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eXtra Botany
COMMENTARY
Mechanisms for maintenance,
replication, and repair of the
chloroplast genome in plants
Brent L. Nielsen*, John D. Cupp and
Jeffrey Brammer
Department of Microbiology and Molecular Biology, Brigham
Young University, Provo, Utah 84602, USA
* E-mail: brentnielsen@byu.edu
Journal of Experimental Botany, Vol. 61, No. 10, pp. 2535–2537,
2010
doi:10.1093/jxb/erq163
Photosynthesis is a complex process that occurs in chlor-
oplasts of higher plants, and requires a large number of
proteins to assemble the photosynthetic machinery. Many
chloroplast-localized proteins are nuclear-encoded and must
be imported into the chloroplasts from the cytoplasm. A
considerable number of genes for photosynthesis and other
chloroplast functions, including transcription and trans-
lation, are encoded in the chloroplast genome (ctDNA),
which ranges in size from about 130–160 kbp in most higher
plants. CtDNA replication is not linked with the plant
cell cycle and the chloroplast genome can be amplified to
a very high copy number per cell in rapidly dividing leaf
tissue. Later in leaf development and plant growth, the
ctDNA levels reduce to very low levels (Oldenburg and
Bendich, 2004b). The controls that regulate ctDNA replica-
tion initiation, replication, and copy number are not
understood. From earlier publications on a number of plant
species it appears that ctDNA may replicate by more than
one mechanism, including a recombination-dependent
replication mechanism (Rowan et al., 2010, this issue;
Oldenburg and Bendich, 2004b;Marechal and Brisson,
2010), a double D-loop mechanism (Chiu and Sears, 1992;
Kunnimalaiyaan and Nielsen, 1997a,b), and rolling circle
replication (Kolodner and Tewari, 1975).
In this issue, Rowan et al. (2010) report on the role of
chloroplast-targeted RecA (cpRecA) in the maintenance
of ctDNA in Arabidopsis. Previously published reports
provide evidence that some ctDNA molecules may be
recombination intermediates as shown by the presence of
branched DNA molecules in some DNA preparations
(Oldenburg and Bendich, 2004a,b;Scharff and Koop,
2007). As summarized in a review by Marechal and Brisson
(2010), recombination has been shown to be involved in the
repair of double-strand breaks and point mutations in
ctDNA. It has been known for some time that a plant
homologue of bacterial RecA is localized in chloroplasts
(Cerutti et al., 1992), but, to date, little is known about the
role of DNA recombination in the maintenance of ctDNA.
Rowan et al. (2010) show clear evidence that cpRecA is
involved in the maintenance of the chloroplast genome copy
number in plants, as T-DNA insertions (from the Agro-
bacterium Ti plasmid) in the nuclear gene encoding this
protein led to a reduction in ctDNA copy number in the
mutant plants relative to wild-type plants and to a change
in the structure of the ctDNA. The levels of detectable
single-stranded DNA increased in the mutants, which is
compatible with the decreased amount of cpRecA which
would normally coat the single-stranded DNA regions and
thus block its detection. After a few generations the mutants
began to show significant signs of distress and reduced
chloroplast function, including variegation and necrosis.
These findings represent a significant advance in our
understanding of the mechanisms involved in the mainte-
nance of ctDNA integrity. The authors suggest that the role
of cpRecA is primarily in DNA repair, as supported by the
analysis of wild-type plants that have been treated with
ciprofloxacin, which induces double-strand DNA breaks. In
these plants, altered ctDNA structures were observed as
in the cpRecA plants. Similar experiments with insertions
in the DRT 100 homologue, which has only very weak
homology to bacterial RecA but can partially complement
E. coli recA mutants showed no effect, suggesting that DRT
100 may not be directly involved in the repair of ctDNA.
The role of cpRecA in DNA repair is clearly supported
by these experiments; it is also possible that cpRecA may
be involved in recombination-mediated replication of the
chloroplast genome.
CpRecA and DRT 100 are not the only RecA homo-
logues localized to chloroplasts. A dual-targeted (to both
chloroplasts and mitochondria) RecA (distinguished from
the others as RecA2) has been identified in the Arabidopsis
nuclear genome (Christensen et al., 2005). T-DNA inser-
tions in this gene lead to non-viable plants (BL Nielsen,
JD Cupp, unpublished observations; Shedge et al., 2007),
suggesting that RecA2 may be essential for ctDNA and/or
mtDNA maintenance and plant development. However, at
this point in time there are no data to determine whether
the lethal phenotype is due to the disruption of chloroplast
or mitochondrial DNA maintenance mechanisms, or both.
The RecA2 gene was not included in the current study by
Rowan et al. (2010, this issue) but its role in ctDNA
replication should be evaluated. The observation that
T-DNA insertions in cpRecA were not lethal may be due
to functional (at least partial) complementation by RecA2.
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But because RecA2 insertions appear to be lethal, RecA2
may play some specific and essential role in ctDNA and/or
mtDNA maintenance. It will be important to obtain and
analyse mutants of RecA2, which may need to be generated
by other approaches such as inducible RNAi, microRNA or
some other technique. If such mutants can be obtained,
then similar approaches to those used by Rowan et al.
should be used to analyse the role of RecA2 in maintaining
ctDNA copy number.
It is possible that early ctDNA replication during
germination and seedling development may be initiated
by one mechanism, such as the double D-loop mechanism,
and high level amplification of ctDNA may occur by
rolling circle replication. Alternatively, perhaps rolling circle
replication occurs initially, maybe in combination with
recombination-dependent replication, and the double D-loop
mechanism is then used as a way of maintaining ctDNA
later in plant development. Rolling circle replication is used
by many bacteriophages to produce large numbers of new
DNA molecules for progeny phage very quickly. Some
bacteriophages, including lambda phage, initiate replication
bidirectionally from a specific origin, similar to bacterial and
eukaryotic chromosomal DNA replication, but after com-
pletion of a unit circle shift to rolling circle DNA repli-
cation. Other phages such as T4 and T7 also have multiple
replication mechanisms, including replication from a specific
origin for both, and a recombination-dependent mechanism
for T4 (Mosig, 1998) and replication as concatamers for
T7 (as described in Oldenberg and Bendich, 2004a;Scharff
and Koop, 2006). Thus there is ample evidence from
other organisms and bacteriophages for multiple replication
mechanisms for individual genomes. This possibility should
be seriously considered for ctDNA, as many aspects of
chloroplast genome structure, transcription and replication
share similarities with bacteriophage mechanisms.
A double D-loop mechanism for ctDNA replication was
reported more than 30 years ago for Pisum sativum
(Kolodner and Tewari, 1975), which lacks the large inverted
repeat common to most higher plant chloroplast genomes.
In P. sativum, the two origins map within the rRNA spacer
region and just downstream of the 5S rRNA gene, about
6 kbp apart. The rRNA operon is present in the large
inverted repeat, so in species that have the inverted repeat
the two replication origins are duplicated. For example,
in tobacco ctDNA there are two identical pairs (one pair
in each of the large inverted repeats) of replication
origins implicated by both in vivo and in vitro analysis
(Kunnimalaiyaan and Nielsen, 1997a,b). However, there is
growing evidence that these are not the only replication
origins in ctDNA.
Support for the involvement of more than one mecha-
nism for ctDNA replication and/or additional replication
origins can be inferred from the results of the Koop
laboratory on insertions in the oriA and oriB replication
origins in tobacco ctDNA (Scharff and Koop, 2006,2007).
Targeted inactivation of either or both of these origins was
not lethal, although some deletions resulted in reduced
growth rate of the plants and reduced ctDNA copy number,
particularly later in leaf development (Scharff and Koop,
2007). This may suggest that the double D-loop mechanism
involving these origins is involved in ctDNA replication
during the transition from rapidly dividing cells to maturing
cells. Scharff and Koop (2006) reported the presence of a
significant amount of linear ctDNA molecules with defined
ends in tobacco, a substantial portion of which mapped to
previously reported ctDNA replication origins, and some of
which mapped to novel specific locations. Similar results
were reported earlier for maize ctDNA by Oldenberg and
Bendich (2004a). The Bendich laboratory reported that the
majority of ctDNA in maize is linear, and that the structure
and copy number of ctDNA molecules change during
development (Oldenburg and Bendich, 2004b). The earlier
published work of Kolodner and Tewari (1975) suggested
that a rolling circle replication initiation site may be present
at a different location from the D-loop origins in the
chloroplast genome. This rolling circle replication site
would be in the single-copy region in species with the
inverted repeat.
Another factor to consider in the control of ctDNA
maintenance is the presence of two nuclear-encoded DNA
polymerases that are both dual targeted to chloroplasts and
mitochondria (Christensen et al., 2005; Carrie et al., 2009).
These plant DNA polymerases share significant homology
with bacterial DNA polymerase I rather than with the
animal mitochondrial DNA polymerase c(Ono et al.,
2007). In Arabidopsis and tobacco the coding regions
for these genes are very highly conserved, suggesting that
they may be functionally redundant. However, the up-
stream promoter regions share no homology (BL Nielsen,
J Brammer, unpublished results), raising the possibility that
the two genes are differentially regulated and may be
expressed at different times and have different roles in
organelle DNA replication and maintenance. Indeed, our
preliminary data suggest that the two enzymes are not
expressed at equal levels or at the same time during plant
development (J Brammer, BL Nielsen, unpublished results).
One or both of these DNA polymerases would be essential
for any of the above-mentioned replication mechanisms.
Future work should examine the involvement of these two
DNA polymerases with cpRecA in the maintenance of
ctDNA.
An origin-binding protein or specificity factor, similar to
dnaA for the bacterial chromosome or rep proteins involved
in plasmid DNA replication initiation, or an enzyme that
nicks the DNA to initiate rolling circle replication, is also
likely to be required for ctDNA replication. While an
origin-binding activity has been characterized for ctDNA
replication in Chlamydomonas (Wu et al., 1989), which
shares some similarity with ctDNA replication in higher
plants, no such protein has been identified in higher plants.
Given the wide range in ctDNA levels in different tissues
during plant development, it seems clear that one or more
protein(s) involved in controlling initiation of ctDNA
replication must be present in plants.
From this new report and previous work from a number
of laboratories, there is strong support for the presence of
2536 |Commentary
at Serials Dept., Harold B. Lee Library, Brigham Young University on June 29, 2010 http://jxb.oxfordjournals.orgDownloaded from
multiple replication origins and/or replication mechanisms,
suggesting that the maintenance of ctDNA is more complex
than in bacteria, the endosymbiotic ancestor (Scharff and
Koop, 2007). There is growing evidence that more than one
mechanism is involved in replication of the chloroplast
genome, and each may function at different times during
chloroplast development and on the different forms of
ctDNA (linear and circular DNA). The majority of ctDNA
in most plant tissues is linear, with a varying proportion
of branched and/or circular molecules, providing support
for the premise that a recombination-mediated replication
mechanism may be involved in ctDNA replication, compat-
ible with the results reported by Rowan et al. (2010, this
issue). It is exciting that some new progress is being made
on ctDNA repair and replication, but there is still much to
be learned about the process of ctDNA maintenance in
plants during the various stages of plant development.
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