Download Identification of Major Proteins in Maize Egg Cells

Plant Cell Physiol. 45(10): 1406–1412 (2004)
JSPP © 2004
Identification of Major Proteins in Maize Egg Cells
Takashi Okamoto 1, 2, 4, Kanako Higuchi 1, Takashi Shinkawa 3, Toshiaki Isobe 3, Horst Lörz 2, Tomokazu
Koshiba 1 and Erhard Kranz 2
1
Department of Biological Sciences, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji, Tokyo, 192-0397 Japan
Biozentrum Klein Flottbek und Botanischer Garten, Entwicklungsbiologie und Biotechnologie, Universität Hamburg, Ohnhorststr 18, 22609
Hamburg, Germany
3
Department of Chemistry, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji, Tokyo, 192-0397 Japan
2
;
Keywords: Annexin — Egg cell — Glycolysis — Major protein — Mass spectrometry.
Abbreviation: LC-MS/MS, liquid chromatography with tandem
mass spectrometry.
Introduction
Egg cells in higher plants are highly differentiated haploid cells, which are fertilized with sperm cells and undergo
subsequent early embryogenesis. In angiosperms, the female
gametophyte, also referred to as the embryo sac or the megagametophyte, develops in an ovule embedded within the ovary.
Although among angiosperms the female gametophyte has a
4
variety of forms, the most common consists of seven cells composed of four cell types: one egg cell, one central cell, two synergid cells and three antipodal cells (Huang and Russell 1992,
Drews and Yadegari 2002). Upon double fertilization, one
sperm cell from the pollen grain fuses with the egg cell, and the
resulting zygote develops into an embryo. The central cell fuses
with the second sperm cell to form a triploid primary
endosperm, which develops into the endosperm (Nawaschin
1898, Guignard 1899, Russell 1992).
In general, the composition of cellular proteins differs depending on cell type. For example, mesophyll cells have a large
amount of ribulose-1,5-bisphosphate carboxylase/oxygenase for
the fixation of carbon dioxide, while the cotyledon cells of nonendospermic seeds such as legume seeds abundantly contain
storage globulins and albumins, which supply the nutrient
source for hypocotyl growth during seed germination and seedling growth (Bewley and Black 1994). These indicate that the
major proteins in such highly differentiated cells reflect the
biological function of the cells. Therefore, identification of the
major protein components in egg cells will provide basic
knowledge of their character. In addition, identification of the
major proteins in the egg cell will give a cue for analyzing the
mechanisms of female gametogenesis, fertilization and early
embryogenesis in higher plants.
Unlike in animals and lower plants, higher plant egg cells
are located in the embryo sac, which is deeply embedded in
ovular tissue. Methods were developed for the isolation of
embryo sacs and egg cells in a wide range of higher plant species (for review see Theunis et al. 1991). However, biochemical analyses of egg cells of higher plants at the protein level
have not been performed to our knowledge due to the limited
amount of such isolated cells. Nevertheless, in maize, routinely
20–40 egg cells can be isolated/experienced by one experimenter per day, and, under optimal conditions up to 60 egg
cells can be obtained by one person per day (Kranz 1999).
Despite this relatively small amount of plant material, recent
advances in proteomics technologies provide the possibility of
identifying proteins in such cells.
In this study, we detected traceable amounts of proteins in
a small number of the egg cells by minimizing the size of gels
for one- and two-dimensional polyacrylamide gel electrophoresis and identified major protein components by highly sensi-
Corresponding author: E-mail, [email protected]; Fax, +81-426-77-2559.
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In most flowering plants, the female gametophyte
develops in an ovule deeply embedded in the ovary.
Through double fertilization, the egg cell fuses with the
sperm cell, resulting in a zygote, which develops into the
embryo. In the present study, we analyzed egg cell lysates
by polyacrylamide gel electrophoresis and subsequent mass
spectrometry-based proteomics technology, and identified
major protein components expressed in the egg cell. The
identified proteins included three cytosolic enzymes of the
glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and triosephosphate isomerase, two mitochondrial proteins, the ATP synthase βsubunit and an adenine nucleotide transporter, and annexin
p35. In addition, expression levels of these proteins in the
egg cell were compared with those in the early embryo, the
central cell and the suspension cell. Annexin p35 was highly
expressed only in the egg cell, and glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and the
adenine nucleotide transporter were expressed at higher
levels in egg cells than in central and cultured cells. These
results indicate that annexin p35 in the egg cell and zygote
is involved in the exocytosis of cell wall materials, which is
induced by a fertilization-triggered increase in cytosolic
Ca2+ levels, and that the egg cell is rich in an enzyme subset
for the energy metabolism.
Major proteins in maize egg cells
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Fig. 1 SDS-PAGE and 2D-PAGE of maize egg cell proteins. (A) Isolated egg cell. Bar = 50 µm. (B) Proteins from 75 egg cells were separated
by SDS-PAGE followed by a modified silver-staining procedure. Numbers to the right of the arrowheads indicate the protein bands subjected to
in-gel tryptic digestion and subsequent LC-MS/MS. Numbers to the right of the bracket indicates the gel region subjected to in-gel tryptic digestion and LC-MS/MS. (C) Identification of annexin p35 as a major protein in maize egg cells. The doubly charged ions of the tryptic peptides (m/z
= 518.34) from a major protein in the egg cells (band 5 in Fig. 1A) were analyzed by LC-MS/MS. The amino acid sequences were verified by
interpreting the b-type (italics) and y-type (normal text) production series as indicated in the figure. (D) Proteins from 180 egg cells were separated by 2D-PAGE followed by modified silver staining. Numbers around the arrowheads indicate the protein spots subjected to in-gel tryptic
digestion and LC-MS/MS.
tive liquid chromatography with tandem mass spectrometry
(LC-MS/MS) technology. We show here that the egg cell abundantly contains three cytosolic glycolytic enzymes, mitochondrial ATP synthase β-subunit, adenine nucleotide translocator
and annexin p35, and discuss possible functions of these proteins in the cell.
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Major proteins in maize egg cells
Table 1 Major proteins of maize egg cells identified by SDS-PAGE and subsequent tandem mass spectrometry
Band
Protein
number
Accession
Peptide
Charge
number (GI) m/z
Mitochondrial ATP synthase β-chain
114420
3
Cytosolic 3-phosphoglycerate kinase
28172915
4
Cytosolic glyceraldehyde-3-phosphate
dehydrogenase
6016075
5
Annexin P35
7441507
6
Mitochondrial adenine nucleotide translocator
22166
Results
Identification of major proteins by SDS-PAGE and subsequent
mass spectrometry
Isolated egg cells (Fig. 1A) were extensively washed to
eliminate contamination of proteins in the enzymic solutions,
which were used during isolation of the cells as described in
Materials and Methods. Proteins from 75 egg cells were separated by 12.5% SDS-PAGE and the gel was silver stained. Protein bands were successfully detected possibly due to the
small-sized gel, in which proteins are concentrated more efficiently than in a normal-sized gel. The band pattern was almost
identical in repetitive experiments. By comparing the intensity
of protein bands from the egg cells with that of the molecular
weight marker co-migrated on the gel, the amount of protein in
an egg cell was roughly estimated to be 100–200 pg (data not
shown). Major protein bands, assigned as bands 1–7 in Fig. 1B,
were excised from the gel, in-gel digested with trypsin, and the
resulting peptide mixtures were analyzed by direct nano-flow
LC-MS/MS. Two doubly charged peptide ions with m/z 518.34
and 696.38 were observed in band 5. Database analysis of the
MS/MS spectrum of the peptide ion with m/z 518.34 showed
that it corresponded to the LIISILAHR sequence of maize
annexin p35 at residues 33–40 (Table 1). Manual assignment of
the fragment ions also yielded the same sequence (Fig. 1C).
Likewise, the MS/MS spectrum of the other peptide ion with
m/z 696.38 was assigned to the ADPKDEFLSTLR sequence of
maize annexin p35 at residues 222–233 (Table 1), confirming
that band 5 corresponds to annexin p35, which is thought to be
involved in exocytosis and vesicle trafficking in plant cells
(Carroll et al. 1998, Battey et al. 1999, Clark et al. 2001).
The results obtained from LC-MS/MS analysis for bands
2–6 are summarized in Table 1. The proteins of 39 kDa (band
4) and 42 kDa (band 3) were identified as cytosolic glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate
kinase, respectively, which are known to be responsible for glycolysis (Plaxton 1996). Bands 2 and 6 corresponded to mitochondrial ATP synthase β-subunit and adenine nucleotide
residues
705.41
729.43
868.01
694.89
559.66
2
2
2
2
2
VLNTGSPITVPVGR
147–160
TVLIMELINNVAK
235–247
LAAALPEGGVLLLENVR 31–48
ELDYLVGAVANPK
105–117
TLLFGEKPVTVFGIR
68–82
749.94
518.34
696.38
723.89
2
2
2
2
VPTVDVSVVDLTVR
LIISILAHR
ADPKDEFLSTLR
YFPTQALNFAFK
237–250
33–40
222–233
161–172
translocator, respectively. Although mitochondrial adenine
nucleotide translocator was identified on the basis of a single
peptide (Table 1), the molecular weight of the protein has been
estimated as 30.5 kDa by the electrophoretic mobility in the
SDS-PAGE gel (Winning et al. 1992), which is consistent with
the mobility of band 6 (Fig. 1B). This supports the possibility
that band 6 corresponds to mitochondrial adenine nucleotide
translocator. Four of the seven major proteins analyzed were
thought to be involved in energy metabolism, such as glycolysis and ATP production/transport, within the cell. Database
analysis of the LC-MS/MS spectrum of peptides from band 1
indicated that this protein has a SSVLESLAGISLPR sequence,
which is identical to the Arabidopsis hypothetical protein
(At1g60500.1) at residues 79–92, however, no maize protein
was detected by the database search (data not shown). The protein of band 7 could not be identified.
In addition to bands 1–7, an attempt was made to determine the first structure of the protein bands with weak intensity. The gel region between bands 1 and 2 (indicated by
blanket, No 8 in Fig. 1B) were excised and trypsin-digested,
and the resulting peptides were analyzed with LC-MS/MS. But
the proteins could not be identified by our LC-MS/MS system,
although the system has extremely high sensitivity. This indicates that only major proteins can be analyzed using LC-MS/
MS when proteins from 75 egg cells are used as materials.
However, this analytical limitation confirms that the identified
proteins listed in Table 1 are not derived from minor proteins
overlapping with the major proteins in the gel, but from major
proteins themselves.
Identification of major proteins by 2D-PAGE and subsequent
mass spectrometry
Proteins from 180 egg cells were separated by isoelectric
focusing and subsequent SDS-PAGE. Protein spots stained
with silver were successfully detected, and eight spots were
selected for in-gel tryptic digestion and subsequent analysis by
LC-MS/MS (Fig. 1D). The profile of protein spots in the gel
was similar in repetitive experiments. The results from LC-MS/
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2
Sequence determined
Major proteins in maize egg cells
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Table 2 Major proteins of maize egg cells identified by 2D-PAGE and subsequent tandem mass spectrometry
Spot
Protein
number
Accession
Peptide
Charge
number (GI) m/z
1
Cytosolic glyceraldehyde-3phosphate dehydrogenase
6016075
5
6
Cytosolic triosephosphate isomerase
136063
Cytosolic 3-phosphoglycerate kinase 28172915
Sequence determined
Residues
870.46
2
VIHDNFGIIEGLMTTVHAITATQK 165–188
749.93
684.36
868.01
694.88
787.42
2
2
2
2
2
VPTVDVSVVDLTVR
IIYGGSVTAANCK
LAAALPEGGVLLLENVR
ELDYLVGAVANPK
GVTTIIGGGDSVAAVEK
Comparison of protein profiles from egg cells with those from
early embryos, central and cultured cells
The modified silver staining method was approximately
five times less sensitive than the conventional method, since
fixative in the modified procedures does not contain glutaraldehyde (Taoka et al. 2000). Although 75 egg cells were subjected
to SDS-PAGE for subsequent LC-MS/MS analyses in Fig. 1B,
15 egg cells were enough to visualize the proteins in SDSPAGE gels with conventional silver staining (Fig. 2A–C).
The protein profiles of the egg cells in the SDS-PAGE gel
were compared with those of two-celled or multicellular
embryos produced in vitro to see whether the expression levels
of the five identified proteins (bands 2–6 in Fig. 1B and
Table 1) change after in vitro fertilization and during early
embryogenesis. Annexin p35 was strongly expressed in the egg
cells, but largely decreased in the two-celled and multicellular
embryos (band 5 in Fig. 2A). Expression levels of the other
proteins remained unchanged after fertilization and during
early embryogenesis (bands 2–4 and 6 in Fig. 2A).
Next, protein profiles were compared between the egg and
central cells. In the central cells, the band intensities for
cytosolic 3-phosphoglycerate kinase and cytosolic glyceraldehyde-3-phosphate dehydrogenase were weaker than those in
the egg cells (bands 3 and 4 in Fig. 2B). Furthermore, the protein corresponding to annexin p35 was hardly detected in the
central cells (band 5 in Fig. 2B). Finally, we compared the profile with cultured maize cells, which are neither gametophytic
nor embryonic. Annexin p35 was not observed in the cultured
cells (band 5 in Fig. 2C). Moreover, cytosolic 3-phosphoglycerate kinase and mitochondrial adenine nucleotide translocator
were hardly detected in the cultured cells (bands 3 and 6 in Fig.
2C).
Discussion
Three cytosolic enzymes for the glycolytic pathway, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate
kinase and triosephosphate isomerase, and two mitochondrial
proteins, an ATPase β-subunit and an adenine nucleotide transporter, and annexin p35 were identified as major proteins in
maize egg cells (Tables 1, 2). Of these six proteins, annexin
p35 was strongly expressed only in the egg cells (Fig. 2A–C).
Annexins are Ca2+ and phospholipid binding proteins, and
extensive studies of the proteins in animal cells have shown
their multifunctional roles in essential cellular processes such
as membrane trafficking, ion transport, mitotic signaling,
cytoskeleton rearrangement and DNA replication (reviewed in
Gerke and Moss 2002). Plant annexins share the basic properties of Ca2+-dependent membrane binding molecules and are
structurally similar to their animal counterparts (Pirck et al.
1994, Clark and Roux 1995, Battey et al. 1996).
Exocytosis and the Golgi-mediated secretion of newly
synthesized plasma membranes and cell wall materials have
been reported as the function of annexin in plant cells (Carroll
et al. 1998, Battey et al. 1999, Clark et al. 2001). It has been
demonstrated that cell wall formation around the zygote starts
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MS analyses are summarized in Table 2. Spot 1 was determined as cytosolic glyceraldehyde-3-phosphate dehydrogenase,
which is identical to band 4 in Fig. 1B, and spot 6 was determined as cytosolic 3-phosphoglycerate kinase, which corresponds to band 3 in Fig. 1A. Spot 5 was identified as cytosolic
triosephosphate isomerase, which also belongs to the enzymes
of the glycolytic pathway. Calculated molecular mass and isoelectric point of cytosolic triosephosphate isomerase (accession
number GI136063) are 27,292 and 5.52, respectively. These
values fit the position of the gel where spot 5 was detected (Fig.
1D), supporting the conclusion that the protein spot corresponds to cytosolic triosephosphate isomerase although only a
single peptide was detected by LC-MS/MS analysis (Table 2).
A doubly charged peptide ion with m/z 617.85 was observed in
spot 2, and database analysis of the LC-MS/MS spectrum of
this ion showed a KIYETKILVK sequence (data not shown),
which is identical to tomato cystatin at residues 226–235 (PIR
accession number T06323). However, database analysis did not
hit with maize cystatin. The result suggests that spot 2 corresponds to a novel maize cystatin, which has not been identified,
or to an unknown maize protein containing the KIYETKILVK
sequence. For spots 3, 4, 7 and 8, proteins could not be identified. Some major proteins detected in the SDS-PAGE gel were
not detected in that of 2D-PAGE (Fig. 1B, D) probably due to
the narrow pI range of isoelectric focusing (pI 4.5–7).
237–250
207–219
31–48
105–117
277–293
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Major proteins in maize egg cells
30 s after in vitro fusion of the egg with a sperm cell (Kranz et
al. 1995). This rapid formation of the cell wall around the
zygote suggests that cell wall materials are stored in the egg
cells before fertilization, and are secreted via possible exocytosis after fertilization. It is well known that Ca2+ exerts the regulation of exocytosis in plant and animal cells (Bush 1995,
Battey et al. 1999), and Carroll et al. (1998) reported that Ca2+stimulated exocytosis in root cap cells is enhanced by exogenously applied annexin p35, suggesting that annexin is
involved in Ca2+-stimulated exocytosis. It has also been
revealed that concentrations of cytosolic Ca2+ in maize egg cell/
zygote increase after fertilization (Digonnet et al. 1997) possi-
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Fig. 2 Comparison of the protein profiles of the egg cells with those
of the early embryo (A), the central cell (B) and cultured cell (C). (A)
Proteins from 15 egg cells (lane 1), 15 two-celled embryos (lane 2) and
15 multicellular embryos (lane 3) were separated by SDS-PAGE. Proteins in the gel were visualized with conventional silver staining. Numbers to the left of the arrowheads are equivalent to those in Fig. 1B. (B)
Proteins from 15 egg cells (lane 1) and four central cells (lane 2) were
separated by SDS-PAGE, and the gel was silver stained. (C) Proteins
from 15 egg cells (lane 1) and maize cultured cells (lane 2; Kranz et al.
1991) were separated by SDS-PAGE followed by silver staining.
bly via influxes of extracellular Ca2+ (Antoine et al. 2000).
When a fluorescent Ca2+ indicator (Kao et al. 1989) was used
to monitor intracellular Ca2+ levels in egg and zygote cells, it
was observed that levels reached a maximum 85 s after in vitro
fertilization (Digonnet et al. 1997). Annexin p35, existing
abundantly in egg cells, might play a role in exocytosis, which
is stimulated by fertilization-induced increases in Ca2+ levels in
the zygote, for rapid cell wall formation around the zygote.
In contrast to animal mitochondria, which respire fatty
acids and glycolytically derived pyruvate, plant mitochondria
rarely respire fatty acids (reviewed in Plaxton 1996). This indicates that glycolysis is of crucial importance in plants because
it is the predominant pathway supplying ‘fuels’ for plant
respiration. Recently, it was revealed that seven glycolytic
enzymes, including glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, are associated with the
outer membranes of mitochondria, suggesting that such microcompartmentation of glycolysis allows pyruvate to be provided
directly into the mitochondrion (Giege et al. 2003). In mitochondria, ATPase synthesizes ATP, which is the principal
energy source for the cells, via an H+ gradient between the
inner and outer membranes, and the resultant ATP is exchanged
with cytosolic ADP by adenine nucleotide transporters (Vignais
1976, Mozo et al. 1995). Giant and polymorphic mitochondria
have been observed in egg cells of maize (Faure et al. 1992)
and geranium (Kuroiwa and Kuroiwa 1992), indicating that
identification of two mitochondrial proteins as major proteins
in maize egg may reflect such well-developed mitochondria.
Five of the six major egg proteins identified in this study
are thought to be involved in the cytosolic and mitochondrial
energy production pathways, suggesting that the egg cell has
sufficient enzymes and transporters to produce and transport an
energy source. After in vitro fusion of the maize egg with a
sperm cell, the majority of cytoplasmic organelles migrate
towards the zygote nucleus, cell wall is actively formed, and
duplication and division of the nucleus occur as part of the
early cytological events in the zygote (Kranz et al. 1995).
These energy-consuming serial zygotic events might explain
why these cells abundantly contain proteins for energy production. Interestingly, it has been reported that glycolysis in the
mouse oocyte is activated by fertilization (Urner and Sakkas
1999). Activation of glycolysis may occur in maize zygote after
fertilization of the egg cell with the sperm cell.
Expression levels of the three glycolytic enzymes and two
mitochondrial proteins in the egg cells were identical to those
in two-celled and multicellular embryos (Fig. 2A). This is
probably the result of early embryogenesis, which requires a
large quantity of energy for embryonic development. Glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate
kinase were expressed at a higher level in the egg cells than
in the central cells (Fig. 2B), while expression levels of 3phosphoglycerate kinase and adenine nucleotide transporter
was low in the cultured cells (Fig. 2C). These results might
indicate that early embryos, as well as egg cells, are rich in the
Major proteins in maize egg cells
Materials and Methods
lsolation and selection of egg and central cells
Ears from the inbred maize (Zea mays) line A188 (courtesy of A.
Pryor, CSIRO, Canberra, Australia) were used for isolating egg and
central cells. Egg cells were isolated as described previously (Kranz et
al. 1991, Kranz 1999). Isolated egg cells were washed four times by
transferring the cells into fresh droplets of mannitol solution
(650 mosmol kg–1 H2O) on coverslips. Between 10 and 50 isolated egg
cells were transferred into a 1 µl droplet of SDS-sample buffer (2%
SDS, 25 mM Tris-HCl pH 6.8, 30% glycerol, 5% 2-mercaptoethanol)
for SDS-PAGE, or into a 1 µl droplet of lysis buffer [8 M urea, 5% 2mercaptoethanol, 2% ampholine pH 3.5–10 (Amersham), 2% Nonidet
P40] for 2D-PAGE. These samples were stored at –80°C until use.
Central cells were isolated according to the method previously
described (Kranz et al. 1998). Isolated central cells were washed as
above, and four isolated central cells were transferred into a 6 µl droplet of SDS-sample buffer.
Seventy five or 15 egg cells dissolved in 6 µl of SDS-sample buffer
were applied to the SDS-PAGE gel. The isoelectric focusing gel [8 M
urea, 3.5% acrylamide, 0.18% bis-acrylamide, 5% (v:v) ampholine pH
3.5–10 and 2% Nonidet P40] was prepared using a thin glass capillary
(length, 5 cm; diameter, 1 mm). One hundred and eighty egg cells dissolved in 6 µl of lysis buffer were applied to the capillary gel. After
isoelectric focusing, the proteins in the capillary gel were separated
further by 12.5% SDS-PAGE. The proteins in the gel used for in-gel
tryptic digestion and subsequent analysis with LC-MS/MS were visualized by modified silver staining according to Taoka et al. (2000). In
other cases, the proteins in the SDS-PAGE gel were detected by conventional silver staining procedures (Oakley et al. 1980).
Identification of proteins by tandem mass spectrometry
Protein bands or spots were excised from the SDS-PAGE gel, ingel digested with trypsin, and subjected to direct nano-flow LC-MS/
MS analysis for protein identification. The chromatography was performed on a nano ESI column (inside diameter, 150 µm × 30 mm)
packed with a C18 reversed phase medium (Mightysil-C18, 3 µm;
Kanto Chemicals, Tokyo, Japan) using a linear gradient from 0 to 70%
acetonitrile in 0.1% formic acid for 35 min at a flow rate of 100 nl/
min, and the separated peptides were directly sprayed into a hybrid
mass spectrometer equipped with an electrospray source (Q-Tof
ultima; Micromass-Waters, Milford, MA, U.S.A.). Electrospray ionization was carried out at a voltage of 1.5 kV, and MS/MS spectra were
automatically acquired in data-dependent mode during the entire run.
All MS/MS spectra were correlated by the search engine, Mascot program (Matrixscience, London, U.K.), against the non-redundant protein sequence database at the National Center for Biotechnology
Information (National Institutes of Health). Each high-scoring peptide
sequence was confirmed by manual inspection of the corresponding
MS/MS spectrum to ensure that the match was correct.
Acknowledgments
We thank Marlis Nissen and Petra von Wiegen for their excellent
technical help in the isolation of ovular tissues and gametes. We thank
Dr. Stefan Scholten for the discussions and the suggestion about the
protein analysis. This work was supported in part by Grants-in-Aid
from the Ministry of Education, Science, Sport, and Culture of Japan
(grants 15031222 to T.K. and 16027242 to T.O.). T.O. was supported
by a JSPS Postdoctoral Fellowship for Research Abroad.
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Electrofusion and culture procedures
Sperm and egg cells were isolated from pollen grains and the
ears of maize, respectively, as described (Kranz et al. 1991, Kranz
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LC-MS/MS analyses of the protein spots, which are detected
only in zygotes or egg cells, will reveal newly synthesized,
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1411
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(Received May 19, 2004; Accepted July 18, 2004)
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