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Keywords:

  • Con A (concanavalin agglutinin) binding site;
  • female cell;
  • fertilization;
  • Nicotiana tabacum (tobacco);
  • polarity

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowlegements
  7. References
  • • 
    The presence of mosaicism in the organization of concanavalin agglutinin (Con A) binding sites on murine egg cells was first reported 30 year ago. This discovery has triggered extensive studies into the roles of glycoproteins in gamete interactions in animals.
  • • 
    This report comprises the first account of the existence of the mosaicism in higher plants.
  • • 
    The distribution of Con A binding sites on both egg cells and central cells of tobacco (Nicotiana tabacum) was found to be polar and apparently determined by the location of the nucleus of the cell. On central cells, Con A binding sites were distributed on the section of the plasma membrane surface near the nucleus. By contrast, the binding sites on egg cells were concentrated away from the nucleus. Therefore, polarity of the plasma membrane component of female cells was confirmed for the first time.
  • • 
    It is proposed that such polarized ConA binding sites could be involved in sperm recognition.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowlegements
  7. References

Lectins are glycoproteins that bind reversibly to specific mono- or oligosaccharides. Data from animals and lower plants show that interactions between lectins and their binding sites (receptors) play an important role in gamete recognition and subsequent events in fertilization (Kim & Kim, 1999; Diekman, 2003).

Johnson et al. (1975) first reported that Con A binding sites are polarly distributed on the membrane surface of murine egg cells. Lectin binding signals were weak or absent on one-fifth of the egg's surface. The authors indicated that the negative-signal area was associated with the underlying second metaphase spindle. Later, Grabel et al. (1979) proposed that the interaction of cell surface lectins and the appropriate cell surface saccharide receptors contributes to cell-to-cell adhesion. An asymmetric distribution of Con A binding sites was also found on the plasma membrane of a fertilized sea urchin egg, and the distribution changed during the first and second cleavage (McCaig & Robinson, 1982). These results indicated a broad role for lectin–receptor interactions in animal fertilization and early embryogenesis, and they triggered extensive study in this field.

In angiosperm plants, fertilization involves two fusion events, the fusion of egg and sperm resulting in embryogenesis and the fusion of central cell and sperm resulting in endosperm formation. The mechanism of the fusion, particularly the membrane component responsible for gamete adhesion and fusion, is unknown. Two alternative hypotheses have been proposed (Knox & Singh, 1987). One hypothesis proposes that gametes fuse because they have unique recognition molecules on their membranes that enable them to interact, paralleling the situation in animals. The other hypothesis proposes that because gametes are the only cells lacking a cell wall, they fuse when brought together in an environment that promotes fusion, paralleling fusion of somatic protoplasts. The actual mechanism may include gamete-specific molecules plus nonspecific membrane interactions.

Based on these hypotheses, Faure (1999) further suggested three possible mechanisms of gamete fusion, as follows. (1) Sperm cells, having the same morphology and recognition molecules on their cell membranes, fuse randomly with eggs or central cells. The prevention of polyspermy that occurs after the first egg-sperm fusion compels subsequent sperm cells to fuse with other female cells. (2) Preferential fertilization, in which sperm cells fuse selectively with their female targets, occurs in any of the plants in which sperm cells differ in morphology but not in recognition molecules. (3) Finally, preferential fertilization arises from specific recognition molecules on the sperm membrane but not from sperm dimorphism. These mechanisms remain to be confirmed. Faure et al. (1994) reported that 80% of sperm–egg pairs fused, 2% of sperm–mesophyll protoplast pairs fused and 18% of sperm–sperm pairs fused with in vitro gamete fusions. In Plumbago zeylanica, the paired sperm cells are dimorphic, with Sua (sperm cell unassociated with vegetative nucleus) containing more plastids tending to fuse with the egg cell and Svn (sperm cell associated with vegetative nucleus) containing more mitochondria tending to fuse with the central cell (Russell, 1985). These results favor the proposal that the gamete cell surface exhibits some specificity that is responsible for gamete recognition.

Plant egg cells exhibit polarity in the location of the nucleus and cytoplasm. In tobacco plants, egg cells are polarized; the vacuole occupies the micropylar part of the cell and the nucleus, with a majority of the cytoplasm in the chalazal part. The polarity of the central cell is identified by the location of the nucleus at the micropylar part and the vacuole at the chalazal half. In short, the polarity of female cells is embodied by the distribution of the nucleus, cytoplasm and organelles. Polarity data in regard to the organization of the gamete membrane is not yet available.

Previously, few researchers studied the distribution of glycoproteins on the plasma membrane due to the difficulty of obtaining female cells. However, Pennell et al. (1991) found that arabinogalactan protein (AGP) was distributed on the egg cell surface but not on the central cell surface. This result confirmed for the first time the obvious differences in glycoprotein distribution in egg cells vs central cells, both of which can fuse with sperm during double fertilization.

With the successful isolation of female cells, it has become possible to perform in vitro investigations with living plant gametes. Sun et al. (2002a) found that fertilization induced the appearance of Phaseolus vulgaris agglutinin (PHA) binding sites and enhanced WGA binding sites on maize zygotes. Con A was found on both egg cells and zygotes, suggesting that it plays a role in fertilization of higher plants. In the present study, extensive investigations revealed the distributive pattern of Con A binding sites on egg cells and central cells of tobacco. Con A binding sites were found to be distributed on these cells in a manner surprisingly similar to that of animal cells, and the binding sites exhibited a distinctly polar distribution on both kinds of female cells. Although modern techniques make the term ‘mosaicism’ archaic in describing this observation, we intend to use this term to emphasize the similarity between animal and plant egg cells.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowlegements
  7. References

Plant materials

Plants of Nicotiana tabacum L. were grown in a glasshouse under a 16-h photoperiod and 50% humidity at 25°C. At 2 d of anthesis, the embryo sacs were fully mature and were collected for preparation.

Reagents

Fluorescein isothiocyanate (FITC)-Con A was purchased from Sigma (Steinheim, Germany) and dissolved in 13% mannitol, pH 5.7. The concentration of Con A stock was 500 µg ml−1.

Isolation of female cells

Female cells were isolated according to the method described by Sun et al. (2000). Tobacco plant ovules were dissected from ovaries and placed into an enzyme solution composed of 13% mannitol, 1% cellulase R-10 and 0.8% macerozyme R-10, pH 5.7, and incubated for 3 h with agitation. The enzymes have been tested and confirmed that they do not influence the distribution and binding ability of lectin binding sites (Burgess & Linstead, 1976; Walko et al., 1987; Sun et al., 2002a; Fang et al., 2003). Three washes in 13% mannitol were then performed to remove any remaining enzymes. The female cells were isolated from the ovules with glass microneedles and transferred into another drop of mannitol solution. Some of the isolated female cells were labeled by Con A directly. As a control, other cells were incubated briefly in 1% paraformaldehyde to fix the membrane protein before labeling. Some of the labeled female cells were observed directly; the remaining cells were re-incubated for a sufficient amount of time before observation as another control (for details, see the Results and Discussion section).

Labeling

Labeling was conducted according to the protocol described by Sun et al. (2002b) and Johnson et al. (1975), with some modifications. Isolated female cells were immediately immobilized on the bottom of a Petri dish in a drop of 13% mannitol covered by a thin layer of mineral oil to prevent evaporation. The volume of the drops containing the materials was approx. 200 nl, and the same volume of labeling solution was added to make the final concentration of Con A 250 µg ml−1; the mixture was then incubated for 40 min. Unbound lectin was removed by thorough washing in 13% mannitol before observation.

Fluorescein diacetate (FDA, Sigma) was dissolved in acetone to produce a 5 mg ml−1 stock solution. For cell viability tests, the FDA stock was added to the medium to a final concentration of 2.5 µg ml−1, and the cells were incubated for 5 min in the dark at room temperature. Calcofluor white ST (CW) staining and the cell fusion technique (Sun et al., 2000) were employed to determine if any cell wall remnants existed.

Controls

Somatic protoplast control  Somatic protoplasts (primarily integument tissue protoplasts) were isolated during female cell isolation and labeled and observed using the procedures described above. Additional protoplasts were treated similarly but without FITC-Con A as another control.

Binding specificity test  Binding specificity was tested using the procedures described above, except that the incubation medium contained competing monosaccharides, primarily α-methyl-D-mannoside. In this case, the materials were preincubated with 0.1 mα-methyl-D-mannoside for at least 30 min before the cells were labeled (Walko et al., 1987).

Membrane stability test  Various factors that may influence membrane stability were tested to confirm the distribution pattern of Con A binding sites on female cells (for details, see the Results and Discussion section).

Measurement and microscopy

The isolation of ovules and female cells and labeling manipulation were performed under an Olympus IMT-2 inverted microscope (Tokyo, Japan). The fluorescent signal was observed under a Leica DM IRB inverted microscope (Wetzlar, Germany). A Cooled-CCD (Roper Scientific, Trenton, NJ, USA) was used to view and record the images. To compare the fluorescence intensities of different parts of cells and among different cells, the integration time of the CCD was kept constant during image collection. Relevant data were collected, and relative fluorescence intensities of different cells were calculated and compared using MetaMorph software (Universal Imaging Corporation, Inc., Downingtown, PA, USA). On the labeled membranes, six sites were chosen for calculating the average fluorescence intensity of each cell (Fig. 1). The average fluorescence intensity of each kind of labeled cell was calculated from 10 separate measurements (Fig. 2).

image

Figure 1. Schematic diagram of the sites chosen for calculation of fluorescence intensity on the cell surface. Six lines each cross the cell membrane twice. The datum on each of the intersecting points (1–6 and 1′−6′) was recorded for the calculation of average intensity and for comparison between the two poles of the cell.

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image

Figure 2. Comparison of fluorescence intensities of polar ends of central cells and egg cells in tobacco (Nicotiana tabacum). The open and closed columns represent the mean fluorescence intensities on the strongly and weakly fluorescing ends of the cells, respectively. The value shown is the average value of 10 randomly selected cells. Error bars indicate the standard deviation of the means for 10 independent experiments.

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Results and Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowlegements
  7. References

Isolation of viable female cells

Isolated, unfertilized female protoplasts were viable, as indicated by strong FDA fluorescence and active streaming of the cytoplasm several hours after isolation. Because FITC-Con A is a marker for protoplast membrane integrity, the presence of the fluorescent signal only on the plasma membrane and not in the cytoplasm served to indicate further the viability of the female cells. A cell wall was not present, as judged by the absence of Calcofluor white fluorescence and easy cell fusion, indicating that the isolated female cells were complete protoplasts. Intact embryo sacs with female cells were also isolated. In this case, both the labeled and unlabeled female cells could be clearly observed in situ.

The distribution of the nucleus, cytoplasm and vacuoles in the female cells was highly polarized. Two nuclei of the central cell were located at the micropylar end, whereas a large vacuole occupied most of the central cell at the opposite end (Fig. 3, panel 1a). The egg protoplast was characteristically smaller than the two synergids, and its nucleus in situ was located at the chalazal end, whereas the nucleus of the synergid was located at its micropylar end (Fig. 3, panel 2a). This polarity persisted throughout the isolation procedure.

image

Figure 3. Polar distribution of Con A binding sites on plasma membranes of female Nicotiana tabacum cells. (1a) An enzymatically isolated central cell. (1b) Fluorescence image of the cell shown in (1a). Stronger fluorescence (arrow) was present on the plasma membrane near the nucleus in the central cell, whereas weak fluorescence (arrowhead) occurred opposite to the nucleus. (2a) An enzymatically isolated egg cell. (2b) Fluorescence image of the cell shown in (2a). Stronger fluorescence (arrow) was present on the plasma membrane opposite to the nucleus in the egg cell. The arrowhead indicates the plasma membrane where weak or no fluorescence was present. (3a) A mature embryo sac. (3b) Fluorescence image of the embryo sac shown in (3a). Fluorescence on the micropylar section was stronger than that of the chalazal section. Stronger fluorescence (arrowhead) was present on the plasma membrane apart from the nucleus on the egg cell in the embryo sac. The arrow indicates the dark pole of the central cell. Bar, 10 µm.

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Polar distribution of Con A binding sites on the plasma membranes of female cells

Of the 110 FITC-Con A labeled central cells that were analyzed, 79.1% showed a polar distribution of Con A binding sites, 12.7% showed no obvious polarity and 8.2% showed no fluorescence (Table 1). The polarity of binding site distribution was indicated by the presence of strong fluorescence on the part of the membrane near the nucleus, whereas weak or no fluorescence was detected on the opposite part of the cell membrane (Fig. 2; Fig. 3, panel 1b). In 77.4% of the egg cells, Con A binding sites were also found to have a polar distribution on the plasma membrane. The distribution of Con A sites differed from that observed for central cells; in egg cells, denser fluorescence was found on the section of plasma membrane located opposite to the nucleus (Fig. 3, panel 2b; Table 1).

Table 1.  Frequency of diverse distribution of Con A binding sites on the plasma membranes of female Nicotiana tabacum cells
Cell typeCells measuredCells with polar-distributed Con A binding sites (%)Cells with undetermined fluorescence polarity (%)Cells with no fluorescence (%)
  1. Con A, concanavalin agglutinin.

Central cells11087 (79.1%)14 (12.7%)9 (8.2%)
Egg cells 31 24 (77.4%) 4 (12.9%)3 (9.7%)

Under the same experimental conditions, isolated female cells exhibited no autofluorescence. Furthermore, both egg cells and central cells that were not labeled with FITC-Con A or FITC-Con A pretreated with α-methyl-D-mannoside under the same conditions lacked a fluorescent signal (data not shown). Thus, binding was specifically identified in our competitive inhibition experiments. In another control experiment, the plasma membranes of somatic protoplasts exhibited a smooth, uniform fluorescent ring that was observable from any orientation, as previously described by Walko et al. (1987).

To confirm further that Con A binding sites were not redistributed during the isolation and labeling procedure, isolated, intact embryo sacs were also labeled with FITC-Con A. In these embryo sacs, egg cells and central cells generally maintained their original positions (Fig. 3, panel 3a). Both types of cells yielded the same fluorescence distribution pattern in situ, as was observed for isolated female cells (Fig. 3, panel 3b). As previously reported, enzymatic treatment did not noticeably change the distribution of lectin binding sites (Burgess & Linstead, 1976; Walko et al., 1987; Fang et al., 2003). Furthermore, some egg cells and central cells were placed in 1% paraformaldehyde dissolved in 9% mannitol for fast surface fixation immediately after isolation. After labeling, the briefly fixed cells yielded the same distribution pattern of Con A sites as did the just isolated female cells (Fig. 4, panels 1a, 1b).

image

Figure 4. Fluorescence images of female Nicotiana tabacum cells treated with brief fixation, prolonged re-incubation or high temperature. (1a) A central cell. (1b) Fluorescence image of the central cell shown in (1a), briefly fixed before fluorescein isothiocyanate (FITC)-concanavalin agglutinin (Con A) labeling. Fluorescence was stronger on the section of the membrane near the nucleus (arrowhead). The arrow indicates the section of the membrane with weak fluorescence. (2a) A central cell. (2b) Fluorescence image of the central cell shown in (2a), re-incubated for 17 h at room temperature after FITC-Con A labeling. The fluorescence was stronger on the section of the membrane near the nucleus (arrowhead). The arrow indicates the section of the membrane with weak fluorescence. (3a) An egg cell. (3b) Fluorescence image of the egg cell shown in (3b), re-incubated for 17 h at room temperature after FITC-Con A labeling. The arrowhead indicates the section of the membrane with a strong fluorescent signal. The arrow indicates the plasma membrane where the fluorescence was much weaker or absent. (4a) An egg cell. (4b) A fluorescence image of the cell shown in (1a), labeled with FITC-Con A at 37°C. The cells exhibit a patch pattern of fluorescence, which is different from that obtained at room temperature. Bar, 10 µm.

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To test the possible influence of membrane fluidity on Con A binding site distribution, labeled female cells were incubated at 25°C for 2–24 h. The polar distribution pattern was stable, and no obvious change was observed even after 17 h of incubation (Fig. 4, panels 2a, 2b, 3a, 3b). The effect of temperatures ranging from 4 to 37°C was also examined. At or below room temperature, no influence on binding pattern or fluorescent intensity was observed. However, patch fluorescence was present on the plasma membrane of egg cells at 37°C (Fig. 4, panels 4a, 4b).

We tested the influence of azide and colchicine on FITC-Con A binding and Con A binding site distribution according to the methods of Johnson et al. (1975). The female cells were treated with 5–15% azide and 0.06% colchicum, and no notable difference in binding site distribution was found between the cells with or without treatment (data not shown). Taken together, our results indicate that the polar distribution of Con A binding sites is a stable and typical feature of both egg cells and central cells. The membrane of unfertilized female cells was a mosaic of two regions, one with a strong fluorescent signal and the other with little or no fluorescence. The presence of this phenomenon in tobacco is surprisingly similar to that found in animals.

The polar distribution of Con A binding sites is important in animals. Mosaicism in the organization of Con A binding sites on the plasma membrane of unfertilized egg cells was first reported for murine egg cells (Johnson et al., 1975). Subsequently, fertilized sea urchin egg cells were also found to exhibit a polar distribution of Con A binding sites on their plasma membranes (McCaig & Robinson, 1982). In lower plants, the interaction of lectins with their binding sites is essential for gametic recognition, adhesion and fusion. In Fucus, Con A binds strongly to the egg surface but not to sperm, and this binding to the eggs inhibits fertilization (Callow, 1985). In the red alga Aglaothamnion oosumiense, Con A binds to the entire spermatial surface except the spermatial appendages; during fertilization, Con A binding sites on the spermatial surface move toward the area contacting the trichogyne and accumulate on the surface of the fertilization canal. Spermatial binding to trichogynes is inhibited by preincubation of the spermatia with Con A.

These experiments suggest that Con A binding sites on the spermatial surface are involved in gamete recognition (Kim & Kim, 1999). In maize, Con A binding sites are also found on egg and zygote cell surfaces and accumulate on the cell plate where the new cell wall was formed during the first zygotic division (Sun et al., 2002a). This report presents the first evidence that Con A binding sites are polarly distributed on both central cells and egg cells. Therefore, Con A binding sites may be defined as another parameter of plasma membrane polarity. The distribution of Con A binding sites on animal and plant female gametes is obviously similar, implying the existence of similar fertilization mechanisms for animals and plants.

The central cells and egg cells were mature in their preparation for fertilization before they were isolated. Central cells and egg cells in situ have incomplete cell walls with naked plasma membranes for fusing with a sperm cell. In previous studies, the morphological polarity of the female cells before fertilization was identified by the location of the nucleus and organelles. The two polar nuclei of each mature central cell were located at the cell's micropylar end, whereas the nucleus of each mature egg cell was located at the cell's chalazal end. In the present study, the polarity of Con A binding site distribution was found to be highly consistent with this morphological polarity. The Con A binding sites obviously accumulated on the section of the plasma membrane near the polar nuclei. However, in the egg cell, Con A binding sites accumulated on the section of the plasma membrane that was located opposite to the nucleus. The results are evidence for distinct differences in the membrane surfaces of central cells and egg cells regarding the location of the nucleus and of sperm cell entry. These findings offer new data helpful for understanding the hypothesis that male–female gamete recognition exists in higher plants, and that glycoproteins play a key role in this recognition (Dumas et al., 1984). Although experimental confirmation of the significance of the opposite polarities of Con A binding site distribution in egg cells vs central cells is not yet possible, it is proposed that such polarized ConA binding sites could be involved in gamete recognition.

Acknowlegements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowlegements
  7. References

This research is supported by the National Natural Science Foundation of China (30370743, 90408002), National Outstanding Youth Science Fund (30225006) and ‘PCSIRT’.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowlegements
  7. References
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