Pollen tube guidance is a unique navigating system that is required for the successful sexual reproduction of plants. As plant sperm cells are non-motile and egg cells are embedded deep inside the female tissues, a pollen tube delivers the two sperm cells that it contains by growing towards the ovule, in which the egg cell resides. Pollen tube growth towards the ovule is precisely controlled and divided into two stages, preovular and ovular guidance. In this Cell Science at a Glance article and accompanying poster, we provide a comprehensive overview of pollen tube guidance and highlight some of the attractant peptides used during ovular guidance. We further discuss the precise one-to-one guidance system that exists in multi-ovular plants. The pollen tube-blocking system, which is mediated by male–female crosstalk communication, to avoid attraction of multiple pollen tubes, is also reviewed.

An efficient and successful fertilization is important to flowering plants, which have limited time for fertilization and a restricted supply of water. Here, the egg cell is deeply embedded inside the ovule to avoid damage (see poster and glossary). The pollen tube of the male gametophyte includes a vegetative nucleus and two sperm cells. Sperm cells are non-motile (Berger et al., 2008), so they need to be delivered through the pollen tube to the egg cell in the ovule (Zhang et al., 2017). The pollen tube is a polarized cell that grows at the tip in a manner that is controlled by cytoskeleton dynamics (Cheung and Wu, 2008). The pollen tube grows through the pistil and reaches the ovule, where it invades the synergid cell and subsequently ruptures; this leads to the discharge of its cytoplasmic content, including the sperm cells, and double fertilization occurs (see glossary) (Hamamura et al., 2011). In order to reach the micropyle, the opening of the ovule, precisely and quickly, a plant-specific navigation system that is called ‘pollen tube guidance’ has evolved. Here, and in the accompanying poster, we summarize the molecular mechanisms of pollen tube guidance and highlight its chemical attractant molecules.

Glossary

Double fertilization: one sperm cell fertilizes the egg cell to form the embryo and another sperm cell fertilizes the central cell to produce the endosperm (in flowering plants).

Egg cell: the female reproductive cell that fuses with a male reproductive cell (sperm cell) to form the embryo.

Embryo sac: a female gametophyte in flowering plants. The mature embryo sac contains two female gametes (the egg and central cell) and adjacent accessory cells (the synergid and antipodal cells).

Filiform apparatus: fibrous membrane-enriched cell wall region at the micropylar end in the synergid cell.

Funiculus: a stalk-like structure that connects ovules to the placenta in the ovary.

Funicular guidance: a mechanism of pollen tube guidance from the surface of the septum to the funiculus.

Gametophytic tissues: reproductive cells (gametes) are generated in haploid gametophytic tissues. The male gametophytic tissue is the pollen and, for the female plant, it is the embryo sac.

Generative cell: see ‘vegetative nucleus’.

Integument: the outer layer that surrounds the ovule in diploid female tissues.

Male germ unit: an assembly of sperm cells and the vegetative nucleus, which is formed by extracellular matrix of the sperm cells and the physical association of sperm cell and the vegetative nucleus.

Microgametogenesis: a reproductive process that produces a male gametophyte (pollen) in the anther of flowering plants.

Micropylar guidance: a mechanism of pollen tube guidance from the funiculus to the micropyle.

Micropyle: a small hole that is formed without closing the edge of the integument in the ovule. The pollen tube penetrates the embryo sac through this hole.

Microspore: a spore that develops into the male gametophytes (pollen) in flowering plants.

Microsporogenesis: the process by which the diploid nucleus of the microspore mother cell undergoes meiosis to form four haploid microspores.

Ovary: a reproductive organ that contains the ovules to produce the seeds. Fruits are mature ovaries after fertilization.

Ovule: a maternal seed tissue before fertilization in flowering plants. The ovule contains the integument and the embryo sac.

Pistil: a female reproductive organ of flowering plants. The pistil contains the stigma, the style, and the ovary.

Pollen: the male gametophyte of plants that contains either two sperm cells or a generative cell and a vegetative cell.

Pollen tube: a protuberant and tip-growing cylindrical cell of the pollen that contains sperm cells or generative cells.

Pollen tube emergence: pollen tubes emerge from the transmitting tract tissue inside the septum to the septum surface.

Pollen tube rupture: the rupture that occurs when the pollen tube arrives at the synergid cells. Pollen tube discharges the two sperm cells to the egg and central cells.

Semi-in vitro fertilization assay: method to co-cultivate and analyze pollen tubes and ovules on solid medium. Pollinated stigma is cut and placed on the medium, which enables observation of pollen tubes that grow from the end of the cut style to the ovule.

Septum: a planar tissue between each space inside the pistil. In Arabidopsis, two carpels are fused to make spaces that contain the ovules.

Stigma: the part of the pistil that receives the pollen. After the pollen is attached to the stigma, the pollen is hydrated and germinates the pollen tube, which allows penetration into the pistil.

Style: a structure that connects the stigma and the ovary in the pistil of flowering plants.

Synergid cells: accessory cells that are adjacent to the egg cell in the embryo sac of flowering plants. The functions of synergid cells are pollen tube attraction and reception.

Transmitting tract: In Arabidopsis, the tissue that includes cylindrical cells and the extracellular matrix to form a passage for the pollen tube inside the pistil.

Vegetative nucleus: the pollen tube nucleus. During microgametogenesis, the microspore divides asymmetrically to give rise to the vegetative nucleus and the generative cell through a first round of mitosis. Following a second round of mitosis, the generative cell divides into two sperm cells in the pollen or pollen tube of flowering plants.

In flowering plants, male microsporogenesis and microgametogenesis occur in the anther (Suzuki, 2009). Following meiosis, the microspore develops into pollen, the male gametophyte. Within the anther, the microspore undergoes a first round of mitosis, which results in the formation of two unequal cells that contain a haploid nucleus: the large vegetative cell and the small generative cell. The generative cell divides during pollen development or pollen tube elongation in a second round of mitosis to form the two sperm cells (see glossary). The vegetative nucleus and two sperm cells are then transported passively in the pollen tube as the male germ unit (McCue et al., 2011, see poster and glossary). When pollen lands on the stigma, the pollen hydrates and this causes a protuberance of the tip of the pollen tube (pollen tube germination) before it then penetrates into the pistil tissue (Sze et al., 2014). In flowering plants, pistils can be categorized as single-ovular and multi-ovular types. Multi-ovular pistils, such as in Arabidopsis thaliana, enclose ∼60 ovules (Cucinotta et al., 2014). Over 100 pollen tubes grow inside the pistil (Pagnussat et al., 2007). However, it is rare that multiple pollen tubes enter one ovule; the frequency is ∼2% in maize (Kato, 2001) and ∼1% in A. thaliana (Huck et al., 2003; Rotman et al., 2003). Therefore, ‘pollen tube guidance’ as a plant-specific navigation system is needed to facilitate the exact one-to-one coupling between sibling ovules and numerous individual pollen tubes.

Pollen tubes elongate within the pistil to find the ovule through multiple steps: (1) stigma penetration, (2) elongation into the transmitting tract, (3) emergence from the transmitting tract, (4) funicular guidance, and (5) micropylar guidance (see glossary) (Johnson and Lord, 2006). Pollen tubes grow through various female tissues, such as the stigma, style, transmitting tract, septum and the surface of the funiculus (Cheung, 1996). The precision in pollen tube guidance thereby results from crosstalk between these female tissues and the pollen tube (Vogler et al., 2016), that is incompatible pollen tubes are rejected by cell-to-cell communication between the pollen tubes and the female tissues (Takayama and Isogai, 2005; Chapman and Goring, 2010; Swanson et al., 2004). These multistep controls can be categorized into preovular and ovular guidance (Higashiyama and Takeuchi, 2015; Kanaoka and Higashiyama, 2015). Preovular guidance is the navigation of any compatible pollen tube from the stigma to the ovary, whereas ovular guidance refers to the precise navigation of a particular pollen tube to one ovule (see poster) (Higashiyama and Hamamura, 2008; Shimizu and Okada, 2000). In the following sections, we summarize the current knowledge of preovular guidance.

Several mechanisms have been proposed to explain the precise growth of pollen tubes towards the ovules. These include mechanical, chemotropic, geometrical and physiological guidance cues (Heslop-Harrison, 1987; Lush, 1999; Chebli and Geitmann, 2007). In pistils, a combination of these mechanisms is observed. The lily flower pollen tube adheres to the aligning epidermal cells and grows along their surface towards the ovary in the style (Iwanami, 1959). In Torenia fournieri, in vitro fertilization experiments have suggested that the guidance system is not unidirectional, as pollen tubes were able grow towards both ends of the style (Higashiyama and Hamamura, 2008). Furthermore, the female gametophyte is considered unnecessary for pollen tube growth from stigma to style (Hülskamp et al., 1995; Sogo and Tobe, 2005). These findings suggest that pollen tubes grow along tissue structures without any directional cues, whereas some molecules for chemical preovular guidance have been reported. Lily stigma chemocyanin, a 9.9 kDa basic plantacyanin, induces pollen tube chemotropism in vitro (Kim et al., 2003). A. thaliana plantacyanin, which is most abundantly localized in the transmitting tract, shows 86.8% similarity to lily chemocyanin and has been proposed to promote directional pollen tube growth (Dong et al., 2005). Lily-secreted stigma cysteine-rich adhesins (SCAs) bind to the pollen tube and function in the formation of an adhesive pectin matrix at the tip of the pollen tube (Mollet et al., 2000; Park et al., 2000; Park and Lord, 2003). SCAs cannot induce chemotropism by themselves, but binding to the pollen tube is proposed as being important for physical access of the chemocyanin to the plasma membrane to control the directional growth of the pollen tube (Kim et al., 2003). A. thaliana SCA-like lipid transfer protein 5 (LTP5) might also interact with pectin in the pollen tube and facilitate pollen tube guidance (Chae et al., 2009). In the tomato, the cysteine-rich stigma-specific protein 1 (STIG1) has also been shown to stimulate pollen tube growth (Huang et al., 2014). STIG1 interacts with pollen receptor kinase 2 (LePRK2) and phosphatidylinositol 3-phosphate. STIG1 is found in the secreted exudate at the stigma surface and style, and accumulates at the pollen tube surface, acting as a signaling peptide to promote pollen tube growth (Tang et al., 2004; Huang et al., 2014). Pistil brassinosteroids promote pollen tube growth (Vogler et al., 2014) and γ-aminobutyric acid (GABA) modulates Ca2+ channels on the plasma membranes as part of the regulation of signaling pathways for pollen tube growth (Palanivelu et al., 2003; Yu et al., 2014). Thus, not only chemical molecules, but also other mechanisms ensure pollen tube growth during preovular guidance.

The transmitting tract is the path from the stigma to the ovary through which the pollen tube grows and the place where it reacts to the guidance signals (see poster) (Mascarenhas, 1975; Heslop-Harrison, 1987; Lord and Sanders, 1992). Two types of transmitting tract are recognized: the open and hollow style, as seen in lilies, or the solid style, as in A. thaliana (At) (Erbar, 2003; Lennon et al., 1998). Proteins such as NO TRANSMITTING TRACT (NTT), HECATE 1 (HEC1), HEC2, HEC3, HALF FILLED (HAF), SPATULA (SPT) and water-soluble chlorophyll proteins (AtWSCP) are important for transmitting tract development, because they induce a developmentally required programmed cell death (PCD). In Arabidopsis, the transmitting tract is formed by PCD of cells in the septum, which ensures intercellular space and a large amount of extracellular matrix (ECM) for pollen tube growth (Alvarez and Smyth, 1999; Boex-Fontvieille et al., 2015; Crawford et al., 2007; Crawford and Yanofsky, 2011; Gremski et al., 2007). The cylindrical transmitting tract cells are surrounded by extensive amounts of fibrous extracellular matrix (ECM) (see poster); (Lennon et al., 1998). The pollen tube passes through the ECM, which is extremely rich in glycoproteins, polysaccharides, glycolipids and abundant arabinogalactan proteins (AGPs) (Pereira et al., 2015). In Nicotiana, transmitting tissue-specific protein (TTS) and 120K proteins, which belong to the AGP family, are responsible for promoting pollen tube growth and ovular guidance (Cheung et al., 1995; Schultz et al., 1997; Wu et al., 2000). Highly glycosylated TTS proteins are incorporated into the pollen tube, which induces the promotion of pollen tube growth and guidance (Wu et al., 2000). Furthermore, pollen tube AGPs that are localized in the plasma membrane act as a Ca2+ capacitor that regulates intercellular Ca2+-dependent actin polymerization for pollen tube growth (Cárdenas et al., 2008; Lamport et al., 2014; Lamport and Várnai, 2013; Pereira et al., 2015; Suárez et al., 2013). In T. fournieri, the terminal 4-O-methyl-glucuronosyl residue of arabinogalactan polysaccharide (AMOR) was identified as a style molecule (Mizukami et al., 2016; Okuda et al., 2013). The action mechanism of AMOR remains unclear, but AMOR induces competency in the pollen tube to react to LURE-dependent guidance cues (LUREs are peptides that act as pollen tube attractants to render ovular guidance), as described below. Therefore, the transmitting tract is not only a pathway for pollen tube growth, but is also an indispensable tissue conferring the ability of the pollen tube to precisely move toward the ovule.

In Arabidopsis, the pollen tube must emerge from the transmitting tract at some point before reaching the ovule (see poster) (Hülskamp et al., 1995). To exit from the transmitting tract, the pollen tube passes through a very narrow space between the septum cells by means of tip growth; this phenomenon is called pollen tube emergence. After pollen tube emergence, it elongates on the surface of the septum, grows along the surface of the funiculus (funicular guidance) and finally moves toward the ovule when it reaches the micropyle (micropylar guidance) (Palanivelu and Tsukamoto, 2012) (see glossary). It is difficult to visualize pollen tube emergence in situ (Cheung et al., 2010), because these steps take place deep inside pistils that are surrounded by tissues that emit strong autofluorescence, masking the signal of fluorescently tagged pollen tube proteins (Mizuta et al., 2015). The K+ transporters cation/H+ exchanger family genes, CHX21 and CHX23 are localized on the endoplasmic reticulum (ER) of the pollen tube and are the only identified regulators of pollen tube emergence (Lu et al., 2011). CHX21 and CHX23 are proposed to regulate local pH that, in turn, alters actin polymerization for the reorientation of the pollen tube growth direction. However, many open questions concerning pollen tube emergence remain, and it is unclear which tissue even triggers emergence (Higashiyama and Takeuchi, 2015). Known ovular guidance mechanisms, such as the LURE-dependent guidance cue, are too locally restricted to attract the pollen tube from the transmitting tract. In Arabidopsis, the distance between the micropyle and the transmitting tract is ∼100 µm, but LURE-dependent guidance has an effective range of only ∼20 µm (Takeuchi and Higashiyama, 2016). Thus, ovule-derived long-range signals have been proposed to function in this process (Horade et al., 2013; Hülskamp et al., 1995). However, despite the absence of identified factors, it is clear that pollen tube emergence is important as a transition phase from preovular to ovular guidance in order for the pollen tube to reach the ovule.

After emergence, the pollen tube switches to ovular guidance, which can be categorized into funicular and micropylar guidance (see poster and glossary). Our knowledge in this field has been deepened by the use of transcriptomics, advanced imaging and in vitro analyses (Higashiyama and Yang, 2017). Here, some gametophytic mutants, such as mutants in magatama (MAA3; Shimizu and Okada, 2000), myb98 (Kasahara et al., 2005), central cell guidance (CCG; Chen et al., 2007), gamete-expressed 3 (GEX3; Alandete-Saez et al., 2008) and ccg-binding protein1 (CBP1; Li et al., 2015) show a mistargeting of the pollen tube to the ovule. For the semi-in vitro assay, the stigma is pollinated in vivo, and its pistil is cut at the style. Afterwards, dissected ovules are placed on a solid culture medium and the emergence of the pollen tube that growths through the pistil is monitored (see poster) (Cheung et al., 1995; Kandasamy and Kristen, 1987). This allows for both an easier observation of pollen tube growth towards an individual ovule and maintenance of the pollen tube competence for growth and guidance, because they are still exposed to pistil tissue (Higashiyama et al., 1998). With this assay and additional laser ablation analysis, it was shown that T. fournieri ovules, especially synergid cells, provide a diffusible chemical guidance cue (Higashiyama and Hamamura, 2008; Higashiyama et al., 1998, 2001; Horade et al., 2013). Traditionally, synergid cells have been considered to be important for pollen tube guidance based on their secretory function and location. MYB98 is a transcription factor that is specifically expressed in the synergid cells (Kasahara et al., 2005; Punwani et al., 2007; Punwani et al., 2008). The myb98 mutant shows an abnormal filliform apparatus, and this inhibits the secretion of attractants; thus, the pollen tube fails to find the micropyle. In contrast, CCG, CBP1 and GEX3 are predominantly expressed in the central and/or egg cell. CCG and CBP1 co-regulate cysteine-rich peptides (CRPs) in the central cell and the synergid cells as regulators of transcription initiation (Chen et al., 2007; Li et al., 2015). GEX3, a plasma membrane-localized protein expressed in the egg cell, plays a role in micropylar guidance (Alandete-Saez et al., 2008). These results suggest that the interaction between central cells and egg cells is also important for the function of ovular guidance (Susaki et al., 2015).

To identify the true pollen tube attractants that act in ovular guidance, egg and synergid cells were isolated and transcriptome analysis was performed (Dresselhaus et al., 1994; Márton et al., 2005; Okuda et al., 2009). Based on this, expression of the Zea mays EGG APPARATUS 1 (ZmEA1) (Dresselhaus et al., 1994), which encodes a highly hydrophobic small membrane protein (Márton et al., 2005), was identified in the egg cell. In vitro and knockdown analyses have indicated that ZmEA1 is an attractant peptide for micropylar guidance (Márton et al., 2012). Other small peptides, especially some CRPs, were highly expressed in synergid cells (Jones-Rhoades et al., 2007; Silverstein et al., 2007; Okuda et al., 2009). Interestingly, many CRPs are related to cell-to-cell communication (Jones-Rhoades et al., 2007; Higashiyama, 2010), such as pollen tube guidance (defensin-like proteins; Okuda et al., 2009) and pollen tube rupture (thionin-like proteins, Leydon et al., 2013); and pectinmethylesterase inhibitor (PMEI) (Woriedh et al., 2013).

T. fournieri (Tf)LURE1 and TfLURE2 are defensin-like CRPs and were identified as pollen tube attractants that are responsible for micropylar guidance in T. fournieri (Okuda et al., 2009). TfLUREs are small polypeptides that contain six cysteine residues and are predominantly expressed in synergid cells (Okuda et al., 2009). Gelatin beads that contain recombinant TfLUREs are able to attract pollen tubes (Higashiyama, 2010). Interestingly, a very low amount (40 pM) of TfLURE2 in beads (corresponding to only ∼1000 molecules) is sufficient to give rise to pollen tube attraction (Goto et al., 2011). Mechanistically, the terminal disaccharide, 4-O-methyl-glucuronosyl residue of AMOR conveys competence to the pollen tube to enable it to react to TfLUREs (Mizukami et al., 2016). LUREs have also been identified in other species, including A. thaliana (AtLUREs), A. lyrata (AlLUREs) (Takeuchi and Higashiyama, 2012) and T. concolor (TcCRP1) (Kanaoka et al., 2011). The amino acid sequences of TfLURE1 and TcCRP1 only differ in eight of 62 residues; strikingly, these confer the species-preferential attraction (Kanaoka et al., 2011). Therefore, LUREs have evolved rapidly, as shown by the high species preference for the guidance cue.

How these attractants overcome interspecific hybridization barriers has also been examined. Maize ZmEA1, which is expressed in Arabidopsis ovules can attract maize pollen tubes (Márton et al., 2012). Arabidopsis LURE1 peptides are also able to attract Arabidopsis pollen tubes, even if they are expressed in a Torenia ovule (Takeuchi and Higashiyama, 2012). These results demonstrate that attractant peptides alone are sufficient to overcome reproductive barriers in ovular guidance, even between distant species.

In Arabidopsis, receptors for LURE1 were identified by means of yeast two-hybrid and phylogenetic analysis with knockout mutants, and comprise the leucine-rich repeats (LRR) receptor-like kinases MALE DISCOVERER 1 (MDIS1), MDIS1-INTERACTING RECEPTOR LIKE KINASE (MIK1), MIK2 (Wang et al., 2016) and POLLEN RECEPTOR-LIKE KINASE 6 (PRK6) (Takeuchi and Higashiyama, 2016). Recombinant AtLURE1 is capable of inducing heterodimerization of MDIS1, and both MIKs. Furthermore, MDIS1, MIK1 and MIK2 localize to the plasma membrane, and knockout mutants for these genes show defective micropylar guidance (Wang et al., 2016). PRK6 is one of eight PRKs in A. thaliana (Chang et al., 2013), and is specifically expressed in the pollen tube (Qin et al., 2009). PRK6 localizes to the plasma membrane of the pollen tube tip and this localization changes in an asymmetric fashion towards AtLURE1 before the pollen tube tip growth direction turns (Takeuchi and Higashiyama, 2016). Recently, pollen tubes of Capsella rubella that express PRK6 or MDIS1 from Arabidopsis have been shown to be attracted to the Arabidopsis ovule, which indicates that MDIS1, MIK1, MIK2 and PRK6 are receptors for species-preferential ovular attractants (Takeuchi and Higashiyama, 2016; Wang et al., 2016). Among the eight PRKs in A. thaliana, PRK6 interacts with PRK1, PRK3 and PRK8, because combinations of mutants show more severe attraction defects (Takeuchi and Higashiyama, 2016). PRK6 also interacts with pollen-expressed Rho of plant guanine nucleotide-exchange factors (ROPGEFs) and the Rho GTPase ROP1 at the plasma membrane, which may promote intracellular tip growth through the activation of the signaling switch. LOST IN POLLEN TUBE GUIDANCE 1 (LIP1) and LIP2 are also receptor-like kinases that are localized at the pollen tube tip plasma membrane (Liu et al., 2013). The lip1 lip2 double mutant is defective in pollen tube guidance that is triggered by AtLURE1; however, LIPs are not direct receptors because they lack an extracellular domain. Ca2+ gradients in pollen tube tips are also essential for micropylar guidance, which is controlled by the plasma membrane-localized cyclic nucleotide-gated channel CNGC18 (Gao et al., 2016).

Several proteins that localize to the ER have also been reported to be involved in pollen tube guidance signaling. For example, POLLEN DEFECTIVE IN GUIDANCE 1 (POD1) interacts with the chaperone CALRETICULIN 3 (CRT3) and controls the folding of membrane proteins to regulate the pollen tube response to signaling (Li et al., 2011). ABNORMAL POLLEN TUBE GUIDANCE 1 (APTG1) was also identified as an ER-localized mannosyltransferase (Dai et al., 2014) that participates in glycosylphosphatidylinositol (GPI) synthesis of COBRA-LIKE PROTEIN 10 (COBL10) (Li et al., 2013). COBL10 localizes to the pollen tube tip, and ovular guidance is defective in its mutant (Li et al., 2013). MITOGEN-ACTIVATED PROTEIN KINASES 3 and 6 (MPK3 and MPK6) are also involved in funicular guidance through the control of competence of pollen tubes for signaling (Guan et al., 2014). Although pollen tube receptors for LUREs have been identified, the full picture of pollen tube guidance is still unclear and further analysis of the signaling mechanisms is needed.

After the pollen tube reaches the micropyle, it enters one synergid cell and pollen tube rupture occurs (see poster and glossary). Three receptor-like kinases, ANXURE 1 and 2 (ANX1 and ANX2) in the pollen tube, and FERONIA (FER) in the synergid cells, have been proposed as triggers of this rupture (Huck et al., 2003; Boisson-Dernier et al., 2009; Miyazaki et al., 2009) through PCD of the receptive synergid cell and additional Ca2+ responses (Boisson-Dernier et al., 2013; Ngo et al., 2014). LORELEI (LRE), which encodes a CRP with a putative GPI-anchor addition domain, and LORELEI-like GPI anchored proteins 1 and 3 (LLG1 and LLG3) are proposed to have functions in chaperoning as co-receptors with FER (Hafidh et al., 2016; Liu et al., 2016). LRE and LLG1 interact with FER in the ER lumen and function as chaperones to bring FER to the filiform apparatus and regulate pollen tube rupture (Li et al., 2015).

In the Arabidopsis ovary, only about one in 60 ovules attracts two pollen tubes (Pagnussat et al., 2007). In contrast, some mutants, for example, maa (Shimizu and Okada, 2000), myb98 (Kasahara et al., 2005) and aptg1 (Dai et al., 2014), show attraction of multiple pollen tubes to a single ovule. Recent experiments have shown that the number of pollen tubes that a single ovule can accept is strictly controlled (Kasahara et al., 2012; Maruyama et al., 2015). If double fertilization fails in an ovule, a fertilization recovery system is triggered; a second pollen tube is attracted to the other synergid cell, but only several hours later (Kasahara et al., 2012; von Besser et al., 2006). This delay suggests that there is a transient signal that blocks the attraction of multiple pollen tubes, such as the degradation of attractants (Shimizu and Okada, 2000). Recently, γ subunit of adaptor protein 1 (AP1G) and V-ATPases were identified as causing synergid cell degradation, which may act as the pollen tube-blocking signal (Wang et al., 2017).

Subsequently, fertilization of the egg cell by the sperm cell triggers ethylene signaling, whereas the fertilization of central cell by sperm induces fusion between the synergid and endosperm (Maruyama et al., 2015; Völz et al., 2013). These two pathways induce PCD of the remaining synergid cell (see poster), which is the second blocking signal that prevents the attraction of multiple pollen tubes. In mammals, polyspermy is prevented by (1) an electric change in the egg plasma membrane, and (2) exocytosis of cortical granules (Bianchi et al., 2014; Cheeseman et al., 2016). It is interesting that the female gametophyte monitors its cellular condition through cell-to-cell communication between male and female gametes to control seemingly opposing mechanisms of attraction and rejection of the pollen tube.

Even after two decades of research, the mechanisms of pollen tube guidance remain to be fully understood. It has been demonstrated that pollen tube attractants (LUREs) and their receptors (PRK6 and MDIS–MIK1) play a key role in micropylar guidance in A. thaliana by means of loss-of-function mutants and binding assays (Takeuchi and Higashiyama, 2016; Wang et al., 2016). However, loss-of-function mutants for these genes showed only partial defects on pollen tube guidance in vivo, which suggests that there are yet to be discovered mechanisms at play. In addition, the relationship between MDIS1–MIK and PRK6 remains unknown (Vogler et al., 2016). Furthermore, the signaling cascade downstream of LUREs, and the interaction partners of AMOR also remain elusive. The use of CRISPR/Cas9 genome-editing techniques may help to reveal the function of other candidates and elucidate the underlying molecular mechanisms (Tsutsui and Higashiyama, 2017). It will be important to establish live-cell imaging approaches, such as two-photon excitation microscopy (Cheung et al., 2010; Mizuta et al., 2015), to determine the roles of these molecules at the subcellular level. Microfluidic devices have also been useful in analyzing pollen tube guidance in vivo and in vitro (Horade et al., 2013; Shamsudhin, et al., 2016; Yanagisawa et al., 2017) at the single-cell level (Agudelo et al., 2013; Sanati Nezhad et al., 2014). These methods will enable us to answer many open questions regarding the mechanisms of pollen tube guidance regulation, such as how only one pollen tube is selected among multiple candidates in the transmitting tract and how the attraction of other pollen tubes is prevented.

We thank Dr Nagahara and Dr Takeuchi for providing the Aniline Blue staining image. We thank Dr Kurihara and members of the Higashiyama laboratory for valuable discussions and comments.

Funding

This work was supported by grants from the Japan Science and Technology Agency (JST; PRESTO grant number JPMJPR15QC to Y.M. and ERATO grant number JPMJER1004 to T.H.), the Research Foundation for Opto-Science and Technology (2015-2016 to Y.M.) and the Ministry of Education, Culture, Sports, Science and Technology, Japan (JP16H06465 to T.H.).

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Competing interests

The authors declare no competing or financial interests.