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 saka@rib.okayama-u.ac.jp.The 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(saka@rib.okayama-u.ac.jp).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

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Supplemental Data /content/suppl/2011/04/21/tpc.111.084012.DC1.html

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