| Although cell walls contribute greatly to the unique properties and diversity of plants, major aspects of the structure and function of plant cell walls remain unresolved. The proteins associated with cell walls are likely to have roles in the biosynthesis and modification of wall polymers and in wall assembly. We are taking advantage of our Pine EST Sequencing Project to identify and characterize major cell wall associated proteins that play a role in the formation of wood. From a pilot-scale survey of 1097 ESTs (expressed sequence tags), 7 clusters of ESTs were identified as putative cell wall associated proteins by sequence homology, including proline or glycine rich sequences, extensin-like proteins, AGP-like proteins and phytocyanins. Representative clones have been chosen to obtain the full length cDNA sequences in order to further characterize these proteins. Gene expression studies using Northern blotting were performed to evaluate the differential expression of these proteins in different tissues and under gravitational stimuli. Statistical analysis with the data of the number of EST occurrences in the five EST sequencing libraries from the ongoing full-scale Pine EST Sequencing Project (so-called Virtual Northerns) were conducted to investigate the digital gene expression profiles of the seven proteins, plus four additional cell wall associated proteins identified by others. The comparison between results from experimental Northerns and Virtual Northerns revealed both consistency in some case, as well as inconsistency in others. Multiple sequence alignments were used to discover the sequence variations within the nucleotide sequences of these proteins.; From these seven putative cell wall related ESTs, PtaAGP6, encoding an arabinogalactan protein (AGP), was chosen for further investigation due to its unique structural features and high abundance in compression wood as compared to normal vertical wood. A polyclonal antibody was raised against the peptide sequence of a basic subdomain of PtaAGP6, and was used to purify an AGP fraction from differentiating xylem and to immunolocalize PtaAGP6 in pine stem tissues. PtaAGP6 epitopes are restricted to a single file of cells that just precede the secondary cell wall thickening of the differentiating xylem, suggesting possible roles in xylem differentiation and wood formation. Multiple SNPs were detected in EST variants. Denaturing HPLC analysis of PCR products suggests that the gene encoding PtaAGP6 has a simple gene family structure and may be encoded by a single gene. |
