MALE GAMETOPHYTE DEFECTIVE 1, Encoding the FAd
Subunit of Mitochondrial F1F0-ATP Synthase, is Essential
for Pollen Formation in Arabidopsis thaliana
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193,
PR China
∗Corresponding author: E-mail, [email protected]; Fax, +86-10-62734839
(Received April 12, 2010; Accepted April 26, 2010)
In flowering plants, pollen formation is a complex process
and strictly controlled by genetic factors. Although thousands
of genes have been identified to be highly or specifically
expressed in pollen grains, little is known about the functions
and regulatory mechanisms of the genes in pollen formation.
Here we report the characterization of a novel gene,
MALE GAMETOPHYTE DEFECTIVE 1 (MGP1), that is essential
for pollen formation in Arabidopsis thaliana. MGP1 encodes
the FAd subunit of mitochondrial F1F0-ATP synthase in
Arabidopsis. It was highly expressed in pollen grains at
the later developmental stage. Mutation in MGP1 led to
destruction of the mitochondria in pollen grains at the
dehydration stage and subsequently death of the pollen
grains. These results suggested that MGP1 plays important
roles in pollen formation, possibly by regulating the activity
of mitochondrial F1F0-ATP synthase in Arabidopsis pollen
grains.
Keywords: Arabidopsis • ATP synthase • Gametogenesis •
MGP1 • Mitochondria • Pollen.
Abbreviations: CaMV, cauliflower mosaic virus; DAPI,
4′,6-diamidino-2-phenylindole; Ds, dissociation; GFP, green
fluorescent protein; GUS, β-glucuronidase; IF1 (INH1),
F1-ATPase inhibitor; MS, Murashige and Skoog; PI, propidium
iodide; RT–PCR, reverse transcription–PCR; SEM, scanning
electron microscopy; STF1, F1-ATPase inhibitor stabilizer 1;
TEM, transmission electron microscopy; TAIL-PCR, thermal
asymmetric interlaced PCR.
Introduction
In flowering plants, the male gametophyte or pollen grain is
a multicelled life unit. It is produced in the male sexual organ,
the anther. The process comprises several important steps.
First, the reproductive cells in an anther primordium divide and
differentiate into microspore mother cells. Then, the microspore
mother cells undergo meiosis to give rise to haploid microspores.
In the angiosperms including Arabidopsis thaliana, before
anthesis, the individual microspore further undergoes two
rounds of mitosis to form a three-celled pollen grain that
consists of a larger vegetative cell and two sperm cells. The
three-celled pollen grain further undergoes dehydration to
form a mature pollen grain (McCormick 2004). The dehydration of pollen grains is crucial for maximum maintenance
of pollen viability and then the pollen grains can tolerate various environmental stresses after they are released from the
anther (Twell 2002, Swanson et al. 2004). A defect in any of
the steps above will interrupt pollen formation or affect male
gametophytic function (Scott et al. 1991, McCormick 1993,
McCormick 2004).
Pollen formation is a highly energy-consuming process (Lee
and Warmke 1979). As in other non-photosynthetic tissues
with undifferentiated plastids and amyloplasts, the energy is
supplied by mitochondria exclusively in the developing pollen
grains (De Paepe et al. 1993). Dysfunction of mitochondria in
pollen grains will drastically affect pollen development (Hanson
and Bentolila 2004). In mitochondria, the energy-bearing compound, ATP, is synthesized by F1F0-ATP synthase (Pedersen
and Carafoli 1987). Furthermore, as well as ATP synthesis, the
mitochondrial F1F0-ATP synthase also engages in ATP hydrolysis, depending on the cell’s physiological pH (Cabezón et al.
2000, Cabezón et al. 2002). The purpose of ATP hydrolysis is to
restore the impaired mitochondrial membrane potential.
In yeast, the ATP hydrolysis activity of the mitochondrial
F1F0-ATP synthase is regulated by F1-ATPase inhibitor (INH1/
IF1) and F1-ATPase inhibitor stabiliser 1 (STF1) proteins. They
inhibit the ATP hydrolysis activity of mitochondrial F1F0-ATP
synthase when the impaired membrane potential is rectified
and ATP hydrolysis is not needed to ensure an adequate supply
of ATP for cell physiological activity, which plays important
roles in cell development (Hashimoto et al. 1987, Ichikawa et al.
1990, Cabezón et al. 2001, Venard et al. 2003). To date, however,
little is known about the roles of the related components of
mitochondrial F1F0-ATP synthase in pollen grains of higher
plants although it is important for sexual plant reproduction.
Regular Paper
Wen-Qing Li, Xue-Qin Zhang, Chuan Xia, Yi Deng and De Ye∗
Plant Cell Physiol. 51(6): 923–935 (2010) doi:10.1093/pcp/pcq066, available online at www.pcp.oxfordjournals.org
© The Author 2010. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Plant Cell Physiol. 51(6): 923–935 (2010) doi:10.1093/pcp/pcq066 © The Author 2010.
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W.-Q. Li et al.
Here we report the isolation and characterization of
a Ds-inserted mutation in a nuclear gene, MALE GAMETOPHYTE
DEFECTIVE 1 (MGP1), that encodes the FAd subunit of mitochondrial F1F0-ATP synthase in Arabidopsis (Heazlewood et al.
2003). MGP1 was highly expressed in pollen grains at the later
developmental stage. The mgp1 mutation led to destruction
of mitochondria and subsequently death of pollen grains at
the dehydration stage.
Results
A
C
B
D
I
100
Isolation and genetic analysis of the mgp1 mutant
90
E
G
F
H
Viable pollen grains (%)
mgp1 was identified by its distorted segregation ratio of
kanamycin resistance in a screen for the gametophyte-defective
mutants from the gene- and enhancer-trap Ds insertion lines
in Arabidopsis ecotype Landsberg erecta (Ler) (Sundaresan
et al. 1995). The progeny seedlings from the self-pollinated
mgp1 heterozygous plants (mgp1/+) segregated in a ratio of
0.70 (1,333) kanamycin-resistant (KanR) to 1 (1,917) kanamycin-sensitive (KanS). No mgp1 homozygous plants (mgp1/mgp1)
were obtained in the progeny (n = 3,250) from the self-pollinated mgp1/+ plants. When the mgp1/+ plants were used as
the male in crosses with wild-type plants, the resulting F1 progeny (n = 1,264) were all sensitive to kanamycin. When the
mgp1/+ plants were used as the female to cross with wild-type
plants, the F1 progeny segregated in a ratio of 0.48 (858) KanR to
1 (1,799) KanS, lower than the expected ratio of 1 : 1. These
results showed that the Ds insertion could be transmitted
through the female gametophyte, but not through the male
gametophyte. Therefore, the mgp1 mutation disrupted the
male gametophytic function completely and only partially
impaired the female gametophytic function.
80
70
60
50
40
30
20
10
0
WT
mgp1/+
Fig. 1 Morphological observation and viability assay of the pollen
grains from wild-type and mgp1/+ plants. (A, B) SEM images of pollen
grains from wild-type (A) and mgp1/+ (B) plants. Arrows indicate the
collapsed pollen grains. (C, D) Alexander-stained pollen grains from
wild-type (C) and mgp1/+ (D) plants. Arrows indicate the collapsed
pollen grains. (E, F) PI-stained pollen grains from wild-type (E) and
mgp1/+ (F) plants. (G, H) The bright field images of E and F. Arrows
indicate the unviable pollen grains that were stained by PI and had
normal morphology. (I) Viability statistics of pollen grains from
wild-type and mgp1/+ plants. Bar = 20 µm.
The mgp1 mutant is defective in pollen
formation
To study how the mgp1 mutation affected male gametophytic
function, we performed phenotypic characterization of mgp1/+
plants. First, we examined the morphological structure of the
pollen grains from mgp1/+ plants. Scanning electron microscopy (SEM) and light microscopy showed that 21.6% pollen
gains from mgp1/+ plants were collapsed at the later anther
development stage (Fig. 1B, D). The Alexander staining assay
(Alexander 1969) showed that these collapsed pollen grains
had lost their cytoplasm (Fig. 1D). Alexander staining can distinguish only those dead cells that have lost their cytoplasm
(Alexander, 1969). To investigate further whether there were
any physiologically dead pollen grains that still contained cytoplasm, we stained the mgp1 pollen grains with propidium iodide
(PI). PI can stain dead cells containing cytoplasm, but not living
cells (Huang et al. 1986). In this assay, 26.4% of the pollen grains
from mgp1/+ plants could be labeled by PI (Fig. 1F), suggesting
that these pollen grains had also lost their viability although
they contained cytoplasm. Taking these findings together,
the percentage of dead pollen grains from mgp1/+ plants was
924
48% (n = 837), very close to the expected 50%, indicating that
96% (48/50) of the mgp1 mutant pollen grains died before
maturation, much more than the 9.3% (n = 622) in the wild
type (Fig. 1I).
We next investigated the process of microsporogenesis
and microgametogenesis in mgp1/+ flowers by staining with
4′,6-diamidino-2-phenylindole (DAPI). The tetrad from mgp1/+
plants consisted of four identical microspores (Fig. 2A),
indicating that the microspore mother cells, heterozygous for
the mgp1 mutation, could undergo meiosis. All the individual
microspores could be released from the tetrads (Fig. 2B) and
underwent the first pollen mitotic division to give rise to a twocelled pollen grain that consisted of a vegetative cell and a generative cell (Fig. 2C). The generative cell in all two-celled pollen
grains could proceed to normal generative cell migration and
complete pollen mitosis II to form the three-celled pollen
grains (Fig. 2D). At the anther developmental stage 14, all
wild-type pollen grains had undergone dehydration (Fig. 2E, F).
In contrast, at the same stage, a large number of pollen grains
Plant Cell Physiol. 51(6): 923–935 (2010) doi:10.1093/pcp/pcq066 © The Author 2010.
MGP1 is essential for pollen formation
A
C
B
D
VN
E
G
F
H
GN
VN
SN
Fig. 2 Analysis of mgp1 pollen development. (A–D) The DAPI-stained pollen grains from mgp1/+ plants at the tetrad stage (A), the one-celled
stage (B), the two-celled stage (C) and the three-celled stage before dehydration (D). (E) The DAPI-stained mature pollen grains from wild-type
plants, which had undergone desiccation. (F) The bright field image of E. (G) The DAPI-stained mature pollen grains from mgp1/+ plants. (H) The
bright field image of G. The arrows in G indicate the pollen grains arrested at the three-celled stage, which did not undergo desiccation.
The arrowheads in G and H indicate the pollen grains that lose their viability completely and could not be stained by DAPI. VN, vegetative
nucleus; GN, generative nucleus; SN, sperm nuclei. Bar = 20 µm.
from mgp1/+ plants had not undergone dehydration. Some of
them were arrested at the initial stage of three-celled pollen
grains. The others collapsed and were devoid of cytoplasm
(Fig. 2G, H).
Genetic analysis indicated that mgp1 mutation also
affected the function of the female gametophyte (Table 1).
To investigate whether the mgp1 mutation impaired the development of the embryo sac, we first examined the ratio of
aborted ovules in mgp1/+ siliques (Supplementary Fig. S1A),
compared with that in wild-type siliques (Supplementary
Fig. S1B). A total of 11.7% (n = 3,808) of the ovules were aborted
in the mgp1/+ siliques, which was higher than the 4% (n = 875)
found in the wild type (Supplementary Fig. S1F). We then
studied ovule development in mgp1/+ siliques using confocal
laser scanning microscopy (Christensen et al. 1998) by comparison with wild-type ovules. In wild-type siliques, most of the
embryo sacs reached the FG7 stage, which contained one
secondary central cell, one egg cell and two synergid cells
Table 1 Genetic transmission of the mgp1 mutation
Cross
(female × male)
KanR
mgp1/+ × mgp1/+
KanS
KanR/
KanS
TE
(female)
TE
(male)
1,333
1,917
0.70
NA
NA
mgp1/+ × +/+
858
1,799
0.48
47.7%
NA
+/+ × mgp1/+
0
1,264
0
NA
0
KanR,
KanS,
kanamycin-resistant seedlings;
kanamycin-sensitive seedlings; NA, not
applicable; TE, transmission efficiency = (KanR/KanS) × 100%.
(Supplementary Fig. S1E). In the mgp1/+ siliques, roughly
6% (n = 235) of the embryo sacs could not proceed to the terminal developmental stage. They were arrested at the early
developmental stages, the FG1 stage (Supplementary Fig. S1D)
or FG2 stage. Therefore, we concluded that the mgp1 mutation
slightly affected the development of female gametophytes or
embryo sacs.
Plant Cell Physiol. 51(6): 923–935 (2010) doi:10.1093/pcp/pcq066 © The Author 2010.
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W.-Q. Li et al.
Identification of the MGP1 gene and phenotypic
complementation of the mgp1 mutant
To identify the MGP1 gene, thermal asymmetric interlaced
(TAIL)-PCR (Liu et al. 1995) was applied to obtain the flanking
sequence adjacent to the 3′ end of the Ds element in the
mgp1 genome. Sequence analysis of the flanking sequences
indicated that the Ds was inserted in the fourth intron of an
Arabidopsis gene, At2g21870 (Fig. 3A). The MGP1 cDNA was
cloned by reverse transcription–PCR (RT–PCR) using the
At2g21870-specific primers: 5′-GTCAACTCCACAGCGACTCG
ATTC-3′ and 5′-CAGCAGCAAGGGAACGATCTTC-3′. The
cloned At2g21870 cDNA was 1,097 bp in length and was nearly
identical to the annotated cDNA sequence (accession No.
NM_127756).
To confirm that Ds insertion into At2g21870 caused the
male gametophyte-defective phenotype of mgp1, a 4.9 kb
MGP1 genomic fragment containing the predicted promoter
region, coding region and 3′-untranslated region of the
At2g21870 gene was amplified by PCR using the primer pair,
5′-GATCAGTCAAGAGGTTGTGCTTG-3′ and 5′-GAGAAGGA
GCTTTGAGAGGAATC-3′, and subcloned into pCAMBIA1300
(CAMBIA, Canberra, Australia). After sequence verification,
the resulting complementation construct was introduced into
mgp1/+ plants by an Agrobacterium-mediated transformation
method (Bechtold and Pelletier 1998). In total, 37 independent
transgenic lines were obtained in a screen using hygromycin
and kanamycin double selection. In the T2 generation, 24 out
of the 37 seedling families showed a segregation ratio of
approximately 2 KanR to 1 KanS, compared with the original
segregation ratio of 0.70 KanR to 1 KanS (Supplementary
Table S1), indicating that the 4.9 kb fragment could complement the gametophyte-defective phenotype of the mgp1
mutant (Feldmann et al. 1997). Furthermore, the mgp1 homozygous plants (mgp1/mgp1) identified in the T3 generation were
fully fertile. All the pollen grains from these transgenic mgp1
homozygous plants (mgp1/mgp1; MGP1/MGP1) were morphologically normal and the pollen tubes could elongate normally
in vitro (Fig. 4B, D, G, J). The germination ratio of the pollen
grains from the complemented mgp1/mgp1 plants reached
82.3% (n = 320), which was close to that (84.7%, n = 255) of
wild-type pollen grains and much higher than that (41.5%,
n = 277) of the pollen grains from the untransformed mgp1
plants (Fig. 4K). These results demonstrated that the gametophyte-defective phenotype of mgp1 was caused by the Ds
insertion in At2g21870.
MGP1 encodes the FAd subunit of the mitochondrial
F1F0-ATP synthase complex in Arabidopsis
The MGP1 mRNA encoded the FAd subunit of the mitochondrial F1F0-ATP synthase complex in Arabidopsis, which consisted of 240 amino acids with an estimated pI of 6.59 and
a molecular mass of 27.6 kDa, and exhibited hydrophilic
characteristics (Supplementary Fig. S2). There were several
proteins with >50% amino acid sequence identity to MGP1
926
in higher plants. Specifically, MGP1 had 73% (177/240) identity
and 87% (210/240) similarity to XP_002279389 from Vitis
vinifera, 70% (169/240) identity and 85% (204/240) similarity
to XP_002310038 from Populus trichocarpa, 66% (119/179)
identity and 82% (149/179) similarity to CAA52349 from
Glycine max, and 65% (158/240) identity and 80% (192/240)
similarity to NP_001045790 from rice (Fig. 3B).
MGP1 is expressed ubiquitously in different tissues
RT–PCR analysis revealed that MGP1 was expressed ubiquitously in many different tissues including seedlings, roots, stems,
leaves, inflorescences and siliques (Fig. 5A). To investigate the
expression pattern of MGP1 in more detail, the promoter region
of MGP1 was fused to a β-glucuronidase (GUS) reporter gene
and introduced into wild-type plants. In the T2 generation,
GUS activity was detected in roots, stems, leaves, inflorescences,
siliques and developing pollen grains, consistent with the results
obtained from RT–PCR assay. During pollen development,
the GUS stain could not be detected in the pollen grains until
the initial stage of three-celled pollen grains (Fig. 5D–F) and
became strongest at the mature stage (Fig. 5G).
MGP1–GFP fusion protein is localized in
mitochondria
To investigate the subcellular localization of MGP1 protein,
a green fluorescent protein (GFP) reporter was fused to the
C-terminus of MGP1 and expressed in Arabidopsis wild-type
plants under the control of the MGP1 promoter or the cauliflower mosaic virus (CaMV) 35S promoter. In the transgenic
plants expressing the MGP1–GFP fusion protein under the control of the CaMV 35S promoter, the GFP signal was detected
strongly in the roots. In the transgenic plants expressing the
MGP1–GFP fusion protein under the control of the MGP1
promoter, the GFP signal could be detected in the mature
pollen grains. In both the mature pollen grains and root hair
cells of such transgenic plants, the fluorescence was restricted
to the subcellular structures with morphology typical of mitochondria (0.5–1 µm round or elliptical particles that were
distributed throughout the cytoplasm) (Fig. 6A, C). To verify
that the GFP signal observed was indeed localized in mitochondria, the roots of the transgenic plants expressing the MGP1–
GFP fusion protein under the control of the CaMV 35S promoter
were stained with a mitochondrial-specific dye for living cells,
Mito-Tracker Red CM-H2XRos (Haugland 1996). The GFP signal
and dye fluorescence signal were compared in the same cells.
As shown in Fig. 6E, the GFP signal (green) and dye fluorescence signal (red) were clearly overlapping in mitochondria.
These data showed that the MGP1–GFP fusion protein was
localized in mitochondria.
In addition, we also predicted the existence of signal peptides for mitochondrial targeting in the MGP1 sequence. In rat,
a core motif of ΦXXΦΦ has been identified as being crucial
for protein import into mitochondria. In the motif, Φ is any
hydrophobic/aromatic amino acid and X is any amino acid with
a long aliphatic side chain and often with a polar group at the
Plant Cell Physiol. 51(6): 923–935 (2010) doi:10.1093/pcp/pcq066 © The Author 2010.
MGP1 is essential for pollen formation
A
3’
mgp1
Ds
5’
9329814
9327275
9328623
ATG
TAA
100bp
B
MGP1
MGP1
MGP1
MGP1
MGP1
MGP1
Fig. 3 Molecular characterization of the MGP1 gene. (A) Schematic diagram of MGP1 structure, showing the Ds insertion site in the mgp1 mutant.
Black boxes indicate the coding sequences, lines indicate the intron and white boxes indicate the untranslated regions. The nucleotide numbers
are consistent with those of bacterial artificial chromosome (BAC) clone F7D8. (B) Sequence alignment of the MGP1 protein and the proteins
similar to MGP1 from different species. Identical amino acids are highlighted in black and similar amino acids are shaded in gray. MGP1,
Arabidopsis MGP1 protein; XP_002279389 Vitis, V. vinifera homolog; XP_0023100038 Populus, P. trichocarpa homolog; CAA52349 Glycine,
G. max homolog; NP_001045790 Oryza, rice homolog. The underlined characters indicate the predicted motif that is crucial for protein import
into mitochondria.
end (Abe et al. 2000, Muto et al. 2001). Previous results indicate
that the motif is also adapted to plant protein. For example,
in the FAd homolog of soybean, the motif (LSSRL) lies in the
N-terminal end of the pre-sequence (Lee and Whelan, 2004).
We also found a similar motif (YASRF) in MGP1 (Fig. 3B), in
which Y (tyrosine) and F (phenylalanine) were aromatic amino
acids, while A (alanine) and S (serine) were the amino acids
with an aliphatic side chain (Fig. 3B).
Plant Cell Physiol. 51(6): 923–935 (2010) doi:10.1093/pcp/pcq066 © The Author 2010.
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W.-Q. Li et al.
B
A
G
100
C
Viable pollen grains (%)
90
D
E
F
80
70
60
50
40
30
20
10
0
WT
H
Comp
J
100
Germination rates of pollen
grains (%)
K
I
mgp1/+
90
80
70
60
50
40
30
20
10
0
WT
mgp1/+
Comp
Fig. 4 Phenotypic complementation of mgp1 homozygous plants carrying an MGP1 transgene (Comp). (A–G) Viability assays of pollen grains.
(A, B) Alexander-stained pollen grains from the wild type (A) and Comp (B). (C, D) PI-stained pollen grains from wild type (C) and Comp (D)
plants. (E, F) The bright field images of C and D, respectively. (G) Viability statistics of pollen grains from wild-type (WT), mgp1/+ and Comp
plants. (H–J) Germination in vitro of pollen grains from wild-type (H), mgp1/+ (I) and Comp (J) plants. (K) Germination rate statistics of pollen
grains from wild-type (WT), mgp1/+ and Comp plants. Bar = 20 µm.
The mgp1 mutation might alter the ATP
hydrolysis activity and lead to the destruction
of mitochondria in pollen grains
To investigate the ATP hydrolysis activity of mitochondrial
F1F0-ATP synthase, we labeled the Mg2+-ATPase in pollen grains
at the dehydration stage. Hydrolysis of ATP leads to accumulation of Pi that can react with the ectogenous Pb2+ to form
Pb3(PO4)2. The Pb3(PO4)2 can be viewed as black particles under
transmission electron microscopy (TEM) (Xie et al. 2006).
928
Therefore, whether the mgp1 mutation affected the ATP hydrolysis activity of mitochondrial F1F0-ATP synthase could be
determined by comparing the density of black particles in
the mitochondria of mgp1 pollen grains with those in the
mitochondria of wild-type pollen grains.
In all of the wild-type pollen grains (n = 40), as shown in
Fig. 7A, the black particles were found mainly in the cytoplasm
and few or no black particles were found in mitochondria.
In contrast, in 45.4% of mgp1 pollen grains (n = 40) from the
Plant Cell Physiol. 51(6): 923–935 (2010) doi:10.1093/pcp/pcq066 © The Author 2010.
MGP1 is essential for pollen formation
A
ed
Se
lin
gs
o
Ro
ts
em
St
s
a
Le
ve
s
In
fl o
re
c
en
sc
es
li
Si
qu
es
MGP1
TUBULIN8
B
D
C
E
G
F
A
C
Fig. 5 Expression pattern of MGP1. (A) Expression pattern
of MGP1 revealed by RT–PCR. (B–G) The GUS stain in
pMGP1::GUS transgenic plants. (B) A transgenic seedling.
(C) A transgenic inflorescence. (D) A transgenic anther
with pollen grains at the one-celled stage. (E) A transgenic
anther with pollen grains at the two-celled stage.
(F) A transgenic anther with the pollen grains at the
three-celled stage. (G) Mature transgenic pollen grains.
Bar = 0.5 cm in B, C; 400 µm in D–F; and 20 µm in G.
B
D
E
F
Fig. 6 MGP1 is localized in mitochondria. (A) MGP1–GFP fusion protein in pollen grains. (B) Phase-contrast image of A. (C) MGP1–GFP fusion
protein in root hair. (D) The mitochondria in the root hair, which were stained with the mitochondria-specific dye Mito-Tracker Red (Haugland
1996). (E) GFP and Mito-Tracker Red fluorescence signals overlap in the root hair. (F) Phase-contrast image of C–E. Bar = 10 µm in A, B; 5 µm in C–F.
Plant Cell Physiol. 51(6): 923–935 (2010) doi:10.1093/pcp/pcq066 © The Author 2010.
929
W.-Q. Li et al.
A
B
C
D
E
by TEM. In all of the wild-type pollen grains (n = 40), as shown
in Fig. 8A, the mitochondria had a great number of cristae
that were arranged intensely and in an orderly manner
(parallel to the long axis of mitochondria). In contrast, the
mitochondria in 26.7% of pollen grains (n = 40) from the
mgp1/+ plants had few cristae that were scattered in a disorderly manner in the cavum enclosed by the mitochondrial
outer membrane (Fig. 8B, E). In addition, 20% of the pollen
grains (n = 40) from the mgp1/+ plants had lost their cytoplasm
and mitochondria. This finding implied that mgp1 mutation
might impair the structure of the mitochondria of the pollen
grains at the late developmental stage.
To investigate whether MGP1 was related to the alteration
of the ATP hydrolysis activity and mitochondrial structure in
the mgp1 pollen grains, we also examined the ATP hydrolysis
activity and mitochondrial structure in the complemented
mgp1 pollen grains. The results showed that all the pollen
grains (n = 40) at the dehydration stage from the complemented mgp1/mgp1 plants had the same stained pattern
of mitochondrial ATP hydrolysis activity as wild-type pollen
grains (Fig. 7B). The mitochondria in all the complemented
mgp1/mpg1 pollen grains (n = 40) at stage 14 also appeared
morphologically as normal as the mitochondria in wild-type
pollen grains (Fig. 8C, F). These results further demonstrated
that MGP1 could complement the gametophyte-defective
phenotype of the mgp1 mutant.
Pollen grains with mitochondria
stained by Pb 2+ (%)
100
90
80
70
60
50
40
30
20
10
0
WT
mgp1/+
Comp
Fig. 7 Assays for ATP hydrolysis activity of mitochondrial F1F0-ATP
synthase in pollen grains at the dehydration stage. (A, B) The pollen
grains from the wild-type (A) and Comp (B) anthers, showing that
a number of black particles (Mg2+-ATPase) were found mainly in the
cytoplasm (arrowheads) and fewer or no black particles could be
found in mitochondria (arrows). (C) A pollen grain from the mgp1/+
anther, showing that the black particles aggregated in mitochondria
at a much higher density (arrows) compared with those in the
wild-type (A) and Comp (B) pollen grains. (D) The mitochondria in
the pollen grains from mgp1/+ anthers, showing the impact on the
mitochondrial structure. The arrowhead indicates the remaining
normal structure of the mitochondria in which ATP hydrolysis
occurred. (E) Statistics of abnormal pollen grains with the mitochondria
stained by Pb2+ in wild-type (WT), mgp1/+ and Comp plants.
Bar = 500 nm in A–C; 200 nm in D.
mgp1/+ plant, the black particles aggregated in mitochondria
at a much higher density, in comparison with those in the cytoplasm (Fig. 7C). This finding indicated that the mgp1 mutation
could enhance the ATP hydrolysis activity in the mitochondria
of mgp1 mutant pollen grains.
To examine whether the mgp1 mutation affected the mitochondrial structure, we compared the mitochondrial structure
in mgp1 pollen grains with that in wild-type pollen grains
at the anther developmental stage 14 (Sanders et al. 1999)
930
Discussion
MGP1 is a novel gene that is involved in pollen formation.
It encodes the FAd subunit of mitochondrial F1F0-ATP synthase
in plants (Heazlewood et al. 2003). Mutation in MGP1 caused
lethality of pollen grains at the late developmental stage in
Arabidopsis. Therefore, MGP1 could be a mitochondrial ATP
synthase essential for pollen formation.
The genetic analysis showed that the mgp1 male gametophytes could not be transmitted to the next generation, but
most of the female gametophytes were not impaired, indicating that the mutation mainly affected the male gametophytic
function, but had much less impact on the female gametophytic function. The phenotypic characterization showed that
the mgp1 pollen grains could develop normally up to the
three-celled stage (Fig. 2A–D). Thus, it was unlikely that
the mutation impaired pollen mitosis. The most significant
defect occurred at the dehydration stage before maturation
(Fig. 2G, H), indicating that MGP1 was important for pollen
dehydration at the late pollen developmental stage.
Dehydration of the developing pollen grains starts at the
late anther developmental stage 12 to form a mature pollen
grain before anthesis (McCormick 2004). The dehydration is
required for maximum maintenance of pollen viability and
then the pollen grains can tolerate the various environmental
stresses after they are released from the anther (Twell 2002,
Swanson et al. 2004). Dehydration is similar to the process of
physiological drought stress, which could lead to oxygen
Plant Cell Physiol. 51(6): 923–935 (2010) doi:10.1093/pcp/pcq066 © The Author 2010.
MGP1 is essential for pollen formation
mgp1
WT
Comp
A
B
C
D
E
F
Pollen grains
with abnormal structures (%)
G
100
90
80
Pollen grains without mitochondria
Pollen grains with abnormal mitochondria
70
60
50
40
30
20
10
0
mgp1/+
WT
Comp
Fig. 8 Morphological observation of mitochondria in wild-type and mgp1 mutant pollen grains at the anther developmental stage 14. (A, D) The
structures of the mitochondria in a wild-type pollen grain. (B, E) The structures of the mitochondria in an mgp1 pollen grain. (C, F) The structures
of the mitochondria in a pollen grain from an mgp1 homozygous plants carrying an MGP1 transgene (Comp). (G) Statistics of pollen grains with
abnormal mitochondria and without mitochondria in wild-type (WT), mgp1/+ and Comp plants. Bar = 1 µm in A, C; 200 nm in B and D–F.
deprivation (Kowaltowski and Vercesi 1999, Hsu et al. 2007,
Pastore et al. 2007). Therefore, the dehydrating pollen grains
may need a mechanism for responding to the temperate stress
to ensure that dehydration can proceed normally. However,
the underlying mechanism still remains unclear.
Pollen dehydration is a highly energy-consuming process.
Recently, it has been reported that the mutation in the energyrelated gene succinate dehydrogenase 1 (sdh1) disrupted male
gametophyte development (León et al. 2007). The development of the sdh1 mutant male gametophytes was interrupted
at the one-celled stage (León et al. 2007), which died much
earlier than the mgp1 male gametophytes. Furthermore,
SDH1 encodes a flavoprotein (a subunit of mitochondrial
complex II) that acts as a component of the electron transport
chain and is directly required for oxidative phosphorylation
(León et al. 2007). Therefore, SDH1 is directly involved in ATP
biosynthesis. The mutation in SDH1 disrupts ATP synthesis,
resulting in the death of pollen grains at the early development
stage due to ATP deficiency. MGP1 encodes the FAd subunit of
mitochondrial F1F0-ATP synthase. It has been proposed that
the FAd in plants may act like the F1 inhibitor stabilizers STF1/
STF2 in yeast (Heazlewood et al. 2003). Therefore, MGP1 is
unlikely to act in the same physiological process as SDH1. It may
be related to inhibition of ATP hydrolysis instead.
ATP hydrolysis is another function of mitochondrial
F1F0-ATP synthase besides ATP synthesis. As regards a cell in
normal physiological conditions, ATP synthesis is carried out
by coupling with energy release from driving back of an electrochemical proton gradient across the inner membrane into
the matrix through the membrane domain of the F1F0-ATP
synthase (Cabezón et al. 2002). When a cell is deprived of
oxygen (subjected to stresses), its electrochemical gradient is
destroyed, the mitochondrial membrane potential is lost and
the enzyme is switched from ATP synthesis to ATP hydrolysis
Plant Cell Physiol. 51(6): 923–935 (2010) doi:10.1093/pcp/pcq066 © The Author 2010.
931
W.-Q. Li et al.
(Cabezón et al. 2002). By ATP hydrolysis, the protons in the
matrix can be transferred to the outer edge of the membrane
and thus the membrane potential is restored (Crompton
et al. 1999). When the impaired membrane potential is rectified, ATP hydrolysis needs to be inhibited and then the function of the enzyme is switched to ATP synthesis. In yeast,
STF1 and IF1 (INH1) function as inhibitors of ATP hydrolysis
(Ichikawa et al. 1990, Hashimoto et al. 1990). So far, however,
there is no direct biochemical evidence to demonstrate that
FAd or other subunits from the complex could inhibit ATP
hydrolysis in plants. The inhibitory mechanism of ATP hydrolysis in plants still remains unclear. Therefore, the inhibitory function of the FAd encoded by MGP1 also remains unclear.
Nevertheless, the characterization of the mgp1 mutant in
this study at least provided a new insight into the biological
function of FAd in plant development.
The causes of the death of mgp1 mutant male gametophytes
at the dehydration stage were unknown. One possible explanation is that the FAd encoded by MGP1 may be involved in the
regulation of the activity of F1F0-ATP synthase during pollen
dehydration (Heazlewood et al. 2003). Dehydration at the later
developmental stage of pollen grains (drought stress) may lead
to the loss of mitochondrial membrane electronic potential.
To restore the impaired membrane electronic potential, the
function of mitochondrial F1F0-ATP synthase could be shifted
from ATP synthesis to ATP hydrolysis. When the membrane
electronic potential is rectified, the FAd subunit may be involved
in inhibiting ATP hydrolysis. In the mgp1 mutant pollen grains,
the FAd encoded by MGP1 had lost its function and then ATP
hydrolysis could not be inhibited. As a result, there was not
enough ATP to maintain the normal membrane electronic
potential. Subsequently the membrane electronic potential
was destroyed, which led to destruction of mitochondria
and eventually collapse of pollen grains. Nevertheless, more
evidence is required to address this question.
In conclusion, MGP1 plays important roles in pollen formation at the late developmental stage, possibly by regulating the
activity of mitochondrial F1F0-ATP synthase.
Materials and Methods
Plant materials and mutant isolation
All Arabidopsis plants used in this study were of the Landsberg
erecta background. The seeds were pre-germinated on MS
(Murashige and Skoog) salt agar plates with or without
50 µg ml−1 kanamycin at 22°C under a light cycle of 16 h light/8 h
dark. The plants were grown in soil at 22°C under the same
light cycle as for germination. The generation of Ds insertion
lines and screen of mutants were performed as previously
described (Sundaresan et al. 1995). The selected mutant
plants were backcrossed with wild-type plants to purify the
mgp1 mutation. The F3 plants with a single Ds insertion linked
to the mgp1 phenotype were selected for further phenotypic
characterization (Supplementary Fig. S3).
932
Genetic analysis of the mgp1 mutant
All crosses of mgp1 plants with wild-type plants were performed
as described previously (Yang et al. 1999, Yang et al. 2003).
Assay for viability of pollen grains
The Alexander assay was used to investigate the viability of
mature pollen grains. The mature pollen grains were collected
in the 20% Alexander staining solution prepared according to
Alexander (1969) and squashed with a slide coverslip. Staining
of pollen grains with 1 µg ml−1 PI was performed as described by
Huang et al. (1986). DAPI assay for pollen grains at different
anther development stages was performed as described previously (Kang et al. 2003).
Observation of mitochondrial structures
in wild-type and mgp1 pollen grains
For TEM, the pollen grains were fixed overnight in a fixation
solution of 3% (v/v) glutaraldehyde in 0.05 M cacodylate
(pH 7.2) and then post-fixed in 1% osmium tetroxide for 3–4 h.
The samples were dehydrated through 30 min exposure to
a series of ethanol/water mixtures (10, 30 50, 70, 90, 95 and
100% ethanol) and 100% acetone three times. Subsequently,
the samples were transferred to a 1 : 1 dry acetone : resin mix
(Spurr, 1969) on a rotator (12 h) and infiltrated in a 1 : 3 dry
acetone : resin mix for an additional 12 h before transferring to
freshly mixed resin. Following two fresh resin changes, the
material was polymerized in molds (70°C for 8–16 h) (Spurr
1969). Ultrathin sections (90–100 nm thick) were cut using
diamond knives on a Leica-UCTR microtome (Leica, Wetzlar,
Germany), mounted on 200 mesh copper grids and stained
with 4% (w/v) uranyl acetate and lead citrate (Reynolds 1963).
The sections were viewed in a HITACHI S-7500 transmission
electron microscope (Hitachi, Tokyo, Japan).
Assay for the hydrolysis activity of Mg2+-ATPase
Analysis of the hydrolysis activity of Mg2+-ATPase in pollen
grains was performed as described by Xie et al (2006).
Molecular cloning of the MGP1 gene and
complementation of the mgp1 mutant
Isolation of the flanking sequences adjacent to the Ds element
by TAIL-PCR (Liu et al. 1995) was performed as described previously with the mgp1 genomic DNA and Ds3/AD6 primers
(Yang et al. 1999, Yang et al. 2003). The full-length MGP1
genomic DNA fragment was amplified using a Taq LA DNA
polymerase PCR kit (TaKaRa, Dalian, China) with the genespecific primers 5′-GATCAGTCAAGAGGTTGTGCTTG-3′ and
5′-GAGAAGGAGCTTTGAGAGGAATC-3′. The cDNA fragment
was amplified by RT–PCR with the gene-specific primers
5′-GTCAACTCCACAGCGACTCGATTC-3′ and 5′-CAGCAGC
AAGGGAACGATCTTC-3′. All resulting DNA fragments were
cloned into the pMD18-T vector (TaKaRa, Dalian, China)
for sequencing. For complementation experiments, the fulllength MGP1 genomic DNA fragment was subcloned into
Plant Cell Physiol. 51(6): 923–935 (2010) doi:10.1093/pcp/pcq066 © The Author 2010.
MGP1 is essential for pollen formation
a pCAMBIA1300 vector (http://www.cambia.org.au) and introduced into mgp1/+ plants using the Agrobacterium-mediated
infiltration method (Bechtold and Pelletier 1998). The transformants were selected in MS medium containing 25 mg l−1
hygromycin (Roche, Shanghai, China) and 50 mgl−1 kanamycin.
The transformants homozygous for mgp1 were selected in the
T3 generations and used for further analysis.
Bioinformatic analysis
For the sequence alignment, the sequences of MGP1homologous genes from different species in plants were
obtained from the National Center for Biotechnology information by BLASTP searches. Multiple sequence alignments were
performed using the DNAMAN software package.
Expression of MGP1
For RT–PCR assay of the expression of MGP1, total RNAs
were extracted from different tissues of wild-type Landsberg
erecta plants using CTAB (cetyltrimethylammonium bromide)
solution (Yu and Goh 2000). The root samples were collected
from 10-day-old seedlings; the leaves, stems, inflorescences
and siliques were harvested separately from flowering plants.
A 2 µg aliquot of total RNA was treated with DNase I (TaKaRa,
Dalian, China) before being used for cDNA synthesis. The firststrand cDNA was synthesized using a Reverse Transcription Kit
(Invitrogen, Shanghai, China) with oligo(dT)18 primer according
to the supplier’s instructions. cDNA was amplified with the
primer pair 5′-GTCAACTCCACAGCGACTCGATTC-3′ and
5′-GATGAAGTTACGGATGTCTAG-3′. Tubulin β-8 cDNA,
amplified with the primers 5′-CTTCGTATTTGGTCAATCCG
GTGC-3′ and 5′-GAACATGGCTGAGGCTGTCAAGTA-3′, was
used as an internal control to normalize the quantity of
the total RNA samples. Each reaction contained 10 µl of 2×
RCR mixture (TIAN GEN, Beijing, China), 2 µl of the first-strand
cDNA mixture and 0.3 µl of gene-specific primers (5 pM) in
a final volume of 20 µl. The cycling program was 3 min at 94°C
followed by 25 cycles of 45 s at 94°C, 1 min at 52°C and 1 min
at 72°C, followed by 72°C for 10 min. The PCR products were
analyzed by electrophoresis in a 1% agarose gel.
For the assay on the activity of the MGP1 promoter, a 2.3 kb
fragment upstream of the ATG start codon was amplified
using the Taq LA DNA polymerase PCR kit (TaKaRa, Dalian,
China) with the gene-specific primers MGP1-prom-1
(5′-GATCAGTCAAGAGGTTGTGCTTG-3′) and MGP1-prom-4
(5′-TTCCTTGATCGAATCGAGTCGC-3′) and cloned into the
pMD18-T vector. After sequence verification, the fragment
was subcloned upstream of the GUS reporter gene in the
pCAMBIA1300 Ti-derived binary vector. Then, the resulting
construct was introduced into Arabidopsis wild-type plants as
described above. GUS staining was performed as described
previously (Sundaresan et al. 1995, Yang et al. 1999).
stop codon and the MGP1 promoter were amplified using the
a Taq DNA polymerase PCR kit (TaKaRa, Dalian, China) with
gene-specific primer pairs: MGP1-cDNA-1 (5′-GTCAACTCCA
CAGCGACTC GATTC-3′)/MGP1-cDNA-KpnI (5′-GGGGTACC
GATGAAGTTACGGATGTCTAG-3′) and MGP1-prom-1
(5 ′ -GATCAGTCAAGAGGTTGTGCTTG-3 ′ )/MGP1-prom-4
(5′-TTCCTTGATCGAATCGAGTCGC-3′), respectively. The
resulting fragments were cloned into pMD18-T vector and
verified by DNA sequencing. The MGP1-coding fragment was
excised from pMD18-T vector with BamHI and KpnI restriction
enzymes and subcloned into the pGFP-2 vector in front of
the start codon of the GFP-coding sequence, resulting in an
MGP1–GFP fusion protein-coding construct. The MGP1 promoter fragment was excised with PstI and BamHI restriction
enzymes. The MGP1–GFP fusion-coding fragment was excised
from the pGFP-2 vector with BamHI and SacI restriction
enzymes. Then, both fragments were subcloned in front of
the NOS terminator sequence in a modified pCAMBIA1300
Ti-derived binary vector. The resulting pMGP1::MGP1-GFP-TNOS
construct was introduced into Arabidopsis wild-type plants
using the Agrobacterium-mediated infiltration method
(Bechtold et al. 1998). The transformants were selected using
20 mg l−1 hygromycin. In the T2 transgenic plants, the subcellular localization of the MGP1–GFP fusion protein in the pollen
grain was observed with a Zeiss LSM510 META laser scanning
microscope (Zeiss, Jena, Germany) with a 488 nm argon laser.
To construct the p35S::MGP1-GFP-TNOS expression cassette,
the MGP1 promoter in the pMGP1::MGP1-GFP-TNOS construct
was replaced by the CaMV 35S promoter derived from pBI121
at the sites of PstI and BamHI. Plant transformation was
performed as described above. The subcellular localization of
the MGP1–GFP fusion protein in the roots was observed by
a Zeiss LSM510 META laser scanning microscope (Zeiss, Jena,
Germany) with a 488 nm argon laser.
To investigate whether the MGP1–GFP fusion protein was
localized in mitochondria, the mitochondria were visualized
in the MGP1–GFP transgenic plant cells with Mito-Tracker
staining methods (Haugland 1996). The whole 14-day-old
transgenic plants were submerged in a solution containing
500 nM Mito-Tracker Red CM-H2XRos in MS medium for
10–15 min. After washing three times in MS medium, the roots
from the transgenic plants were sliced with razor blades and
mounted between a slide and coverslip in distilled water. Cells
were examined with a 543 nm argon laser.
All the images were edited with Zeiss LSM Image Browser
software and Photoshop version 7.0 software.
Supplementary data
Supplementary data are available at PCP online.
Funding
Subcellular localization of MGP1 protein
To generate the construct for the subcellular localization of
MGP1 protein, the coding region of MGP1 cDNA without the
This work was supported by the Ministry of Sciences and
Technology [973 project number: 2007CB108700]; the Natural
Plant Cell Physiol. 51(6): 923–935 (2010) doi:10.1093/pcp/pcq066 © The Author 2010.
933
W.-Q. Li et al.
Science Foundation of China (NSFC) [project number:
30530060]; the Ministry of Education, PR China [111 project
number: B06003].
Acknowledgements
We thank Dr. V. Sundaresan and Dr. Weicai Yang for their kind
help with the mutant screens.
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