[current topics in developmental biology] current topics in developmental biology volume 34 volume...

Gametes and Fertilization in Flowering Plants

Darlene Southworth Department of Biology Southern Oregon State College Ashland, Oregon 97520

I . Introduction 11. Male Gametes

A. Meiosis to Gametogenesis B . Sperm Structure C. Pollen Tube Growth

A. Meiosis to Embryo Sac Formation B . Egg Structure C. Central Cell Structure

IV. Double Fertilization

111. Female Gametes

A. Fertilization in Vivo B . Fertilization in V i m

V. Summary

References

1. Introduction

The field of flowering plant gametes and fertilization is moving from descriptive ultrastructure to precise descriptions of development, experimental investiga- tions, and molecular approaches, although the molecular and cellular activities that regulate gametogenesis and fertilization are as yet unknown. The purpose of this article is to provide an overview of plant gametes and fertilization, including current research and reviews on structure and function, and to compare plant gametes and fertilization events with those in animals.

The process of fertilization in flowering plants is not well known, in part because the gametes are not free in the environment, but are embedded in other cells or tissues. Gametes in flowering plants are relatively sparse compared to gamete production by marine invertebrates or by brown algae. Neither sperm nor eggs are readily released as in vertebrates or ferns.

Flowers enclose male and female organs in which meiosis occurs: anthers (“male gonads”) and ovaries (“female gonads”). In anthers, meiosis produces haploid cells that develop into pollen. In contrast to animal meiosis, in which sperm are produced directly, male haploid cells in flowering plants divide, even-

Current Topics in Developmenral Bioloxy, Val 34 Copyright 0 1996 by Academic Press. Inc. All rights of reproduction in any form reserved. 259

260 Darlene Southworth

tually producing two sperm cells per pollen grain. Ovaries enclose ovules or immature seeds. In each ovule is one meiotic cell surrounded by protective tissues. In the most common pattern, three of the four meiotic products die, and the one surviving haploid cell divides three times. One of the resulting haploid cells becomes the egg.

Pollen lands on the stigma, an extension of the ovary, and grows a long pollen tube into an ovule. Sperm are moved through the pollen tube and deposited near the egg. Fertilization, a fusion of gametes, takes place, resulting in a zygote that grows into an embryo. In addition, a second fertilization between a sperm and the central cell also occurs, giving rise to endosperm, a nutritive tissue.

II. Male Gametes

Sperm cells in flowering plants are structurally unique among plant cells. Spe- cialized features of sperm cells include the absence of cell walls (a departure from the textbook definition of plant cells as having cell walls), a small cytoplasmic volume, condensed chromatin, microtubule bundles, and a spindle shape with long extensions. Reviews have emphasized particular aspects of flowering plant sperm structure including the cytoskeleton of sperm and generative cells (Palevitz and Tiezzi, 1992), association of two sperm cells with the vegetative nucleus in the male germ unit (Mogensen, 1992), evolution of double fertilization (Knox et al . , 1993), and isolation of sperm cells (Russell, 1991; Theunis et al . , 1991; Chaboud and Perez, 1992).

A. Meiosis to Gametogenesis

1. Pollen Formation

Meiosis takes place in anthers of very young flower buds. Following meiosis, haploid cells differentiate into pollen (Bedinger, 1992) (Fig. 1). They develop a complex cell wall, take up nutrients, and differentiate. Each haploid cell divides asymetrically into a larger vegetative cell and a smaller generative cell, both enclosed within the pollen grain wall. The vegetative cell will not divide again, but will develop a long extension that is the pollen tube. The vegetative cell acts as a nurse or Sertoli cell surrounding the generative cell and later the sperm.

Contact between the vegetative cell and the generative cell is close, forming a type of cell-cell junction in which membranes closely parallel each other (South- worth, 1992). No membrane bridges link vegetative and generative cells. Al- though the separation distance between the cell membranes of the vegetative and generative cells is uneven in aldehyde-fixed thin sections (Fig. l), in freeze- fractured or freeze-substituted pollen, the separation distance is uniform, and

7. Gametes and Fertilization in Flowering Plants 261

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POLLEN

DISCHARGE FROM ANTHER GENERATIVE CELL D I V I S I O N

BICELLULAR POLLEN

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I POLLEN TUBE GROWTH

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V

TRICELLULAR POLLEN

GENERATIVE CELL D I V I POLLEN TUBE GROWTH

POLLEN TUBE WITH

Fig. 1 Sperm development pathways in flowering plants. Pollen mother cells in the anther (A) divide by meiosis, producing a tetrad of haploid cells (B) that grow and divide (C) to form the generative cell ( G ) and the vegetative cell (v). In bicellular pollen (D), division of the generative cell occurs in the pollen tube (E). In tricellular pollen (F) the generative cell divides before discharge of the pollen; the pollen tube develops with sperm cells already present (G). Both bicellular and tricellular pollen produce a pollen tube containing two sperm cells (H).


plasma membranes are parallel (Cresti et al . , 1987; Southworth et al., 1989a). In freeze-fractured pollen, the surface of the generative cell is indented. Distinctive fracture patterns of parallel ridges occur on the inner vegetative cell membrane at the indentations (Fig. 2). The ridged pattern suggests a junction, either for maintaining cell contact or for passage of materials, but does not precisely resemble tight or gap junctions.

Undoubtedly, the vegetative cell is responsible for nourishment of the generative cell and for controlling its cell cycle; however, there is no direct evidence for transfer of molecules between them. The vegetative cell stores food during its development within the anther and later takes up nutrients from the style during pollen tube growth.

The generative cell divides to form two sperm (Fig. 1). When pollen grains are released from the anther, they are either bicellular with one vegetative cell and one generative cell or tricellular with one vegetative cell and two sperm cells, depending on the timing of generative cell division. An old terminology inac- curately referred to pollen as “binucleate” or “trinucleate,” and failed to recognize that these nuclei were in separate cells.

2. Development of the Generative Cell

The generative cell differentiates and develops structural properties later found also in sperm. After the first pollen mitosis, the wall around the generative cell decreases and in some species disappears completely. The generative cell rounds up and lies in a cavity or pocket within the vegetative cell. A generative cell is surrounded by its own cell membrane appressed against an inner cell membrane of the vegetative cell pocket. Generative cells are closely associated with the vegetative nucleus, often with a cellular extension entwined through the vegetative nucleus but always separated from it by the two cell membra,ies (Mogensen, 1992; Yu and Russell, 1993).

Chromatin condenses to make a densely packed nucleus (Fig. 3). In lily, generative cells and sperm form male-specific histones that are variants of histones H2B and H3 (Ueda and Tanaka, 1995a,b). These altered histones are not found in vegetative cell nuclei. No specific function has been demonstrated yet, although they correlate with condensation of chromatin in generative cells and in sperm and suggest a diminished capacity for transcription.

The cytoskeleton of generative cells is distinct from that of somatic cells. Immature generative cells are spheroidal with a meshwork of microtubules (Zhou and Yang, 199 1). As microtubules elongate, the generative cell becomes ellipsoi- dal and finally spindle shaped with cytoplasmic extensions at one or both ends of the cell (Fig. 3). Parallel microtubule bundles form a slightly spiraled cage or basket of microtubule bundles in the thin cytoplasmic layer around the nucleus (Del Casino et al . , 1992; Bohdanowicz et al . , 1995). The extensions vary in precise shape and location, but consistently include ends of microtubule bundles.


Fig. 2 Electron micrographs of the generative cell of Amaryllis belladonna. (A) Thin section across the sperm cell, through the nucleus and microtubule bundles in the cytoplasm. The interface between the generative cell and vegetative cell is convoluted (at right). GN, Generative cell nucleus; VC, vegetative cell. Bar: 0.2 pm. (B) Freeze-fracture face of the vegetative cell membrane appressed to the generative cell. Bar: 0.5 pm. (C) Ridges on the fracture face of the vegetative cell at indentations into generative cell. Bar: 0.2 pm. [Adapted from Southworth er al. (1994), Fig. 1 . p. 539; Figs. 5 and 6 , p. 541.1


Fig. 3 Light micrographs of generative cells. (A) Generative cell of lily; phase contrast. Bar: 10 pm. (B) Fluorescence of the generative cell nucleus of lily as in (A), stained with DAPI. (C) Microtubule bundles in the generative cell in a pollen tube of Nicoriana tubacum stained with FITC- labeled antibodies to a-tubulin (Del Casino et al . , 1993). confocal scanning laser microscopy (Y.-Q. Li, unpublished). Bar: 5 pm.

Microtubule bundles are not regularly bridged to the plasma membrane or to the nuclear envelope (Cresti et al., 1990) (Fig. 2).

Evidence for actin microfilaments in generative cells is conflicting (Taylor er al., 1989; Palevitz and Liu, 1992; Knox et al., 1993) although microfilaments have been observed in vegetative cells (Lancelle et al., 1987; Palevitz and Liu, 1992). In Brassica, actin filaments encircled the generative cell, but it was not clear whether the actin was in the generative cell or in the vegetative cell surrounding it (Hause er d . , 1992). Knox et al. (1993) showed short actin microfilaments in generative cells of lily in isolated vegetative cell protoplasts in culture. In contrast to the rapid movement of organelles in the vegetative cell, cytoplasmic streaming is minimal in generative cells (Pierson et al., 1990). This is consistent with the absence of actin microfilaments that would be involved in cytoplasmic streaming (Palevitz and Liu, 1992).

Generative cell division occurs either within the pollen grain before shedding


of pollen from anthers or in the pollen tube after pollination and pollen tube germination (Fig. 1). Timing of cell division is hereditary, but regulative mechanisms are unknown. Generative cell division within the pollen grain is a typical mitosis with formation of a cell plate. Division within the pollen tube is unusual in that the cell plate is oblique or longitudinal to the long axis of the pollen tube (Terasaka and Niitsu, 1989). A skewed spindle is formed with chromosomes moving nearly parallel to the metaphase plate. The cell plate is reduced.

The cytoskeleton of the generative cell gives rise to the cytoskeleton of the sperm cell. In tobacco, generative cell microtubule bundles disassemble, then reorganize as a spindle apparatus, followed by formation of new microtubule bundles in sperm (Palevitz, 1993; Yu and Russell, 1993). In Trudescuntiu, generative cell microtubule bundles participate directly in spindle formation by kinetochore fiber capture (Palevitz and Cresti, 1989; Palevitz, 1990; Liu and Palevitz, 199 1, 1992; Palevitz and Tiezzi, 1992). Interphase microtubules do not disassem ble. Microtubule bundles are present during prophase and become part of the spindle apparatus. After cytokinesis, the original microtubule bundles of generative cells form the microtubule bundles of sperm. In sperm, microtubule bundles generally consist of fewer microtubules than in generative cells but not precisely half as many (Cresti er al., 1990; Yu and Russell, 1993).

B. Sperm Structure

1. Morphology

Sperm in pollen grains or pollen tubes are surrounded by two membranes: the sperm cell membrane plus the inner cell membrane of the vegetative cell (Fig. 4). Their shape ranges from spheroidal to highly elongate with one or more thin extensions 30 ym or longer. They are unusually small, as little as 3 ym in diameter, with few organelles, little cytoplasm, and condensed chromatin. The cytoskeleton is similar to that in generative cells, a basket or cage of microtubule bundles arranged around the nucleus.

The small size of sperm cells derives from the small size of the generative cell, from their lack of growth, and from cytoplasmic diminution observed in serial reconstructions (Yu and Russell, 1992). In tobacco, vesicles or cytoplasmic bodies containing membranous organelles and rarely microtubules pinch off from sperm cells (Mogensen and Rusche, 1985; Yu et ul., 1992). Vesicles remain in pockets in the vegetative cell. Further loss of cytoplasm occurs at fertilization when a cytoplasmic body is Ieft outside the egg at fertilization (Mogensen, 1982, 1988).

Sperm nuclei are oval to elongate, with most of the nuclear volume occupied by condensed chromatin in a tightly packed mass that fluoresces brightly with


Fig. 4 Sperm of Gerberujumesonii. (A) Freeze-fracture of sperm in pollen. N, Nucleus. Bar: 0.5 pm. (B) Thin section of sperm; nucleus with condensed chromatin. Bar: 0.5 pm. (C) Isolated sperm with extension (arrowhead); phase contrast. Bar: 5 pm. (D) Isolated sperm as in (C); DAPI staining, fluorescence microscopy. [(A) and (B) from Southworth (1990), Fig. 3, p. 99; Fig. 8, p. 101; (C) and (D) from Southworth and Knox (1989), Fig. l c and d, p. 275.1

7 . Gametes and Fertilization in Flowering Plants 267

DNA-fluorochromes (Fig. 4). In thin sections, condensed chromatin is unevenly stained (Fig. 4). Nucleoli are difficult to identify. Occasional nuclear “vacuoles,” spheroidal zones free of chromatin, are observed. Mitochondria have been observed in nuclei of sperm cells, apparently trapped there during reconstitution of the nuclear envelope under close condition!: in the pollen tube (Yu and Russell, 1994~). The nuclear envelope lies in contact with condensed chromatin, and nuclear pores are sparse to absent (Southworth et al., 1989b; Southworth, 1990) (Fig. 4). Sperm organelles include mitochondria, plastids, ribosomes, endoplasmic reticulum, dictyosomes, and vesicles. No structure comparable to an acrosome has been detected.

As in generative cells, bundles of microtubules, with a predominantly axial orientation, branch and rejoin to form a slightly twisted basket or cage of microtubule bundles surrounding the nucleus (Cresti et al., 1992; Palevitz and Tiezzi, 1992; Pierson and Cresti, 1992; Knox et a/. , 1993). Microtubule bundles show no particular association with the plasma membrane or nuclear envelope, although the small volume of cytoplasm leads to close proximity of the bundles to both nucleus and plasma membrane (Fig. 5). Similar patterns of microtubule bundles are found in sperm cells located in tricellular pollen at anthesis and in pollen tubes derived from bicellular pollen after mitosis. Microtubule bundles terminate in the cellular extensions. Because microtubule bundles join and di- verge, the number of bundles and the microtubules per bundle change along the length of the sperm so that a single cross-section provides little quantitative information.

Microtubules in flowering plant sperm are particularly labile and depolymerize when sperm are released from the pollen tube by osmotic shock. Isolated sperm change shape from spindle shaped to spheroidal within a few minutes (Russell, 1991; Theunis er al., 1991; Palevitz and Tiezzi, 1992; Zee, 1992). This corre- lates with loss of microtubule bundles and of polymerized microtubules. Only in high osmotica was the spindle shape maintained (Southworth and Knox, 1989).

The presence of actin in sperm, as in generative cells, is uncertain. Taylor et al. (1989) reported actin in sperm in pollen tubes of Rhododendron. However, Palevitz and Liu (1992) were unable to replicate these results. Actin was not detected in sperm cells of Brussica (Hause et al. , 1992).

Sperm of some species are dimorphic with respect to size and number of organelles (Russell, 1991; Zhu et al., 1992; Yu and Russell, 1994b). Other species show slight differences in size and shape, but no distinct dimorphism (Mogensen and Rusche, 1985). In Plumbago, with the most dimorphic sperm of any described, sperm closest to the vegetative nucleus had no plastids and more than 200 mitochondria whereas the other sperm contained fewer mitochondria and more than 20 plastids (Russell, 1984). Reports of sperm dimorphism are of particular interest because of their possible relationship to double fertilization (see Section IV).

Fig. 5 Sperm of Brussicu sp. (A) Microtubule bundles in cross-section of sperm in the pollen tube of B. oleruceu. A gap, probably induced by aldehyde fixation, separates the sperm cell from the surrounding vegetative cell (M. Cresti, unpublished). Bar: 0.1 krn. (B) Sperm and vegetative nucleus in pollen tube of B. campestris. This constitutes the male germ unit. DAPI staining; fluorescence microscopy. Bar: 10 km. (D) Isolated sperm cells as in (C); DAPI staining, fluorescence microscopy.

7 . Gametes and Fertilization in Flowering Plants 269

2. Male Germ Unit

Sperm in pollen tubes remain in close association with each other and with the vegetative nucleus. In many species, one sperm is attached, via a long extension, to the vegetative cell membrane appressed to the vegetative nucleus. The second sperm is attached to the opposite end of the first either by membrane contact or by extracellular matrix. This tripartite structure, two sperm plus vegetative nucleus, is called the male germ unit (Dumas et al . , 1984; Mogensen, 1992; Yu et al., 1992) (Fig. 5). Contacts between vegetative cell and generative cell are transient, separating during division of the generative cell and reforming as a contact between one sperm and the vegetative cell (Yu and Russell, 1994a). The two sperm cells and vegetative nucleus remain together during pollen tube growth and passage of sperm through the pollen tube, but separate quickly when the pollen tube is ruptured. This association serves to deliver two sperm cells simultaneously to the embryo sac for double fertilization.

3. Sperm in Vitro

Sperm cells have been isolated from pollen grains and pollen tubes (Southworth and Knox, 1989; Theunis et al., 1991; Russell, 1991) (Fig. 5). Methods include osmotic shock, gentle grinding, and enzymatic digestion of the pollen tube cell wall. Sperm and generative celis apparently contain a lower osmoticum than the vegetative cell, so a solution that is hypotonic to the vegetative cell may be isotonic or hypertonic to sperm (Southworth and Morningstar, 1992). Isolated sperm retain membrane integrity as measured by osmotic response, exclusion of charged dyes, and the fluorochromatic reaction (Southworth and Knox, 1989). Isolated sperm can function in in vitro fertilization experiments (see Section IV, B) .

C. Pollen Tube Growth

Pollen is carried by animals, primarily insects, or by wind to the stigma, the receptive portion of female flower parts. This process is pollination, but not fertilization. Pollen and stigma recognize each other, and the stigmatic cells signal the pollen to germinate. The vegetative cell in the pollen grain hydrates and elongates, forming a pollen tube (Mascarenhas, 1993). Sperm cells are in the pollen grain and move into the pollen tube (Fig. 5). Sperm reach the egg by passing through the pollen tube as it grows toward the egg.

1. Mechanisms of Pollen Tube Growth

Pollen germinates on the surface of the stigma, penetrates between stigmatic cells, and grows within the cell walls of stigmatic cells and between cells of the

2 70 Darlene Southworth

style (Heslop-Hamson and Heslop-Harrison, 1994). Often stylar tissue is loosely compacted, and pollen tube growth proceeds through a gellike extracellular matrix that nourishes the pollen tube and determines the route of growth. Pollen tubes grow out the base of the style into the ovary cavity, where they grow along tissue surfaces and into the micropyles of ovules. Pollen tubes burst in cells adjacent to the egg, freeing the sperm at a distance of a few microns from the egg and central cells (Fig. 6).

Pollen tubes grow at their tips, away from the pollen grain (Pierson and Cresti, 1992). They develop osmotic pressure, and with the constraint of cell wall polysaccharides, retain plasticity only at the tip where new wall material is added from dictyosome vesicles. Pollen tubes have an extensive cytoskeleton including microtubules, actin microfilaments, myosin on organelles and on the generative cell surface, and a number of motor proteins including dynein and kinesin (Lan- celle et d., 1987; h t r o m et d., 1995; Miller et d., 1995; Tirlapur et d., 1995). The presence of the microtubule motor protein kinesin could be detected in the pollen tube, but not in the generative cell (Cai et al., 1993).

Sanders and Lord (1989, 1992) observed that polystyrene latex beads placed on whole or cut stigmas were translocated in and through the style, suggesting that the extracellular matrix of stylar tissues interacts with cell surfaces to pro-

Hg. 6 Organization of the embryo sac in an ovule. The tip of the pollen tube has entered one of the two synergids and ruptured, releasing two sperm (S). One sperm will fuse with the egg and the other with the central cell.


mote pollen tube extension. A molecule recognized by antibodies to vitronectin, a surface adhesion molecule, was found in styles (Sanders et al., 1991). Sanders and Lord (1992) argue that growth of the pollen tube by cellular extension of pollen tube resembles the directed cell movements of animal cells, particularly the cells of the neural crest.

2. Movement of Sperm Cells

Sperm and generative cells travel through the pollen tube; however, spontaneous movements, of sperm or generative cells have not been reported (Pierson et al., 1990; Pierson and Cresti, 1992). Flowering plant sperm are not flagellated. They lack autonomous motility, exhibiting no ameboid motion or twisting. It is likely that sperm are moved by the actions of the vegetative cell surrounding it. The cytoskeleton of sperm cells is not organized to promote movement. Microtubules are infrequently linked to the plasma membrane (Cresti et al., 1990; Del Casino et aE., 1992). Because actin is usually a component of ameboid motion, the lack of actin microfilaments in sperm cells is consistent with lack of self-generated motility.

By contrast, the pollen tube cytoplasm of the surrounding vegetative cell is well suited for intracellular movements (Palevitz and Tiezzi, 1992; Pierson and Cresti, 1992). Cytoplasmic streaming is a major feature of the pollen tube cytoplasm. A myosin-like protein has been observed at the interface of vegetative and generative cells (Pierson and Cresti, 1992; Bohdanowicz et al . , 1995; Tirlapur et al., 1995), and capitate projections on the inner plasma membrane of vegetative cells are sometimes associated with microtubules or microfilaments in vegetative cell cytoplasm (Van Went and Gori, 1989; Cresti et al., 1991; Southworth et al., 1994; Bohdanowicz et al., 1995). Microtubules in the pollen tube form a net- work around the generative cell and vegetative nucleus. Movement is slowed after oryzalin treatment, which disrupts microtubules (Astrom et al., 1995).

This evidence suggests that microfilaments in vegetative cell cytoplasm have a key role in sperm movement within the pollen tube, with the direction of movement determined by polarity of microfilaments or microtubules (Pierson and Cresti, 1992). Sperm movement depends on a connection between the sperm plasma membrane and the inner vegetative cell plasma membrane lining the pocket in which the sperm is located. In turn, the inner vegetative cell membrane may be attached to the pollen tube cytoskeleton that provides the motive force (Lancelle et al., 1987; Southworth et al., 1989a,b; Southworth, 1990, 1992).

111. Female Gametes

Meiosis leading to egg formation occurs within the ovary of a flower bud. In each ovary are one to many ovules. Each ovule is an immature seed consisting of one


meiocyte surrounded by several tissue layers and attached by a vascular connection to the placenta or inner lining of the ovary (Fig. 6).

A. Meiosis to Embryo Sac Formation

Events beginning with meiosis and ending in differentiation of an egg cell vary among flowering plants. In the most common pattern (called the Polygonum type), three of the four meiotic products die, leaving one functional haploid cell per meiocyte, a situation roughly comparable to the loss of polar bodies in animal oogenesis (Gifford and Foster, 1989; Huang and Russell, 1992). After meiosis, the functional haploid nucleus divides three times to produce eight nuclei. The cytoplasm then cleaves to form the cells of an embryo sac (Fig. 6) . One of these differentiates into an egg. The factors that determine which haploid cell will become the egg appear to be related to the position of the cell within the embryo sac. Two cells adjacent to the egg become synergids that function as the destination of the pollen tube. Adjacent to the egg in the center of the embryo sac, two nuclei fuse before cytokinesis to form a diploid central cell. An embryo sac thus consists of seven cells: one egg, two synergids, one diploid central cell, and three other haploid cells that do not function in fertilization.

Following meiosis and several cycles of mitosis in an ovule, one haploid cell becomes the egg, and one cell becomes the central cell. Both are fertilized by sperm, usually from the same pollen tube. This is the process of double fertilization that is characteristic of flowering plants.

B. Egg Structure

Distinguishing features of flowering plant eggs include the location in the embryo sac, polarity of cell shape and organelle distribution, and incompleteness of the cell wall (Russell et al . , 1990; Huang and Russell, 1992). The egg cell differentiates at the end of the embryo sac nearest the opening (micropyle) through which the pollen tube will enter (Fig. 6). The egg itself is polarized, with the nucleus either central or at one end of the egg and with a large vacuole either surrounding the nucleus or at the opposite end of the cell (Zhu el al., 1993). A partial cell wall encloses the egg, leaving a portion of the plasma membrane adjacent to the plasma membrane of the synergids with no wall between them.

The organelles of egg cytoplasm suggest a cell that is quiescent but potentially active. The nucleus is relatively large with a large nucleolus. Ribosomes are abundant in the cytoplasm, but not as polysomes. Dictyosomes and endoplasmic reticulum are present but with few vesicles. Plastids contain few internal membranes and infrequently starch. The location of plastids within the egg varies


from an even distribution to a polarized distribution away from the micropylar end of the egg (Zhu et al. , 1993). Mitochondria are often clustered around the nucleus.

Embryo sacs and eggs have been isolated from ovules in 27 genera by a combination of enzymatic digestion and squashing or micromanipulation (Huang and Russell, 1992; Huang et al., 1992). Egg cells can be distinguished from other cells of the embryo sac by their location in partially intact isolated embryo sacs and by their relative size among the cells freed from embryo sacs. These eggs are alive as judged by the fluorochromatic reaction and by their ability to fuse with isolated sperm in vitro (see Section IV,B).

C. Central Cell Structure

The central cell is distinguished by its location in the embryo sac, the incompleteness of the cell wall, the number of nuclei and their state of fusion, and the large size of the vacuole (Huang and Russell, 1992; Huang et al., 1992). Cyto- plasmic strands traverse the vacuole, and organelles pass along these strands. Central cells are commonly uninucleate (diploid) or binucleate (haploid). Usually nuclei fuse before fertilization.

IV. Double Fertilization

Double fertilization is a characterizing feature of flowering plants (Gifford and Foster, 1989; Russell, 1992; Knox et al., 1993). The primary event of fertilization is the fusion of egg and sperm, producing a zygote that develops into an embryo. A second fertilization or fusion event occurs between the second sperm and the central cell adjacent to the egg. This second fertilization forms endosperm, a nutritive tissue that supports growth of the immature embryo. Both fertilizations are essential for reproductive success.

Ultrastructural details of fertilization are available for few species. The events of fertilization in flowering plants are not easy to follow because the egg is embedded in tissues of the ovule.

A. Fertilization in Vivo

A pollen tube with two sperm enters the ovule through an opening called the micropyle (Fig. 6). The pollen tube generally ruptures in one synergid, but in plants lacking synergids, it bursts between cells of the embryo sac. Sperm are released from the pollen tube near the wall-less portions of the egg and central cell.


Fertilization occurs with fusion of egg and sperm membranes. Membranes make contact and fuse at numerous sites along the contact zone. Vesicles form as fusion occurs. Russell (1983) estimated that the time for fusion was less than 1 min in Plumbago because partially fused gametes were rare in ovules sampled at 5-min intervals in a precisely timed system. Fusion of sperm and central cell membranes proceeds similarly.

The sperm nucleus enters the egg or central cell. Entry of organelles is more variable. In barley (Mogensen, 1982, 1988) and in Plumbago, a cytoplasmic body derived from the sperm cell is eliminated or prevented from entering the egg, and only a portion of the sperm plasma membrane incorporates into the zygote membrane. Sperm mitochondria and plastids may be excluded from the egg during the fusion process. This explains the maternal inheritance pattern of mitochondria and plastids in most species.

Because there are two events of fertilization and two sperm per pollen tube, the question arises whether there is preferential fertilization; that is, whether the two sperm cells differ in such a way that each sperm has a predetermined destination. The sperm of Plumbago have the most extreme dimorphic distribution of any sperm yet observed (Russell, 1984). In Plumbago, the sperm that is not attached to the vegetative nucleus and that contains more plastids and fewer mitochondria fuses with the egg in 94% of cases (Russell, 1985). This observa- tion suggests some mechanism for preferential fertilization in this species; however, preferential fertilization may not occur in plants that do not have dimorphic sperm.

B. Fertilization in Vitro

Because sperm cells are encased in pollen tubes, and egg cells are embedded in ovular tissue, in vitro fertilization cannot be achieved readily. With the development of sperm isolation techniques and advances in the separation of eggs from ovules and embryo sacs in the 1980s, experimental approaches to in vitro fertilization of flowering plants have been successful. Kranz et al. (1991) pioneered fusion of isolated gametes by electrofusion. Sperm isolated by hypoosmotic shock were brought into surface contact with eggs isolated by enzymatic digestion and micromanipulation. They fused rapidly (in 1 sec) by electrofusion. These in vitro zygotes divided and gave rise to masses of cells (Kranz et ul. , 1992). While these results were exciting, they most likely did not represent the method of fusion of plant gametes in vivo.

Faure et al. (1994) demonstrated fusion of plant gametes in mannitol with 5 mM calcium. Gametes, isolated as by Kranz et al. (1992), were brought into contact by manipulation with needles in a fusion medium under mineral oil. After 4 min of adhesion, gametes fused within 10 sec. There was no fusion of a


second sperm in 20 trials. In controls, sperm fused with other sperm cells in 2% of trials and with leaf mesophyll protoplasts in 17% of trials. This method may more closely imitate conditions in vivo. These results support the hypothesis that there is a block to polyspermy in flowering plant eggs (Faure et al., 1994).

V. Summary

Flowering plant sperm resemble animal sperm with condensed chromatin, reduced cell volume, and specialized cytoskeleton. The structure of plant eggs is unlike that of animal eggs; however, egg activation by fertilization is a similar phenomenon requiring sperm-egg fusion, migration of gamete nuclei in the egg, and nuclear fusion followed by cell division. Sperm can be isolated readily from pollen grains or pollen tubes by osmotic shock. Eggs are more difficult to isolate, requiring both enzymatic digestion and micromanipulation. Research on fertilization, including blocks to polyspermy and egg activation, is at its inception. The tools for experimentation on fertilization are complex and not widely available. Simpler methods with which to test in vitro fertilization would be valuable.

The events of gametogenesis, fertilization, and activation in animals provide useful hypotheses for work on plant gametes. For example, it should be possible to characterize gamete cell surfaces, especially of sperm, and to raise antibodies to surface molecules that function in recognition and fertilization. Second, it ought to be possible to identify membrane dimorphism between the two sperm in one pollen grain or tube to determine the underlying basis for double fertilization.

The microtubular cytoskeleton is a highly distinctive feature of sperm. Quan- titative comparisons of the cytoskeleton of generative cells and sperm in the same species, in bi- and tricellular pollen, e.g., counts of microtubule bundles and microtubules per bundle and quantitative comparison of cytoskeletons of generative cells and sperm in pollen grains and pollen tubes, would determine the stability of the cytoskeleton. A more complete description of the cytoskeleton, including location of y-tubulins (Palevitz et al., 1994), of microtubule-associated proteins (e.g., dynein and kinesin), and of components of centrioles and basal bodies, could identify dispersed microtubule-organizing centers and help us to understand the evolution of the distinctive sperm cytoskeleton.

There is a growing base of information on the molecular biology of anther development (Mascarenhas, 1992; Twell, 1994). Currently, gene expression can be identified at stages of anther development and in tissues of the anther, including pollen grains. Because sperm and generative cells represent such a small fraction of the protein and RNA of anthers and pollen, most research on molecular biology of pollen is concerned with the vegetative cell. Further research resolving gene expression in developing sperm would be useful.


Acknowledgments

This work was supported by NSF Grants IBN-9305453 and IBN-9418178 through Research at Undergraduate Institutions.

References

Astrom, H., Som, O., and Raudaskoski, M. (1995). Role of microtubules in the movement of the vegetative nucleus and generative cell in tobacco pollen tubes. Sex. Plant Reprod. 8, 61-69.

Bedinger, P. A. (1992). The remarkable biology of pollen. Plant Cell 4, 879-887. Bohdanowicz, J., Ciampolini, F., and Cresti, M. (1995). Striped projections of the outer mem-

brane of the generative cell of Convallariu majalis pollen. Sex. Plant Reprod. 8, 223-227. Cai, G., Bartalesi, A., Del Casino, C., Moscatelli, A., Tiezzi, A , , and Cresti, M. (1993). The

kinesin-immunoreactive homologue from Nicotiana rabacum pollen tubes: Biochemical properties and subcellular localization. Planta 191, 496-506.

Chaboud, A., and Perez, R. (1992). Generative cells and male gametes: Isolation, physiology, and biochemistry. Znr. Rev. Cytol. 140, 205-232.

Cresti, M., Lancelle, S. A,, and Hepler, P. K. (1987). Structure of the generative cell wall complex after freeze substitution in pollen tubes of Nicotiana and Impariens. J . Cell Sci. 88, 373- 388.

the pollen tubes of Brassica olerucea L. Protoplasma 154, 151-156.

plasmic face of the outer membrane of the generative cell in Amaryllis belladonna. Ann. Bor. 68, 105-107.

Cresti, M., Blackmore, S . , and Van Went, J. L. (1992). “Atlas of Sexual Reproduction in Flow- ering Plants,” pp. 62-83. Springer, New York.

Del Casino, C., Tiezzi, A,, Wagner, V. T., and Cresti, M. (1992). The organization of the cytoskeleton in the generative cell and sperms of Hyacinthus orientalis. Protoplasma 168, 41-50.

Del Casino, C., Li, Y. Q., Moscatelli, A., Scali, M. , Tiezzi, A., and Cresti, M. (1993). Distr- bution of microtubules during the growth of tobacco pollen tubes. Biol. Cell 79, 125-132.

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

Faure, J. E., Diggonett, C., and Dumas, C. (1994). An in vzrro system for adhesion and fusion of gametes. Science 263, 1598-1600.

Gifford, E. M., and Foster, A. S. (1989). “Morphology and Evolution of Vascular Plants.” W. H. Freeman, New York.

Hause, G., Hause, B., and Van Lammeren, A. A. M. (1992). Microtubular and actin filament configurations during microspore and pollen development in Brassica nupus cv. Topas. Can. J .

Cresti, M., Murgia, M., and Theunis, C. H. (1990). Microtubule organization in sperm cells in

Cresti, M., Ciampolini, F., and Van Went, J. L. (1991). Strip-shaped projections at the cyto-

Bot. 70, 1369-1376. Heslop-Hanison, J., and Heslop-Hanison, Y. (1994). Intracellular movement and pollen physiol-

ogy: Progress and prospects. In “Molecular and Cellular Aspects of Plant Reproduction” (R. J. Scott and A. D. Stead, eds.), pp. 191-201. Cambridge University Press, Cambridge.

Huang, B.-Q., and Russell, S. D. (1992). Female germ unit: Organization, isolation and function. Int. Rev. Cyrol. 140, 233-293.

Huang, B.-Q., Pierson, E. S., Russell, S. D., Tiezzi, A , , and Cresti, M. (1992). Video micro- scopic observations of living, isolated embryo sacs of Nicotiana and their component cells. Sex. Plant Reprod. 5 , 156-162.

7. Gametes and Fertilization in Flowering Plants

Knox, R. B., Zee, S . Y., Blomstedt, C., and Singh, M. B. (1993). Male gametes and fertilization in angiosperms. New Phytol. 125, 679-694.

Kranz, E., Bautor, H. , and Lorz, H. (1991). In vitro fertilization of single, isolated gametes of maize mediated by electrofusion. Sex. Plant Reprod. 4, 12-16.

Kranz, E., Lorz, H., Digonnet, C., and Faure, J.-E. (1992). In vitro fusion of gametes and production of zygotes. In?. Rev. Cytol. 140, 407-423.

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

Liu, B . , and Palevitz, B. A. (1991). Kinetochore fiber formation in dividing generative cells of Tradescantia. Kinetochore reorientation associated with the transition between lateral microtubule interactions and end-on kinetochore fibers. J . Cell Sci. 98, 475-482.

cells of Tradescantia. Changes in microtubule organization and kinetochore distribution visu- alized by antitubulin and CREST immunocytochemistry. Protoplusma 166, 122- 133.

Mascarenhas, J. P. (1992). Pollen gene expression: Molecular evidence. In?. Rev. Cytol. 140, 3-18.

Mascarenhas, J. P. (1993). Molecular mechanisms of pollen tube growth and differentiation. Sex. Plant Reprod. 5 , 1303-1314.

Miller, D. D., Scordilis, S. P., and Hepler, P. K. (1995). Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana a h a . J . Cell Sci. 108,

277

Liu, B., and Palevitz, B. A. (1992). Anaphase chromosome separation in dividing generative

2549-2653. Mogensen, H. L. (1982). Double fertilization in barley and the cytological explanation for hap-

loid embryo formation, embryoless caryopses, and ovule abortion. Cariesberg Res. Commun. 47, 313-354.

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

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

Mogensen, H. L., and Rusche, M. L. (1985). Quantitative analysis of barley sperm: Occurrence and mechanism of cytoplasm and organelle reduction and the question of sperm dimorphism. Protoplasma 128, 1-13.

Palevitz, B. A. (1990). Kinetochore behavior during generative cell division in Tradescantia virginiana. Protoplasma 157, 120-127.

Palevitz, B. A. (1993). Organization of the mitotic apparatus during generative cell division in Nicotianu tabucum. Protoplustnu 174, 25-35.

Palevitz, B. A., and Cresti, M. (1988). Microtubule organization in the sperm of Tradescantia virginiana. Protoplusma 146, 28-34.

Palevitz, B. A , , and Cresti, M. (1989). Cytoskeletal changes during generative cell division and sperm formation in Tradescantia virginiuna. Protoplasma 150, 54-7 1.

Palevitz, B. A, , and Liu, B. (1992). Microfilaments (F-actin) in generative cells and sperm: An evaluation. Sex. Plant Reprod. 5, 89-100.

Palevitz, B. A, , and Tiezzi, A. (1992). The organization, composition and function of the generative cell and sperm cytoskeleton. Int. Rev. Cytol. 140, 149-185.

Palevitz, B. A, , Liu, B., and Joshi, H. C. (1994). y-Tubulin in tobacco pollen tubes. Association with generative cell and vegetative microtubules. Sex. Plant Reprod. 7 , 209-214.

Pierson, E. S., and Cresti, M. (1992). Cytoskeleton and cytoplasmic organization of pollen and pollen tubes. Int. Rev. Cytol. 140, 73-125.

Pierson, E. S . , Lichtscheidl, I . K., and Derkson, J. (1990). Structure and behaviour of organelles in living pollen tubes of Lilium longiporum. J . Exp. Bot. 41, 1461-1468.

Russell, S. D. (1984). Ultrastructure of the sperm of Plumbago zeylanica. 2. Quantitative cytology and three-dimensional reconstruction. Planta 162, 385-391.


Russell, S. D. (1985). Preferential fertilization in Plumbago zeylanica: Ultrastructural evidence for gamete recognition in an angiosperm. Proc. Natl. Acad. Sci. U . S . A . 82, 6129-6134.

Russell, S. D. (1991). Isolation and characterization of sperm cells in flowering plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 189-204.

Russell, S. D. (1992). Double fertilization. Int. Rev. Cytol. 140, 357-388. Russell, S. D., Rougier, M., and Dumas, C. (1990). Organization of the early post-fertilization

megagametophyte of Popufus deltoides. 1 . Ultrastructure and implications for male cytoplasmic transmission. Protoplasma 155, 153- 165.

three species of flowering plants. Science 243, 1606-1608.

tension. Int. Rev. Cytol. 140, 297-318.

strate adhesion molecule, vitronectin, occurs in four species of flowering plants. Plant Cell 3,

Sanders, L. C., and Lord, E. M. (1989). Directed movement of latex particles in the gynoecia of

Sanders, L. C., and Lord, E. M. (1992). A dynamic role for the stylar matrix in pollen tube ex-

Sanders, L. C., Wang, C.-S., Walling, L. L., and Lord, E. M. (1991). A homolog of the sub-

629-635. Southworth, D. (1990). Membranes of sperm and vegetative cells of Gerbera jamesonii. J .

Southworth, D. (1992). Freeze fracture of male reproductive cells. Int. Rev. Cytol. 140, 187-204. Southworth, D., and Knox, R. B. (1989). Isolation of sperm cells from Gerbera jamesonii pol-

Southworth, D., and Morningstar, P. A. (1992). Isolation of generative cells from pollen of

Southworth, D., Platt-Aloia, K. A., DeMason, D. A., and Thomson, W. W. (1989a). Freeze-

Struct. Biol. 103, 97-103.

len. Plant Sci. 60, 273-277.

Phoenix dactylifera. Sex. Plant Reprod. 5 , 270-274.

fracture of the generative cell of Phoenix dactylifera (Arecaceae). Sex. Plant Reprod. 2, 270- 276.

Southworth, D., Platt-Aloia, K. A., and Thomson, W. W. (1989b). Freeze-fracture of sperm and vegetative cells in Zea mays pollen. J . Ultrasrruct. Mol. Struct. Res. 101, 165-172.

Southworth, D., Salvatici, P., and Cresti, M. (1994). Freeze fracture of membranes at the interface between vegetative and generative cells in Amaryllis pollen. Inr. J . Plant Sci. 155, 538- 544.

Taylor, P., Kenrick, J., Li, Y., Kaul, V., Gunning, B. E. S. , and Knox, R. B. (1989). The male germ unit of Rhododendron: Quantitative cytology, three-dimensional reconstruction, isolation and detection using fluorescent probes. Sex. Plant Reprod. 2, 254-264.

Terasaka, O., and Niitsu, T. (1989). Peculiar spindle configuration in the pollen tube revealed by the anti-tubulin immunofluorescence method. Bot. Mag. (Tokyo) 102, 143-147.

Theunis, C. H., Pierson, E. S. , and Cresti, M. (1991). Isolation of male and female gametes in higher plants. Sex. Plant Reprod. 4, 145-154.

Tirlapur, U. K., Cai, G . , Faleri, C., Moscatelli, A, , Scali, M., Del Casino, C., Tiezzi, A., and Cresti, M. (1995). Confocal imaging and immunogold electron microscopy of changes in distribution of myosin during pollen hydration, germination and pollen tube growth in Nicotiana tabacum L. Eur. J . Cell Biol. 67, 209-217.

Twell, D. (1994). The diversity and regulation of gene expression in the pathway of male ga- metophyte development. In “Molecular and Cellular Aspects of Plant Reproduction” (R. J. Scott and A. D. Stead, eds.), pp. 83-135. Cambridge University Press, Cambridge.

Ueda, K . , and Tanaka, I. (1995a). The appearance of male gamete-specific histones gH2B and gH3 during pollen development in Lifium long.$orum. Dev. B i d . 169, 210-217.

Ueda, K., and Tanaka, I. (1995b). Male-specific H2B and H3 histones, designated gH2B and gH3 in Lilium fong.$orum. Planta 197, 289-295.

Van Went, J., and Gori, P. (1989). The ultrastructure of Capparis spinosa pollen grains. J . Sub- microsc. Cyrol. Pathol. 21, 149-156..

Yu, H.-S., and Russell, S . D. (1992). Male cytoplasmic diminution and male germ unit in young


and mature pollen of Cymbidium goeringii: A 3-dimensional and quantitative study. Sex. Plant Reprod. 5, 169-181.

Yu, H.-S., and Russell, S. D. (1993). Three-dimensional ultrastructure of generative cell mitosis in the pollen tube of Nicotiana tabacum. Eur. J . Cell Biol. 61, 338-348.

Yu, H.-S., and Russell, S. D. (1994a). Male reproductive cell development in Nicotiana tab- mum: Male germ unit associations and quantitative cytology during sperm maturation. Sex. Plant Reprod. I, 324-332.

Yu, H.-S., and Russell, S. D. (1994b). Populations of plastids and mitochondria during male reproductive development in Nicoriana tabacum L. : A cytological basis for occasional biparental inheritance. Planta 193, 115-122.

sperm cells. Plant Cell 6, 1477-1484.

ucum L.: Three-dimensional reconstruction, cytoplasmic diminution, and quantitative cytology. Protoplasma 168, 172- 183.

isolated generative cells of Allernandra neriifoliu during mitosis. Sex. Plant Reprod. 5, 182- 188.

Zhou, C., and Yang, H.-Y. (1991). Microtubule changes during the development of generative cells in Hippeastrum vittatum pollen. Sex. Plant Reprod. 4, 293-297.

Zhu, T., Mogensen, H. L., and Smith, S. E. (1992). Heritable paternal cytplasmic organelles in alfalfa sperm cells: Ultrastructural reconstruction and quantitative cytology. Eur. J . Cell Biol. 59, 211-218.

Zhu, T., Mogensen, H. L., and Smith, S. E. (1993). Quantitative, three-dimensional analysis of alfalfa egg cells in two genotypes: Implications for biparental plastid inheritance. PIantu 190, 143-150.

Yu, H.-S., and Russell, S. D. (1994~). Occurrence of mitochondria in the nuclei of tobacco

Yu, H.-S., Hu, S.-Y., and Russell, S. D. (1992). Sperm cells in pollen tubes of Nicotiana tab-

Zee, S. Y. (1992). Confocal laser scanning microscopy of microtubule organizational changes in

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