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A Conserved, Mg2+-Dependent Exonuclease DegradesOrganelle DNA during Arabidopsis Pollen Development C W
Ryo Matsushima,a,1 Lay Yin Tang,a,1 Lingang Zhang,a Hiroshi Yamada,a David Twell,b and Wataru Sakamotoa,2
a Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama 710-0046, Japanb Department of Biology, University of Leicester, Leicester LE1 7RH, United Kingdom
In plant cells, mitochondria and plastids contain their own genomes derived from the ancestral bacteria endosymbiont.
Despite their limited genetic capacity, these multicopy organelle genomes account for a substantial fraction of total cellular
DNA, raising the question of whether organelle DNA quantity is controlled spatially or temporally. In this study, we
genetically dissected the organelle DNA decrease in pollen, a phenomenon that appears to be common in most angiosperm
species. By staining mature pollen grains with fluorescent DNA dye, we screened Arabidopsis thaliana for mutants in which
extrachromosomal DNAs had accumulated. Such a recessive mutant, termed defective in pollen organelle DNA degrada-
tion1 (dpd1), showing elevated levels of DNAs in both plastids and mitochondria, was isolated and characterized. DPD1
encodes a protein belonging to the exonuclease family, whose homologs appear to be found in angiosperms. Indeed, DPD1
has Mg2+-dependent exonuclease activity when expressed as a fusion protein and when assayed in vitro and is highly active
in developing pollen. Consistent with the dpd phenotype, DPD1 is dual-targeted to plastids and mitochondria. Therefore, we
provide evidence of active organelle DNA degradation in the angiosperm male gametophyte, primarily independent of
maternal inheritance; the biological function of organellar DNA degradation in pollen is currently unclear.
INTRODUCTION
Mitochondria and plastids originate from the endosymbiosis of
rickettsia-like a-proteobacteria and cyanobacteria-like photo-
synthetic bacteria, respectively (Gray et al., 1999; Dyall et al.,
2004; Keeling, 2010). Most genes in the primitive endosymbi-
onts were transferred to the plant nuclear genome, yet both
organelles retain remnant genomes and carry out DNA repli-
cation, transcription, and translation (Gray, 1999; Kleine et al.,
2009). For example, coordinated expression of nuclear and
organelle genes is a central subject in the studies of chloroplast
biogenesis.
Despite its smaller genome size and limited genetic informa-
tion, organelle DNA sometimes account for a substantial amount
of total DNA because it is present as multiple copies (for review,
see Sakamoto et al., 2008). For example, total DNAs from leaf
tissues in higher plants often contain >20% of plastid DNAs
(ptDNAs) (Bennet and Smith, 1976; Lamppa and Bendich, 1979;
Arabidopsis Genome Initiative, 2000; Rauwolf et al., 2010). The
copy number of ptDNA appears to correlate with nuclear ploidy
and appears to vary among species or even among different
tissues and during developmental stages (Herrmann andKowallik,
1970; Kowallik andHerrmann, 1972; Lamppa andBendich, 1979;
Scott and Possingham, 1980; Kuroiwa et al., 1981; Boffey and
Leech, 1982; Tymms et al., 1983). Multiplication of plastids by
division during leaf development further complicates the ptDNA
amount per organelle. Such a complex polyploid nature of the
plastid genome (also of mitochondrial genome) has raised the
question of whether organelle DNA levels are controlled spatially
or temporally. Although several proteins are known to play roles
in maintaining the configuration of plant organelle genomes
(Abdelnoor et al., 2003; Edmondson et al., 2005; Zaegel et al.,
2006; Shedge et al., 2007; Marechal et al., 2008, 2009; Rowan
et al., 2010), very little is understood about DNA degradation at
the molecular level.
ptDNAs (and also mitochondrial DNAs [mtDNAs]) are cytolog-
ically detectable as nucleoids by staining tissues with DNA
fluorescent dye, such as 49,6-diamidino-2-phenylindole (DAPI) or
SYBR green I (SYBR) (Kuroiwa, 1991, 2010). The ptDNAs exist as
a complex with proteins that constitute plastid nucleoids (Sato
et al., 1998, 2001, 2003; Murakami et al., 2000; Jeong et al.,
2003). The cytological detection of organelle DNAs is frequently
used to estimate DNA levels together with DNA gel blot hybrid-
ization, quantitative PCR, and colorimetric detection of DNA
hydrolysates. These methods enable us to investigate the num-
ber, morphologies, and behavior of plastid nucleoids during leaf
development (Rauwolf et al., 2010 and references therein). In
Arabidopsis thaliana, cytological observation demonstrated that
amounts of ptDNA increase more than 10-fold during leaf de-
velopment (Fujie et al., 1994). By contrast, contradictory results
were reported for ptDNA levels in mature leaves: constant or
declining ptDNA amounts have been reported by different lab-
oratories (Rowan et al., 2004, 2009; Li et al., 2006; Zoschke et al.,
2007). Studies of numbers of mtDNAs per cell have revealed that
1 These authors contributed equally to this work.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Wataru Sakamoto([email protected]).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.111.084012
The Plant Cell, Vol. 23: 1608–1624, April 2011, www.plantcell.org ã 2011 American Society of Plant Biologists
mtDNA levels also fluctuate during leaf development. More
importantly, some mitochondria might lack a complete genome
(Preuten et al., 2010). A decrease in mtDNAs has also been
reported during pollen development (Wang et al., 2010). These
circumstantial observations prompted us to study organelle DNA
degradation by a forward genetic approach.
Here, we specifically examine organelle DNA levels in mature
pollen of Arabidopsis. Much attention has been given to organelle
DNA levels in male reproductive organs because the decrease of
organelle DNAs in male tissues is suggested to correlate with
their maternal inheritance: ptDNAs that are detectable by DAPI
stains in male germ cells are often associated with biparental
inheritance of ptDNAs (Hagemann and Schrodoer, 1989; Nagata
et al., 1999; Birky, 2001; Hagemann, 2004; Kuroiwa, 2010).
Irrespective of the inheritance mode, however, we noticed that
organelle DNAs are cytologically absent in pollen vegetative cells
(Matsushima et al., 2008a; Sakamoto et al., 2008). A survey of
numerous mature pollen grains using DAPI staining has revealed
that almost all angiosperm species lack cytologically detect-
able organelle DNAs in pollen vegetative cells (Corriveau and
Coleman, 1988;Mogensen, 1996; Zhang et al., 2003;Wang et al.,
2010). It is noteworthy that pollen vegetative cells do not con-
tribute to fertilization, but they do contain numerous plastids and
mitochondria, which might be necessary for a pollen tube to
germinate, elongate, and deliver spermcells into the embryo sac.
Given that the lack of DAPI signals in pollen vegetative cells is so
clear and consistent in many species (irrespective of inheritance
mode), we reasoned that organelle DNA levels are strictly down-
regulated by a dominant mechanism. We also exploited the
relative ease in examining organelle DNAs in mature pollen,
rather than in leaf tissues, which require time-consuming sec-
tioning or protoplast isolation.
For this study, we performed extensive forward genetic anal-
ysis and isolated Arabidopsis mutants in which organelle DNA
was retained in mature pollen grains. Characterization of the
gene responsible for the mutants led us to identify a nuclease
that is expressed preferentially during pollen development. It is
particularly interesting that this DNase is localized in both the
plastid andmitochondria, providing evidence for the existence of
an organelle nuclease in eukaryotes. Our data reveal an active
mechanism of organelle DNA degradation in a tissue-specific
manner, which is primarily independent of maternal inheritance.
RESULTS
IsolationofArabidopsisMutantsDefective inOrganelleDNA
Degradation of Pollen Grains
Our cytological analysis of pollen grains in Arabidopsis revealed
that when mature pollen was stained with DAPI, almost no
signals corresponding to organelle DNAs were detected (Figures
1 and 2; see also Sakamoto et al., 2008). Numerous plastids and
mitochondria exist in pollen vegetative and sperm cells. There-
fore, we inferred that organelle DNAs decrease during pollen
development and that it is feasible to screen mature pollen for
mutants that exhibit altered levels of organelle DNAs. This
strategy presents the additional advantage that the pollen phe-
notype will segregate in M1 pollen and thus can be found by
screening mature pollen grains from M1 flowers (Chen and
McCormick, 1996). In our screening method, pollen grains col-
lected from M1 or M2 flowers were fixed briefly with glutaralde-
hyde with subsequent gentle squashing over a cover slip and
DAPI staining, which allowed careful observation of DAPI sig-
nals (Figure 1A). Mature pollen grains of ;2000 individual ethyl
methanesulfonate–mutagenized M1 and 2000 M2 Arabidopsis
plants were screened using this method. As a consequence, we
isolated five mutant lines that exhibited unusual DAPI signals
within the cytoplasmof vegetative cells. TheseDAPI signalswere
distinct from those corresponding to vegetative and spermnuclei
and rather resembled organelle DNAs (Figure 1B). Mutants
isolated in M1 population were further characterized to obtain
M2 individuals where all pollen showed the phenotype. These
mutants were designated as defective in pollen organelle DNA
degradation (dpd). This dpd phenotype was characterized ge-
netically in the subsequent generations. Overall, our screening
and genetic analysis identified two recessive mutations, dpd1
and dpd2; this work specifically examined dpd1. All mutants
except for dpd2 were the alleles of dpd1 (designated as dpd1-1
to dpd1-4; see below). The recessive nature of dpd mutations
Figure 1. Experimental Strategy to Isolate Mutants Defective in Organ-
elle DNA Degradation in Arabidopsis Pollen.
(A) Schematic representation of the experiment. Arabidopsis wild-type
Col were mutagenized using ethyl methanesulfonate. Mature pollen
grains from M1 or M2 plants were screened using the pollen squash
method (middle) for easier observation of DAPI signals derived from
organelle DNAs. Examples of DAPI-stained pollen grains before and after
the squash are shown on the right. Bars = 20 mm.
(B) Isolation of dpd mutants. DAPI-stained squashed pollen from Col
(wild type), dpd1, and dpd2 are shown.
Organelle DNA Degradation in Pollen 1609
Figure 2. Observation of Organelle DNAs in Developing Pollen and Leaf Mesophyll Cells of Col and dpd1-1.
(A) Schematic representation of pollen development in Arabidopsis.
(B) DAPI-stained Technovit sections of bicellular and tricellular pollens. Strong signals are indicated by arrows in tricellular pollen and sperm nuclei;
other signals in dpd1 correspond to extrachromosomal DNAs. Bars = 5 mm.
(C) Electron micrographs of mature pollen grains from wild-type ecotype Col and dpd1-1. Bars = 2 mm.
(D) DAPI-stained mesophyll protoplasts from Col and dpd1-1. Protoplasts were gently squashed to detect DAPI signals within chloroplasts. Bars =
20 mm.
1610 The Plant Cell
implied a dominant mechanism to decrease organelle DNAs in
mature pollen, as we expected.
Organelle DNAs Do Not Decrease during dpd1
Pollen Development
To observe organelle DNA levels more carefully, we prepared
Technovit-embedded thin sections (0.5mm) of developing pollen
grains fromwild-type Columbia (Col) and dpd1 and stained them
with DAPI. In Arabidopsis, mature pollen is formed after two
characteristic mitoses (Figure 2A) (Borg et al., 2009). Asymmetric
division of uninucleate microspores at pollen mitosis I produces
bicellular pollen comprising a vegetative cell and a germ cell.
Subsequent division of the germ cell at pollen mitosis II (PMII)
produces a pair of sperm cells forming tricellular pollen. Our thin
section analysis showed that, in Col, cytoplasmic DAPI signals
start to decrease in the late bicellular stage. They disappear
completely at the tricellular stage in Col, indicating that the
decrease (or degradation) of organelle DNA normally occurs
during PMII (Figure 2B). By contrast, dpd1 showed strong
cytoplasmic DAPI signals in pollen, which were retained even
at the tricellular stage. Segregation of the dpd phenotype in the
F2 population from a cross between dpd1 and Col revealed that
dpd1 behaved as a single recessive trait and that the dpd
phenotype showed complete penetrance in the selfed progeny
(see Supplemental Tables 1 and 2 online). No ultrastructural
abnormality was observed in dpd1 organelles (Figure 2C),
suggesting that the membrane integrity of both mitochondria
and plastids is maintained. Examination of in vitro–germinated
pollen by DAPI also revealed that organelle DNAs were retained
even after germination in dpd1 (see Supplemental Figure 1 on-
line). These results demonstrated that dpd1 appears to compro-
mise DNA reduction during pollen development.
Vegetative and Reproductive Growth of dpd1
We next examined whether dpd1 displays any visible pheno-
types not only in reproductive growth but also in vegetative
growth. None of the dpd1 alleles showed differences in their
vegetative growth under normal conditions (Figure 3A), suggest-
ing that DPD1 primarily affects organelle DNA levels in pollen
grains but not those in other tissues. To test this possibility, we
examined organelle DNA levels by staining protoplasts derived
from 6-week-old mature leaves with DAPI. Results showed that,
unlike mature pollen grains, no apparent difference in DAPI-
detectable organelle DNAs was detected in chloroplasts of leaf
cells (Figure 2D). These results imply that, in dpd1, organelle DNA
levels are increased in pollen grains but not in other somatic
tissues. Subsequently, visible phenotypes in dpd1 male repro-
ductive organs were also examined. Visual inspection showed
Figure 3. Plant Architecture and Reproductive Organs of dpd1 Mutants.
(A) Five-week-old dpd1-1. Bar = 1 cm.
(B) Immature seeds from wild-type Col and dpd1-1. Bars = 1 mm.
(C) Floral organs from Col and dpd1-1. Bars = 2 mm.
(D) Single flower from Col and dpd1-1. Bars = 2 mm.
(E) Single anther from Col and dpd1-1 stained with Alexander solution. Bars = 100 mm.
Organelle DNA Degradation in Pollen 1611
that flowers and seed set appeared normal in dpd1 (Figures 3B
to 3D). The dpd phenotype was completely penetrant in dpd1
selfed progeny and segregated normally in the F2 (see Supple-
mental Tables 1 and 2 online). Mature pollen grains in dpd1
exhibitedmorphology and viability (Alexander stain, Figure 3E; see
Supplemental Table 3 online) that were indistinguishable from
those of the wild type. No difference in pollen size was found
between the wild type and dpd1 (see Supplemental Table 4
online). Collectively, we inferred that the dpd phenotype does
not significantly influence overall plant growth and pollen
morphology.
Both ptDNA and mtDNA Are Retained in dpd1 Pollen
The dpd phenotype in dpd1 raised the question of which DNA-
containing organelles––plastids, mitochondria, or both––emitted
the DAPI signals in pollen vegetative cells. To address this
question, we visualized plastids and mitochondria in mature
pollen by expressing organelle-targeted fluorescent proteins
(green fluorescent protein in plastids [ptGFP] and red fluorescent
protein in mitochondria [mtRFP]; Figure 4). We reported previ-
ously that these organelle-targeted GFP/RFPs, when expressed
under the control of a vegetative cell-specific promoter LAT52
from tomato (Solanum lycopersicum; Twell et al., 1990), can show
plastids or mitochondria in pollen vegetative cells (Matsushima
et al., 2008b; Tang et al., 2009). We introduced the correspond-
ing transgenes (Lat52pro:PTS:GFP and Lat52pro:MTS:RFP) into
dpd1. Mature pollen grains from these transgenic lines were
examined using DAPI or SYBR stain (depending on the fluores-
cent protein used) and simultaneous detection of fluorescent
proteins. The results demonstrated that DAPI signals in dpd1
(Lat52pro:PTS:GFP) colocalized with ptGFP. Similarly, SYBR
signals in dpd1 (Lat52pro:MTS:RFP) colocalized with mtRFP
(Figure 4). These results indicate that the extrachromosomal
DNA signals in dpd1 mutants were derived from both plastids
and mitochondria.
Both ptDNA and mtDNA Increased in Pollen but Not in
Somatic Cells
To confirm that both ptDNA and mtDNA levels were altered in
pollen but not in somatic tissues of dpd1, we performed a PCR-
based assay. Total DNAs were prepared from pollen and young
seedlings and assayed by quantitative real-time PCR. Levels of
ptDNAs (psbA) or mtDNAs (cox1) in these tissues of Col and
dpd1 were normalized based on the level of nuclear DNAs (18S
rDNA; see Methods and Rowan et al., 2009). As expected,
ptDNAs andmtDNAs had significantly increased in dpd1-1 com-
pared with Col (n = 3, Welch’s t test, P = 0.0041 and 0.0226,
respectively) (Figures 5A and 5B). A large difference in mtDNA
levels was detected, probably because of an extremely low level
of mtDNA in Arabidopsis wild-type pollen grains (Wang et al.,
2010). By contrast, young seedlings showed no significant
differences between Col and dpd1-1 in the level of ptDNA and
mtDNA (P = 0.2887 and 0.2692, respectively) (Figures 5C and
5D). Together, these results verified our cytological analysis of
dpd1 pollen stained with DAPI. We concluded that both ptDNA
and mtDNA levels are increased in dpd1 pollen.
Map-Based Cloning of the DPD1 Locus
We identified the DPD1 gene based on conventional map-based
cloning. Wemapped the dpd1-1mutation within a 623-kb region
on chromosome 5 (summarized in Supplemental Figure 2 online).
A survey of genes that potentially encode proteins targeted to
plastids and/ormitochondria allowed us to select possible genes
responsible for the dpd1 phenotype. Subsequent sequencing of
the candidate genes identified a base change in the At5g26940
gene, which results in an amino acid substitution (Figure 6A).
Figure 4. Colocalization of DNA Signals with Plastids and Mitochondria in dpd1 Pollen.
A transgene that expresses plastid-targeted GFP or mitochondria-targeted RFP in pollen vegetative cells (presented on the left) was introduced into
dpd1-1. Colocalization of plastidial GFP signals with DAPI signals (top panels) or of mitochondrial RFP signals with SYBR (bottom panels) was
examined. Arrowheads indicate colocalization of fluorescent organelles and DAPI-stained or SYBR-stained signals. The asterisk denotes nuclear-
derived DAPI signals. Bars = 5 mm.
1612 The Plant Cell
Sequencing of this gene in three other dpd1 alleles (dpd1-2,
dpd1-3, anddpd1-4; Figure 6B) revealed that all these alleles had
base changes in At5g26940 (Figure 6). The base changes in
dpd1-1, dpd1-2, and dpd1-3 caused amino acid substitutions
(see Supplemental Figure 3 online), whereas dpd1-4 had a base
change close to the border of intron1/exon2. Furthermore, we
obtained two T-DNA insertion mutants (dpd1-5 and dpd1-6) in
At5g26940 and examined whether they showed the dpd1 phe-
notype. As expected, they all exhibited the dpd phenotype that
was indistinguishable from other dpd1 alleles.
To prove that At5g26940 encodes DPD1, a genomic fragment
encompassing At5g26540 was transformed into the dpd1-1
mutant, we generated five transgenic lines. One of the lines
was homozygous for the transformed sequence. It fully comple-
mented the dpd phenotype (Figure 7A) when mature pollen
grains were examined using our squash method. Moreover,
we conducted a PCR-based assay to verify the altered or-
ganelle DNA levels in dpd1 and the complemented line. PCR
products corresponding to plastid and mitochondria DNAs
were more abundant in dpd1-1 pollen than in Col and the
complemented line. Collectively, we concluded that At5g26940
encodes DPD1.
DPD1 Encodes a Protein Belonging to the Exonuclease
Family and Dual Targeted to Plastids and Mitochondria
DPD1 is present in a single copy inArabidopsis, where it encodes
a protein (316 amino acids) belonging to the exonuclease family
(Pfam: PF00929, ExonulX-T), which is included in the large
ribonuclease H-like superfamily (Clan CL0219). Three domains
included within the protein family members, ExoI, ExoII, and
ExoIII«, appeared to be conserved in DPD1 and other members
(see Supplemental Figure 3 online). A BLAST search and sys-
tematic analysis of DPD1 homologs using the entire DPD1 amino
acids and the SALAD Database (http://salad.dna.affrc.go.jp/
salad/en/) revealed that DPD1 homologs are present in small
green algae (Ostreococcus and Micromonas), moss (Physcomi-
trella), and higher plants (Figure 6C). By contrast, no suchhomolog
was detectable in a green alga (Chlamydomonas), a red alga
(Cyanidioschyzon), or a fungus (Saccharomyces). Although pro-
teins detectedas related toDPD1 in the green algae andmosshad
conserved exonuclease domains, they were much larger (>1260
amino acids) and apparently had additional DNA helicase do-
mains, suggesting a role that is distinct from organelle DNA
degradation. These results imply that DPD1 had evolved with
the appearance of anisogamous male reproductive organs in the
plant lineage.
It was tempting to speculate that DPD1 is targeted to both
plastids andmitochondria. To examine the cellular localization of
DPD1, we transiently expressed DPD1-GFP in Arabidopsis pro-
toplasts prepared from mesophyll cells, as described previously
(Miura et al., 2007). TheDPD1-GFP fusion protein colocalized not
only with chlorophyll autofluorescence, but also with the mito-
chondria-specific dyeMitotracker Red (see Supplemental Figure
4 online). These results indicate that DPD1 is localized in plastids
and mitochondria, each of which shows defective organelle
DNA degradation in dpd1 mutants.
DPD1 Is a Mg2+-Dependent Exonuclease
Sequence information and thedpd1phenotype together strongly
suggest that DPD1 has exonuclease activity (Figure 6A). To study
this possibility in vitro, a recombinant DPD1-His protein (6 3histidine tagged at C terminus) was generated in Escherichia coli
and affinity purified as described in Methods. In addition to the
wild-type DPD1-His gene, we expressed twoDPD1-His genes in
which a point mutant was introduced (Figure 8A). Because of a
nonsense mutation at the 186th amino acid, DPD1-His(Y186*)
has DPD1 that is C-terminally truncated and lacks His. Similarly,
DPD1-His(A236V) has an amino acid change equivalent to
dpd1-1 (Figure 6A; see Supplemental Figure 3 online). A majority
of these DPD1 proteins expressed in E. coliwere detected as ag-
gregated, although we purified DPD1-His and DPD1-His(A236V)
from soluble fractions (Figures 8B to 8D). Purified DPD-His
proteins were detected as two bands: The lower band was likely
a degradation product. Actually, DPD1-His(Y186*) was not pu-
rified because of the absence of the His tag, but we used this
fraction as a negative control.
Figure 5. Quantitative Analysis of Organelle DNA Levels in Pollen Grains
and Young Seedlings.
Levels of organelle DNAs per nuclear DNAs in Col and dpd1-1 were
determined using real-time PCR. Mean values of normalized organelle
DNA levels of Col were determined as 1.0. The relative values of dpd1-1
were calculated (n = 3, SD as error bars).
(A) ptDNA in pollen grains.
(B) mtDNA in pollen grains.
(C) ptDNA in 17-d-old seedlings.
(D) mtDNA in 17-d-old seedlings.
Organelle DNA Degradation in Pollen 1613
Nuclease activity of these recombinant proteins was assayed
in a standard buffer withmagnesiumbivalent cation as a cofactor
and DNA fragments (PCR-amplified ptDNA). The result showed
that DNAs were degraded by DPD-His during the first 20 min of
incubation (Figure 8E). By contrast, neither heat-denatured
DPD1-His, DPD1-His(A236V), nor the negative control fraction
degraded PCR fragments, even after 60 min. Also, EDTA inhib-
ited nuclease activity of DPD1-His, indicating that magnesium is
a necessary cofactor. By contrast, none of three other bivalent
cations (copper, manganese, or zinc) was observed to act as a
cofactor for DPD1 DNA degradation (Figure 8F). These data
indicate that DPD1-His has magnesium-dependent nuclease
activity. Complete degradation of PCR fragments indicates that
DPD1-His has exonucleolytic activity, but its endonucleolytic
activity was not ruled out completely. To test this possibility, we
performed the same nuclease assays with circular plasmids.
Intact plasmids were stable even after 60-min incubation with
DPD1-His. However, once nicked or linearized by restriction
enzymes (EcoRI, EcoRV, and KpnI), the plasmids became com-
pletely degradable by DPD1-His (Figure 8G). These data reflect
that DPD1 is an exonuclease that requires accessible ends to
degrade double-stranded DNAs.
Pollen-Enhanced Expression of DPD1
We next performed RT-PCR analysis to examine tissue-specific
expression of DPD1 (Figure 9A). RNAs were prepared from
Figure 6. Identification of the DPD1 Gene.
(A) Schematic representation of At5g26940 gene (DPD1) and the predicted protein encoded. Gray boxes show coding regions. Adenine of the
translation start codon (ATG) is designated as +1. Arrows indicate positions of mutations in dpd1 alleles. Here, dpd1-1, dpd1-2, and dpd1-3 are located
within the coding region; dpd1-4 is located at the splicing acceptor site of the first intron. In addition, dpd1-5 and dpd1-6 are T-DNA–inserted alleles.
Red boxes (ExoI, ExoII, and ExoIIIe) are functional domains conserved among 39-59 exonucleases. a.a., amino acids.
(B) DAPI-stained squashed pollen grains in dpd1 mutant alleles other than dpd1-1. A part of the squashed pollen cytoplasm is shown. Bars = 10 mm.
(C) A phylogenetic tree of DPD1 and its homologs. Multiple alignment was performed using ClustalW as described inMethods (see Supplemental Figure
7 and Supplemental Data Set 1 online).
1614 The Plant Cell
seedlings, the shoot apex, flowers, and various stages of pollen
development; RT-PCR was conducted as described previously
(Honys and Twell, 2004). As expected, DPD1 was highly ex-
pressed in flowers compared with whole seedlings and the shoot
apex. Further dissection of pollen development at the stage of
unicellular microspore, bicellular pollen, tricellular pollen, and
mature pollen grains revealed that DPD1 starts to express at the
bicellular pollen stage. It reaches a peak at the tricellular pollen
stage. This temporal expression of DPD1 in pollen coincides
perfectly with the disappearance of DAPI-stained organelle
DNAs (Figure 2B). It also coincides with the fact that organelle
DNA levels in vegetative tissues are not altered in dpd1. To
further characterize DPD1 expression in vivo, transgenic Arabi-
dopsis plants were generated that expressed the previously
described DPD1-GFP fusion under the DPD1 promoter. Char-
acterization of these transgenic plants showed that the DPD1
promoter is highly active in mature pollen grains (Figures 9B and
9C). Furthermore, we crossed this transgenic plant with another
line harboring Lat52pro:MTS:RFP, thereby expressing mtRFP in
vegetative cells. Careful observation of this line revealed smaller
GFP signals colocalized withmtRFP and additional larger signals
corresponding to plastids (Figure 9D). This observation con-
firmed the results of our transient assay in mesophyll cells (see
Supplemental Figure 4 online) that DPD1 is dual-targeted to both
organelles. When characterized by confocal microscopy, we
occasionally found smaller GFP mitochondrial signals that did
not merge with mtRFP in a hollow area that likely corresponds to
sperm cells (Figure 9E). The size and distribution of these small
GFP signals resembled those observed in sperm mitochondria
(Matsushima et al., 2008b), indicating that DPD1 is expressed
not only in vegetative but also in sperm cells. Collectively, these
results suggest that DPD1 is predominantly expressed in the
male gametophyte.
Pollen Viability and Transmission Efficiency in dpd1
Our results thus far indicated that DPD1 is a pollen-specific
exonuclease responsible for organelle DNA degradation. Sup-
porting this assumption, dpd1 showed no detectable phenotype
in plant vegetative growth (Figure 3). To characterize the effect of
organelle DNA degradation in pollen vegetative cells, we further
examined dpd1 pollen viability. We first examined the transmis-
sion efficiency (TE) of the dpd1mutation. A heterozygous DPD1/
dpd1-1 plant was subjected to a reciprocal cross with Col. The
genotype of F1 seeds was determined using PCR-based geno-
typing, as described in Methods, to estimate TE of dpd1 in the
next generation. This result indicated that TE of dpd1 through the
male (TEmale) was not reduced to a statistically significant degree
(Table 1). We also performed conditional TE tests to examine
whether TEmale is affected by the position of seeds in fertilized
siliques. After a reciprocal cross betweenCol andDPD1/dpd1-1,
fertilized siliques were cut into upper and lower halves. Then, the
genotype for DPD1 was determined separately (Table 2). Again,
these results showed no significant difference in TEmale. We
subsequently examined pollen germination ability in vitro. De-
spite the unaffected TE in dpd1, we found that the germination
rate is slightly lower in dpd1-1 than in Col and the complemented
line (Table 3). This reduction, which is likely to be too slight to
Figure 7. Complementation of the dpd1 Phenotype.
(A) Squashed mature pollen stained with SYBR from dpd1, Col, and
dpd1 complemented with At5g26940 genomic sequence (Comp.). A
trace of the picture from dpd1 is shown at the top right. VN, vegetative
nucleus; SN, sperm nucleus; PC, pollen coat; Ext, extrachromosomal
DNAs. Bars = 10 mm.
(B) Comparison of organelle DNAs in Col, dpd1-1, and dpd1-1
+At5g26940 pollen grains. Band intensities were quantified; each value
is shown as the average of three biological repeats6 SD. Different letters
denote significant differences between samples (P = 0.05, Tukey–
Kramer’s HSD).
[See online article for color version of this figure.]
Organelle DNA Degradation in Pollen 1615
Figure 8. DPD1-His Fusion Protein Has Exonuclease Activity in Vitro.
(A) Schematic view of the constructions employed in DPD1-His expression in E. coli. In order: bacteriophage T7 promoter, DPD1 cDNA, and His-tag
sequence. Relative positions of the point mutations are indicated by vertical arrows (numbers denote corresponding amino acids).
(B) Expression of DPD1-His in E. coli. SDS-PAGE of cell lysate with (+) or without (�) isopropyl b-D-1-thiogalactopyranoside is shown along with
molecular mass markers. Asterisks denote the bands corresponding to the fusion proteins.
(C) SDS-PAGE of DPD1-His protein purified by HiTrap Chelating HP column. TP, total soluble protein fraction; P, purified fraction. Bands corresponding
to DPD1-His are indicated by an arrow.
(D) Immunoblot analysis of DPD1-His. Same protein fractions in (C) were probed with anti-His antibody.
(E) Nuclease assay of recombinant DPD1-His proteins. Effects of heat treatment and EDTA were tested simultaneously as negative controls.
(F) Requirements of bivalent cations: magnesium (Mg2+), copper (Ca2+), manganese (Mn2+), and zinc (Zn2+) were tested.
(G) Digestion of supercoiled and linearized plasmids with DPD1-His. Plasmid pGreen0229 (4454 bp), circular or linearized by digestion with restriction
enzymes as indicated (EcoRI, EcoRV, and KpnI) was subjected to DPD1-His nuclear assay. Open and closed arrowheads indicate positions of
linearized and supercoiled plasmids, respectively.
Data in (A) to (C) are representative of three independent experiments.
[See online article for color version of this figure.]
1616 The Plant Cell
affect pollen fertilization to a considerable degree, was the only
phenotypewe observed consistently in dpd1. By contrast, pollen
tube growth and delivery into the embryo sac appeared to
proceed normally in bothCol and dpd1 (see Supplemental Figure
5 online). Based on these careful investigations, we concluded
that organelle DNA degradation controlled by DPD1 in pollen
vegetative cells has very limited influence on pollen viability,
although we cannot completely rule out the possibility that it
affects pollen grain germination.
Inheritance Mode of Organelle DNAs in dpd1
Next, we raised the question of whether dpd1 exhibits altered
organelle DNA inheritance. We therefore designed the following
genetic analysis in which several other ecotypes were employed
along with dpd1 to validate organelle DNAs transmitted from the
male or female parent. First, we tested ptDNAs. When crossed
with ecotype Cape Verde Islands (Cvi), with polymorphic ptDNA
(Martınez et al., 1997), dpd1 (Col background) showed normal
Figure 9. Pollen-Specific Expression of DPD1.
(A) RT-PCR analysis to determine tissues and developmental stages expressing DPD1 gene. UNM, uninucleate microspore; BCP, bicellular pollen;
TCP, immature tricellular pollen; MPG, mature pollen grain. Histone variant H3.3 gene (At4g40040) was used as a control.
(B) and (C) In vivo expression analysis of DPD1. DPD1 promoter was fused to the DPD1-GFP fusion protein used in Supplemental Figure 4 online.
Transgenic lines expressing the transgene (DPD1pro-DPD1-GFP) were subjected to detection of GFP in flowers (B) and pollen grains (C).
(D) Detailed observation of DPD1-GFP in mature pollen. Pollen grains from a transgenic plant expressing mtRFP in the vegetative cell and DPD1pro-
DPD1-GFP were examined using confocal microscopy. Signals corresponding to DPD1-GFP and mtRFP are shown along with the merged and
differential interference contrast (DIC) images.
(E) A confocal section showing small GFP signals that do not merge with mtRFP in a hollow area (indicated by the arrow).
Bars = 1 mm in (B), 20 mm in (C), and 5 mm in (D) and (E).
Organelle DNA Degradation in Pollen 1617
maternal inheritance of the plastid genome, as did the wild type
(n = 65; see Supplemental Figure 6 online). Plastids have not
been detected inside Arabidopsis sperm cells (Tang et al., 2009;
Wang et al., 2010). Therefore, exclusion of plastids from themale
gamete is perhaps the dominant mechanism for plastid maternal
inheritance in Arabidopsis.
We subsequently investigated the mode of mitochondrial
inheritance in dpd1. This experiment required careful investi-
gation because the Arabidopsis nuclear genome contains se-
quences identical to mtDNA integrated in the pericentromeric
region of chromosome 2 (Stupar et al., 2001). Therefore, we
were compelled to identify a rare single-nucleotide polymor-
phism that is specific to mtDNAs, based on the available
sequence data (Unseld et al., 1997). Our search identified one
polymorphism between Col and C24 (Figure 10A). Based on
this polymorphism, we created a degenerate cleaved amplified
polymorphic sequence (dCAPS) marker. Our PCR analysis re-
vealed that this polymorphism (C to A in C24 matR gene) was
specific for the mitochondrial genome but not for the matR
sequence integrated in chromosome 2 (Figures 10B and 10D). To
test the paternal leakage of mtDNAs in dpd1, a tester dpd1 line
that had aC24-derived cytoplasmwas generated and crossed to
Col female (Figures 10C and 10D). The result showed that no
paternal transmission of mitochondria occurs in our experimen-
tal scale (n = 300). We conclude that DPD1 is independent of the
inheritance mode of organelle DNA, at least under our experi-
mental scale, highlighting the turnover of extrachromosomal
DNAs as a unique biological process rather than a mechanism
for maternal inheritance.
DISCUSSION
Identification of Organelle Nuclease and Its
Evolutional Implication
We conducted a genetic study of organelle DNA decrease of
pollen grains. Isolation of recessive dpd mutations, which
retained more organelle DNAs than Col, implies that some genes
control organelle DNA degradation in the male gametophyte.
Molecular cloning enabled us to identify DPD1, which resides in
both plastids and mitochondria and has exonuclease activity in
vitro. Enhanced expression in male reproductive organs, partic-
ularly at PMII, coincided perfectly with our cytological obser-
vations. Collectively, we concluded that DPD1 is a DNase
controlling organelle DNA levels in a tissue-specific manner (in
pollen development and particularly at PMII). The decline of
organelle DNAs in pollen vegetative cells has been reported
consistently, not only in Arabidopsis but in almost all angiosperm
species (Mogensen, 1996; Nagata et al., 1999). Our data and
circumstantial evidence strongly imply that organelle DNA levels
decrease in pollen vegetative cells through a conserved mech-
anism that involves DPD1. Our expression analysis further im-
plies that the DPD1 promoter is also active in sperm cells of
Arabidopsis. Species showing biparental inheritance of ptDNAs
(e.g.,Medicago truncatula; Matsushima et al., 2008a) are known
to have strong DAPI signals in male germ cells. In such species,
altered DPD1 expression in male germ cells might account for
biparental inheritance.
A database search for DPD1 homologs revealed several inter-
esting aspects of how DPD1 emerged during evolution. First, a
DPD1 homolog is not found in cyanobacteria, suggesting that
DPD1 is not of endosymbiotic origin. Second, it is not found in
lower eukaryotes, including fungi and green alga. Finally, a closely
related homolog is not found in moss but is found specifically in
angiosperm species. Based on these observations, we consider
that DPD1 evolved along with the appearance of anisogamous
male reproductive organs (i.e., male and female gametes that are
not identical in morphology and the male gamete is smaller and
contains vegetative cells). The exonuclease family to which DPD1
belongs includes various proteins fromprokaryotes to eukaryotes.
Examples includeDnaQprotein inE. coliasa proofreading subunit
of the DNA polymerase III holoenzyme (Scheuermann and Echols,
1984) and TREX1 that degrades retroelements and exogenous
DNAs inmammalian cells (Lehtinenet al., 2008). It canbeassumed
Table 1. Genetic Transmission Analysis of dpd1-1 Mutations
Parental Cross No. of Wild Type (+/+) No. of dpd1-1 (+/�) Total TE (%)a x2 (P Value) for 1:1
dpd1-1+/� 3 Col 99 77 176 77.8 2.8 (0.097)
Col 3 dpd1-1+/� 71 65 136 91.5 0.26 (0.61)
The genotype of each progeny was determined using PCR.aTE (%) = number of mutant/number of wild-type progenies 3 100.
Table 2. Conditional Genetic Transmission Analysis of dpd1-1 Mutations
Parental Crossa No. of Wild Type (+/+) No. of dpd1-1 (+/�) Total TE (%)b x2 (P Value) for 1:1
Col 3 dpd1-1+/�-U 49 39 88 80 1.1 (0.29)
Col 3 dpd1-1+/�-L 34 32 66 94 0.061 (0.81)
dpd1-1+/� 3 Col-U 39 48 87 123 0.93 (0.33)
dpd1-1+/� 3 Col-L 32 44 76 138 1.9 (0.17)
The genotype of each progeny was determined using PCR.aSiliques were cut in the middle and separated into upper (U) and lower (L) parts. Seeds collected from each part were subjected to genotyping
analyses.bTE (%) = number of mutant/number of wild-type progenies 3 100.
1618 The Plant Cell
that one of these nuclease members evolved into DPD1 to
degrade organelle DNAs, along with the emergence of pollen
vegetative cells in angiosperms. Conserved presence of DPD1
homologs in species with anisogamy is consistent with our result
that DPD1 expression is limited predominantly to pollen. Collec-
tively, these data demonstrate that organelle DNA degradation is
common in angiosperm pollen and that it is controlled by the
exonuclease DPD1.
Regulation of DPD1 Expression, Degradation, and
Relevance to General Organelle DNA Degradation
In addition to the circumstances described previously, it is
notable that most of the dpd mutants we isolated through our
extensive screening were dpd1 alleles. We therefore consider
that DPD1 plays a central role in DNA degradation in both
plastids and mitochondria. Apparently, several questions arose
from our findings. We first asked about whether the role of DPD1
in organelle DNA degradation can be generalized in other so-
matic tissues, such as those of leaves. This is apparently not the
case because of (1) a lack of detectable phenotype in dpd1
vegetative growth, (2) pollen-specific expression of DPD1, and
(3) a lack of detectable change of organelle DNA levels in dpd1
leaf tissues. Moreover, DPD1 does not appear in plastid pro-
teome data prepared from nonpollen tissues (e.g., The Plant
Protein Database, http://ppdb.tc.cornell.edu/). Actually, DPD1
has never been associated with plastid nucleoids (or transcrip-
tionally active chromosomes) derived from nonpollen tissues
(Pfalz et al., 2006). Based on these observations, we infer that
DPD1 functions specifically in pollen and does not participate in
general organelle DNA metabolism, including DNA replication
and/or degradation. However, it is possible that misregulation of
DPD1 expression affects organelle DNA levels in somatic cells.
Additional investigations are necessary to elucidate the function
of DPD1 in organelle degradation in tissues other than pollen.
We next examined the question of whether DPD1 acts on DNA
degradation by itself or requires additional factor(s). Given its
exonucleolytic activity, it is presumed that linear DNAs are the
substrate of DPD1, but circular DNAs are not. Although predom-
inant forms of both ptDNAs and mtDNAs are believed to consist
of circular DNAs, nicked or linear molecules have been found in
both plastid and mitochondria (Bendich, 2004; Oldenburg and
Bendich, 2004); those molecules can be DPD1 substrates. It is
therefore possible that DPD1 alone can degrade some, but not
all, of the organelle DNA population. A significant difference in
organelle DNA levels between Col and dpd1 pollen (Figure 5)
implies that most of the organelle DNA population might in fact
be nicked or linear molecules in pollen vegetative cells (Bendich,
2004). Alternatively, DPD1 degrades organelle DNAs coopera-
tively with an unidentified endonuclease. In either case, DPD1
nuclease requires magnesium as a cofactor. In a green alga,
Chlamydomonas (showing isogamy with identical male and
female gametes), sex-specific degradation of chloroplast ge-
nomes putatively correlates with a Ca2+-dependent nuclease
(Nishimura et al., 1999, 2002). A Mg2+-dependent property
distinguishes DPD1 from this Chlamydomonas nuclease. Again,
our results obtained in this study are consistent with our pre-
sumption that DPD1 had emerged in angiosperms.
Table 3. In Vitro Germination Rate of Wild-Type and dpd1-1 Pollen
Grains
Repeat
No. of Germinated Pollen/Total Pollen (%)a
Col dpd1-1
dpd1-1
(Complemented)b
Experiment 1 397/794 (50.0) 322/767 (42.0) 583/1068 (54.6)
Experiment 2 289/421 (68.6) 267/501 (53.3) 166/266 (62.4)
Experiment 3 811/1278 (63.5) 765/1328 (57.6) 600/1030 (58.3)
aPollen grains were collected from at least three plants in each exper-
iment.bTransgene containing wild-type DPD1 allele is homozygous.
Figure 10. Genetic Analysis of mtDNA Transmission Confirming a Lack
of Paternal mtDNA Transmission in dpd1.
(A) A rare single-nucleotide polymorphism identified between Col and
C24 ecotypes of matR gene (highlighted in red). A dCAPS marker was
generated to distinguish C or A polymorphism, which was detectable by
digesting the PCR fragment with ScaI.
(B) Presence of thematR polymorphisms in different ecotypes and dpd1.
Because of the presence of mtDNA sequence in the Arabidopsis nuclear
genome, corresponding polymorphism was shown for nuclear DNA (n)
and mtDNA (mt).
(C) A genetic strategy to test paternal leakage of mtDNA in dpd1.
Because dpd1 is in the Col background, a cross was made between C24
(female) and dpd1 (male). The resulting F2 individual recessive for dpd1
(highlighted green) with mitochondria derived from C24 [dpd1(C24)] was
used as a tester line. Three hundred F1 plants from a cross between Col
(female) and the tester line were subjected to dCAPS analysis; no
paternal leakage was detected.
(D) Examples of dCAPS analysis detecting A or C polymorphism. In
crosses between Col and C24, the band corresponding to A polymor-
phism is only detected when C24 is a maternal parent.
Organelle DNA Degradation in Pollen 1619
An important remaining question related to organelle DNA
quantity in pollen is whether plastids and mitochondria that
contain no DNAs exist. We have not performed detailed mea-
surements of the organelle DNA quantity. Therefore, this ques-
tion remains to be resolved in this study. Our work specifically
examined the existence of a dual-targeted organelle DNase
rather than the degree of DNA degradation. It is noteworthy,
however, that several reports have described that the copy
numbers of organelle genomes were less than the organelle
numbers. For example, Wang et al. (2010) recently characterized
mtDNA levels in both somatic and gametic tissues using quan-
titative PCR. Their results showed that in both tissues, mtDNA
copy numbers were insufficient for eachmitochondrion to have a
complete copy of the genome. Preuten et al. (2010) also showed,
based on quantitative PCR analysis, that mtDNA copy numbers
are insufficient in various stages of leaf tissues. Accordingly,
frequent fusion of mitochondria has been proposed to account
for such genome insufficiency (Arimura et al., 2004; Sheahan
et al., 2005). Consistent with our current study,Wang et al. (2010)
revealed that mtDNA levels are much lower in pollen than in
mesophyll cells. Based on these observations, we consider that
mitochondria in pollen vegetative cells are functional even with
low mtDNA levels to support pollen tube elongation and fertili-
zation. Assessment of precise genome copy numbers in pollen
vegetative cells, particularly for plastid genomes, awaits further
study, which might require a new methodology.
Maternal Inheritance and Possible Role of Organelle DNA
Degradation in Pollen Function
Because DPD1 is predominantly expressed in pollen, we care-
fully examined pollen viability in dpd1. The loss of organelle DNA
degradation in dpd1 does not significantly affect pollen viability,
although dpd1 showed lower TEmale than the wild type did. A
difference was also observed in the pollen germination rate in
vitro. Nevertheless, this differencewas too little to influence TE to
any great degree. Therefore, our results leave open the question
of the relevance of DPD1 to pollen germination or other pollen
functions. This work identified the molecule that governs the
organelle DNA decrease in pollen through the dpd phenotype:
upregulated organelle DNAs in pollen vegetative cells. To unravel
the physiological significance of organelle DNA degradation,
future analysis of dpd1 phenotypes is necessary under various
conditions. Given that mature pollen grains are the only tissue
that is physically isolated from parental plant bodies, it is pre-
sumed that organelle DNA can be salvaged for nucleotide
recycling.
As described in the Introduction, the decline of organelle DNAs
in pollen development has been documented in many species,
particularly in relation to uniparental inheritance of organelle
genomes. The cytological detection of ptDNAs in generative or
sperm cells is well correlated with species showing biparental
inheritance, suggesting that DNA quantity in male germs is rel-
evant to organelle inheritance (Corriveau and Coleman, 1988;
Mogensen, 1996; Nagata et al., 1999; Zhang et al., 2003). These
observations raise the possibility that a DNase such as DPD1
excludes extra organelle DNAs in male germ cells and assures
their uniparental inheritance. In Arabidopsis, no plastids are
found in sperm cells (Tang et al., 2009; Wang et al., 2010), and
DPD1 is unlikely to affect maternal ptDNA inheritance. Therefore,
plastid maternal inheritance is determined primarily by the ex-
clusion of plastids in germ cells rather than ptDNA amounts in
Arabidopsis (Martınez et al., 1997; Azhagiri and Maliga, 2007). In
fact, our F1 analysis between Cvi and dpd1 showed that DPD1
did not affect plastid inheritance (see Supplemental Figure 6
online).
In contrast with plastids, there are;10mitochondria in sperm
cells that can be transmitted into fertilized egg and central cells
(Matsushima et al., 2008b; Wang et al., 2010). The overwhelming
number of mitochondria in egg cells (;800) appears to account
for stochastic propagation of maternal mitochondria (Birky,
2001). In fact, results of our genetic analyses suggest that
DPD1 has no effect on the inheritance mode of mtDNAs and
ptDNAs (Figure 10). Again, we must emphasize that a large
survey revealed that nonfertilizing pollen vegetative cells lack
cytologically detectable organelle DNAs in many species, irre-
spective of the inheritance mode of plastids and mitochondria.
We therefore consider that organelle DNA degradation by DPD1
is a priori independent of maternal inheritance in Arabidopsis.
Given the common function of DPD1 at PMII, it is possible that
the amplification of organelle DNAs, rather than their degrada-
tion, in generative cells is important for their inheritance mode.
This possibility is in fact implied by Nagata et al. (1999). We
propose that the control of organelle DNA degradation by DPD1
evolved with the formation of the anisogamous angiospermmale
gametophyte and that it is primarily independent of organelle
DNA inheritance in Arabidopsis. Future studies of DPD1 homo-
logs in species showing biparental plastid inheritance can help
us understand the role of organelle DNA degradation in pollen.
METHODS
Plant Materials, Mutant Screening, and Mapping
Arabidopsis thaliana ecotypes Col and Nossen were used as wild-type
plant materials for mutagenesis. The T-DNA insertion alleles in DPD1
(dpd1-5 and dpd1-6) were obtained from the ABRC (Salk_091621 and
Salk_015164, respectively). For detecting the inheritance mode of ptDNA
and mtDNA, ecotypes Cvi and C24 were used, respectively. The tester
line for mtDNA inheritance was generated by crossing dpd1-1 or dpd1-6
(Col background) to C24 female (Figure 10). For assessing paternal
mtDNA leakage, pollen from this tester line was used to cross with Col
female. Transgenic plants expressing ptGFP and mtRFP in pollen veg-
etative cells have been described in our previous works (Matsushima
et al., 2008b; Tang et al., 2009).
For mutagenesis, wild-type seeds were mutagenized by soaking them
initially for 16 h in 0.1 or 0.2% (v/v) methanesulfonic acid ethyl ester
(Sigma-Aldrich). Approximately 2000 flowers from 857 M1 lines were
collected, and pollen grains were examined using fluorescence micros-
copy. In addition, ;2000 M2 individual plants were used for screening
pollen grains. Mature pollen grains were placed on a glass slide and
immersed in a drop of deionized water that had been supplemented with
3% (w/v) glutaraldehyde and 1 to 10 mg mL21 DAPI (Invitrogen) in TAN
buffer (20 mM Tris-HCl, pH 7.65, 0.5 mM EDTA, 7 mM 2-mercaptoeth-
anol, 0.4 mM phenylmethyl sulfonyl fluoride, and 1.2 mM spermidine). In
some cases, 1:1000 diluted SYBR Green I (Invitrogen) was used also,
instead of DAPI, especially for detecting mtDNAs. Pollen grains were
squashed by putting gentle pressure on a cover slip that had been placed
1620 The Plant Cell
on the glass slide. They were examined using a fluorescence microscope
(BX51; Olympus) and a confocal laser scanning microscope (FV1000;
Olympus).
To determine themap position of theDPD1 locus, dpd1-1 and the wild-
type ecotype Landsberg erectawere crossed. Pollen of F2 progenies was
stained with DAPI to determine their genotype. Genomic DNAs from F2
progenies were isolated and analyzed using simple sequence length
polymorphism markers with data obtained from The Arabidopsis Infor-
mation Resource (http://www.Arabidopsis.org).
Thin Sections of Technovit 7100 Resin of Pollen Grains
Pollen grains were fixed in 2.5% (v/v) glutaraldehyde and 1% (w/v)
paraformaldehyde in cacodylate buffer, pH 7.4, for at least 24 h at room
temperature. Samples were subsequently dehydrated through a graded
ethanol series (20% [v/v], 40%, 60%, 80%, and 100%) and then embed-
ded in resin (Technovit 7100; Heraeus Kulzer). The embedded samples
were cut into 0.5-mm sections using an ultramicrotome (Ultracut N;
Reichert-Nissei) and diamond knives. Then they were dried on cover
slips. Thin sections were stained with 1 mgmL21 DAPI. To prevent fading,
1 mg mL21 n-propyl gallate in 50% (v/v) glycerol was added to the
samples before fluorescence microscopic examination.
For transmission electron microscopy, pollen grains were fixed in 4%
glutaraldehyde and 5% paraformaldehyde in cacodylate buffer, pH 7.4,
for at least 24 h at room temperature. A second fixation was performed in
5% (w/v) potassium permanganate solution at room temperature for 20
min. After rinsing in distilled water, the fixed pollen grains were dehy-
drated through a graded ethanol series (20%, 40%, 60%, 80%, and
100%) and embedded in Spurr’s resin (Polysciences). Ultrathin (70 to 90
nm) sections were stained in 1% (w/v) uranyl acetate and 0.5% (w/v) lead
citrate and examined using an electron microscope (H-7100; Hitachi)
operating at 75 kV.
Plasmid Construction and Nuclease Assay
To construct pG002926940 for complementation of dpd1, a DNA frag-
ment containing the At5g26940 genomic sequence was amplified using
PCR with primers: 59-GTTGGTACCTTGTAGCTCTGTTTTGGCCTA-39
(KpnI site underlined) and 59-GCAGAGCTCATGATGTTCCCTTATAAT-
TAG-39 (SacI site underlined). The fragment was cloned into the KpnI and
SacI sites of pGreen0029. To construct p3526940TP55GFP, a DNA frag-
ment corresponding to the putative transit peptide and the C-terminal 15
amino acids was amplified using PCR with primers: 59-GAGCTCGAGA-
TGTGTATCTCAATCTCG-39 (XhoI site underlined) and 59-ACCTT-
GCATGGGAGACCACACGTTACGTCT-39 (BsaI site underlined). The
fragment was digested with XhoI and BsaI and cloned into the SalI
and NcoI sites of p35S-sGFP, as described previously (Sakamoto et al.,
2003).
To construct pTopoCT26940w/o40 for expressing recombinant DPD1-
His protein in Escherichia coli, aDPD1 cDNA containing the entire reading
frame, except for the region corresponding to transit peptide (N-terminal
40 amino acids), was amplified by PCR using full-length DPD1 cDNA
(U82439; ABRC) as a template with primers 59-ATGGCTTCTTCTGT-
TGATGGTAAAGCA-39 and 59-GGCCTTCTTGTTCTTGGCCATGGC-39.
Mutagenesis of pTopoCT26940w/o40 to express DPD1-His(Y186*) and
DPD1-His(A236V) was conducted using a QuickChange Multi site-
directed mutagenesis kit (Stratagene).
To purify DPD1-His, pTopoCT26940w/o40was transformed into E. coli
strain BL21 (Invitrogen). Protein induction was performed with 0.8 mM
isopropyl b-D-1-thiogalactopyranoside in 300 mL of culture. DPD1-His
was purified using HiTrap Chelating HP (GE Healthcare) according to the
manufacturer’s instructions. Anti-His antibody kit (Qiagen) was used to
detect DPD1-His by immunoblotting. Purified DPD1-His was made
imidazole-free and concentrated using a centrifugal filter (Centricon
YM-10; Millipore). The protein concentration was determined using a kit
(Bio-Rad protein assay; Bio-Rad Laboratories). The PCR fragments
derived from ptDNA (1658 bp) were amplified using the following primers:
59-GCTTCAGCGGCTGCAATTGCTAT-39 and 59-GCTTGTGAAGTATGT-
GTTCGAG-39. The nuclease assay reaction (100 mL) consisted of 40 mM
Tris-HCl, pH 7.5, 2 mM MgCl2, and 1 mg DNA substrate; purified DPD1-
His (2.5 mg protein) was finally added to initiate the reaction. For inhibition
analyses, DPD1-His were incubated at 988C for 5 min prior to nuclease
assay, or reactions were conducted with 10 mM EDTA. Reactions were
completed by immediately adding stopping buffer (1% [w/v] SDS, 50%
[v/v] glycerol, and 0.05% [w/v] bromophenol blue) and were subjected to
1% (w/v) agarose gel electrophoresis. After staining with ethidium bro-
mide, remaining undigested PCR fragments were image captured and
quantified using software (ImageJ; NIH).
Nucleic Acid Extraction and PCR Analysis
To prepare total DNAs, mature pollen grains were collected by rubbing a
silicon bar (;2 mm width and 2 cm length) onto dehiscent anthers from
seven flowers and by putting the bar into an Eppendorf tube containing 45
mL of distilled water. The pollen suspension was incubated at 958C for 5
min and centrifuged for 5 min at 16,000g. Then, 9 mL of the supernatant
was subjected to the PCR. Total DNAs from seedlings (17 d old) were
isolated as described in a previous report (Miura et al., 2007).
For real-time PCR, the following primers were used according to
Rowan et al. (2009): psbA (ptDNA), 59-AGAGACGCGAAAGCGAAAG-39
and 59-CTGGAGGAGCAGCAATGAA-39; cox1 (mtDNA), 59-CCACG-
CATGTTGAAGATAGTTG-39 and 59-AGTAGGTAGCGGCACTGGGT-39;
and 18S rRNA (nuclear DNA), 59-AAACGGCTACCACATCCAAG-39 and
59-ACTCGAAAGAGCCCGGTATT-39. Amplification was conducted using
THUNDERBIRD SYBR qPCR Mix kit (TOYOBO) and Light Cycler 2.0
(Roche Diagnostics), with 50 cycles of a denaturation at 958C for 5 s and
an extension at 608C for 30 s. LightCycler Software (version 4.0; Roche
Diagnostics) was used to quantify PCR reactions. The amount of organ-
elle DNA was normalized by the value for 18S rRNA (n = 3).
To evaluate organelle DNA contents in mature dpd1 and wild-type
pollen (Figure 7B), extracts from five pollen grains were subjected to PCR.
Primers used to amplify ptDNA (region corresponding to ndhG) were
59-GGCCCCCACATAAATAAGGAGTTG-39 and 59-TCACCTCAAACAAA-
AAATGGGGTAAA-39. Primers used to amplify mtDNA (region corre-
sponding to nad9) were 59-ATGGAAAGATCGGAACATGGGAAT-39 and
59-GGGTCATCTCAATGGGTTCAGAA-39. Both primer sets were de-
signed according to the Arabidopsis chloroplast and mitochondrial
genome sequences (AP000423 and NC_001284). For detecting a single
nucleotide polymorphism between Col and C24 in the mitochondrial
matR gene, a dCAPS primers were generated as follows: 59-GTCAA-
GGCTGCCACTCGGTCCTAAGACG-39 and 59-CAACTCCTACGAGTCG-
TCCGGCGGAAAG-39. The polymorphism (C or A at nucleotide +297)was
estimated by ScaI digestion of the PCR fragment (Figure 10A).
Total RNAs were purified from isolated spores at four developmental
stages, as described in an earlier study (Honys and Twell, 2004). The
cDNA synthesis from 750 ng DNase-treated total RNA was primed with
oligo(dT) in a 21-mL reaction using a SuperScript first-strand synthesis
system for RT-PCR (Invitrogen). Histone variant H3.3 (At4g40040) was
used as a control as described (Brownfield et al., 2009). The RT-PCR
primers used for DPD1 were 59-GCATCGGAAAAATGAGCGGAT-39 and
59-CACCCTCCCTTGTAAGACTATAG-39 and for histone-H3 59-AGCTC-
CCTTTCCAGAGGCTA-39 and 59-TCCAAGTCTCCTACACCCAAA-39.
Characterization of Pollen and Other Phenotypes
For the pollen viability test, anthers were collected from stage 13 Arabi-
dopsis flowers and stained with Alexander stain for at least 2 h. In vitro
germination of pollen was performed as described previously (Matsushima
Organelle DNA Degradation in Pollen 1621
et al., 2008b). For pollen tube guidance, pistils were fixed in 10% (v/v)
acetic acid in ethanol overnight, incubated in 1 N NaOH overnight, washed
three times with 50 mM potassium phosphate buffer, pH 7.5, and stained
for at least 5 min in 0.05% (w/v) aniline blue. Pistils were supplemented
with 50% (v/v) glycerol and squashed lightly under a cover slip. For ob-
serving organelle DNAs in mesophyll cells, protoplasts were prepared
from mature leaves (7 weeks old) and stained with DAPI as described
previously (Kato et al., 2007).
Phylogenetic Analysis
Multiple alignment was performed using ClustalW (gap open penalty, 10;
gap extension penalty, 0.05; selected weight matrix, BLOSUM) and
manually adjusted to optimize alignment (shown in Supplemental Data
Set 1 online). The tree was generated as unrooted using the neighbor-
joiningmethod. The confidence of nodes in the tree was supported by the
value from 1000 bootstrap replicates.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: Arabidopsis DPD1, At5g26940; Zea mays, ACG42491; Oryza
sativa, Os4g0623400; Ostreococcus tauri, CAL55462; Micromonas sp
RCC299. XP002507456; Physcomitrella patens, XP001752327; Populus
trichocarpa, XP002330239; Sorghum bicolor, XP002468000; Vitis vin-
ihera, XP002282861; and Ricinus communis, XP002512483.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Organelle DNAs Detected in the Pollen
Tube.
Supplemental Figure 2. Mapping of the DPD1 Gene on Chromo-
some 5.
Supplemental Figure 3. Predicted Amino Acid Sequence of DPD1
and Alignment with Other Homologs.
Supplemental Figure 4. Subcellular Localization of DPD1.
Supplemental Figure 5. Pollen Phenotype in dpd1.
Supplemental Figure 6. Genetic Analysis of ptDNA Transmission.
Supplemental Figure 7. Multiple Sequence Alignment of DPD1 and
Its Homologs by ClustalW.
Supplemental Table 1. Penetrance of dpd1 Mutation on the Pollen
Phenotype.
Supplemental Table 2. Segregation of dpd1-1 Mutation in F2
Population.
Supplemental Table 3. Viability of Mature Pollen Grains Tested Using
Alexander Staining.
Supplemental Table 4. Measurement of Pollen Area Size.
Supplemental Data Set 1. Text File of Alignment Corresponding to
the Phylogenetic Analysis in Figure 6.
ACKNOWLEDGMENTS
We thank Sodmergen for assisting us with electron microscopy and for
useful discussion. We also thank the ABRC for providing the T-DNA
mutant lines, Mizuki Takenaka for providing information related to
Arabidopsis mtDNA polymorphism, and Rie Hijiya, Yumiko Kaji, Nami
Sakurai-Ozato, Chieko Hattori, and Said Hafidh for their technical
assistance. This work was supported by Grants-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science, and
Technology (No. 16085207 and No. 22112516 to W.S.) and by the
Oohara Foundation (to W.S.).
Received February 2, 2011; revised April 1, 2011; accepted April 11,
2011; published April 26, 2011.
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1624 The Plant Cell
DOI 10.1105/tpc.111.084012; originally published online April 26, 2011; 2011;23;1608-1624Plant Cell
Ryo Matsushima, Lay Yin Tang, Lingang Zhang, Hiroshi Yamada, David Twell and Wataru SakamotoDevelopment
PollenArabidopsis-Dependent Exonuclease Degrades Organelle DNA during 2+A Conserved, Mg
This information is current as of July 17, 2020
Supplemental Data /content/suppl/2011/04/21/tpc.111.084012.DC1.html
References /content/23/4/1608.full.html#ref-list-1
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