The fusion of sperm cells and the function of male germ unit (MGU) of tobacco (Nicotiana tabacum L.)

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1 Sex Plant Reprod (1998) 11: Springer-Verlag 1998 ORIGINAL PAPER selor&:hui Qiao Tian Scott D. Russell The fusion of sperm cells and the function of male germ unit (MGU) of tobacco (Nicotiana tabacum L.) csim&:received: 25 August 1997 / Revision accepted 16 March p&:Abstract Sperm cells released from in vivo-in vitro grown pollen tubes of tobacco are associated in pairs and initially enclosed by the plasma membrane of the pollen tube. When the sperm cells are placed together, using glass microinjector needles, in an enzymatic solution, up to half undergo cellular fusion with subsequent nuclear fusion. The frequency of sperm cell fusion decreases with time during the elongation of the pollen tube, suggesting that mechanisms inhibiting self-fusion of sperm cells may develop as the pollen tube elongates through the style toward the ovule. This tendency may play an important role in inhibiting fusion of the two sperm cells inside the calcium-rich synergid where the male germ unit dissociates and sperm cells are transported to their target cells - the egg and central cell. dwk&:key words Fusion Nicotiana tabacum Male germ unit Sperm cell&bdy: Numerous publications have now emerged on isolated sperm cells (reviews: Southworth and Knox 1988; Russell 1991; Theunis et al. 1991). Experimental work has established that isolated sperm cells may be used to form successful fusion products with egg cells of the same species (maize: Kranz et al. 1991; Faure et al. 1994; and wheat: Kovács et al. 1995), with egg cells of other species (Kranz et al. 1995) and with somatic cells (Mo and Yang 1992). In addition to these more conventional fusions, Mo and Yang (1992) have fused sperm cells of the same and different species using polyethylene glycol (PEG) solutions in several bicellular pollen species, and Kranz et al. (1995) have fused maize sperm cells with electrofusion. We report here on the fusion of isolated sperm cells of tobacco with sperm cells from the same and different pollen tubes with the goal of enhancing our understanding of sperm cell development and the function of the male germ unit. Introduction Flowering plant sperm cells are structurally simple haploid cells that are essentially entirely dependent on the surrounding cytoplasm for nutrition and transport. Although these cells closely resemble ordinary protoplasts, in vitro manipulation of flowering plant gametes has only begun to be explored, despite the potential attractiveness of reproductive cells for plant cell engineering (Keijzer et al. 1988). Using the special biological and genetic characteristics of sperm cells, new methods of plant breeding may be possible in the future that allow double fertilization mechanisms to be better understood. H.Q. Tian S.D. Russell ( ) Department of Botany and Microbiology, University of Oklahoma, Norman, OK , USA srussell@ou.edu Tel ; Fax Permanent address: H.Q. Tian, College of Life Sciences, Wuhan University, Wuhan, China&/fn-block: Materials and methods To obtain the highest quality sperm cells from the bicellular pollen of tobacco, pollen tubes were grown using the in vivo-in vitro technique (Shivanna et al. 1988). Styles were pollinated in vivo, and pollen tubes were allowed to grow for a predetermined amount of time. Then styles were cut near the growing pollen tubes at predetermined positions on the style (Fig. 1) and floated in a culture medium of 0.01% (w/v) H 3 BO 3, 0.01% (w/v) KH 2 PO % (w/v) CaCl 2 2H 2 O and 15% (w/v) sucrose for several hours until tubes emerged from the cut tips (Tian and Russell 1997b). Normally, pollen tube growth through the 4-cm style of tobacco requires 2 days from pollination to fertilization (Tian and Russell 1997a). Four stages were sampled to examine maturational changes in sperm cells over time. Style lengths of 1, 2, 3 and 4 cm (cut at 13, 20, 27 and 34 h after pollination, respectively) were grown in culture medium 4 8 h, until numerous pollen tubes emerged from the cut end of a style. The style was then immersed in a 9% (w/v) mannitol solution to burst the pollen tubes and release the paired, intact sperm cells into the solution. Sperm cell fusion was induced using solutions containing 0 2% (w/v) cellulase (Onozuka R-10), 0 2% (w/v) pectinase (Serva), 0 25 mm CaCl 2, 1 mm MgSO 4, 5 mm KH 2 PO 4, 3 mm 2[N-morpholino]ethanesulfonic acid and 1% (w/v) bovine serum albumin, ph 5.4. Osmolality of the fusion solution was adjusted to

2 172 Fig. 1 Tobacco gynoecium showing the position of stylar excision 13, 20, 27 and 34 h after pollination, respectively. Styles are immersed in a growth buffer until pollen tubes emerge from the cut end, 4 8 h after excision&/f :c.gi mosmol/kg H 2 O using % (w/v) mannitol. Between 30 and 50 µl of fusion solution was placed on a slide and covered with mineral oil. Sperm cells were collected and transferred into the fusion solution with a microinjector and brought into contact for fusion. In some cases, 5% PEG (molecular weight 3350) was added to induce fusion. Fusion products were maintained in a KM8p medium (Kao and Michayluk 1975). Results Newly isolated sperm cells Release of sperm cells from emergent pollen tubes occurs nearly as soon as the style is transferred to the 9% mannitol solution. Of the two sperm cells, the one initially associated with the vegetative nucleus is almost always the smaller. The larger sperm cell is appressed to the smaller one and is not directly associated with the vegetative nucleus (Fig. 2a). Within several minutes of isolation, the vegetative nucleus usually broke down; however, the two sperm cells remained associated, with pollen cytoplasmic material apparently wrapped around the two sperm cells, maintaining their ellipsoidal to elongated shape in the 9% mannitol solution (Fig. 2b). Achieving adhesion of the two sperm cells from a single pollen tube was apparently inhibited by this surrounding material. Sperm cells from different pollen tubes have the additional pollen plasma membrane that has to be removed before adhesion. Sperm cells from the same pollen tube are already in contact with each other within the pollen membrane, although at this stage they do not fuse. When the pairs of sperm cells were transferred to the enzymecontaining solution, however, the adhesion between the two sperm cells almost immediately disappeared, the two sperm cells detached, and they quickly assumed a rounder shape (Fig. 2c). If paired sperm cells were kept in the Fig. 2a f Isolation and manipulation of sperm cells. Bars 10 µm, except 2d, 30 µm. a Two sperm cells (S ua and S vn ) and vegetative nucleus (VN) soon after release from the pollen tube b Two sperm cells remain associated by cytoplasmic material (arrows). Vegetative nucleus has already disintegrated c Two separated sperm cells in enzyme solution d Sperm cells were positioned next to egg cells (e) using a glass needle e Pair of adherent sperm cells in enzyme solution f Sperm cells in high concentrations of enzyme (2% cellulase and 2% pectinase) solution degenerate after 20 min. 950&/f :c.gi

3 173 Fig. 3 Effect of different concentrations of cellulase and pectinase on the fusion frequency of paired isolated sperm cells after 26 h of in vitro-in vivo pollen tube growth. The standard error of each of three experiments is indicated above the corresponding bar&/f :c.gi Fig. 4 Effect of different calcium concentrations on fusion frequency of paired isolated sperm cells after 26 h of in vitro-in vivo pollen tube growth. The standard error of each of three experiments is indicated above the corresponding bar&/f :c.gi mannitol bursting solution for 30 min without enzymes, some of the connected sperm cells detached, but these single sperm cells did not adhere or fuse without further manipulation. Sperm cell fusion Sperm cells placed together using a glass needle (Fig. 2d) adhere tightly (Fig. 2e). After 3 5 min, some of the adhered sperm cells fuse, although the effectiveness of fusion is strongly modified by the concentration of enzymes, calcium and the age of sperm cells. Cellulase and pectinase were both critical for fusion and the frequency of sperm cell fusion increased with increasing enzymatic concentrations (Fig. 3). When the concentration of both enzymes was increased to 2%, 52.4% of the sperm cells fused; however, the high enzyme concentration has a deleterious effect on sperm cells and fusion products that increases with duration. After 20 min of 2% enzyme treatment, sperm cells become swollen and lose their integrity (Fig. 2f). Sperm cells could be maintained longer (50 min) in a 1% cellulase and 1% pectinase solution with only a slight decrease in fusion. Use of a 0.5% cellulase and 0.5% pectinase solution results in just one-quarter of the cell pairs fusing. Without enzymes, none of the sperm cells fused (Fig. 3). Use of either 1% cellulase or pectinase separately did not induce fusion of sperm cells, suggesting that both enzymes are required to induce fusion. In all of the enzymatic solutions containing cellulase and pectinase, some sperm cells fused without the addition of calcium, but the fusion frequency increased when calcium was added (Fig. 4). In addition to increasing the frequency of sperm fusion, calcium also increased the speed of the fusion process. In enzyme solutions without calcium, the fusion process was slow and after 5 min the fusion products were still in their original ellipsoidal shape. With the addition of calcium, only about 20 s elapsed from the onset of fusion to the rounding of the fusion products (Fig. 5a h). Figure 6 illustrates 15 fusion products collected into KM8p medium. Each of these appeared to remain intact, as viewed with phase contrast microscopy, for at least 3 h after transfer into the medium, some with apparent organelle movement in the fusion products. Spontaneous self-fusion of sperm cell pairs was uncommon, occurring only in newly formed sperm cells. Eleven hours after pollination (ca. 2 h after generative cell division; 0.7-cm stylar length), 7.3% of the newlyformed sperm cells displayed self-fusion (n=76) in the 9% mannitol solution. Nineteen hours after pollination (stage 1) and at later stages, no spontaneous fusion of related sperm cells occurred. At subsequent stages, an enzymatic treatment was required for fusion of any harvested sperm cells. Although sperm cells collected 19 h after pollination did not display spontaneous fusion in the bursting solution, they displayed the highest frequency of fusion in the enzyme solution (63.6%, n=89; Fig. 7). At later stages, the frequency decreased to 23.3% (n=60) 40 h after pollination (stage 4). The change in the frequency of fusion between stages 3 (34 h after pollination) and 4 (1.6% overall) is small enough to suggest that the mechanism preventing self-fusion of sperm cells is fully formed before stage 3.

4 174 Fig. 5a h Isolated sperm cells and fusion products. a g Fusion of sperm cells (arrows) at 3 s intervals. Egg (e) and adherent sperm cell remain unfused Bars 30 µm. h Fusion product containing two sperm nuclei Bar 10 µm&/f :c.gi Fig. 6 Population of 15 sperm fusion products. Arrowhead indicates sperm cell Bar 10 µm&/f :c.gi Fig. 7 Effect of developmental stage on fusion frequency of paired isolated sperm cells. Developmental stages (1 4) are 19, 26, 33, and 40 h after pollination using in vitro-in vivo culture, respectively. Fusion medium included 1% cellulase, 1% pectinase and 25 mm calcium. The standard error of each of four experiments is indicated above the corresponding bar&/f :c.gi In a previous study, sperm cells occasionally fused in up to 15% PEG (Tian and Russell 1997c). Sperm cells also fused during the first 2 min after the addition of 5% PEG to the enzymatic solution. When three or four sperm cells adhered, they usually fused to form a single multinucleated product. After 5 min, fusion of sperm cells sharply decreased as cells adhered to the slide. Prolonged exposure to PEG has a strongly deleterious effect on sperm cells and fusion products, similar to the effect described for high concentrations of enzymes.

5 Discussion Requirements for sperm cell fusion Prior studies of sperm cells have mainly been conducted en masse. The concentration of sperm cells was one of the important factors for the fusion of sperm cells of Zephyranthes candida according to Mo and Yang (1992): using cells/ml in a 10 30% PEG solution the fusion frequency of sperm cells was 15%, which increased with increasing concentration. At cells/ml, the frequency of bicellular fusion was 26%; at cells/ml, this decreased to 18%, with many fusions involving more than two cells. Large multinucleated fusion products were also reported in experiments using maize sperm cells in calcium-containing media (Roeckel and Dumas 1993). Recently, Zhang et al. (1997) reported calcium-induced fusion of maize sperm cells, and also found that the rates of fusion increased with increasing concentrations of calcium. Electrofusion is also effective for fusing sperm cells (Kranz et al. 1995), but detailed information about these fusions is unavailable. In our study, we used only pairs of tobacco sperm cells (from the same or different pollen tubes) to form fusion products, thereby forming only binucleated cells. Both cellulase and pectinase were required in the fusion solution for fusion to occur. Although these may remove an adherent material from the sperm cell surface, an alternative explanation is that the membrane is altered in a manner that may induce fusion. The presence of periodic acid-sensitive materials on the surface of sperm cells (Russell and Cass 1981), and the increasing inhibition of sperm-sperm fusion during development supports the presence of insoluble polysaccharides on the gamete surface. That sperm cells rounded more rapidly in media containing both of the enzymes suggests that a structural component involved in maintaining cell shape has been altered or removed. Insoluble polysaccharidic materials on the outside of sperm cells, however, likely exist in the form of a highly modified periplasm rather than as a cell wall, since later fusion with the egg cell is imperative to later reproductive success (Dumas et al. 1984). Cellulase and pectinase have been shown to be deleterious to cells of the female gametophyte (Leduc et al. 1995), and in that study they inhibit sperm fusion with the egg cell. Apparently, once inhibitors to fusion have been removed, the fusion of two sperm cells in tight contact is possible. Roeckel and Dumas (1993) and Zhang et al. (1997) report that calcium is a critical element in the fusion of sperm cells of maize. Although sperm cells of tobacco could be fused in enzymatic solutions without calcium, calcium increased fusion efficiency and decreased the time required for the fusion process from 5 min to 20 s. Our results indicate that the sperm cells of tobacco are strongly fusigenic under these conditions, but that spontaneous fusion is normally prevented in vivo. Male germ unit function 175 In angiosperms, it has been confirmed that the two sperm cells are normally physically associated with the vegetative nucleus, in both the pollen grain and tube. This association of sperm cells and the vegetative nucleus forms a structure termed the male germ unit (MGU) which functions as a unit transmitting all of the DNA of the male gametophyte - cytoplasmic and nuclear - during sexual reproduction in flowering plants (Dumas et al. 1984; Matthys-Rochon and Dumas 1988; Hu 1990; Russell et al. 1990; Mogensen 1992). In tobacco, when the pollen tube ruptures, the two sperm cells and the vegetative nucleus are released along with cytoplasmic materials of the pollen tube (Tian and Russell 1997b). Some of the cytoplasmic materials of the pollen tube appear to remain associated with the two sperm cells, linking them and the vegetative nucleus together. The MGU associations therefore still exist after the release of the MGU from the pollen tube, supporting the function of the MGU as a transmission unit. When the sperm cells are placed in an enzymatic solution, however, the material associated with the sperm cells is digested, and the sperm cells display a strongly fusigenic ability, especially at stages 1 and 2 (Fig. 7). This suggests that developmentally dependent changes occur in the MGU during pollen tube growth that strengthen the barrier to sperm cells fusing with each other during passage in the pollen tube. Upon the division of the generative cell, which occurs at approximately 9 h using the in vivo-in vitro technique, the two sperm cells are separated by a cell wall of apparently callosic material (Yu and Russell 1993) which could provide an early and rapidly formed barrier to fusion. After this time, apparently the surface of the sperm cells itself is modified by the environment of the MGU to prevent self-fusion in the very restricted space available in the pollen tube and to prevent self-fusion as the cells are ejected from the pollen tube. Inside the female gametophyte, the pollen tube and its cytoplasmic contents, including the two sperm cells, are released into one of the two synergids, which are cells characterized by an unusually high concentration of calcium (Russell 1992). Newly-released sperm cells are initially associated with each other and with the cytoplasm of the pollen tube, which stabilizes and facilitates transport of the MGU during pollen tube elongation; boundaries between the two sperm cells prevent the sperm from fusing with each other, but may also inhibit fusion with the female target cells. An additional function of the synergid may therefore be to alter the sperm cell surface, enhance the fusigenic ability of the sperm cells, and release the sperm cells from their association in the MGU. Physiological changes in one of the synergids of tobacco are evident in the cytoskeleton (Huang and Russell 1994) and in antimonate-labeled calcium (Tian and Russell 1997a) before and during the arrival of the pollen tube, and these changes appear to be pollination-dependent. This suggests that changes in the synergid prior to the

6 176 entrance of the pollen tube are functional, and not degenerative, changes, and that the synergid may have a specifically timed role in the modification of the embryo sac to facilitate the fusion of the sperm with their target cells - the egg and central cell - during fertilization. 2.p&:Acknowledgement This research was supported by US Department of Agriculture grant References Dumas C, Knox RB, McConchie CA, Russell SD (1984) Emerging physiological concepts in fertilization. What s New Plant Physiol 15:17 20 Faure JE, Digonnet C, Dumas C (1994) An in vitro system for adhesion and fusion of maize gametes. Science 263: Hu SY (1990) Male germ unit and sperm heteromorphism: the current status. Acta Bot Sin 32: Huang BQ, Russell SD (1994) Fertilization in Nicotiana tabacum: cytoskeletal modifications in the embryo sac during synergid degeneration. Planta 194: Kao KN, Michayluk MR (1975) Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126: Keijzer CJ, Wilms HJ, Mogensen HL (1988) Sperm cell research: the current status and applications for plant breeding. In: Wilms HJ, Keijzer CJ (eds) Plant sperm cells as tools for biotechnology. Pudoc, Wageningen, pp 3 8 Kovács M, Barnabás B, Kranz E (1995) Electro-fused isolated wheat (Triticum aestivum L.) gametes develop into multicellular structures. Plant Cell Rep 15: Kranz E, Bautor J, Lörz H (1991) In vitro fertilization of single, isolated gametes of maize mediated by electrofusion. Sex Plant Reprod 4:12 16 Kranz E, Wiegen P, Lörz H (1995) Early cytological events after induction of cell division in egg cells and zygote development following in vitro fertilization with angiosperm gametes. Plant J 8:9 23 Leduc N, Matthys-Rochon E, Dumas C (1995) Deleterious effect of minimal enzymatic treatments on the development of isolated maize embryo sacs in culture. Sex Plant Reprod 8: Matthys-Rochon E, Dumas C (1988) The male germ unit: retrospect and prospects. In: Wilms HJ, Keijzer CJ (eds) Plant sperm cells as tools for biotechnology. Pudoc, Wageningen, pp Mo YS, Yang HY (1992) Isolation and fusion of sperm cells in several bicellular pollen species. Acta Bot Sin 34: Mogensen HL (1992) Male germ unit: concept, composition and significance. Int Rev Cytol 140: Roeckel P, Dumas C (1993) Survival at 20 C and cryopreservation of isolated sperm cells from Zea mays pollen grains. Sex Plant Reprod 6: Russell SD (1991) Isolation and characterization of sperm cells in flowering plants. Ann Rev Plant Physiol 42: Russell SD (1992) Double fertilization. Int Rev Cytol 140: Russell SD, Cass DD (1981) Ultrastructure of the sperms of Plumbago zeylanica. l. Cytology and association with the vegetative nucleus. Protoplasma 107: Russell SD, Cresti M, Dumas C (1990) Recent progress on sperm characterization in flowering plants. Physiol Plant 80: Shivanna KR, Xu H, Taylor P, Knox RB (1988) Isolation of sperms from the pollen tubes of flowering plants during fertilization. Plant Physiol 87: Southworth D, Knox RB (1988) Methods for isolation of sperm cells from pollen. In: Wilms HJ, Keijzer CJ (eds) Plant sperm cells as tools for biotechnology. Pudoc, Wageningen, pp Theunis CH, Pierson ES, Cresti M (1991) Isolation of male and female gametes in higher plants. Sex Plant Reprod 4: Tian HQ, Russell SD (1997a) Calcium distribution in fertilized and unfertilized ovules and embryo sacs of Nicotiana tabacum L. Planta 202: Tian HQ, Russell SD (1997b) Micromanipulation of male and female gametes of Nicotiana tabacum: I. Isolation of gametes. Plant Cell Rep 16: Tian HQ, Russell SD (1997c) Micromanipulation of male and female gametes of Nicotiana tabacum: II. Preliminary attempts for in vitro fertilization and egg cell culture. Plant Cell Rep 16: Yu HS, Russell SD (1993) Three-dimensional ultrastructure of generative cell mitosis in the pollen tube of Nicotiana tabacum. Eur J Cell Biol 61: Zhang G, Liu D, Cass DD (1997) Calcium-induced sperm fusion in Zea mays L. Sex Plant Reprod 10:74 82