CELL SCIENCE AT A GLANCE SPECIAL ISSUE: PLANT CELL BIOLOGY

Chemical signaling for pollen tube guidance at a glanceYoko Mizuta1,2,* and Tetsuya Higashiyama1,3

ABSTRACTPollen tube guidance is a unique navigating system that is required forthe successful sexual reproduction of plants. As plant sperm cells arenon-motile and egg cells are embedded deep inside the femaletissues, a pollen tube delivers the two sperm cells that it contains bygrowing towards the ovule, in which the egg cell resides. Pollen tubegrowth towards the ovule is precisely controlled and divided intotwo stages, preovular and ovular guidance. In this Cell Scienceat a Glance article and accompanying poster, we provide acomprehensive overview of pollen tube guidance and highlightsome of the attractant peptides used during ovular guidance. We

further discuss the precise one-to-one guidance system that existsin multi-ovular plants. The pollen tube-blocking system, which ismediated by male–female crosstalk communication, to avoidattraction of multiple pollen tubes, is also reviewed.

KEY WORDS: Pollen tube, Sexual plant reproduction, One-to-onepollen tube guidance, Intercellular communication, Chemicalattractants, LUREs

IntroductionAn efficient and successful fertilization is important to floweringplants, which have limited time for fertilization and a restrictedsupply of water. Here, the egg cell is deeply embedded inside theovule to avoid damage (see poster and glossary). The pollen tube ofthe male gametophyte includes a vegetative nucleus and two spermcells. Sperm cells are non-motile (Berger et al., 2008), so they needto 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 atthe tip in a manner that is controlled by cytoskeleton dynamics(Cheung and Wu, 2008). The pollen tube grows through the pistiland reaches the ovule, where it invades the synergid cell and

1Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. 2Precursory Research forEmbryonic Science and Technology (PRESTO), Japan Science and TechnologyAgency (JST), Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan. 3GraduateSchool of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan.

*Author for correspondence (mizuta.yoko@f.mbox.nagoya-u.ac.jp)

Y.M., 0000-0002-8086-2297

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subsequently ruptures; this leads to the discharge of its cytoplasmiccontent, including the sperm cells, and double fertilization occurs(see glossary) (Hamamura et al., 2011). In order to reach themicropyle, the opening of the ovule, precisely and quickly, a plant-specific navigation system that is called ‘pollen tube guidance’ hasevolved. Here, and in the accompanying poster, we summarize themolecular mechanisms of pollen tube guidance and highlight itschemical attractant molecules.

One-to-one pollen tube guidanceIn flowering plants, male microsporogenesis andmicrogametogenesis occur in the anther (Suzuki, 2009).Following meiosis, the microspore develops into pollen, the malegametophyte. Within the anther, the microspore undergoes a firstround of mitosis, which results in the formation of two unequal cellsthat contain a haploid nucleus: the large vegetative cell and the smallgenerative cell. The generative cell divides during pollendevelopment or pollen tube elongation in a second round ofmitosis to form the two sperm cells (see glossary). The vegetativenucleus and two sperm cells are then transported passively in thepollen tube as the male germ unit (McCue et al., 2011, see posterand glossary). When pollen lands on the stigma, the pollen hydratesand this causes a protuberance of the tip of the pollen tube (pollen

tube germination) before it then penetrates into the pistil tissue (Szeet al., 2014). In flowering plants, pistils can be categorized as single-ovular and multi-ovular types. Multi-ovular pistils, such as inArabidopsis 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; thefrequency is ∼2% in maize (Kato, 2001) and ∼1% in A. thaliana(Huck et al., 2003; Rotman et al., 2003). Therefore, ‘pollen tubeguidance’ as a plant-specific navigation system is needed tofacilitate the exact one-to-one coupling between sibling ovules andnumerous individual pollen tubes.

Multistep control of pollen tube guidancePollen tubes elongate within the pistil to find the ovule throughmultiple steps: (1) stigma penetration, (2) elongation into thetransmitting 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 femaletissues, such as the stigma, style, transmitting tract, septum and thesurface of the funiculus (Cheung, 1996). The precision in pollen tubeguidance thereby results from crosstalk between these female tissuesand the pollen tube (Vogler et al., 2016), that is incompatible pollentubes are rejected by cell-to-cell communication between the pollen

GlossaryDouble fertilization: one sperm cell fertilizes the egg cell to form the embryo and another sperm cell fertilizes the central cell to produce the endosperm (inflowering 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 adjacentaccessory 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, forthe 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 physicalassociation 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 andcentral 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 themedium, 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, whichallows 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 tubeattraction and reception.Transmitting tract: InArabidopsis, 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. Duringmicrogametogenesis, themicrospore divides asymmetrically to give rise to the vegetative nucleus andthe 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 pollentube of flowering plants.

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tubes and the female tissues (Takayama and Isogai, 2005; Chapmanand Goring, 2010; Swanson et al., 2004). These multistep controlscan be categorized into preovular and ovular guidance (Higashiyamaand Takeuchi, 2015; Kanaoka and Higashiyama, 2015). Preovularguidance is the navigation of any compatible pollen tube from thestigma to the ovary, whereas ovular guidance refers to the precisenavigation of a particular pollen tube to one ovule (see poster)(Higashiyama and Hamamura, 2008; Shimizu and Okada, 2000). Inthe following sections, we summarize the current knowledge ofpreovular guidance.

Preovular guidanceSeveral mechanisms have been proposed to explain the precisegrowth of pollen tubes towards the ovules. These includemechanical, chemotropic, geometrical and physiological guidancecues (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 cellsand grows along their surface towards the ovary in the style(Iwanami, 1959). In Torenia fournieri, in vitro fertilizationexperiments have suggested that the guidance system is notunidirectional, as pollen tubes were able grow towards both endsof the style (Higashiyama and Hamamura, 2008). Furthermore, thefemale gametophyte is considered unnecessary for pollen tubegrowth from stigma to style (Hülskamp et al., 1995; Sogo and Tobe,2005). These findings suggest that pollen tubes grow along tissuestructures without any directional cues, whereas some molecules forchemical preovular guidance have been reported. Lily stigmachemocyanin, a 9.9 kDa basic plantacyanin, induces pollen tubechemotropism in vitro (Kim et al., 2003). A. thaliana plantacyanin,which is most abundantly localized in the transmitting tract, shows86.8% similarity to lily chemocyanin and has been proposed topromote directional pollen tube growth (Dong et al., 2005). Lily-secreted stigma cysteine-rich adhesins (SCAs) bind to the pollentube and function in the formation of an adhesive pectin matrix at thetip of the pollen tube (Mollet et al., 2000; Park et al., 2000; Park andLord, 2003). SCAs cannot induce chemotropism by themselves, butbinding to the pollen tube is proposed as being important forphysical access of the chemocyanin to the plasma membrane tocontrol the directional growth of the pollen tube (Kim et al., 2003).A. thaliana SCA-like lipid transfer protein 5 (LTP5) might alsointeract with pectin in the pollen tube and facilitate pollen tubeguidance (Chae et al., 2009). In the tomato, the cysteine-rich stigma-specific protein 1 (STIG1) has also been shown to stimulate pollentube growth (Huang et al., 2014). STIG1 interactswith pollen receptorkinase 2 (LePRK2) and phosphatidylinositol 3-phosphate. STIG1 isfound in the secreted exudate at the stigma surface and style, andaccumulates at the pollen tube surface, acting as a signaling peptide topromote 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 onthe plasmamembranes as part of the regulation of signaling pathwaysfor pollen tube growth (Palanivelu et al., 2003; Yu et al., 2014). Thus,not only chemical molecules, but also other mechanisms ensurepollen tube growth during preovular guidance.

The role of the transmitting tractThe transmitting tract is the path from the stigma to the ovarythrough which the pollen tube grows and the place where it reactsto the guidance signals (see poster) (Mascarenhas, 1975; Heslop-Harrison, 1987; Lord and Sanders, 1992). Two types of transmittingtract 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 celldeath (PCD). In Arabidopsis, the transmitting tract is formed byPCD of cells in the septum, which ensures intercellular space and alarge amount of extracellular matrix (ECM) for pollen tube growth(Alvarez and Smyth, 1999; Boex-Fontvieille et al., 2015; Crawfordet al., 2007; Crawford and Yanofsky, 2011; Gremski et al., 2007).The cylindrical transmitting tract cells are surrounded by extensiveamounts 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-specificprotein (TTS) and 120K proteins, which belong to the AGP family,are responsible for promoting pollen tube growth and ovularguidance (Cheung et al., 1995; Schultz et al., 1997;Wu et al., 2000).Highly glycosylated TTS proteins are incorporated into the pollentube, which induces the promotion of pollen tube growth andguidance (Wu et al., 2000). Furthermore, pollen tube AGPs that arelocalized in the plasma membrane act as a Ca2+ capacitor thatregulates intercellular Ca2+-dependent actin polymerization forpollen 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 ofarabinogalactan polysaccharide (AMOR) was identified as a stylemolecule (Mizukami et al., 2016; Okuda et al., 2013). The actionmechanism of AMOR remains unclear, but AMOR inducescompetency in the pollen tube to react to LURE-dependentguidance cues (LUREs are peptides that act as pollen tubeattractants to render ovular guidance), as described below.Therefore, the transmitting tract is not only a pathway for pollentube growth, but is also an indispensable tissue conferring the abilityof the pollen tube to precisely move toward the ovule.

Emergence of pollen tubes from the transmitting tractIn Arabidopsis, the pollen tube must emerge from the transmittingtract at some point before reaching the ovule (see poster) (Hülskampet al., 1995). To exit from the transmitting tract, the pollen tubepasses through a very narrow space between the septum cells bymeans of tip growth; this phenomenon is called pollen tubeemergence. After pollen tube emergence, it elongates on the surfaceof the septum, grows along the surface of the funiculus (funicularguidance) and finally moves toward the ovule when it reaches themicropyle (micropylar guidance) (Palanivelu and Tsukamoto,2012) (see glossary). It is difficult to visualize pollen tubeemergence in situ (Cheung et al., 2010), because these steps takeplace deep inside pistils that are surrounded by tissues that emitstrong autofluorescence, masking the signal of fluorescently taggedpollen tube proteins (Mizuta et al., 2015). The K+ transporterscation/H+ exchanger family genes, CHX21 and CHX23 arelocalized on the endoplasmic reticulum (ER) of the pollen tubeand are the only identified regulators of pollen tube emergence (Luet al., 2011). CHX21 and CHX23 are proposed to regulate local pHthat, in turn, alters actin polymerization for the reorientation of thepollen tube growth direction. However, many open questionsconcerning pollen tube emergence remain, and it is unclear whichtissue even triggers emergence (Higashiyama and Takeuchi, 2015).Known ovular guidance mechanisms, such as the LURE-dependent

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guidance cue, are too locally restricted to attract the pollen tube fromthe transmitting tract. In Arabidopsis, the distance between themicropyle 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 theabsence of identified factors, it is clear that pollen tube emergence isimportant as a transition phase from preovular to ovular guidance inorder for the pollen tube to reach the ovule.

Ovular guidanceAfter 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 beendeepened by the use of transcriptomics, advanced imaging andin vitro analyses (Higashiyama and Yang, 2017). Here, somegametophytic mutants, such as mutants in magatama (MAA3;Shimizu and Okada, 2000), myb98 (Kasahara et al., 2005), centralcell 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 theovule. For the semi-in vitro assay, the stigma is pollinated in vivo,and its pistil is cut at the style. Afterwards, dissected ovules areplaced on a solid culture medium and the emergence of the pollentube that growths through the pistil is monitored (see poster)(Cheung et al., 1995; Kandasamy and Kristen, 1987). This allowsfor both an easier observation of pollen tube growth towards anindividual ovule and maintenance of the pollen tube competence forgrowth and guidance, because they are still exposed to pistil tissue(Higashiyama et al., 1998). With this assay and additional laserablation analysis, it was shown that T. fournieri ovules, especiallysynergid cells, provide a diffusible chemical guidance cue(Higashiyama and Hamamura, 2008; Higashiyama et al., 1998,2001; Horade et al., 2013). Traditionally, synergid cells have beenconsidered to be important for pollen tube guidance based on theirsecretory function and location. MYB98 is a transcription factor thatis specifically expressed in the synergid cells (Kasahara et al., 2005;Punwani et al., 2007; Punwani et al., 2008). The myb98 mutantshows an abnormal filliform apparatus, and this inhibits thesecretion of attractants; thus, the pollen tube fails to find themicropyle. In contrast, CCG, CBP1 and GEX3 are predominantlyexpressed in the central and/or egg cell. CCG and CBP1 co-regulatecysteine-rich peptides (CRPs) in the central cell and the synergidcells as regulators of transcription initiation (Chen et al., 2007; Liet al., 2015). GEX3, a plasma membrane-localized proteinexpressed in the egg cell, plays a role in micropylar guidance(Alandete-Saez et al., 2008). These results suggest that theinteraction between central cells and egg cells is also importantfor the function of ovular guidance (Susaki et al., 2015).

Pollen tube attractant peptides – the LUREsTo identify the true pollen tube attractants that act in ovularguidance, egg and synergid cells were isolated and transcriptomeanalysis was performed (Dresselhaus et al., 1994; Márton et al.,2005; Okuda et al., 2009). Based on this, expression of the Zea maysEGG APPARATUS 1 (ZmEA1) (Dresselhaus et al., 1994), whichencodes a highly hydrophobic small membrane protein (Mártonet al., 2005), was identified in the egg cell. In vitro and knockdownanalyses have indicated that ZmEA1 is an attractant peptide formicropylar guidance (Márton et al., 2012). Other small peptides,especially someCRPs, 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 tubeguidance (defensin-like proteins; Okuda et al., 2009) and pollentube rupture (thionin-like proteins, Leydon et al., 2013); andpectinmethylesterase inhibitor (PMEI) (Woriedh et al., 2013).

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

How these attractants overcome interspecific hybridizationbarriers has also been examined. Maize ZmEA1, which isexpressed in Arabidopsis ovules can attract maize pollen tubes(Márton et al., 2012). Arabidopsis LURE1 peptides are also able toattract Arabidopsis pollen tubes, even if they are expressed in aTorenia ovule (Takeuchi and Higashiyama, 2012). These resultsdemonstrate that attractant peptides alone are sufficient to overcomereproductive barriers in ovular guidance, even between distantspecies.

Pollen tube receptors for LUREs and related signalingfactorsIn Arabidopsis, receptors for LURE1 were identified by means ofyeast two-hybrid and phylogenetic analysis with knockout mutants,and comprise the leucine-rich repeats (LRR) receptor-like kinasesMALE DISCOVERER 1 (MDIS1), MDIS1-INTERACTINGRECEPTOR LIKE KINASE (MIK1), MIK2 (Wang et al., 2016)and POLLEN RECEPTOR-LIKE KINASE 6 (PRK6) (Takeuchiand Higashiyama, 2016). Recombinant AtLURE1 is capable ofinducing heterodimerization of MDIS1, and both MIKs.Furthermore, MDIS1, MIK1 and MIK2 localize to the plasmamembrane, and knockout mutants for these genes show defectivemicropylar guidance (Wang et al., 2016). PRK6 is one of eightPRKs in A. thaliana (Chang et al., 2013), and is specificallyexpressed in the pollen tube (Qin et al., 2009). PRK6 localizes to theplasma membrane of the pollen tube tip and this localizationchanges in an asymmetric fashion towards AtLURE1 before thepollen tube tip growth direction turns (Takeuchi and Higashiyama,2016). Recently, pollen tubes of Capsella rubella that expressPRK6 or MDIS1 from Arabidopsis have been shown to be attractedto the Arabidopsis ovule, which indicates that MDIS1, MIK1,MIK2 and PRK6 are receptors for species-preferential ovularattractants (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 moresevere attraction defects (Takeuchi and Higashiyama, 2016). PRK6

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also interacts with pollen-expressed Rho of plant guanine nucleotide-exchange factors (ROPGEFs) and the Rho GTPase ROP1 at theplasma membrane, which may promote intracellular tip growththrough the activation of the signaling switch. LOST IN POLLENTUBE GUIDANCE 1 (LIP1) and LIP2 are also receptor-like kinasesthat are localized at the pollen tube tip plasma membrane (Liu et al.,2013). The lip1 lip2 double mutant is defective in pollen tubeguidance that is triggered by AtLURE1; however, LIPs are not directreceptors because they lack an extracellular domain. Ca2+ gradients inpollen tube tips are also essential for micropylar guidance, which iscontrolled by the plasmamembrane-localized cyclic nucleotide-gatedchannel 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 withthe chaperone CALRETICULIN 3 (CRT3) and controls the foldingof membrane proteins to regulate the pollen tube response tosignaling (Li et al., 2011). ABNORMAL POLLEN TUBEGUIDANCE 1 (APTG1) was also identified as an ER-localizedmannosyltransferase (Dai et al., 2014) that participates inglycosylphosphatidylinositol (GPI) synthesis of COBRA-LIKEPROTEIN 10 (COBL10) (Li et al., 2013). COBL10 localizes to thepollen tube tip, and ovular guidance is defective in its mutant (Liet al., 2013). MITOGEN-ACTIVATED PROTEIN KINASES 3 and6 (MPK3 and MPK6) are also involved in funicular guidancethrough the control of competence of pollen tubes for signaling(Guan et al., 2014). Although pollen tube receptors for LUREs havebeen identified, the full picture of pollen tube guidance is stillunclear and further analysis of the signaling mechanisms is needed.

Pollen tube reception and prevention of attracting multiplepollen tubesAfter the pollen tube reaches the micropyle, it enters one synergidcell and pollen tube rupture occurs (see poster and glossary). Threereceptor-like kinases, ANXURE 1 and 2 (ANX1 and ANX2) in thepollen tube, and FERONIA (FER) in the synergid cells, have beenproposed as triggers of this rupture (Huck et al., 2003; Boisson-Dernier et al., 2009; Miyazaki et al., 2009) through PCD of thereceptive synergid cell and additional Ca2+ responses (Boisson-Dernier et al., 2013; Ngo et al., 2014). LORELEI (LRE), whichencodes a CRP with a putative GPI-anchor addition domain, andLORELEI-like GPI anchored proteins 1 and 3 (LLG1 and LLG3) areproposed to have functions in chaperoning as co-receptors with FER(Hafidh et al., 2016; Liu et al., 2016). LRE and LLG1 interact withFER in the ER lumen and function as chaperones to bring FER to thefiliform 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, forexample, maa (Shimizu and Okada, 2000), myb98 (Kasahara et al.,2005) and aptg1 (Dai et al., 2014), show attraction of multiplepollen tubes to a single ovule. Recent experiments have shown thatthe number of pollen tubes that a single ovule can accept is strictlycontrolled (Kasahara et al., 2012; Maruyama et al., 2015). If doublefertilization fails in an ovule, a fertilization recovery system istriggered; 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 blocksthe attraction of multiple pollen tubes, such as the degradation ofattractants (Shimizu and Okada, 2000). Recently, γ subunit ofadaptor protein 1 (AP1G) and V-ATPases were identified ascausing 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 celltriggers ethylene signaling, whereas the fertilization of central cellby sperm induces fusion between the synergid and endosperm(Maruyama et al., 2015; Völz et al., 2013). These two pathwaysinduce PCD of the remaining synergid cell (see poster), which is thesecond blocking signal that prevents the attraction of multiple pollentubes. In mammals, polyspermy is prevented by (1) an electricchange in the egg plasma membrane, and (2) exocytosis of corticalgranules (Bianchi et al., 2014; Cheeseman et al., 2016). It isinteresting that the female gametophyte monitors its cellularcondition through cell-to-cell communication between male andfemale gametes to control seemingly opposing mechanisms ofattraction and rejection of the pollen tube.

Conclusions and future directionsEven after two decades of research, the mechanisms of pollen tubeguidance remain to be fully understood. It has been demonstratedthat pollen tube attractants (LUREs) and their receptors (PRK6 andMDIS–MIK1) play a key role in micropylar guidance in A. thalianaby means of loss-of-function mutants and binding assays (Takeuchiand Higashiyama, 2016; Wang et al., 2016). However, loss-of-function mutants for these genes showed only partial defects onpollen tube guidance in vivo, which suggests that there are yet to bediscoveredmechanisms at play. In addition, the relationship betweenMDIS1–MIK and PRK6 remains unknown (Vogler et al., 2016).Furthermore, the signaling cascade downstream of LUREs, and theinteraction partners of AMOR also remain elusive. The use ofCRISPR/Cas9 genome-editing techniques may help to reveal thefunction of other candidates and elucidate the underlying molecularmechanisms (Tsutsui and Higashiyama, 2017). It will be importantto establish live-cell imaging approaches, such as two-photonexcitation microscopy (Cheung et al., 2010; Mizuta et al., 2015), todetermine the roles of these molecules at the subcellular level.Microfluidic devices have also been useful in analyzing pollen tubeguidance in vivo and in vitro (Horade et al., 2013; Shamsudhin, et al.,2016; Yanagisawa et al., 2017) at the single-cell level (Agudeloet al., 2013; Sanati Nezhad et al., 2014). These methods will enableus to answer many open questions regarding the mechanisms ofpollen tube guidance regulation, such as how only one pollen tube isselected amongmultiple candidates in the transmitting tract and howthe attraction of other pollen tubes is prevented.

AcknowledgementsWe thank Dr Nagahara and Dr Takeuchi for providing the Aniline Blue stainingimage. We thank Dr Kurihara and members of the Higashiyama laboratory forvaluable discussions and comments.

Competing interestsThe authors declare no competing or financial interests.

FundingThis work was supported by grants from the Japan Science and Technology Agency(JST; PRESTO grant number JPMJPR15QC to Y.M. and ERATO grant numberJPMJER1004 to T.H.), the Research Foundation for Opto-Science and Technology(2015-2016 to Y.M.) and the Ministry of Education, Culture, Sports, Science andTechnology, Japan (JP16H06465 to T.H.).

Cell science at a glanceA high-resolution version of the poster and individual poster panels are available fordownloading at http://jcs.biologists.org/lookup/doi/10.1242/jcs.208447.supplemental.

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