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.
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 type||Cells measured||Cells with polar-distributed Con A binding sites (%)||Cells with undetermined fluorescence polarity (%)||Cells with no fluorescence (%)|
|Central cells||110||87 (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).
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.