Pollen Proteins Bind to the C-terminal Domain of Nicotiana alata Pistil Arabinogalactan Proteins

Abstract

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.