After pollination, the first stage of the pollen-pistil interactions occurs right at the surface of the stigma ( Figure 1C ). The stigma is the receptive terminal of the pistil (Bedinger et al., 2017) and can be classified into two broad groups based on their structure and size: wet stigmas and dry stigmas (Heslop-Harrison and Shivanna, 1977). Wet stigmas, such as in Nicotiana and Petunia (Solanaceae), are covered by a viscous secretion, while dry stigmas, e.g. in Brassicaceae (Edlund et al., 2004), lack a surface exudate and possess a layer of intact papilla cells. There are also semi-dry stigmas, such as those found in the Asteraceae family, where a small amount of exudate is present when the stigma is mature (Hiscock et al., 2002).

Once the pollen grain is deposited on the stigma surface, it must be hydrated to become metabolically activated before germinating and developing a pollen tube that grows through the pistil to deliver the sperm cells to the ovary for fertilisation. Wet stigmas tend to hydrate pollen indiscriminately, while pollen hydration on dry stigmas tends to be more selective (Heslop-Harrison, 2000). In Brassicaceae, the dry stigma surface is a highly discriminative first point of contact with the desiccated pollen grains (Huang et al., 2023), which are reliant on the stigma papilla cells to provide water and other components required for pollen hydration and germination (Broz and Bedinger, 2021). The evolution of the dry stigma made pollen hydration a crucial checkpoint in the reproductive process. At this stage, a molecular dialogue occurs between the pollen grain and the stigmatic papilla cell to determine their compatibility. Therefore, it is at this stage where the earliest post-pollination prezygotic barrier can be established, contributing to the prevention of fertilisation between different plant species.

The pollen coat, which is the outermost layer of the pollen grain, plays a central role in mediating molecular recognition events during pollen-stigma interactions. Accumulating evidence has demonstrated that the pollen coat carries small secreted cysteine-rich proteins (CRPs) that are involved in cell-cell communication during pollen–stigma interactions ( Figure 1C ) (Bircheneder and Dresselhaus, 2016; Kim et al., 2021; Wang et al., 2023). Despite being diverse and mostly functionally uncharacterised, CRPs have been identified as key players in plant reproductive signalling (Silverstein et al., 2007; Marshall et al., 2011; Kim et al., 2021). Genes encoding these signalling proteins are often highly polymorphic and species- or genus-specific, suggesting their important roles in establishing and maintaining reproductive barriers between species. Among these CRPs are the pollen coat protein B class (PCP-Bs), which regulate compatible pollen hydration (Wang et al., 2017) by competing with stigmatic ligands for binding to the stigmatic receptor FERONIA (FER) complex, leading to a reduction in stigmatic reactive oxygen species (ROS) that facilitates pollen hydration ( Figure 1C ) (Liu et al., 2021; Huang et al., 2023). PCP-B are polymorphic among species in Brassicaceae, and their binding with FER occurs in a species-preferential manner. Arabidopsis PCP-Bγ outcompetes PCP-B from Brassica rapa for binding to FER, thereby serving as a ligand-receptor pair in establishing an interspecific reproductive barrier during pollen-stigma interaction (Huang et al., 2023).

A recently identified Arabidopsis stigma-specific transmembrane protein, Stigma privacy 1 (SPRI1), confers interspecies incompatibility by rejecting pollen from distantly related species in Brassicaceae (Fujii et al., 2019). This finding proposes a novel SI-independent mechanism for promoting intraspecific pollen germination, that in turn, facilitates reproductive isolation. However, the pollen ligands that interact with SPRI1 are yet to be identified. Recent proteomic studies of the pollen coat of three Brassicaceae species revealed numerous CRPs with uncharacterised functions (Wang et al., 2023), which provides possible candidates for mediating both intra- and interspecific pollen-stigma recognition by interacting with SPRI1 and other unknown stigmatic receptors ( Figure 1C ). Some of the genes encoding these pollen coat CRPs, including PCP-Bs, are undergoing rapid diversification and evolving under positive selection (Wang et al., 2023). Evolutionary analysis revealed that SPRI1 function was lost multiple times during the evolution of A. thaliana (Fujii et al., 2019). Impaired hydration or germination of pollen due to lower ligand-receptor-binding affinity can lead to a delay in pollen tube growth. Thus alternations in either ligands or receptors apply selective pressure on genes encoding the binding partners, suggesting that the coevolution between PCP-B and FER, as well as between SPRI1 and its as-yet-unidentified pollen ligand, may play a more significant role in shaping the species-specificity of their interaction, rather than the evolution of the ligands alone. Consequently, this coevolution could contribute substantially to the establishment of prezygotic barriers and the process of speciation.

In tomato (Solanum lycopersicum), a small pistil secreted CRP, STIGMA-SPECIFIC PROTEIN1 (STIG1), was identified as a signalling ligand that binds to the pollen receptor kinase (LePRK1/2) ( Figure 1C ). This interaction promotes pollen tube growth by regulating cellular reactive oxygen species (ROS) production (Goldman et al., 1994; Verhoeven et al., 2005; Huang et al., 2014). Although it is not clear whether STIG1 and LePRK1/2 interact in a species or family-preferential manner, studies suggested that STIG1 homologues have diverged functions in Solanaceae species (Goldman et al., 1994; Verhoeven et al., 2005; Huang et al., 2014), indicating the potential role of STIG1 in establishing a prezygotic reproductive barrier during early pollen tube growth.

Pollen tube growth through the style towards the ovary is another important phase contributing to interspecific reproductive barriers. The styles vary widely in size and structure, making the speed and navigation of pollen tube growth through the style crucial for reproductive success. Multiple (mostly not yet identified) molecular factors involved in pollen tube-style interactions appear to contribute to interspecific hybridisation barriers.

In Solanum, silencing the genes encoding pistil SI proteins, HT proteins, eliminates the stylar barrier in S. lycopersicum to heterospecific pollen from S. habrochaites and S. arcanum. However, this silencing only weakens but does not eliminate the barrier for S. pennellii pollen, implying the presence of additional female barriers (Tovar-Mendez et al., 2017). S-RNase-based self-incompatibility in Solanaceae is known to play an important role in the prevention of interspecific hybridisation (Chetelat and Deverna, 1991; Murfett et al., 1996; Baek et al., 2015; Bedinger et al., 2017). On the pollen side, multiple genetically identified loci confer pollen resistance to S-RNase-based hybridisation barriers between S. pennellii and S. lycopersicum (Chetelat and Deverna, 1991). Some of these genes have been identified and functionally characterised. In particular, SpSLF-23 gene linked to the S-locus and Cullin1 gene encoding CUL1 that is part of Skp1-Cullin-F-box ubiquitin E3 ligase complex in S. pennellii target pistil SI factor for degradation to unilaterally overcome the interspecific barrier (Chetelat and Deverna, 1991; Li and Chetelat, 2010; Li and Chetelat, 2014; Li and Chetelat, 2015). Overexpression of a gene encoding farnesyl pyrophosphate synthase in the pollen of S. lycopersicum has been shown to cause resistance to S-RNase-independent interspecific incompatibility (Qin et al., 2018).

Some plants, such as lilies, have open styles and the pollen tube grows through mucilage covering the surface of the central canal. In Lilium longiflorum, stigma/style cysteine-rich adhesin (SCA) peptides, which are members of lipid transfer proteins (LTPs) belonging to CRPs, play a crucial role in the adhesion of pollen tubes to the stylar transmitting tract epidermis (TTE) ( Figure 1C ) (Park et al., 2000; Chae et al., 2007). The TTE secrets SCA peptides, which facilitate the adhesion of pollen tubes to the TTE wall surface by forming an adhesive matrix with the pollen tube through binding to stylar pectin in a pH-dependent manner (Mollet et al., 2000). In A. thaliana, an SCA-like LTP5 is secreted from both pollen and the pistil and is essential for maintaining normal pollen tube growth and fertility (Chae et al., 2009) ( Figure 1C ). Although no protein receptor has been identified for the SCA/pectin complex, interspecific differences in the density of extracellular matrix and functions of enzymes produced by the pollen tube suggest that this pollen tube-style adhesion regulatory system may provide a platform for the establishment of reproductive barriers by promoting or opposing pollen tube growth, favouring conspecific pollen.

The interaction between the growing pollen tube and the ovary marks the final stage at which post-pollination prezygotic interspecific barriers can act. Once a pollen tube grows through the transmitting tract (TT) and emerges onto the septum surface, it must be precisely guided towards the micropyle for sperm cell delivery. Studies in various species have shown that attraction of pollen tubes to the embryo sac and ovules can occur in a species-specific manner (Higashiyama et al., 2006; Uebler et al., 2013; Zhong et al., 2019), which may play a crucial role in establishing reproductive barriers between plant species. Several molecular factors involved in pollen-ovary interactions appear to contribute to interspecific hybridisation barriers. In Zea mays, a small peptide, EGG APPARATUS1 (ZmEA1), is expressed specifically in the synergid cells and filiform apparatus (FA), and it attracts pollen tubes while arresting their growth at higher concentrations ( Figure 1C ) (Márton et al., 2005; Márton et al., 2012). Moreover, it binds to the pollen tube in a species-preferential manner. When expressed in Arabidopsis ovules, ZmEA1 guides maize pollen tubes to grow towards the micropylar opening of the ovule in vitro (Márton et al., 2012). A group of synergid-expressed defensin-like CRPs identified in Torenia and Arabidopsis, LURE (TfLUREs and AtLUREs), in vitro demonstrated activity in attracting pollen tube growth in a species-specific manner (Higashiyama et al., 2006; Okuda et al., 2009; Takeuchi and Higashiyama, 2012). Pollen tube tip-localised receptor-like kinase 6 (PRK6) acts as an essential receptor for perceiving the signalling of the LURE1 peptides in A. thaliana (AtLURE1), guiding the pollen tube towards the ovule (Takeuchi and Higashiyama, 2016). A recent study has identified four LURE1-related peptides, XIUQIU1-4, as pollen tube attractants that act irrespective of the species and independently of PRK6 in Brassicaceae (Zhong et al., 2019) ( Figure 1C ). The AtLURE1s-PRK6 interactions were revealed to be not essential that fertilisation but rather facilitate the emergence of pollen tubes onto the septum surface, promoting the selection of conspecific pollen (Zhong et al., 2019). Phylogenetic profiling demonstrated that each XIUQIU peptide has ortholog(s) in the analysed species of Brassicaceae, while AtLURE1s form species-specific clusters within Arabidopsis species, reflecting their distinct functions in species-specificity and roles in reproductive isolation (Takeuchi and Higashiyama, 2012; Zhong et al., 2019).

The species-specificity of molecular crosstalk during pollen tube reception may also contribute to interspecific reproductive barriers. Incongruity of molecular mechanisms at this stage can result in pollen tube overgrowth caused by failure of growth arrest and tip rupture to release sperm cells. This phenotype has been observed in interspecific crosses in Arabidopsis (Escobar-Restrepo et al., 2007) and Rhododendron (Williams et al., 1986). In maize, pollen tube rupture upon arrival at the micropyle is mediated by cysteine-rich defensin-like proteins ZmES4 (Amien et al., 2010), which are released from vesicles in the egg apparatus ( Figure 1C ). In Arabidopsis, the regulation of pollen tube rupture is different, and it involves a group of signalling peptides called Rapid Alkalinisation Factors (RALFs), which also belong to the CRPs. RALFs and members of Catharanthus roseus RLK1-LIKE (CrRLK1L) receptors regulate pollen tube reception, tip rupture and the prevention of polytubey (Ge et al., 2017; Mecchia et al., 2017; Galindo-Trigo et al., 2020; Zhong et al., 2022) ( Figure 1C ). As the pollen tube reaches the micropyle, a synergid receptor complex comprising FER, LORELEI (LRE), ANJEA (ANJ) and HERCULES RECEPTOR KINASE 1 (HERK1) controls pollen tube reception through perceiving unidentified pollen ligands (Galindo-Trigo et al., 2020). Autocrine pollen ligands RALF4/19 interact with the pollen tube receptor complex anxur/budda’s paper seal (ANX/BUPS), maintaining cell wall integrity and preventing premature rupture. Synergid-secreted RALF34 competes with RALF4/19 for binding to the ANX/BUPS-LLG2/3 (LRE-LIKE GPI-ANCHORED PROTEIN2/3) complex, triggering the rupture of the pollen tube and the release of sperm cells (Ge et al., 2017; Ge et al., 2019). Septum-localised FER-ANJ-HERK1 receptor complex interacts with pollen ligands RALF6, 7, 16, 36 and 37 to regulate polytubey blocking (Zhong et al., 2022) ( Figure 1C ). While this mechanism appears to differ between distantly related species, it is not clear whether it contributes significantly to interspecific reproductive barriers between closely related species.