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.

Nicotiana section Alatae contains eight species with variable flower sizes and morphologies. Section members readily hybridize in the glasshouse, but no hybrids have been observed in natural sympatric and parapatric populations. To investigate interspecific crossing relationships with respect to mechanisms preventing hybridization, all members of section Alatae were intercrossed in a complete diallel. We found positive correlation between the pistil length of the pollen donor and interspecific seed set relative to the conspecific cross. Pollen tube growth rate and pollen donor pistil length were positively correlated as well. Furthermore, pollen from short-pistil members of section Alatae could only grow a maximum distance proportional to, but greater than, their own pistil lengths. Our results show that pollen tube growth capacity (i.e., rate and distance), provides a hybridization barrier in long-pistil species 9 short-pistil species crosses. We also found another hybridization barrier not specifically related to pollen tube growth capacity in short-pistil species 9 long-pistil species. Taken together, these barriers can generally be described by a 'pistil-length mismatch' rule; in section Alatae, pollen has the most success fertilizing ovules from species with pistil lengths similar to their own. This rule could contribute to hybridization barriers in Section Alatae because the species display dramatically different pistil lengths.

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.

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.

This paper describes the introduction of synchrotron-based macromolecular crystallography (MX) into an undergraduate laboratory class. An introductory 2 week experimental module on MX, consisting of four laboratory sessions and two classroom lectures, was incorporated into a senior-level biochemistry class focused on a survey of biochemical techniques, including the experimental characterization of proteins. Students purified recombinant protein samples, set up crystallization plates and flash-cooled crystals for shipping to a synchrotron. Students then collected X-ray diffraction data sets from their crystals the remote interface of the Molecular Biology Consortium beamline (4.2.2) at the Advanced Light Source in Berkeley, CA, USA. Processed diffraction data sets were transferred back to the laboratory and used in conjunction with partial protein models provided to the students for refinement and model building. The laboratory component was supplemented by up to 2 h of lectures by faculty with expertise in MX. This module can be easily adapted for implementation into other similar undergraduate classes, assuming the availability of local crystallographic expertise and access to remote data collection at a synchrotron source.