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 NaPCCPgreen 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.

Pollen proteins bind to the C-terminal domain of Nicotiana alata pistil arabinogalactan proteins.

Pollen tube growth is influenced by interaction between pollen proteins and the pistil extracellular matrix. The transmitting tract-specific glycoprotein (NaTTS) and 120-kDa glycoprotein (120K) are two pistil arabinogalactan proteins (AGPs) that share a conserved C-terminal domain (CTD) and directly influence pollen tubes in Nicotiana alata. 120K and other extracellular matrix proteins are taken up and transported to vacuoles of growing pollen tubes. We hypothesize that signaling and trafficking processes inside pollen tubes are important for controlling pollen tube growth. We performed a yeast two-hybrid screen of pollen cDNAs using sequences from 120K and NaTTS as baits. We found that an S-RNase-binding protein (SBP1), a C2 domain-containing protein (NaPCCP), and a putative cysteine protease bound to the AGP baits. SBP1 from Petunia hybrida and Solanum chacoense is a putative E3 ubiquitin ligase that binds to S-RNase and other proteins. C2 domain-containing proteins bind lipids and can regulate myriad cellular processes. Cysteine proteases are often associated with the degradation of vacuolar proteins. Expression analysis revealed that transcripts for these proteins are expressed in mature pollen. NaPCCP and NaSBP1 were characterized further because of their potential roles in signaling and trafficking. In vitro pull-down assays verified binding between maltose-binding protein (MBP) fusions, MBP::NaPCCP or MBP::NaSBP1 and glutathione S-transferase (GST), GST::AGP CTD fusions. NaSBP1 binds to the AGP CTDs through its helical and RING domains. NaPCCP binds through its C-terminal region. Binding between NaPCCP and NaSBP1 and the pistil AGPs may contribute to signaling and trafficking inside pollen tubes growing in planta.

Compartmentalization of S-RNase and HT-B degradation in self-incompatible Nicotiana.

Pollen-pistil interactions are crucial for controlling plant mating. For example, S-RNase-based self-incompatibility prevents inbreeding in diverse angiosperm species. S-RNases are thought to function as specific cytotoxins that inhibit pollen that has an S-haplotype that matches one of those in the pistil. Thus, pollen and pistil factors interact to prevent mating between closely related individuals. Other pistil factors, such as HT-B, 4936-factor and the 120 kDa glycoprotein, are also required for pollen rejection but do not contribute to S-haplotype-specificity per se. Here we show that S-RNase is taken up and sorted to a vacuolar compartment in the pollen tubes. Antibodies to the 120 kDa glycoprotein label the compartment membrane. When the pistil does not express HT-B or 4936-factor, S-RNase remains sequestered, unable to cause rejection. Similarly, in wild-type pistils, compatible pollen tubes degrade HT-B and sequester S-RNase. We suggest that S-RNase trafficking and the stability of HT-B are central to S-specific pollen rejection.