Abstract

Plant fertilization is achieved through the involvement of various pollen–pistil interactions. Self-/non-self-recognition in pollination is important to avoid inbreeding, and directional and sustainable control of pollen tube growth is critical for the pollen tube to deliver male germ cells. Recently, various secreted peptides (polypeptides) have been reported to be involved in cell–cell communication of pollen–pistil interactions. These include determinants of self-incompatibility, factors for pollen germination and tube growth, and pollen tube attractants. Interestingly, many of them are cysteine-rich peptides/polypeptides (CRPs). In this review, I focus on the peptides involved in pollen–pistil interactions and discuss properties of peptide signaling in each step from pollination to fertilization.

Introduction

Sexual reproduction in flowering plants requires complicated intercellular communication among male and female cells. Female reproductive cells of flowering plants are deeply embedded in the pistil (female reproductive organ), and thus both sporophytic and gametophytic cells are involved in the interactions (Suzuki 2009). In brief, the fertilization process begins when pollen, the male gametophyte produced by anthers (male reproductive organ), reaches the stigma of the pistil (Fig. 1). The pollen tube, which is a tip-growing cell, germinates from a hydrated pollen grain. In plants having self-incompatibility mechanisms, self-/non-self-pollen grains or tubes are recognized by female sporophytic tissue. The pollen tube growth occurs directionally thorough the stigma and style to enter the ovary. Appropriate cell–cell communication is critical for the pollen tube to reach the ovary. The tube eventually arrives at the target embryo sac (female gametophyte) in the ovule. Pollen tube guidance by the female gametophyte is critical for this step. Finally, two sperm cells within the pollen tube are discharged into the embryo sac through an interaction between the pollen tube and the embryo sac. One of these two sperm cells fuses with the egg cell to form the embryo and the other fuses with the central cell to form the endosperm via gametic interactions. This fertilization process, called double fertilization, is achieved when all of the above cell–cell communication stages are successful.

Fig. 1

Schematic representation of secreted peptides and other molecules involved in pollen–pistil interactions. Non-peptide (polypeptide) molecules are indicated in parentheses. See the text for details.

Fig. 1

Schematic representation of secreted peptides and other molecules involved in pollen–pistil interactions. Non-peptide (polypeptide) molecules are indicated in parentheses. See the text for details.

Recent studies have revealed that various secreted peptides are involved in pollen–pistil interactions (Table 1). Higher plants use various types of peptides for different steps and mechanisms of pollen–pistil interactions (Figs. 1, 2). Unlike small peptides such as systemin, phytosulfokine (PSK) and CLAVATA3/ESR (CLE) peptides, which are involved in plant development and defense (Matsubayashi 2003, Miwa et al. 2009), most of the peptides identified in pollen–pistil interactions are not likely to be processed into smaller peptides after cleavage of the signal peptide. Many of these peptides are CRPs, which might have originally evolved from antimicrobial peptides that are common in prokaryotes and eukaryotes (Yeaman and Yount 2007). CRPs are classified into various groups including defensin-like proteins and lipid transfer proteins, but they all have an N-terminal signal peptide and a divergent charged or polar mature peptide with conserved cysteine residues (Silverstein et al. 2007). Plants possess many classes of antimicrobial CRPs that do not appear to have homologs outside the plant kingdom, such as thionins, lipid transfer proteins and snakins. Genes of CRPs form a large gene family; 825 genes in Arabidopsis and 598 genes in rice (Silverstein et al. 2007). In this review article, I will summarize and discuss peptide signaling in intercellular communication of the pollen–pistil interactions. Previous related review articles have described more comprehensive mechanisms and molecules involved in self-incompatibility (Takayama and Isogai 2005), intracellular signaling in pollen tube growth (Cheung and Wu 2008), pollen tube guidance and double fertilization (Johnson and Lord 2006, Higashiyama and Hamamura 2008, Berger et al. 2009), peptide signaling of plant development (Matsubayashi 2003, Miwa et al. 2009) and chemoattraction in plant reproduction (Sekimoto 2005).

Fig. 2

Alignment of secreted peptides involved in pollen–pistil interactions. Group description of CRPs is indicated in parentheses according to a previous suggestion (Silverstein et al. 2007). Peptides without signal peptides are shown. For peptides of which the N-terminal amino acid sequences have not been determined, signal peptides were predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP/). No information related to the signal peptide of Ole e I and ScHT-B1 was available. Representative sequences were aligned with ClustalW and modified manually as described in Okuda et al. (2009). Conserved cysteines (yellow), glycines of the γ-core motif in defensin/defensin-like protein (DEFL) (purple) and amino acids involved in copper binding of plantacyanin (green) are shaded and displayed in open boxes below the alignments. Sequence identifiers are prefixed with initials to designate the plant species; At, Arabidopsis thaliana; Bo, Brassica oleracea; Br, Brassica rapa; Le, Lycopersicon esculentum; Ll, Lilium longiflorum; Nt, Nicotiana tabacum; Na, Nicotiana alata; Ole, Olea europaea; Os, Oryza sativa; Pr, Papaver rhoeas; Rs, Raphanus sativus; Sc, Solanum chacoense; Tf, Torenia fournieri; Zm, Zea mays. For alignment of all TfCRP1–TfCRP16 expressed in the synergid cell of Torenia, see Okuda et al. (2009).

Fig. 2

Alignment of secreted peptides involved in pollen–pistil interactions. Group description of CRPs is indicated in parentheses according to a previous suggestion (Silverstein et al. 2007). Peptides without signal peptides are shown. For peptides of which the N-terminal amino acid sequences have not been determined, signal peptides were predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP/). No information related to the signal peptide of Ole e I and ScHT-B1 was available. Representative sequences were aligned with ClustalW and modified manually as described in Okuda et al. (2009). Conserved cysteines (yellow), glycines of the γ-core motif in defensin/defensin-like protein (DEFL) (purple) and amino acids involved in copper binding of plantacyanin (green) are shaded and displayed in open boxes below the alignments. Sequence identifiers are prefixed with initials to designate the plant species; At, Arabidopsis thaliana; Bo, Brassica oleracea; Br, Brassica rapa; Le, Lycopersicon esculentum; Ll, Lilium longiflorum; Nt, Nicotiana tabacum; Na, Nicotiana alata; Ole, Olea europaea; Os, Oryza sativa; Pr, Papaver rhoeas; Rs, Raphanus sativus; Sc, Solanum chacoense; Tf, Torenia fournieri; Zm, Zea mays. For alignment of all TfCRP1–TfCRP16 expressed in the synergid cell of Torenia, see Okuda et al. (2009).

Table 1

Secreted peptides and proteins involved in pollen–pistil interactionsa

Name Type of peptides and proteinsb Group description of CRPc Mol wt. of mature peptides and proteinsd Expressed tissues Plant families, genera or species Function 
SCR/SP11 CRP (8 Cys) DEFL 6 kDa Tapetum and pollen Brsassicaceae Male determinant of self- incompatibility 
SLG Glycoprotein – ∼60 kDa Stigma Brsassicaceae To enhance the activity of the receptor of SCR/SP11 
S-protein Small glycoprotein (4 Cys)e – 17 kDa Stigma Papaveraceae Female determinant of self- incompatibility 
S-RNase RNase (glycosylated) – ∼30 kDa Stigma and transmitting tissue Solanaceae, Rosaceae, Scrophulariaceae (Plantaginaceae) Female determinant of self- incompatibility 
HT-B Asparagine-rich protein (6 Cys)e, possibly with post-translational modification – ∼18 kDa Stigma and style Nicotiana and Solanum Non-S-specific factor required for self-incompatibility 
GRP17 Oleosin, a glycine-rich protein with an oil-binding domain – 49 kDa Stamen Arabidopsis Involved in pollen hydration 
Chemocyanin Plantacyanin (3 Cys) – 10 kDa Stigma Lily In vitro pollen tube attraction (re-orientation) activity 
LAT52 CRP (4 Cys, glycosylated) Pollen Ole e I allergen and extensin family protein ∼20 kDa Pollen Tomato Involved in hydration and tube growth of pollen 
LeSTIG1 CRP (16 Cys) STIG1 13 kDa Stigma Tomato Stimulating activity in pollen tube growth 
SCAs CRP LTP 9 kDa Stigma and transmitting tissue Lily Involved in adhesion of pollen tubes to the transmitting tissue 
120K AGP (glycosylated) – ∼120 kDa Trasmitting tissue Nicotiana alata Non-S-specific factor required for self-incompatibility 
TTS AGP (glycosylated) – ∼100 kDa trasmitting tissue Nicotiana Necessary for pollen tube growth 
ZmEA1 EA1-like – – Synergid and egg cells Maize Necessary for micopylar pollen tube guidance 
LUREs CRP (6 Cys) DEFL 9 and 10 kDa Synergid cells Torenia fournieri Pollen tube attractants 
Name Type of peptides and proteinsb Group description of CRPc Mol wt. of mature peptides and proteinsd Expressed tissues Plant families, genera or species Function 
SCR/SP11 CRP (8 Cys) DEFL 6 kDa Tapetum and pollen Brsassicaceae Male determinant of self- incompatibility 
SLG Glycoprotein – ∼60 kDa Stigma Brsassicaceae To enhance the activity of the receptor of SCR/SP11 
S-protein Small glycoprotein (4 Cys)e – 17 kDa Stigma Papaveraceae Female determinant of self- incompatibility 
S-RNase RNase (glycosylated) – ∼30 kDa Stigma and transmitting tissue Solanaceae, Rosaceae, Scrophulariaceae (Plantaginaceae) Female determinant of self- incompatibility 
HT-B Asparagine-rich protein (6 Cys)e, possibly with post-translational modification – ∼18 kDa Stigma and style Nicotiana and Solanum Non-S-specific factor required for self-incompatibility 
GRP17 Oleosin, a glycine-rich protein with an oil-binding domain – 49 kDa Stamen Arabidopsis Involved in pollen hydration 
Chemocyanin Plantacyanin (3 Cys) – 10 kDa Stigma Lily In vitro pollen tube attraction (re-orientation) activity 
LAT52 CRP (4 Cys, glycosylated) Pollen Ole e I allergen and extensin family protein ∼20 kDa Pollen Tomato Involved in hydration and tube growth of pollen 
LeSTIG1 CRP (16 Cys) STIG1 13 kDa Stigma Tomato Stimulating activity in pollen tube growth 
SCAs CRP LTP 9 kDa Stigma and transmitting tissue Lily Involved in adhesion of pollen tubes to the transmitting tissue 
120K AGP (glycosylated) – ∼120 kDa Trasmitting tissue Nicotiana alata Non-S-specific factor required for self-incompatibility 
TTS AGP (glycosylated) – ∼100 kDa trasmitting tissue Nicotiana Necessary for pollen tube growth 
ZmEA1 EA1-like – – Synergid and egg cells Maize Necessary for micopylar pollen tube guidance 
LUREs CRP (6 Cys) DEFL 9 and 10 kDa Synergid cells Torenia fournieri Pollen tube attractants 

aSmall proteins (polypeptides) of ∼20 kDa (or <150 amino acids) are called ‘peptides’ in this review article. Silverstein et al. (2007) also searched for plant CRPs with a limit of 150 amino acids.

bNotes in parentheses indicate the numbers of cysteines after the (predicted) cleavage site and the existence of glycosylation.

cThe group description by Silverstein et al. (2007) is indicated.

dEstimated by various methods including SDS–PAGE, mass spectrometry and calculation from predicted amino acid sequences. Molecular weights differ by haplotypes, isoforms and extent of glycosylation.

eS-protein homolog 1 (SPH1, At4g16295) in Arabidopsis and HT-B of Nicotiana alata are not included as CRPs (Silverstein et al. 2007).

Determinants of self-incompatibility

Self-incompatibility is the system used to avoid self-fertilization (Suzuki 2009). In flowering plants, different mechanisms of self-incompatibility have evolved independently in various plant species. In many species, the self-/non-self-recognition of self-incompatibility is governed by a single polymorphic locus, the S-locus. Three kinds of male and female determinant sets on the S-locus have been identified and intensively studied (Takayama and Isogai 2005, Wheeler et al. 2009). In all three cases, one of the determinants is diffusible and can act on cells of the opposite sex.

In the Brassicaceae, the male determinant is a small (∼6 kDa), secreted CRP, SCR/SP11, encoded by the S-locus (Table 1; Schopfer et al. 1999, Takayama et al. 2000). SCR/SP11 is a CRP with eight cysteines belonging to a subclass of defensin-like proteins (Fig. 2) and is expressed specifically in the tapetum and developing pollen within the anther, where it accumulates in the pollen coat of mature pollen. SCR/SP11 is perceived by the female determinant in the plasma membrane of the stigma, the S-locus receptor kinase (SRK) (Stein et al. 1991, Takayama et al. 2001). Scatchard analysis showed that the two determinants interact directly with high affinity (Kd = 0.7 nM; Takayama and Isogai 2003). SRK (∼110 kDa) is a serine/threonine kinase with one transmembrane domain, as commonly found in plant receptor kinases (Matsubayashi 2003). S-locus glycoproteins (SLGs, Table 1; ∼60 kDa), which are secreted proteins coded in the S-locus, are similar to the extracellular domain of SRK (Nasrallah et al. 1987, Takayama et al. 1987) and enhance the activity of SRK in some S-haplotypes (Takasaki et al. 2000, Silva et al. 2001). A particular characteristic of the ligand–receptor pair involved in self-compatibility is that both genes exist together on the S-locus, and both are transmitted to descendents as a set. Both SCR/SP11 and SRK have a hypervariable region, which allows specific binding in ‘self’ combinations among multiple alleles.

SCR/SP11 is a defensin-like protein with eight cysteines, but the arrangement of cysteines differs from that of true plant defensins, including AFP1 of radish (Fig. 2). The tertiary structure of SP11 of the S8 haplotype (S8-SP11) was resolved using nuclear magnetic resonance (NMR) (Mishima et al. 2003). S8-SP11, as well as other possible SCR/SP11 proteins, has three antiparallel β-sheets and one α-helix, stabilized by four disulfide bonds (C1–C8, C2–C5, C3–C6 and C4–C7; the same combinations as those of AFP1). The structure of the loop between a β-sheet and the α-helix is suggested to be a hypervariable domain for S-haplotype-specific interactions. The global fold of S8-SP11 made by β-sheets and an α-helix is similar to that of AFP1. S8-SP11 can induce self-incompatibility at concentrations as low as 50 fmol per stigma (Takayama et al. 2001), and self-pollen shows impaired hydration or arrested tube growth.

Another defensin-like protein, PCP-A1, which is similar to plant defensins such as AFP1 with regard to the arrangement of cysteines (Fig. 2), is also expressed in the pollen of Brassica oleracea (Doughty et al. 1998). The PCP-A1 gene is not linked to the S-locus, and the function of PCP-A1 in self-incompatibility is unknown. However, PCP-A1 has been suggested to interact with SLG, and might be involved in self-incompatibility and/or pollination reactions. Floral defensins that show antifungal activity are expressed in the developing pistil of Nicotiana alata and Petunia (Lay et al. 2003). Since their expression is regulated developmentally, they might be involved not only in defense, but also in intercellular signaling.

In the Papaveraceae, the female determinant of self- incompatibility was identified as an S-protein based on an in vitro bioassay (Foote et al. 1994; recently renamed ‘PrsS’ by Wheeler et al. 2009). S-proteins are small secreted glycoproteins of ∼17 kDa specifically expressed in the stigma (Table 1). Some populations of native S-proteins are thought to be N-glycosylated. The backbone peptide (∼15 kDa) is polymorphic throughout the S-protein and has four conserved cysteines. Recombinant S-proteins expressed in Escherichia coli inhibit pollen germination in an S-haplotype-specific manner, suggesting that the S-protein is the sole determinant and that glycan chains are not required for induction of self-incompatibility. Recently, a novel 20 kDa plasma membrane protein, Papaver rhoeas pollen S (PrpS), was identified as the male determinant and is encoded by a highly polymorphic pollen-expressed gene on the S-locus (Wheeler et al. 2009). S-protein-binding protein (SBP) might also work as an accessory receptor (Hearn et al. 1996, Jordan et al. 1999). SBP is a pollen-specific integral membrane proteoglycan of 70–120 kDa that specifically binds to stigmatic S-proteins. Identification of the male determinant PrpS should clarify the signaling mechanism leading to rejection of self-pollen that involves programmed cell death of the pollen (Thomas and Franklin-Tong 2004).

In the Solanaceae, the Rosaceae and the Scrophulariaceae (Plantaginaceae), the diffusible determinant is a catalytic protein of ∼30 kDa, unlike the other two self-incompatibility systems, SCR/SP11 of the Brassicaceae and the S-protein of the Papaveraceae. The female determinant in these families is an S-RNase having regions similar to fungal RNase T2 (Table 1; McClure et al. 1989). The S-RNase is specifically and abundantly expressed in the style and is N-glycosylated. Unlike the other two diffusible determinants, the S-RNase is not the ligand received by a corresponding receptor on the plasma membrane but is taken up by both self- and non-self-pollen tubes (Luu et al. 2000). In self-pollen tubes, the RNA is degraded and RNase activity is necessary for pollen rejection (McClure et al. 1990, Huang et al. 1994). A high level of expression of S-RNase has been suggested to be critical for rejection of self-pollen (e.g. Lee et al. 1994, Murfett et al. 1994). S-RNase levels, 0.25–5 mg ml−1 in the style of Solanum chacoense, are suggested to determine the incompatible phenotype (Qin et al. 2006).

The male determinant in the pollen tube is an F-box protein, S-locus F-box (SLF)/S-haplotype-specific F-box (SFB) that is generally involved in protein degradation (Lai et al. 2002, Entani et al. 2003, Ushijima et al. 2003, Yamane et al. 2003, Sijacic et al. 2004). The S-RNase has been proposed specifically to avoid ubiquitin-mediated protein degradation in self-pollination. Highly variable regions in SLF/SFB are expected to be involved in some haplotype-specific interactions, but the mechanism of the interactions is unknown. Other stylar extracellular matrix (ECM) factors are suggested to be necessary for full functioning of the S-RNase (McClure et al. 2000). Such non-S-RNase factors involve a small, asparagine-rich protein (Table 1, HT-B; McClure et al. 1999, O’Brien et al. 2002) and a 120 kDa glycoprotein (Table 1, 120K; Schultz et al. 1997, Hancock et al. 2005), although their action mechanisms in self-incompatibility remain unknown (McClure 2006). HT-B is a secreted peptide of ∼18 kDa containing six cysteines (McClure et al. 1999). Some post-translational modification of HT-B has been suggested because the molecular weight of HT-B is predicted to be 8.6 kDa. The glycoprotein 120K is one of S-RNase-binding proteins, which include a transmitting tissue-specific protein (TTS), a pistil extensin-like protein III (PELPIII) and a plantacyanin (McClure et al. 2000, Cruz-Garcia et al. 2005). Among S-RNase-binding proteins, 120K, TTS and PELPIII are arabinogalactan proteins (AGPs) that form the most abundant class of S-RNase-binding proteins (Cruz-Garcia et al. 2005). The three AGPs possess a cysteine-rich Ole e I-like domain at the C-terminal. Note that TTS proteins and plantacyanins are also involved in other pollen–pistil interactions, as described below.

Directional cues for pollen tube growth on the stigma

After pollination, compatible pollen grains hydrate and extrude a pollen tube. In tobacco, in which the stigma is covered with a lipid-rich exudate, lipids, including triacylglycerides, are sufficient and essential for pollen hydration and directional tube growth (Wolters-Arts et al. 1998), although the mechanism of how the lipids control these processes remains unclear (Wolters-Arts et al. 2002). Lipids (Wolters-Arts et al. 1998) and glycine-rich proteins (GRPs) capable of lipid binding (e.g. GRP17, Table 1; Mayfield and Preuss 2000) in the pollen coat of Arabidopsis are also involved in pollination reaction with the dry stigma. However, the existence of chemotropic compounds has been reported in various plant species (Tsao 1949, Zeijlemaker 1956), and pollen tubes that germinate on medium grow toward the excised stigma or its exudates in the above plants.

Many years ago, chemotropic compounds were also predicted in the sugar-rich stigma exudate of lilies (Tsao 1949, Miki 1954, Zeijlemaker 1956, Welk et al. 1965). Kim et al. (2003) recently confirmed this chemotropic activity in a fraction from lily stigma proteins. When the active fraction of stigma proteins (1.5 μg μl−1) was applied to a 2 mm diameter agarose well, most pollen tubes germinating from pollen grains that were manually arranged 2–3 mm from the well reoriented toward the well. Using biochemical and proteomics approach, these authors finally identified the chemotropic compound as a chemocyanin, a small, secreted peptide showing homology to plantacyanins with a molecular mass of 9.9 kDa, as measured by mass spectrometry (Table 1). Plantacyanins are basic cell wall proteins of unknown function, many of which are capable of redox reactions. Plantacyanins belong to a subfamily of blue copper proteins; however, one important amino acid for copper binding is substituted in chemocyanin, and whether chemocyanin can bind copper is not known. Chemocyanin is expressed abundantly in the stigma and style, and to a lesser extent in other vegetative organs. Plantacyanins of other plant species are also expressed in various tissues, implying that they might be multifunctional proteins. In the in silico search of CRPs of higher plants by Silverstein et al. (2007), plantacyanins were encountered but were not further examined due to scarcity and a non-conserved arrangement of cysteines. The gene structure of plantacyanins and CRPs might be relatively close.

A fraction peak containing chemocyanin showed chemotropic activity at 0.23 μg μl−1 (∼23 μM). Attraction activity was higher at 0.69 μg μl−1 (∼69 μM) or when mixed with another peptide that is a major compound in the active fraction of stigma proteins. This peptide is a stigma/stylar cysteine-rich adhesin (SCA), which was identified as an ECM protein involved in the adhesion of pollen tubes to the ECM of the pistil tissue, as described below. SCA itself does not have chemotropic activity and might be an accessory protein important for the function of the chemocyanin. SCA binds to pollen tubes (Park et al. 2000) and might facilitate physical access of chemocyanin to pollen tubes. The minimum concentration of chemocyanin necessary was estimated to be 0.05 μg μl−1 (∼5 μM) in the presence of SCA.

The function of chemocyanin in pollination remains to be elucidated because down-regulation experiments in lily are still difficult (Kim et al. 2003). Chemocyanin may be the directional cue for germinating pollen tubes to grow toward openings in the hollow stylar canal of the lily. Arabidopsis possesses only one plantacyanin, which is expressed abundantly in the transmitting tissue and to a lesser extent in other vegetative organs, as in the case of lily chemocyanin (Dong et al. 2005). A T-DNA knock-down line, but not a knock-out line, is available for the plantacyanin gene of Arabidopsis, but no phenotype has been observed. Overexpression of the plantcyanin led to some pollen tubes aberrantly growing on papilla cells of the stigma, suggesting that the plantcyanin might be involved in pollen tube guidance. Further studies on chemocyanin and its homologs in other species will provide insights in elucidating the long-discussed issue of chemotropism induced by the stigma.

Peptides for pollen germination and tube growth

Autocrine signaling has been suggested to occur in tomato pollen. A putative ligand secreted from pollen, LAT52, is received by a receptor kinase of the pollen, LePRK2. LAT52 is a CRP with four cysteines expressed in the vegetative (tube) cell of tomato pollen (Table 1; Muschietti et al. 1994) and is likely to be secreted and glycosylated at an N-linked glycosylation site. Its apparent molecular weight is ∼20 kDa, while that of the backbone peptide without the signal sequence is 16 kDa. LAT52 shows sequence similarity to Kunitz trypsin inhibitors, but the arrangement and number of cysteines are not conserved between Kunitz trypsin inhibitors and LAT52 (Muschietti et al. 1994). LAT52 does not include an active site domain for the proteinase inhibitor. LAT52 is classified in a subgroup of CRPs (Silverstein et al. 2007), namely pollen Ole e I allergen and extensin family protein, together with its homologs in other plant species, including the pollen-specific proteins Zm13 of maize (Hanson et al. 1989), PS1 of rice (Zou et al. 1994) and Ole e I of olive (Villalba et al. 1993). Ole e I is the major pollen allergen. Down-regulation experiments with antisense RNA showed that LAT52 is involved in pollen hydration and tube growth (Muschietti et al. 1994). The LAT52 gene is well known because its promoter is effective in many plant species as a pollen vegetative cell-specific promoter.

LAT52 in the pollen coat interacts with the pollen receptor kinase LePRK2, which possesses a leucine-rich repeat (LRR) (Tang et al. 2002) and is involved in pollen germination and polarized tube growth (Kaothien et al. 2005, Zhang et al. 2008). LAT52 was identified as the partner of LePRK2 through a yeast two-hybrid screen (Tang et al. 2002). LAT52 can interact with LePRK2 before but not after germination of pollen (Tang et al. 2002). Moreover, LePRK2 also interacts with LeSTIG1, a CRP specifically expressed in the stigmatic secretory zone (Tang et al. 2004). LeSTIG1 has been suggested to displace binding of LAT52.

LeSTIG1 is a CRP with 16 cysteines (Table 1). The molecular weight of mature LeSTIG1 without the putative signal peptide is 13 kDa; the apparent molecular mass of STIG1 in petunia is 12–16 kDa and it is possibly N-glycosylated (Verhoeven et al. 2005). Glutathione S-transferase (GST) fusion of LeSTIG1 lacking the signal peptide, expressed in E. coli, can stimulate pollen tube growth at 100 nM but not at 50 nM, although LAT52–GST does not promote pollen tube growth in vitro (Tang et al. 2004). Chemotropic activity was not observed for LeSTIG1 (Tang et al. 2004). STIG1 was originally found in tobacco (Goldman et al. 1994) and later in Petunia, and is likely to be involved in the regulation of stigma exudate secretion (Verhoeven et al. 2005).

Factors for pollen tube growth from the stigma to the ovary

Pollen tubes are thought to perceive many molecules in the ECM of the pistil. The growth rate of in vivo grown pollen tubes is much higher than that of in vitro grown tubes. External molecules include not only nutrients of low molecular weight compounds such as water, calcium, sugars (Sivitz et al. 2008) and boron (Iwai et al. 2006), but also various proteins, including CRPs and glycoproteins.

SCA was identified using an in vitro adhesion assay, wherein adhesion of pollen tubes to an artificial matrix was examined (Jauh et al. 1997). SCA is a CRP of 9 kDa specifically expressed in the stigma and style of lilies, and is classified as a lipid transfer protein (LTP) with eight cysteines (Table 1; Park et al. 2000). SCA has three isoforms, all of which show pollen tube adhesion activity, although the level of adhesion varies among them (Chae et al. 2007). These enhance the chemotropic activity of chemocyanin (Kim et al. 2003), suggesting that adhesive molecules in the ECM are involved in perceiving the external signal by the pollen tube.

Tobacco TTS proteins are AGPs in the ECM of the style that stimulate pollen tube growth in vitro and are necessary for pollen tube growth in vivo (Table 1; Cheung et al. 1995, Wu et al. 1995). TTS proteins are not small and have molecular weights of 50–100 kDa (the backbone peptide is 30 kDa). TTS proteins are reported not only to have nutritive functions (at >1 μg ml−1) but also to have chemotropic activity in vitro (at >200 μg ml−1; Cheung et al. 1995), although this is still debatable (Higashiyama and Inatsugi 2006). In addition, a TTS protein of Nicotiana alata interacts with S-RNase possibly to facilitate interactions with pollen tubes. The extent of glycosylation shows a gradient toward the ovary (Wu et al. 1995). AGPs are interesting molecules showing a characteristic localization (Coimbra et al. 2007), and xylogen, an AGP identified in an in vitro bioassay in Zinnia, is a bioactive molecule for differentiation of tracheary elements (Motose et al. 2004).

Guidance cues in the ovary

In many plant species, pollen tube guidance in the stigma and style is governed by sporophytic tissues (Higashiyama and Hamamura 2008). After entering the ovary, pollen tube guidance transits from sporophytic control to gametophytic control. In the Arabidopsis ovary, for example, pollen tubes emerge from the transmitting tract to the surface of the septum. The timing of this emergence is related to the development of the embryo sac (Hülskamp et al. 1995). Emerged pollen tubes grow to the nearest or adjacent ovule. Pollen tube guidance to the ovule is governed by the female gametophyte (Hülskamp et al. 1995, Ray et al. 1997), and control involves at least two steps (Shimizu and Okada 2000). The first step is funicular guidance from the placenta to the funiculus of the ovule, and the second step is micropylar guidance from the entrance of the micropyle to the embryo sac.

Since the 1860s, many plant biologists expected that the ovule, or, more precisely, the micropyle of the ovule, would emit some diffusible attractant(s). Earlier studies by Van-Tieghem (1869) showed that pollen tubes grow toward excised ovules in vitro. Various molecules, including external calcium ions (Mascarenhas and Machlis 1962), were proposed to be the attractant (Reger et al. 1992, Higashiyama and Hamamura 2008); however, no true attractant was identified in these classical studies, possibly due to difficulties associated with in vitro bioassays and embryo sacs hidden by sporophytic cells. With regard to bioassay systems, discriminating between attraction and growth stimulation is difficult. Attractant proteins for axon guidance, netrins, were successfully identified as secreted proteins of ∼78 kDa that are partly homologous with laminin because neurons could only grow on medium when netrin was present (Kennedy et al. 1994, Serafini et al. 1994). Pollen tubes can grow in medium in the absence of an attractant. Attempts to identify the pollen tube attractant have been unsuccessful, but observation of pollen tubes in vivo (e.g. Shimizu and Okada 2000) and in vitro (e.g. Willemse et al. 1995) still implies the presence of an attractant derived from the micropyle of the ovule.

Recent studies in a unique plant, Torenia fournieri, which has a protruding embryo sac, showed that pollen tubes are attracted to the micropylar end of the embryo sac in vitro (Higashiyama et al. 1998, Higashiyama and Hamamura 2008). Pollen tubes were required to grow through a cut style, suggesting that female sporophytic tissue promotes pollen tubes to become competent to be attracted. Pollen tubes of Arabidopsis are also attracted to excised ovules more frequently when growing through a cut style (Palanivelu and Preuss 2006). Classical assay systems did not use pollen tubes growing through a style, which might be one reason why ovular attractants were not previously found. In the in vitro Torenia system, pollen tubes do not leave the embryo sac, even when their tips slip off the micropylar end of the sac (Higashiyama et al. 1998). In such cases, pollen tubes form a narrow coil to grow continuously toward the micropylar end of the embryo sac. When an embryo sac attracting a pollen tube is moved using a micromanipulator, the pollen tube precisely chases the micropylar end of the embryo sac (Higashiyama and Hamamura 2008). This in vitro Torenia system suggests that some diffusible signal is derived from the micropylar end of the embryo sac.

The diffusible attraction signal in Torenia is derived from two synergid cells on the side of the egg cell, as shown by laser cell ablation (Higashiyama et al. 2001). The attraction signal is species preferential or species specific, even in closely related species (Higashiyama et al. 2006). The distance of pollen tube attraction by the synergid cell is 100–200 μm at most in vitro. In Arabidopsis, a synergid cell-specific transcription factor gene, MYB98, is involved in micropylar guidance, which is likely to be effective for a distance of ∼100 μm (Kasahara et al. 2005, Punwani et al. 2007). In maize, an egg apparatus-specific gene, ZmEA1, is involved in pollen tube guidance at the micropyle of the ovule and is likely to be effective for ∼50 μm (Table 1; Márton et al. 2005).

Attractant molecule LUREs (LURE1 and LURE2) were identified by expressed sequence tag (EST) analysis of the synergid cell of Torenia (Table 1; Okuda et al. 2009). Synergid cells released by cell wall degradation enzymes were collected under a microscope, and 25 synergid cells were used to establish a cDNA library via 30 PCR cycles. LUREs were the focus in the EST data because the number of ESTs of LUREs was extremely high due to their abundant expression, which might be an important property for the attractant gene that would retain a sharp concentration gradient around the embryo sac. Consistent with severe specificity, CRPs generally show rapid molecular evolution (Silverstein et al. 2007). Moreover, LUREs are expressed specifically in the synergid cell. LURE proteins are secreted toward the micropylar end of the synergid cell and detected in the filiform apparatus, a characteristic cell wall structure at the base of the two synergid cells.

LUREs are CRPs of ∼9 kDa (∼65 amino acids) with six cysteines, belonging to a subgroup of defensin-like proteins that differs from that of SCR/SP11, ZmES1, and true plant defensins such asRsAFP1, according to the arrangement of cysteine residues (Fig. 2; Okuda et al. 2009). EST analysis of the synergid cell of Torenia revealed that various CRP genes (TfCRP1–TfCRP16) are abundantly expressed in the synergid cell (Okuda et al. 2009). Among the three major CRPs, TfCRP1 and TfCRP3 were identified as LURE1 and LURE2, respectively. Another gene, TfCRP2, was not shown to possess activity to attract pollen tubes. TfCRP2 is a smaller defensin-like protein (48 amino acids) with eight cysteines, predominantly expressed in the synergid cell among ovular cells like LUREs but also expressed in anthers and developing fruits. Whether the remaining 13 TfCRPs (TfCRP4–TfCRP16) are attractants is still not known. Down-regulation of each LURE by morpholino antisense oligos decreases the frequency of pollen tube attraction, suggesting that LUREs are involved in pollen tube attraction by the synergid cell.

Recombinant proteins of both LURE1 (TfCRP1) and LURE2 (TfCRP3) expressed in E. coli have strong activity to attract pollen tubes of their own species in vitro (Fig. 3). Neither non-competent pollen tubes nor pollen tubes of other species (Lindernia micrantha) are attracted by these recombinant proteins. As in the case of SCR/SP11, further processing or modification has not been suggested, apart from cleavage of the signal peptide. The tertiary structure of LUREs is likely to be stabilized by intramolecular disulfide bonds between the six cysteines and might be critical for pollen tube attraction because a skip of refolding processes or heat treatment drastically reduces the activity. When recombinant LUREs are embedded in gelatin beads of ∼40 μm and settled ∼50 μm in front of pollen tubes, the tubes are attracted to the beads. At optimum concentration, both LUREs attract about 60% of pollen tubes, which is equivalent to the frequency of attraction by the synergid cell. The optimum concentrations in beads for LURE1 and 2 are 40 and 4 nM, respectively, although the ratios of appropriately refolded LUREs in the protein solution have not been determined. Unexpectedly, both LUREs show attraction activity at 40 pM in a bead, which means that only ∼1,000 molecules in a bead are sufficient to generate an attraction signal. This implies the possibility that single-molecule imaging of LUREs might be performed to reveal their action mechanism on the pollen tube.

Fig. 3

Attraction of pollen tubes by recombinant LURE2. A pollen tube growing through a cut style (competent pollen tube) was attracted by LURE2 mixed with 10 kDa Alexa Fluor. LURE2 was injected in front of the pollen tube (asterisk) through a micropipet at time points of 00:16 (min:s) and 10:20. Spectral colors correspond to the intensity of fluorescence of the Alexa Fluor dye, with white representing the highest level (see color scales). This figure was reproduced with permission from Nature Publishing Group (Okuda et al. 2009). Scale bar, 20 mm.

Fig. 3

Attraction of pollen tubes by recombinant LURE2. A pollen tube growing through a cut style (competent pollen tube) was attracted by LURE2 mixed with 10 kDa Alexa Fluor. LURE2 was injected in front of the pollen tube (asterisk) through a micropipet at time points of 00:16 (min:s) and 10:20. Spectral colors correspond to the intensity of fluorescence of the Alexa Fluor dye, with white representing the highest level (see color scales). This figure was reproduced with permission from Nature Publishing Group (Okuda et al. 2009). Scale bar, 20 mm.

Many CRPs of Arabidopsis are also suggested to be expressed in the synergid cell because CRPs are down-regulated in the ovule of a knock-out mutant of the MYB98 gene, a synergid cell-specific transcription factor (Jones-Rhoades et al. 2007, Punwani et al. 2007). Using green fluorescent protein (GFP) fusion, some CRPs were shown to be secreted toward the filiform apparatus (Punwani et al. 2007). In EST analysis of the isolated egg cell (Cordts et al. 2001) and embryo sac (Yang et al. 2006) of maize, various CRPs were shown to be expressed specifically in the female gametophyte. Some of defensin-like proteins including ZmES (Cordts et al. 2001) and those with characteristic triple cysteines at the C-terminus (Yang et al. 2006) were predominantly expressed in the synergid cell, although they showed a different arrangement of cyteines from that of LUREs. Expression of various CRPs might be a characteristic of the synergid cell. LUREs might have evolved from antimicrobial peptides of the synergid cell located at the gateway of the embryo sac. LUREs possess cysteine-stabilized αβ (CSαB) and γ-core motifs conserved in antimicrobial peptides. Whether CRPs are attractant molecules in other plant species is still unknown. The knock-out mutant myb98 of Arabidopsis was impaired not only in pollen tube guidance but also in development of the filiform apparatus of the synergid cells. The possibility exists that myb98 is defective in the expression of attractant genes and/or secretion of attractants through the filiform apparatus. Micropylar pollen tube guidance of Arabidopsis is also defective in mutants of magatama (maa; Shimizu and Okada 2000, Shimizu et al. 2008), central cell guidance (ccg; Chen et al. 2007) and gex3 (Alandete-Saez et al. 2008). In the ovule, MAA genes are likely to be expressed ubiquitously, and CCG and GEX3 are specifically expressed in the central cell and the egg cell, respectively. Clarifying whether the egg and central cell are involved directly and/or indirectly in micropylar guidance would be of interest.

Zea mays EGG APPARATUS 1 (ZmEA1) is a gene specifically expressed in the egg and synergid cells (egg apparatus) and is necessary for micropylar guidance of maize (Márton et al. 2005). ZmEA1 is a small plasma membrane protein (94 amino acids) with a transmembrane domain and is a member of the EA1-like (EAL) gene family in plants (Gray-Mitsumune and Matton 2006). ZmEA1 fused with GFP was shown to diffuse from the egg apparatus as the ovule develops, suggesting that ZmEA1 may be further processed on the plasma membrane. ZmEA1 has been proposed to be an attractant protein (Dresselhaus and Márton 2009), although further detailed studies are needed.

Other guidance cues, such as the funicular guidance of Arabidopsis, are completely unknown. Funicular guidance is governed by the embryo sac, but the origin of the guidance cue is not known. The guidance cue might be directly derived from the embryo sac or indirectly derived from sporophytic funiculus cells. Species specificity observed in funicular guidance implies that the guidance cue might be a molecule showing rapid molecular evolution (Shimizu 2002). However, γ-aminobutyric acid (GABA) has been reported to form a concentration gradient toward the ovule and is involved in ovular sporophytic guidance (Palanivelu et al. 2003). The search for the attractant for funicular guidance is a major issue in the study of pollen tube guidance, as is the search for receptors of LUREs in the pollen tube.

Pollen tube discharge and double fertilization

After arrival of the pollen tube at the embryo sac, it enters the embryo sac through the micropylar end. Pollen tubes usually enter the embryo sac by growing between the two halves of the filiform apparatus of the synergid cells (Higashiyama 2002). Just after passing the filiform apparatus, wall deposition at the tip of the pollen tube appears disordered, resulting in pollen tube discharge. Complicated interaction between male and female cells has been suggested in pollen tube discharge (Dresselhaus and Márton 2009). An LRR-type receptor-like kinase of the synergid cell, FERONIA, is involved in cessation of pollen tube growth in the embryo sac (Escobar-Restrepo et al. 2007). FERONIA localizes on the plasma membrane of the micropylar end of the synergid cell. Consistent with severe species specificity observed in this step of the gametophytic interaction, FERONIA has an extracellular variable domain. FERONIA is believed to interact with an unidentified ligand on the surface of the pollen tube to sense the arrival of the pollen tube. Identifying the male ligand is of interest, as well as analyzing the complicated pollen tube–synergid cell interaction to receive the tube contents (Berger et al. 2008). Notably, ANXUR 1 and 2, two sister receptor kinases of FERONIA, are expressed in pollen and are necessary for pollen tube growth (Miyazaki et al. 2009). ZmES genes, which encode defensin-like proteins of eight cysteines (Fig. 2) predominantly expressed in the synergid cell of maize, might be involved in pollen tube discharge (Dresselhaus and Márton 2009).

The two discharged sperm cells then fuse with their respective targets. GCS1/HAP2 is a plasma membrane protein with one transmembrane domain involved in membrane fusion (Mori et al. 2006, von Besser et al. 2006). Note that GCS1/HAP2 homologs are widely distributed not only in the plant kingdom but also in other eukaryotes and are involved in fertilization (gamete fusion) (Hirai et al. 2008, Liu et al. 2008). The partner on the female side has not been identified and its detection would help in understanding the evolution and fundamental mechanisms of sexual reproduction.

Properties of CRPs in pollen–pistil interactions

As summarized above, several CRPs are involved in pollen–pistil interactions. Plant CRPs are likely to be constitutively produced as a frontline defense in peripheral cell layers of nutrient-rich structures, namely flowers and seeds (Silverstein et al. 2007). Not only CRPs but other pathogenesis-related (PR) proteins are expressed in the pistil (e.g. Kuboyama et al. 1998, Edreva 2005). Some populations of CRPs are suggested to have evolved to acquire non-defense but reproductive regulatory roles.

The self-incompatibility determinant SCR/SP11 and pollen tube attractant LUREs are both defensin-like proteins. Defensins of eukaryotes have been suggested to share a common ancestor, and plant defensins, including radish AFP1, show structural similarities to invertebrate and insect defensins (Carvalho and Gomes 2009). Genes of defensin-like proteins form a large gene family, i.e. 323 genes in Arabidopsis and 93 genes in rice (Silverstein et al. 2005, Silverstein et al. 2007). SCR/SP11 and LUREs differ from plant defensins with regard to the arrangement of cysteines (Fig. 2); however, both possess a γ-core and CSαβ motifs that are conserved in antimicrobial peptides (Silverstein et al. 2005, Yeaman and Yount 2007). Determining whether these peptides have antimicrobial activity would be of great interest.

The tertiary structure stabilized by disulfide bonds has been suggested to be critical for both SCR/SP11 and LUREs. These CRPs are not further processed into small peptides after cleavage of the signal peptide. Human β-defensin 3 requires appropriate disulfide bonds for chemotactic activity to attract T lymphocytes and immature dendritic cells, but not for antimicrobial activity (Wu et al. 2003). This might be due to different action mechanisms of β-defensin 3 for these two activities: β-defensin 3 attacks lipid layers of the plasma membrane of microorganisms for defense but is received by a chemokine receptor on the plasma membrane of attracted cells. In the plant defensin AFP2, synthetic oligopeptides of the flanking loop between two β-sheets show antifungal activity (Schaaper et al. 2001). The significance of the tertiary structure of CRPs in pollen–pistil interactions suggests that they might be received by receptors on the plasma membrane of the target cell.

Two kinds of defensin-like protein, SCR/SP11 and LUREs, control the onset and final step of pollen tube growth. Despite the similarity of these molecules, the action mechanisms are suggested to show considerable differences. What is the main difference? SCR/SP11 is the sole determinant that governs strict self-/non-self-recognition, while LUREs are plural factors. Two LUREs have been identified to date, and four other candidates have a similar cysteine arrangement (Okuda et al. 2009). Plural attractants may have two important functions: they might allow molecular evolution of each gene, while possible differences in function (e.g. reorientation and trapping) might generate an accurate guidance signal. Note also that the concentration gradient of LUREs is probably critical for the directional signal, unlike SCR/SP11. This is indirectly supported by the fact that pollen tube attraction to the protruding embryo sac of Torenia can be observed in semi-solid medium but not in liquid medium, and that the micro-bead assay is achieved using gelatin but not agarose. Extremely abundant expression of LUREs in the synergid cell might also be important in retaining the sharp and long-distance concentration gradient.

Why are several CRPs involved in the pollen–pistil interaction? Some populations of CRPs expressed in plant reproductive organs might be utilized as diffusible signaling molecules in cell–cell communication due to properties such as rapid molecular evolution, stabilized tertiary structure, small molecular weight and defense ability. Studies on the molecular evolution of identified CRPs, including LUREs, as well as identification of CRPs involved in signaling of other steps of plant reproduction, will provide insights into the evolution of peptide signaling in flowering plants. Indeed, flowering plants have several hundred receptor-like kinases (Arabidopsis Genome Initiative 2000, Shiu and Bleecker 2001). Some populations of CRPs expressed in floral organs could be ligands of orphan receptors.

Finally, S-RNase and chemocyanin require other ECM molecules, such as SCA and glycoproteins, for full functioning (McClure et al. 2000, Kim et al. 2003). The requirement for accompanying ECM molecules might be characteristic of intercellular signaling in plant reproduction, which would be another interesting issue.

Funding

This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology, Japan [No. 18075004 and 19370017]; PRESTO project, Japan Science and Technology Agency, Japan; Yamada Science Foundation; the Mitsubishi Foundation.

Acknowledgments

I thank collaborators who worked with me to identify LUREs. They provided me with insights to write this review article.

Abbreviations

    Abbreviations
  • AGP

    arabinogalactan protein

  • CRP cysteine-rich peptide/polypeptide; DEFL

    defensin-like protein

  • ECM

    extracellular matrix

  • EST

    expressed sequence tag

  • GABA

    γ-aminobutyric acid

  • GFP

    green fluorescent protein

  • GST

    glutathione S-transferase

  • LRR

    leucin-rich repeat

  • LTP

    lipid transfer protein

  • SCA

    stigma/stylar cysteine-rich adhesin

  • SLG

    S-locus glycoprotein

  • SRK

    S-locus receptor kinase

  • TTS

    transmitting tissue-specific protein.

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