Elevation of Pollen Mitochondrial DNA Copy
Number by WHIRLY2: Altered Respiration and
Pollen Tube Growth in Arabidopsis1
Qiang Cai 2, Liang Guo 2, Zhao-Rui Shen, Dan-Yang Wang, Quan Zhang*, and Sodmergen*
Key Laboratory of Ministry of Education for Cell Proliferation and Differentiation, College of Life Sciences,
Peking University, Beijing 100871, China (Q.C., L.G., Z.-R.S., Q.Z., S.); and Key Laboratory of Ministry of
Education for Resource Biology and Biotechnology in Western China, School of Life Science, Northwest
University, Xi’an 710069, China (D.-Y.W.)
ORCID IDs: 0000-0001-5871-2519 (Q.C.); 0000-0002-1954-1179 (L.G.); 0000-0001-8735-0422 (D.-Y.W.).
In plants, the copy number of the mitochondrial DNA (mtDNA) can be much lower than the number of mitochondria. The
biological significance and regulatory mechanisms of this phenomenon remain poorly understood. Here, using the pollen
vegetative cell, we examined the role of the Arabidopsis (Arabidopsis thaliana) mtDNA-binding protein WHIRLY2 (AtWHY2).
AtWHY2 decreases during pollen development, in parallel with the rapid degradation of mtDNA; to examine the importance of
this decrease, we used the pollen vegetative cell-specific promoter Lat52 to express AtWHY2. The transgenic plants (LWHY2) had
very high mtDNA levels in pollen, more than 10 times more than in the wild type (ecotype Columbia-0). LWHY2 plants were
fertile, morphologically normal, and set seeds; however, reciprocal crosses with heterozygous plants showed reduced
transmission of LWHY2-1 through the male and slower growth of LWHY2-1 pollen tubes. We found that LWHY2-1 pollen
had significantly more reactive oxygen species and less ATP compared with the wild type, indicating an effect on mitochondrial
respiration. These findings reveal that AtWHY2 affects mtDNA copy number in pollen and suggest that low mtDNA copy
numbers might be the normal means by which plant cells maintain mitochondrial genetic information.
Reflecting their endosymbiotic origin, mitochondria
contain DNA genomes (mtDNA) encoding several key
proteins for oxidative phosphorylation. As most genes
identified in the mitochondrial genome are indispensable for mitochondrial function, it is generally believed
that each mitochondrion must possess at least one full
copy of the genome. Indeed, this seems to be the case in
animals. For example, although the number of mitochondria per cell varies in human, mouse, rabbit, and
rat cell lines, the mtDNA copy number per mitochondrion remains constant at 2.6 6 0.3 (Robin and Wong,
1988). Also, in mouse egg cells, each mitochondrion
1
This work was supported by the National Basic Research Program of China (grant no. 2013CB126900).
2
These authors contributed equally to the article.
* Address correspondence to [email protected] and
[email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Sodmergen ([email protected]).
Q.C. and L.G. performed the experiments and analyzed the data;
Z.-R.S. contributed to the data analysis; D.-Y.W. contributed technical
assistance to the single pollen quantification of mtDNA; Q.C. prepared the article; Q.Z. supervised and complemented the writing;
Q.Z. conducted the experiments; S. conceived the project and revised
the article.
www.plantphysiol.org/cgi/doi/10.1104/pp.15.00437
660
contains an estimated one to two copies of the mtDNA
(Pikó and Matsumoto, 1976).
Plant cells, however, have very few copies of the
mtDNA compared with the number of mitochondria.
For example, in the Cucurbitaceae, cells containing 110
to 140 copies of the mtDNA have 360 to 1,100 mitochondria (Bendich and Gauriloff, 1984). In Arabidopsis
(Arabidopsis thaliana), leaf cells each contain approximately 670 mitochondria (Sheahan et al., 2005) and
approximately 50 copies of the mtDNA (Draper and
Hays, 2000). Thus, in plant cells, each mitochondrion
does not possess one complete copy of the mtDNA, a
phenomenon that occurs commonly in somatic cells of
plants (Preuten et al., 2010). In addition, work in Arabidopsis, barley (Hordeum vulgare), and tobacco (Nicotiana
tabacum) showed that cells in leaves, stems, and roots
contain few copies of the mtDNA (40–160), whereas
cells in root tips contain more copies (300–450; Preuten
et al., 2010). This is consistent with the mitochondrial
nucleoid diminishment previously observed in developing root and shoot tips (Fujie et al., 1993, 1994),
which suggests that the low copy numbers in plant cells
result from a decrease in the mtDNA copy number in
nondividing cells during development.
One question raised by these findings is whether
some mitochondria have complete mtDNAs while
others have no mtDNA or whether mitochondria have
partial mtDNAs. Using techniques for the direct visualization of small amounts of DNA, our group revealed
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WHIRLY2 Affects mtDNA Levels in Pollen
that up to two-thirds of mitochondria in Arabidopsis
mesophyll cells totally lack mtDNA and the remaining
one-third of mitochondria possess mtDNA of about
100 kb on average (Wang et al., 2010). This agrees well
with a previously reported value for mtDNA copy
number (about 50 copies per cell; Draper and Hays,
2000) and is consistent with the idea that plant mitochondrial genomes exist as submolecules smaller than
the total genomic sizes (Satoh et al., 1993; Kubo and
Newton, 2008). Among plant cells possessing low mtDNA
copy numbers, the vegetative cell in the pollen grains is
an extreme case; a mature pollen grain of Antirrhinum
majus, containing many more mitochondria than a somatic cell, possesses only 16 copies of the mtDNA
(Wang et al., 2010). Similar to the changes observed in
somatic cells, this extremely low level of mtDNA in
pollen vegetative cells results from a rapid decrease
in mtDNA copy number during pollen development
(Sodmergen et al., 1991; Nagata et al., 1999). In A. majus,
the vegetative cell in its initial developmental stage has
482.7 copies of the mtDNA per cell, indicating a 30-fold
decrease (482.7/16) during development (Wang et al.,
2010). These results from both somatic and reproductive cells led to the intriguing idea that the mtDNA
copy number in plants decreases in parallel with cell
differentiation, to a very low value, and thus that several mitochondria must share the genetic information
carried on a single copy of the mtDNA. Plant cell mitochondria undergo frequent and coupled fusions and
divisions, which may explain how mitochondria share
this information (Arimura et al., 2004). However, the
biological significance of why plant cells lose their
mtDNA, and how this benefits these cells, remains
unknown. Given that pollen germination, pollen tube
elongation, and sperm cell delivery all require energy
conversion, the extremely low mtDNA copy numbers,
such as in pollen vegetative cells, must not compromise mitochondrial function.
The mtDNA copy numbers remain constant in various
tissues, however, indicating that cellular mechanisms
accurately regulate the levels of mtDNA in relation to
cell type (Robin and Wong, 1988; Preuten et al., 2010). In
yeast and animals, this regulation involves the core enzymes of mtDNA replication, such as DNA polymerase-g
(Sharief et al., 1999), RNA polymerase (Wanrooij et al.,
2008), and mitochondrial helicase (Liu et al., 2009), as
well as a group of DNA-binding proteins such as
ARS-binding factor2 protein in yeast (Saccharomyces
cerevisiae; Newman et al., 1996), MITOCHONDRIAL
TRANSCRIPTION FACTOR A (TFAM) in human (Alam
et al., 2003), and mitochondrial single-stranded DNA
binding protein in Drosophila spp. (Maier et al., 2001).
Overexpression of TFAM causes an increase in the
mtDNA copy number, and RNA interference of TFAM
decreases the mtDNA copy number (Ekstrand et al.,
2004; Kanki et al., 2004). Also, the homozygous knockout of TFAM in mouse results in embryos that lack mtDNA
and thus fail to survive (Larsson et al., 1998). Clearly,
protein factors within mitochondrial nucleoids play a
crucial role in the regulation of mtDNA copy number.
Recent investigation in Arabidopsis revealed that,
similar to the case in yeast and animal cells, DNA polymerase, the core enzyme of mtDNA replication,
functions to maintain mtDNA levels in plants. Mutation of Arabidopsis PolIA or PolIB (homologs of bacterial DNA polymerase I) causes a reduction in mtDNA
copy number, and double mutation of these proteins is
lethal (Parent et al., 2011). Also, an Mg2+-dependent
exonuclease, DEFECTIVE IN POLLEN ORGANELLE
DNA DEGRADATION1 (DPD1), degrades organelle
DNA, helping to produce the proper amounts of
mtDNA in pollen cells (Matsushima et al., 2011; Tang
et al., 2012). These results provide insights into the
molecular control of mtDNA levels in plants, via both
mtDNA replication and mtDNA degradation. Except
for these enzymes, however, other protein factors (such
as TFAM in animals) have not been identified in plants.
The DNA-binding proteins, such as MutS Homolog1
(MSH1), Organellar Single-Strand DNA Binding Protein1 (OSB1), Recombinase A1 (RecA1), RecA3, and
WHIRLY2 (WHY2), identified so far in plant mitochondria likely participate in genomic maintenance by
affecting substoichiometric shifting (Abdelnoor et al.,
2003), stoichiometric transmission (Zaegel et al., 2006),
genomic stability (Shedge et al., 2007; Odahara et al.,
2009), and DNA repair (Cappadocia et al., 2010). None
of these plant nucleoid factors (DNA-binding proteins)
has been implicated in the control of mtDNA copy
number; thus, the mechanisms by which nonenzyme
protein factors regulate mtDNA copy number in plants
remain obscure.
To test whether nucleoid DNA-binding proteins can
affect mtDNA copy number, we examined the effect of
producing Arabidopsis WHY2, a single-stranded DNAbinding protein (Cappadocia et al., 2010), in the pollen
vegetative cell, which generally does not express WHY2
(Honys and Twell, 2004). We found that expression of
WHY2 resulted in a 10-fold increase in mtDNA copy
number in the pollen vegetative cell. This increase affected mitochondrial respiration, mitochondrial size,
and pollen tube growth. Thus, our results uncover a
novel function for WHY2, a member of the plant Whirly
protein family, in regulating mtDNA amounts and indicate that, in plants, low mtDNA copy number does
not compromise mitochondrial function but rather promotes proper mitochondrial function.
RESULTS
Arabidopsis Pollen Vegetative Cells Have Few Copies of
the mtDNA
In flowering plants, the pollen vegetative cell generally has an extremely low mtDNA copy number, and
the amount of mtDNA can be directly observed by
fluorescence microscopy of cells stained for DNA. In
Arabidopsis, we found that the fluorescent signals for
the mtDNA were undetectable in the vegetative cell
of the mature pollen grain (Fig. 1, A and B), showing
that this cell has very little mtDNA. We did observe
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Cai et al.
Figure 1. The pollen vegetative cells of Arabidopsis have a very low mtDNA copy number. A, Flowers and pollen of ecotype
Columbia-0 (Col-0) Arabidopsis at bicellular pollen (BCP) and mature pollen grain (MPG) stages. The pollen grains were stained
with 49,6-diamino-phenylindole (DAPI). Arrows indicate dotted signals of cytoplasmic DNA. GN, Generative nucleus; SN, sperm
nucleus; VN, vegetative nuclei. Bars = 10 mm. B, Pollen vegetative cells with transgenic expression of mitochondrial GFP (Lat52:
Dips-GFP). The pollen grains were stained with DAPI. Arrows indicate mitochondria having mtDNA signals. Double arrowheads
indicate the signals of plastid DNA. Bars = 1 mm. C, Copy number of mtDNA per pollen. The values were acquired from three
individual quantifications (Supplemental Fig. S2). Error bars indicate SD. D, Number of mitochondria per pollen. The values were
acquired from 10 individual pollen grains of each stage. Error bars indicate SD.
fluorescent signals for mtDNAs in the BCP (Fig. 1, A and
B) and other, earlier developmental stages (Supplemental
Fig. S1), indicating a decrease of mtDNA levels during
pollen development, as indicated previously (Nagata
et al., 1999; Matsushima et al., 2011; Tang et al., 2012).
Using a competitive PCR technique developed for
determining the copy number of mtDNA in single cells
(Wang et al., 2010), we measured 133.3 6 11.5 copies of
the mtDNA per BCP and 10.3 6 2.1 per mature pollen
grain (Fig. 1C; Supplemental Fig. S2), indicating a
more than 10-fold decrease during pollen development. We also counted 486 6 107 mitochondria per
BCP and 1,708 6 100 mitochondria per mature pollen
grain (Fig. 1D). Thus, each mitochondrion in the vegetative cell at the BCP stage contains an average of
0.274 (133.3/486) copies of the mtDNA, but each mitochondrion at the mature pollen stage contains only
0.006 (10.3/1,708) copies, equivalent to about 170 mitochondria per mtDNA. Since the vegetative cell does
not divide during pollen development, the decrease of
mtDNA likely results from mtDNA degradation.
AtWHY2 Levels Decrease with the mtDNA Copy Number
A previous microarray analysis of pollen showed that
AtWHY2 transcription decreases during pollen development and is absent in mature pollen grains (Honys
and Twell, 2004). Our reverse transcription (RT)-PCR
assays verified the array data (Supplemental Fig. S3A).
To examine the levels of AtWHY2 in pollen, we used
immunoblots with anti-AtWHY2 antibodies against total
proteins from pollen at different developmental stages.
Our results showed a decrease in AtWHY2 during pollen
development and an undetectable level of AtWHY2 in
mature pollen (Supplemental Fig. S3B). This is consistent
with the transcriptional profiling and with the expression
from the AtWHY2 promoter analyzed by fusion with the
GUS reporter gene (Supplemental Fig. S3C), indicating
that transcriptional regulation causes the decrease in
AtWHY2 in wild-type vegetative cells from pollen mitosis I. Thus, the decrease of AtWHY2 occurs at the same
time as the degradation of mtDNA during pollen development, consistent with our hypothesis that WHY2
may affect mtDNA levels.
To further examine the role of AtWHY2 in regulating
mtDNA levels, we next tested whether AtWHY2 in
cells could cause an increase in mtDNA copy numbers.
We tested this by expressing AtWHY2 in vegetative
cells under the control of the Lat52 promoter, which
drives consistent, high-level expression in pollen vegetative cells (Twell et al., 1989); we termed the resulting
transgene LWHY2 (Fig. 2A). We also constructed a
transgene expressing an AtWHY2-GFP fusion under the
control of the Lat52 promoter (LWHY2-GFP) and a Lat52GFP control transgene (LGFP). We succeeded with this
strategy (Fig. 2B) and obtained homozygous LWHY2
transgenic lines showing stable transcription of AtWHY2
(Fig. 2C), accumulation of AtWHY2 (Fig. 2D), and, most
interestingly, mtDNA signals in the vegetative cells of
mature pollen (Fig. 2E). The signals appeared in independent transgenic lines for LWHY2 and LWHY2-GFP
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WHIRLY2 Affects mtDNA Levels in Pollen
cells. Quantification of mtDNA levels showed 124 6 11.1,
135.7 6 16.6, and 128.7 6 18.4 copies of the mtDNA per
mature pollen grain in LWHY2-1, LWHY2-2, and LWHY2-3
plants, respectively, in contrast to 9 6 2.6 copies per
pollen in LGFP control plants (Fig. 2F; Supplemental Fig.
S4, B and C). Thus, the pollen vegetative cells expressing
AtWHY2 have more than 10-fold more copies of the
mtDNA, indicating a role for AtWHY2 in the elevation of
mtDNA levels. The transgenic plants of all lines showed
no abnormal phenotypes in plant growth, flowering, or
seed set (Supplemental Fig. S5; see below).
AtWHY2 Localizes to the Mitochondrial Nucleoid
Figure 2. The mtDNA copy number is highly elevated in mature pollen
grains that express AtWHY2. A, Construction of the transgenic vectors. B,
WHY2-GFP signals in mature pollen grains of LWHY-GFP plants and
absence of signal in WWHY2-GFP, indicating the successful driving of
AtWHY2 expression in mature pollen vegetative cells by Lat52. Bar =
10 mm. C, RT-PCR showing the increased transcript levels of AtWHY2 in
pollen of the transgenic plant. MSP, Microspore; TCP, tricellular pollen. D,
Immunoblot showing the accumulation of AtWHY2 in mature pollen
grains of a transgenic plant in comparison with the undetectable level in
pollen of Col-0. HSP is the loading control. E, Appearance of dotted DAPI
(mtDNA) signals (arrows) in mature pollen grains of LWHY2-1 plants and
absence of the signal in LGFP control plants. SN, Sperm cell nucleus; VN,
vegetative nuclei. Bar = 10 mm. F, Copy number of mtDNA per mature
pollen grain determined with three independent transgenic lines of
LWHY2 (LWHY2-1, LWHY2-2, and LWHY2-3). Error bars indicate SD.
(Fig. 2E; Supplemental Fig.S4A) but not in the LGFP lines
(Fig. 2E), implying that transgenic expression of AtWHY2
causes a higher level of mtDNA in the pollen vegetative
To understand how AtWHY2 affects the level of
mtDNA in cells, we next examined its subcellular localization. Previous work using transient expression
systems showed that AtWHY2 localizes to the mitochondria in potato (Solanum tuberosum) and onion (Allium
cepa) cells (Krause et al., 2005). We examined the mitochondrial localization of AtWHY2 in pollen vegetative
cells of Arabidopsis using a homozygous transgenic
plant expressing AtWHY2-GFP and mitochondrialocalized RFP (for red fluorescent protein; Fig. 3, A and B),
from a cross between the transgenic lines LWHY2-GFP-1
and LDips-RFP (a construct containing Lat52:Dips-RFP,
where Lat52 is the promoter and Dips is the mitochondrial localization signal; Matsushima et al., 2008). Fluorescence microscopy showed that the AtWHY2-GFP
signals appeared dimorphic, with strong and weak signals (Fig. 3A). With normal and controlled (reduced) exposures for GFP, we show that both the strong and weak
signals colocalize with the RFP signal (Fig. 3, A and B).
The mtDNA signals, however, colocalized with the
strong AtWHY2-GFP signals (Fig. 3C). These results indicate that AtWHY2 incorporates unequally into mitochondria, with the mitochondria-possessing mtDNA
having significantly more AtWHY2. In the mitochondria of LWHY2-1 plants, electron microscopy revealed
electron-dense nucleoid-like structures (Fig. 3D;
Supplemental Fig. S6A), and immunogold electron
microscopy showed that gold signals of mtDNA and
AtWHY2 colocalize within the nucleoid-like structure (Fig. 3E). These observations indicate that AtWHY2
forms a DNA-protein complex in the nucleoid of mitochondria. The mitochondria in the LWHY2-1 pollen
vegetative cells were also larger than those in the cells of
Col-0 (Fig. 3D; Supplemental Fig. S6, A and B). Given
that we observed fewer mitochondria per pollen grain in
LWHY2-1 plants (Supplemental Fig. S6C), AtWHY2 may
compromise mitochondrial division during pollen development via an unknown mechanism.
The WHY2 DNA-Binding Motif Is Required to Affect
mtDNA Levels
Structural study revealed a KGKAAL motif in Whirly
proteins, and this motif is required for Whirly proteins
to bind to single-stranded DNA (Desveaux et al., 2002).
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Figure 3. AtWHY2 localizes in mitochondria and aggregates with mtDNA. A, Signals of AtWHY2-GFP and mitochondrial RFP in
mature pollen grains in normal exposure. Large and small arrows indicate strong and weak signals of AtWHY2-GFP, respectively.
The blocked area was enlarged (bottom) to show localization of the weak signals in mitochondria. Bar = 3 mm. B, Signals of
AtWHY2-GFP and mitochondrial RFP in mature pollen grains in reduced exposure. The blocked area was enlarged (bottom)
to show localization of the strong AtWHY2-GFP signals in mitochondria. Bar = 3 mm. C, Signals of mtDNA (DAPI staining)
merged with the strong signals of AtWHY2-GFP. Bar = 3 mm. D, Electron microscopy of mitochondria in mature pollen vegetative cells of Col-0 and LWHY2-1 plants. Arrows indicate the electron-dense nucleoid-like structure appearing in mitochondria of LWHY2-1 cells. Bars = 200 nm. E, Immunogold electron microscopy of mitochondria in mature pollen vegetative
cells of Col-0 and LWHY2-1. The signals of mtDNA (5-nm gold) and AtWHY2 (10-nm gold) localize, and they colocalize into
the electron-dense nucleoid-like structure. Bars = 200 nm.
The motif in AtWHY2 appears at amino acids 62 to 67
from the N terminus. To see if the elevation of mtDNA
levels requires this putative DNA-binding motif, we
mutated this motif in AtWHY2 by replacing KGKAAL
with QGQGGV (Fig. 4A). We prepared a transgenic
construct consisting of the Lat52 promoter and the
mutated AtWHY2 (AtWHY2M62-67) and GFP (Lat52:
AtWHY2M62-67-GFP), obtained a homozygous transgenic
plant, and crossed it with LDips-RFP. In pollen cells
expressing AtWHY2M62-67-GFP and mitochondrial RFP,
AtWHY2M62-67-GFP localized to mitochondria, as expected, but we observed no mtDNA signals in the mature pollen vegetative cell (Fig. 4B) and no nucleoid-like
structures in the mitochondria (Fig. 4C). Quantification
revealed 15.3 6 3.5 copies of mtDNA per pollen of
LWHY2M62-67-GFP, similar to the value detected for Col-0
plants (10.3 6 2.1 copies per pollen) and much smaller
than the value for LWHY2-1 plants (124 6 11.1 copies per
pollen). We thus conclude that AtWHY2 elevation of
mtDNA levels requires the DNA-binding motif. Interestingly, similar to the LWHY2-1 plants, mitochondria in
the LWHY2M62-67-GFP cells also appeared larger than
Col-0 and LGFP mitochondria (Fig. 4C; Supplemental
Fig. S6B), and LWHY2M62-67-GFP cells also had fewer
mitochondria per cell (Supplemental Fig. S6C). This
suggests that AtWHY2 may inhibit mitochondrial division, but this inhibition does not require the DNAbinding motif.
AtWHY2 Decreases Transmission through the Male
Germ Line
The homozygous transgenic lines set seeds that
appeared normal in morphology and number (Fig.
5A). This indicates that expression of WHY2 in pollen
vegetative cells and the resulting high levels of mtDNA
do not affect the function of pollen in sexual reproduction. We observed normal arrival of the pollen tube
at the embryo sac (Fig. 5B), in agreement with this idea.
However, segregation analysis of self-crosses using
three heterozygous transgenic lines of LWHY2 showed
skewed ratios of segregation, 2.17:1, 2.4:1, and 2.28:1
kanamycin-resistant (KanR) to kanamycin-sensitive
(KanS) progeny (Table I), all deviating from the expected ratio of 3:1 and indicating unequal transmission
of the transgene. As significant differences were observed in two independent transgenic lines, LWHY2-1 and
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WHIRLY2 Affects mtDNA Levels in Pollen
Figure 4. The DNA-binding motif in AtWHY2 is necessary for the
regulation of mtDNA levels. A, Mutation of the motif KGKAAL to
QGQGGV. B, Mature pollen grains carrying AtWHY2M62-67-GFP (GFP)
and mitochondrial RFP (mtRFP). The pollen grains were stained with
DAPI. There was no detectable signal for mtDNA in the pollen vegetative cell. SN, Sperm cell nucleus; VN, vegetative nuclei. Bar = 10 mm.
C, Electron microscopy of mitochondria in the vegetative cells of
LWHY2M62-67 and LGFP. Bar = 200 nm.
LWHY2-3, we conclude that the change in segregation did
not result from an effect of the insertion site of the transgene. We performed reciprocal crosses between heterozygous Col-0 and LWHY2-1 to quantify the male and
female transmission efficiencies and found a significant
reduction in transmission through the male lineage but no
difference in transmission through the female (Table II).
To understand how transmission through the male
parent is affected in the transgenic plants, we first examined the viability of mature pollen grains freshly
collected from dehiscing anthers of LWHY2-1 plants by
Alexander staining and in vitro germination. Our results revealed 98.3% (n = 580) and 98.7% (n = 628) positive staining of pollen grains in LWHY2-1 and Col-0,
respectively, and 43% (n = 500) and 38.6% (n = 500)
germination (after 4 h in germination medium) of pollen in LWHY2-1 and Col-0, respectively. This indicates
that pollen of LWHY2-1 and Col-0 shows no significant
difference in viability or germination (Fig. 5, C and D).
Next, we inspected the growth of the pollen tube by
Aniline Blue staining using Col-0 flowers pollinated
with LWHY2-1 and Col-0 pollen. We found that the
leading pollen tubes of LWHY2-1 were shorter than
those of Col-0 (Fig. 5E). This indicates that pollen tubes
of LWHY2-1 may grow more slowly than those of Col-0.
Averaging the length of four leading pollen tubes
within an ovary showed significant differences between the pollen tubes at 4 h (1,433 6 57 mm for Col-0
and 1,196 6 42 mm for LWHY2-1; P = 0.049) and 8 h
(2,243 6 100 mm for Col-0 and 1,893 6 115 mm for
LWHY2-1; P = 0.038) after pollination but showed
similar lengths at 2 h (553 6 116 mm for Col-0 and 540 6
125 mm for LWHY2-1; P = 0.926) and 10 h (2,263 6 75 mm
for Col-0 and 2,213 6 49 mm for LWHY2-1; P = 0.185)
after pollination (Fig. 5F). These results imply that pollen
grains of LWHY2-1 show reduced pollen tube growth
from 500 to 1,000 mm in length. The similar length of
pollen tubes at 2 h after pollination indicates that
LWHY2-1 and Col-0 pollen grains show little or no difference in germination and initial growth of the pollen
tube. Also, the pollen tubes exhibit a similar length at 10 h
after pollination, having reached the end of the ovary.
The reduced growth of LWHY2 pollen tubes may
reduce the chance for the pollen tube to reach unfertilized ovules located in the lower half of the ovary when
competing with pollen tubes of Col-0. This may explain
the abnormal segregation in self-crosses and the lower
male transmission in reciprocal crosses (Tables I and II).
To test this, we performed limited pollinations and
observed that the segregation ratio of KanR to KanS
approached 1:1 for pollination of Col-0 with pollen
from heterozygous LWHY2 plants when we limited the
number of pollen grains to no more than the number of
ovules per ovary. Thus, pollination with fewer than 40
pollen grains per stigma yielded a segregation ratio of
1.02 6 0.03, significantly different (P = 0.014) from the
ratio of 0.75 6 0.03 that we observed in pollinations
with more than 200 pollen grains per stigma (Fig. 6A).
We also predicted that the ratio of KanR to total would
vary in seeds harvested from the upper or lower position of the silique in crosses with excess heterozygous
LWHY2 pollen. Indeed, when we divided the silique
into three equal portions (Fig. 6B), the seeds showed
decreasing ratios of KanR to total, with 48.7% 6 0.7% KanR
in the top, 41% 6 0.7% in the middle, and 34.7% 6 1% in
the bottom portions (Fig. 6C). The percentage of KanR
differed significantly between each two portions (P = 0.003
between the top and the middle, P = 0.009 between the
middle and the bottom, and P = 0.002 between the top and
the bottom), indicating that the growth of most LWHY2
pollen tubes slows when they reach approximately
700 mm in length, one-third of the distance from the top
of the ovary. Thus, AtWHY2 causes a disadvantage in
male competence owing to reduced pollen tube growth.
AtWHY2 appears to inhibit mitochondrial division
and increase the copy number of the mtDNA. To examine which of these cellular events, or both, causes the
reduced strength of pollen tube growth, we examined
segregation ratios in the heterozygous transgenic lines
of LWHY2M62-67, as these derive from homozygous lines
that show inhibition of mitochondrial division without
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Figure 5. Seed and pollen phenotypes of plants expressing AtWHY2.
A, Developing seeds of homozygous
LWHY2-1 plants compared with Col-0.
Bar = 1 mm. B, Arrival of pollen tubes
to the embryo sac 8 h after fertilization
observed with Aniline Blue staining of
pollen tubes. Arrows indicate the top
of the pollen tube. Bars = 50 mm. C,
Mature pollen grains of Col-0 and
LWHY2-1 stained with Alexander
solution. Bars = 50 mm. D, In vitro
germination of pollen. Bars = 30 mm.
E, In vivo growth of pollen tubes 4 and
8 h after pollination observed with
Aniline Blue staining. Arrows indicate the positions of leading pollen
tubes. Bar = 500 mm. F, Length of the
leading pollen tubes 2, 4, 8, and
10 h after pollination. For both the
genotypes, four leading pollen tubes
were measured at each time point.
Error bars indicate SD (Student’s
t test: *, P , 0.05).
increased mtDNA copy number (Fig. 4, B and C;
Supplemental Fig. S6, B and C). Our results for three
independent transgenic lines showed KanR:KanS ratios
of 2.91:1, 3.03:1, and 2.97:1, which fits the expected ratio
of 3:1 (Table III). This indicates equal transmission of
gametes in a self-cross of heterozygous LWHY2M62-67,
revealing that the transgenic alterations of mitochondrial size and number do not affect male competence
(pollen tube growth). Therefore, we conclude that the
high copy number of mtDNA in pollen vegetative cells
causes the reduced pollen tube growth.
High mtDNA Copy Number Causes
Respiratory Abnormalities
Pollen germination and pollen tube growth require
rapid and ample mitochondrial respiration (Rounds
et al., 2011). Given that neither pollen viability nor morphology is aberrant in LWHY2 plants (Fig. 5C), the reduced
pollen tube growth may indicate defects in energy production or consumption in LWHY2 pollen. We measured
the ATP level in mature anthers of Col-0 and LWHY2-1 plants
and found 19.98 6 1.58 mmol ATP g21 (fresh weight) in
LWHY2-1 and 30.84 6 3.61 mmol g21 in Col-0 anthers
(Fig. 7A). Thus, pollen grains of LWHY2-1 harbor less
ATP, 65% of that in Col-0 (19.98/30.84) as measured in
the entire anther. For a better understanding of the mitochondrial condition that causes this reduction in cellular
ATP, we further measured ADP, NADH, and NAD+,
finding 236.8 6 40.1 nmol ADP g21 fresh weight in
LWHY2-1 and 285.6 6 68.2 nmol g21 in Col-0 anthers
(Fig. 7A). This reveals an imbalance in the ATP-ADP ratio,
which was 84.4 (19.98/0.2368) for LWHY2-1 and 107.9
(30.84/0.2856) for Col-0 (Fig. 7A), indicating possible mitochondrial dysfunction. NADH, at 89.5 6 13.5 nmol g21 for
Table I. Analysis of segregation in self crosses of different LWHY2 transgenic lines
KanR and KanS were observed with T1 seeds. Asterisks indicate significant difference from the expected
KanR-total ratio (75%; x 2, P , 0.05).
Line (Female 3 Male)
KanR
KanS
KanR:KanS
KanR:Total (Expected)
x2 (P) for 3:1
LWHY2-1(+/2) 3 LWHY2-1(+/2)
LWHY2-2(+/2) 3 LWHY2-2(+/2)
LWHY2-3(+/2) 3 LWHY2-3(+/2)
195
202
221
90
84
97
2.17
2.40
2.28
68.4% (75%)
70.6% (75%)
69.5% (75%)
6.57* (0.010)
2.91 (0.088)
5.14* (0.023)
666
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WHIRLY2 Affects mtDNA Levels in Pollen
Table II. Analysis of segregation in reciprocal crosses of Col-0 and heterozygous LWHY2-1
KanR and KanS were observed with T1 seeds. TE (transmission efficiency of gametes) = number of KanR/
number of KanS 3 100%. Double asterisks indicate highly significant difference from the expected TE
(100%; x 2, P , 0.01).
Transmission
Male
Female
Cross (Female 3 Male)
KanR
KanS
KanR:Total
TE (Expected)
x 2 (P) for 100%
Col-0 3 LWHY2-1(+/2)
LWHY2-1(+/2) 3 Col-0
206
228
276
221
42.7%
50.8%
74.6 (100)
103.1 (100)
10.2** (0.001)
0.11 (0.741)
LWHY2-1 anthers, exhibited a 246% increase compared
with 36.4 6 2.4 nmol g21 for Col-0. NAD+, at 351.1 6 18.9
nmol g21 for LWHY2-1 anthers compared with 410.3 6
23.1 nmol g21 for Col-0, and the imbalanced ratio of
NADH to NAD+, at 0.255 (89.5/18.9) for LWHY2-1 compared with 0.089 (36.4/410.3) for Col-0 (Fig. 7B), support
the idea that defective respiration occurs in the pollen
grains of LWHY2 possessing more copies of the mtDNA.
Mitochondrial dysfunction generally also involves
the generation of excess reactive oxygen species (ROS;
Alandijany et al., 2013). We detected the accumulation of extra ROS in the pollen grains of LWHY2-1 (Fig.
8, A and B), corroborating the abnormality of LWHY2-1
mitochondria. To test if this increased ROS level
might cause oxidative stress in pollen cells, we examined the expression of ALTERNATIVE OXIDASE
(AOX) and ALTERNATIVE INTERNAL NAD(P)H
DEHYDROGENASE (NDA), which encode proteins that
participate in the alternative pathway of plant mitochondria known to reduce the formation of ROS
(Maxwell et al., 1999; Svensson and Rasmusson, 2001).
We also examined AOX1 protein and observed increased AOX1 levels (Fig. 8C). Our results showed increased AOX1a, AOX1d, NDA1, and NDA2 transcript
levels (Fig. 8D) in the pollen grains of LWHY2-1, indicating that genes in this pathway express at higher
levels. In contrast, we observed no increase in proteins in
the ordinary pathway of mitochondrial respiration in
the LWHY2-1 pollen (Supplemental Fig. S7). The enhanced levels of ROS might cause cellular stress, thus
activating the alternative pathway, the cellular mechanism involved in reducing the level of ROS, to eliminate
the stress. Our further inspection showed significant
and rapid loss of pollen viability during storage (Fig. 8, E
and F), supporting the idea that pollen grains of LWHY2-1
likely experience a certain degree of cellular stress.
mtDNA than the number of mitochondria in their cells.
This is a special property of plant cells, because animal
cells have more than one copy of the mtDNA per mitochondrion (Pikó and Matsumoto, 1976; Robin and
Wong, 1988) and reduction in the mtDNA copy number
in animal cells causes mitochondrial defects and disease
(for review, see Montier et al., 2009). Given that the
insufficiency of mtDNA in the somatic or the pollen
cells of plants did not compromise cell respiration
(Preuten et al., 2010; Wang et al., 2010), we predict that
plant and animal cells may have evolved different
mechanisms to maintain and use the information in the
mitochondrial genome. A previous study observed
frequent and coupled fusions and divisions of mitochondria in cultured tobacco cells (Arimura et al., 2004),
implying that plant mitochondria may have a dynamic
DISCUSSION
Low mtDNA Copy Numbers May Be Sufficient for Plant
Cell Viability
Direct observation revealed that approximately twothirds of mitochondria in Arabidopsis mesophyll cells
lack mtDNA (Wang et al., 2010). This insufficiency of
mtDNA occurs not only in Arabidopsis but also in
different tissues of various plants (Preuten et al., 2010).
Thus, although the universality of this phenomenon
remains to be examined in many plant clades, we can
conclude that plants can have fewer copies of the
Figure 6. Decreased transmission by pollen expressing AtWHY2. A,
Segregation KanR-KanS ratios in F1 progeny harvested with pollination
of heterozygous LWHY2-1 pollen grains onto Col-0 pistils. EnPG, Excess pollen; LnPG, limited number of pollen grains; n, number of
seedlings inspected. Error bars indicate SD (Student’s t test: *, P , 0.05).
B, Equal division of a mature fertilized silique into top (Top), middle
(Mid.), and bottom (Bot.) portions. C, The ratios of KanR to total in F1
progeny harvested from each of the silique portions with pollination
with excess heterozygous LWHY2-1 pollen grains onto Col-0 pistil.
n, Number of seedlings inspected. Error bars indicate SD (Student’s t test:
**, P , 0.01).
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Cai et al.
Table III. Analysis of segregation in self crosses of different LWHY2 M62-67 transgenic lines
KanR and KanS were observed with T1 seeds.
Line (Female 3 Male)
D62-67
M62-67
LWHY2
-1(+/2) 3 LWHY2
-1(+/2)
LWHY2 D62-67-2(+/2) 3 LWHY2 M62-67-2(+/2)
LWHY2 D62-67-3(+/2) 3 LWHY2 M62-67-3(+/2)
KanR
KanS
KanR:KanS
KanR:Total (Expected)
x 2 (P) for 3:1
215
191
202
74
63
68
2.91
3.03
2.97
74.4% (75%)
75.2% (75%)
74.8% (75%)
1.73 (0.812)
3.60 (0.942)
3.65 (0.944)
mechanism to share the insufficient copies of the
mtDNA within the cell.
To test whether the low mtDNA copy number is
appropriate for plant cells to maintain and use mitochondrial genetic information, here we used a direct
experimental strategy to elevate the amount of mtDNA
per cell and examined the result. No homolog of TFAM,
the well-known regulator of mtDNA levels in animals
(Larsson et al., 1998; Alam et al., 2003; Ekstrand et al.,
2004; Kanki et al., 2004), has been identified in plants;
therefore, we used a plant-specific mtDNA-binding
protein, WHY2, which may similarly affect mtDNA levels
in plants. Our analysis targeted the pollen vegetative cell
because it has a very low mtDNA copy number (about 10
copies per pollen; Wang et al., 2010) and WHY2 does not
express in pollen (Honys and Twell, 2004), making the
pollen vegetative cell a natural knockout cell line of
WHY2. As shown above, our results provide experimental evidence for the prediction that, in plants, having
more mtDNA than the insufficient level does not benefit
mitochondrial function; rather, it compromises mitochondrial function. The low copy number of mtDNA,
therefore, is conducive to proper mitochondrial function
and growth of plant cells. This indicates that the mtDNA
copy number also matters in plant cells, as is observed in
animal cells (Moraes, 2001; Montier et al., 2009).
The mechanism by which mtDNA copy number affects mitochondrial function, however, remains unknown. With the transgenic LWHY2-1 plants, we detected
abnormal activation of mitochondrial transcription
(Supplemental Fig. S8). This was abnormal because
the level of mitochondrial proteins remained unvaried
(Supplemental Fig. S7). We propose that this abnormality may occur as part of the cellular responses to
the abnormally high mtDNA copy number and may
require future research.
AtWHY2 Alters mtDNA Copy Numbers
Another purpose of this study was to test whether
AtWHY2, an Arabidopsis mtDNA-binding factor, could
affect the copy number of mtDNA. As one of the ubiquitous plant-specific Whirly family proteins, WHY2 localizes in plant mitochondria, unlike the other family
members, WHY1 and WHY3, which localize in plastids
(Krause et al., 2005). These Whirly family proteins share
a typical DNA-binding structure (Desveaux et al., 2002)
and participate in the maintenance of organellar genomes.
Recent work demonstrated that a double knockout of
AtWHY1 and AtWHY3 results in chloroplast defects due
to illegitimate recombination in the plastid genome
(Maréchal et al., 2009; Lepage et al., 2013), and AtWHY1
and AtWHY3 appear to stabilize the plastid genome by
repressing short-range rearrangements (Zampini et al.,
2015). Structural analysis also indicated that AtWHY2
binding may stabilize mtDNA to favor accurate repair of
DNA double-strand breaks (Cappadocia et al., 2010).
Figure 7. Respiratory abnormality in mature pollen
grains of LWHY2-1. A, Amounts of ATP and ADP
quantified in mature LWHY2-1 anthers in comparison
with Col-0. Error bars indicate SD (Student’s t test:
*, P , 0.05). B, Amounts of NADH and NAD+ quantified in mature LWHY2-1 anthers in comparison with
Col-0. Error bars indicate SD (Student’s t test: *, P ,
0.05; and **, P , 0.01). FW, Fresh weight.
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WHIRLY2 Affects mtDNA Levels in Pollen
Figure 8. ROS accumulation and reduced pollen viability over time in LWHY2-1. A, Mature pollen grains of Col-0 and heterozygous and homozygous LWHY2-1 incubated with 29,79-dichlorodihydrofluorescein diacetate. Green fluorescence indicates
the level of ROS. Bar = 40 mm. B, Relative levels of ROS measured by quantification of the fluorescence from each pollen grain.
The values are averages of five measurements. Error bars indicate SD. C, Relative amount of AOX1 shown by immunoblot. HSP is
the loading control. D, Relative transcript levels of AOX1a, AOX1d, NDA1, and NDA2, which encode proteins in the alternative
pathway. The values were normalized to the nucleus-encoded UBIQUITIN5 and averaged from three independent experiments,
with the transcript abundance of Col-0 adjusted as 1. Error bars indicate SD. E, Viability staining of pollen after 4 and 48 h of
storage. Red fluorescence indicates pollen grains that lost viability. Bar = 60 mm. F, Percentage of living pollen grains after
various times of storage. The values were acquired by averaging three individual tests with 300 pollen grains per test. Error bars
indicate SD.
Taken together, the evidence acquired so far shows the
great importance of WHY proteins in maintaining the
stability, rather than affecting the quantity, of organelle
genomes. Here, we show a significant increment of
mtDNA copy number (10-fold or greater) with accumulation of AtWHY2 in the pollen vegetative cells, indicating a remarkable effect of AtWHY2 upon mtDNA
quantity. Given that high copy numbers of mtDNA occurred in independent transgenic lines and that transgenic expression of GFP with the equivalent vector
(LGFP) did not affect the copy number of mtDNA, we
conclude that AtWHY2 affects the mtDNA levels in the
pollen vegetative cell.
Thus, our result reveals a potential novel function
for this Whirly family protein in regulating mtDNA
levels. In addition, our investigation reports, to our
knowledge, the first nonenzyme protein factor that
affects mtDNA levels in plants. In fact, except for the
core enzymes of mtDNA replication, PolIA and PolIB,
which increase the amount of mtDNA (Parent et al.,
2011), and an exonuclease, DPD1, which degrades
mtDNA (Matsushima et al., 2011; Tang et al., 2012),
few plant factors have been experimentally shown to
affect mtDNA quantity. The DNA-binding factors so
far identified in plants, such as OSB, RecA, and MSH,
likely affect mtDNA via its recombination and repair
rather than regulate mtDNA levels (for review, see
Maréchal and Brisson, 2010).
Intriguingly, however, our result in pollen appears to
contradict the results obtained in somatic cells. In
Arabidopsis, transgenic overexpression of AtWHY2
using the 35S promoter does not cause an increase in
mtDNA or mitochondrial transcript levels but rather a
decrease in their levels (Maréchal et al., 2008). To verify
this, we repeated the previous transgenic approach and
obtained identical results (Supplemental Fig. S9). The
exact reason for such a conflict is unclear. However,
given that the complete knockout of AtWHY2 results in
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Cai et al.
no phenotypes in the plant or in mitochondria, this
suggests that functional homologs of AtWHY2, or other
compensating mechanisms, may exist in somatic mitochondria (Maréchal et al., 2008). If this were the case,
the result of this report may imply that the homologs or
mechanisms probably do not function in the pollen
vegetative cell.
AtWHY2 May Impede mtDNA Degradation
The regulation of mtDNA copy number is of particular interest in animal cells because imbalance in
this regulation, causing either increases or decreases in
copy number, has been implicated in human disease
(Moraes, 2001; Montier et al., 2009). Work in animal cells
demonstrated that continuous, well-balanced replication and turnover (degradation) maintain the levels of
mtDNA in cells (Berk and Clayton, 1974; Kai et al.,
2006). Accordingly, factors affecting either degradation or replication likely function as key regulators of
mtDNA levels. TFAM, as one of the key regulators of
mtDNA levels in animals, is proposed to regulate the
copy number of mtDNA by limiting the rate of mtDNA
turnover (Ekstrand et al., 2004; Matsushima et al., 2004).
TFAM associates with the full mtDNA and packages it
into protein-DNA aggregates (nucleoids; Alam et al.,
2003; Garrido et al., 2003), thus protecting the mtDNA
from degradation. This depends on TFAM’s tight but
nonspecific DNA binding and high abundance in mitochondria (Fisher et al., 1992; Ekstrand et al., 2004). The
other regulatory factors found in animals, TWINKLE
helicase and mtSSB, facilitate mtDNA replication by helix
destabilization (Korhonen et al., 2003, 2004; Tyynismaa
et al., 2004), a different mechanism from that of TFAM.
The mechanism by which AtWHY2 affects mtDNA
copy number is not yet clear. However, as a nonenzyme
DNA-binding factor without sequence specificity
(Maréchal et al., 2008; Cappadocia et al., 2010), AtWHY2
may affect mtDNA copy number in a way similar to
TFAM, by impeding mtDNA degradation by pollen
mitochondrial DNases such as DPD1 (Matsushima et al.,
2011; Tang et al., 2012). This agrees with a previous
study showing that AtWHY2 protects DNA against
nuclease digestion in vitro (Cappadocia et al., 2010).
Our results showing the appearance of nucleoid-like
structures containing mtDNA (Fig. 3) and that knockout of the DNA-binding ability of AtWHY2 results in
the loss of its effect on mtDNA copy numbers (Fig. 4)
support this idea.
Another important observation indicating that
AtWHY2 may act by protecting mtDNA from degradation, rather than by promoting mtDNA replication, is
that AtWHY2 expression results in an equivalent
amount of mtDNA to that detected in BCPs. Our results
show that, relative to the mature pollen of Col-0, which
contain approximately 10 copies of mtDNA per pollen
cell, the LWHY2 transgenic lines contain approximately
120 to 130 copies per pollen grain. This is equivalent to
that detected in the BCPs (approximately 130 copies;
Fig. 1). The elevation of mtDNA by AtWHY2 may occur
through a mechanism maintaining the mtDNA copy
number during pollen development. Given that the
degradation of mtDNA in pollen vegetative cells starts
at the early BCP stage (Nagata et al., 1999) and that the
Lat52 promoter drives gene expression in pollen vegetative cells beginning at the microspore stage (Twell
et al., 1989, 1990; Eady et al., 1995), earlier accumulation
of AtWHY2 in the vegetative cell may prevent the
degradation of mtDNA in the transgenic lines. To solidify this inference, we expressed AtWHY2:GFP in the
generative cell of Arabidopsis using the Histone Three
Related10 promoter, which drives gene expression beginning at the late BCP stage (Ingouff et al., 2007). Our
results showed the correct import of AtWHY2 into the
generative mitochondria but undetectable signals of
mtDNA in the sperm cells of TCP (Supplemental Fig.
S10). This observation indicates that AtWHY2 fails to
maintain mtDNA levels when imported to mitochondria after the initiation of mtDNA degradation, agreeing with the idea that AtWHY2 affects the copy number
of mtDNA by protecting the mtDNA from degradation.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
Arabidopsis (Arabidopsis thaliana) ecotype Col-0 was used as wild-type plant
material and for transgenic screening. Seeds were surface sterilized and sown
on 0.7% (w/v) agarose plates containing one-half-strength Murashige-Skoog
medium (Sigma-Aldrich) supplemented with 3% (w/v) Suc. For transgenic
screening, the medium above was supplemented with kanamycin to 30 mg L21.
Arabidopsis seedlings were grown at 23°C under 16 h of light/8 h of darkness
and transferred to sterilized nutrient soil 14 d after imbibition.
Constructs and Plant Transformation
All plasmids constructed in this study were modified from pGreen-Lat52:
Dips-GFP (Matsushima et al., 2008). A 717-bp region of AtWHY2 was amplified
from wild-type leaf complementary DNA (cDNA) using the primers WHY2SalF and WHY2-PstR. The product was cloned into pGreen-Lat52:Dips-GFP to
replace the Dips coding sequence and generate pGreen-Lat52:AtWHY2-GFP
(LWHY2-GFP), which was then verified by sequencing. AtWHY2 amplified by
primers WHY2-SalF and WHY2-XbaR was cloned into pGreen-Lat52:Dips-GFP
to replace Dips-GFP and generate pGreen-Lat52:AtWHY2 (LWHY2), which
was then verified by sequencing. Subsequently, GFP was amplified with the
primers GFP-SalF and GFP-XbaR and cloned into pGreen-Lat52:Dips-GFP to
replace Dips-GFP and create pGreen-Lat52:GFP, which was used as a control. To
create pGreen-WHY2pro:GUS and pGreen-WHY2pro:WHY2-GFP (WWHY2-GFP),
which were used to control the expression of GUS and WHY2-GFP, a 2,010-bp
fragment of the AtWHY2 promoter was amplified from wild-type genomic
DNA using the primers proWHY2-KpnF and proWHY2-SalR and cloned into
the pGreen vector.
The generative cell-specific promoter HTR10 was amplified using the
primers proHTR10-KpnF and proHTR10-SalI and cloned to pGreen-Lat52:
AtWHY2-GFP to replace Lat52 and create pGreen-HTR10:AtWHY2-GFP. To
generate AtWHY2M62-67, two separate mutant fragments were amplified from
pGreen-Lat52:AtWHY2 using the primers WHY2-SalI and WHY2M62-67-R and the
primers WHY2M62-67-F and WHY2-PstI. Then, the target AtWHY2M62-67 was
amplified using the previous two products as templates and the primers
WHY2-SalF and WHY2-PstR via overlapping PCR. The resulting product was
cloned into pGreen-Lat52:AtWHY2-GFP to replace the native AtWHY2 and
generate pGreen-Lat52:AtWHY2M62-67-GFP (LWHY2M62-67-GFP). All of the
primers used are listed in Supplemental Table S1. Plants were stably transformed with Agrobacterium tumefaciens (GV3101) via the floral dip method as
described previously (Zhang et al., 2006).
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WHIRLY2 Affects mtDNA Levels in Pollen
Determination of mtDNA Copy Number per Pollen Grain
A competitive PCR method that quantifies the copy number of the mtDNA
in single plant cells, including single pollen grains (Wang et al., 2010), was
used in this study to measure mtDNA copy number per pollen grain. All
procedures of the quantification, including the pretreatment of pollen to release mtDNA from the cell, the preparation of competitor template, and the
settings for PCR, were performed exactly as described by Wang et al. (2010).
Briefly, five pollen grains for each quantification were gently crushed and
pretreated by rapid freezing, thawing, and proteinase K digestion. The
resulting mixture was divided equally into five PCR tubes. The mtDNA copy
number per pollen grain was obtained after two rounds of competitive PCR
with mitochondrial matR as target. The efficiency coefficient for target/
competitor amplification (0.8 for AtMatR6; Wang et al., 2010) was used in
the elaboration of the value.
RNA and DNA Extraction and RT-Quantitative PCR
Total RNA was extracted from different Arabidopsis tissues using TRIzol
reagent (Invitrogen). The concentrations of total RNA were quantified spectrophotometrically by the Nanodrop ND-2000c system (Thermo Fisher Scientific). A total of 5 mg of RNA was reverse transcribed using the PrimeScript
First-Strand cDNA Synthesis Kit (TaKaRa) according to the manufacturer’s
instructions. The quantitative PCR amplification containing 5 mL of 23SYBR
premix ExTaq (TaKaRa), 0.2 mM of each primer, and 80 ng of cDNA in a 10-mL
mixture was performed on the LightCycler II Real-Time PCR System (Roche).
The primers used for RT-quantitative PCR are listed in Supplemental Table S3.
The PCR conditions consisted of an initial denaturation at 95°C for 10 min
followed by 45 cycles of 95°C for 15 s and 60°C for 45 s. Data were analyzed
using LightCycler3 software (Roche). The expression levels of target transcripts were normalized to the level of the internal standard UBIQUITIN5
using the DCT method (22DCT = relative amount of transcripts; DCT = CTTarget –
CTInternal standard).
The quantification of mtDNA levels in leaves used quantitative PCR as
described (Preuten et al., 2010). Total DNA was extracted from fresh leaves
using a cetyl-trimethyl-ammonium bromide protocol (Murray and Thompson,
1980). The mtDNA levels in total DNA were measured by amplification of
mitochondrial matR, which was measured by quantitative real-time PCR.
Quantitative real-time PCR was performed in the LightCycler II Real-Time PCR
System (Roche) using the SYBR premix ExTaq kit (TaKaRa). Real-time PCR was
performed in a 10-mL reaction mixture containing 5 mL of 23SYBR premix
ExTaq, 0.2 mM of each primer, and 40 ng of genomic DNA. The primers used for
quantitative analysis of mtDNA are listed in Supplemental Table S1. The following amplification was used: 95°C for 10 min and 45 cycles of 95°C for 20 s
and 60°C for 45 s. Data were analyzed using LightCycler3 software (Roche). All
mtDNA levels were normalized to the internal standard nucleus-encoded
single-copy gene phage-type RNA polymerase as described previously (Preuten
et al., 2010) using the DCT method (see above).
Polyclonal Antibody Production and Immunoblot Analysis
To produce polyclonal antibodies against WHY2, ATP1, ATP3, apocytochrome b, and succinate dehydrogenase1-1, the synthetic polypeptides of
these proteins were used to immunize rabbits, and the resulting polyclonal
antibodies were purified from antisera using antigen-antibody affinity chromatography (Abgent). The sequences of polypeptides used for immunization
are listed in Supplemental Table S2.
The mature pollen grains from newly opened flowers at stage 13, according
to flower development in Arabidopsis (Smyth et al., 1990), were collected and
ground in ice-cold extraction buffer (100 mM Tris-HCl, 15 mM EDTA, and
200 mM NaCl, pH 7.5, supplemented with 0.5% [v/v] Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, and 0.1% [v/v] b-mercaptoethanol), and then
the homogenized slurry was centrifuged at 10,000g and 4°C for 15 min. The
supernatant was mixed with SDS-PAGE loading buffer, boiled, and separated
by gel electrophoresis. Then, the gel was transferred to a polyvinylidene
difluoride membrane (Millipore) in transfer buffer (25 mM Tris, 190 mM Gly,
and 20% [v/v] methanol) for 1.5 h at 18 V. The polyvinylidene difluoride
membrane was blocked for 30 min at room temperature in 13 Tris-buffered
saline (TBS; 0.2 M Tris-HCl, pH 7.5, and 5 M NaCl) containing 5% (w/v) skim
milk and probed with the corresponding polyclonal antibodies (1:1,000 in
13 TBS containing 0.1% [v/v] Tween 20) for 1 h at room temperature. After
three washes in 13 TBS containing 0.1% (v/v) Tween 20, the membranes
were incubated with the secondary antibody, donkey anti-rabbit IgGIRDye
(dilution, 1:10,000; ODYSSEY; Li-Cor), for 1 h at room temperature. The immunoblots were detected with the ODYSSEY (Li-Cor) according to the manufacturer’s instructions.
Fluorescence Microscopy
For direct observation of mtDNA signals from the pollen vegetative cell,
fresh pollen grains placed onto a glass slide were immersed in TAN buffer
(Nemoto et al., 1988) supplemented with 3% (v/v) glutaraldehyde and 10 mg
mL21 DAPI (Invitrogen). After covering with a coverslip, the samples were
examined with an epifluorescence microscope (DMI 6000B; Leica). For mature
pollen grains, to remove the interference of the pollen walls, the coverslip was
pressed down, bursting the pollen grains and releasing the vegetative cytoplasm, thus allowing clear detection of mtDNA signals. Photomicrographs of
squashed pollen grains were captured with a CCD camera (DFC480; Leica)
attached to the microscope.
Electron Microscopy
For transmission electron microscopy, fresh pollen grains from open
flowers were fixed with 5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate
buffer (pH 7.4) at room temperature for 4 h and then postfixed overnight in
1% (w/v) osmium tetroxide at 4°C. The samples were dehydrated through a
series of alcohol solutions and embedded in Spurr’s resin. Ultrathin sections
were incubated with 1% (w/v) uranyl acetate and lead citrate and examined
with an electron microscope (TECNAI G2 20; FEI) at an accelerating voltage
of 120 kV.
Immunoelectron microscopy for detecting mtDNA and AtWHY2 was performed according to the standard method (Johnson and Rosenbaum, 1990).
Briefly, fixation of pollen grains was performed as described above, except that
the postfixation step was omitted. The fixed samples were embedded in LR
White resin (Sigma-Aldrich). For localization of mtDNA, the sections were incubated first with a mouse anti-DNA antibody (1:20 dilution; Sigma-Aldrich)
and then with goat anti-mouse IgM conjugated to 5-nm colloidal gold (1:60
dilution; British BioCell International). As a negative control, sections were
pretreated with DNase and processed as above. For the localization of
AtWHY2, the sections were incubated first with a rabbit anti-AtWHY2 antibody prepared as described above (1:50 dilution) and then with goat anti-rabbit
IgG conjugated to 10-nm colloidal gold (1:60 dilution; British BioCell International). The colocalization of mtDNA and AtWHY2 was performed with sections treated with the above procedures in series. All sections were examined
with an electron microscope (TECNAI G2 20; FEI) at the accelerating voltage of
120 kV after a brief staining with 1% (w/v) uranyl acetate.
Phenotypic Analyses of Pollen and Pollen Tubes
For in vitro germination, mature pollen grains from newly opened flowers
were spread on culture plates prepoured with germination medium (1.5% [w/v]
agar, 0.01% [w/v] H3BO3, 5 mM KCl, 5 mM CaCl2, 1 mM MgSO4, and 18% [w/v]
Suc, pH 7.5; Boavida and McCormick, 2007). The plates were sealed with
Parafilm and incubated at 23°C. To assay in vivo pollen tube growth, wild-type
pistils were pollinated with wild-type and LWHY2-1 pollen. After specific periods of tube growth, the pistils were fixed in acetic acid:ethanol (1:3) solution,
cleared in 8 M NaOH, and stained with Aniline Blue as described previously
(Mori et al., 2006). The images of germinated pollen and pollen tubes stained by
Aniline Blue in the pistils were taken on a microscope (DMI 6000B; Leica)
equipped with Leica Application Suite software, and the length of pollen tubes
was measured with ImageJ software (National Institutes of Health; http://rsb.
info.nih.gov/ij).
The viability of mature pollen grains was investigated after propidium iodide
(50 mg mL21; Sigma-Aldrich) staining for 3 min, and pollen was examined with
a fluorescence microscope (DMI 6000B; Leica; Hashida et al., 2013). The mature
pollen grains were preprocessed by storage for various periods (4, 12, 24, 36, 48,
60, and 72 h) in dry conditions at 23°C.
The ROS level of mature pollen grains was indicated by staining with
29,79-dichlorodihydrofluorescein diacetate (10 mM; Sigma-Aldrich) in 13 PBS
(pH 7.4) for 5 min using a fluorescence microscope (DMI 6000B; Leica) with
absorption and emission wavelengths at 488 and 530 nm, respectively
(Matsushima et al., 2008). Image capture and quantification of the pollen
fluorescence intensity were performed by the Leica software. Each detection
was repeated three times.
Plant Physiol. Vol. 169, 2015
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
Cai et al.
ATP, ADP, NAD+, and NADH Measurements
To assay ATP and ADP concentrations in mature pollen, pollen from four
newly opened flowers was harvested and ground in 200 mL of distilled,
deionized water, the mixture was centrifuged at 10,000g at 4°C for 5 min, and
the supernatant was divided equally into four microfuge tubes. The ATP levels
were measured by the luciferin-luciferase method according to the instructions
of the ENLITEN ATP Assay System (Promega). The ADP levels were tested
using the ADP-Glo Kinase Assay Kit (Promega) for converting ADP to ATP,
and total ATP was quantified by luminescence in the Multifunctional Microplate Reader (FlexStation3; Molecular Devices).
For the measurement of NAD+ and NADH, mature pollen from eight newly
opened flowers was collected and ground in 400 mL of NAD+/NADH Extraction buffer using the NAD+/NADH Quantification Kit (Sigma-Aldrich),
and then the homogenized slurry was centrifuged at 10,000g at 4°C for 3 min
and the supernatant was divided equally into eight microfuge tubes. The
amounts of NAD+ and NADH were measured for the A450 using the Multifunctional Microplate Reader (FlexStation3; Molecular Devices) according to
the manufacturer’s instructions.
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession numbers: AtWHY2, At1g71260; AtHTR10, At1g19890; mitochondrial maturase of
Arabidopsis, Y08501.
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Pollen mtDNA decreases to low levels during
pollen development.
Supplemental Figure S2. Quantification of mtDNA per BCP and mature
pollen grain of Col-0 using competitive PCR.
Supplemental Figure S3. Pattern of AtWHY2 expression in Col-0 Arabidopsis.
Supplemental Figure S4. Pollen mtDNA levels in different transgenic lines.
Supplemental Figure S5. Plant and flower phenotypes of LWHY2-1.
Supplemental Figure S6. Characteristics of mitochondria from Col-0 and
transgenic lines.
Supplemental Figure S7. Proteins crucial for the ordinary pathway of
mitochondrial respiration in pollen do not differ significantly between
Col-0 and LWHY2-1.
Supplemental Figure S8. Mitochondrial transcription was significantly enhanced in the pollen of LWHY2-1 plants.
Supplemental Figure S9. Overexpression of AtWHY2 driven by the 35S
promoter did not substantially affect the copy number of mtDNA in
somatic cells.
Supplemental Figure S10. AtWHY2 localization in the mitochondria of generative cells when expressed under the control of the AtHTR10 promoter.
Supplemental Table S1. Primers used in this study.
Supplemental Table S2. Sequences of polypeptides used for polyclonal
antibody production.
ACKNOWLEDGMENTS
We thank Wataru Sakamoto for generously providing the transgenic
Arabidopsis seeds (Lat52:Dips-GFP and Lat52:Dips-RFP) and Dr. Ying-Chun
Hu for technical assistance in electron microscopy.
Received March 24, 2015; accepted July 15, 2015; published July 20, 2015.
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