Plant Cell Physiol. 49(7): 1074–1083 (2008)
doi:10.1093/pcp/pcn084, available FREE online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Mitochondrial Dynamics in Plant Male Gametophyte Visualized by
Fluorescent Live Imaging
Ryo Matsushima 1, Yuki Hamamura 2, 3, Tetsuya Higashiyama 3, Shin-ichi Arimura 4, Sodmergen 5,
Nobuhiro Tsutsumi 4 and Wataru Sakamoto 1, *
1
Research Institute for Bioresources, Okayama University, Kurashiki, Okayama, 710-0046 Japan
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo, 113-0033 Japan
3
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan
4
Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo,
113-8657 Japan
5
College of Life Sciences, Peking University, Beijing 100871, PR China
Editor-in-Chief’s choice
2
Visualization of organelles in living cells is a powerful
method for studying their dynamic behavior. Here we
attempted to visualize mitochondria in angiosperm male
gametophyte (pollen grain from Arabidopsis thaliana) that
are composed of one vegetative cell (VC) and two sperm cells
(SCs). Combination of mitochondria-targeted fluorescent
proteins with VC- or SC-specific expression allowed us to
observe the precise number and dynamic behavior of
mitochondria in the respective cell types. Furthermore, live
imaging of SC mitochondria during double fertilization
confirmed previous observations, demonstrated by electron
microscopy in other species, that sperm mitochondria enter
into the egg and central cells. We also attempted to visualize
mutant mitochondria that were elongated due to a defect in
mitochondrial division. This mutant phenotype was indeed
detectable in VC mitochondria of a heterozygous F1 plant,
suggesting active mitochondrial division in male gametophyte.
Finally, we performed mutant screening and isolated a putative
mitochondrial protein transport mutant whose phenotype was
detectable only in haploid cells. The transgenic materials
presented in this work are useful not only for live imaging but
also for studying mitochondrial functions by mutant analysis.
Keywords: Arabidopsis thaliana — Fertilization —
Fluorescent protein — Mitochondria — Pollen grain —
Sperm cell.
Abbreviations: CaMV, cauliflower mosaic virus; DAPI,
40 ,6-diamidino-2-phenylindole; GC, generative cell; GFP, green
fluorescent protein; mtDNA, mitochondrial DNA; PMI, pollen
mitosis I; PMII, pollen mitosis II; RFP, red fluorescent protein;
SC, sperm cell; VC, vegetative cell.
Introduction
Male gametogenesis in angiosperms occurs in anthers
and proceeds with several cell divisions (Southworth and
Russell 2001). A tetrad, resulting from meiosis of a pollen
mother cell, is separated and becomes four microspores.
Each microspore synchronously undergoes pollen mitosis I
(PMI) to form a larger vegetative cell (VC) and a smaller
generative cell (GC). Subsequently, the GC undergoes
pollen mitosis II (PMII) to form two sperm cells (SCs).
The timing of the initiation of PMII is species dependent
and occurs either before or after pollination (Brewbaker
1967). Thus, two or three haploid cells constitute a male
gametophyte as pollen grains, where only SCs contribute to
fertilization. After pollination on the stigma, a pollen tube
elongates from pollen grains. Tip growth from the VC
delivers two SCs to the ovule for double fertilization
(Batygina and Vasilyeva 2001, Lord and Russell 2002).
Once the pollen tube arrives and penetrates into the
micropyle, SCs are discharged into one of two synergids,
which degenerates upon pollen tube discharge. A mixed
cytoplasm of the VC and the degenerated synergid forms
the environment that enables the non-motile SCs to be
dually fused with the egg and central cells. Following
fertilization, the egg cell gives rise to the seed’s embryo,
which is the beginning of sporophyte generation, and the
central cell gives rise to the seed’s endosperm, which
surrounds the developing embryo and provides it with
nutrients (Drews and Yadegari 2002).
Processes such as pollen development, pollination and
double fertilization have been well studied in many species
(Berger 2008); however, the behavior and fate of cellular
organelles other than nuclei are rather unclear. In the
present study, we focus on studying mitochondria. This
organelle contains its own DNA and originated from the
endosymbiosis of a-proteobacteria (Gray 1992, Nass and
Nass 1963a, Nass and Nass 1963b). With only a few
exceptions, mitochondrial DNA (mtDNA) is transmitted
from the maternal parent in angiosperms (Forsthoefel et al.
1992, Martinez-Zapater et al. 1992, Testolin and Cipriani
1997). Based on conventional microscopic analyses, several
mechanisms which promote the maternal inheritance of
mitochondria have been suggested (Mogensen 1996). A key
*Corresponding author: E-mail, [email protected]; Fax, þ81-86-434-1206.
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Mitochondrial dynamics in plant male gametophyte
to understanding the mode of inheritance is direct
visualization of mitochondria during pollen development
and double fertilization. Prior to this study, however, the
live imaging of mitochondria in pollen grains has not been
described.
In this study, we generated transgenic Arabidopsis
plants expressing fluorescence-tagged mitochondria within
living pollen grains that were successfully visualized in vivo
with fluorescence microscopy. The establishment of this
transgenic material allowed us to assess (i) the precise size,
shape and number of mitochondria in SCs; (ii) the
incorporation of SC mitochondria into the egg and central
cells upon fertilization; and (iii) mitochondrial division in
the VC. Furthermore, we demonstrate that a recessive
heterozygous phenotype that is not detectable in diploid
cells can be observed in haploid cells.
Results
Visualization of mitochondria in living pollen grains
A common promoter such as the cauliflower mosaic
virus (CaMV) 35S promoter is inactive in pollen grains
(Mitsuhara et al. 1996). Therefore, the LAT52 promoter
from tomato and the DUO1 promoter from Arabidopsis
that have been shown to drive specific expression in VCs
and SCs, respectively, were used (Twell et al. 1990, Eady
et al. 1995, Rotman et al. 2005). We used these two promoters to create constructs which expressed mitochondriatargeted green fluorescent protein (GFP; Haseloff et al.
1997) or red fluorescent protein (RFP; Campbell et al.
2002). To target fluorescent proteins into mitochondria, the
N-terminal targeting signal from the gene encoding a
mitochondrial F1-ATPase d subunit was used (Sakamoto
and Wintz 1996). The resulting constructs were designated
as VC-mtGFP for mitochondria-targeted GFP in the VC,
VC-mtRFP for mitochondria-targeted RFP in the VC, and
SC-mtGFP for mitochondria-targeted GFP in the SC. We
selected transgenic lines that segregated kanamycin resistance (selective marker) in a Mendelian fashion for further
analysis. Detection of GFP and RFP in transgenic pollen
was performed at the T3 or successive homozygous
generations with fluorescence microscopy.
Mature pollen grains from VC-mtGFP plants exhibited
a number of globules corresponding to mitochondria
(Fig. 1A). Their size was estimated to be 0.64 0.046 mm
(n ¼ 24) in diameter. These data are in good accordance
with previous observations by electron microscopy
(Yamamoto et al. 2003). Transgenic pollen grains from
SC-mtGFP emitted green fluorescence that was arranged
as two rings (Fig. 1B). This is consistent with the fact
that PMII occurs in anthers in Arabidopsis and a mature
pollen grain is tricellular with two SCs (Owen and Makaroff
1995). Staining with a DNA-specific fluorescent dye,
1075
40 ,6-diamidino-2-phenylindole (DAPI), showed that GFP
signals surrounded the SC nucleus (Fig. 1C, E). It should be
noted that mtDNA was not stained by DAPI due to the
decrease in mtDNA during pollen development (Nagata
et al. 1999). We crossed the SC-mtGFP and VC-mtRFP
plants to visualize mitochondria in the VC and SCs with
different fluorescent proteins (Fig. 1F). These results
suggest that both VCs and SCs have an active mitochondrial protein transport system.
The average number of mitochondria included in one
SC was calculated to be 8.3 1.6 (n ¼ 174). The average
difference in number of mitochondria between two SCs was
1.9 1.3 (n ¼ 86). Mitochondrial size in an SC was
comparable with that in a VC. Unlike species such as
Plumbago zeylanica that have highly dimorphic SCs
(Russell 1984), we did not find dimorphism or a significant
difference in numbers of mitochondria between the two
SCs. Time-lapse movies to capture mitochondrial movements in the pollen tube are shown as Movies S1 and S2 in
the Supplementary material available online (150 speed).
During pollen germination, VC mitochondria were detectable throughout the pollen tube except for the tip region
(Fig. 1G, H). This region probably corresponds to a clear
zone that was previously suggested to be free of large
organelles (Supplementary Movie S1; Rosen et al. 1964,
Pierson et al. 1990). Mitochondria in the pollen tube were
within the cytoplasmic streaming referred to as ‘reverse
fountain-like’ (Supplementary Movie S2; Iwanami 1956).
This appears to comprise a central stream directed toward
the tip and return streams along the cortex. At the same
time, mitochondria in SCs were also traveling through
pollen tubes (Fig. 1I, J). SCs exhibited slower migration
compared with the intense cytoplasmic streaming inside the
pollen tube (Movie S3 in the Supplementary material
available online, 150 speed).
Live imaging of paternal mitochondria during double
fertilization revealed their incorporation into the egg and
central cells
Since we were successfully able to visualize mitochondria in SCs, we next tested their behavior during double
fertilization. The mtGFP fluorescence driven by the DUO1
promoter successfully enabled their visualization after
pollen germination (Fig. 1I). By dissecting wild-type pistils
that were pollinated by pollen of SC-mtGFP plants, timelapse images were captured upon double fertilization
in vitro as described previously (Palanivelu and Preuss
2006, Ingouff et al. 2007). Prior to fertilization and before
the pollen tube discharged its contents toward the ovule,
SCs were inside the pollen tube and localized near the
female gametophyte (Fig. 2A, B). The time 0 h is defined as
the point in time when sperm cells are discharged into the
ovule. After the discharge of sperm cells and just prior to
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Mitochondrial dynamics in plant male gametophyte
Fig. 1 Fluorescent images of pollen grains of the transgenic plants. (A) GFP-labeled mitochondria in a vegetative cell of a VC-mtGFP
plant. (B) GFP-labeled mitochondria in two sperm cells of a SC-mtGFP plant. (C–E) Co-visualization of a nucleus and mitochondria in the
same pollen grain by DAPI staining. (C) GFP-labeled mitochondria in sperm cells (D) DAPI-stained sperm cell nuclei (arrowheads) and
vegetative cell nucleus (asterisk). (E) The merged image of (C) and (D). (F) Mitochondria labeled with different fluorescent proteins
depending on the vegetative cell and sperm cells by crossing SC-mtGFP and VC-mtRFP plants. (G) Fluorescent GFP-labeled mitochondria
in vegetative cells during pollen germination. (H) Differential interference contrast image of the same field of (G). (I) Fluorescent
GFP-labeled mitochondria in sperm cells during pollen germination. (J) Differential interference contrast image of the same field of (I).
Scale bars ¼ 5 mm.
double fertilization, paternal mitochondria appeared to
cluster as two distinct spots (Fig. 2C). Two hours later, the
mitochondrial clusters dispersed at the micropylar end
(Fig. 2D). A time-lapse movie during double fertilization is
shown as Movie S4 in the Supplementary material (450
speed). This dispersion probably indicates that the SC
released mitochondria from its narrow cytoplasm, and that
one of the two SCs fused with an egg cell and the other with
a central cell. Similar dispersion patterns were observed in
eight of 11 samples. In the remaining three samples,
dispersion was observed in an egg cell but was not clear
in a central cell. Thus, our data indicate that the paternal
mitochondria enter the egg and central cells upon double
fertilization. Paternal mitochondria became barely detectable within 5 h after fertilization (Fig. 2G, H).
Contribution of mitochondrial division-related protein,
DRP3A, to the VC but not to the SC
In contrast to nuclear divisions, very little is known
about mitochondrial division during the maturation
of pollen. Creation of our transgenic plants allowed us to
test this possibility in VCs and SCs. DRP3A (At4g33650)
encodes a dynamin-like protein which contains a GTPase
domain and plays a central role in mitochondrial division
(Arimura et al. 2004, Logan et al. 2004, Mano et al. 2004).
A T-DNA insertion allele of drp3a resulted in elongation of
mitochondria in somatic cells (e.g. leaf and root tissues)
(Logan et al. 2004). This mutant was crossed with
VC-mtGFP and SC-mtGFP plants to monitor the morphology of mitochondria in pollen grains. Nearly all pollen
grains from homozygous drp3a mutants contained a
VC with elongated mitochondria (Fig. 3A, B). On the
other hand, this morphology was never detected in the
parental VC-mtGFP plants (Fig. 3C). In F1 plants from
a cross between drp3a and VC-mtGFP, the ratio of
pollen with normal or elongated mitochondria was approximately 1 : 1 (Table 1 and Fig. 3D). These results indicated
that mitochondrial division indeed occurs in the VC
and involves DRP3A in a gametophytic fashion.
In contrast, elongation of mitochondria was not observed
Mitochondrial dynamics in plant male gametophyte
1077
Fig. 2 Behavior of mitochondria in sperm cells during double fertilization. (A, B) GFP-labeled mitochondria in sperm cells of a SC-mtGFP
plant prior to pollen tube discharge (arrowheads). The embryo sac is indicated with a solid line. (C) GFP-labeled mitochondria in sperm
cells after pollen discharge but before double fertilization (arrowheads). (D–H) GFP-labeled sperm mitochondria in the egg and central
cells after double fertilization (arrows). The time points after pollen tube discharge are indicated in each panel. Scale bar ¼ 50 mm.
in SCs (Fig. 3E–G). The average number of mitochondria
included in one SC of homozygous drp3a mutants was
calculated to be 8.5 2.3 (n ¼ 34) and was not significantly
different from that of the wild type (Welch’s t-test, twotailed, P ¼ 0.68). The different morphologies of mitochondria that were observed between the VC and SC in the drp3a
mutant suggest that the contribution of DRP3A differs
between the VC and SC.
The potential of VC-mtGFP plants for forward genetic
analysis
Successful observation of elongated mitochondria in
the drp3a heterozygote implies that screening for organellerelated mutants is feasible using heterozygous M1 plants.
Homozygous VC-mtGFP seeds were mutagenized by ethyl
methane sulfonate, and pollen grains from 649 M1 plants
were screened. We obtained one mutant line with diffuse
GFP signals in the entire VC (Fig. 4A, B). The fluorescent
pattern in the mutant is similar to that of pollen expressing
cytoplasmic GFP (Fig. 4C), suggesting that the mitochondria-targeted GFP accumulated in the cytosol. When we
crossed this mutant with a VC-mtRFP plant, pollen grains
from F1 plants exhibited a diffuse RFP signal in the cytosol
(Fig. 4D). No mutation was detected in the mitochondriatargeted pre-sequence of the transgene VC-mtGFP (data not
shown). These results apparently excluded the possibility
that the mistargeting of GFP was due to a cis-acting
mutation in the transgene, and thus we named the mutant
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Mitochondrial dynamics in plant male gametophyte
Fig. 3 Fluorescent images of mitochondria in pollen grains of the DRP3A knockout mutant. (A) Elongated mitochondria in the vegetative
cell of a homozygous drp3a mutant transformed with the VC-mtGFP gene. (B) Magnified image of the elongated mitochondria in the
vegetative cell of a homozygous drp3a pollen grain. (C) Magnified image of wild-type mitochondria in a VC-GFP pollen grain. (D) Pollen
grains of a heterozygous drp3a mutant. Approximately 50% of pollen grains had elongated mitochondria (arrowhead) and the remainder
exhibited a wild-type phenotype (arrow). (E) Mitochondria in sperm cells of the homozygous drp3a mutant transformed with the SC-mtGFP
gene. (F) Magnified image of sperm cell mitochondria of the homozygous drp3a mutant. (G) Magnified image of wild-type mitochondria in
a SC-mtGFP pollen grain. Scale bars ¼ 10 mm in (A, D, E) and 2 mm in (B, C, F, G).
dmt1 (defective in mitochondrial protein transport1). We also
crossed dmt1 with the plant that expressed mitochondrial
GFP in leaf tissues (driven by the CaMV 35S promoter).
Some F1 plants included the pollen grains showing the dmt
phenotype; however, mitochondrial fluorescent patterns in
the leaves of the F1 plants were indistinguishable from that
of the wild type (Fig. 4F, G). These results suggest that the
dmt1 phenotype is recessive and detectable only in pollen
Mitochondrial dynamics in plant male gametophyte
grains but not in somatic diploid tissues. Although cloning
awaits further characterization, we demonstrate the versatility of our transgenic materials to identify novel genes, the
mutant phenotype of which can be difficult to see in somatic
diploid cells.
Discussion
Despite previous work using electron microscopy and
other means, our understanding of mitochondrial behavior
Table 1 Ratio of wild-type and mutant pollen grains of
F1 plants from a cross between a drp3a mutant and a
VC-mtGFP transgenic plant
a
F1 plant
No.
Wild-type
pollen
drp3a
pollena
Total
w2 (P-value)
for wild-type:
mutant ¼ 1 : 1
F1-1
F1-2
F1-3
Total
25
28
22
75
26
24
29
79
51
52
51
154
0.02 (0.89)
0.31 (0.58)
0.96 (0.33)
Mutant pollen had elongated mitochondria as shown in Fig. 3D.
1079
in the male gametophyte is limited and has not been
comprehensively studied. Many unanswered questions such
as the precise shape and number, division status during
pollen development and transmission of mitochondria in
subsequent fertilization remained to be solved. In the
present study, the successful visualization of fluorescencetagged mitochondria in Arabidopsis pollen grains allowed
these questions to be answered for the first time. Moreover,
we also employed a novel forward genetics approach of
isolating the mutant defective in mitochondrial protein
transport. The live imaging of the mitochondria in pollen
tissues makes both cytological and genetic characterization
possible.
Our interest in the mitochondria of pollen tissues
comes from the fact that their DNA is principally
maternally transmitted in angiosperms. The maternal
inheritance implies that mtDNA, or the corresponding
mitochondria themselves, is eliminated during pollen development and fertilization. In the case of another endosymbiotic organelle, the plastid, approximately 80% of
angiosperm species display maternal inheritance (Corriveau
and Coleman 1988, Zhang et al. 2003). Depending on the
species, either PMI via unequal plastid distribution
(Lycopersicon type) or GC/SC development via plastid
Fig. 4 Isolation of a dmt1 mutant defective in mitochondrial protein transport. (A, B) Fluorescent images of dmt1 pollen grains. Asterisks
and arrows indicate mutant and wild-type pollen grains, respectively. (C) A pollen grain of the transgenic plant expressing cytoplasmic
GFP. (D) Wild-type and dmt1 pollen grains of the VC-mtRFP plants. Asterisks and arrows indicate the mutant and wild-type pollen grains,
respectively. (E) Leaf mitochondria of the transgenic plant expressing mitochondrial GFP under the control of the cauliflower mosaic virus
35S promoter (35S-mtGFP). (F) Leaf mitochondria of the dmt1 mutant in the 35S-mtGFP background. Scale bars ¼ 20 mm.
1080
Mitochondrial dynamics in plant male gametophyte
degeneration (Solanum type) prevents paternal transmission
of plastids (Mogensen 1996, Hagemann 2002). Therefore,
the GC and SCs in mature pollen grains tend to be plastidfree. We performed the visualization of plastids in living
pollen using a plastid-targeted GFP (unpublished data).
Our attempt to detect such plastids in the SC failed with the
DUO1 promoter, again demonstrating that plastids are not
present in the SC. Similarly, our electron microscopic
observation did not show plastids in SCs, suggesting that
Arabidopsis belongs to the Lycopersicon type (unpublished
data). However, plastid discrimination may not occur in a
strict manner. A low frequency transmission of paternal
plastid DNAs has been reported in several plant species that
were originally defined to exhibit strict maternal plastid
inheritance (Azhagiri and Maliga 2007, Ruf et al. 2007,
Svab and Maliga 2007). In the case of Arabidopsis, the
frequency of the exceptional paternal transmission to the
progeny is 3.910–5 (Azhagiri and Maliga 2007), therefore
with microscope-based methods it would be difficult to
capture infrequent plastids in SCs. In the case of mitochondria, however, almost all angiosperms show maternal
inheritance (Forsthoefel et al. 1992, Martinez-Zapater
et al. 1992, Testolin and Cipriani 1997). Unlike plastids, a
number of mitochondria were localized in SCs (Fig. 1B),
but mtDNAs were undetectable by DNA staining reagents
due to an active degradation process (Sodmergen et al.
1992, Nagata et al. 1999).
How are mitochondria situated in SCs during the double
fertilization of Arabidopsis? It is of particular interest to
understand whether paternal mitochondria enter the zygote.
Based on previous electron microscopic observations,
mitochondrial behavior during double fertilization seems to
vary among angiosperms (Jensen and Fisher 1968, Russell
1980, Russell 1983, Russell et al. 1990). In barley, most SC
mitochondria are pinched off from SC cytoplasm as a form of
enucleated cytoplasmic bodies before fertilization and do not
enter the egg cell (Mogensen and Rusche 1985, Mogensen
1990). In contrast, transmission of SC mitochondria into
both the egg and central cells has been reported in tobacco
(Nicotiana tabacum. L) (Yu et al. 1994). In mouse, GFPlabeled sperm mitochondria enter a zygote (Shitara et al.
2001). However, these mitochondria are selectively eliminated from the cytoplasm of embryonic cells during the twocell stage in early embryogenesis. In mammals, sperm
mitochondria are ubiquitinated in the pre-zygotic stage and
selectively degraded by the proteasome pathway in fertilized
eggs (Sutovsky et al. 1999). We demonstrated that paternal
mitochondria enter the egg and central cells in Arabidopsis
upon fertilization (Fig. 2). The mitochondrial fluorescent
signals became undetectable within 5 h after fertilization. The
disappearance may be due to the active degradation of
paternal mitochondria; however, GFP-specific degradation
cannot be ruled out. Further experiments with organelle
markers other than fluorescent proteins are necessary to
examine the active degradation process.
Using drp3a pollen, mitochondria were found to be
elongated in the VC but not in the SC (Fig. 3), therefore
demonstrating that mitochondrial division proceeds at least
in the VC and depends on dynamin-like proteins. Regarding
the SC, we predict two possible scenarios relating to
mitochondrial division. First, mitochondrial division may
proceed in the SC but depends on another mitochondrial
dynamin, DRP3B (At2g14120) (Arimura and Tsutsumi
2002, Hong et al. 2003). However, available transcriptomic
data (Honys and Twell 2004) imply that expression of
DRP3B is not reliably detected after PMII. Alternatively,
mitochondrial division may no longer be necessary and may
not proceed in the SC. Consistent with the latter hypothesis,
previous electron microscopic observation in barley
(Hordeum vulgare L.) has shown that the size of mitochondria does not change during sperm maturation (Mogensen
and Rusche 1985).
Our transgenic plant material is useful for forward
genetic studies aiming to isolate novel mitochondria-related
mutants. In addition, the combination of RFP and GFP,
together with simple detection of the pollen mutant
phenotype in F1, allows us to shorten the period of genetic
analysis. This novel screening system (haploid-based screening) should give us a particular advantage for isolating
mutants whose defect cannot be detected in somatic
diploids, possibly due to their lethality. In fact, we were
unable to obtain homozygous dmt1 mutants, and the
phenotype in pollen grains always segregated. Additional
mutants such as those related to mitochondrial morphologies should be obtained by increasing the screening scale.
Future genetic studies using this system should lead to a
better understanding of novel mitochondrial functions.
Materials and Methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Columbia was used as the wildtype plant material. The mutant drp3a, containing a T-DNA
insertion within the fifth intron of the DRP3A gene, was obtained
through the Arabidopsis Biological Resource Center
(Salk_066958). Seeds were sown on 0.5% (w/v) Gelrite gellan
gum (Sigma, St Louis, MO, USA) or 0.8% (w/v) agar TC-6
(Funakoshi, Tokyo, Japan) plates containing Murashige–Skoog
medium (Sigma) supplemented with 1.0% (w/v) sucrose. Seedlings
were transferred onto soil. All plants were maintained under 12 h
light at a constant temperature of 228C. For crossing VC-mtGFP
and SC-mtGFP plants with drp3a, the genotype of the drp3a
mutation was verified by PCR using the following primers:
DRP3A exon5 fw, 50 -AACCGCACAGTCAAGGAAGC-30 ;
DRP3A exon7 rv, 50 -TATCCGCGACTTCAAATCCG-30 ; and
Salk T-DNA LBb1 primer, 50 -GCGTGGACCGCTTGCTG
CAACT-30 . Transgenic plants expressing mitochondrial GFP
under the control of the CaMV 35S promoter (35S-mtGFP) have
been constructed previously (Feng et al. 2004).
Mitochondrial dynamics in plant male gametophyte
Plasmid construction
All plasmids that were constructed in this study were
based on the LAT52-GFPN plasmid. LAT52-GFPN is a plasmid
containing the GFP gene inserted between the LAT52 promoter
and the nopaline synthase terminator. In this vector, SalI and NcoI
restriction sites are located between the LAT52 promoter and the
GFP gene. A KpnI site is located at the 50 end of the LAT52
promoter and an XbaI site is located at the 30 end of the GFP gene.
To construct VC-mtGFP, a DNA fragment containing the
mitochondria-targeted pre-sequence of the mitochondria F1ATPase d0 subunit was produced by PCR amplification using
Columbia genomic DNA and the following primers: 50 -GCA
GTCGACATGGCTAATCGTTTCAG-30 and 50 -TAGCCATGG
TTTGAGCAGAAGCAG. The fragment produced from this
amplification was digested with SalI and NcoI, and inserted into
the corresponding SalI and NcoI sites of LAT52-GFPN.
To construct VC-mtRFP, a DNA fragment containing the
RFP gene was PCR amplified by using mRFP in pRSETB as a
template and the following primers: 50 -GGGCCATGGCCTC
CTCCGAGGAC-30 and 50 -CCCTCTAGATTAGGCGCCGGTG
GAGTG-30 . The amplified RFP fragment was digested with NcoI
and XbaI, resulting in two fragments due to an internal NcoI site.
Both fragments were inserted together into the NcoI and XbaI sites
of VC-mtGFP to replace the GFP gene with the RFP gene. To
construct SC-mtGFP, the promoter region of the DUO1 gene
(At3g60460, from –1,232 to –1) was PCR amplified using
Columbia genomic DNA and the following primers: 50 -TCC
GGTACCGTAGTAAACTAATGAGGAGGA-30 and 50 -CGCG
TCGACTTTCCTCATCGCTAATCGATC-30 . The amplified
fragment was treated with KpnI and SalI, and inserted into the
KpnI and SalI sites of VC-mtGFP to replace the LAT52 promoter
with the DUO1 promoter. To generate transgenic Arabidopsis
plants, Columbia wild-type plants were transformed with the
aforementioned chimeric genes by the in planta method (Clough
and Bent 1998).
Fluorescence microscopy
Pollen grains in 5% (w/v) mannitol (Nacalai Tesque, Kyoto,
Japan) on glass slides were examined with a fluorescence
microscope equipped with a Disk Scanning Unit (DSU-BX61,
Olympus, Tokyo, Japan). Pollen grains were germinated on
medium [10 mM HEPES-KOH (pH 7.0), 18% (w/v) sucrose,
0.01% (w/v) H3BO3, 1 mM CaCl2, 1 mM Ca(NO3)2, 1 mM MgSO4]
for 2 h at 228C. To stain DNA, pollen grains were placed on a glass
slide and immersed in a drop of deionized water that was
supplemented with 10 mg ml–1 DAPI (Invitrogen, Tokyo, Japan).
Immediately after being covered with a coverslip, the pollen grains
were squashed by exerting an appropriate degree of pressure
through the coverslip. To detect DAPI signals, an excitation filter
XF1076 (385–415 nm, Omega Optical, Brattleboro, VT, USA), a
dichroic mirror XF2059 (Omega Optical) and a barrier filter
XF3002 (420–485 nm) were used. To detect GFP signals, a filter set
(U-MGFPHQ, Olympus), an excitation filter (460–480 nm), a
dichroic mirror (DM485) and a barrier filter (495–540 nm) were
used. To detect RFP signals, a filter set (U-MWIG3, Olympus), an
excitation filter (530–550 nm), a dichroic mirror (DM570) and a
barrier filter (570 nm long pass) were used. Photomicrographs
were captured with a cooled CCD camera (DP30BW, DP30
Monochrome Digital Camera, Olympus) that was attached
to the microscope. Images were analyzed with Metamorph
(Molecular Devices, Downingtown, PA, USA) and Adobe
Photoshop (Adobe Systems, Tokyo, Japan).
1081
Live imaging of the double fertilization process
Live imaging analysis of the double fertilization process was
performed according to a previous method (Palanivelu and Preuss
2006, Ingouff et al. 2007). Principally, emasculated pistils of wildtype Columbia were crossed with pollen from the SC-mtGFP
plants. Pollinated pistils were dissected soon after pollination to
cultivate ovules and a cut pistil together. Pollen tubes growing
through the cut style were guided to the ovules in vitro. Time 0 h is
defined as the point in time when SCs are being discharged into the
ovule. Images were captured by using an inverted microscope
(IX-71, Olympus, Japan) with a disk-scan confocal system
(CSU10, Yokogawa, Japan), an LD laser (excitation 488 nm), a
piezo Z-drive (Physik Instrumente, Karlsruhe, Germany), a dualview system (Roper Bioscience, Pleasanton, CA, USA) and an
EM-CCD camera (Cascade II, Roper Bioscience, USA) that was
controlled by Metamorph (Molecular Devices).
Mutant screening
Seeds of VC-mtGFP seeds were mutagenized by initially
soaking them for 16 h in 0.1 or 0.2% (v/v) methanesulfonic acid
ethyl ester (Sigma). Seeds were subsequently washed with water
and sown on plates or soil. Two flowers from distinct branches of
individual M1 lines were collected and pollen grains were examined
with a fluorescence microscope to isolate the mutants exhibiting
abnormal organelle phenotypes. When pollen exhibited some
abnormal phenotypes, M2 seeds obtained from the branch were
collected. Inheritance of phenotypes was confirmed with pollen of
M2 and M3 plants.
Supplementary material
Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.
oxfordjournals.org.
Funding
The Ministry of Education, Culture, Sports, Science
and Technology Grant-in-Aid for Scientific Research (No.
16085207 to W.S. and No. 18870018 to R.M.); The Japan
Science Society Sasakawa Scientific Research Grant
(No.19-415); the Oohara Foundation.
Acknowledgments
The authors would like to thank Drs. Sheila McCormick
(UC Berkeley) and Shinichi Nishikawa (Nagoya University) for
providing the LAT52-GFPN plasmid, Dr. Alice Y. Cheung
(University of Massachusetts) for providing the seeds of the
transgenic plant expressing cytoplasmic GFP, Drs. Phil
Mullineaux and Roger Hellens (John Innes Centres and the
Biotechnology and Biological Sciences Research Council) for
providing the pGreen kit, Dr. Roger Tsien (University of
California, San Diego) for his gift of mRFP1 in pRSETB, and
the ABRC for providing the T-DNA mutant lines. We would also
like to thank Chieko Hattori, Rie Hijiya and Nami Sakurai-Ozato
for their technical assistance.
1082
Mitochondrial dynamics in plant male gametophyte
References
Arimura, S.-i, Aida, G.P., Fujimoto, M., Nakazono, M. and Tsutsumi, N.
(2004) Arabidopsis dynamin-like protein 2a (ADL2a), like ADL2b,
is involved in plant mitochondrial division. Plant Cell Physiol. 45:
236–242.
Arimura, S.-i and Tsutsumi, N. (2002) A dynamin-like protein (ADL2b),
rather than FtsZ, is involved in Arabidopsis mitochondrial division.
Proc. Natl Acad. Sci. USA 99: 5727–5731.
Azhagiri, A.K. and Maliga, P. (2007) Exceptional paternal inheritance of
plastids in Arabidopsis suggests that low-frequency leakage of plastids
via pollen may be universal in plants. Plant J. 52: 817–823.
Batygina, T.B. and Vasilyeva, V.E. (2001) In vivo fertilization.
In Current Trends in the Embryology of Angiosperm. Edited by
Bhojwani, S.S. and Soh, W.Y. pp. 101–142. Kluwer Academic
Publishers, Dordrecht.
Berger, F. (2008) Double-fertilization, from myths to reality. Sex. Plant
Reprod. 21: 3–5.
Brewbaker, J.L. (1967) The distribution and phylogenic significance of
binucleate and trinucleate pollen grains in the angiosperm. Amer. J. Bot.
54: 1069–1083.
Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S.,
Zacharias, D.A. and Tsien, R.Y. (2002) A monomeric red fluorescent
protein. Proc. Natl Acad. Sci. USA 99: 7877–7882.
Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J.
16: 735–743.
Corriveau, J.L. and Coleman, A.W. (1988) Rapid screening method to
detect potential biparental inheritance of plastid DNA and results for
over 200 angiosperm species. Amer. J. Bot. 75: 1443–1458.
Drews, G.N. and Yadegari, R. (2002) Development and function of the
angiosperm female gametophyte. Annu. Rev. Genet. 36: 99–124.
Eady, C., Lindsey, K. and Twell, D. (1995) The significance of microspore
division and division symmetry for vegetative cell-specific transcription
and generative cell differentiation. Plant Cell 7: 65–74.
Feng, X., Arimura, S.-i., Hirano, H.-Y., Sakamoto, W. and Tsutsumi, N.
(2004) Isolation of mutants with aberrant mitochondiral morphology
from Arabidopsis thaliana. Genes Genet. Syst. 79: 301–305.
Forsthoefel, N.R., Bohnert, H.J. and Smith, S.E. (1992) Discordant
inheritance of mitochondria and plastid DNA in diverse alfalfa
genotypes. J. Hered. 83: 342–345.
Gray, M.W. (1992) The endosymbiont hypothesis revisited. Int. Rev. Cytol.
141: 233–357.
Hagemann, R. (2002) Milestones in plastid genetics of higher plants. Prog.
Bot. 63: 5–51.
Haseloff, J., Siemering, K.R., Prasher, D.C. and Hodge, S. (1997) Removal
of a cryptic intron and subcellular localization of green fluorescent
protein are required to mark transgenic Arabidopsis plants brightly. Proc.
Natl Acad. Sci. USA 94: 2122–2127.
Hong, Z., Bednarek, S.Y., Blumwald, E., Hwang, I., Jurgens, G.,
Menzel, D., Osteryoung, K.W., Raikhel, N.V., Sinozaki, K.,
Tsutsumi, N. and Verma, D.P.S. (2003) A unified nomenclature for
Arabidopsis dynamin-related large GTPases based on homology and
possible functions. Plant Mol. Biol. 53: 261–265.
Honys, D. and Twell, D. (2004) Transcriptome analysis of haploid male
gametophyte development in Arabidopsis. Genome Biol. 5: R85.
Ingouff, M., Hamamura, Y., Gourgues, M., Higashiyama, T. and
Berger, F. (2007) Distinct dynamics of HISTONE3 variants between
the two fertilization products in plants. Curr. Biol. 17: 1032–1037.
Iwanami, Y. (1956) Protoplasmic movement in pollen grains and tubes.
Phytomorphology 6: 288–295.
Jensen, W.A. and Fisher, D.B. (1968) Cotton embryogenesis: the
entrance and discharge of the pollen tube in the embryo sac. Planta 78:
158–183.
Logan, D.C., Scott, I. and Tobin, A.K. (2004) ADL2a, like ADL2b, is
involved in the control of higher plant mitochondrial morphology.
J. Exp. Bot. 55: 783–785.
Lord, E.M. and Russell, S.D. (2002) The mechanisms of pollination and
fertilization in plants. Annu. Rev. Cell Dev. Biol. 18: 81–105.
Mano, S., Nakamori, C., Kondo, M., Hayashi, M. and Nishimura, M.
(2004) An Arabidopsis dynamin-related protein, DRP3A, controls both
peroxisomal and mitochondrial division. Plant J. 38: 487–498.
Martinez-Zapater, J.M., Gil, P., Capel, J. and Somerville, C.R. (1992)
Mutations at the Arabidopsis CHM locus promote rearrangements of the
mitochondrial genome. Plant Cell 4: 889–899.
Mitsuhara, I., Ugaki, M., Hirochika, H., Ohshima, M., Murakami, T., et al.
(1996) Efficient promoter cassettes for enhanced expression of foreign
genes in dicotyledonous and monocotyledonous plants. Plant Cell
Physiol. 37: 49–59.
Mogensen, H.L. (1990) Fertilization and early embryogenesis.
In Reproductive Versatility in the Grasses. Edited by Chapman, G.P.
pp. 76–99. Cambridge University Press, Cambridge.
Mogensen, H.L. (1996) The hows and whys of cytoplasmic inheritance in
seed plants. Amer. J. Bot. 83: 383–404.
Mogensen, H.L. and Rusche, M.L. (1985) Quantitative ultrastructural
analysis of barley sperm I. Occurrence and mechanism of cytoplasm and
organelle reduction and the question of sperm dimorphism. Protoplasma
128: 1–13.
Nagata, N., Saito, C., Sato, A., Kuroiwa, H. and Kuroiwa, T. (1999) The
selective increase or decrease of organellar DNA in generative cells just
after pollen mitosis one controls cytoplasmic inheritance. Planta 209:
53–65.
Nass, M.M.K. and Nass, S. (1963a) Intramitochondrial fibers with DNA
characteristics. I. Fixation and electron staining reactions. J. Cell. Biol.
19: 593–611.
Nass, S. and Nass, M.M.K. (1963b) Intramitochondrial fibers with DNA
characteristics. II. Enzymatic and other hydrolytic treatments. J. Cell.
Biol. 19: 613–629.
Owen, H.A. and Makaroff, C.A. (1995) Ultrastructure of microsporogenesis and microgametogenesis in Arabidopsis thaliana (L.) Heynh. ecotype
Wassilewskija (Brassicaceae). Protoplasma 185: 7–21.
Palanivelu, R. and Preuss, D. (2006) Distinct short-range ovule signals
attract or repel Arabidopsis thaliana pollen tubes in vitro. BMC Plant Biol.
6: 7–15.
Pierson, E.S., Lichtscheidl, I.K. and Derksen, J. (1990) Structure and
behaviour of organelles in living pollen tubes of Lilium longiflorum.
J. Exp. Bot. 41: 1461–1468.
Rosen, W.G., Gawlik, S.R., Dashek, W.V. and Siegesmund, K.A. (1964)
Fine structure and cytochemistry of Lilium pollen tubes. Amer. J. Bot. 51:
61–71.
Rotman, N., Durbarry, A., Wardle, A., Yang, W.C., Chaboud, A.,
Faure, J.-E., Berger, F. and Twell, D. (2005) A novel class of MYB
factors controls sperm-cell formation in plants. Curr. Biol. 15: 1–20.
Ruf, S., Karcher, D. and Bock, R. (2007) Determining the transgene
containment level provided by chloroplast transformation. Proc. Natl
Acad. Sci. USA 104: 6998–7002.
Russell, S.D. (1980) Participation of male cytoplasm during gamete fusion
in an angiosperm, Plumbago zeylanica. Science 210: 200–201.
Russell, S.D. (1983) Fertilization in Plumbago zeylanica: gametic fusion and
fate of the male cytoplasm. Amer. J. Bot. 70: 416–434.
Russell, S.D. (1984) Ultrastructure of the sperm of Plumbago zeylanica II.
Quantitative cytology and three-dimensional organization. Planta 162:
385–391.
Russell, S.D., Rougier, M. and Dumas, C. (1990) Organization of the early
post-fertilization megagametopyte of Populus deltoides—ultrastructure
and implications for male cytoplasmic transmission. Protoplasma 155:
153–165.
Sakamoto, W. and Wintz, H. (1996) Nucleotide sequence of cDNAs
encoding gamma, delta, delta-prime, and epsilon subunits of mitochondrial F1-ATPase in Arabidopsis thaliana. Plant Physiol. 112:
1735–1736.
Shitara, H., Kaneda, H., Sato, A., Iwasaki, K., Hayashi, J.-I., Taya, C. and
Yonekawa, H. (2001) Non-invasive visualization of sperm mitochondria
behavior in transgenic mice with introduced green fluorescent protein
(GFP). FEBS Lett. 500: 7–11.
Sodmergen, Suzuki, T., Kawano, S., Nakamura, S., Tano, S. and
Kuroiwa, T. (1992) Behavior of organelle nuclei (nucleoids) in generative
cell and vegetative cells during maturation of pollen in Lilium longiflorum
and Pelargonium zonale. Protoplasma 168: 73–82.
Mitochondrial dynamics in plant male gametophyte
Southworth, D. and Russell, S. (2001) Male gametohenesis—development
and structure of sperm. In Current Trends in the Embryology of
Angiosperm. Edited by Bhojwani, S.S. and Soh, W.Y. pp. 1–16. Kluwer
Academic Publishers, Dordrecht.
Sutovsky, P., Moreno, R.D., Ramalho-Santos, J., Dominko, T., Simerly, C.
and Schatten, G. (1999) Ubiquitin tag for sperm mitochondoria. Nature
402: 371–372.
Svab, Z. and Maliga, P. (2007) Exceptional transmission of plastids
and mitochondria from the transplastomic pollen parent and its
impact on transgene containment. Proc. Natl Acad. Sci. USA 104:
7003–7008.
Testolin, R. and Cipriani, G. (1997) Paternal inheritance of chloroplast
DNA and maternal inheritance of mitochondrial DNA in the genus
Actinidia. Theor. Appl. Genet. 94: 897–903.
1083
Twell, D., Yamaguchi, J. and McCormick, S. (1990) Pollen-specific gene
expression in transgenic plants: coordinate regulation of two different
tomato gene promoters during microsporogenesis. Development 109:
705–713.
Yamamoto, Y., Nishimura, M., Hara-Nishimura, I. and Noguchi, T. (2003)
Behavior of vacuoles during microspore and pollen development in
Arabidopsis thaliana. Plant Cell Physiol. 44: 1192–1201.
Yu, H.-S., Huang, B.-Q. and Russell, S.D. (1994) Transmission of male
cytoplasm during fertilization in Nicotiana tabacum. Sex. Plant Reprod. 7:
313–323.
Zhang, Q., Liu, Y. and Sodmergen (2003) Examination of the cytoplasmic
DNA in male reproductive cells to determine the potential for
cytoplasmic inheritance in 295 angiosperm species. Plant Cell Physiol.
44: 941–951.
(Received April 13, 2008; Accepted May 29, 2008)