© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs208447. doi:10.1242/jcs.208447

CELL SCIENCE AT A GLANCE SPECIAL ISSUE: CELL BIOLOGY Chemical signaling for tube guidance at a glance Yoko Mizuta1,2,* and Tetsuya Higashiyama1,3

ABSTRACT further discuss the precise one-to-one guidance system that exists Pollen tube guidance is a unique navigating system that is required for in multi-ovular . The pollen tube-blocking system, which is the successful sexual reproduction of plants. As plant cells are mediated by male–female crosstalk communication, to avoid non-motile and egg cells are embedded deep inside the female attraction of multiple pollen tubes, is also reviewed. tissues, a pollen tube delivers the two sperm cells that it contains by growing towards the , in which the egg cell resides. Pollen tube KEY WORDS: Pollen tube, Sexual , One-to-one pollen tube guidance, Intercellular communication, Chemical growth towards the ovule is precisely controlled and divided into attractants, LUREs two stages, preovular and ovular guidance. In this Cell Science at a Glance article and accompanying poster, we provide a Introduction comprehensive overview of pollen tube guidance and highlight An efficient and successful fertilization is important to flowering some of the attractant peptides used during ovular guidance. We 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 1Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo- cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. 2Precursory Research for the male includes a vegetative nucleus and two sperm Embryonic Science and Technology (PRESTO), Japan Science and Technology cells. Sperm cells are non-motile (Berger et al., 2008), so they need Agency (JST), Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. 3Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464- to be delivered through the pollen tube to the egg cell in the ovule 8602, Japan. (Zhang et al., 2017). The pollen tube is a polarized cell that grows at the tip in a manner that is controlled by dynamics *Author for correspondence ([email protected]) (Cheung and Wu, 2008). The pollen tube grows through the pistil Y.M., 0000-0002-8086-2297 and reaches the ovule, where it invades the synergid cell and Journal of Cell Science

1 CELL SCIENCE AT A GLANCE Journal of Cell Science (2018) 131, jcs208447. doi:10.1242/jcs.208447

Glossary : one sperm cell fertilizes the egg cell to form the and another sperm cell fertilizes the central cell to produce the (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 (the egg and central cell) and adjacent accessory cells (the synergid and antipodal cells). Filiform apparatus: fibrous membrane-enriched region at the micropylar end in the synergid cell. Funiculus: a stalk-like structure that connects to the placenta in the . 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 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. : a that develops into the male (pollen) in flowering plants. Microsporogenesis: the process by which the diploid nucleus of the microspore mother cell undergoes meiosis to form four haploid . Ovary: a reproductive organ that contains the ovules to produce the . are mature ovaries after fertilization. Ovule: a maternal 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 , 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 , 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 . Following a second round of mitosis, the generative cell divides into two sperm cells in the pollen or pollen tube of flowering plants.

subsequently ruptures; this leads to the discharge of its cytoplasmic tube ) before it then penetrates into the pistil tissue (Sze content, including the sperm cells, and double fertilization occurs et al., 2014). In flowering plants, pistils can be categorized as single- (see glossary) (Hamamura et al., 2011). In order to reach the ovular and multi-ovular types. Multi-ovular pistils, such as in micropyle, the opening of the ovule, precisely and quickly, a plant- , enclose ∼60 ovules (Cucinotta et al., 2014). specific navigation system that is called ‘pollen tube guidance’ has Over 100 pollen tubes grow inside the pistil (Pagnussat et al., 2007). evolved. Here, and in the accompanying poster, we summarize the However, it is rare that multiple pollen tubes enter one ovule; the molecular mechanisms of pollen tube guidance and highlight its frequency is ∼2% in (Kato, 2001) and ∼1% in A. thaliana chemical attractant molecules. (Huck et al., 2003; Rotman et al., 2003). Therefore, ‘pollen tube guidance’ as a plant-specific navigation system is needed to One-to-one pollen tube guidance facilitate the exact one-to-one coupling between sibling ovules and In flowering plants, male microsporogenesis and numerous individual pollen tubes. microgametogenesis occur in the anther (Suzuki, 2009). Following meiosis, the microspore develops into pollen, the male Multistep control of pollen tube guidance gametophyte. Within the anther, the microspore undergoes a first Pollen tubes elongate within the pistil to find the ovule through round of mitosis, which results in the formation of two unequal cells multiple steps: (1) stigma penetration, (2) elongation into the that contain a haploid nucleus: the large vegetative cell and the small transmitting tract, (3) emergence from the transmitting tract, generative cell. The generative cell divides during pollen (4) funicular guidance, and (5) micropylar guidance (see glossary) development or pollen tube elongation in a second round of (Johnson and Lord, 2006). Pollen tubes grow through various female mitosis to form the two sperm cells (see glossary). The vegetative tissues, such as the stigma, style, transmitting tract, septum and the nucleus and two sperm cells are then transported passively in the surface of the funiculus (Cheung, 1996). The precision in pollen tube pollen tube as the male germ unit (McCue et al., 2011, see poster guidance thereby results from crosstalk between these female tissues and glossary). When pollen lands on the stigma, the pollen hydrates and the pollen tube (Vogler et al., 2016), that is incompatible pollen and this causes a protuberance of the tip of the pollen tube (pollen tubes are rejected by cell-to-cell communication between the pollen Journal of Cell Science

2 CELL SCIENCE AT A GLANCE Journal of Cell Science (2018) 131, jcs208447. doi:10.1242/jcs.208447 tubes and the female tissues (Takayama and Isogai, 2005; Chapman the solid style, as in A. thaliana (At) (Erbar, 2003; Lennon et al., and Goring, 2010; Swanson et al., 2004). These multistep controls 1998). Proteins such as NO TRANSMITTING TRACT (NTT), can be categorized into preovular and ovular guidance (Higashiyama HECATE 1 (HEC1), HEC2, HEC3, HALF FILLED (HAF), and Takeuchi, 2015; Kanaoka and Higashiyama, 2015). Preovular SPATULA (SPT) and water-soluble proteins guidance is the navigation of any compatible pollen tube from the (AtWSCP) are important for transmitting tract development, stigma to the ovary, whereas ovular guidance refers to the precise because they induce a developmentally required programmed cell navigation of a particular pollen tube to one ovule (see poster) death (PCD). In Arabidopsis, the transmitting tract is formed by (Higashiyama and Hamamura, 2008; Shimizu and Okada, 2000). In PCD of cells in the septum, which ensures intercellular space and a the following sections, we summarize the current knowledge of large amount of extracellular matrix (ECM) for pollen tube growth preovular guidance. (Alvarez and Smyth, 1999; Boex-Fontvieille et al., 2015; Crawford et al., 2007; Crawford and Yanofsky, 2011; Gremski et al., 2007). Preovular guidance The cylindrical transmitting tract cells are surrounded by extensive Several mechanisms have been proposed to explain the precise amounts of fibrous extracellular matrix (ECM) (see poster); growth of pollen tubes towards the ovules. These include (Lennon et al., 1998). The pollen tube passes through the ECM, mechanical, chemotropic, geometrical and physiological guidance which is extremely rich in glycoproteins, polysaccharides, cues (Heslop-Harrison, 1987; Lush, 1999; Chebli and Geitmann, glycolipids and abundant arabinogalactan proteins (AGPs) 2007). In pistils, a combination of these mechanisms is observed. (Pereira et al., 2015). In Nicotiana, transmitting tissue-specific The lily pollen tube adheres to the aligning epidermal cells protein (TTS) and 120K proteins, which belong to the AGP family, and grows along their surface towards the ovary in the style are responsible for promoting pollen tube growth and ovular (Iwanami, 1959). In Torenia fournieri, in vitro fertilization guidance (Cheung et al., 1995; Schultz et al., 1997; Wu et al., 2000). experiments have suggested that the guidance system is not Highly glycosylated TTS proteins are incorporated into the pollen unidirectional, as pollen tubes were able grow towards both ends tube, which induces the promotion of pollen tube growth and of the style (Higashiyama and Hamamura, 2008). Furthermore, the guidance (Wu et al., 2000). Furthermore, pollen tube AGPs that are female gametophyte is considered unnecessary for pollen tube localized in the plasma membrane act as a Ca2+ capacitor that growth from stigma to style (Hülskamp et al., 1995; Sogo and Tobe, regulates intercellular Ca2+-dependent polymerization for 2005). These findings suggest that pollen tubes grow along tissue pollen tube growth (Cárdenas et al., 2008; Lamport et al., 2014; structures without any directional cues, whereas some molecules for Lamport and Várnai, 2013; Pereira et al., 2015; Suárez et al., 2013). chemical preovular guidance have been reported. Lily stigma In T. fournieri, the terminal 4-O-methyl-glucuronosyl residue of chemocyanin, a 9.9 kDa basic plantacyanin, induces pollen tube arabinogalactan polysaccharide (AMOR) was identified as a style in vitro (Kim et al., 2003). A. thaliana plantacyanin, molecule (Mizukami et al., 2016; Okuda et al., 2013). The action which is most abundantly localized in the transmitting tract, shows mechanism of AMOR remains unclear, but AMOR induces 86.8% similarity to lily chemocyanin and has been proposed to competency in the pollen tube to react to LURE-dependent promote directional pollen tube growth (Dong et al., 2005). Lily- guidance cues (LUREs are peptides that act as pollen tube secreted stigma cysteine-rich adhesins (SCAs) bind to the pollen attractants to render ovular guidance), as described below. tube and function in the formation of an adhesive pectin matrix at the Therefore, the transmitting tract is not only a pathway for pollen tip of the pollen tube (Mollet et al., 2000; Park et al., 2000; Park and tube growth, but is also an indispensable tissue conferring the ability Lord, 2003). SCAs cannot induce chemotropism by themselves, but of the pollen tube to precisely move toward the ovule. binding to the pollen tube is proposed as being important for physical access of the chemocyanin to the plasma membrane to Emergence of pollen tubes from the transmitting tract control the directional growth of the pollen tube (Kim et al., 2003). In Arabidopsis, the pollen tube must emerge from the transmitting A. thaliana SCA-like transfer protein 5 (LTP5) might also tract at some point before reaching the ovule (see poster) (Hülskamp interact with pectin in the pollen tube and facilitate pollen tube et al., 1995). To exit from the transmitting tract, the pollen tube guidance (Chae et al., 2009). In the tomato, the cysteine-rich stigma- passes through a very narrow space between the septum cells by specific protein 1 (STIG1) has also been shown to stimulate pollen means of tip growth; this phenomenon is called pollen tube tube growth (Huang et al., 2014). STIG1 interacts with pollen receptor emergence. After pollen tube emergence, it elongates on the surface kinase 2 (LePRK2) and phosphatidylinositol 3-phosphate. STIG1 is of the septum, grows along the surface of the funiculus (funicular found in the secreted exudate at the stigma surface and style, and guidance) and finally moves toward the ovule when it reaches the accumulates at the pollen tube surface, acting as a signaling peptide to micropyle (micropylar guidance) (Palanivelu and Tsukamoto, promote pollen tube growth (Tang et al., 2004; Huang et al., 2014). 2012) (see glossary). It is difficult to visualize pollen tube Pistil promote pollen tube growth (Vogler et al., emergence in situ (Cheung et al., 2010), because these steps take 2014) and γ-aminobutyric acid (GABA) modulates Ca2+ channels on place deep inside pistils that are surrounded by tissues that emit the plasma membranes as part of the regulation of signaling pathways strong autofluorescence, masking the signal of fluorescently tagged for pollen tube growth (Palanivelu et al., 2003; Yu et al., 2014). Thus, pollen tube proteins (Mizuta et al., 2015). The K+ transporters not only chemical molecules, but also other mechanisms ensure cation/H+ exchanger family genes, CHX21 and CHX23 are pollen tube growth during preovular guidance. localized on the endoplasmic reticulum (ER) of the pollen tube and are the only identified regulators of pollen tube emergence (Lu The role of the transmitting tract et al., 2011). CHX21 and CHX23 are proposed to regulate local pH The transmitting tract is the path from the stigma to the ovary that, in turn, alters actin polymerization for the reorientation of the through which the pollen tube grows and the place where it reacts pollen tube growth direction. However, many open questions to the guidance signals (see poster) (Mascarenhas, 1975; Heslop- concerning pollen tube emergence remain, and it is unclear which Harrison, 1987; Lord and Sanders, 1992). Two types of transmitting tissue even triggers emergence (Higashiyama and Takeuchi, 2015). tract are recognized: the open and hollow style, as seen in lilies, or Known ovular guidance mechanisms, such as the LURE-dependent Journal of Cell Science

3 CELL SCIENCE AT A GLANCE Journal of Cell Science (2018) 131, jcs208447. doi:10.1242/jcs.208447 guidance cue, are too locally restricted to attract the pollen tube from Rhoades et al., 2007; Silverstein et al., 2007; Okuda et al., 2009). the transmitting tract. In Arabidopsis, the distance between the Interestingly, many CRPs are related to cell-to-cell communication micropyle and the transmitting tract is ∼100 µm, but LURE- (Jones-Rhoades et al., 2007; Higashiyama, 2010), such as pollen tube dependent guidance has an effective range of only ∼20 µm guidance (defensin-like proteins; Okuda et al., 2009) and pollen (Takeuchi and Higashiyama, 2016). Thus, ovule-derived long- tube rupture (thionin-like proteins, Leydon et al., 2013); and range signals have been proposed to function in this process pectinmethylesterase inhibitor (PMEI) (Woriedh et al., 2013). (Horade et al., 2013; Hülskamp et al., 1995). However, despite the T. fournieri (Tf)LURE1 and TfLURE2 are defensin-like CRPs absence of identified factors, it is clear that pollen tube emergence is and were identified as pollen tube attractants that are responsible for important as a transition phase from preovular to ovular guidance in micropylar guidance in T. fournieri (Okuda et al., 2009). TfLUREs order for the pollen tube to reach the ovule. are small polypeptides that contain six cysteine residues and are predominantly expressed in synergid cells (Okuda et al., 2009). Ovular guidance Gelatin beads that contain recombinant TfLUREs are able to attract After emergence, the pollen tube switches to ovular guidance, pollen tubes (Higashiyama, 2010). Interestingly, a very low amount which can be categorized into funicular and micropylar guidance (40 pM) of TfLURE2 in beads (corresponding to only ∼1000 (see poster and glossary). Our knowledge in this field has been molecules) is sufficient to give rise to pollen tube attraction (Goto deepened by the use of transcriptomics, advanced imaging and et al., 2011). Mechanistically, the terminal disaccharide, 4-O- in vitro analyses (Higashiyama and Yang, 2017). Here, some methyl-glucuronosyl residue of AMOR conveys competence to the gametophytic mutants, such as mutants in magatama (MAA3; pollen tube to enable it to react to TfLUREs (Mizukami et al., 2016). Shimizu and Okada, 2000), myb98 (Kasahara et al., 2005), central LUREs have also been identified in other species, including cell guidance (CCG; Chen et al., 2007), -expressed 3 A. thaliana (AtLUREs), A. lyrata (AlLUREs) (Takeuchi and (GEX3; Alandete-Saez et al., 2008) and ccg-binding protein1 Higashiyama, 2012) and T. concolor (TcCRP1) (Kanaoka et al., (CBP1; Li et al., 2015) show a mistargeting of the pollen tube to the 2011). The amino acid sequences of TfLURE1 and TcCRP1 only ovule. For the semi-in vitro assay, the stigma is pollinated in vivo, differ in eight of 62 residues; strikingly, these confer the species- and its pistil is cut at the style. Afterwards, dissected ovules are preferential attraction (Kanaoka et al., 2011). Therefore, LUREs placed on a solid culture medium and the emergence of the pollen have evolved rapidly, as shown by the high species preference for tube that growths through the pistil is monitored (see poster) the guidance cue. (Cheung et al., 1995; Kandasamy and Kristen, 1987). This allows How these attractants overcome interspecific hybridization for both an easier observation of pollen tube growth towards an barriers has also been examined. Maize ZmEA1, which is individual ovule and maintenance of the pollen tube competence for expressed in Arabidopsis ovules can attract maize pollen tubes growth and guidance, because they are still exposed to pistil tissue (Márton et al., 2012). Arabidopsis LURE1 peptides are also able to (Higashiyama et al., 1998). With this assay and additional laser attract Arabidopsis pollen tubes, even if they are expressed in a ablation analysis, it was shown that T. fournieri ovules, especially Torenia ovule (Takeuchi and Higashiyama, 2012). These results synergid cells, provide a diffusible chemical guidance cue demonstrate that attractant peptides alone are sufficient to overcome (Higashiyama and Hamamura, 2008; Higashiyama et al., 1998, reproductive barriers in ovular guidance, even between distant 2001; Horade et al., 2013). Traditionally, synergid cells have been species. considered to be important for pollen tube guidance based on their secretory function and location. MYB98 is a transcription factor that Pollen tube receptors for LUREs and related signaling is specifically expressed in the synergid cells (Kasahara et al., 2005; factors Punwani et al., 2007; Punwani et al., 2008). The myb98 mutant In Arabidopsis, receptors for LURE1 were identified by means of shows an abnormal filliform apparatus, and this inhibits the yeast two-hybrid and phylogenetic analysis with knockout mutants, of attractants; thus, the pollen tube fails to find the and comprise the leucine-rich repeats (LRR) receptor-like kinases micropyle. In contrast, CCG, CBP1 and GEX3 are predominantly MALE DISCOVERER 1 (MDIS1), MDIS1-INTERACTING expressed in the central and/or egg cell. CCG and CBP1 co-regulate RECEPTOR LIKE KINASE (MIK1), MIK2 (Wang et al., 2016) cysteine-rich peptides (CRPs) in the central cell and the synergid and POLLEN RECEPTOR-LIKE KINASE 6 (PRK6) (Takeuchi cells as regulators of transcription initiation (Chen et al., 2007; Li and Higashiyama, 2016). Recombinant AtLURE1 is capable of et al., 2015). GEX3, a plasma membrane-localized protein inducing heterodimerization of MDIS1, and both MIKs. expressed in the egg cell, plays a role in micropylar guidance Furthermore, MDIS1, MIK1 and MIK2 localize to the plasma (Alandete-Saez et al., 2008). These results suggest that the membrane, and knockout mutants for these genes show defective interaction between central cells and egg cells is also important micropylar guidance (Wang et al., 2016). PRK6 is one of eight for the function of ovular guidance (Susaki et al., 2015). PRKs in A. thaliana (Chang et al., 2013), and is specifically expressed in the pollen tube (Qin et al., 2009). PRK6 localizes to the Pollen tube attractant peptides – the LUREs plasma membrane of the pollen tube tip and this localization To identify the true pollen tube attractants that act in ovular changes in an asymmetric fashion towards AtLURE1 before the guidance, egg and synergid cells were isolated and transcriptome pollen tube tip growth direction turns (Takeuchi and Higashiyama, analysis was performed (Dresselhaus et al., 1994; Márton et al., 2016). Recently, pollen tubes of Capsella rubella that express 2005; Okuda et al., 2009). Based on this, expression of the Zea mays PRK6 or MDIS1 from Arabidopsis have been shown to be attracted EGG APPARATUS 1 (ZmEA1) (Dresselhaus et al., 1994), which to the Arabidopsis ovule, which indicates that MDIS1, MIK1, encodes a highly hydrophobic small membrane protein (Márton MIK2 and PRK6 are receptors for species-preferential ovular et al., 2005), was identified in the egg cell. In vitro and knockdown attractants (Takeuchi and Higashiyama, 2016; Wang et al., 2016). analyses have indicated that ZmEA1 is an attractant peptide for Among the eight PRKs in A. thaliana, PRK6 interacts with PRK1, micropylar guidance (Márton et al., 2012). Other small peptides, PRK3 and PRK8, because combinations of mutants show more especially some CRPs, were highly expressed in synergid cells (Jones- severe attraction defects (Takeuchi and Higashiyama, 2016). PRK6 Journal of Cell Science

4 CELL SCIENCE AT A GLANCE Journal of Cell Science (2018) 131, jcs208447. doi:10.1242/jcs.208447 also interacts with pollen-expressed Rho of plant guanine nucleotide- Subsequently, fertilization of the egg cell by the sperm cell exchange factors (ROPGEFs) and the Rho GTPase ROP1 at the triggers ethylene signaling, whereas the fertilization of central cell plasma membrane, which may promote intracellular tip growth by sperm induces fusion between the synergid and endosperm through the activation of the signaling switch. LOST IN POLLEN (Maruyama et al., 2015; Völz et al., 2013). These two pathways TUBE GUIDANCE 1 (LIP1) and LIP2 are also receptor-like kinases induce PCD of the remaining synergid cell (see poster), which is the that are localized at the pollen tube tip plasma membrane (Liu et al., second blocking signal that prevents the attraction of multiple pollen 2013). The lip1 lip2 double mutant is defective in pollen tube tubes. In mammals, polyspermy is prevented by (1) an electric guidance that is triggered by AtLURE1; however, LIPs are not direct change in the egg plasma membrane, and (2) exocytosis of cortical receptors because they lack an extracellular domain. Ca2+ gradients in granules (Bianchi et al., 2014; Cheeseman et al., 2016). It is pollen tube tips are also essential for micropylar guidance, which is interesting that the female gametophyte monitors its cellular controlled by the plasma membrane-localized cyclic nucleotide-gated condition through cell-to-cell communication between male and channel CNGC18 (Gao et al., 2016). female gametes to control seemingly opposing mechanisms of Several proteins that localize to the ER have also been reported to attraction and rejection of the pollen tube. be involved in pollen tube guidance signaling. For example, POLLEN DEFECTIVE IN GUIDANCE 1 (POD1) interacts with Conclusions and future directions the chaperone CALRETICULIN 3 (CRT3) and controls the folding Even after two decades of research, the mechanisms of pollen tube of membrane proteins to regulate the pollen tube response to guidance remain to be fully understood. It has been demonstrated signaling (Li et al., 2011). ABNORMAL POLLEN TUBE that pollen tube attractants (LUREs) and their receptors (PRK6 and GUIDANCE 1 (APTG1) was also identified as an ER-localized MDIS–MIK1) play a key role in micropylar guidance in A. thaliana mannosyltransferase (Dai et al., 2014) that participates in by means of loss-of-function mutants and binding assays (Takeuchi glycosylphosphatidylinositol (GPI) synthesis of COBRA-LIKE and Higashiyama, 2016; Wang et al., 2016). However, loss-of- PROTEIN 10 (COBL10) (Li et al., 2013). COBL10 localizes to the function mutants for these genes showed only partial defects on pollen tube tip, and ovular guidance is defective in its mutant (Li pollen tube guidance in vivo, which suggests that there are yet to be et al., 2013). MITOGEN-ACTIVATED PROTEIN KINASES 3 and discovered mechanisms at play. In addition, the relationship between 6 (MPK3 and MPK6) are also involved in funicular guidance MDIS1–MIK and PRK6 remains unknown (Vogler et al., 2016). through the control of competence of pollen tubes for signaling Furthermore, the signaling cascade downstream of LUREs, and the (Guan et al., 2014). Although pollen tube receptors for LUREs have interaction partners of AMOR also remain elusive. The use of been identified, the full picture of pollen tube guidance is still CRISPR/Cas9 genome-editing techniques may help to reveal the unclear and further analysis of the signaling mechanisms is needed. function of other candidates and elucidate the underlying molecular mechanisms (Tsutsui and Higashiyama, 2017). It will be important Pollen tube reception and prevention of attracting multiple to establish live-cell imaging approaches, such as two-photon pollen tubes excitation microscopy (Cheung et al., 2010; Mizuta et al., 2015), to After the pollen tube reaches the micropyle, it enters one synergid determine the roles of these molecules at the subcellular level. cell and pollen tube rupture occurs (see poster and glossary). Three Microfluidic devices have also been useful in analyzing pollen tube receptor-like kinases, ANXURE 1 and 2 (ANX1 and ANX2) in the guidance in vivo and in vitro (Horade et al., 2013; Shamsudhin, et al., pollen tube, and FERONIA (FER) in the synergid cells, have been 2016; Yanagisawa et al., 2017) at the single-cell level (Agudelo proposed as triggers of this rupture (Huck et al., 2003; Boisson- et al., 2013; Sanati Nezhad et al., 2014). These methods will enable Dernier et al., 2009; Miyazaki et al., 2009) through PCD of the us to answer many open questions regarding the mechanisms of receptive synergid cell and additional Ca2+ responses (Boisson- pollen tube guidance regulation, such as how only one pollen tube is Dernier et al., 2013; Ngo et al., 2014). LORELEI (LRE), which selected among multiple candidates in the transmitting tract and how encodes a CRP with a putative GPI-anchor addition domain, and the attraction of other pollen tubes is prevented. LORELEI-like GPI anchored proteins 1 and 3 (LLG1 and LLG3) are proposed to have functions in chaperoning as co-receptors with FER Acknowledgements (Hafidh et al., 2016; Liu et al., 2016). LRE and LLG1 interact with 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 FER in the ER lumen and function as chaperones to bring FER to the valuable discussions and comments. filiform apparatus and regulate pollen tube rupture (Li et al., 2015). In the Arabidopsis ovary, only about one in 60 ovules attracts two Competing interests pollen tubes (Pagnussat et al., 2007). In contrast, some mutants, for The authors declare no competing or financial interests. example, maa (Shimizu and Okada, 2000), myb98 (Kasahara et al., 2005) and aptg1 (Dai et al., 2014), show attraction of multiple Funding This work was supported by grants from the Japan Science and Technology Agency pollen tubes to a single ovule. Recent experiments have shown that (JST; PRESTO grant number JPMJPR15QC to Y.M. and ERATO grant number the number of pollen tubes that a single ovule can accept is strictly JPMJER1004 to T.H.), the Research Foundation for Opto-Science and Technology controlled (Kasahara et al., 2012; Maruyama et al., 2015). If double (2015-2016 to Y.M.) and the Ministry of Education, Culture, Sports, Science and fertilization fails in an ovule, a fertilization recovery system is Technology, Japan (JP16H06465 to T.H.). triggered; a second pollen tube is attracted to the other synergid cell, Cell science at a glance but only several hours later (Kasahara et al., 2012; von Besser et al., A high-resolution version of the poster and individual poster panels are available for 2006). This delay suggests that there is a transient signal that blocks downloading at http://jcs.biologists.org/lookup/doi/10.1242/jcs.208447. the attraction of multiple pollen tubes, such as the degradation of supplemental. attractants (Shimizu and Okada, 2000). Recently, γ subunit of References adaptor protein 1 (AP1G) and V-ATPases were identified as Agudelo, C. G., Sanati Nezhad, A., Ghanbari, M., Naghavi, M., Packirisamy, M. causing synergid cell degradation, which may act as the pollen tube- and Geitmann, A. (2013). TipChip: a modular, MEMS-based platform blocking signal (Wang et al., 2017). for experimentation and phenotyping of tip-growing cells. Plant J 73, 1057-1068. Journal of Cell Science

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