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Arabinogalactan proteins (AGPs) are abundant plant proteoglycans implicated in plant growth and development. Here, we report the genetic characterization, partial purification and immunolocalization of a classical AGP (PtaAGP6, accession number AF101785) in loblolly pine (Pinus taeda L.). A PtaAGP6 full-length cDNA clone was expressed in bacteria. PtaAGP6 resembles tomato LeAGP-1 and Arabidopsis AtAGP17-19 in that they all possess a subdomain composed of basic amino acids. The accessibility of this domain in the glycoprotein makes it possible to label the PtaAGP6 epitopes on the cell surface or in the cell wall with polyclonal antibodies raised against this subdomain. The antibodies recognize the peptide of the basic subdomain and bind to the intact protein molecule. A soluble protein-containing fraction was purified from the differentiating xylem of pine trees by using beta-glucosyl Yariv reagent (beta-glcY) and was recognized by antibodies against the basic subdomain. Immunolocalization studies showed that the PtaAGP6 epitopes are restricted to a file of cells that just precede secondary cell wall thickening, suggesting roles in xylem differentiation and wood formation. The location of apparent labeling of the PtaAGP6 epitopes is separated from the location of lignin deposition. Multiple single nucleotide polymorphisms (SNPs) were detected in EST variants. Denaturing HPLC analysis of PCR products suggests that PtaAGP6 is encoded by a single gene. Mobility variation in denaturing gel electrophoresis was used to map PtaAGP6 SNPs to a site on linkage group 5.

Two genes preferentially expressed in differentiating xylem of loblolly pine (Pinus taeda L.) were cloned from cDNA and genomic libraries and designated PtX3H6 and PtX14A9. Transcripts of PtX3H6 and PtX14A9 are very abundant in differentiating xylem, less abundant in needles, and very low or non-detectable in embryos and megagametophytes. PtX3H6 contains a putative signal peptide, a threonine-rich region, a proline-rich region, and a hydrophobic tail. Repeats of Pro-Pro-Pro-Val-X-X are similar to repeats found in proline-rich cell wall proteins. The amino acid compositions of PtX3H6 and PtX14A9 are similar to those of arabinogalactan proteins (AGPs). PtX14A9 contains an 8 amino acid sequence similar to amino terminal sequences of ryegrass, carrot and rose AGPs. Upstream sequences have been determined from genomic clones encoding PtX3H6 and PtX14A9. A 7 bp sequence found in the 5' flanking regions of both genes has previously been shown to be involved in the vascular-specific expression of GRP 1.8, a glycine-rich protein found in bean. The sequence is also present upstream of another glycine-rich protein from bean, GRP 1.0, and may be partially responsible for the xylem-specific expression of pTx3H6 and PtX14A9.

DNA transfer using Agrobacterium tumefaciens has been demonstrated in sugar pine, Pinus lambertiana Dougl. Shoots derived from cytokinin-treated cotyledons formed galls after inoculation with A. tumefaciens strains containing the plasmid pTiBo542. A selectable marker, neomycin phosphotransferase II, conferring resistance to kanamycin, was transferred into sugar pine using a binary armed vector system. Callus proliferated from the galls grew without hormones and in some cases, kanamycin-resistant callus could be cultured. Southern blots provided evidence of physical transfer of T-DNA and the nptII gene. Expression of the nptII gene under control of the nos promoter was demonstrated by neomycin phosphotransferase assays. Several aspects of DNA transfer were similar to those previously observed in angiosperms transformed by A. tumefaciens. This is the first evidence for DNA transfer by Agrobacterium in this species and the first physical evidence for transfer in any pine. These results bring us closer to genetic engineering in this commercially important genus of forest trees.