Pollen tube growth and guidance toward the female gametes are controlled at various stages by chemotactic signals and growth support molecules derived from the sporophytic and gametophytic tissues of the female flower organs. Pollen grains placed on the stigma (Figure 1A) by contact, wind or different pollinators stick to the papilla cells and start to hydrate followed by their germination. The efficient adhesion of the pollen grain to the papilla cell is regulated by interaction events between these cells and may activate thereby inter- and intra-species barriers to prevent unsuccessful pollination and fertilization events already at this early time point during reproduction. Angiosperms possess different strategies to recognize self from alien pollen and evolved independent self-incompatibility (SI) mechanisms to prevent self-fertilization. Early SI mechanisms are based on cell-cell communication events between the papilla cells and the pollen grains, whereas later SI mechanisms occur while the growing pollen tube interacts with the cells of the transmitting tract. Species of the Solanaceae, for example, use a pistil-expressed S-RNase, which penetrates the pollen tube (McClure et al., 1989; Luu et al., 2000). A compatible pollen tube expresses the S-locus F-box protein (SLF), which leads to the degradation of the S-RNase (Hua and Kao, 2006; Kubo et al., 2010), while in incompatible interactions intact S-RNase degrades RNAs resulting, for example, in the disruption of the actin cytoskeleton and other cellular processes (Liu et al., 2007; Roldán et al., 2012). In Papaveraceae SI depends on the small pistil secreted protein Papaver rhoeas style S (PrsS), which binds to the S-locus pollen tube membrane protein P. r. pollen S (PrpS) and activates a Ca²⁺-dependent signaling cascade resulting in pollen inhibition and programmed cell death (Wheeler et al., 2009; Wu et al., 2011). SI is best understood in Brassicaceae, which use a surface-localized S-locus receptor kinase (SRK) in papilla cells (Takasaki et al., 2000) and a pollen coat localized cysteine-rich protein (SP11/SCR) (Schopfer, 1999; Shiba et al., 2001) to distinguish self from alien pollen. Their successful interaction leads to proteasome dependent degradation of Exo70A1, an essential component of the exocyst complex. It is thought to be involved in secretion of essential pollen germination factors necessary for pollen hydration (Synek et al., 2006; Samuel et al., 2009). Rejection of pollen in Brassicaceae thus occurs already during pollen hydration and germination at the surface of papilla cells. Little is known about SI in the economically important grasses (reviewed in Dresselhaus et al., 2011). Pollen hydration and germination appear not to be affected, although only grass pollen tubes are capable of penetrating the style and reach the transmitting tract. This indicates that SI in the grasses depends on successful interaction of the pollen tube with the sporophytic cells of the style and transmitting tract. The signaling events involved in this recognition process still await their discovery. More details about SI mechanisms can be found in Iwano and Takayama (2012), Watanabe et al. (2012) and Dresselhaus and Franklin-Tong (2013).

After adhesion and hydration, compatible pollen germinates, penetrates the style and grows through the extracellular space of stylar cells toward the transmitting tract (Figure 1B). The growth direction of the pollen tube is regulated by the formation of different gradients including water, γ-amino butyric acid (GABA), calcium and other small molecules such as D-serine. The water flow during hydration forms an external gradient specifying the site of pollen tube outgrowth and was shown to be controlled by triacylglyceride (Lush et al., 1998; Wolters-Arts et al., 1998). Ca²⁺ influx into the pollen tube tip region is known to be essential for germination and tube growth (Brewbaker and Kwack, 1963; for a review see Steinhorst and Kudla, 2013a) and leads to the generation of an oscillating apex-based cytoplasmic Ca²⁺ (Ca²⁺_cyto) gradient (Miller et al., 1992; Calder et al., 1997). Initially, papilla cells export Ca²⁺_cyto by the auto-inhibited Ca²⁺-ATPase13 (ACA13) at the pollen grain adhesion site (Iwano et al., 2004, 2014). Extracellular Ca²⁺ is then imported into the pollen tube by glutamate receptor-like channels (GLRs), which can be stimulated by D-serine (Michard et al., 2011). In animal systems it was shown that GLRs are non-selective cation channels catalyzing Na⁺ and/or Ca²⁺ influx into cells. Binding of the agonist D-serine to GLRs should thus lead to channel opening resulting in a Ca²⁺_cyto increase (Gilliham et al., 2006). D-serine is produced by Serine-Racemase1 (SR1), which shows an expression peak in the style indicating D-serine availability. The induced changes in Ca²⁺_cyto concentration in the pollen tube might thereafter regulate and coordinate many different signaling events like actin polymerization and thus influence pollen tube growth behavior and growth direction. Ca²⁺_cyto-sensors, belonging to the protein families of calmodulin (CaM), calmodulin-like proteins (CMLs), calcium-dependent protein kinases (CDPKs), and calcineurin B-like proteins (CBLs) are expressed in the pollen tube and are thought to control different cell-cell communication events indicated by their localization and overexpression phenotypes. The presence of these different Ca²⁺_cyto receptors around the sperm cells and at the pollen tube tip indicate an essential role of Ca²⁺ signals both during pollen tube growth and double fertilization (Zhou et al., 2009; Steinhorst and Kudla, 2013b).

During pollen tube growth the tip needs to modulate the surrounding cell wall of stylar cells enabling its penetration through the extra-cellular space, most likely by interaction with extensin-like proteins and arabinogalactan proteins as well as the secretion of cell wall softening enzymes and inhibitors such as polygalacturonases and pectin methylesterase inhibitors (Cosgrove et al., 1997; Grobe et al., 1999; Stratford et al., 2001; Ogawa et al., 2009; Nguema-Ona et al., 2012; Woriedh et al., 2013). The transmitting tract is composed of small cylindrical cells that are surrounded by an extracellular matrix (ECM), which contains a mixture of glycoproteins, glycolipids, and polysaccharides (Lennon et al., 1998). The ECM provides essential nutrients as well as components for an accelerated, extended and guided pollen tube growth (Palanivelu and Preuss, 2006). Without an intact transmitting tract like in the NO TRANSMITTING TRACT (NTT) mutant or its target HALF FILLED (HAF), pollen tube growth is severely affected and either slowed down or prematurely terminated. NTT encodes a C2H2/C2HC zinc finger transcription factor involved in ECM production and is essential for programmed cell death in the transmitting tract upon pollination (Crawford et al., 2007). HAF encodes a bHLH transcription factor and is involved in NTT dependent transmitting tract regulation (Crawford and Yanofsky, 2011). The transmitting tract-specific arabinogalactan glycoproteins TTS1 and TTS2 have a positive effect on in vitro grown tobacco pollen and show a gradient of increased glycosylation correlating with pollen tube growth direction inside the transmitting tract (Cheung et al., 1995; Wu et al., 1995). Another factor which has a positive effect on pollen tube growth and guidance is chemocyanin, a small secreted peptide in the style of lily (Kim et al., 2003). The different sporophyte-derived signals do not only guide or increase pollen tube growth rate, but rather lead to a change in the pollen transcriptome and thereby activate the pollen for female gametophyte-derived attraction signals (Higashiyama et al., 1998; Palanivelu and Preuss, 2006). Recently, de novo expression of closely related MYB transcription factors and other genes were reported to be induced during pollen tube growth through the style regulating themselves a number of downstream genes. Hence pollen tubes maturate during their growth through the sporophytic tissue and thereby become competent for fertilization (Leydon et al., 2013, 2014).

The signaling events that control pollen tube exit from the transmitting tract and guidance toward the ovule are not known. In Arabidopsis this process was shown to be tightly regulated and usually only a single pollen tube exits the transmitting tract in proximity of an unfertilized ovule. The pollen tube grows on the septum surface toward the funiculus, the tissue connecting the ovule with the septum (Figures 1B–D; Supplemental Movies 1, 2). At the funiculus the pollen tube is directed through the micropyle inside the ovule by a mechanism known as micropylar guidance (Shimizu and Okada, 2000). In Arabidopsis a gradient of GABA was reported in front of the ovule. The transaminase POLLEN ON PISTIL2 (POP2) forms this gradient through GABA degradation. At moderate concentrations GABA stimulates pollen tube growth and thus likely supports growth toward the ovule (Palanivelu et al., 2003). Another candidate involved in micropylar guidance is D-serine, which was already described above. Its synthesizing enzyme gene SR1 is also expressed in the ovule indicating the presence of D-serine (Michard et al., 2011). Semi-in vitro fertilization experiments revealed an oscillation of Ca²⁺_cyto levels in growing pollen tubes depending on their distance from an unfertilized ovule and especially from the synergid cells (Shi et al., 2009; Iwano et al., 2012). The connection between Ca²⁺_cyto and D-serine by GLR channels in growing pollen tubes was already described above. The observed changes in the Ca²⁺_cyto level depending on its distance from the synergid cells might again result from this interplay.

Recently, two pollen-expressed mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, were identified in Arabidopsis, which are part of the ovular guidance network. In vivo pollination assays revealed that mpk3/6 double mutant pollen tubes were not capable of growing along the funiculus after transmitting tract exit but micropylar guidance (see below) was not effected in the double mutants (Guan et al., 2014). MPK3/6 are two cytoplasmic protein kinases, which seem to be part of the signaling cascade mediating extracellular stimuli to changes in pollen tube growth direction.

In summary, our current understanding of ovular pollen tube guidance is very limited, but a whole orchestra of small molecules derived from the ovule seem to be involved in pollen tube growth support and attraction, and multiple signaling networks are required in pollen tubes to respond to the diverse set of signals and to direct their growth behavior.

After arrival at the surface of the ovule, the pollen tube reaches the last phase of its journey, which is known as micropylar pollen tube guidance. It enters the micropyle, an opening between the two integuments, and directly grows toward the egg apparatus in species such as Arabidopsis (Figure 2A). In grasses the pollen tube first has to overcome a few layers of nucellus cells (Márton et al., 2005) before it also gets in contact with the filiform apparatus of the synergid cells, a thickened and elaborated cell wall at their micropylar pole, where the cell surface is extensively invaginated (Willemse and van Went, 1984; Huang and Russell, 1992). It was believed for a long time that the pollen tube grows through the filiform apparatus to enter one synergid cell, leading to pollen tube burst and cell death of the receptive synergid cell. Recently, it was shown that the pollen tube is repelled by the filiform apparatus and instead grows along the cell wall of the synergid cells until it reaches a certain point after the filiform apparatus where its growth is arrested and burst occurs explosively (Leshem et al., 2013). Pollen tube burst results in the discharge of its cytoplasmic contents including the two sperm cells. The synergid cells represent the main source for chemo-attractants required for micropylar pollen tube guidance. Moreover, laser ablation experiments in Torenia fournieri have demonstrated that a single synergid cell is sufficient and necessary to attract pollen tubes (Higashiyama et al., 2001). The major function of the filiform apparatus may thus be to considerably increase the micropylar surface of the synergid cells, which represent glandular cells of the egg apparatus. Many known components required for pollen tube growth and guidance are membrane-associated and accumulate at the filiform apparatus, which gives it the additional role of a signaling platform. It contains, for example, a high Ca²⁺ concentration, which is known to play a key role during the regulation of pollen tube growth (Brewbaker and Kwack, 1963; Chaubal and Reger, 1990; Iwano et al., 2004; Michard et al., 2011) and also seems to trigger pollen tube burst afterwards (see below). In Arabidopsis the formation of the filiform apparatus as well as the expression of different attractants in the synergid cells depend on the activity of the R2R3-type Myb transcription factor MYB98 (Kasahara et al., 2005; Punwani et al., 2007). Among other genes, MYB98 regulates the expression of genes encoding cysteine-rich proteins (CRPs), including those representing a subgroup of defensin-like (DEFL) polypeptides (Punwani et al., 2008; Takeuchi and Higashiyama, 2012).

In Torenia it was shown that a DEFL subgroup of CRPs called LUREs are secreted from the synergid cells and accumulate at the filiform apparatus (Okuda et al., 2009; Kanaoka et al., 2011). LUREs attract pollen tubes in a species-preferential manner from a distance of about 100–150 μm and were recently shown to bind to the tip region of pollen tubes (Okuda et al., 2009, 2013). Due to their rapid molecular evolution it was difficult to identify orthologs in other plant species, but finally the DEFL subgroup CRP810/AtLURE1 of Arabidopsis was discovered to be involved in micropylar pollen tube guidance (Takeuchi and Higashiyama, 2012). In Zea mays, EGG APPARATUS1 (ZmEA1), a small hydrophobic precursor protein of 94 aa was reported as an egg apparatus-specific protein required for micropylar pollen tube guidance (Márton et al., 2005, 2012). ZmEA1 was shown to bind in a species-specific manner to the apical region of the pollen tube, where it is quickly internalized and degraded, likely keeping the pollen tube susceptible to pollen tube attractants while growing through the micropylar nucellus cell layers (Márton et al., 2012; Uebler et al., 2013).

More puzzling is the role of the central cell in micropylar guidance of the pollen tube. For example magatama (maa) mutants show defects in central cell maturation; both haploid nuclei are smaller and often fail to fuse. Pollen tubes grow in the direction of an unfertilized maa ovule but loose their way just before entering the micropyle. Moreover, mutant female gametophytes attracted two pollen tubes at a high frequency (Shimizu and Okada, 2000). MAA3 was recently shown to encode a helicase required for general RNA metabolism, which could explain the central cell maturation defect but not the defect in pollen tube guidance (Shimizu et al., 2008). Another example of central cell-dependent defects in micropylar pollen tube guidance is the transcriptional regulator CENTRAL CELL GUIDANCE (CCG), which is expressed exclusively in the central cell of the female gametophyte (Chen et al., 2007). These guidance defects may be indirect and caused by non-functional or immature central cells influencing maturation of egg apparatus cells and thus the generation of guidance components in these cells. It might also be possible that molecules generated by the MAA3 and CCG pathways directly regulate the generation of guidance molecules in the neighboring cells. Also the egg cell seems to be involved in micropylar guidance. GAMETE EXPRESSED 3 (GEX3) is a plasma membrane-localized protein, which is expressed in the unfertilized egg cell. Down-regulation of GEX3 by antisense RNA in the egg cell leads to defects in micropylar guidance by an unknown mechanism (Alandete-Saez et al., 2008).

Until recently, male factors and signaling pathways reacting to attractants secreted from the egg apparatus were unknown. The receptor-like kinases (RLKs) LOST IN POLLEN TUBE GUIDANCE1 (LIP1) and 2 (LIP2) have been identified, which are preferentially expressed in the pollen tube. Both proteins show membrane localization due to a palmitoylation site and are involved in the AtLURE1-dependent guidance mechanism. lip1/2 double mutant pollen reach the funiculus but fail to grow through the micropyle inside the ovule, and the pollen tube shows a reduced attraction toward AtLURE1 (Liu et al., 2013). However, it is unclear whether LIP1/2 are directly involved in LURE perception.

Once released, the two sperm cells are delivered to the so-called gamete fusion site between egg and central cell (Figure 2B). It is controversial whether this requires active transport or is solely based on cytoplasmic flow associated with burst of both pollen tube and receptive synergid cell and/or the architecture of the egg apparatus. Most flowering plants, like Arabidopsis, generate isomorphic sperm cells and therefore fusion of sperm cell appears to be random, either with the egg or the central cell (Berger et al., 2008; Ingouff et al., 2009; Liu et al., 2010). Some reports suggest that fertilization of the egg cell is preferred, which was demonstrated, for example, in mutants of CYCLIN DEPENDENT KINASE A1 (CDKA;1), which generate only one sperm-like germ cell (Iwakawa et al., 2006; Nowack et al., 2006). Experiments with photo-labeled sperm cells have demonstrated that there is no preference for either female gamete (Hamamura et al., 2011). The differentiation into two equal sperm cells depends on the activity of the MYB transcription factor DUO POLLEN 1 (DUO1), which is required for correct male germ cell differentiation by regulating key genes essential for fertilization such as GAMETE EXPRESSED 2 (GEX2) and GENERATIVE CELL SPECIFIC 1 (GCS1), also known as HAPLESS 2 (HAP2) (Brownfield et al., 2009). GEX2 encodes a single-pass transmembrane protein with filamin repeats exposed to the extracellular space. GEX2 localizes to the sperm cell plasma membrane and contains extracellular immunoglobulin-like domains, similar to gamete interaction factors reported in algae and mammals (Misamore et al., 2003; Inoue et al., 2005). In the presence of GEX2 the two gametes adhere to the egg and central cell. gex2 mutant sperm cells show reduced adhesion to female gametes, likely causing cell fusion failure (Mori et al., 2014). GCS1/HAP2 is another factor required for gamete interaction in Arabidopsis. After pollen tube burst, both sperm cells of gcs1/hap2 loss of function mutants remain at the fusion site and fail to fuse with female gametes, leading to the attraction of additional pollen tubes (polytubey). It was further shown that in the absence of the potential fusogen GCS1/HAP2, attachment of male to the female gamete occurs but no membrane fusion is visible, implying that the protein mediates membrane fusion as a component of signaling events, or more likely that it is directly involved in the fusion event (Wong and Johnson, 2010; Mori et al., 2014). GCS1/HAP2 is a conserved protein and has been identified in genomes of all major eukaryotic taxa except fungi. Gcs1/hap2 mutants in protozoan and algal gametes result in fusion failure, suggesting that this protein is required for a common mechanism of membrane fusion in eukaryotes (Mori et al., 2006; Hirai et al., 2008; Liu et al., 2008; Steele and Dana, 2009; Wong and Johnson, 2010). Upon sperm cell arrival at the gamete fusion site (Figure 2B) the egg cell starts to secrete small cysteine-rich proteins of the EGG CELL 1 (EC1) family. EC1 leads to the relocalization of HAP2/GCS1 from the endomembrane system to the sperm cell plasma membrane and thus activates sperm cells enabling them to fuse with the female gametes (Sprunck et al., 2012). The egg cell appears to require activation itself and calcium may play a key role in this process; this is indicated by a single strong Ca²⁺_cyto transient in the egg cell associated with pollen tube burst and sperm delivery (Denninger et al., 2014), which thus precedes EC1 secretion.

The relocalization of a transmembrane fusogen was also described for the mammalian-specific fusogen IZUMO1 (Inoue et al., 2005). Female components, which are directly involved in gamete fusion are so far unknown in higher plants. In mammals, CD9-like membrane spanning proteins of the tetraspanin family are located at the plasma membrane of eggs and were shown to be required for gamete fusion (Kaji et al., 2000; Le Naour et al., 2000; Miyado et al., 2000). In Arabidopsis the conserved tetraspanin family consists of 17 members. While TET11 and especially TET12 are located at the surface of sperm cells and reach high concentrations in the membrane region connecting both sperm cells, TET9 appears at the surface of female gametophyte cells including the egg and central cell (Boavida et al., 2013). Arabidopsis tetraspanins were shown to form homo- and heterodimers, but so far functional studies are missing. However, their presence at the surface of plant gametes and structural homology to mammalian CD9-like proteins suggest that they may possess a similar role during gamete interaction.

In general polyspermy blocks prevent multiple fertilization events that would otherwise lead to abnormal development or even embryo lethality, and thus reproductive failure. In Chlamydomonas FUS1, a single-pass transmembrane protein with a high similarity to prokaryotic invasion and adhesion molecules, mediates membrane fusion (Ferris et al., 1996; Misamore et al., 2003). In Chlamydomonas both GCS1/HAP2 and FUS1 are rapidly degraded after cell fusion, resulting in a fast membrane block to prevent polygamy (Misamore et al., 2003; Liu et al., 2010). In mammals, it was recently shown that IZUMO1 is recognized by the GPI-anchored protein JUNO on the egg cell surface. Rapid degradation of JUNO after fertilization suggests an additional mechanism for membrane block to prevent polyspermy (Bianchi et al., 2014).

In Arabidopsis it was shown that the polyspermy block only functions in the egg cell and not in the central cell, which is capable of fusing with more than one sperm cell, as demonstrated in the tetraspore (tes) mutant. This mutant produces more than one sperm pair, which is released simultaneously at the gamete fusion site. After fertilization, polyploidy resulting from multiple fertilization events was observed in the developing endosperm, but not in the embryo (Scott et al., 2008). The cause of the egg cell-specific fast block to polyspermy is unclear. In vitro fertilization experiments with maize egg and sperm cells have shown that cell wall material is detectable already within 30 sec after fusion (Kranz et al., 1995) and thus may prevent further gametic membrane interactions. Additionally, a quick block to polyspermy may also depend on the degradation of fusogens as described above. Calcium may play a role in immediate signaling of successful plasmogamy and release of cell wall material as an extended Ca²⁺_cyto transient is observed in the egg cell associated with successful gamete fusion (Denninger et al., 2014). However, the precise cellular function of calcium signaling during gamete interaction is currently unclear and will require further experimentation.

Another way to prevent polyspermy is the deactivation of pollen tube guidance and the activation of repelling mechanisms. In Arabidopsis usually only a single pollen tube is guided inside the ovule to execute double fertilization. After unsuccessful fertilization events, for example by failure of cell-cell fusion in gcs1/hap2, duo1, duo3, gex2, cdka;1, or ec1-RNAi gametes (Beale et al., 2012; Kasahara et al., 2012; Sprunck et al., 2012; Maruyama et al., 2013; Mori et al., 2014), secondary pollen tubes are attracted by the remaining synergid cell by a process named as polytubey. This process is delayed by a couple of hours (Kasahara et al., 2012), suggesting that pollen tube repellents are released upon sperm cell discharge and require degradation until additional pollen tubes can be attracted by the remaining synergid cell. After successful fertilization this cell quickly disintegrates, but remains viable for significantly longer times upon fertilization failure (Beale et al., 2012). A recent report showed that both female gametes independently control successful fertilization thus maximizing reproductive success (Maruyama et al., 2013). The key to prevent polytubey is the quick degeneration of the 2nd synergid cell. In order to investigate its death, it was recently reported that an Ethylene-Insensitive (EIN3-EIN2)/Ethylene-Insensitive3-like2 (EIL2)-dependent, ethylene-response cascade is activated after fertilization. Its artificial activation results in premature synergid cell disintegration and thus a block to pollen tube attraction (Völz et al., 2013). The degeneration of the 2nd synergid cell thus leads to the stop of attractant secretion and ultimately prevents polyspermy.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.