Plant Molecular Biology 52: 91–102, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 91 An arabinogalactan protein associated with secondary cell wall formation in differentiating xylem of loblolly pine Yi Zhang 1,2,7 , Garth Brown 3 , Ross Whetten 1 , Carol A. Loopstra 4 , David Neale 3 , Marcia J. Kieliszewski 5 and Ronald R. Sederoff 1,2,6,∗ 1 Forest Biotechnology Group, 2500 Partners II, Centennial Campus, Box 7247, North Carolina State University, Raleigh, NC 27695-7247, USA ( ∗ author for correspondence; e-mail ron_sederoff@ncsu.edu); 2 Department of Genetics, North Carolina State University, Raleigh, NC 27695-7614, USA; 3 Department of Environmental Horti- culture, University of California, Davis, CA 95616, USA; 4 Department of Forest Science and Crop Biotechnology Center, Texas A & M University, MS 2123, College Station, TX 77843, USA; 5 Department of Chemistry and Bio- chemistry, Ohio University, Athens, OH 45701-2979, USA; 6 Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA; 7 Current address: Johnson and Johnson PRD, 3210 Merryfield Row, San Diego, CA 92121, USA Received 24 April 2002; accepted in revised form 23 October 2002 Key words: arabinogalactan proteins (AGPs), Pinus taeda, plant cell wall biosynthesis, xylem differentiation Abstract 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 β -glucosyl Yariv reagent (β -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. Abbreviations: AGPs, arabinogalactan proteins; β -glc-Y, Yariv reagent Introduction AGPs represent a large family of plant proteoglycans that constitute part of a larger class of hydroxyproline- rich plant glycoproteins (HRGPs) (reviewed by Showalter, 1993; Nothnagel, 1997; Majewska-Sawka and Nothnagel, 2000). AGPs are widely distributed throughout the plant kingdom and reside predomi- nantly in the plasma membrane, cell wall and ex- tracellular matrix (Ding and Zhu 1997; Gao et al. 1999). They are found less frequently in certain cy- toplasmic vesicles (Chapman et al., 2000). AGPs are typically composed of ca. 90% carbohydrate, predom- inantly galactose and arabinose (Fincher et al., 1983), and 10% protein, rich in hydroxyproline (Hyp), Ser, Ala and Thr (Showalter and Varner, 1989; Du et al.,