Multiple Signals Guide Pollen Tubes to Their Targets

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provided by Elsevier - Publisher Connector Developmental Cell, Vol. 2, 273–281, March, 2002, Copyright 2002 by Cell Press Plotting a Course: Multiple Signals Review Guide Pollen Tubes to Their Targets

Mark A. Johnson and Daphne Preuss1 cell, undergoes meiosis (Figure 1). The four haploid Department of Molecular Genetics and Cell Biology products of meiosis are initially held together in a tetrad. Howard Hughes Medical Institute After the tetrads disassemble, an asymmetric mitotic The University of Chicago division occurs in which the larger daughter cell com- 1103 East 57th Street pletely engulfs its smaller sister. The fates and gene Chicago, Illinois 60637 expression programs of these two daughter cells are different: the larger somatic (or vegetative) cell undergoes high levels of transcription and ultimately produces a pollen tube, while the smaller, generative cell divides Pollen plays a critical role in the life cycle of all flow- mitotically to produce two sperm with condensed nuclei ering plants, generating a polarized pollen tube that that form the male germ line. delivers sperm to the eggs in the interior of the flower. Throughout its development, the pollen grain acquires Pollen tubes perceive multiple extracellular signals components that prepare it for communication with fe- during their extended growth through different floral male cells. Some of these components come from the environments; these environments discriminate among vegetative cell itself, while others are derived from the pollen grains, allowing only those that are appropri- secretory cells (tapetum) that surround the grain during ately recognized to invade. The phases of pollen tube its development. These tapetal cells provide much of growth include interactions that establish pollen polar- the raw material required for building the multilayered ity, entry of pollen tubes into female cell walls, and pollen cell wall and the protein- and lipid-rich pollen adhesion-based pollen tube motility through a carbo- coat; these layers protect the grain during the time it hydrate-rich matrix. Recent studies have identified is a free-living organism and provide essential signals cells within the female germ unit as important sources necessary for pollen recognition. of pollen guidance cues. Other signals undoubtedly exist, and their discovery will require genetic screens that target diploid tissues as well as haploid male and Phase I, The Stigma Surface: Pointing Tubes female cells. in the Right Direction The initiation of pollen tube growth requires adhesion of a desiccated pollen grain to a female stigma cell, A Lengthy Journey: Pollen Tube Migration followed by hydration of the grain, allowing it to initiate through Floral Tissues metabolism (Figure 2). These processes represent Fertilization of flowering plants requires the delivery of checkpoints established by the female cells; they pro- sperm cells to ovules embedded deep within the flower. vide important fertility barriers, particularly in plants with Higher plant cells do not have flagella, and most flowers dry stigmas, such as Arabidopsis thaliana. The pollen do not have an open channel through which sperm could grain monitors these interactions, reorganizes its polar- travel; instead, sperm cells migrate within a pollen tube, ity, and begins tube growth only if appropriate contacts a polarized cell extension that responds to guidance are made. Genetic and biophysical investigations of cues, digests intervening female tissues, and navigates these processes have characterized the role of genes, through five distinct environments (Phases I –V, below). expressed in diploid cells that encode components on Recent investigations using genetic, biochemical, and the pollen and the stigma surfaces. In particular, signifi- cell ablation approaches have demonstrated that each cant effort has been focused on understanding the inter- of these environments presents the pollen tube with play that leads to hydration, defining the surface mole- distinct extracellular signals; the intracellular responses cules that regulate water transfer. Although significant to these signals result in polarized growth and precise progress has been made, there are many exciting oppor- guidance of pollen tubes through compatible flowers. tunities for discovery: Just how do pollen grains reorga- Unlike animal gametes that have nearly quiescent nuclei, nize their polarity in response to this water traffic? Are haploid pollen tubes express many genes essential for these events solely under the control of genes ex- their growth and guidance. Consequently, pollen tube pressed by diploid cells, or is the haploid pollen grain guidance demonstrates a harmony between haploid and itself an active participant? diploid gene expression programs coordinated to direct The surfaces of stigma cells and pollen grains are the migration of a single cell. coated with hydrophobic molecules, a waxy cuticle on dry stigmas and a lipid- and protein-rich coat on the pollen grain (Elleman et al., 1992). Under these surface Pollen Assembly: Building a Gamete layers are cell walls; the outer pollen wall (the exine) Delivery Machine regulates an initial adhesion step, leading to a species- The construction of a pollen grain begins in male diploid specific interaction (Zinkl et al., 1999). The binding force tissues when a specialized cell, the microspore mother between a pollen grain and a compatible stigma cell is remarkably strong; it is one of the few examples of cell adhesion that can occur in a dry environment. Most 1Correspondence: [email protected] likely, each species has its own set of male and female Developmental Cell 274

Figure 1. Development of Pollen Grains and Tubes (A) Male gametes develop in the anther where diploid microspore mother cells (yellow) un- dergo meiosis. The four haploid microspores (red) are initially held in a tetrad that disperses before the first pollen mitosis (PM I). This mitosis is asymmetric, and one product, the vegetative cell, engulfs the other (generative cell, white). In a second pollen mitosis (PM II), the generative cell divides to form two sperm cells (white). Mature pollen is encased in a multilayered wall (black) and coat (yellow), synthesized by surrounding diploid tapetal cells. (B) An Arabidopsis pollen tube grown in vitro and stained to highlight callose (aniline blue) and nuclei (DAPI); Nomarski optics (left), fluorescence (right). Pollen tubes contain callose plugs that divide the tube cell into an active growing region at the tip and a vacated region nearest the grain. Sperm cells (intense fluorescence) and the nucleus of the vegetative cell (diffuse fluorescence) are associated with one another and migrate near the tip. adhesion factors—identifying these components will re- apparatus await discovery, and it remains to be seen veal how diversity among adhesion molecules deter- whether similar recognition systems are involved in in- mines the choice of mating partners. terspecies recognition. Interestingly, it has been shown After adhesive contacts are firmly established, the that the Arabidopsis genome contains remnants of the extracellular pollen coat begins a remarkable migration Brassica SCR and SRK genes, indicating that self-com- to the site of stigma contact—a process known as coat patibility may have been acquired in Arabidopsis when conversion (Elleman et al., 1992) (Figure 2B). This reorga- this self-recognition system was lost (Kusaba et al., nization is immediately followed by pollen hydration. 2001). Pollen coat lipids play a role in establishing a conduit Self-compatible pollinations also rely on proteins to that allows transport of water from the stigma cell; plants regulate hydration. GRP17 is an abundant Arabidopsis with wet stigmas have a very similar mechanism, except pollen coat protein that is required for the rapid onset lipids are supplied by the hydrophobic stigma coating of pollen hydration (Mayfield and Preuss, 2000). This (the exudate) (Wolters-Arts et al., 1998). In Arabidopsis protein is glycine rich and also contains an oil binding cer mutants, deficiencies in pollen coat lipids lead to oleosin domain; it is a member of a family of pollen coat hydration defects (Fiebig et al., 2000; Hu¨ lskamp et al., proteins encoded by a cluster of similar GRP genes. 1995a; Preuss et al., 1993). Similarly, mutants with ge- Arrangement of the GRP genes in a cluster may have netically ablated stigmas are defective in pollen hydra- served to facilitate diversification at this locus, leading tion, and the addition of exogenous lipid to these mutant to models that changes in GRP sequences may contrib- stigmas restores pollen function (Wolters-Arts et al., ute to the process of speciation (Mayfield et al., 2001). The sequences of GRP genes are widely diverged be- 1998). tween Arabidopsis and Brassica oleracea, yet there ap- Lipids, however, are not the sole factors important in pears to be a strong selection for the maintenance of pollen hydration. In self-incompatible Brassica species, each GRP gene. Thus, the entire set of GRP proteins is proteins found on the pollen coat and stigma surface likely required for pollen hydration, contributing to the prevent hydration of incompatible pollen. The genes that regulation of water transport or to the structure of the encode these specificity determinants are located at a pollen coat. highly polymorphic, multigenic locus (the self-incompat- Following hydration, reorganization of the pollen grain ibility or S locus). The extensive investigations of S locus cytoplasm determines the polarity of pollen tube exten- proteins has been summarized in several recent reviews sion, which, in many species, emerges through the pore (Dickinson, 2000; Dixit and Nasrallah, 2001); conse- adjacent to the stigma surface (Figure 2B). Although the quently, we only briefly summarize this system here. nature of the signals that direct polarized growth is not SCR (S locus cysteine-rich protein) and SRK (S locus understood, there is evidence that cell polarity is orga- receptor kinase) are interacting specificity determinants nized in response to a gradient of water established by from pollen and the stigma, respectively (Schopfer et pollen coat lipids (Wolters-Arts et al., 1998). For exam- al., 1999; Takasaki et al., 2000). Their interaction, en- ple, pollen tubes can orient toward the aqueous phase hanced by the stigma-expressed S locus glycoprotein of in vitro generated gradients. In addition, signals from (SLG) (Takayama et al., 2001), initiates a signaling cas- the stigma are required for tube orientation: removing cade that results in the exclusion of water from self stigmas from Arabidopsis pistils causes tubes to grow pollen, blocking initiation of pollen tube growth. The in random directions and prevents migration into subse- components that are downstream of this recognition quent layers (Kandasamy et al., 1994). This defect can Review 275

Figure 2. The Pollen Tube Path (A) Pollen tubes grow through five distinct environments as they navigate toward the egg (Kandasamy et al., 1994). Pollen grains germinate on finger-like stigma cells (Phase I), penetrate these cells by growing through their cell walls (Phase II), and reach the transmitting tract (TT, Phase III). The ovary houses the ovules (Ov), which contain female gametes. After exiting the transmitting tract, pollen tubes grow on the inner surface of the ovary until they reach a funiculus (Phase IV). Finally, a pollen tube targets the synergid cell (panel D) where it releases the sperm (Phase V). (B) Determination of initial course of pollen tube growth. (1) A desiccated pollen grain binds to a stigma cell (green); (2) the pollen coat (yellow) flows to the site of contact and directs water transfer to the grain, resulting in hydration; (3) the pollen tube emerges from the pore (red bar) closest to the site of attachment. (C) A cross-section of the ovary illustrates the routes taken by pollen tubes toward the funiculus, which emerges from the junction of the septum and the ovary wall. Pollen tubes may exit the transmitting tract, growing within the septum and emerging in the ovary onto the funiculus surface (left); alternatively, they may enter the ovary directly from the tract, growing on the surface of the septum toward the funiculus. In this phase of pollen tube growth, the tubes are exposed to air (white). (D) Diagram of an Arabidopsis embryo sac, the female gamete-bearing structure contained within each ovule. Each embryo sac consists of seven haploid cells: an egg, two synergids, a central cell, and three antipodal cells (Christensen et al., 1997). The central cell is homozygous diploid, containing two fused haploid nuclei. Following fertilization, one sperm cell fuses with the egg to produce the zygote, and the other fuses with the central cell to form the endosperm. Two outer and two inner integuments encase the embryo sac, but do not fuse, leaving a gap called the micropyle. be overcome by the addition of specific lipids, strength- molecular requirements are poorly understood. In Arabi- ening the hypothesis that the regulated transfer of water dopsis and other members of the Brassica family, the through lipids is necessary to set the initial course of pollen tube burrows into the wall of a stigma cell, grow- tube growth (Wolters-Arts et al., 1998). ing within this dense matrix until it reaches connective tissues below the stigma (Elleman et al., 1992). It is clear Phase II, The Stigma Cells: Pollen Tubes that pollen is well adapted to this phase of growth; in Invade Female Tissues contrast, successful fungal or bacterial invasion of the After germination, pollen tubes penetrate the floral tis- stigma is rare (Atkinson et al., 1993). Most likely, both sue and begin their approach toward the eggs. The the pollen tube and stigma contribute signals necessary invasive growth of pollen tubes into female cells has for triggering cell wall degradation after a compatible been characterized at a morphological level, but the pollination (Hiscock et al., 1994). Mutations that affect Developmental Cell 276

the ability of pollen tubes to invade the stigma have not that ensure proper transduction of those signals within been isolated, despite extensive screening for sterile the tube. mutants in Arabidopsis. To date, such screens have While the process of pollen tube guidance is similar focused on genes expressed in diploid cells; haploid- to the adhesion-based outgrowth of neurons, proteins specific screens (see below) will target enzymes that homologous to abundant components of the neural are instead expressed by the haploid pollen tube. ECM, including collagen, laminins, vitronectin, or fibro- nectin (Tessier-Lavigne and Goodman, 1996), have not Phase III, The Transmitting Tract: The Pollen been identified in the Arabidopsis genome (AGI, 2000; Tube Superhighway Palanivelu and Preuss, 2000). Instead, flowering plants After pollen tubes exit the stigma cell wall, they enter may have evolved a separate set of ECM molecules for the transmitting tract, a specialized tissue that connects adhesion-based translocation. One such factor, SCA the stigma, style, and the ovary (Figure 2A). The cells (stigma/stylar cysteine-rich adhesin), is a 9 kDa protein that line the transmitting tract are coated with a nutrient- purified from lily transmitting tract exudates. In combi- rich extracellular matrix (ECM); this matrix completely nation with pectin, purified fractions containing SCA fills the intercellular space of the transmitting tract in support pollen tube adhesion in vitro (Jauh et al., 1997; species with closed styles (Lord, 2000). Pollen tubes Mollet et al., 2000; Park et al., 2000). The pollen receptor grow faster, longer, and more precisely through this for the putative SCA/pectin complex is not known; iden- matrix than in vitro, indicating that pollen tubes respond tification of this receptor would help to clarify whether to signaling factors supplied by female tissues. Several the motility of pollen tubes mechanistically resembles searches for chemotactic molecules have identified nu- that of axons. tritional components (Brewbaker and Kwak, 1963), as In many species, the transmitting tract is housed well as some candidate signaling molecules (see below). within a septum that divides the ovary into two or more In addition, the transmitting tract ECM may directly mo- compartments, separating the tract from the ovules (Fig- bilize pollen tubes. Sanders and Lord (1989) showed ure 2C). Consequently, in order to reach the embryo that, in three different species, inert beads can traverse sac, pollen tubes must cross the placental cell layer that the transmitting tract at the same rate as pollen tubes; surrounds the transmitting tract, migrate on the surface this activity was only observed when bead diameters of the septum until reaching the funiculus, and enter the were similar to pollen cells. Similarly, the tips of severed micropyle (Hu¨ lskamp et al., 1995b; Kandasamy et al., pollen tubes have been observed to move through the 1994; Lennon et al., 1998). Signals are apparently re- style and enter the ovary (Jauh and Lord, 1995). Although quired to direct the exit of pollen tube from the tract it is not clear how the transmitting tract ECM causes inert at specific locations. For example, when Arabidopsis particles to move, pollen tube traffic may be directed pistils are pollinated with a limiting number of pollen through interactions with the pollen cytoskeleton, much grains, pollen tubes emerge from the transmitting tract like the migration of animal cells in contact with a sub- at the first unfertilized ovule (Hu¨ lskamp et al., 1995b). strate (Jauh and Lord, 1995; Palanivelu and Preuss, Mutations that disrupt embryo sac development com- 2000). Consistent with this idea, star-shaped foci of pletely eliminate this bias, but do not affect earlier stages F-actin have been detected at points of contact between of tube growth, indicating separate signals control mi- pollen tubes and the transmitting tract ECM (Jauh and gration through and emergence from the transmitting Lord, 1995). tract (Hu¨ lskamp et al., 1995b). These mutant phenotypes In many plant species, heavily glycosylated arabino- implicate diploid ovule tissue or the haploid female germ galactan proteins (AGPs) are the predominant class of unit as potential sources of the transmitting tract exit transmitting tract ECM components. Several lines of signal. Intriguingly, the pollen tube must perceive this evidence implicate AGPs in supporting and directing signal when it is separated from the embryo sac by pollen tube growth (Cheung et al., 2000; Jauh and Lord, several cells and considerable space. How such signals 1996; Lennon et al., 1998). Specific AGPs from tobacco are transduced through apparently solid plant tissues (TTS proteins) have been implicated in pollen tube guid- remains mysterious. Pollen-specific mutations that af- ance (Cheung et al., 1995). Directed growth toward TTS fect the patterns of transmitting tract exit would be help- protein in vitro was apparent in medium lacking sugar, ful, yet genetic screens for such defects are challenging, but less clear when a competing patch of medium con- requiring isolation of genes expressed at the haploid taining 0.1% sucrose was also provided. Consequently, stage, coupled with the ability to rapidly track pollen because pollen tubes must take up carbohydrates as tube behaviors. a source of energy (Labarca and Loewus, 1973), it is possible that the sugars attached to TTS provide nutri- Phase IV, The Septum Surface: Traversing tional support for pollen tube growth rather than guid- the Path to an Ovule ance signals. A gradient of TTS glycosylation has been After pollen tubes exit the transmitting tract, their envi- observed in the tobacco style, with higher levels of car- ronment shifts dramatically—no longer are they sur- bohydrate near the ovary; this gradient may serve to rounded by a nutrient-rich connective tissue but instead lead pollen tubes toward their targets (Wu et al., 1995). are exposed to an air-filled chamber. On the placenta, However, a gradient of any individual molecule, includ- the tubes adhere tightly to the surface of the septum ing TTS, is unlikely to be active over a concentration cells, typically growing over the junction of two cell walls. range sufficient to govern pollen tube guidance through Migration on the funiculus involves the same adhesive the entire transmitting tract (Lush, 1999); more likely, behavior, but upon approaching an ovule this pattern multiple signals are required, as well as mechanisms again changes, with the tubes breaking contact with Review 277

the surface and extending through open space to the (Higashiyama et al., 1998). Laser ablation was used to micropyle (Figure 2D). Both genetic and cell ablation determine which of the eight embryo sac cells provides experiments have shown that pollen tubes perceive sig- a micropyle-targeting signal. Intriguingly, destruction of nals that direct growth through each of these stages. diploid ovule cells, the egg cell, and the central cell had While the molecular nature of these signals is not under- no affect on micropyle targeting in vitro (Higashiyama stood, they appear to be derived from both haploid and et al., 2001), while dramatic defects were observed when diploid female tissues. the synergid cells were ablated. Micropyles completely As described above, Arabidopsis pollen tubes emerge lost their capacity to attract pollen tubes when both -of em %70ف from the transmitting tract near the first available ovule; synergid cells were destroyed, and only this exit point subsequently directs the tubes to the bryo sacs with one ablated synergid attracted a pollen nearest funiculus, with only one pollen tube approaching tube, indicating that both synergid cells emit micropylar each micropyle (Hu¨ lskamp et al., 1995b; Wilhelmi and attractants. This short-range signal is likely chemotactic; Preuss, 1996). Genetic evidence indicates that a combi- the distances through which it acts are consistent with nation of signals from the diploid ovule cells and the theoretical estimates for the effective range of chemical haploid embryo sac is required for targeted growth along gradients (Lush, 1999). the funiculus. Mutations that disrupt these signals cause While the mechanisms that direct the final stage of pollen tubes to migrate aberrantly, growing past funiculi pollen tube growth are likely universal, the molecules and extending to the outer surfaces of ovary (Hu¨ lskamp that regulate these steps may vary between species. et al., 1995b; Ray et al., 1997). Strong support for an Foreign pollen tubes that manage to circumvent all ear- embryo sac signal comes from analysis of mutants that lier barriers to interspecies pollination are often blocked carry a heterozygous chromosomal translocation; this at the micropyle targeting step (Shimizu and Okada, rearrangement leads to genetic imbalances and devel- 2000). Thus, the modes of perception, the effective con- opmental arrest in half of the ovules but no apparent centrations, or the signals themselves may be distinct, defect in the surrounding diploid tissues. Intriguingly, even when closely related species are compared. The pollen tubes selectively bypass the ovules that carry diversity of these signals and their perception mecha- defective embryo sacs yet grow toward those that are nisms promises to be a fascinating area for future morphologically normal (Ray et al., 1997). Other muta- studies. tions clearly point to diploid tissues as a source of important guidance signals. For example, pollen tubes fail to migrate along or adhere to the funiculus of pop2/pop3 Phase V, Releasing Sperm into the Micropyle: mutants, where a diploid-specific function essential for The End of the Journey pollen tube guidance is altered yet other aspects of After reaching a synergid cell, the pollen tube releases development are normal (Wilhelmi and Preuss, 1996). the contents of its cytoplasm, including the two sperm Gene products encoded by POP2/POP3 are required in cells, into the embryo sac (Higashiyama et al., 2000). both male and female tissues, indicating that communi- The synergid also degenerates, leaving the sperm cells cation between both sexes is needed to direct funicular without an enclosing extracellular membrane for the first choice. Such guidance signals might rely on an adhesion time. To achieve double fertilization, one sperm fuses mechanism, elicited in the cells of the septum and funic- with the egg cell and the other with the central cell, ulus in response to signals from the embryo sac (Ray giving rise to the embryo and endosperm, respectively et al., 1997). (Russell, 1992). The sperm cells of flowering plants are The signals that direct pollen tubes to the funiculus nonmotile, and the mechanisms that are responsible for are distinct from downstream cues that promote guid- migration to the egg and central cell are poorly under- ance to the micropyle. The Arabidopsis MAA gene prod- stood. Cytological studies suggest bands of actin from ucts are expressed in the haploid embryo sac and are the synergids might play a role (Russell, 1996). Although required during late stages of its development; these in vitro fertilization conditions have been developed genes may contribute to the production of a signal that (Faure and Dumas, 2001), molecules mediating this fu- directs the last stage of pollen tube growth (Shimizu sion event have not been identified. It is likely that factors and Okada, 2000). In wild-type, pollen tubes follow the expressed by the haploid sperm, pollen tube, and egg borders between files of funiculus cells and then turn genomes are required for this process. abruptly toward the micropyle when they are less then 10 ␮m away. In contrast, pollen tubes approach maaϪ ovules, but fail to reach the micropyle, wandering along Intracellular Signaling Networks Continually the funiculus and growing on the outer ovule surface Redefine the Pollen Tube Tip (Shimizu and Okada, 2000). Along the path of pollen tube growth, extracellular sig- Recent investigations of pollination in Torenia four- nals are transduced to an intracellular response system, nieri have identified the synergid cells of the embryo redirecting tube migration by redefining the tip. The pol- sac as a likely source of the short-range micropylar len tube extends by depositing vesicles carrying new targeting signal (Figure 2D) (Higashiyama et al., 1998, membrane and cell wall components via an actin-myo- 2001). In an in vitro system, pollinated pistils were dis- sin delivery system (Hepler et al., 2001; Vidali and Hepler, sected and placed on medium, and the guidance of 2001). Although few extracellular guidance cues have emerging pollen tubes toward dissected ovules was ob- been elucidated, the availability of in vitro assays, cou- served. Targeting to the micropyle was specific; 98% pled with parallels to other systems, have identified a of intact ovules attracted pollen tubes, while less than long list of candidate intracellular signaling molecules. 0.1% of the pollen tubes arrived at heat-treated ovules Intracellular messengers that direct tip growth include Developmental Cell 278

cyclic AMP, plant-specific Rho-type GTPases, phosphatidylinositol monophosphate kinase, and calcium (Feijo et al., 2001; Hepler et al., 2001; Kost et al., 1999; Moutinho et al., 2001; Zheng and Yang, 2000). Identifying components that connect the intracellular and extracellular guidance signaling networks remains one of the more exciting challenges for the field.

Gene Expression from Haploid Pollen Nuclei The haploid pollen grain and diploid plant can be considered independent organisms that share a genome. Al- though basic cellular functions are performed by a common set of proteins, the haploid pollen tube also contains a unique set of gene products, some of which potentially perceive guidance cues and direct tip growth. Mascarenhas and his colleagues used mRNA hybridization kinetics to demonstrate that Tradescantia and maize pollen grains each contain 20,000–24,000 unique mRNAs, 10%–20% of which are pollen specific (Mascarenhas, 1990). Gene expression from haploid pollen nuclei depends on a unique set of pollen-specific promoter elements, as well as pollen-specific master regulatory factors that control transcription of suites genes at specific times in pollen development and tube growth (Lohrmann et al., 2001; Mitsuda et al., 2001; Yang Figure 3. Pollen Tube Genetics et al., 2001). Expression of pollen-specific messages is (A) Mutations in genes essential for pollen tube or embryo sac function result in distorted marker gene segregation when two heterozy- highly active following the first mitotic division; Lat52 is gous individuals are crossed. For example, a mutant allele (blue) the archetype of a “late” class of promoters, directing that disrupts pollen function is not transmitted to progeny, resulting specific expression from the vegetative nucleus after in a 1:1 segregation of heterozygous and wild-type. pollen mitosis I (Figure 1A) (Twell et al., 1990). Biochemi- (B) An insertion element, containing a ␤-glucuronidase (GUS) re- cal analysis indicates such late transcripts are likely porter gene driven by the postmeiotic Lat52 promoter, simultane- loaded onto polysomes in desiccated pollen grains, ously mutagenizes and marks the pollen tube cell. This reporter can be detected in pollen grains or pollen tubes; consequently, in priming the grain for rapid translation immediately fol- heterozygotes, only half of the pollen cells stain (left). During the lowing hydration (Mascarenhas, 1975). To date, only final stages of fertilization, GUS activity can be detected at the tip about 100 pollen-expressed genes have been cloned; of the pollen tube and near the synergid cell in the embryo sac microarray technology, coupled with novel genetic (right). assays promises to tremendously expand this set.

Pollen Genetics: The Search for Haploid-Specific growth, but are impractical when assessing pollen tube Functions functions obscured by floral tissues. Because haploid- A complete understanding of the role of the haploid specific defects prevent the formation of homozygous pollen genome will require a concerted effort—relying plants, such screens require assays that detect aberrant on genetic screens for gametophytic mutations that spe- formation of half of the pollen produced by heterozygous cifically disrupt pollen function in vivo, morphological plants (Figure 3A). This approach has identified genes assays that rapidly pinpoint the growth phase affected that regulate pollen mitosis and cytokinesis, as well as by each mutation, and biochemical assays that distin- genes that determine vegetative and generative cell guish between defects in pollen tube growth and altered fates (Chen and McCormick, 1996; Johnson and McCor- responses to extracellular guidance cues. Screens for mick, 2001; Magnard et al., 2001; Park et al., 1998). haploid-specific defects encompass genes transcribed In Arabidopsis, screens for aberrant transmission of before tube germination as well as those transcribed mutant alleles can be conducted to saturation. These during tube growth; several lines of evidence confirm surveys are not limited to male defects but also can that both classes are critical for pollen tube function identify essential female gametophyte functions. In or- (Kindiger et al., 1991; Mascarenhas, 1975). Two broad der to track transmission of the mutant alleles, they must strategies have been employed in systematic surveys be tightly linked to markers that are easily scored. The for haploid-specific genes: (1) visual screens for mor- simplest method for achieving this is to mutagenize phological defects or (2) transmission assays for altered plant populations with insertion elements carrying se- rates of inheritance. These screens have the potential lectable markers, such as genes encoding drug resis- to isolate mutants that disrupt each phase of pollen tube tance. Commonly, heterozygous transgenic plants are growth and guidance; they may identify embryo sac- self-fertilized, and their progeny are screened to identify derived signals as well as pollen-expressed signal per- families that deviate from the expected Mendelian ratio ception factors. of 3:1 for drug resistance and sensitivity, respectively Visual screening methods are ideal for examining pol- (Bonhomme et al., 1998; Christensen et al., 1998; Feld- len grain development or early phases of pollen tube mann et al., 1997; Howden et al., 1998; Procissi et al., Review 279

Table 1. Haploid-Specific Mutations that Disrupt Pollen Tube Growth or Guidance Affected Phase Mutant Percent T* Mutant Phenotype of Pollen Tube Cell (Figure 2) Mutagen Reference tip1 50 (top) Germination is reduced, 1, 2, 3 EMS Ryan et al. (1998) 14 (bot) Slow/short growth relative to wild-type†‡ ttd34 1 Short twisted† 3 T-DNA Procissi et al. (2001) ttd41 4 Indistinguishable from wild-type† 4, 5 T-DNA Procissi et al. (2001) ttd42 1 Variable: short, twisted, branched† 3 T-DNA Procissi et al. (2001) mad4 ND Slow/short growth relative to wild-type‡ 1, 2 EMS Grini et al. (1999) pgp1 0 Postgermination 3 or 4 Tn David Twell, personal communication pgp2 1.6 Postgermination 3 or 4 Tn David Twell, personal communication pgp3 0.8 Postgermination 3 or 4 Tn David Twell, personal communication

* Percentage of F1 plants fathered by mutant pollen in a cross of heterozygous mutants to wild-type females. For tip1, heterozygous flowers were self-fertilized and seeds from the top and bottom (bot) half of F1 ovaries were scored for the tip1 phenotype. Fifty percent of transmission occurs only if the mutant grains compete effectively with wild-type. † Determined in vitro. ‡ Determined in vivo. Abbreviations: tip1, defect in root tip growth; ttd, T-DNA transmission defect; mad, male gametophyte defective; pgp, progamic phase defect; EMS, ethanelmethyl sulfonate; Tn, transposon.

2001; D. Twell, personal communication). Segregation the mutant tubes cannot be distinguished morphologi- of the marker gene in a 1:1 pattern indicates a strong cally from their wild-type siblings. While construction of gametophytic defect, and reciprocal crosses to wild- homozygous strains would simplify this process, such type are used to distinguish male- and female-specific homozygotes can only be generated when the mutations functions. Three classes of mutants have been identified are leaky. Alternatively, a cell-autonomous, pollen-spe- in these screens: pollen specific, embryo sac specific, cific reporter could be linked to the mutant defect, dra- and those that affect both gametophytes to some matically enhancing the power of these screens (Figure degree. 3B). Such reporters could be included when insertion Interestingly, several mutants identified in these elements are used as mutagens; alternatively, a collec- screens specifically disrupt male transmission but have tion of strains, marked throughout the genome with such no apparent affect on pollen grain development. These reporters, could be used as a starting point for chemical mutations alter different phases of tube migration and mutagenesis. implicate candidate genes that play a role in pollen tube The field of pollen biology is clearly poised for an growth or guidance functions (Table 1). ttd34 and ttd42 explosion of information as genes identified through show altered tube morphologies when grown in vitro, haploid-specific genetic screens are cloned. In particu- indicating that these genes may be involved in tip speci- lar, the ease of isolating genes tagged by insertion ele- fication or in organization of the pollen tube cytoplasm ments promises to provide exciting breakthroughs in (Procissi et al., 2001). ttd41 cannot be distinguished the near future. Genetic analyses of haploid-specific from wild-type pollen tubes when grown in vitro, yet defects present special challenges—homozygotes can- genetic analysis indicates that ttd41 pollen are almost not be produced and trans-heterozygotes that sort al- completely nonfunctional in vivo. Thus, this mutant may leles into complementation groups cannot be gener- represent a particularly interesting class of late defects ated. These drawbacks are less significant when the that alter guidance within the ovary or sperm cell delivery promise of identifying signaling components and their (Procissi et al., 2001). receptors is considered. As an alternative to random insertional mutagenesis, specific regions of the Arabidopsis genome can be sur- Conclusions and Future Implications veyed if they are linked to a phenotypic marker that From the initiation of pollen adhesion to the fusion of can be conveniently scored. Marked populations are sperm cells with their targets, the analysis of reproduc- mutagenized with a chemical mutagen or ionizing radia- tion in flowering plants provides important opportunities tion, and the offspring of individual plants are screened for revealing how cells perceive signaling information for aberrant segregation of the marker. This approach across cell wall boundaries. Genetic screens have dem- was applied to a large region of chromosome I, identi- onstrated that pollen tube growth requires a diverse fying seven loci essential for gametophyte function. Four array of signals, and biochemical analyses have re- of these mutations affect transmission through the male vealed some candidate signaling molecules, although and three reduce inheritance through both gameto- many additional components are yet to be discovered. phytes (Grini et al., 1999). Because this approach can The challenge of identifying extracellular signaling fac- yield point mutations and subtle alleles, it is an important tors and their receptors will require an integration of complement to insertional mutagenesis. techniques, including cell ablation, in vitro assays, and Phenotypic characterization of heterozygous mutants novel genetic approaches. The analysis of plant cell with late defects in pollen growth is challenging because signaling throughout pollination will continue to provide Developmental Cell 280

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