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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 ; 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. 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. 1406 Major proteins in maize egg cells 1407 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. 1408 Table 1 Major proteins in maize egg cells 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 2 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 Sequence determined 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/ Major proteins in maize egg cells 1409 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 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). 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 237–250 207–219 31–48 105–117 277–293 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 1410 Major proteins in maize egg cells 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. 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- 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 enzyme subset for energy metabolism. However, in the present study, the expression levels of the identified proteins were compared between egg cells and other cell types on the basis of the intensity of the silver-stained band with the same mobility in the SDS-PAGE gels. This will be insufficient to elucidate the specificity of the protein bands. To know whether the protein bands observed in early embryos, central cells and cultured cells correspond to the identified proteins in egg cells, further analysis such as Western blotting with specific antibody or/and RT-PCR should be conducted in the near future. In fact, we have tried to synthesize cDNA from a small number of the cells. To our knowledge, this is the first report to identify the major proteins in angiosperm egg cells. Using the small-sized 2D-PAGE, comparisons of the protein profiles between egg cells and zygotes are currently underway in our laboratories. LC-MS/MS analyses of the protein spots, which are detected only in zygotes or egg cells, will reveal newly synthesized, modified or rapidly degraded proteins in the zygotes. This will provide a novel insight into zygote development and early embryogenesis in higher plants. 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. 1411 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. References 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 1999). A pair of a sperm and an egg cell protoplast were fused electrically under microscopical observation (Kranz and Lörz 1993). Fusion products were cultured on the transparent, semipermeable membranes of Millicell-CM dishes (diameter 12 mm; Millipore, Bedford, MA, U.S.A.) as described (Kranz et al. 1991). After 40–50 h of culture, two-celled embryos were harvested and washed three times by transferring them into fresh droplets of culture medium. Two to five isolated two-celled embryos were transferred into a 1 µl droplet of SDSsample buffer. The zygotes developed into multicellular embryos after 3 d of culture, and after washing, three to five of these embryos were transferred into a 1 µl droplet of SDS-sample buffer. 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