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.

Arabinogalactan Proteins in Arabidopsis thaliana Pollen Development

“…We are committed to search for AGP-specific ligands recently identified for tobacco pistil AGPs (Lee et al, 2008), reason why we performed microarrays in the double null mutant, agp6 agp1 pollen tubes, to try to bring some clarification for the biological way of action of this ubiquitous class of plant proteoglycans.…”

NaStEP: A Proteinase Inhibitor Essential to Self-Incompatibility and a Positive Regulator of HT-B Stability inNicotiana alataPollen Tubes

“…120K forms complexes with S-RNases and other proteins (CruzGarcia et al, 2005) in vitro, and suppression of 120K expression prevents S-specific pollen rejection . Protein-protein interaction assays demonstrate that 120K interacts with the pollen-specific protein NaPCCP (a pollen C2 domain-containing protein), a protein that binds phosphatidylinositol 3-phosphate and is associated with the pollen tube endomembrane system (Lee et al, 2008(Lee et al, , 2009). …”

Protein Interactions in S-RNase-Based Gametophytic Self-Incompatibility

“…Hua & Kao (2006) further showed that SBP1 interacted with PiSLF 2 and PiSLF 1 in both two-hybrid and pull-down assays, as well as with Cullin-1 and PhUBC1 (Sims, unpublished), an E2 conjugation enzyme protein from Petunia hybrida. Lee et al (2008) used C-terminal domains of the style-transmitting-tract proteins TTS and 120K to screen a pollen two-hybrid library from Nicotiana alata, and also pulled out NaSBP1 from this screen. All of these reports (Hua & Kao, 2006;Lee et al, 2008;O'Brien et al, 2004;Sims & Ordanic 2001) showed that SBP1 was not pollen-specific, but was expressed in all tissues examined.…”

“…Lee et al (2008) used C-terminal domains of the style-transmitting-tract proteins TTS and 120K to screen a pollen two-hybrid library from Nicotiana alata, and also pulled out NaSBP1 from this screen. All of these reports (Hua & Kao, 2006;Lee et al, 2008;O'Brien et al, 2004;Sims & Ordanic 2001) showed that SBP1 was not pollen-specific, but was expressed in all tissues examined. SBP1 is non-allelic as well, as SBP1 isolated from S 1 S 1 and S 3 S 3 lines of Petunia hybrida are 100% identical.…”

“…Biochemical data suggests that these proteins and the SRNase may form a complex that is taken up into pollen tubes. In an extension of these experiments, Lee et al, (2008) used the C-terminal domains of the TTS and the 120 K proteins in yeast two-hybrid screens of pollen cDNA libraries. In addition to interaction with SBP1 (see section 4.2) a putative cysteine protease, NaPCCP, interacted with both TTS and 120K.…”