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Annu. Rev. Cell Dev. Biol. 2002. 18:81–105 doi: 10.1146/annurev.cellbio.18.012502.083438 c 2002 by Annual Reviews. All rights reserved Copyright ° First published online as a Review in Advance on May 9, 2002

THE MECHANISMS OF POLLINATION AND FERTILIZATION IN PLANTS Elizabeth M. Lord1 and Scott D. Russell2 1

Department of Botany and Plant Sciences, University of California, Riverside, California 92521; e-mail: [email protected] 2 Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019; e-mail: [email protected]

Key Words double fertilization, embryo sac, gametes, pistil, pollen tube guidance ■ Abstract In flowering plants, pollen grains germinate to form pollen tubes that transport male gametes (sperm cells) to the egg cell in the embryo sac during sexual reproduction. Pollen tube biology is complex, presenting parallels with axon guidance and moving cell systems in animals. Pollen tube cells elongate on an active extracellular matrix in the style, ultimately guided by stylar and embryo sac signals. A well-documented recognition system occurs between pollen grains and the stigma in sporophytic self-incompatibility, where both receptor kinases in the stigma and their peptide ligands from pollen are now known. Complex mechanisms act to precisely target the sperm cells into the embryo sac. These events initiate double fertilization in which the two sperm cells from one pollen tube fuse to produce distinctly different products: one with the egg to produce the zygote and embryo and the other with the central cell to produce the endosperm. CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . COMPATIBLE POLLEN/PISTIL INTERACTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . Adhesion and Hydration on the Stigma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Germination and Chemotropism on the Stigma . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pollen Tube Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pollen Tube Guidance in the Style and Ovary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SELF-INCOMPATIBILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sporophytic—Brassicaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gametophytic–Papaveraceae and Solanaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . POLLINATION ECOLOGY AND EVOLUTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mate Choice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pollen Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FERTILIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Male Gamete Lineage Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Vivo Studies of Fertilization in Flowering Plants . . . . . . . . . . . . . . . . . . . . . . . . . 1081-0706/02/1115-0081$14.00

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In Vitro Studies of Fertilization in Flowering Plants . . . . . . . . . . . . . . . . . . . . . . . . . POST-FERTILIZATION EVENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear Migration and Fusion in Angiosperms . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endosperm Initiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EVOLUTION OF DOUBLE FERTILIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONCLUSIONS AND PROSPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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INTRODUCTION The earliest record we have suggesting a human interest in the mechanism of pollination and fertilization in plants is that of a relief carving from the palace of Syrian King Ashuirnasir-pal II, 883–859 BC, which shows a winged man with a male inflorescence from a palm tree distributing pollen over a female inflorescence (Stanley & Linskens1974). It was not until 1824 that an Italian mathematician and astronomer, Amici, first observed pollen tubes germinating on a stigma. He later proposed that the pollen tube carried the sperm cells to the ovule where the egg resided. In 1884, Strasburger (Maheshwari 1950) demonstrated fertilization as fusion of egg and sperm nuclei in the flowering plants (angiosperms), a difficult task because of the enclosure of the egg inside the ovule within the ovary. By 1898, double fertilization was recognized as well, a feature unique to flowering plants (see below). Our present effort is to provide an overview of the field of pollination and fertilization and propose directions for new research. Progress in our understanding of plant reproduction has been particularly good in the past ten years. Here we attempt to cover events from pollination to fertilization in a single chapter, including some discussion of the ecology and evolution of breeding systems. To do so, we must focus on the most recent literature. A major review covering the molecular and genetic basis of pollen/pistil interaction has just been published (Wheeler et al. 2001).

COMPATIBLE POLLEN/PISTIL INTERACTIONS Adhesion and Hydration on the Stigma Pollen, which contains the sperm cells, develops in the stamen, the male organ of the flower. Here it undergoes dehydration to some extent before being released from the anther to the environment. The pistil, the female organ of the flower, is comprised of the stigma (where pollen first alights), the style (a conduit between the stigma and ovary), and the ovary, which contains the ovules. When pollen comes in contact with the stigma, it adheres, hydrates, and germinates. The result is a pollen tube cell that carries the sperm cells, endocytosed into the larger tube cell, thus conveying them to the embryo sac and egg in the ovule. A flower can produce pollen that penetrates its own pistil and fertilizes its own ovules in most angiosperms, a predominantly self-compatible (SC) group. These early events of adhesion and hydration of pollen on the stigma have been studied intensively because they are the initial steps of pollination and because they are readily observed.

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Stigmas are of two broad types, wet and dry, with respect to the amount of secreted matrices that the pollen encounters on their surface. The pollen grain has a tough outer wall, like a spore, which typically is sculptured. It provides a porous surface containing the many substances that are deposited on it by the parent sporophyte while it is inside the anther or that are secreted into it by the male gametophyte during pollen development (Dickinson et al. 2000, Mayfield et al. 2001). The function of these pollen coat substances is still a mystery for the most part, even though they are the major allergens of humans and of medical interest. One exception to this is the recent discovery of the male recognition factor in self-incompatibility (SI) in the Brassicaceae that resides in the pollen coat (see below). This coat may have some role in adhesion of pollen to vectors such as insects and birds because wind-pollinated species have thin pollen coats and may also have evolved as a food source for biotic vectors. The pollen coat is assumed to have a role in adhesion to the stigma (Doughty et al. 2000, Luu et al. 1999, Takayama et al. 2000), but data in Arabidopsis leave this in doubt (Zinkl et al. 1999). Hydration of the pollen grain is somehow aided by the presence of the coat, which in Arabidopsis contains predominantly lipases and oleosins (Mayfield et al. 2001). The loss of one oleosin protein from the coat (GRP 17–1) impairs pollen hydration. These data from only one family, the Brassicaceae, suggest a complex series of events in both adhesion and hydration of pollen on dry stigmas. Another role for the pollen coat substances is that of facilitating penetration into the style. Enzymes residing in the coat play a role in modification of the stigma extracellular matrix (ECM) to allow for penetration into the pistil (Bih et al. 1999, Wheeler et al. 2001). On the stigma side, there is evidence for a plasma membrane–localized aquaporin-like protein called MIP-MOD in the Brassicaceae (Dixit et al. 2001). The aquaporins are water-transporting molecular channels, and in plants they occur in either the plasma membrane or the vacuole. A crucial first step in acceptance of non-self pollen on dry stigmas occurs at pollen hydration, and presumably this aquaporin-like protein is a major regulator of hydration by controlling water flow from the stigma to the pollen grain. On wet stigmas, lipids (trilinoleins) have been implicated in hydration as well (Wolters-Arts et al. 1998), but little is known about initial steps in wet stigmas having exudates low in these lipids. The pollen coat is full of proteins that could act as ligands for receptors in the stigma, preparing the way for adhesion, hydration, and germination of compatible pollen, but so far the only known recognition event at the stigma surface is that of SI, where active rejection ensues when self pollen lands on the stigma (Dixit & Nasrallah 2001). There are a plethora of receptor-like kinases in plants but a paucity of known ligands. Glycine-rich proteins (GRPs), of which oleosins are a member, are common cell wall proteins of unknown function. WAKs (wall-associated kinases) are receptors (serine-threonine kinases) (Kohorn 2001) that occur throughout the plant, and an Arabidopsis GRP (AtGRP-3) regulates WAK-1 by binding to its cell wall domain (Park et al. 2001). It would be interesting to know whether WAKs and oleosins interact in the stigma. To date no receptors or ligands have

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been described for self-compatible processes at the stigma surface even though this first step in pollination is such a critical one for effective fertilization. When the pollen grain is hydrated, the tube cell cytoplasm becomes activated and some genes, such as MAP kinases of unknown function, may act specifically at this stage (Heberle-Bors et al. 2001). Many of the genes necessary for germination have been transcribed, and the pollen tube cell is primed for the next events leading to fertilization, which can take hours or days to occur (Taylor & Hepler 1997).

Germination and Chemotropism on the Stigma After hydration, the pollen grain produces an outgrowth from an aperture or thin area in the wall. This is the site of tip growth that results in the production of a pollen tube, which will ultimately convey the sperm cells to the embryo sac. The initial process of germination is under complex control, but a major regulator is Rop, a GTPase that shares the same ancestor as Cdc42, Rac, and Rho in the RHO family of small GTPases (Fu & Yang 2001). The function of Rop in germination was studied by using Rop1 overexpression and rop mutants in Arabidopsis. Expression of a dominant-negative (DN) rop1 mutant inhibits pollen germination (Li et al. 1999), whereas overexpression of WT Rop1 promotes germination (V. Vernoud & Z. Yang, unpublished results). Once germination occurs, maintenance of polar growth in the pollen tube is also controlled by Rop in a complex signaling network that includes regulation of a tip-focused Ca+2 gradient and F-actin polymerization (see below). Hundreds of pollen grains are usually deposited on the stigma, and germination there is known to be density dependent. This is called the mentor effect, and has recently been attributed to a peptide growth factor, phytosulfokine-α (Chen et al. 2000). This five amino acid–sulfated peptide, first confirmed as a mitogen in plant cell cultures, was subsequently found to be capable of promoting germination of pollen cultured at low density. There are only four known peptides in higher plants that play a role in cell-cell communication via receptors, and phytosulfokine is a recent addition to this group (Matsubayashi et al. 2001). After pollen germination, penetration into the style is the next step, and here the processes vary considerably across species, stigma, and style types. Entrance is always into a specialized ECM of the pistil that appears to guide pollen tubes to the ovules (Lord 2000). In solid styles that have dry stigmas, pollen tubes have to penetrate an outer lipidic cover of the stigmatic papilla, the cuticle, whereas in wet stigmas, entrance is directly into the ECM of a degenerating papilla. In open styles, typically the stigma is also open and covered with a secreting epidermis that is continuous with that of the stylar canal so no penetration is necessary, just guidance along a secretory dermal layer into the style and then to the ovary. On the stigma, pollen tubes germinate in an ECM that is usually a combination of both pollen coat secretions and stigmatic exudates. In the Solanaceae, lipids (triglycerides) in the stigma exudate were shown to be essential for pollen germination oriented toward the style (Wolters-Arts et al. 1998). In this case, the cue for directional growth of the pollen tube is a physical factor, a gradient of water set up

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by the stigmatic lipids over an aqueous component of the exudate. In other stigma types, pollen tube guidance is required for entry into the style. The open stigma and style of lily is an example where pollen germinates over a large stigma surface, and the tubes must be guided toward the central stylar canal. An abundant, secreted peptide in lily, SCA (stigma/stylar cysteine-rich adhesin) has been purified from the transmitting tract and shown to be involved, with a pectic polysaccharide, in pollen tube adhesion and guidance in the style (Mollet et al. 2000, Park et al. 2000). Binding of SCA to the pectin is necessary for adhesion of pollen tubes to this specialized ECM. On the stigma, this peptide appears to act alone as a chemotropic compound. Pollen tubes were shown to reorient their growth toward a chemical gradient of SCA in vitro establishing SCA as the first chemotropic peptide identified in plants (S. Kim, S.-Y. Park & E.M. Lord, unpublished results). Thus in the stigma, SCA may act alone as a chemotropic molecule, whereas in the style it acts with a pectin in haptotactic (adhesion-mediated) guidance. Allurin, a vertebrate sperm chemo-attractant, is related to sperm adhesion molecules (Olson et al. 2001). So this combination of chemo-attractant and adhesion function is not unique to SCA. These few studies document the interplay between the pollen tube and the stigma for compatible pollinations. Pollen-stigma interactions that lead to the rejection of self pollen is much better understood (see below). How recognition between pollen and stigma in a compatible situation regulates intracellular events is a far more mysterious subject. Given the importance of Rop signaling in the activation of pollen germination and maintenance of pollen tube growth, it would be of great interest to see if recognition regulates intracellular Rop signaling.

Pollen Tube Cell Biology The pollen tube cell is the fastest growing plant cell, with rates of up to 1 cm · h−1. It shows other unusual features that make it unique as a plant cell (Figure 1). In particular, it carries the sperm cells inside it as it grows and is guided by specialized ECMs to the ovule where the egg resides. The pollen tube grows by tip growth, producing new membrane and wall at the front. The tube cell and sperm cells move along with the advancing tip, a movement that is unique in plants where cell walls normally confine the protoplast in one location (Lord 2000). The cell biology of tip-growing cells in general, and of pollen tubes in particular, has been studied intensively as systems of polarized growth. Pollen can be readily grown in vitro, and the early stages of tube formation are now studied using a variety of sophisticated tools. The exciting new findings on tip-focused calcium gradients, on proton, potassium, and chloride fluxes and oscillating growth have recently been reviewed by Hepler et al. (2001), as are aspects of the pollen tube cytoskeleton and membrane trafficking. Here, we focus on aspects of pollen cell biology related to its function in sperm cell transport over long distances in vivo. The initial growth of the pollen tube is probably regulated primarily by internal cues. Once the tube cell has fully exited the pollen grain and the first cell wall (callose plug) has formed, external cues in the transmitting tract likely play a larger tole in pollen tube growth. The most important recent discoveries about pollen tube cell biology

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Figure 1 Pollination in the lily pistil. The pollen grains (PG) germinate on the stigma (St) and produce a pollen tube (PT) that carries the male germ unit (MGU), including sperm cells, to the ovule (Ov). Lily has a hollow style lined with a transmitting tract epidermis (TTE) on which pollen tubes are guided to the ovary (O). An enlargement shows the pollen tube tip on the TTE, which secretes an extracellular matrix (ECM) including adhesion molecules. Golgi vesicles secrete wall material and membrane at the tip where a dynamic F-actin occurs. Behind the tip, cytoplasmic streaming occurs on F-actin cables, and a cortical cytoskeleton keeps the MGU near the tip.

relevant to growth and guidance in vivo include the discovery of a form of dynamic actin at the growing tip (Fu et al. 2001); the machinery for actin polymerization, including ARP2 (Klahre & Chua 1999, Hepler et al. 2001); and the master switch, Rop GTPase (Fu & Yang 2001). A prolonged debate on pollen tube cell biology concerned the presence or absence of F-actin at the leading edge or growing tip of the pollen tube. For various reasons, including fixation artifacts, the presence of F-actin was questionable until an actin-binding domain of a mouse Talin tagged with an enhanced GFP mutant was used (Fu et al. 2001). The same group had

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discovered a subfamily of Rho GTPases in plants and Rop (Zheng & Yang 2000) and localized a pollen-specific Rop to the pollen tube tip plasma membrane. They were able to block Rop signaling and induce constitutively active Rop to confirm that polar tip growth in pollen was controlled by this GTPase. Because the RHO family of small GTPases is known to control the actin cytoskeleton in mammals and yeast, the assertion that pollen tube tips were devoid of F-actin was puzzling. The use of the enhanced GFP mutant was necessary to detect the very dynamic form of tip-localized F-actin in pollen tubes (Fu et al. 2001; video available at http://www.jcb.org/). This dynamic F-actin oscillates (as does growth at the pollen tube tip), appearing before the growth spurt, and its presence was found to be controlled by Rop, as is the tip-focused cytosolic calcium gradient (Fu & Yang 2001). The realization that a population of F-actin at the very tip of the pollen tube, not involved in cytoplasmic streaming, was essential for growth strongly suggests a role for the machinery of cell motility at the pollen tube tip (Lord 2000). A role for F-actin polymerization in animal cell movement is accepted, but the mechanism is only now being deciphered (Borisy & Svitkina 2000, Higgs & Pollard 2001) (http://www.cellmigration.org/). Several of the major players exist in pollen tube tips. The fact that the tube cell and the sperm cells it carries move through the style to the ovary is not debatable, but the mechanism of movement for this cell complex is still unknown. The discovery of the F-actin machinery for cell motility at the tube cell tip begins to address mechanism, but the large size of the tube cell (1–5 mm) and the presence of other cytoskeletal components such as myosin and microtubules (MTs) probably means a large cast of characters is involved. A role for MTs in pollen tube growth was dismissed initially because colchicine, which breaks down MTs, had no effect on pollen tube tip growth in young in vitro–grown pollen tubes. Subsequent studies using older pollen tubes and performed in vivo established that MTs were essential for maintenance of polarity in the tube cell and particularly for movement of the male germ unit (tube cell nucleus and two sperm cells), which insures its retention at the tip (Joos et al. 1994). The MTs are oriented longitudinally in the tube cell cortex, and several motor proteins (kinesins and dyneins) are associated with them (Cai et al. 2001). Breakup of MTs leads to a lack of forward movement of the male germ unit and displacement of the vacuole into the tip, so these MTs may play a role in movement of the tube cell protoplast along the pollen tube wall. The motor molecules associated with the MTs are prominent in the cortex and may be involved in this movement. This important aspect of pollination has been neglected because protoplast movement is most apparent only after hours of in vitro culture when the first callose plug, which sequesters the living part of the pollen tube toward the tip, is formed at the rear of the tube cell. In culture, these pollen tubes are considered old and difficult to work with, but they represent just a fraction of the mature tube lengths that occur in vivo. Vesicle secretion rates at the tip of the pollen tube may also be a controlling factor for growth rate in concert with endocytosis behind the tip (Parton et al. 2001). Other important findings indicate that cAMP occurs in pollen tubes, that modulation of its

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concentration affects growth (Moutinho et al. 2001), and that transient adhesions between the plasma membrane and wall occur at the tip (Parton et al. 2001).

Pollen Tube Guidance in the Style and Ovary A prevalent model for pollen tube guidance is based on an analogy with adhesion and cell movement in animals, in particular with the cell biology of neuronal navigation (Lord 2000, Palanivelu & Preuss 2000, Wheeler et al. 2001). Migration of either neurons or their nerve growth cones occurs by mechanisms similar to those responsible for chemotaxis in non-neuronal cells (Song & Poo 2001). The importance for proper guidance of adhesion molecules in the ECM and their receptors on the moving cell is now well documented. These extracellular factors occur along the path and, by selective adhesion and intracellular signaling, are responsible for guiding cells or their extensions to a goal. A hierarchy of guideposts is proposed so that these target-derived factors can act in succession. Some are diffusible, others bind to matrix molecules, but all are secreted into a specialized ECM at the right time and place to effect growth and /or guidance. How these ligand-receptor complexes control axon guidance is not known, but signaling is thought to occur through the actin cytoskeleton via the Rho family of small GTPases (Song & Poo 2001). The cast of characters is present in both the pollen tube and the pistil for a similar mechanism to be operating in pollen tube guidance. The ECM molecules involved are no doubt unique to plants, as no animal ECM homologs were found in the Arabidopsis genome (the Arabidopsis Genome Initiative 2000); however, many of the pollen tube cytoskeleton components and their associated signaling complexes have animal homologs or at least have conserved functional domains (Wu et al. 2001, Hepler et al. 2001). The components of the stylar-transmitting tract ECM are a complex mixture and vary depending on the species studied (Wheeler et al. 2001). AGPs (arabinogalactan proteins) are abundant in many styles, and in one case, the TTS protein occurs in a glycosylation gradient in the tobacco style. TTS is one of the very few molecules described that forms a gradient in vivo and induces chemotropism of pollen tubes in vitro (Wheeler et al. 2001). On the pollen tube side, there are pollen-specific glycoproteins in the walls of several species, the Pex proteins, that have conserved leucine-rich repeats and N-terminal variable regions suggestive of specific ligand binding (Stratford et al. 2001). Receptor kinases in tomato pollen tubes that are phosphorylated by contact with the stylar matrices are also good candidates for pollen/pistil cross talk (Wheeler et al. 2001). We have very little information about the cell biology of pollen tubes growing in vivo. The use of GFP as a reporter system allows live imaging of pollen tubes, and two-photon microscopy should be capable of penetrating the pistil tissues, which until now have obscured the pollen tube path in vivo, without having to resort to fixations and staining (Cheung 2001). Pollen tubes adhere to transmitting tract surfaces and appear to be guided along them, especially in hollow styles such as those in lily, but also in solid styles as in Arabidopsis (Lord 2000). In lily, two molecules, a pectic polysaccharide and a

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peptide, SCA, were found to be necessary for pollen tube adhesion to and growth on an in vitro stylar matrix (Park et al. 2000, Mollet et al. 2000). Both molecules are secreted into the lily stylar ECM where pollen tubes adhere. In the assay, SCA binds to the pectin in a pH-dependent manner, and this binding is necessary for pollen tube adhesion to the SCA/pectin matrix. Recombinant SCA was shown to be active in the adhesion assay (Park & Lord 2002). Pollen tubes grown in vitro do not adhere to one another. Only when they are in contact with the pistil SCA/pectin matrix in vitro will they adhere to the matrix and to each other and grow. A mechanism for this adhesion is not known, but the assumption is that SCA binds a receptor in the pollen tube plasma membrane. Growth, which occurs near the pollen tube tip where plasma membrane may be exposed to the stylar matrix, is necessary for adhesion. SCA’s activity in chemotropism on the stigma (S. Kim, S.-Y. Park & E.M. Lord, unpublished results), where free diffusion would be necessary to set up a gradient, and its activity in adhesion in the style, where binding to pectin is necessary, suggests a dual role for this peptide in the lily pistil. The pollen receptors for SCA in the stigma and in the style may be different. Pollen tubes adhere best in the in vitro assay when they are at least two hours old and have achieved the length they would in vivo as they enter the style, which suggests developmental regulation of the capacity to respond to the SCA/pectin complex in the style (Mollet et al. 2000). The effect on the cytoskeleton of pollen tube adhesion to the stylar ECM needs to be studied in vivo and preferably with live pollen tubes. An exciting new finding about pollen tube guidance concerns the last step in the process, entrance into the ovule. A variety of genetic studies have revealed the importance of the ovule and, in particular, the female gametophyte or embryo sac in guiding a single pollen tube into the ovule opening or micropyle where contact can occur with the embryo sac and sperm can be released (Higashiyama et al. 2001). In vitro assays using isolated ovules demonstrated clearly that a cue attractive to pollen tubes emanated from the ovule, and laser ablation studies on the embryo sac show definitively that the cue comes from the embryo sac synergid cells that flank the egg cell. These elegant studies provide experimental support for an old proposal that when the pollen tube is in the vicinity of the ovule, the secretory synergid cells, one of which will receive the pollen tube contents including the sperm cells (see below), guide the pollen tube to the micropyle. The signal is not yet known, but heat treatment of the ovule prevents it from attracting pollen tubes. This suggests that either active secretion, a protein factor, or both are necessary for chemotropism. One intriguing aspect of this work is that pollen tubes must pass through the stigma and style in order to be capable of chemotropism in the isolated ovule assay (Higashiyama et al. 1998). When pollen is cultured in the presence of the ovules, no chemotropism to the micropyle occurs even though the generative cell has divided and produced sperm cells (T. Higashiyama, personal communication). This means that contact with the pistil, starting with the stigma and then the style, is essential in some way to prepare the pollen tube to receive the signal from the ovule. This supports the premise that a hierarchy of control points exists in pollination and that study must be done in vivo to decipher the complexities of these signaling pathways. Different receptors may form on the

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pollen tube cell at different stages in its passage from stigma to ovary, and these may be induced to form by contact with specific ligands in the transmitting tract tissues. The complexities here may rival those in the animal nervous system in axon guidance.

SELF-INCOMPATIBILITY The most studied and understood process of pollination in the flowering plants is that of recognition and rejection of self pollen. Several groups have independently evolved unique mechanisms for self-incompatibility (SI) in the angiosperms. There are some recent excellent reviews on this topic (Wheeler et al. 2001) so we cover only the major findings here.

Sporophytic—Brassicaceae The Brassicaceae (mustard family) has sporophytic SI where the outcome of the pollination is determined by the genotype of the pollen parent (Dixit & Nasrallah 2001). The S-gene products in the pistil were discovered some time ago and found to reside in the stigmatic papillae in this dry stigma type, where the site of selfpollen arrest occurs. The essential component is a membrane-spanning serinethreonine kinase (S receptor kinase; SRK), which has a large extracellular domain in the papillar ECM that was presumed to interact with an S-gene-specific pollen ligand. Recently, the long-sought-after male component was discovered and found to be a small, cysteine-rich protein (SCR) that resides in the pollen coat (Schopfer et al. 1999, Shiba et al. 2001). The model now is that interaction between the transmembrane glycoprotein SRK and the peptide ligand SCR induces a cascade of signaling events that lead to rejection of self pollen. Indeed, using tagged proteins, the Nasrallah group and others have shown that SRK and SCR bind to each other and that the binding is allele specific (Kachroo et al. 2001, Takayama et al. 2001). This work on SI in Brassica is one of the few cases where both the ligand and the receptor have been identified for a well-known cell-cell signaling event in plants. The discovery of the pollen component in this story is a major advance in the field of plant cell biology.

Gametophytic–Papaveraceae and Solanaceae In the other major type of SI, gametophytic SI, the outcome of pollination is determined by the haploid pollen genotype (Wheeler et al. 2001). Advances have been made in two families that have evolved quite separate mechanisms for this type of SI: the Papaveraceae (poppy family) and the Solanaceae (tobacco family). In poppy, small pistil S proteins secreted by the stigma interact with the S-gene product in the pollen tube, thought to be a plasma membrane receptor, causing the arrest of incompatible pollen on the stigma (poppy has no style). An in vitro assay system has facilitated study of the cell biology of SI, and a fascinating cascade of events has been described that occur within minutes after contact of pollen

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tubes with the stigmatic S-gene protein. Inhibition is mediated by a calcium-based signal transduction pathway in the pollen tube that results in fragmentation of its F-actin during a process of programmed cell death. Downstream of the Ca+2 signals induced by SI, several proteins are phosphorylated in an S-specific manner, and a MAPK is activated (Wheeler et al. 2001). Positive identification of the male S-gene product would complete the story. In the Solanaceae, the S-gene product on the female side is an RNase that acts on SI pollen tubes in the style by degrading ribosomes (McCubbin & Kao 2000). The male S-gene product is as yet unknown. Pollen tubes endocytose the S-RNase, which is secreted into the stylar ECM, whether they are compatible or not, and in vitro the RNase enters and kills both compatible and incompatible pollen. A recent study in vivo shows that this uptake also occurs in the style, but only SI pollen tubes are arrested in vivo (Luu et al. 2000). It has been shown that the RNase capacity of the S-gene product in the style is necessary and sufficient to cause SI in the Solanaceae. The model for SI in this family is that the pollen S-gene product is an RNase inhibitor that recognizes and inhibits all S-RNases except those of the same allelic specificity. The in vivo data are good evidence that the pistil environment can profoundly affect pollen tube cell biology. In some as yet unknown manner the compatible tube cell can endocytose an RNase that in vitro would arrest its growth, but to which it is immune when growing in vivo. Discovery of the male S-gene products in both the Papaveraceae and Solanaceae will certainly advance the field of pollen tube cell biology in general.

POLLINATION ECOLOGY AND EVOLUTION Mate Choice Two aspects of pollination ecology that have gained attention lately are pollen competition and female choice, both components of sexual selection in plants. Mate choice in plants certainly occurs in cases of self-incompatibility, but whether compatible pollination and fertilization involves a component of female choice by the pistil is still debated. One mechanism for sexual selection would be pollen competition, with the fastest pollen tubes achieving the most seed set. Another mechanism could be choice by the maternal plant, with selection of particular pollen tubes by the pistil, thus causing facilitated tracking to the ovary (Marshall & Diggle 2001). The recent work on rapid evolution of reproductive proteins from both the female and male sides supports the concept of sexual selection in plants (Swanson & Vacquier 2002). It is known that pollen tube growth rates vary in vivo and in vitro and that the ability of a pollen donor to sire seeds varies with the maternal plant in selfcompatible species. Thus nonrandom mating occurs in plants. It is obvious that interactions between pollen tubes of varying genetic background in one pistil, as well as pollen/pistil-specific interactions, create a complex environment so that cellular mechanisms that produce nonrandom matings will be difficult to decipher. The major lesson for cell biologists from these population studies is clear,

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however; the pollen tube’s function in delivering viable sperm cells to the embryo sac involves a close partnership with the transmitting tract of the pistil that must include a myriad of recognition and signaling events.

Pollen Flow Gene flow in the environment affects genetic structure of populations, a fact of some importance to evolutionists, conservationists, and agriculturalists (Ellstrand 2001, Schulke & Waser 2001). Pollen-mediated gene movement can be studied using paternity analysis. This method has allowed us to study the effects of pollinator activity, both biotic and abiotic. Recently, due to concerns about gene flow from transgenic crop plants, pollen flow has become a hot topic. Earlier concerns, which are ongoing, regard how crop-to-wild-plant gene flow could endanger native plants and create new weeds. Basic knowledge of pollination is essential today to adequately address the social and economic problems that are inevitable with new technologies, especially those involving food crops.

Evolution Angiosperms, literally meaning vessel seed, are defined by several features, but the one most defining feature is the pistil or vessel. The carpels that form the pistil arise from a floral meristem, and during development some form of epidermal fusions take place to close the carpels around the ovules. Typically, the carpel fusion event results in formation of the transmitting tract of the style with the stigma formed at the top. Thus the transmitting tract, including the placental epidermis in the ovary where the ovules form, is formed from a dermal layer. In primitive angiosperms, the receptive surface of the sometimes open carpels can be less distinct, with no style formed. Primitive flowers are typically bisexual (though the extant Amborella, at the base of all angiosperms, is unisexual), insect pollinated (beetles, flies, and bees), and self compatible, although SI occurs in some taxa (Thien et al. 2000). A more thorough study of the mechanisms of pollination in the primitive angiosperms, including genetic analyses, is needed especially now that we have the results of the Deep Green Consortium, the Angiosperm Phylogeny Group (1998).

FERTILIZATION Male Gamete Lineage Gene Expression Genetic expression in the male gametophyte has increasingly focused on a differentiation of cellular programs between the pollen vegetative or tube cell and the male germ lineage within it. The male gametes themselves are nonmotile, and thus the metabolic needs and control of their position are ceded to the surrounding pollen. Sperm cells move through the pollen tube by association with cytoskeletal components on the perispermatic envelope (inner pollen plasma membrane that encloses the sperm cells) and cortical arrays of actin filaments and MTs that line

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the interior of the pollen tube. Superficial myosin on the vegetative nucleus and perispermatic envelope may have a role in translocating the male germ unit in the pollen tube; the sperm cells, although moving, remain passive. A lower number of nuclear pores in the sperm (Straatman et al. 2000) would also suggest passivity, but interestingly this lower number does not prevent the male germ cells from expressing their own genetic program. Expression studies of pollen indicate a diversity of wall-producing genes and a variety of housekeeping genes that presumably provide for the metabolic needs of the male gametophyte. Genes expressed in the male germ (generative and sperm) cells emphasize the unique nature of these cells. Histones of the generative cell lineage are substituted with germ-line-specific gene products of H2A and H3 (Xu et al. 1999a). The vegetative nucleus, in contrast, displays decreased quantities of H1 protein (Tanaka et al. 1998), hypoacetylation of H4, and increased methylation, especially prior to germination (Janousek et al. 2000), presumably related to heterochromatin formation. Nuclei of the male germ lineage become increasingly distinct from the vegetative nucleus. Differences in chromatin condensation distinguish the vegetative and germ line nuclei, presumably reflecting commitment to a reproductive program. Differentiation of expression programs is provided by occurrence of germ-line-specific promoters, for instance the LGC1 promoter, which is apparently activated only after the separation of the cell from the intine (Xu et al. 1999b). The actual divergence in the developmental program, however, appears to coincide with the eccentric placement of the cell wall during generative cell formation (Eady et al. 1995). Additional germ-line-specific expression is evidenced in the activation of cyclin, DNA repair, and ubiquitin genes in the generative and sperm cells (Xu et al. 1998, Singh et al. 2002). In addition to germ-line-unique polyubiquitins, there is also RT-PCR and in situ hybridization evidence of differences in the two sperm cells of Plumbago (Singh et al. 2002). These specific ubiquitin genes are turned on in each of these cells in succession prior to their final differentiation, presumably purging remaining cytoplasmic signals. If there is a specific surface protein or glycoprotein displayed on the surface of the cell, it is likely to be formed in such cells or under their control. Further evidence for an independent developmental program in sperm cells is the presence of specific arabinogalactan proteins on the sperm cell surface (Southworth & Kwiatkowski 1996) and heterogeneity of cell surface determinants in different flowering plant species (Southworth et al. 1999). Nonrandom positioning of B chromosomes in the nuclei of maize sperm cells also points to independent developmental control (Rusche et al. 2001).

In Vivo Studies of Fertilization in Flowering Plants The past several years have seen a resurgence of in vivo approaches using the remarkable plant Torenia fournieri (Scrophulariaceae), in which the embryo sac protrudes from the tip of the ovule (Erdelsk´a 1974) exposing the egg apparatus. Higashiyama et al. (1997) recorded the kinetics of fertilization by direct

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observation of the two synergids, egg cell and central cell (which form the socalled female germ unit). One item of contention given the apparent participation of sperm-associated myosin with a pre-existing actin corona is how long the unfused sperm cells remain in the synergid. According to Higashiyama et al. (1997), both sperm cells are present only transiently inside the degenerated synergid, but Wallwork & Sedgley (2000) report durations in excess of an hour. Reconciling these observations is problematical. The rapid kinetics of nuclear changes may reflect decay of the synergid and vegetative nuclei in the degenerated synergid after pollen tube discharge. The release of enzymes, potentially including nucleases, may be a consequence of synergid penetration that may protect the interior of the embryo sac from transmission of foreign, including viral, DNA (Heslop-Harrison et al. 1999). Nuclear migration within the central cell precedes fertilization. Initially, the polar fusion nucleus occupies a mid-chalazal position, migrating toward the egg apparatus just before pollen tube arrival. After the sperm nucleus enters the central cell, it adheres to the polar fusion nucleus, fuses, and migrates to a far chalazal position where division of the primary endosperm nucleus occurs at 15 h after pollination. Consistent with prior microcinematographic observations, the sperm nucleolus enlarges to the size of the polar nucleoli and fuses during migration (Higashiyama et al. 1997). Migration of the polar fusion nucleus will also occur without pollination, but it is delayed 24 h. Symplastic communication between the cells of the female germ unit changes dramatically during embryo sac maturation and early embryogenesis as well. One day prior to anthesis, rhodamine-labeled dextran of 10 kDa passes freely between the central cell and egg apparatus cells. At anthesis, ∼40% of ovules still permit passage of the 10-kDa tracer, but near fertilization, the exclusion barrier drops below 3 kDa in 80% of the ovules (12 h after pollination) and 90% in the zygote (24 h after pollination). Without pollination, the exclusion barrier drops to 3 kDa in nearly half of the ovules at two days after anthesis (Han et al. 2000). Growth of the integument and multiplication of endosperm nuclei unfortunately inhibit a more direct observation of fertilization. CELL CYCLE AND FERTILIZATION The role of the cell cycle during fertilization has recently gained renewed interest. Animal fertilization, with rare exceptions, involves gametes with a 1C complement of DNA corresponding to the G1 phase and without intervening DNA synthesis. Early results in angiosperms, however, provided a baffling variety of data and DNA complements. Recent evidence suggests three major patterns of pollen dissemination in the bicellular (generative cell–containing) and tricellular (sperm cell–containing) pollen of flowering plants: 1C, 2C, and intermediate values (Friedman 1999). Furthermore, fusion occurs in two conditions: 1C (G1 fusion) or 2C (G2 fusion). Only in Arabidopsis and the gnetophytes have data on both the egg cell and sperm cell been followed through development to fusion, although these may be inferred in others by zygotic measurements (Friedman 1999).

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Phase synchrony appears to be an important factor governing gamete fusion. Gametes that are in S phase, even if appressed to one another, will presumably not fuse until both reach a 2C complement, indicative of G2. In tobacco, 1C sperm in the style remain at a G1 until arriving near the ovule. Only when the sperm completes S phase in the degenerate synergid and enters G2 does fusion occur (H.Q. Tian, T. Yuan & S.D. Russell, unpublished data). Interestingly, progression in the cell cycle in the male and female gametes occurs more or less in concert, as female gametes require an extra 24 h to reach G2 without pollination. Complex communication between the male and female gametes is also evidenced by receptivity related responses in maize egg cells (Mol et al. 2000) and tobacco female germ cells (Tian & Russell 1997a) in response to the presence of pollen tubes in the style. ATTRACTION AND DELIVERY OF MALE GAMETES WITHIN THE EMBRYO SAC Evidence for pollen tube attraction has been an elusive target until recently, and efforts to demonstrate it using living male and female gametophytes have been inconclusive. Pollen tubes in Arabidopsis were attracted over distances of little more than 100 µm to cross the septum and fertilize the ovule (H¨ulskamp et al. 1995). No attraction was observed in incompletely formed ovules or those lacking embryo sacs. These, and observations with other mutants (Ray et al. 1997), suggest that embryo sac cells may themselves govern pollen tube guidance. A direct demonstration of this (see above) required use of a remarkable member of the Scrophulariaceae family, Torenia fournieri. A semi in vitro system of culturing mature ovules and pollen tubes was painstakingly developed so that fertilization could be directly observed in some ovules (Higashiyama et al. 1998). Higashiyama et al. (2000), using only light microscopy, described the pollen tube entering the embryo sac at the micropylar end “thrusting its way between the two synergid cells.” Although an exact mode of entry will presumably reveal that the pollen tube enters one synergid directly (van der Pluijm 1964), rupture clearly occurs at the pollen tube tip, with the discharged contents of the pollen tube restricted to the degenerated synergid (Figure 2). When the contents of the pollen tube were discharged into the synergid, they arrived at a peak velocity estimated to be 12,000 ± 5800 µm3 s−1, decreasing rapidly during the first 0.1 s (Higashiyama et al. 2000). A remarkable video of this event is available as supplementary data on the journal web site (http://www.plantphysiol.org/cgi/content/full/ 122/1/11/DC1). MIGRATION OF MALE GAMETES WITHIN THE EMBRYO SAC Upon the discharge of the pollen tube within the embryo sac, pollen tube cytoskeletal arrays are rapidly depolymerized, the myosin-coated perispermatic envelope disintegrates, and the sperm are typically immersed in the micropylar end of the degenerate synergid. Although neither sperm cell is typically in contact with the egg or central cell at this stage, the male gametes nonetheless are conveyed chalazally to their respective fusion site. Male gametes often are in contact with both the egg and central cell immediately prior to fusion (Russell 1992). The means of this movement—a

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nonmotile cell traversing the lifeless cytoplasm of two former cells—defies conventional mechanisms, but both chemical elements of an actomyosin movement system are present. Actin becomes organized as a corona, tracing the anticipated pathway of gamete travel. Coronas extend from the middle of the synergid, one terminating near the egg nucleus and the other near the polar nuclei (Huang & Russell 1994, Huang & Sheridan 1998, Huang et al. 1999, Fu et al. 2000, Ye et al. 2002). Myosin released from the pollen tube binds with the surface of the sperm cells (Zhang et al. 1999), presumably through electrostatic force (Zhang & Russell 1999). Microinjected Alexa 488-phalloidin in living embryo sacs of Torenia reveals a dramatic reorganization of F-actin in the synergid and egg cells during pollen tube approach, arrival, and discharge. An actin cap is located at the base of the synergid, and with degeneration, fluorescent label accumulates in patches on the cortex of the egg cell. An actin corona becomes conspicuous upon pollen tube arrival and discharge, when sperm cells are deposited near the base of the corona. Sperm cells in the synergid subsequently migrate chalazally toward the point of fusion. The actin corona disappears soon after male gamete transmission (Figure 3). That actin coronas have a role in fertilization is indicated by the association of actin coronas with each fertilized egg in maize plants with supernumerary female gametes (Huang & Sheridan 1998). Recent work on the nun orchid provides evidence that living egg and central cells control these events; cellular indentations complementary in shape to the sperm cells are presented as target sites near the termination of coronas (Ye et al. 2002). In tobacco, nun orchid, and possibly Torenia, transit in the synergid appears to be a protracted process.

In Vitro Studies of Fertilization in Flowering Plants Isolation of living, viable gametes late in the 1980s led to the combination of male and female gametes using electrofusion (Kranz et al. 1991), chemical induction (Kranz & L¨orz 1994a), and more subtle means (Faure et al. 1994). Ultimately, some products resulted in the regeneration of plants (Kranz & L¨orz 1994b) and formation of in vitro endosperm (Kranz et al. 1998). These studies have focused on a small group of monocots that includes grasses such as Zea, Coix, Sorghum, Hordeum, and Triticum (reviewed previously by Kranz & Kumlehn 1999). ADHESION AND CHARACTERISTICS OF IN VITRO FUSION In electrofusion and chemical induction, the combination of gametes is coerced; however, in an example by Faure et al. (1994), the medium, which included low concentrations of calcium, did not appear to coerce fusion. Polyspermy was apparently successfully blocked, and chemical signals, including a calcium influx, have been recorded (see below). Attempts to combine dicot gametes have met with some success in Nicotiana tabacum. (Sun et al. 1995, 2000, 2001; Tian & Russell 1997b). In each of these cases, fusion has been stimulated using chemical means with polyethylene glycol (PEG). Despite the fusigenic nature of PEG, however, multiple fusions seemed to be inhibited within 30 min after fusion. This inhibition of fusion applied

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to various combinations of gametes, somatic cells, and gametic-somatic combinations, as well. This refractory period is attributed to a requirement of the cells to complete cytoplasmic reorganization prior to further fusion (Sun et al. 2000), casting doubts on claims of polyspermy block. The greater apparent efficiency of gamete-to-gamete fusion has also been challenged. Recently, Sun et al. (2001), fusing somatic cells (and even chloroplasts) with cells of various sizes, found that efficiency of fusion in each size class did not appear to differ with that found in male gamete lineage cells of similar size. Disparities in volume could also be a mechanism for preferentiality during fertilization when sperm cells differ in volume (Tian et al. 2001). Combination of distant hybrids and genetic modification of the artificial zygotes have been a frequently cited goal of in vitro fertilization. This goal may not be distant, as Scholten & Kranz (2001) have now demonstrated the expression of transgenes in gametes and in vitro fertilized zygotes. With the use of germ-linespecific promoters (for instance, the LCG1 promoter; Xu et al. 1999b), alterations could be rapidly incorporated in the germ line for transmission or reimplantation in host plants to produce seed. GAMETE MATURATION, POLARITY DETERMINATION AND REGENERATION Gamete maturation and the competence of gametes to respond appropriately to gametes of the opposite sex are incompletely known in angiosperms. Maturation of the sperm cell surface can be demonstrated by attempting to induce fusion of sperm cells during maturation (Tian & Russell 1998). When placed in apposition, newly formed sperm cells of tobacco often fuse spontaneously, but within minutes they lose this tendency. At 20 h after sperm formation, fusion could be induced only by exposing cells to dilute solutions of cellulase and pectinase (interestingly both were required). Apparently, sperm maturation involves important carbohydrate determinants on the cell surface, but not a wall in any conventional sense because this would impede fusion. Polarity of the male germ unit appears to be initiated prior to generative cell separation from the intine and determined by the position of the vegetative nucleus (Russell et al. 1996). For female gametes, the best understood model is maize, in which egg maturation is categorized into three classes (Mol et al. 2000). Type A egg cells are small, densely cytoplasmic and have a central nucleus, suggestive of a nonpolar condition. Type B egg cells are larger and have numerous small vacuoles surrounding a nearly centrally located perinuclear cytoplasm. Type C cells are highly polarized, containing the largest cells, a prominent apical vacuole, and a mid-basal perinuclear cytoplasm. As silks emerge from their husks, type A egg cells are prevalent in the ovules (88%), but they decrease to nearly half (58%) at optimum silk length, and types B and C become more common near the time of fertilization. Cross talk from pollen seems to stimulate maturation of female gametes in ways that include calcium accumulation (Tian & Russell 1997a), cell organization (Mol et al. 2000), nuclear migration (Higashiyama et al. 1997), and cell cycle progression (H.Q. Tian, T. Yuan, S.D. Russell unpublished data). Conversely, signals from the

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embryo sac serve to stimulate pollen tube growth and maturation. However, the nature of this communication is unknown. In many biological systems, the polarity of the zygote can be altered by illumination source or site where gamete fusion occurred and can be induced by factors as subtle as electrical charge, touch, or changes in membrane permeability. In angiosperms, however, there is no evidence that such manipulations have succeeded in reorienting or establishing polarity, nor is there evidence of a particular region of the egg cell being more receptive to fusion than another (Antoine et al. 2001). If egg cells possess inborn polarity signals, their placement presumably occurs in reference with pre-cellular stages of embryo sac development. Normally, egg cells occur within the embryo sac, which presumably provides a structural context for the determination of the egg’s cellular polarity. CALCIUM WAVES, SITE OF FUSION AND EGG ACTIVATION As in animal fertilization, a calcium influx is associated with in vitro fertilization in plants. Using maize, Antoine et al. (2000) demonstrate influx rates peaking at more than 10 pmol · cm−2 · s−1 that result in a homogeneous influx over the surface of the cell that may continue up to 2 h. Although the calcium influx is initiated at the fusion site and propagated in a wave-like progression, unlike that in some other models, polarity is not evident past the first several minutes after fusion. The most visible manifestation of egg activation is a contraction of the egg cell/zygote, causing a wrinkling of the cell surface that apparently accompanies calcium influx (Figure 4). This is followed by resumption of a smooth cell surface with turgid appearance and the subsequent deposition of cell wall as detected by Calcofluor white. Artificial influx induction can be triggered by 20 µM A23187 ionophore or 5 µM ionomycin, resulting in calcium influx and egg events that mimic activation. In contrast, influx can be inhibited by the addition of gadolinium at a functional concentration of 10 µM GdCl3, thereby inhibiting cell wall formation.

POST-FERTILIZATION EVENTS Nuclear Migration and Fusion in Angiosperms The matter of nuclear migration once the zygote has fused is still an understudied phenomenon of zygote activation. Cytoskeletal elements such as microtubules and F-actin will undoubtedly play a role in the positioning and movement of the male and female nuclei. Studies to date, however, seem to suggest that rather than extensive cytoskeletal rebuilding immediately prior to nuclear fusion, there is a phase of cytoplasmic re-establishment coinciding with the establishment of the cell wall (Antoine et al. 2001). Only with greater cellular stability are the usual mechanisms of nuclear positioning conspicuous.

Endosperm Initiation Endosperm forms a determinate, polarized structure with considerable complexity and a distinct developmental program, as demonstrated by the fate of cultured

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Figure 4 The chart represents Ca2+ flux measurements during maize in vitro fertilization (time 0 is gametic fusion). Arrow indicates the onset of a Ca2+ influx after fusion. Figures illustrate (A) the egg cell before fusion (male gamete position is shown by a white arrowhead); (B) egg cell after fusion, after cell contraction has started; (C ) strong egg cell contraction; and (D) post-fusion egg cell reshaping. Figure from Antoine et al. (2000) reproduced with permission.

endosperm derived from in vitro fertilization of the central cell (Kranz et al. 1998). The uniqueness of cell origins and architecture in free-nuclear endosperm has been recognized just recently to consist of nuclear cytoplasmic domains (Brown et al. 1999) that form compartments without a conventional cell wall (Ortegui & Staehelin 2000) in a highly atypical manner compared with the other generations. In the scheme of alternation of generations, endosperm truly does form a third generation of flowering plants.

EVOLUTION OF DOUBLE FERTILIZATION Apparently, angiosperm predecessors may have produced female gametophytes with more than one fertilizable nucleus. Presumably, each sperm fused with a female target nucleus. The genetic benefit of the second nutritive fertilization increasingly outweighed the terminal nature of the endosperm lineage sufficiently that the central cell no longer competes with the egg for survival in modern plants. Instead, endosperm development precedes the zygote, providing an aggressively nutritive tissue that assures the success of its sibling embryo (Friedman 1995).

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Based on examinations of basal angiosperms, the following seem to be plesiomorphic characteristics of endosperm: (a) initial division of the primary endosperm nucleus to form two cells; (b) polar development of micropylar and chalazal domains with different patterns of development (initially uniseriate development of the micropylar domain and more random cellular development of the chalazal domain), with the micropylar domain being the larger of the two; and (c) copious nutritive development, with proteins and lipids of endosperm forming the primary food source for the embryo (Floyd & Friedman 2000). In fact, early endosperm lineages can sometimes display strikingly similar patterns in polarity and organization compared with early embryo lineages.

CONCLUSIONS AND PROSPECTS In comparison with animals, sexual reproduction in seed plants appears far more complex, subtle, and painstakingly accurate in the production and delivery of gametes. Reproductive interactions in flowering plants are largely restricted to those between the pistil (stigma, style, and ovary, which are actually sporophyte tissues) and pollen tubes, which are a separate three-celled male gametophytic stage of the life cycle. Just before fertilization, the female gametophyte exerts an effect on the pollen tube, guiding it to the micropyle. These differences make the mechanism of pollination and fertilization in plants a fundamentally interesting problem in cell biology that should stimulate research for many years to come. The Annual Review of Cell and Developmental Biology is online at http://cellbio.annualreviews.org

LITERATURE CITED Angiosperm Phylogeny Group. 1998. An ordinal classification for the families of flowering plants. Ann. MO. Bot. Gard. 85:531–53 Antoine AF, Dumas C, Faure JE, Feijo JA, Rougier M. 2001. Egg activation in flowering plants. Sex. Plant Reprod. 14:21–26 Antoine AF, Faure JE, Cordeiro S, Dumas C, Rougier M, et al. 2000. A calcium influx is triggered and propagates in the zygote as a wavefront during in vitro fertilization of flowering plants. Proc. Natl. Acad. Sci. USA 97:10643–48 Arabidopsis Genome Initiative. 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796– 813 Bih FY, Wu SSH, Ratnayayake C, Walling LL,

Nothnagel EA, et al. 1999. The predominant protein on the surface of maize pollen is an endoxylanase synthesized by a tapetum mRNA with a long 50 leader. J. Biol. Chem. 274:22884–94 Borisy GG, Svitkina TM. 2000. Actin machinery: pushing the envelope. Curr. Opin. Cell Biol. 12:104–12 Brown RC, Lemmon BE, Nguyen H, Olsen OA. 1999. Development of endosperm in Arabidopsis thaliana. Sex. Plant Reprod. 12:32– 42 Cai G, Romagnoli S, Cresti M. 2001. Microtubule motor proteins and the organization of the pollen tube cytoplasm. Sex. Plant Reprod. 14:27–34 Chen Y-F, Matsubayashi Y, Sakagami Y. 2000.

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PLANT POLLINATION AND FERTILIZATION Peptide growth factor phytosulfokine-α contributes to the pollen population effect. Planta 211:752–55 Cheung AY. 2001. Imaging elongating pollen tubes by green fluorescent protein. Sex. Plant Reprod. 14:9–14 Dickinson HG, Elleman CJ, Doughty J. 2000. Pollen coatings—chimaeric genetics and new functions. Sex. Plant Reprod. 12:302– 9 Dixit R, Nasrallah JB. 2001. Recognizing self in the self-incompatibility response. Plant Physiol. 125:105–8 Dixit R, Rizzo C, Nasrallah M, Nasrallah J. 2001. The Brassica MIP-MOD gene encodes a functional water channel that is expressed in the stigma epidermis. Plant Mol. Biol. 45:51– 62 Doughty J, Wong HY, Dickinson HG. 2000. Cysteine-rich pollen coat proteins (PCPs) and their interactions with stigmatic S (incompatibility) and S-related proteins in Brassica: putative roles in SI and pollination. Ann. Bot. 85:161–69 Eady C, Lindsey K, Twell D. 1995. The significance of microspore division and division symmetry for vegetative cell-specific transcription and generative cell differentiation. Plant Cell 7:65–74 Ellstrand NC. 2001. When transgenes wander, should we worry? Plant Physiol. 125:1543– 45 Erdelsk´a O. 1974. Contribution to the study of fertilization in the living embryo sac. In Fertilization in Higher Plants, ed. HF Linskens, pp. 191–95. Amsterdam: North Holland Faure JE, Digonnet C, Dumas C. 1994. An in vitro system for adhesion and fusion of maize gametes. Science 263:1598–600 Floyd SK, Friedman WE. 2000. Evolution of endosperm developmental patterns among basal flowering plants. Int. J. Plant Sci. 161: S57–81 Friedman WE. 1995. Organismal duplication, inclusive fitness theory, and altruism: understanding the evolution of endosperm and the angiosperm reproductive syndrome. Proc. Natl. Acad. Sci. USA 92:3913–17

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Figure 2 Discharge of the contents of a pollen tube into the embryo sac in Torenia fournieri with time after initiation of discharge. Arrows indicate the expanding contents of the pollen tube in the embryo sac. Thermal pseudocolor images display change in images. Small arrow indicates the rupturing plasma membrane of the synergid cell. Bar is 10 µm; ec, egg cell; es, embryo sac; pt, pollen tube; sy, synergid cell. Figure from Higashiyama et al. (2000) reproduced with permission of American Society of Plant Biologists.

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Figure 3 Diagrammatic reconstruction of actin organization (green) and nuclear position (red ) during fertilization in Torenia fournieri. (a) Mature embryo sac with dense actin present near filiform apparatus (arrows); (b) onset of synergid degeneration with first evidence of corona formation; (c) actin reorganization just prior to pollen tube penetration; (d ) pollen tube arrival, discharge, and transport of sperm nuclei (arrowheads); and (e) completion of fusion, dissolution of actin corona, initiation of post-fertilization actin pattern. CC, central cell; dSy, degenerated synergid; E, egg cell; EN, endosperm nucleus; PT, pollen tube; SN, secondary nucleus; Z, zygote. Figure from Fu et al. (2000) reproduced with permission of Springer-Verlag.

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