fertilization in nicotiana tabacum

Planta (1993) 191:256-264 P l ~ ' l ' ~

�9 Springer-Verlag 1993

Fertilization in Nicotiana tabacum: ultrastructural organization of propane-jet-frozen embryo sacs in vivo B.-Q. Huang*, G. W. Strout, S. D. Russell

Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019~0245, USA

Received: 5 January 1993 / Accepted: 23 February 1993

Abstract. Ovules of Nico t iana tabacum L. were cryofixed with a propane-jet freezer and freeze-substituted in acetone to examine technique-dependent changes in pre- and post-fertilization embryo sacs using rapidly frozen material. Freezing quality was acceptable in 10% of the embryo sacs in the partially dissected ovules, with ice- crystal damage frequently evident in vacuoles and nuclei. One of the two synergids begins to degenerate before pollen-tube arrival in cryofixed material, with breakdown of the plasma membrane and large chalazal vacuole delayed until the penetration of the pollen tube. Ear- ly synergid degeneration involved characteristic increases in cytoplasmic electron density and the generation of cytoplasmic bodies to the intercellular space through "pinching-off'. Upon pollen-tube arrival, the male gametes are released through a terminal aperture into the degenerate synergid. Sperm cells undergo morphological alteration before gametic fusion: their mitochondrial electron density increases, the endoplasmic reticulum di- lates, cytoplasm becomes finely vacuolated and the surrounding pollen plasma membrane is lost, causing the sperm cells and vegetative nucleus to dissociate. Dis- charge of the pollen tube results in the formation of numerous enucleated cytoplasmic bodies which are either stripped or shed from sperm cells and pollen-tube cytoplasm. Two so-called X-bodies are found in the degenerate synergid after pollen-tube penetration: the presumed vegetative nucleus occurs at the chalazal end and the presumed synergid nucleus near the micropylar end.

Key words: Cryofixation - Embryo sac Fertilization Freeze substitution - Nico t iana (fertilization)

* Present address: Department of Biology, Box 8238, University Station, University of North Dakota, Grand Forks, ND 58202, USA Abbreviations: DSy=degenerate synergid; ES=embryo sac, PSy=persistent synergid; PT=pollen tube Correspondence to: S. D. Russell; FAX: 1 (405) 325 7619

Introduction

Although cryofixation has been shown to be superior to chemical fixation in preserving cell structures of plants in their native states (Tiwari and Polito 1988; Lancelle et al. 1986, 1987; Lancelle and Hepler 1992), reports using cryofixation on female gametophytes and fertilization prob- lems have been few and restricted to light-microscopic observations (Fisher and Jensen 1969; Cass and Jensen 1970; Russell 1982; Russell and Cass 1988). Because of the rapid and dynamic nature of fertilization events, the seconds to minutes required for adequate chemical preservation may induce changes in the structure of the tissue, especially when fixed with glutaraldehyde. In cotton, changes in the receptive synergid have been reported to be fixation dependent; glutaraldehyde fixation ap- peared to trigger significant morphological changes in cell integrity and organelle structure (Jensen and Fisher 1968), whereas cryofixed material displayed no changes in the degenerate synergid (DSy) until after pollen-tube (PT) penetration (Fisher and Jensen 1969). Cryofixation may be useful in determining the native condition of the female cells and refining our understanding of the com- plex cytological nature of fertilization. In this study we employed cryofixation using a propane-jet freezer fol- lowed by freeze substitution to investigate the ultrastructural organization of the megagametophyte before, during and after fertilization. This approach provides a basis for revising concepts of synergid degeneration and evalu- ating morphological changes involved in receptivity.

Nicotiana tabacum (tobacco) is typical of angio- sperms in the organization and function of its female cells according to light microscopy (Goodspeed 1947; Jos and Singh 1968) and transmission-electron-microscopy stud- ies (Mogensen and Suthar 1979). The mature female gametophyte or embryo sac (ES) contains an egg cell, a central cell with two polar nuclei, two synergid cells and three antipodal cells. As has been reported in other flowering plants, the PT of tobacco enters the ovule through the micropyle, penetrates one of the synergids, which are cells specialized for PT reception, and the sperm cells are

B.-Q. Huang et al.: Fertilization in tobacco 257

transmitted from the synergid to the female target cells. The cells of the ES each contain a full complement of organelles and also a prominent vacuole. The synergid is highly polarized. The micropylar end contains the filiform apparatus - a highly modified cell wall at the micropylar end of the ES - which is associated with numerous mitochondria and vesicles. The center of the cell contains the nucleus surrounded by profiles of dense, paral- lel, stacked rough ER. A conspicuous vacuole occupies the chalazal end of the synergid. Cell wall protuberances on the exterior of the synergid extend into the central cell in the synergid 'hook' region forming a transfer cell wall (Mogensen and Suthar 1979). Each synergid maintains direct connections with the egg cell, central cell, other synergid and ES wall.

After pollination, but prior to the arrival of the PT, one of the two synergids begins to degenerate. This is more evident in chemically fixed material observed using transmission electron microscopy (Mogensen and Suthar 1979) than using light microscopy with living material (Huang and Russell 1992). The current study addresses the organization and fertilization-induced changes in the ES of N. tabacum using physical fixation methods to re- duce the artifacts attributable to normal chemical fixation. This paper addresses those ES structures particular- ly susceptible to alteration during chemical fixation. General information on the organization of the ES is available in the classical literature cited previously.

Materials and methods

Plants of Nicotiana tabacum L. were grown as described previously (Huang et al. 1992; Huang and Russell 1992). Flowers were hand- pollinated and collected 48 h later. The ovary was cut longitudinally into several pieces and incubated in 20 mM 2-(N-morpholino)- ethanesulfonic acid (Mes) buffer (pH 5.5) containing 2 mM CaC12, 2 mM KCl and 0.2 M sucrose during further dissection and for at least 10-30 min prior to freezing (Ding et al. 1991). Ovules were then removed from the placenta, loaded in the specimen holder and frozen using an MF-7200 propane-jet freezing device (RMC Corp., Tucson, AZ, USA). After freezing, the material was substituted in a solution of 1% tannic acid in absolute acetone at -85~ for 2 d, fixed 1 d in a mixture of 2% uranyl acetate and 1% OsO4 in anhydrous acetone at -20~ warmed to 4~ overnight, transferred to fresh anhydrous acetone for 4 h at 4~ (three changes), infiltrated in Spurr's resin (Spurr 1969) for 3 d at room temperature and polymer- ized at 70~ overnight. Material was sectioned at 70-90 nm using an ultramicrotome (Ultracut; Reichert-Jung, Vienna, Austria). Sec- tions were stained 30 min in 2% uranyl acetate in 70% methanol and 10 rain in Sato's lead citrate as modified by Hanaichi et al. (1986). Sections were examined using a Zeiss 10 transmission electron microscope (Zeiss, Oberkochen, FRG) operated at 60 kV.

Results and discussion

Condition of gametophytic cells before fertilization and quality of cryopreservation. The condition of the cytoplasm was frequently very good using propane-jet-frozen and freeze-substituted material (Figs. 1-3). The normal depth of good freezing using propane-jet freezing - in which the sample is cooled on both sides of the specimen simultaneously is estimated at approx. 40 ~tm (Gilkey

and Staehelin 1986). The buffer used by Ding et al. (1991), which contains Mes, CaCI2, KCI and 0.2 M sucrose provides adequate freezing to a depth of as much as 80 ~tm or more. The principal artifacts introduced through propane-jet freezing involve the physical deformation of specimens. The loading of the holder and then its movement as the jets fire can impart appreciable physical damage. If the specimen is not kept moist during handling, incidental dehydration can also occur. The artifacts attributable to the cryoprotectant are minor for the materials in this buffer; high concentrations of sucrose may induce plasmolysis. It is unclear how penetrating each of the components may be, and cells of the ES in particular are thought to possess a higher osmotic value than so- matic cells (Huang and Russell 1989).

In the current study, material was trimmed to less than 200 ~tm; about 10% of the specimens displayed acceptable amounts of freezing damage (Figs. 1-3), with little evidence of ice-crystal formation except in the nuclei and vacuoles. None of the obvious artifacts of osmotic damage such as plasmolysis were observed, nor was there any unequivocal evidence of physical deformation in the best-frozen samples. Although some cell materials ap- peared to be oval-shaped rather than round, chemically fixed materials have yielded similar results. The most conspicuous artifact of freezing was the presence of a coarse reticulum of electron-dense material in the vacuoles (e.g., large vacuole in Fig. i). The formation of this reticulum presumably is the result of solutes separating from the solvent during ice-crystal development causing a eutectic to form.

Cryofixation appears to improve the preservation of rough-ER lumina and mitochondria (Figs. 1-2); presumably the increases in electron density compared to chemically fixed specimens are a result of reduced extrac- tion in rapid-frozen freeze-substituted tissues. Dic- tyosomes also appear to be better formed (Fig. 1) and are associated with more numerous aggregations of vesicles. The transfer cell wall of the synergid which extends into the central cell contains two mixed phases: one is electron-lucent and the other contains a denser fibrillar material; the plasma membrane is tightly appressed (Figs. 2, 3). Boundaries between the internal cell walls of the ES indicate that only a narrow cell wall exists in the chalazal boundaries between the synergid, central cell and egg cell (Mogensen and Suthar 1979); these boundaries are nar- rower in cryofixed, freeze-substituted ovules (Fig. 2). Some features, including the distribution of ribosomes, which are found both in association with ER and clumped in small aggregates in the cytoplasm, appear similar in chemical fixation (Mogensen and Suthar 1979).

Senescent cells, in particular, seem to have their degenerate appearance accelerated by chemical fixation. Cells at the chalaza appear to have graduated degeneracy at the edge of the ES, indicating that this process occurs independently of fixation method. In Fig. 1, a gradient is evident from intact cells near the chalaza (right) to in- creasingly degenerate cells at the edge of the ES (left), with cellular remnants evident in the wail of the ES at late stages (arrows). The degeneration of chalazal cells is characterized by: (1) increasing irregularities in the out-

258 B.-Q. Huang et al.: Fertilization in tobacco

Fig. 1-3. Ultrastructure of a mature cryofixed embryo sac of Nico- tiana tabacum before fertilization

Fig. 1. Antipodal cells (AC) and chalazal cells (Ch) in an unfertilized ovule. Antipodal cells contain abundant endoplasmic reticulum (ER), mitochondria (M) and dictyosomes with vesicles (single arrowhead). Remnants of degenerate chalazal cells are evident in the wall of the ES (arrows); neighboring chalazal cells show stages of degeneration. Irregular cell walls (CW) and vesicle-containing bodies (double arrowheads) are seen in chalazal cells. N, nucleus; V, vacuole. x 10000; ba r= 1 gm

Fig. 2. 'Hook' region near the attachment of the synergid (Sy) with the wall of the ES displays biphasic cell wall (14/} projections extend- ing into the central cell (CC). Abundant endoplasmic reticulum (ER) and mitochondria (M) are found in synergid cytoplasm. 1nt, integument cell; V, vacuole, x 5000; ba r= 1 gm

Fig. 3. Numerous plasmodesmata (Pd) in a cell wall (CW) of the antipodal cell (AC). N, nucleus; M, mitochondria, x 20000; bar = 1 gm


Fig. 4--6. Morphological changes in the degenerated synergid (DSy) before the arrival of the PT

Fig. 4. Beginning of DSy degeneration is indicated by increasing electron density compared to the persistent synergid (PSy). Al- though plasma membrane, tonoplast of vacuole (F), endoplasmic reticulum (ER) and other membrane systems appear intact at higher magnification, the nucleus (N) displays a modified structure and the cytoplasm becomes nearly electron opaque. Intercellular spaces in the egg apparatus may contain fragments of DSy cytoplasm (arrowheads). The PSy, central cell (CC) and integument cells (Int) remain unchanged during this process. M, mitochondria, x 8000; bar = 1 gm

Fig. 5. Cytoplasm of DSy contains intact endoplasmic reticulum (ER), mitochondria (M) and irregular lobes of cytoplasm (large arrow). Slender evaginations (arrowhead) and numerous dense vesicles are present at the edge of the DSy (small arrows), x 20000; bar =0.5 ~tm

Fig. 6. Cytoplasmic bodies and vesicles aggregate in the intercellular space between the central cell (CC), egg cell (E) and the PSy (not shown) forming electron-dense clusters (arrowheads) in this region. ER, endoplasmic reticulum; M, mitochondria; V, vacuole, x 20000; bar = 1 ~m

line of the nucleus, (2) increasing cytoplasmic electron density within mitochondria in particular, (3) formation of vesicle-containing bodies in the cytoplasm, (4) in- creased irregularity of cellular membranes and cell walls, (5) breakdown of internal membranes and ultimately cellular membranes, and finally (6) crushing of degenerated cells.

Synergid degeneration, P T arrival and P T discharge. In chemically fixed ovules of Nicotiana, the degeneration of the synergid begins when the PT is within 100 Jam, but is apparently not completed until the PT arrives (Mogensen and Suthar 1979; Huang and Russell 1992). In frozen

material, the DSy is characterized by conspicuous increases in the electron density of the cytoplasmic matrix, nuclear breakdown, dilation of ER, internal alterations in organelle structure and general osmiophilia; however, membranes appear to remain intact, including the plasma membrane, tonoplast, ER, mitochondria and plastids (Figs. 4, 5). Cellular evaginations, dense bodies and membrane-bound inclusions occur at the edge of the DSy (Fig. 5). No corresponding changes occur in the persistent synergid (PSy). The deposit of these electron-dense materials may reflect the secretion of DSy products prior to the breakdown of the cell, contributing to materials found between the egg cell, central cell and synergids (Fig. 6).


Fig. 7-8. Pollen-tube discharge and condition of the sperm cell at deposition

Fig. 7. Pollen tube (PT) aperture (arrows) through which the PT cytoplasm and contents are released into the DSy. Arrowheads indicate polysaccharide vesicles of the PT. Cell wall (CI4/) is thickened adjacent to the filiform apparatus (FA). Int, integument cell. x 8000; bar = 1 ~tm

Fig. 8. Single membrane-bound, unfused sperm cell within the retic- ulated matrix of the former vacuole of the DSy. The sperm cell contains a nucleus (N), dictyosomes (D), strongly dilated endoplasmic reticulum (ER), presumed mitochondria (M) and numerous vacuoles (l/) with electron-dense materials. An amorphous, electron- dense body (arrow) is apparently specific to the mature sperm cell. • bar=l ~tm

The position of electron-dense materials in this region and their common occurrence in angiosperm ESs (Rus- sell 1992) suggests that may be involved with one or more of the events of fertilization.

Ultrastructural evidence of cellular integrity of the DSy before PT arrival is in agreement with observations of living embryo sacs of tobacco, in which synergids retained the ability to accumulate fluorescein diacetate until PT penetration (Huang and Russell 1992). As the PT arrives at the DSy, both the plasma membrane and vacuolar membranes appear to break down completely. A pore forms at the tip of the PT (Fig. 7) and the PT releases its contents, including the apical cytoplasm, vegetative nucleus and two sperm cells into the DSy.

Cellular organelles remain largely intact during this initial stage and after PT discharge, surrounding the area formerly occupied by the vacuole (Fig. 9). The region formerly occupied by the synergid vacuole is characterized by an apparent reticulum of electron-dense material created largely by ice-crystal formation during preparation. At first, the discharged cytoplasm of the PT is seen only in the base of the DSy (Fig. 9), but this mixes and soon polysaccharide vesicles are distributed throughout

the cell (Fig. 7). Using chemical fixation, Mogensen and Suthar (1979) reported small differences between two synergids at 48 h after pollination; however, these differences may become extreme even in frozen unfertilized ESs as the PT approaches. Despite these changes the tonoplast and plasma membrane are retained in frozen material (Fig. 4).

The observations of synergid behavior reported in this study strengthen the idea that synergid degeneration is both preprogrammed and dependent on the presence of PTs for full expression. In tobacco, the onset of synergid degeneration occurs before the arrival of the PT, but the breakdown of the cell occurs only after PT penetration. Although the synergids are reported to degenerate completely prior to PT arrival in a number of flowering plants (review: Russell 1992), only indirect evidence suggests that synergid degeneration may necessarily be required for normal PT discharge. In Proboscidea, Mogensen (1978) reported that a PT that abutted the basal wall of the PSy did not enter the ES and produced an abnormal- ly thick cell wall at its tip, causing the cessation of PT growth. Similarly, Cass and Jensen (1970) reported an instance in barley where one PT entered the PSy and


Fig. 9-12. Contents of the DSy after fertilization

Fig. 9. Degenerated synergid (DSy) soon after PT discharge showing condition of the DSy and position of sperm cell (S). Irregular cell walls (14/) are continuous around the periphery of the cell in this oblique section of DSy. Electron-dense organelles and the sperm have penetrated into the region of the DSy formerly occupied by the vacuole. Distorted nuclear profiles (N) presumably represent vegetative nucleus. CC, central cell. x 4000; bar=2 lam

Fig. 10. Chalazal region of DSy after PT penetration and fertilization. The sizable electron-dense body at the chalazal end of DSy presumably represents degenerated vegetative nucleus, termed X-

body (XB) in the classical literature. Persistent synergid (PSy) is still intact at this stage. V, vacuole, x 4000; bar= 2 gm

Fig. 11. Enucleated cytoplasmic body of presumed sperm origin in DSy. This body is characterized by numerous vacuoles (I0, presumed mitochondria (M) seen as inclusions in the ER, dictyosomes (D) and an amorphous, electron-dense body (arrowhead) seen else- where only in mature sperm cells. • 17 600; bar=0.5 ktm

Fig. 12. Enucleated cytoplasmic body of presumed PT origin in DSy. This body is characterized by numerous mitochondria (M), undilated endoplasmic reticulum (ER) and polysaccharide vesicles (PI0. x 22 500; bar=0.5 gm

deposited its two sperm cells, but neither of the sperm cells were apparently able to fuse with the egg or central cell. Jensen and Fisher (1968) also report one observation in cotton where a PT initially entered the PSy and contin- ued to elongate until it entered the DSy; although the PT arrived through the side of the cell, it discharged its contents in an essentially normal manner. In contrast, though, there are a number of plants in which synergid degeneration is never reported prior to the arrival of the PT (review: Russell 1992). The common feature appears to be that all typically organized ESs possess a DSy after PT arrival. Some of the critical events of synergid degeneration including p lasma-membrane breakdown and

vacuolar lysis may be necessary for transmission of the sperm cells in normal ESs.

The condition of the receptive synergid appears to be fixation dependent. In tobacco, the differences are subtle and clearly better preserved using physical fixation, dis- playing a more highly graduated sequence of degeneration events. The most extreme case of fixation-dependent changes is in cotton, in which ovules that were chemically fixed displayed complete synergid degeneration after pollination but prior to PT arrival (Jensen and Fisher 1968), whereas ovules that were freeze substituted did not dis- play synergid degeneration until PT penetration (Fisher and Jensen 1969). The condition of synergids in other


plants appears to be variable and would likely also bene- fit from the use of freeze-substituted material for better preservation.

Condition of unfused sperm cells within the degenerate synergid, migration of sperm cells and dissociation of the male germ unit. Initially, the two sperm cells linked with the vegetative nucleus in a male germ unit (sensu Dumas et al. 1984, reviewed in Mogensen 1992) are injected into the receptive synergid through a terminal aperture in the PT (Fig. 7) and arrive centrally in the DSy in the region formerly occupied by the vacuole (Fig. 9). The force of ejection from the PT appears to dissociate them. A de- generating vegetative nucleus is frequently located near the unfused sperm cell (Fig. 9), although it may still have a prominent nucleolus at this stage (not shown). The vegetative nucleus itself seems distorted (arrowhead, Fig. 9). Apparently, the force of PT rupture may alter the form of the vegetative nucleus (which is a member of a develop- mentally terminal lineage) while not altering the shape of the sperm cell. Near the former male germ unit are located abundant enucleated cytoplasmic bodies (Figs. 11, 12) of sperm and PT origin. These presumably are generated from shear forces brought about by PT discharge and created spontaneously, similar to the formation of mi- celles. The best evidence suggests that the male germ unit association is severed during PT discharge and the sperm cells dissociate from the vegetative nucleus at this time.

Unfused sperm cells within the DSy appear to be strongly modified by their passage in and discharge from the PT (Fig. 9). As opposed to previous stages, the sperm cells become rounder and contain fewer recognizable organelles than at previous stages using either chemical fixation (Yu et al. 1992) or high-pressure freezing (data not shown). The internal membrane systems of sperm cells apparently swell and enclose cytoplasmic bodies within the cell (arrow, Fig. 8), as confirmed by serial sections. A few heritable organelles, apparently mitochondria, are evident in the cytoplasm of the sperm, but these appear to be enclosed within ER inclusions (Fig. 8). Their sequestration appears to add to the numerous mecha- nisms for diminution of potentially heritable male cytoplasmic organelles previously shown to be in effect (Rus- sell et al. 1990; reviews: Mogensen 1992; Russell 1992). Dictyosomes, ER, and a rounded nucleus are also prominent features prior to gametic fusion, as are characteristic sperm-specific bodies (arrow, Fig. 8) consisting of amorphous clusters of electron-dense bodies. These bodies are present only in mature sperm cells, are osmiophilic and pleiomorphic, with an average diameter of about 700 nm and potentially provide a marker for the fate of the male cytoplasm (data not shown).

Vacuolization and the "rounding" of sperm cells are observed in unfused male gametes deposited in the DSy (Fig. 8), but not during normal growth within the PT in tobacco (Yu et al. 1989). Late in PT passage, vacuolization and sperm-cell rounding have been reported in a number of chemically fixed sperm cells (see Rougier et al. 1992; Russell 1992) and in freeze-substituted cells using light microscopy (Cass and Jensen 1970; Russell and Cass 1981); the present study, however, is the first to

confirm these observations using rapidly frozen material and transmission electron microscopy, and supports the idea that these changes are not artifactual. An interesting effect of this trend of vacuolization is that the sperm cells may actually become larger (H.-S. Yu, personal commu- nication) while their cytoplasm becomes more diffuse, as in Populus (Russell et al. 1990).

In tobacco, the exterior of the sperm cell is delimited by a single membrane (the PT membrane apparently hav- ing been removed during PT discharge) and the surface seems to lack any distinctive features (Fig. 8). Apparently once PT discharge occurs, the inner PT plasma membrane is stripped off and releases the sperm and vegetative nucleus triggering the disassociation of the male germ unit and allowing the gametes to separate and fol- low their respective fates, as has been noted in barley (Mogensen 1988), Lycopersicon (Kadej and Kadej 1983), Plumbago (Russell 1983), Populus (Russell et al. 1990) and Spinacia (Wilms 1981). This also reveals the surface of the sperm cells within the synergid at a time during which cellular conditions are presumably changing rapidly pri- or to fertilization and may represent a crucial phase in the preparation of the sperm for fusion (Russell 1983, 1985).

Numerous enucleated cytoplasmic bodies are generated during PT discharge into the DSy (Figs. 11, 12). These appear to have two sources: enucleated cytoplasmic bodies containing dilated ER, dense mitochondria in ER inclusions and sperm-specific bodies originate from sperm cells (Fig. 11), whereas the other enucleated cytoplasmic bodies containing more conventionally organized cytoplasm, ER, mitochondria and polysaccharide vesicles likely originate from the PT (Fig. 12). The sperm-derived enucleated cytoplasmic bodies appear to originate from the dissolution of the physical association of the sperm and the vegetative nucleus as suggested in other species (Russell and Cass 1981; Russell 1992).

The unfused sperm cells move to the chalazal end of the DSy and ultimately enter the intercellular space between the egg and central cell from where they fuse with their respective female target cell. The initial movement of the sperm cells and vegetative nucleus to the chalazal end of the synergid has been theorized to occur by a "fountain" effect in which the center of the synergid remains fluid and its peripheral cytoplasm becomes gel- like, possibly as the result of endogenous aldehydes in the PT (Fisher and Jensen 1969), although the high concen- tration of calcium in the vacuole of the synergid (Chaubal and Reger 1990, 1992) could also induce this effect.

In tobacco, the center of the synergid is occupied by a vacuole that apparently remains intact until the final mo- ments prior to PT discharge (e.g., Fig. 4, 5), and the breakdown of the vacuole is correlated with intense chlorotetracycline fluorescence indicating rapid membrane uptake of calcium (Huang and Russell 1992), which may induce cytoplasmic gelling immediately. Materials ejected from the PT would therefore continue to propel contents of the PT towards the chalazal end of the DSy. In support of this hypothesis, the initial separation of PT contents from the surrounding synergid cytoplasm appears noteworthy (Fig. 9), even though it is temporary; at


mately appressing the sperm to the surfaces of the egg and central cells, or perhaps th rough surface migra t ion by means of the c o m p o u n d s present in the DSy and on the discharged sperm cells.

After the t ransmission of the sperm and fertilization have occurred, electron-dense bodies similar to reported X-bodies have been observed in the chalazal region of the DSy. Presumably, the X-body in Fig. 10 represents the degenerat ing PT vegetative nucleus, which is the larger of the two nuclei expected to be present in the DSy. Reorganization in the zygote. The most notable change in the egg cell after fertilization is the appearance of numerous small vacuoles t h roughou t the cytoplasm of the cell (Fig. 13). I rregular and thick cell wails are seen at the micropylar side of the zygote (Fig. 13); these are associated with electron-dense bodies at the edge of the zygote and may represent fragments of the DSy. Some mito- chondr ia and plastids containing starch grains are observed in the perinuclear region. The zygote remains un- divided for 6-7 d after pollination.

This work was supported by United States Department of Agricul- ture NRICGP grants 88-37261-3761 and 91-37304-6471. We thank Mr. Hong-Shi Yu for technical assistance in preparing the specimens and for helpful discussion during the preparation of the manuscript.

Fig. 13. Zygote contains chalazal nucleus (N), perinuclear mitochondria, plastids with starch grains and a large central vacuole (I/). Abundant vacuoles form in the micropylar cytoplasm after fertilization and the cell wall becomes significantly thicker. Electron-dense bodies (arrowheads) near the DSy are conspicuous. The PSy has apparently begun to degenerate at this time. lnt, integument cell. x 3400; bar=2 gm

later phases the polysacchar ide vesicles of the PT are observed t h roughou t the DSy (Figs. 7, 10), indicating that the contents of the PT are redistributed.

The volume of the synergid m ay initially remain the same as before discharge or increase slightly, a l though it is likely that the volume of the DSy decreases sharply dur ing later stages as reported in co t ton (Jensen and Fisher 1968). Wi th the DSy decreasing in volume and the sperm cells apparent ly increasing in volume, the likeli- h o o d of contact with the plasma membranes of the egg and central cell are presumably enhanced; however, the exact mechanism by which the sperm cells migrate to the edge of the synergid after PT discharge remains to be resolved. If sperm are indeed non-moti le , this can be ac- complished th rough cont inued flow of PT materials, ulti-

References

Cass, D. D., Jensen, W. A. (1970) Fertilization in barley. Am. J. Bot. 57, 62 70

Chaubal, R., Reger, B. J. (1990) Relatively higher calcium is local- ized in synergid cells of wheat. Sex. Plant Reprod. 3, 98-102

Chaubal, R., Reger, B. J. (1992) The dynamics of calcium distribution in the synergid cells of wheat after pollination. Sex. Plant Reprod. 5, 206-213

Ding, B., Turgeon, R., Parthasarathy, M. V. (1991) Routine cryofixation of plant tissue by propane jet freezing for freeze substitution. J. Electron Microsc. Tech. 19, 107-117

Dumas, C., Knox, R. B., McConchie, C. A., Russell, S. D. (1984) Emerging physiological concepts in fertilization. What's New Plant Physiol 15, 17-20

Fisher, D. B., Jensen, W. A. (1969) Cotton embryogenesis: the iden- tification, as nuclei, of the X-bodies in the degenerated synergid. Planta 84, 122-133

Gilkey J. C., Staehelin, L. M. (1986) Advances in ultrarapid freezing for the preservation of cellular ultrastructure. J. Electron Mi- crosc. Tech. 3, 177-211

Goodspeed, T. H. (1947) Maturation of the gametes and fertilization in Nicotiana. Madrofio 9, 11(~120

Hanaichi, T., Sato, T., Hoshino, M., Mizuno, N. (1986) A stable lead stain by modification of Sato's method. Proc. XIth Int. Cong. Electron Microsc. Kyoto, p. 2181

Huang, B.-Q., Russell, S. D. (1989) Isolation of fixed and viable eggs, central cells and embryo sacs from ovules of Plumbago zeylanica. Plant Physiol. 90, 9-12

Huang, B.-Q., Russell, S. D. (1992) Synergid degeneration in Nico- tiana: a quantitative, fluorochromatic and chlorotetracycline study. Sex. Plant Reprod. 5, 151-155

Huang, B.-Q., Pierson, E. S., Russell, S. D., Tiezzi, A., Cresti, M. (1992) Video microscopic observations of living, isolated embryo sacs of Nicotiana and their component cells. Sex. Plant Reprod. 5, 15(~162

Jensen, W. A., Fisher, D. B. (1968) Cotton embryogenesis: The en- trance and discharge of the pollen tube in the embryo sac. Planta 78, 158-183


Jos, J. S., Singh, S. P. (1968) Gametophyte development and em- bryogeny in Nicotiana. J. Indian Bot. Soc. 47, 117-128

Kadej, A., Kadej, F. (1983) The ultrastructure of the egg cell in Lycopersicon esculentum Miller at the moment of fertilization. In: Fertilization and embryogenesis in ovulated plants, pp. 249- 252, Erdelsk~, O. ed. VEDA, Bratislava, Czechoslovakia

Lancelle, S. A., Hepler, P. K. (1992) Ultrastructure of freeze-substituted pollen tubes of Lilium longiflorum. Protoplasma 167, 215- 230

Lancelle, S. A., Callaham, D. A., Hepler, P. K. (1986) A method for rapid freeze fixation of plant cells. Protoplasma 131, 153 165

Lancelle, S. A., Cresti, M., Hepler, P. K. (1987) Ultrastructure of the cytoskeleton in the freeze-substituted pollen tubes of Nicotiana aIata. Protoplasma 140, 141-150

Mogensen, H. L. (1978) Pollen tube-synergid interactions in Pro- boscidea louisianica (Martineaceae). Am. J. Bot. 65, 953-964

Mogensen, H. L. (1988) Exclusion of male mitochondria and plastids during syngamy in barley as a basis for maternal inheri- tance. Proc. Natl. Acad. Sci. USA 85, 2594 2597

Mogensen, H. L. (1992) Male germ unit: concept, composition, and significance. Int. Rev. Cytol. 140, 129-147

Mogensen, H. L., Suthar, H. K. (1979) Ultrastructure of the egg apparatus of Nicotiana tabacum (Solanaceae) before and after fertilization. Bot. Gaz. 140, 168 179

Rougier, M., Jnoud, N., Said, C., Russell, S., Dumas, C. (1992) Inter- specific incompatibility in Populus: inhibition of tube growth and male germ unit behavior in Populus deltoides x P. alba cross. Protoplasma 168, 107-112

Russell, S. D. (1982) Fertilization in Plumbago zeylanica: entry and discharge of the pollen tube into the embryo sac. Can. J. Bot. 60, 2219 2230

Russell, S. D. (1983) Fertilization in Plumbago zeylanica: gametic fusion and the fate of the male cytoplasm. Am. J. Bot. 70, 416 434

Russell, S. D. (1985) Preferential fertilization in Plumbago: ultrastructural evidence for gamete-level recognition in an angiosperm. Proc. Natl. Acad. Sci. USA 82, 6129-6132

Russell, S. D. (1992) Double fertilization. Int. Rev. Cytol. 140, 357- 388

Russell, S. D., Cass, D. D. (1981) Ultrastructure of the sperm of Plumbago zeylanica: 1. Cytology and association with the vegetative nucleus. Protoplasma 107, 85-107

Russell, S. D., Cass, D. D. (1988) Fertilization in Plumbagella mi- crantha. Am. J. Bot. 75, 778 781

Russell, S. D., Rougier, M., Dumas, C. (1990) Organization of the early post-fertilization megagametophyte of Populus deltoides: ultrastructure and implications for male cytoplasmic transmission. Protoplasma 155, 153-165

Spurt, A. R. (1969) A low-viscosity resin embedding medium for electron miscroscopy. J. Ultrastruct. Res. 26, 31-34

Tiwari, S. C. Polito, V. S. (1988) Organization of the cytoskeleton in pollen tubes of Pyrus communis: a study employing conventional and freeze-substitution electron microscopy, immunofluores- cence, and rhodamine-phalloidin. Protoplasma 147, 100-112

Wilms, H. J. (1981) Pollen tube penetration and fertilization in spinach. Acta Bot. Neerl. 30, 101 122

Yu, H.-S., Hu, S.-Y., Russell, S. D. (1992) Sperm cells in pollen tubes of Nicotiana tabacum L.: three-dimensional reconstruction, cytoplasmic diminution, and quantitative cytology. Protoplasma 168, 172-183

Yu, H.-S., Hu, S.-Y., Zhu, C. (1989) Ultrastructure of sperm cells and the male germ unit in pollen tubes of Nicotiana tabacum. Proto- plasma 152, 29-36

fertilization in nicotiana tabacum

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