Download Dynamic Interplay between Nucleoid Segregation

Dynamic Interplay between Nucleoid Segregation and
Genome Integrity in Chlamydomonas Chloroplasts1[OPEN]
Masaki Odahara 2, Yusuke Kobayashi 2, Toshiharu Shikanai, and Yoshiki Nishimura*
Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
(M.O., Y.K., T.S., Y.N.); and Department of Life Science, College of Science, Rikkyo (St. Paul’s) University,
Toshima-ku, Tokyo 171-8501, Japan (M.O.)
The chloroplast (cp) genome is organized as nucleoids that are dispersed throughout the cp stroma. Previously, a cp homolog of
bacterial recombinase RecA (cpRECA) was shown to be involved in the maintenance of cp genome integrity by repairing
damaged chloroplast DNA and by suppressing aberrant recombination between short dispersed repeats in the moss
Physcomitrella patens. Here, overexpression and knockdown analysis of cpRECA in the green alga Chlamydomonas reinhardtii
revealed that cpRECA was involved in cp nucleoid dynamics as well as having a role in maintaining cp genome integrity.
Overexpression of cpRECA tagged with yellow fluorescent protein or hemagglutinin resulted in the formation of giant filamentous
structures that colocalized exclusively to chloroplast DNA and cpRECA localized to cp nucleoids in a heterogenous manner.
Knockdown of cpRECA led to a significant reduction in cp nucleoid number that was accompanied by nucleoid enlargement.
This phenotype resembled those of gyrase inhibitor-treated cells and monokaryotic chloroplast mutant cells and suggested that
cpRECA was involved in organizing cp nucleoid dynamics. The cp genome also was destabilized by induced recombination
between short dispersed repeats in cpRECA-knockdown cells and gyrase inhibitor-treated cells. Taken together, these results
suggest that cpRECA and gyrase are both involved in nucleoid dynamics and the maintenance of genome integrity and that the
mechanisms underlying these processes may be intimately related in C. reinhardtii cps.
Chloroplasts (cps) are plant organelles that are responsible for photosynthesis and the supply of certain
metabolites. Although cps contain their own genomic
DNA, the genes encoding most of the functional cp
proteins were transferred from chloroplast DNA (cpDNA)
to nuclear DNA during the evolutionary process. As a result, cpDNA genomes are now small (generally 100–200 kb
in size) compared with the genomes of ancestrally related
cyanobacteria. The remaining cpDNA is densely packed
and encodes genes essential for cp function and photosynthesis (Bungard, 2004; Green, 2011). In most plants,
cpDNA is composed of four regions: two copies of a large
inverted repeat, and large and small single-copy regions
1
This work was supported by the Funding Program for NextGeneration World-Leading Researchers (NEXT program; grant no.
GS015), a Grant-in-Aid for Challenging Exploratory Research (grant
nos. 26650111 and 16K14768), the Basic Research Projects from the
Sumitomo Foundation, the Core Stage Backup Program from Kyoto
University to Y.N., the Strategic Research Foundation Grant-Aided
Project for Private Universities to Y.N., and a Grant-in-Aid for Japan
Society for the Promotion of Science Fellows (grant no. 26$786 to Y.K.).
2
These authors contributed equally to the article.
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Yoshiki Nishimura ([email protected]).
M.O., Y.K., T.S., and Y.N. conceived and designed the experiments; M.O. and Y.K. performed the experiments; M.O. and Y.N.
wrote the article.
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separated by the inverted repeats (Green, 2011). The cps
usually possess 10 to 100 copies of the genomic DNA, some
of which are organized into a nucleoid similar to that
formed by bacterial chromosomal DNA (Kuroiwa, 1991).
The cp nucleoids are composed mainly of cpDNA and
constitutive nucleoid proteins (together termed the core
nucleoid) and are associated with RNA and proteins involved in DNA and RNA metabolism. The nucleoid is
thereby involved in the regulation of DNA replication and
transcription (Powikrowska et al., 2014).
The cp nucleoids are found as small particles dispersed
throughout the cp stroma in a diverse range of plant and
algal taxa. However, the shape and distribution of cp
nucleoids change dynamically according to cell cycle
phase (Ehara et al., 1990), developmental stage (Kuroiwa
et al., 1981), and metabolic status (Yehudai-Resheff et al.,
2007). However, the mechanisms governing the dynamic
micromorphology of cp nucleoids remain largely unknown. A few studies examined mutants defective in cp
nucleoid segregation. The monokaryotic chloroplast (moc)
mutants exhibiting impaired cp nucleoid dispersion were
obtained by insertional mutagenesis in Chlamydomonas
reinhardtii (Misumi et al., 1999). The moc mutants contained a single large cp nucleoid that was comparable
in size to the nucleus, but the overall amount of cpDNA
was slightly lower than in wild-type cells. The mutants
exhibited aberrant cp nucleoid segregation, which
resulted in cells with lower amounts of cpDNA. However, cpDNA copy number recovered quickly, probably
as a result of accelerated cpDNA replication (Misumi
et al., 1999). The gene responsible for moc phenotypes
remains unidentified. Arabidopsis (Arabidopsis thaliana)
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Odahara et al.
GYRA and GYRB, which are homologs of bacterial-type
topoisomerase II gyrase, modulate the topology of organelle DNA in Arabidopsis (Wall et al., 2004). Knockdown of
GYRA or GYRB resulted in the aggregation of cp nucleoids
into one or a few large particles in Nicotiana benthamiana
(Cho et al., 2004). These studies indicate that there may be
links between cp nucleoid micromorphology and cpDNA
molecular structure that are related to cpDNA replication.
Distinct mechanisms maintain cpDNA integrity in
plants that differ from the mechanisms underlying the
establishment and maintenance of micromorphological
cpDNA structure. Homologous recombination (HR)
repair is a fundamental pathway that is critical in the
recombination, replication, and repair of DNA defects
such as double-strand breaks (DSBs) and stalled replication forks, thereby maintaining genome stability.
RAD51 is a eukaryotic homolog (Shinohara et al., 1992)
of RecA, a bacterial recombinase that plays an essential
role in HR repair (Cox, 1999). In addition to RAD51,
plants characteristically encode bacterial-type RecA
homologs in their nuclear DNA (Lin et al., 2006). These
plant RECAs are targeted to cps and/or mitochondria
(Cerutti et al., 1992; Khazi et al., 2003; Odahara et al.,
2007; Shedge et al., 2007; Inouye et al., 2008). Active HR
occurs between large cp inverted repeats (Kolodner and
Tewari, 1979), and RecA-related strand transfer activity
is observed in cp extracts (Cerutti et al., 1993), suggesting
that RecA-mediated HR mechanisms are active in cp.
Expression of an Escherichia coli dominant-negative RecA
protein in C. reinhardtii affected cp HR efficiency (Cerutti
et al., 1995). Furthermore, transcription and translation
of chloroplast-targeted RECA (cpRECA) is induced by
treatment with DNA-damaging agents in various plants
(Cerutti et al., 1992, 1993; Nakazato et al., 2003; Inouye
et al., 2008). Taken together, these observations suggest that
cpRECA is involved in the repair of damaged cpDNA.
Some mutant studies examined the role of cpRECA
in the maintenance of cp genome integrity. In Physcomitrella patens, only modest growth defects were seen
in knockout mutants of cpRECA (RECA2); however, the
repair of damaged cpDNA was impaired, and mutants
exhibited enhanced sensitivity to methyl methanesulfonate and UV light (Odahara et al., 2015). In addition,
recombination between short dispersed repeats (SDRs;
less than 100 bp) in cpDNA was higher in knockout
mutants than in the wild type (Odahara et al., 2015).
This suggests that cpRECA may suppress aberrant
recombination between SDRs in cp and play a similar
role in maintaining cp genome stability to that of mitochondrial RECA in mitochondrial genome stability
(Odahara et al., 2009). After reproduction for several
generations, Arabidopsis cpRECA mutants exhibited
variegation in leaves and enhanced sensitivity to DSBand reactive oxygen species-inducing agents (Rowan
et al., 2010; Jeon et al., 2013). In addition, compared
with the wild type, mutants exhibit a reduction in
cpDNA copy number, an increase in single-stranded
cpDNA, and cpDNA structural alterations (Rowan
et al., 2010). Recent next-generation sequencing approaches have revealed that Arabidopsis cpRECA acts
to suppress U-turn like rearrangements mediated by
microhomology (Zampini et al., 2015)
In this study, we wished to explore the possible
linkage between cp nucleoid micromorphology and the
molecular structure and topology of cpDNA. In land
plants, cp nucleoid morphology and molecular structure are complex and vary considerably depending on
tissue type, developmental stage, age, and environment
(Oldenburg and Bendich, 2015). Furthermore, as plant
cells contain multiple cps that actively move around in
response to environmental and other cues (Wada, 2013),
monitoring the micromorphology of cp nucleoids at high
resolution can be problematic (Terasawa and Sato, 2005).
Therefore, we used the unicellular green alga Chlamydomonas reinhardtii as a simple model system. C. reinhardtii has
a single cup-shaped cp containing approximately 80 copies
of cpDNA (approximately 203 kb) organized into five- to
10-cp nucleoids that can be readily observed using fluorescence microscopy. C. reinhardtii can be grown in homogenous culture, facilitating pharmacological analysis
with various inhibitors, and abundant genome data
(Merchant et al., 2007) and molecular tools are available.
In this research, we performed detailed functional
analysis of cpRECA, with special focus on its impact on
cp nucleoid dynamics and cp genome stability. We also
analyzed the effect of gyrase inhibition on cp nucleoid
dynamics and cp genome stability. The results demonstrate that cpRECA and gyrase are both involved in
cp nucleoid dynamics and cp genome stability.
RESULTS
Preferential Localization of cpRECA at the Periphery of
cp Nucleoids
The intracellular localization pattern of cpRECA has
been examined in tobacco (Nicotiana tabacum) leaves
(Nakazato et al., 2003) but never in C. reinhardtii. Immunofluorescence microscopy using an antibody raised
against C. reinhardtii cpRECA (Supplemental Fig. S1) confirmed that C. reinhardtii cpRECA localized to cp nucleoids
(Fig. 1A). However, superimposition of the cpRECA signal
and the DAPI-stained DNA signal indicated that cpRECA
was not evenly distributed in cp nucleoids but instead was
found at the cp nucleoid periphery, and only in a subset of
cp nucleoids (Fig. 1A).
Yellow fluorescent protein (YFP) optimized for C.
reinhardtii (Karcher et al., 2009) was used to confirm the
cpRECA localization pattern in living cells. The cpRECA
gene was fused with YFP and overexpressed under the
control of the PSBD promoter in nuclear DNA in C.
reinhardtii (Supplemental Fig. S2A). The cpRECA-YFP
signal was again detected only in parts of the cp nucleoids, and the fluorescent signal indicated the formation of
microfilament-like structures fringing the nucleoids (Fig.
1B). In some cells, however, the micromorphology of cp
nucleoids was drastically deformed, and massive fibrous
structures that traversed the cp were observed (Fig. 1C).
Superresolution microscopy revealed that fibrous structures penetrated interspaces of the thylakoid membranes
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Chloroplast Roles of RecA and DNA Gyrase
Figure 1. Subcellular localization of cpRECA. A,
Immunofluorescence visualization of cpRECA. C.
reinhardtii wild-type cells were immunolabeled
using anti-cpRECA antibody. Fluorescence intensities of colocalized 49,6-diamino-phenylindole
(DAPI) and cpRECA (indicated by the arrow) were
determined (at right). B to D, Subcellular localization of
cpRECA-YFP or cpRECA (K127R)-YFP. YFP fluorescence (B) and anti-YFP immunofluorescence (C and D)
were observed in C. reinhardtii cells ectopically
expressing cpRECA-YFP or cpRECA (K127R)-YFP. Cells
were stained with DAPI. DIC, Differential interference
contrast; N, nucleus. Bars = 5 mm.
(Supplemental Fig. S2B; Supplemental Movies S1 and S2).
The fibrous cp nucleoids were observed more frequently
in cell lines with higher cpRECA expression levels. The
variable cpRECA-YFP distribution patterns may be due
to fluctuations in cpRECA-YFP expression levels within
individual cells.
One possible explanation for the unusual fibrous
structures was that aggregation between YFP molecules might have artificially caused the formation of
fibrous cp nucleoids. To test this possibility, cpRECA
fused to hemagglutinin (HA) instead of YFP expressed
in wild-type cells. The HA tag was detected by immunofluorescence microscopy using anti-HA antibody.
Again, similar cpRECA filamentous structures were
observed that colocalized with cpDNA (Supplemental
Fig. S2), excluding the possibility that the formation of
the filamentous cpRECA-YFP structure was due to YFP
aggregation.
The correspondence between cpRECA-YFP signals
and cpDNA in the fibrous structures raises the possibility that large regions of cpDNA are present as singlestranded DNA (ssDNA), since RecA preferentially
binds to ssDNA (Kowalczykowski et al., 1994). We
tested this by comparing the mung bean (Vigna radiata)
nuclease susceptibility of cpDNA in the wild type and
the cpRECA-YFP-expressing strain, in which approximately 30% of cells showed fibrous cpRECA-YFP
structures. Mung bean nuclease digests ssDNA and
single-stranded RNA nonspecifically, but it does not
digest fully paired duplexes. Total DNA was extracted,
and equal amounts were treated with mung bean
nuclease. Quantitative PCR (qPCR) analysis of two
loci on cpDNA revealed that the sensitivity of cpDNA
to mung bean nuclease treatment in the cpRECAYFP-expressing strain was almost equivalent to that
in the control strain (Supplemental Fig. S3).
To address whether cpRECA activity was required
for the formation of the fibrous cp nucleoids, a K127R
mutation was introduced into cpRECA-YFP that corresponded to a dominant-negative mutation (K72R) in
the ATPase domain of E. coli RecA. RecA K72R is suggested to form filaments with normal RecA and inhibit
RecA function depending on its ratio to normal RecA
(Renzette and Sandler, 2008). Overexpression of
cpRECA (K127R)-YFP resulted in the condensation
of cp nucleoids, and neither fibrous nor wild-typelike cp nucleoids were observed (Fig. 1D), indicating that cpRECA was required for the formation of
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Odahara et al.
fibrous cp nucleoids and the maintenance of wildtype-like cp nucleoid distribution.
1999), which also contain only a single large cp nucleoid (Fig. 2A, b and c).
Condensation of Chloroplast Nucleoids in cpRECAKnockdown Cells
Decrease in Chloroplast Nucleoid Number in Gyrase
Inhibitor-Treated Cells
To further analyze the function of cpRECA, cpRECAknockdown (KD) strains were developed using an artificial microRNA system optimized for C. reinhardtii (Molnar
et al., 2009). Two cpRECA-KD cell lines were generated
by suppressing cpRECA transcription to 12% and 37%
of wild-type levels (Supplemental Fig. S4A). The
knockdown lines exhibited no significant growth defects under standard culture conditions (Supplemental
Fig. S4B). However, the micromorphology of cp nucleoids was substantially altered in knockdown lines.
Fluorescence microscopy using SYBR Green I, a
double-stranded DNA-staining dye, showed remarkable differences in distribution, size, and number of cp
nucleoids in cpRECA-KD cells compared with the wild
type (Fig. 2). The cpRECA-KD cells had considerably
fewer cp nucleoids than the wild type: the modal
number of nucleoids per cp in the wild type was 12, in
contrast with the single nucleoid per cp in cpRECA-KD
cells (Fig. 2B). The decrease in nucleoid number was
associated with an increase in nucleoid size in cpRECA-KD cells (Supplemental Fig. S5), and the size of a
single large cp nucleoid was frequently comparable to
the size of the cell nucleus (Fig. 2Aa). The enlarged cp
nucleoids seen in cpRECA-KD cells were generally
round (Fig. 2Aa). However, the nucleoid structure was
frequently distorted when it was associated with pyrenoids (subcellular microcompartments sequestering
Rubisco). These visible phenotypes of cpRECA-KD
cells are reminiscent of moc mutants (Misumi et al.,
The condensation of cp nucleoids in cpRECA-KD cells
was reminiscent of a phenotype observed in Nicotiana
benthamiana cells with repressed cp-targeted gyrase
genes (Cho et al., 2004). Gyrase is a bacterial type II
topoisomerase that uses ATP to introduce negative
supercoils into DNA. Here, gyrase inhibitors (nalidixic acid and novobiocin) were used to investigate
the possible contribution of gyrases to the regulation
of cp nucleoid micromorphology in C. reinhardtii.
Nalidixic acid and novobiocin have different modes
of action. Nalidixic acid inhibits the dissociation of
covalently bound gyrase from DNA, resulting in the
production of DNA DSBs, whereas novobiocin inhibits ATPase activity and thereby inhibits the covalent binding of gyrase to DNA (Sugino et al., 1978;
Chen et al., 1996). Treatment of C. reinhardtii wildtype cells with nalidixic acid or novobiocin inhibited
growth (Supplemental Fig. S6A). Fluorescence microscopy of the inhibitor-treated cells revealed a
dose-dependent decrease of cp nucleoid number,
and most of the cells contained a single large cp
nucleoid at the highest inhibitor concentration (Fig.
3; Supplemental Fig. S6B). Corresponding with the
decrease in cp nucleoid number, individual cp nucleoids were generally enlarged in inhibitor-treated
cells. Most of the single cp nucleoids were round in shape
and comparable in size to nucleoids in cpRECA-KD
and moc cells (Fig. 3). These results suggest that the
inhibition of gyrase function leads to a dramatic
Figure 2. The cp nucleoid number and size in
cpRECA-KD cells. A, C. reinhardtii cp nucleoids in
cpRECA-KD (a), moc A84 (b), moc G33 (c), and
wild-type (d) cells. DNA was stained using SYBR
Green I. SYBR Green I (green) and chlorophyll (red)
fluorescence signals were observed simultaneously
using fluorescence microscopy. N denotes the nucleus, and arrows indicate examples of cp nucleoids
(cpN). Bar = 5 mm. B, Histograms of cp nucleoid number per cell in cpRECA-KD and wild-type
(WT) cells.
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Chloroplast Roles of RecA and DNA Gyrase
reduction in cp nucleoid number and an associated
increase in nucleoid size.
Taken together, our data suggest that both cpRECA
and gyrases are likely to be involved in the maintenance
of cp nucleoid micromorphology through the regulation
of cpDNA replication, recombination, and topology.
Destabilization of the Chloroplast Genome in
cpRECA-KD Cells
Reductions in the levels of cpRECA and gyrase affected cp nucleoid number and size. Next, we examined whether the molecular structure of the cp genome
is altered in cpRECA-KD lines and gyrase inhibitortreated cells. Previously, we showed that knockout
of P. patens cpRECA2 resulted in cp genome instability
as a consequence of increased recombination between
SDRs (Odahara et al., 2015). Therefore, cp genome
instability in C. reinhardtii cpRECA-KD cells was investigated with a particular focus on recombination
between dispersed repeats. First, the C. reinhardtii cpDNA
sequence was examined for repeat sequences using
REPuter (Kurtz et al., 2001). Numerous repeats were
identified that were consistent with previous analysis
using MultiPipMaker (Maul et al., 2002). Approximately 2,600 pairs of repeats longer than 35 bp (0-bp
mismatch) were identified in the C. reinhardtii cp genome (Supplemental Data Set S1).
Most of the identified repeats were unsuitable for
use in the PCR analysis of recombination shown in
Supplemental Figure S7, as they were palindromic or
were found in large inverted repeats. Although several
suitable loci were identified, recombination analysis
was problematic in many of these for two main reasons.
The design of primers specific to recombination products was hampered, first, by the presence of abundant
repeats and, second, by low copy numbers or no copies
of recombination products. However, through trial and
error, suitable recombination products were identified
from CD5 (77 bp, 1-bp mismatch), CI12 (94 bp, 1-bp
mismatch), CD15 (59 bp, 0-bp mismatch), and CD20
(71 bp, 1-bp mismatch) repeats (Fig. 4F; Supplemental
Table S1). Intramolecular recombination between these
Figure 3. Effects of gyrase inhibitors on cp nucleoids. Wild-type cells
were cultivated in medium containing the gyrase inhibitor nalidixic
acid or novobiocin. DNA was stained using SYBR Green I. SYBR Green I
(green) and chlorophyll (red) fluorescence signals were observed simultaneously using fluorescence microscopy. N and cpN indicate nucleus and cp nucleoids, respectively. Bar = 5 mm.
repeats results in deletion and inversion of the flanking
region when they are oriented as direct repeats and
inverted repeats, respectively (Supplemental Fig. S7;
Supplemental Table S1). Copies of CD5 and CD20
were found at additional loci with similarity to their
surrounding sequences (CD59 and CD209; Fig. 4F;
Supplemental Table S1); therefore, the PCR analysis
detected recombination products from both CD5 and
CD59 and products from CD20 and CD209 simultaneously. Recombination products from CD5 and CD20
were significantly more abundant in the cpRECA-KD
strains than in the wild type, whereas recombination
products from CI12 or CD15 were not significantly
affected (Fig. 4, A–D). The increase in CD5 and CD20
products was more prominent in the knockdown line
with the lowest cpRECA transcript levels (Fig. 4, A and
D). Analysis of cpDNA copy number relative to nuclear DNA using qPCR of the chloroplastic psbB locus
and the nuclear cblp locus showed that cpDNA copy
number in cpRECA-KD cells was 50% to 65% of that in
the wild type (Fig. 4E). These results suggest that
knockdown of cpRECA causes cp genomic instability
by inducing aberrant recombination between SDRs
and that the increase in recombination is accompanied
by a reduction in cpDNA copy number.
Chloroplast Genomic Instability in Gyrase InhibitorTreated Cells
The cp genome instability was observed in cpRECA-KD cells. Because inhibition of gyrase and knockdown of cpRECA had similar effects on cp nucleoid
number and size, it was also possible that gyrase inhibition might similarly have effects on genome stability.
To test this possibility, recombination between SDRs in
cpDNA was analyzed in gyrase inhibitor-treated cells.
First, qPCR analysis was performed for CD5, which
exhibited elevated recombination in cpRECA-KD lines.
The abundance of CD5 recombination products increased in wild-type cells treated with nalidixic acid or
novobiocin, depending on inhibitor concentration (Fig.
5, A and B). Novobiocin-treated cells exhibited a more
pronounced recombination effect than nalidixic acidtreated cells: the abundance of CD5 recombination
products increased approximately 160-fold relative to
the wild type at the highest novobiocin concentration,
compared with approximately 9-fold at the highest
nalidixic acid concentration (Fig. 5, A and B). Recombination products from CI12, CD15, and CD20 also
increased in nalidixic acid- and novobiocin-treated cells
compared with the wild type (Fig. 5, C–E). As with
CD5, the effect was more pronounced with novobiocin
than with nalidixic acid (Fig. 5, C–E).
The cpDNA copy number was determined using
qPCR in wild-type and gyrase inhibitor-treated cells.
The copy number in cells treated with either nalidixic
acid or novobiocin decreased in a dose-dependent
manner, with cpDNA copy numbers decreased to approximately 60% and approximately 20% of the wild
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Odahara et al.
Figure 4. Effects of cpRECA knockdown on cpDNA. A to C, Quantification of CD5, CI12, and CD15 recombination products. qPCR
analysis is shown for relative copy numbers of CD5 (A), CI12 (B), and CD15 (C) cpDNA recombination products. Copy numbers
were normalized to cp psbB. Data represent averages of three replicates. D, PCR analysis of CD20 recombination products in
cpRECA-KD lines using psbB as an amplified internal control. E, qPCR analysis of relative cpDNA psbB copy number, normalized to
nuclear Cblp. F, Positions of CD5, CI12, CD15, and CD20 in C. reinhardtii cpDNA. Large inverted repeats are shown with thick
black lines. SDRs are indicated with triangles. The cpDNA sequence corresponds to accession NC_005353 when taken clockwise
from position 1. Data represent means of three replicates. *, P , 0.01 (compared with the wild type [WT]).
type in cells exposed to 1.6 mM nalidixic acid and 400 mM
novobiocin, respectively (Fig. 5, F and G). Together, these
results indicate that cp genome destabilization and
cpDNA copy number reduction are associated with
the impairment of cp nucleoid segregation in gyrase
inhibitor-treated cells.
DISCUSSION
In this study, we showed that knockdown or overexpression of cpRECA, or treatment of cells with gyrase
inhibitors, resulted in significant alterations to the micromorphology of cp nucleoids in C. reinhardtii. These alterations were accompanied by the impairment of cpDNA
integrity and reductions in cpDNA copy number.
cpRECA is a homolog of bacterial RecA, a recombinase
involved in homologous pairing and strand exchange in
HR repair that has an important role in the repair of stalled
and collapsed replication forks (Lusetti and Cox, 2002).
Here, immunofluorescence staining (Fig. 1) of cpRECA
and the expression of cpRECA tagged with YFP or HA
(Fig. 1; Supplemental Fig. S2) confirmed that cpRECA
localized to cp nucleoids. Notably, cpRECA did not associate with cp nucleoids homogenously but was found at
the periphery of a small subset of cp nucleoids (Fig. 1).
This nonuniform localization of cpRECA suggests that
cpRECA is unlikely to be a constitutive core component of cp nucleoids and may indicate specific binding
of cpRECA to damaged DNA.
In some cells, overexpression of cpRECA resulted in
deformation of the dispersed cp nucleoid particles into
a single thick filamentous structure. These structures
were completely covered with cpRECA, in contrast with
the nonhomogenous associations seen in cells with wildtype-like cp nucleoids (Fig. 1B), suggesting that cpRECA
was the critical factor driving the formation of the fibrous
cp nucleoid structures (Fig. 1D). Bacterial RecA binds
preferentially to ssDNA to form a filamentous nucleoprotein structure (Kowalczykowski et al., 1994), suggesting that cpRECA may similarly bind to cpDNA in C.
reinhardtii overexpressing cpRECA. However, mung bean
nuclease/qPCR analysis revealed that the cpRECA-YFP
overexpression line did not have more ssDNA regions in
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Chloroplast Roles of RecA and DNA Gyrase
Figure 5. Chloroplast genome instability in gyrase inhibitor-treated cells. A
and B, qPCR analysis of relative CD5
copy numbers in wild-type cells treated
with nalidixic acid (A) or novobiocin (B).
Copy numbers were normalized to cp
psbB. C and D, qPCR analysis of CI12
(C) or CD15 (D) recombination products
in cells treated with 1.6 mM nalidixic acid
or 400 mM novobiocin. Copy numbers
were normalized to psbB. E, PCR analysis
of CD20 recombination products in cells
treated with nalidixic acid or novobiocin,
using psbB as an amplified internal control. WT, The wild type. F and G, qPCR
analysis of relative cpDNA psbB copy
number, normalized to nuclear Cblp, in
wild-type cells treated with nalidixic acid
(F) or novobiocin (G). The untreated wildtype ratio was set as 1. Data represent
means of three replicates. *, P , 0.01
(compared with the untreated control).
cpDNA than the control strain (Supplemental Fig.
S3). RecA forms nucleofilament also with doublestranded DNA, which is triggered by nucleation at
ssDNA regions (Lusetti and Cox, 2002). The fibrous
cp nucleoid/cpRECA structure might be largely
composed of double-stranded DNA-cpRECA nucleofilaments, which would be insensitive to mung bean
nuclease treatment.
Compared with the wild type, cpRECA-KD cells
exhibited a decrease in cp nucleoid number and an increase in cp nucleoid size that, in some cases, produced
a single large nucleoid comparable in size to the nucleus
(Fig. 2). This cp nucleoid condensation phenotype was
reminiscent of the moc mutant phenotype, the gene responsible for which has not yet been identified (Misumi
et al., 1999). In bacteria, a recA mutation results in the
production of anucleate cells, suggesting that RecA
may be involved in bacterial chromosome segregation
(Zyskind et al., 1992). In the bacterial recA mutant,
stalled or collapsed replication forks may not be properly processed; therefore, subsequent chromosome
segregation may be prevented. Defects in the repair
of impaired replication forks in cpRECA-KD cps may
similarly perturb cpDNA segregation. The association
of cpRECA with only a subset of cp nucleoids suggests
that cpRECA acts as an accessory protein at the nucleoids; however, it is surprising that overproduction
or knockdown of a single accessory protein would
have such a substantial impact on cp nucleoid micromorphology. Therefore, it may be of interest to investigate
cpRECA functions in other processes, such as cp division,
during which cp nucleoid shape and number undergo
dynamic changes (Kuroiwa et al., 1981; Nakamura et al.,
1986; Ehara et al., 1990).
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Odahara et al.
The condensation of cp nucleoids also was observed in cells treated with the gyrase inhibitors novobiocin and nalidixic acid (Fig. 3). Novobiocin was
more effective at inducing cp nucleoid condensation
than was nalidixic acid, suggesting that condensation
did not occur as a result of DSB formation but rather
by the inhibition of ATPase activity and the resultant
cpDNA structural and topological changes. Bacterial
gyrase is a type II topoisomerase that can introduce
negative supercoils. Gyrase-defective bacterial mutants
exhibit impaired segregation of catenated chromosomes
(Steck and Drlica, 1984). Similarly, cp chromosome segregation was impaired by knockdown of cp gyrase in N.
benthamiana (Cho et al., 2004) and by treatment with
nalidixic acid in the alga Cyanidioschyzon merolae (Itoh
et al., 1997). Therefore, it is likely that gyrase has a
common role in chromosome segregation in bacteria and
cps. Our data suggest that both cpRECA and gyrases are
involved in cpDNA segregation and the maintenance of
cp nucleoid morphology via the control of cpDNA replication, recombination, and topology.
Recombination between cp SDRs was induced by
knockdown of cpRECA. This suggests that cpRECA
is involved in the maintenance of cp genome stability by the suppression of aberrant recombination in
C. reinhardtii, as was shown previously in the land
plant P. patens (Odahara et al., 2015). In contrast with
the cp genomes of land plants, which have few repeats longer than 30 bp, the C. reinhardtii cp genome
contains abundant repeats. However, despite this,
recombination was induced in relatively few of the
C. reinhardtii repeats in cpRECA-KD cells. This suggests
that C. reinhardtii cpRECA may generally suppress recombination between short repeats, but additional suppression pathways that do not require cpRECA activity
may be present in C. reinhardtii cp. In addition, hot spots
or suppressed regions for recombination may be present
in the cpDNA genome (Newman et al., 1992). Alternatively, the presence of abundant repeats might minimize
the accumulation of recombination products from any
two specific repeats.
SDR recombination products also accumulated in cells
treated with gyrase inhibitors (Fig. 5), suggesting that cp
genomes were destabilized as a result of induced recombination between SDRs in the inhibitor-treated cells. While
the accumulation of CD5 recombination products was
similar between inhibitor-treated cells and cpRECA-KD
cells, CI12 and CD15 recombination products accumulated
at higher levels in inhibitor-treated cells than in cpRECA-KD
cells. These results suggest that recombination is induced in
a wider range of repeats in inhibitor-treated cells than in
cpRECA-KD cells. The difference in induced recombination
may reflect differences in the roles of cpRECA and gyrase in
the suppression of recombination.
In this study, we demonstrated that cp genome instability is accompanied by an impairment of nucleoid
segregation in cpRECA-KD cells and cells treated with
gyrase inhibitors. Our previous study suggested that
the roles of mitochondrial RECA1 (Odahara et al., 2009)
and chloroplastic RECA2 (Odahara et al., 2015) in the
repair of stalled or collapsed replication forks in P.
patens corresponded to the activities of bacterial
RecA. RECA proteins may promote homologous recombination during the repair of stalled or collapsed
replication forks, thereby preventing organelle genome instability. C. reinhardtii cpRECA may function
similarly in the repair of stalled or collapsed replication forks, and cpRECA-independent recombination, which is suppressed to a low level in the wild
type, may be induced during the repair of disordered
replication forks in cells with the cpRECA-KD background. Consequently, the impairment of nucleoid
segregation and genome instability in cpRECA-KD
cells may be the consequence of defective replication
repair processes (Supplemental Fig. S8).
Bacteria defective in gyrase experience replication
fork arrest as a result of positive supercoil accumulations ahead of the fork. However, HR repair proteins,
including RecA, are not required for the repair of these
arrested forks (Grompone et al., 2003). This suggests
that replication fork arrest induced by the accumulation
of positive supercoiling is not subject to HR repair in
E. coli. As in E. coli, gyrase in cps is involved in DNA
supercoiling (Wall et al., 2004); therefore, it is likely that
supercoil accumulation induces cp replication fork arrest in C. reinhardtii. Recombination between dispersed
repeats in inhibitor-treated cp, which is probably independent of cpRECA, may occur when arrested replication forks are processed (Supplemental Fig. S8). This
may correspond with recombination-dependent replication in novobiocin-treated C. reinhardtii cp, as proposed by Woelfle et al. (1993). Gyrase may maintain cp
genome stability by reducing replication stresses that
can lead to genome instability.
In summary, results from the induction of cp genome
instability and the impairment of nucleoid segregation in
two different cell types suggested that genome stability
and nucleoid segregation in cp were intimately related.
In addition, overexpression of cpRECA profoundly affected cp nucleoid structure, indicating its potential effects on nucleoid dynamics. Our findings provide further
evidence toward an integrated understanding of nucleoid dynamics and genome integrity in cps.
MATERIALS AND METHODS
Plant Materials and Culture Medium
Chlamydomonas reinhardtii wild-type strains CC-125 (mating type plus) and
CC-124 (mating type minus; Harris, 1989), the cell wall-less mutant cw15
(Harris, 1989), and mutant strains moc A84 and moc G33 (Misumi et al., 1999)
were used in this study. Cells were cultivated in Tris-acetate-phosphate (TAP)
liquid medium (Harris, 1989) at 20°C under constant white light. For growth
comparisons, cells were cultivated in TAP liquid medium and then spotted onto
TAP agar medium after cell counting. For gyrase inhibitor treatment, cells were
cultivated in TAP liquid medium containing novobiocin or nalidixic acid at the
concentrations given in the figure legends.
Knockdown of cpRECA in C. reinhardtii
An artificial microRNA system utilizing pChlami RNA3 (Molnar et al., 2009)
was used to knockdown the C. reinhardtii cpRECA gene (GenBank accession no.
2344
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Chloroplast Roles of RecA and DNA Gyrase
AB048829). Artificial microRNA sequences for cpRECA (oligonucleotide DNA
P1 and P2) were designed using WMD3 (http://wmd3.weigelworld.org/cgibin/webapp.cgi; Molnar et al., 2009). Oligonucleotides were annealed and then
cloned into the SpeI site of pChlami RNA3 (a generous gift from Dr. David
Baulcombe, University of Cambridge). The resulting plasmid was introduced
into C. reinhardtii cc125 cells by electroporation (Shimogawara et al., 1998), and
transformants were selected on TAP agar medium containing paromomycin.
Knockdown of cpRECA was analyzed by quantitative reverse transcriptionPCR (parameters are given below) with primers P3 and P4 for cpRECA and
P5 and P6 for Cblp. Primer sequences are provided in Supplemental Table S2.
was performed using diluted antibody (1:2,500) and the ECL Plus western-blot
detection kit (GE Healthcare). Chemiluminescence was detected with a luminoimage analyzer (LAS4000; Fujifilm) and analyzed using Multi Gauge version
4.0 software (Fujifilm). Gels were also stained using Coomassie Brilliant Blue to
assess loading.
Indirect Immunofluorescence Microscopy
C. reinhardtii genomic DNA was extracted as follows. C. reinhardtii cells were
resuspended in 2% SDS, 400 mM NaCl, 40 mM EDTA, and 100 mM Tris-HCl, pH
8. DNA was then extracted using phenol/chloroform and precipitated by
adding CTAB. RNA was extracted from C. reinhardtii cells using Sepasol
(Nacalai). After DNaseI treatment to remove residual genomic DNA, reverse
transcription was performed using total RNA, random hexamers, and reverse
transcriptase ReverTra Ace (Toyobo).
Proteins were detected by immunofluorescence staining as described previously
(Nishimura et al., 2002). Cells were harvested and fixed in 3.8% formaldehyde on
ice for 10 min. After washing with Tris-buffered saline (TBS), the cells were gently
permeabilized with 0.1% Triton X-100 on ice for 20 min. Cells were then resuspended in TBS buffer containing 5% bovine serum albumin and 0.25% Tween
20 and then kept on ice for 30 min. The cells were then incubated on ice for 1 h with
the primary antibody (anti-YFP mouse IgG [Takara Bio] and anti-HA mouse IgG
[Santa Cruz Biotechnology]) at a 1:1,000 dilution. After washing, the cells were
resuspended in TBS buffer on ice for 30 min. The secondary antibody (Alexa Fluor
488 goat anti-mouse IgG) was added at a 1:1,000 dilution, and cells were incubated
on ice for 1 h. Finally, cells were washed, stained with 1% DAPI, and observed by
fluorescence microscopy under UV light (for DAPI) or blue light (for Alexa).
PCR Analysis of cpDNA
Supplemental Data
Extraction of Nucleic Acids
qPCR analysis of C. reinhardtii cpDNA was performed using total genomic
DNA and primers as follows: P7 and P8 for psbB, P9 and P10 for psbD, P11 and
P12 for DNA recombined between CD5 repeats, P13 and P14 for CD15, P15 and
P16 for CI12, and P17 and P18 for Cblp. qPCR was performed using the
MX3000P QPCR System (Agilent) and FastStart Universal SYBR Green Master
(Rox; Roche). PCR analysis was performed using C. reinhardtii total genomic
DNA and primers P19 and P20 for CD20 and P21 and P22 for psbB.
The following supplemental materials are available.
Supplemental Figure S1. Immunoblotting of cpRECA.
Supplemental Figure S2. Overexpression of cpRECA tagged with YFP or HA.
Supplemental Figure S3. ssDNA nuclease sensitivity of cpDNA.
Supplemental Figure S4. Knockdown of the cpRECA gene in C. reinhardtii.
Supplemental Figure S5. Fluorescence microscopy of cpRECA-KD cells.
Analysis of ssDNA Nuclease Sensitivity
Total genomic DNA was extracted from cells cultivated under standard
culture conditions. For heat denaturation, the extracted genomic DNA was
incubated at 98°C for 5 min and then chilled on ice. One microgram of genomic
DNA was digested with 1 unit of mung bean (Vigna radiata) nuclease (New
England Biolabs) at 30°C for 30 min. qPCR analysis was performed using the
7500 Fast Real-Time PCR System (Applied Biosystems) and primers P21 and
P22 for psbB, P23 and P24 for psbD, and P17 and P18 for Cblp.
Supplemental Figure S6. Effect of gyrase inhibitors on C. reinhardtii
growth and cp nucleoid number.
Supplemental Figure S7. qPCR assay for the amplification of recombination products from direct and inverted repeats.
Supplemental Figure S8. A model for genome instability and the defect in
nucleoid segregation in cps.
Supplemental Table S1. C. reinhardtii cp repeats tested for recombination.
Supplemental Table S2. Oligonucleotide DNAs used in this study.
Fluorescence Microscopy
For YFP-tagging analysis, cpRECA was amplified from genomic DNA using
primers P25 and P26, and YFP, which was a generous gift from Dr. Ralph Bock
(Max-Planck-Institute), was amplified using primers P27 and P28. Fragments were
inserted into NdeI-BamHI and XbaI-EcoRI sites of pGenD-C3HA (Chlamydomonas
Resource Center), respectively. The expression level of cpRECA-YFP was analyzed
by quantitative reverse transcription-PCR with primers P3 and P4 for cpRECA and
P29 and P30 for MAA7. Cells were stained using a 1:1,000 dilution of SYBR Green
or 1 mg mL21 DAPI to stain cp nucleoids. DAPI staining was performed after
fixation with 2.5% glutaraldehyde. Cells were observed with a BX51 fluorescence
microscope (Olympus) for standard observations and with a TCS SP5 confocal
laser scanning microscope (Leica Microsystems) for superresolution analysis.
Supplemental Movie S1. Superresolution 3D image of overexpressed
cpHLP-YFP.
Supplemental Movie S2. Superresolution 3D image of overexpressed
cpRECA-YFP.
Supplemental Data Set S1. C. reinhardtii cp repeats (.35 bp, 0-bp mismatch).
ACKNOWLEDGMENTS
We thank Hitomi Tanaka for technical assistance, Osami Misumi for providing C. reinhardtii strains, and David Baulcombe and Ralph Bock for plasmids.
Received October 5, 2016; accepted October 13, 2016; published October 17,
2016.
Antibody Preparation
Primers P31 and P32 were used to amplify cpRECA cDNA. The resulting fragment was cloned into pQE80l (Qiagen), and the plasmid was transformed into
Escherichia coli strain BL21. The E. coli culture was grown in Luria-Bertani medium at
37°C. Once the culture reached OD600 0.7 to 1, isopropyl b-D-1-thiogalactopyranoside
was added to a final concentration of 1 mM. Expressed protein was purified using
Ni-NTA agarose (Qiagen) according to the manufacturer’s instructions. Antibodies
were raised by injecting the purified recombinant protein into mice every 2 weeks for
a total of five times.
Immunoblot Analysis
Proteins extracted from C. reinhardtii cells were separated using 12.5% SDSPAGE and transferred onto a polyvinylidene fluoride membrane. Detection
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