A Pollen Protein,NaPCCP, That Binds Pistil Arabinogalactan Proteins Also Binds Phosphatidylinositol 3-Phosphate and Associates with the Pollen Tube Endomembrane System

As pollen tubes grow toward the ovary, they are in constant contact with the pistil extracellular matrix (ECM). ECM components are taken up during growth, and some pistil molecules exert their effect inside the pollen tube. For instance, the Nicotiana alata 120-kD glycoprotein (120K) is an abundant arabinogalactan protein that is taken up from the ECM; it has been detected in association with pollen tube vacuoles, but the transport pathway between these compartments is unknown. We recently identified a pollen C2 domain-containing protein (NaPCCP) that binds to the carboxyl-terminal domain of 120K. As C2 domain proteins mediate protein-lipid interactions, NaPCCP could function in intracellular transport of 120K in pollen tubes. Here, we describe binding studies showing that the NaPCCP C2 domain is functional and that binding is specific for phosphatidylinositol 3-phosphate. Subcellular fractionation, immunolocalization, and live imaging results show that NaPCCP is associated with the plasma membrane and internal pollen tube vesicles. Colocalization between an NaPCCP∷green fluorescent protein fusion and internalized FM4-64 suggest an association with the endosomal system. NaPCCP localization is altered in pollen tubes rejected by the self-incompatibility mechanism, but our hypothesis is that it has a general function in the transport of endocytic cargo rather than a specific function in self-incompatibility. NaPCCP represents a bifunctional protein with both phosphatidylinositol 3-phosphate- and arabinogalactan protein-binding domains. Therefore, it could function in the transport of pistil ECM proteins in the pollen tube endomembrane system.Angiosperm sexual reproduction requires pollen transfer to a receptive stigma followed by its hydration, germination, and pollen tube growth. Pollen tubes grow through the stigma and style toward the ovule, where the sperm cells are discharged for fertilization. Pollen tubes do not divide; rather, they extend through tip growth while periodically producing callose plugs, separating highly vacuolated distal regions from the actively growing tip (Taylor and Hepler, 1997). The tip region shows strong zonation. An apical region or clear zone, a subapical, organelle-rich zone, a nuclear zone, and a distal vacuolated zone or plug region that may extend several centimeters are easily recognized (Mascarenhas, 1993). Proper deposition of wall material and rapid tube extension require coordination between GTPase-regulated trafficking pathways, the cytoskeleton, signaling pathways, and oscillatory ion and water fluxes (Li et al., 1999; Fu et al., 2001; Zonia et al., 2002; Camacho and Malhó, 2003; Chen et al., 2003; de Graaf et al., 2005; Gu et al., 2005).Pollen tube endomembrane system dynamics are critical for growth: wall materials are deposited by exocytosis, and the membrane is recovered by endocytosis (Picton and Steer, 1983; Cheung and Wu, 2008). Exocytosis of material synthesized in the Golgi occurs near the tip (Cheung et al., 2002). Additional wall material is produced by membrane-bound callose synthase, but this occurs behind the tip (Brownfield et al., 2007). Distinct endocytosis zones have been identified by pulse-chase membrane labeling, observations of charged nanoparticles, and electron microscopy (Derksen et al., 1995; Moscatelli et al., 2007; Zonia and Munnik, 2008). Clathrin-independent endocytosis occurs at the pollen tube apex; endocytic vesicles clearly contribute to vesicle populations in the clear zone once thought to be composed entirely of exocytic vesicles (Moscatelli et al., 2007; Bove et al., 2008; Zonia and Munnik, 2008). Inhibitor studies suggest that clathrin-dependent endocytosis occurs in the organelle-rich zone a few micrometers back from the tip (Moscatelli et al., 2007). Furthermore, coated vesicles have been observed from 6 to 15 μm from the tip by electron microscopy (Derksen et al., 1995).Pollen-pistil interactions influence pollen tube growth either positively or negatively. Positive effects are evident from the observation that pollen tubes grow as much as 10 times faster and achieve much greater lengths in planta than in culture (Cheung et al., 2000). Self-incompatibility (SI) systems provide the best understood examples of negative effects. In SI, pollen-pistil interactions cause rejection of closely related pollen tubes (de Nettancourt, 2001).Arabinogalactan proteins (AGPs) secreted into the pistil extracellular matrix (ECM) play key roles in both positive and negative interactions, but the underlying molecular interactions with pollen tubes are just beginning to be understood. The transmitting tract-specific (TTS) glycoprotein (Cheung et al., 1995; Wu et al., 1995, 2000) and the 120-kD glycoprotein (120K; Hancock et al., 2005) are pistil AGPs implicated in pollination in Nicotiana. Both are abundant components of the pistil ECM (Cheung et al., 1995; Lind et al., 1996) and share a conserved Cys-rich C-terminal domain (CTD). TTS was first described in Nicotiana tabacum (i.e. NtTTS) as a pollen tube attractant. Pollen tubes grow toward TTS in culture, and its glycosylation levels progressively increase closer to the ovary (Cheung et al., 1995). Pollen tubes deglycosylate TTS, which suggests that TTS may act as a nutritive factor (Wu et al., 1995) and, thus, positively affect pollen tube growth.120K is implicated in SI in Nicotiana alata (Cruz-Garcia et al., 2005; Hancock et al., 2005), a species that displays S-RNase-based gametophytic SI (McClure et al., 1989). In SI, compatibility is controlled by the polymorphic S-locus; pollen is rejected if its S-haplotype matches either of the two S-haplotypes in the diploid pistil (de Nettancourt, 2001). Each S-haplotype is unique and encodes separate pollen- and pistil-specificity genes (Kao and Tsukamoto, 2004). S-RNases determine specificity on the pistil side and directly inhibit the growth of closely related pollen tubes (McClure et al., 1989). S-locus F-box proteins (SLF/SFB) control specificity on the pollen side (Sijacic et al., 2004). SLF/SFB proteins bind S-RNase in vitro and appear to form several distinct complexes with other pollen proteins (Qiao et al., 2004; Hua and Kao, 2006; Huang et al., 2006). SI, therefore, is a clear example of inhibitory pollen-pistil interactions: interaction between a pistil protein, S-RNase, and a pollen protein, SLF/SFB, determines compatibility. However, other pistil factors are also required for SI (McClure et al., 1999; Hancock et al., 2005; McClure and Franklin-Tong, 2006). 120K, for example, is required for SI but does not directly contribute to S-specificity (Hancock et al., 2005).120K was first identified as an abundant component of the transmitting tract ECM that contains both arabinogalactan and extensin-like carbohydrate moieties (Lind et al., 1994). 120K is an S-RNase-binding protein that is taken up by growing pollen tubes (Lind et al., 1996; Cruz-Garcia et al., 2005; Goldraij et al., 2006). Immunolocalization studies show 120K in the pollen tube cytoplasm and associated with pollen tube tonoplast membranes (Lind et al., 1996; Goldraij et al., 2006). Goldraij et al. (2006) also found S-RNase in the lumen of pollen tube vacuoles. In many cases, S-RNase was found in vacuoles with 120K apparently embedded in the surrounding membrane. S-RNase is also found in vacuoles of incompatible pollen tubes, but the breakdown of these vacuoles late in SI and the concomitant release of S-RNase may contribute to the rejection mechanism. Other pistil proteins are also taken up by growing pollen tubes; for example, endocytosis of biotinylated stigma/style Cys-rich adhesin has been reported in lily (Lilium longiflorum) pollen tubes (Kim et al., 2006). Although the uptake of pistil proteins such as 120K and S-RNase has not been well characterized, it is likely that endocytosis and retrograde transport of ECM components occurs on a large scale. Thus, it is important to identify pollen proteins that interact with endocytic cargo from the pistil ECM and that could participate in transport through the pollen tube endomembrane system.We recently described a pollen-specific C2 domain-containing protein, NaPCCP, that interacts with the CTD of the potential cargo proteins, NaTTS and 120K. NaPCCP consists of a short N-terminal domain, an 80-residue C2 domain, and a 79-residue C-terminal region. In vitro pull-down assays showed that the C-terminal region of NaPCCP is sufficient for binding the AGP CTDs (Lee et al., 2008b). Originally implicated in binding mammalian protein kinase C to phosphatidylserine in a calcium-dependent manner (Bazzi and Nelsestuen, 1987, 1990; Brose et al., 1992), C2 domains are now known to contribute to transient membrane association of a variety of proteins with functions that include vesicular transport, lipid modification, GTPase regulation, ubiquitylation, and protein phosphorylation (Coussens et al., 1986; Clark et al., 1991; Brose et al., 1992; Cullen et al., 1995; Dunn et al., 2004). Calcium-independent lipid binding of C2 domain-containing proteins has also been reported (Damer and Creutz, 1994; Fukuda et al., 1994).Here, we report the lipid-binding properties of NaPCCP and its association with the pollen tube endomembrane system. Lipid overlay and liposome-binding experiments show that NaPCCP specifically binds to phosphatidylinositol 3-phosphate (PI3P). Immunolocalization and live imaging studies of compatible pollen tubes show that NaPCCP is associated with the pollen tube plasma membrane (PM) and with punctate structures in the cytoplasm. In SI, incompatible pollen tubes show altered NaPCCP distributions. We speculate that NaPCCP is involved in the uptake and transport of proteins from the ECM.