mediated rearrangements in mtDNA and
important alterations in the relative stoichiometry of these
modified DNA molecules have been associated with the
inability to produce viable pollen and also with spontaneous
cytoplasmic reversion of the plants to a fertile state (Levings
Table 1 Repeated sequences in the organelle genomes of
angiosperms1
Number of repeats
Species
Plastid
Mitochondria
Arabidopsis thaliana
1
69
Carica papaya
1
36
Nicotiana tabacum
1
69
Vitis vinifera
2
114
Oryza sativa (indica)
3
132
Sorghum bicolor
4
63
Triticum aestivum
5
94
Zea mays
5
60
Accession
numbers
NC_000932
NC_001284
NC_010323
NC_012116
NC_001879
NC_006581
NC_007957
NC_012119
NC_008155
NC_007886
NC_008602
NC_008360
NC_002762
NC_007579
NC_007982
NC_007982
1
Direct and inverted perfectly identical repeated sequences with a
size ‡ 50 bp were identified using the REPUTER program and the
published organelle genome sequences of the indicated flowering
plant species (Kurtz et al., 2001). All plants listed here have a pair of
large inverted repeats (IRs) in their plastid genome. These IRs are
included in the total number of plastid repeats.
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& Pring, 1976; Schardl et al., 1985; Janska et al., 1998;
and reviewed in Schnable & Wise, 1998). The rearrangements responsible for CMS can create and amplify novel
open reading frames (ORFs) which encode chimeric proteins able to interfere with mitochondrial function and perturb pollen development (Forde et al., 1978; Dewey et al.,
1986, 1987; Wise et al., 1987; Young & Hanson, 1987;
and reviewed in Budar & Berthomé, 2007). In addition to
spontaneous mtDNA rearrangements which can cure CMS,
introduction of nuclear fertility-restorer genes can also reestablish the production of potent pollen by reducing the
expression level of chimeric ORFs or by countering the
physiological effects of the sterility gene products (reviewed
in Newton et al., 2004; Chase, 2007).
Recombination in mitochondria is not limited to HR
between rather large repeats; it can also, albeit less frequently, use microhomologous repeats to alter mtDNA. For
example, microhomology-mediated recombination has been
reported to promote reversion to fertility in CMS maize
plants and pearl millet (Pennisetum glaucum) (Schardl et al.,
1985; Feng et al., 2009). Many other cases of such microhomology-mediated rearrangements (involving repeats ranging from 6 to 31 bp) have been reported to induce the
nonchromosomal stripe (NCS) phenotype in maize
mutants (Newton et al., 1990; Hunt & Newton, 1991).
Apart from the large inverted repeats, there are very few
repeated regions in the plastid genomes of flowering plants
that could potentially serve as HR substrates (Table 1).
Introduction of foreign repeated sequences by transformation of plastids often results in unintended secondary HR
events (see Gray et al., 2009 and references therein). This
suggests that repeats large enough to recombine efficiently
and induce rearrangements might be selected against as they
have the potential to destabilize the plastid genome of flowering plants. Even though the number of large repeats in
ptDNA is kept to a minimum, perturbations of plastid genome stability do occur and mainly result from illegitimate
recombination events between microhomologous repeats.
For example, in tobacco, Nicotiana plastid extrachromosomal element (NICE1), an extrachromosomal element
formed during transformation, was found to excise from
plastid DNA through recombination between two imperfect repeats of 16 bp (Staub & Maliga, 1994). Accumulation of head-to-tail concatemeric DNA has also been
reported in a photosynthetically incompetent C. reinhardtii
mutant that failed to express atpB RNA and had been transformed with a plasmid carrying a weak atpB construct. In
some of the transformants, the defect in atpB expression
was rescued by an over-amplification of the plasmid, which
was found to be present as an episome of 20–30 copies per
plastid genome and reorganized by recombination in a
head-to-tail configuration (Kindle et al., 1994).
Both mitochondrial and plastid genomes are more frequently subject to alterations under specific environmental
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conditions. In particular, long-term tissue ⁄ cell culture and
albinism can strongly destabilize plant organelle genomes
(Day & Ellis, 1984; Rode et al., 1987; Vitart et al., 1992;
Kanazawa et al., 1994; Kawata et al., 1997; Cahoon et al.,
2003). Overall, the evidence shows that external stress (i.e.
variation in the selective pressure for photosynthesis ⁄ respiration and transformation of foreign DNA in plastids) placed
on the recombination machinery of plant organelles can render it more error-prone. Some of these conditions could also
favour specific rare (substoichiometric) rearranged DNA
molecules that would normally be corrected by the DNA
repair machinery but are conserved because of the useful
traits they confer on organelles. Alternatively, these rearranged molecules might become more abundant when selective pressure is relaxed simply because they replicate better.
Early on, nuclear mutations were also recognized as a
major cause of genomic instability in plant organelles. For
example, in maize, the WF9 nuclear genome is particularly
prone to mitochondrial genome rearrangements which produce an NCS phenotype, implying that some particular
genes in this background destabilize mtDNA (Newton &
Coe, 1986). Other recently reported alleles in the maize P2
line dramatically increase the abundance of modified
mtDNA molecules, which results in leaf striping patterns of
different colours (Kuzmin et al., 2005). In plastids, mutation of the plastome mutator (PM) nuclear locus produces
variegated Oenothera plants as a result of a strongly
increased rate of deletion, duplication and point mutations
in the plastid genome (Chiu et al., 1990; Chang et al.,
1996; Stoike & Sears, 1998). Although the specific alleles
responsible for most cases of organelle genome instability
have not yet been identified, a number of genes that actively
prevent deleterious recombination activity in organelle genomes have recently been characterized. Interestingly, these
genes often encode proteins that are targeted to both organelles or are part of families comprising mitochondrion- and
plastid-localized members, suggesting that at least some
genome maintenance strategies are shared by plastids and
mitochondria. These studies provide us with a preliminary
portrait of the recombination surveillance machinery in
plant organelles. So far these proteins include MutS homologue 1 (MSH1), RecA-like recombinases and two families
of plant-specific single-stranded DNA (ssDNA)-binding
proteins: the organellar ssDNA-binding proteins (OSBs)
and the Whirlies.
2. MSH1
Mutation of the nuclear locus chloroplast mutator
(CHM ⁄ MSH1) in Arabidopsis yields variegated plants bearing sectored green ⁄ white ⁄ yellow leaves symptomatic of dysfunctional chloroplasts. This phenotype is inherited in a
non-Mendelian fashion which led its original discoverer
and Arabidopsis research pioneer Georges Rédei to propose
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that the variegation was caused by mutations in the plastid
genome (Rédei, 1973). Nearly 20 yr later, a surprising correlation between variegation in chm plants and rearrangements of the mitochondrial genome through recombination
was established (Martinez-Zapater et al., 1992). Moreover,
despite extensive investigation, no modifications of the plastid genome could be detected, indicating that genomic
instability in mitochondria probably leads to the observed
chloroplast defects in chm plants (Martinez-Zapater et al.,
1992; Mourad & White, 1992). A subsequent study
described the molecular arrangement of two novel mtDNA
species present in the progeny of a cross between chm1–3
(ecotype Landsberg erecta (Ler)) and WT (ecotype Columbia (Col-0)) plants. One of the rearrangements was ascribed
to an illegitimate recombination event mediated by microhomologous regions of 11 bp (Sakamoto et al., 1996).
However, as this analysis was based on preliminary mitochondrial genome sequences from Arabidopsis ecotype
C24, which differs from the other ecotypes in the regions
implicated in the rearrangements, the recombination events
were later reinterpreted as resulting from HR between larger
dispersed repeats (> 200 bp) (Unseld et al., 1997; Forner
et al., 2005; Zaegel et al., 2006; Shedge et al., 2007; ArrietaMontiel et al., 2009). Further examination of mtDNA rearrangements showed that every repeated sequence of the
mitochondrial genome between 108 and 560 bp was subject to asymmetric recombination in chm plants. The efficiency of recombination correlated with the length and
extent of sequence similarity between individual repeats
(Arrieta-Montiel et al., 2009). The mtDNA rearrangements
found in these plants are also often accompanied by altered
mitochondrial transcript profiles (Sakamoto et al., 1996;
Arrieta-Montiel et al., 2009).
Cloning of the CHM gene revealed that it has significant
sequence similarity with MutS, a bacterial gene with roles in
DNA recombination and mismatch repair(MMR) (Abdelnoor
et al., 2003). Similarly to MSH1 in yeast, this MutS
homologue is targeted to mitochondria. In Saccharomyces
cerevisiae, msh1 mutations cause a petite phenotype indicative of defective respiration that is accompanied by largescale rearrangements and point mutations in the mitochondrial genome (Reenan & Kolodner, 1992). Consequently,
the Arabidopsis CHM gene was renamed MSH1 (Abdelnoor
et al., 2003). So far, mitochondrial MutS homologues have
only been found in yeast, plants and corals (Culligan et al.,
2000). Because msh1 plants do not accumulate point mutations in the mitochondrial genome and their fitness remains
constant in time, plant MSH1 homologues seem to have
specialized in the regulation of recombination instead of
being required for accurate mtDNA MMR (Abdelnoor
et al., 2003). In agreement with the established role of MSH1
in Arabidopsis, a down-regulation of MSH1 expression in
tomato (Solanum lycopersicum) and tobacco was shown to
destabilize the mitochondrial genome. Remarkably, in these
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two plant species the mtDNA rearrangements seem able to
induce a stable (nonreversible) CMS phenotype, suggesting
that this strategy could be used to obtain nontransgenic
male-sterile lines in a variety of crops (Sandhu et al., 2007).
In the bacterial MMR system, MutS homodimers bind
preferentially to mispaired or looped-out DNA in order to
direct repair of mismatched heteroduplexes and prevent
recombination between divergent sequences. They perform
their task with the help of the adaptor protein MutL and
the endonuclease MutH (Rayssiguier et al., 1989 and
reviewed in Jun et al., 2006). Plant MSH1 proteins contain
three recognizable domains in addition to three other conserved domains of unknown function (Abdelnoor et al.,
2006). At the N-terminus, following the transit peptide,
there is a domain similar to the MutS-I DNA-binding and
mismatch recognition motif. Plant MSH1 homologues also
contain an ATPase domain conserved in all MutS-like proteins and a unique GIY-YIG endonuclease domain found in
a number of other nucleases such as the excinuclease component uvrC and the homing endonuclease I-TevI (Fig. 3a)
(Abdelnoor et al., 2003, 2006). Point mutations in both
the ATPase and endonuclease domains of MSH1 induce
variegation and reorganization of mtDNA, highlighting the
functional importance of these domains. There are apparently no plant MutL or MutH homologues predicted to
localize to organelles. This, together with the peculiar
domain architecture of MSH1, suggests that this protein
represents a compact system with mismatch recognition
and endonuclease functions bundled together. The role of
MutS family proteins in preventing recombination between
homeologous DNA regions is broadly conserved in bacteria,
yeast, humans and plants (Rayssiguier et al., 1989; Worth
et al., 1994; Datta et al., 1997; Elliott & Jasin, 2001;
Emmanuel et al., 2006). Although the molecular details
remain fuzzy, combined evidence raises the possibility that
plant MSH1 homologues may function in the early steps of
recombination by favouring heteroduplex rejection, thereby
stabilizing the mitochondrial genome. Interestingly, MSH1
proteins from Arabidopsis and tomato can localize to both
organelles whereas their counterparts from maize and soybean (Glycine max) are targeted solely to mitochondria
(Christensen et al., 2005; Abdelnoor et al., 2006). As no
plastid DNA rearrangements have been observed to date in
msh1 mutants, it is possible that MSH1 proteins could play
additional roles in the plastids of some plant species.
3. RecA homologues
The eubacterial recombinase RecA and its eukaryotic homologues of the Rad51 family are essential proteins for the
central steps of HR (reviewed in Cox, 2007). They are key
factors for the accurate pairing of homologous DNA
sequences, the promotion of strand invasion and the migration of branches during the recombination process. All the
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(a)
(b)
Fig. 3 Protein families of the organelle recombination surveillance machinery. (a) Rectangular representation of proteins with established roles
in preventing aberrant recombination of plant organelle genomes. Arabidopsis MutS homologue 1 (MSH1) is a 120-kDa MutS homologue
fused with a GIY-YIG endonuclease domain. Organellar RecA homologues are 40–45-kDa proteins containing a well-conserved bacteria-like
RecA-fold. Organellar single-stranded DNA (ssDNA)-binding proteins (OSBs) are 30–50-kDa proteins which harbour an oligonucleotide ⁄ oligosaccharide binding (OB)-fold-like domain putatively involved in oligomerization and a variable number of ssDNA-binding PDF domains in their
C-terminal region. Depicted here is the Arabidopsis OSB1 protein with a single PDF domain. Whirlies are c. 24-kDa proteins that bind singlestranded DNA and RNA through their highly-conserved central Whirly domain. TP, transit peptide. (b) Crystallographic three-dimensional
structure of Solanum tuberosum Whirly 1 (StWhy1). Left, cartoon representation of an StWhy1 protomer with b-strands coloured in yellow,
a-helices in red and loops in green. Right, cartoon representation of an StWhy1 tetramer showing its whirligig-like appearance (adapted from
Desveaux et al., 2002). These representations were generated using PYMOL (DeLano, 2002).
different pathways of HR (DSBR, SDSA and BIR ⁄ RDR)
begin by a strand-exchange reaction mediated by RecA family proteins, making RecA and its homologues crucial for
homology-directed DNA repair of a variety of genomedestabilizing lesions, including DSBs and stalled replication
forks (reviewed in Cox et al., 2000).
Very few genes of the RecA family have been found outside prokaryotes. The discovery of a RecA homologue
encoding a protein that localizes to the stroma of pea (Pisum
sativum) chloroplasts was thus somewhat surprising (Cerutti
et al., 1992). The presence of RecA-like strand-exchange
activity in pea chloroplasts and soybean mitochondria also
supports the existence of functional RecA homologues in
plant organelles (Cerutti & Jagendorf, 1993; Manchekar
et al., 2006). Complete sequencing of the nuclear genome
of Arabidopsis revealed that it harbours at least three active
RecA homologues : RecA1, RecA2 and RecA3 (Table 2). A
fourth RecA-like gene exists but as it encodes a heavily truncated protein, it is probably a pseudogene (Khazi et al.,
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2003; Shedge et al., 2007). Fusions between GFP and the
first 80 amino acids of these proteins established that RecA1
localizes to plastids, RecA3 to mitochondria and RecA2 to
both organelles.
Mutation of the mitochondrion-targeted RecA3 leads to
large-scale rearrangements of mtDNA. These rearrangements are caused by HR between repeated sequences
(> 150 bp). The effect of RecA3 mutation on mtDNA
recombination is different from that observed in msh1
plants. Indeed, the affected repeats appear to be a subset of
those perturbed in MSH1 mutant plants (Shedge et al.,
2007; Arrieta-Montiel et al., 2009). These modifications
are partially reversible as reintroducing a functional copy of
RecA3 can result in the loss of aberrant DNA molecules in
the majority of the progeny. Strikingly, recA3 single
mutants have no phenotype that could distinguish them
from WT plants, indicating that mitochondria can cope
with a certain level of rearrangements. The other two RecA
homologues are apparently essential for viability as null
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Table 2 Characterized protein families of the recombination surveillance machinery of plant organelle genomes
Gene name
TAIR code
Function
Localization
Why1
Why2
Why3
At1g14410
At1g71260
At2g02740
Plant-specific
ssDNA-binding
proteins
Plastid
Mitochondrion
Plastid
OSB1
OSB2
OSB3
OSB4
RecA1
RecA2
RecA3
RecA4
At1g47720
At4g20010
At5g44785
At1g31010
At1g79050
At2g19490
At3g10140
At3g32920
Plant-specific
ssDNA-binding
proteins
Mitochondrion
Plastid
Plastid ⁄ mitochondrion
Unknown
Plastid
Plastid ⁄ mitochondrion
Mitochondrion
Pseudogene?
Msh1
At3g24320
RecA homologues
MutS homologue
Plastid ⁄ mitochondrion
Evidence for a role in
organelle RRR
References
Deletion of Why1 ⁄ Why3 increases
illegitimate recombination
between microhomologous
repeats in ptDNA
Deletion of OSB1 increases
recombination between short
homologous sequences in mtDNA
Krause et al. (2005)
Maréchal et al. (2008)
Maréchal et al. (2009)
Deletion of RecA3 in Arabidopsis
thaliana or RecA1 in
Physcomitrella patens increases
recombination between short
homologous sequences in mtDNA
Mutation of Msh1 increases
recombination between short
homologous sequences in mtDNA
Shedge et al. (2007)
Odahara et al. (2009)
Zaegel et al. (2006)
Pfalz et al. (2006)
Abdelnoor et al. (2003)
Martinez-Zapater et al. (1992)
Christensen et al. (2005)
Msh, MutS homologue; mtDNA, mitochondrial DNA; OSB, organellar ssDNA-binding protein; ptDNA, plastid DNA; RRR, DNA replication,
recombination and repair; ssDNA, single-stranded DNA; Why, Whirlye.
alleles could not be recovered, suggesting that they play crucial roles in plant organelles (Shedge et al., 2007).
A similar phenomenon was reported in disruptants of the
mitochondrion-targeted RecA homologue PpRecA1 in the
moss Physcomitrella patens. In this case the rearrangements
involved shorter repeats (> 40 bp, < 100 bp) as there are no
quasi-identical repeats larger than 90 bp in the mitochondrial genome of P. patens (Terasawa et al., 2007; Odahara
et al., 2009). Recombination between shorter repeats in Arabidopsis recA3 mutants cannot be ruled out, as the mtDNA
of these plants has not been systematically assessed for
recombination among sequences < 100 bp. Additionally,
the PpRecA1 gene appears to be important for the accurate
and efficient repair of damaged mtDNA as it is transcriptionally activated when moss is grown on media containing genotoxic agents such as mitomycin C and bleomycin. MtDNA
repair is also slowed down in PpRecA1 mutants following
treatment with the alkylating agent methyl methane sulphonate (Odahara et al., 2007). It is thus clearly established that
organelle-targeted RecA homologues in plants have important roles in the prevention of recombination between short
repeated sequences and in the promotion of DNA repair.
The biochemical properties and the molecular details
underlying the biological roles of RecA recombinases have
been studied extensively (reviewed in Bell, 2005). The
strand-exchange reaction begins with the polymerization of
RecA onto ssDNA overhangs present at lesion sites, thereby
forming a presynaptic filament in the presence of ATP. A
homology search step allows the alignment of the presynaptic RecA–ssDNA complex with an intact homologous
donor double-helix. The presynaptic complex is then able
to destabilize the donor duplex and catalyse strand exchange
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which forms a D-loop. All participants are subsequently dissociated upon ATP hydrolysis (Chen et al., 2008). The central RecA-fold, which also contains the ATP-binding and
hydrolysis domains, is generally well conserved in plant homologues, suggesting a similar mode of action for bacterial
and plant RecA recombinases (Fig. 3a; Shedge et al., 2007).
Interestingly, the RecA3 protein differs from RecA1 and
RecA2 in that it lacks the acidic C-terminal domain also
found in prokaryotic RecA proteins. C-terminal deletions
in bacterial recombinases enhances most in vitro activities of
RecA, supporting an autoregulatory role for this portion of
the protein (Cox, 2007). In vitro assays using purified bacterial proteins have also shown that the antirecombination
activity of MutS is a result of the inhibition of RecA-mediated strand-transfer activity between ssDNA molecules and
homeologous or damaged DNA duplexes (Worth et al.,
1994; Calmann & Marinus, 2004). This suggests that
MSH1 might have a similar regulatory role in the strandexchange activity of RecA homologues in plant organelles.
In accordance with the demonstrated role of mitochondrial
RecA proteins in plants, bacterial RecA by itself also prevents strand-exchange reactions involving substantially
diverged sequences (Hahn et al., 1988).
4. The organellar single-stranded DNA-binding
proteins
The OSBs form a recently discovered family of plantspecific proteins that all share a particular affinity for
ssDNA (Vermel et al., 2002; Zaegel et al., 2006). There are
four different OSBs in Arabidopsis, which localize to mitochondria (OSB1), chloroplasts (OSB2) or both organelles
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(OSB3) as determined using GFP fusion proteins.
Although expressed sequence tags have been reported for
OSB4, the cDNA has not yet been cloned and its precise
location and function have not been determined (Table 2).
T-DNA insertion mutants of the OSB1, OSB2 and OSB3
genes were obtained but a phenotype was observed only in
the progeny of OSB1 mutants. Indeed, some osb1 plants of
the T4 and T5 generations develop variegated and distorted
leaves. The affected plants exhibit rearrangements in multiple regions of the mitochondrial genome. These rearrangements were shown to result from HR between short
repeated sequences (> 200 bp). In some of the plants, the
accumulation of aberrant mtDNA molecules in osb1 plants
is reversible by reintroduction of a WT OSB1 allele. However, when the rearranged DNA becomes too abundant, the
phenotype is irreversible, presumably because the rearranged genome has become the principal component of
mtDNA and compensating recombination cannot happen
to any great extent. This is similar to the early and late msh1
mutant plants where prolonged propagation of msh1 plants
by selfing results in a gradual accumulation of rearrangements (Arrieta-Montiel et al., 2009). Thus, like Msh1 and
RecA3, Osb1 is part of the mitochondrial recombination
surveillance machinery in Arabidopsis and acts by limiting
the occurrence of HR between short repeated sequences.
Limited structure–function analysis of the OSBs has been
performed (Zaegel et al., 2006). All OSBs have a transit
peptide at their N-terminus to ensure their proper targeting
to organelles. Although not extremely well conserved (30–
50% similarity) in terms of amino acid residues, a domain
structurally similar to the oligonucleotide ⁄ oligosaccharide
binding fold (OB-fold), a hallmark of many ssDNA-binding proteins, is found in the middle part of OSBs. Additionally, the C-terminal section of these proteins contains a
variable number of PDF motifs which are specific to plants
(Fig. 3a). As their name implies, these 50 amino acid
domains contain consecutive highly conserved PDF residues. In vitro nucleic acid binding assays revealed that Arabidopsis OSB1 and OSB2 bind preferentially to ssDNA
with a high affinity (Kd 2 nM for OSB2). Interestingly,
the PDF motif is required for the ssDNA-binding activity
of OSB1 while the OB-fold-like motif is dispensable. The
residues required for high-affinity ssDNA binding in canonical OB-fold domains are not found in plant OSBs. It is
possible that the OB-fold-like domain of these proteins has
been transformed to fulfill other roles perhaps in oligomerization as the structural motifs required for this function of
the OB-fold are still present.
5. The Whirlies
Whirlies form another small family of ssDNA-binding proteins that is present mainly in the plant kingdom (reviewed in
Desveaux et al., 2005). The prototypical Whirly, Solanum
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tuberosum Whirly 1 (StWhy1), was first characterized as a
nuclear transcriptional regulator of the pathogenesis-related
gene PR-10a in potato (Matton et al., 1993; Després et al.,
1995; Desveaux et al., 2000, 2004). Whirlies also possess
active transit peptides at their N-terminus which can target
these proteins either to the plastids or to the mitochondria. In
fact, every completely sequenced angiosperm has at least one
mitochondrion- and one plastid-targeted Whirly (mtWhirlies and ptWhirlies), suggesting that they play important roles
in both organelles (Krause et al., 2005; Table 2). Recently,
the dual nuclear and plastid localization of barley (Hordeum
vulgare) Whirly 1 (HvWhy1) was demonstrated by immunolocalization and cellular subfractionation experiments
(Grabowski et al., 2008). The mechanisms and conditions
which might permit and regulate the dual localization of
Whirlies remain poorly characterized at present but investigations are ongoing (Krause & Krupinska, 2009).
In agreement with the multiple localizations of Whirly
proteins, evidence abounds showing that these proteins have
functions related to DNA metabolism in both organelles
and the nucleus. Thus, members of the Whirly family act as
transcriptional regulators for a number of nuclear genes and
are important for optimal resistance to the biotrophic
oomycete Hyaloperonospora parasitica (Desveaux et al.,
2000, 2004; Gonzalez-Lamothe et al., 2008; Xiong et al.,
2009). Whirlies have also been implicated in the maintenance of telomere length in nuclear chromosomes (Yoo
et al., 2007). Moreover, there is ample evidence that Whirlies associate with plastid and mitochondrial genomes in a
non-sequence-specific fashion, supporting a function of
these proteins in the DNA metabolism of plant organelles.
The Arabidopsis ptWhirlies AtWhy1 and AtWhy3 have
been purified in association with the transcriptionally active
chromosome of chloroplasts (Pfalz et al., 2006). In maize,
the plastid-targeted Whirly, ZmWhy1, localizes to the
stroma of chloroplasts and is tethered to thylakoid membranes via its interaction with ptDNA (Prikryl et al., 2008).
In Arabidopsis, DNA immunoprecipitation experiments
revealed that the mitochondrial AtWhy2 binds to many different regions of mtDNA without any obvious sequence
consensus (Maréchal et al., 2008). Similar experiments
showed that the chloroplast-targeted AtWhy1 and AtWhy3
interact with every ptDNA region tested (Maréchal et al.,
2009). This nonspecific binding is also found in other plant
species as ZmWhy1 also seemingly interacts with every
region of ptDNA (Prikryl et al., 2008).
Recently, a role for ZmWhy1 in chloroplast biogenesis
was discovered. Hypomorphic mutants of this gene have
pale green to completely white leaves depending on the
severity of the Zmwhy1 allele, indicating that chloroplasts
become defective following ZmWhy1 abrogation (Prikryl
et al., 2008). The authors of this study isolated ZmWhy1
by co-immunoprecipitation with the splicing factor CRS1,
which is implicated in the maturation of the atpF RNA
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(Jenkins et al., 1997). RNA immunoprecipitation experiments also revealed a specific interaction between ZmWhy1
and the atpF RNA. In accordance with these results, the
splicing efficiency of the atpF intron was diminished in
ZmWhy1 mutants, albeit not as drastically as in CRS1
mutant plants, suggesting an accessory role of ZmWhy1 in
the maturation of this RNA. Additional analysis of these
plants revealed that their plastid ribosomal RNA content
was greatly diminished or even nonexistent in the most
severe mutant. Exactly how ZmWhy1 is involved in the loss
of maize plastid ribosomes remains unclear at present as no
interaction occurs between ZmWhy1 and individual ribosomal RNAs.
To better understand the role of plastid-targeted Whirlies
(ptWhirlies), Arabidopsis single and double knock-out (KO)
lines of the AtWhy1 and AtWhy3 genes were recently characterized (Maréchal et al., 2009). Some of the progeny of
KO1 ⁄ 3 plants (c. 5%) have sectored green ⁄ white ⁄ yellow
leaves containing dysfunctional chloroplasts. This maternally inheritable variegation phenotype was shown to result
from increased instability of the plastid genome in the
absence of ptWhirlies. Plastids of KO1 ⁄ 3 plants accumulate
aberrant ptDNA molecules which correspond to deletion,
duplication and ⁄ or circularization events resulting from illegitimate recombination between microhomologous repeats.
Most of these rearranged molecules are undetectable in WT
plants, suggesting that they arise de novo in Whirly mutant
plants. Very strong amplification of some of the rearranged
DNA molecules correlates with the appearance of defective
chloroplasts. This phenomenon is not unique to Arabidopsis, as similarly rearranged DNA molecules also accumulate
in the plastids of maize lines with reduced levels of ZmWhy1
(Maréchal et al., 2009). The absence of a defect in rRNA
maturation and the relatively benign phenotype of KO1 ⁄ 3
plants compared with ZmWhy1 mutants suggest that Whirlies form a flexible family of single-stranded nucleic acid proteins that can serve different purposes depending on cellular
conditions and ⁄ or plant species. Thus, ptWhirlies prevent
the accumulation of abnormal ptDNA molecules produced
by microhomology-mediated recombination and have a conserved role in the maintenance of plastid genome stability.
A good amount of information is available on the various
domains that compose individual Whirly proteins. Following their N-terminal transit peptides, some Whirlies have a
putative transactivation domain. In StWhy1, this region
contains a stretch of several glutamine residues, and the
transactivation function of this domain has been demonstrated (Desveaux et al., 2004). Polyserine, polyglutamine
and polyproline regions found in a number of transcriptional
activators are also present in other members of the Whirly
family but their transactivation potential has not been
assessed yet (Triezenberg, 1995; Desveaux et al., 2005). As
these domains are often involved in the recruitment of coactivators, they might function generally as protein–protein
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interaction modules depending on the cellular context. A
highly conserved central domain forms the structural core of
Whirly proteins and is necessary for their high-affinity
ssDNA-binding activity (Kd 3 nM; L. Cappadocia and
N. Brisson, unpublished results). Gel filtration analysis of
Whirly–DNA complexes and X-ray crystallography have
established that these proteins adopt a tetrameric quaternary
structure (Desveaux et al., 2002). Finally, the C-terminus of
Whirly proteins in flowering plants comprises a conserved
short acidic ⁄ aromatic domain which has been proposed to
modulate the ssDNA-binding capacity of Whirlies (Desveaux et al., 2005; Fig. 3a).
Resolution of the three-dimensional structure of the
Whirly domain revealed a novel ssDNA-binding fold (Desveaux et al., 2002). Each protomer is composed of two antiparallel b-sheets packed perpendicularly against each other
along with three a-helixes. In the tetramer, the b-sheets
form blade-like extensions radiating from an a-helical core.
The general impression of this peculiar quaternary structure
is that of a whirligig, which suggested the name Whirly for
this family of plant ssDNA-binding proteins (Fig. 3b).
6. Recombination mechanisms involved in organelle
genome rearrangements
Recombination between large repeated sequences of the
mitochondrial genome results in gene conversion accompanied by reciprocal crossing-over, which is the predicted
product of the classical DSBR pathway (Palmer & Shields,
1984; Fauron et al., 1995) (Fig. 1a). The rare HR events
between shorter repeats in mtDNA (> 50 bp, < 1 kbp) produce a very low number of rearranged molecules (sublimons) which are thus usually substoichiometric relative to
the more abundant normal subgenomes (Small et al.,
1987). As described above, environmental conditions or
nuclear mutations in the recombination surveillance
machinery of mitochondria can increase the frequency of
recombination between these shorter repeats and can also
induce rapid amplification of pre-existing rare rearranged
molecules in a process called substoichiometric shifting
(SSS) (see for instance (Arrieta-Montiel et al., 2001;
Woloszynska & Trojanowski, 2009; and review by
Mackenzie, 2007). Recombination events between short
mitochondrial repeats almost always result in the accumulation of only one of the recombination products predicted by
the DSBR model. It has often been argued that the other predicted recombination product did not accumulate substantially because of its putative inability to replicate efficiently.
However, the fact that a single recombination product is the
rule rather than the exception indicates that the recombination pathway that generates this product is asymmetric.
It is thus likely that short repeat-mediated recombination
of the mitochondrial genome occurs via the BIR ⁄ RDR
pathway of HR (Shedge et al., 2007; Fig. 1b). Indeed, the
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final product of BIR ⁄ RDR is gene conversion with nonreciprocal crossing-over, which corresponds to what is
observed in short repeat-mediated mtDNA rearrangements.
The use of such an asymmetric HR mechanism is also more
parsimonious than the postulated general instability of one
of the recombination products resulting from the symmetric DSBR pathway. BIR ⁄ RDR can produce a plethora of
genome rearrangements including duplications, deletions
(a)
Review
and inversions. Additionally, if strand invasion during
BIR ⁄ RDR occurs on the same DNA molecule at a gapped
site upstream of the invading single-stranded 3-OH end, it
can prime rolling-circle replication (McEachern & Haber,
2006; Hastings et al., 2009a). In this way, a single recombination event could rapidly amplify a given rearranged molecule and be important for SSS. A low level of HR using the
DSBR pathway could also produce the reciprocal recombi-
(b)
Fig. 4 Microhomology-mediated break-induced replication. (a) Single-ended double-stranded breaks (DSBs) produced by replication fork
collapse can be nonconservatively repaired by the microhomology-mediated break-induced replication pathway (MMBIR). In the illustrated
case, a single-stranded break on the lagging-strand template leads to replication fork collapse. Then, resection of the 5¢-end on the broken
DNA frees a 3¢-OH single-stranded overhang which can invade a donor duplex at a microhomologous repeat. A replication fork is then
established and DNA synthesis starts. The replication forks resulting from template switching often have low processivity and can ‘jump’ onto
another template using multiple different repeats. Replication stops when polymerization reaches the end of the donor molecule. MMBIR can
produce a wide variety of rearrangements which depend on the position of the template switch relative to the position of the replication fork
collapse. If the switch occurs on a different DNA molecule behind the position of the break, a duplication is produced. Template switching on
the same or a different DNA molecule ahead of the break will produce a deletion. Inversions can also be formed if the switch occurs in the
wrong orientation on the same or a different molecule. A switch on the same molecule but behind the break at a single-stranded gap can produce a rolling circle which can rapidly amplify the rearranged DNA as illustrated in panel (b). This particular type of microhomology-mediated
invasion can occur, for example, between two Okazaki fragments on the lagging strand of a stalled replication fork. Following invasion, a
ligation step allows the formation of a circular molecule and rolling-circle replication ensues. This permits the rapid amplification of the DNA
region between the two microhomologous repeats. Breakage of the circle reforms a single-ended DSB which can then recombine with another
copy of the plastid genome through homologous recombination (HR) or microhomology-mediated recombination. The final product is a
ptDNA molecule containing concatemeric tandem repeats particularly prone to HR. This further recombination may create extrachromosomal
double-stranded circular molecules. In this figure, the coloured rectangles indicate microhomologous repeats and junctions. (b) Modified from
Hastings (2007).
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nation products which are often observed at low levels in
plant mitochondria (Woloszynska et al., 2001; Zaegel
et al., 2006; Woloszynska & Trojanowski, 2009). However,
SSS requires preferential replication of one of these two
products and this is more readily explained through a
BIR ⁄ RDR pathway. Alternatively or additionally, particular
recombination products might be specifically amplified following mtDNA sorting during plant development.
The extensive illegitimate recombination that is triggered
in the plastid genome following removal of ptWhirlies is
also best explained by a special nonconservative BIR-like
pathway called microhomology-mediated break-induced
replication (MMBIR) (Maréchal et al., 2009; Fig. 4). In
this case, the microhomologous repeats (< 30 bp) involved
in recombination of the plastid genome are too short to
serve as HR substrates. Actually, efficient strand exchange
catalysed by bacterial RecA and essential for HR requires
approx. 50 bp of perfect homology (Watt et al., 1985;
Lovett et al., 2002). The MMBIR pathway functions independently of RecA and can account for the observation of
both duplication ⁄ circularization and deletion events in
plants lacking ptWhirlies, whereas other asymmetrical pathways such as microhomology-mediated end-joining, singlestrand annealing and nonhomologous end-joining only
produce deletion events (Slack et al., 2006; McVey & Lee,
2008; and review by Hastings et al., 2009a). Similarly,
MMBIR might also create some of the numerous microhomology-mediated rearrangements previously reported in
plastids and mitochondria. Following replication fork collapse and production of a single-ended DSB, microhomology-mediated template switching occurs and primes DNA
synthesis at a different location, allowing completion of
DNA replication and concomitant production of rearranged DNA molecules.
Interestingly, MMBIR is a highly conserved error-prone
recombination mechanism that is an important source of
genomic rearrangements in humans, yeast and bacteria. In
humans, MMBIR can generate large-scale modifications of
the genome and copy-number variation that are associated
with the development of several diseases (Lee et al., 2007;
Hastings et al., 2009b; Zhang et al., 2009). This type of
microhomology-mediated recombination is also favoured
by DNA-related stresses, notably in response to DSBs, leading to the hypothesis that MMBIR might function as a
back-up repair mechanism when the homologous recombinational capacity of the cell or the organelle is overwhelmed
(Arlt et al., 2009; Hastings et al., 2009a). Such a repair
mechanism could provide plant organelles with an
additional layer of protection against highly deleterious
genotoxic stresses. Additionally, as MMBIR enables DNA
rearrangements and generates copy-number variation, it is
able to rapidly modify the genome. Some of the incurred
changes might improve the survival rate of plants exposed
to unfavourable environmental conditions. A similar mech-
New Phytologist (2010) 186: 299–317
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anism, called stress-induced mutation, is present in bacteria
and also produces fast genomic changes through the errorprone repair of DSBs (Ponder et al., 2005). As described
above, certain plants maintain microhomology-mediated
DNA rearrangements in their organellar genomes, supporting the idea that some of these modifications might prove
beneficial (Ogihara et al., 1988; Hiratsuka et al., 1989;
Kanno et al., 1993; Moeykens et al., 1995).
The interior of organelles is teeming with reactive byproducts of the electron transport machineries which are
potent inducers of DNA damage. Unexpectedly, plant organelle genomes actually have lower synonymous-substitution
rates than nuclear genomes, suggesting that they mutate at a
slower rate (Wolfe et al., 1987; Drouin et al., 2008). One
possible explanation for this is that organelles are particularly efficient at repairing their genomes through conservative pathways including HR. Plastids and mitochondria
contain a large number of genome-equivalents and these
multiple genome copies are brought together in ribonucleoprotein particles called nucleoids. This closeness might
favour error-free DNA repair through intermolecular
recombination. The common use of BIR-like mechanisms
during replication and repair of DNA could enhance the
homogeneity of genomes in individual plant organelles.
Indeed, gene conversion by BIR can extend from the break
site to the end of the donor duplex. This is much larger than
the small regions surrounding the breaks that are converted
during DSBR or SDSA (Fig. 1). Hence, BIR ⁄ RDR would
favour conservation of sequence over long segments of
DNA, thereby contributing to the lower synonymous-substitution rate of plant organelle genomes and overall genome stability.
IV. Conclusion and perspectives
With its documented roles in replication as well as in
the accurate and error-prone repair of plastid and mitochondrial DNA, recombination truly represents a cornerstone of plant organelle genome stability. The large
number of repeats and highly unusual organization and
structure of the plant mitochondrial genome make it
appear quite chaotic compared with the plastid genome,
which is remarkably uniform when examined using classical techniques such as DNA gel blots. However, when
ptDNA is scrutinized using more sensitive techniques,
one uncovers previously invisible layers of complexity.
Indeed, fluorescence in situ hybridization (FISH) shows
that reorganized molecules represent from 0.8 to 2% of
all ptDNA (Lilly et al., 2001). It seems plausible that
some of the atypical molecules detected by FISH represent the low level of MMBIR that was revealed by PCR
in WT Arabidopsis plants (Maréchal et al., 2009). It is
possible that these molecules constitute unavoidable byproducts of essential recombination processes which are
The Authors (2010)
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Phytologist
required for the proper replication and repair of ptDNA.
Thus, under normal conditions, both plastid and mitochondrial genomes are subject to spurious recombination
events that generate a certain amount of variability. For
both plant organelles, some of this variation is preserved
during evolution. The highly dynamic nature of the mitochondrial genome has been proposed to result from selective pressure for frequent spontaneous CMS which favours
outcrossing while preserving the highly efficient self-fertilization in the population (Mackenzie, 2005). By contrast,
evolution seems to counterselect against the presence of
large repeats in the plastid genome, presumably because
of the deleterious effect of ptDNA instability on plant
fitness.
Contrasting with the drastic differences in the plasticity
of their genomes, both organelles use similar mechanisms of
recombination and share some components of the recombination surveillance machinery, supporting a convergence in
the strategies used by plastids and mitochondria to protect
and propagate their genetic material. The precise function
of these factors will be the subject of future investigation.
Although one can easily imagine that MSH1 and RecA proteins in plant organelles might function similarly to their
bacterial homologues, the precise roles of the plant-specific
ssDNA-binding OSBs and Whirlies in the maintenance of
organelle genome stability remain shrouded in mystery.
Although often envisioned as simple DNA-coating proteins,
bacterial and mammalian ssDNA-binding proteins are surprisingly versatile and dynamic and recent studies now
depict them as crucial organizers and mobilizers of genome
maintenance complexes (Richard et al., 2008; Shereda
et al., 2008; Sakaguchi et al., 2009). Studying the interplay
between the various organellar ssDNA-binding proteins
and the rest of the recombination surveillance machinery of
plant organelles will certainly prove highly interesting. Our
understanding of the processes that preserve plant organelle
genomes has progressed considerably in the last decade with
the identification of the first few organelle DNA RRR proteins. This provides a basis for ongoing work that will unravel the detailed mechanisms of recombination, replication
and repair in the small but crucial genomes of plastids and
mitochondria.
Acknowledgements
We thank Laurent Cappadocia for his help with the analysis
of organelle genome repeats. We thank members of the
Brisson lab for inspiring discussions and critical reading of
the manuscript. A.M. was supported by scholarships from
the Natural Science and Engineering Research Council of
Canada (NSERC) and the Fonds Québécois de la Recherche sur la Nature et les Technologies. Research in the Brisson lab is supported by a grant from NSERC.
The Authors (2010)
Journal compilation New Phytologist Trust (2010)
Tansley review
Review
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