The proline rich proteins (PRPs) are a family of structural cell wall proteins that are restricted to the Fabaceae family. Previous work from the Cooper laboratory identified four PRPs (PRP1-4) in Medicago truncatula. PRP4 was cloned as a genomic DNA sequence which was subsequently used to generate a transcriptional fusion to a reporter gene in order to determine its expression pattern in transgenic hairy roots. To analyze the other three PRPs, I screened a BAC library for PRP homologous sequences and showed that the PRPs are closely linked on chromosome 4 and are in the same transcriptional orientation. In addition, I identified three additional PRP4-like genes (PRP4B-D) as well as a PRP4 pseudogene. The sequence homology among these latter PRPs suggest they arose by recent tandem duplication events. Analysis of the pentapeptide repeat content of these seven PRPs suggests they differ in their propensity to form covalent crosslinks within the cell wall. Available genomic and mRNA data from other legumes suggest that the expansion of the PRP family within a given species occurred via local gene duplication events following speciation, some of which appear to have occurred recently. Transcriptional reporter gene fusions for the four PRP promoters described here facilitated analysis of their expression in M. truncatula hairy root cross-sections. Results suggest the PRPs have both divergent and redundant functions. In silico analysis of the cloned promoter sequences shows they share known conserved promoter motifs including some reported to be involved in nodulation, root tissue-specific expression, and response to hormonal signals. Additionally, immunoblot data from the Cooper Lab suggest that PRP4 is also expressed in seed pod walls. Generation of stable transgenic Arabidopsis carrying the transcriptional reporter gene fusion confirmed the activity of the PRP4 promoter in silique pod walls.

The ABI Five binding Proteins (AFPs) are a family of proteins that function in negatively regulating abscisic acid (ABA) signaling in plants. While their exact mechanism of action is unknown, yeast-two-hybrid (Y2H) analysis indicates that they interact with phosphatases that negatively regulate the ABA core signaling pathway (PP2Cs) and YFP-fused AFP2 appears as a doublet on immunoblots when overexpressed in Arabidopsis or Nicotiana benthamiana. Co-overexpression of an AFP2-AB subdomain construct with a PP2C or SnRK2 kinase alters the doublet appearance of the protein. Taken together, these results suggest that AFP2 may be phosphorylated in vivo. An immunoblot probed with a phosphoserine-specific antibody confirmed that this doublet appearance results from differential phosphorylation states. Mass spectrometry experiments searching for post-translational modifications identified one phosphorylation target in the B domain (to date).

Mutational analyses were performed targeting sites within known SnRK2 kinase target motifs and the residue identified by mass spectrometry as well as sites that are conserved within the AFP family and predicted to be phosphorylated. These experiments yielded data suggesting we have successfully identified some of the phosphorylated residues that are responsible for the doublet appearance. However, the mutations had no apparent effect on the ability of AFP2 to interact with known binding partners in planta. Preliminary data assaying the functional significance of these mutations suggest that phosphorylation state may not control AFP activity.

Circumstantial evidence including the predicted disordered structural nature of the AFP proteins, the fact that they interact with components at each level of ABA core signaling and that these interactions occur in nuclear puncta, and the observation that they are subject to phosphorylation suggests that AFPs could act as protein scaffolds that facilitate ABA core signaling (and potentially other responses) by increasing the local concentration of necessary components. Investigations into this new hypothesis are ongoing and hope to shed light onto the exact mechanism of AFP function in regulating ABA signaling.