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Plant and Cell Physiology Advance Access published online on April 19, 2005
Plant and Cell Physiology, doi:10.1093/pcp/pci106
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Plant and Cell Physiology 2005 © The Japanese Society of Plant Physiologists (JSPP); all rights reserved.
Received August 15, 2004
Accepted April 7, 2005

Regular Paper

The Lysine-Rich Arabinogalactan-Protein Subfamily in Arabidopsis: Gene Expression, Glycoprotein Purification and Biochemical Characterization

Wenxian Sun 1, Jianfeng Xu 2, Jie Yang 1, Marcia J. Kieliszewski 3, and Allan M. Showalter 1*

1 Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701-2979; Molecular and Cellular Biology Program, Ohio University, Athens, OH 45701-2979
2 Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701-2979
3 Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701-2979; Molecular and Cellular Biology Program, Ohio University, Athens, OH 45701-2979

* To whom correspondence should be addressed.
Allan M. Showalter, E-mail: showalte{at}ohio.edu


  Abstract

AtAGP17, AtAGP18, and AtAGP19 are homologous genes encoding three putative glycosylphosphatidylinositol (GPI)-anchored classical arabinogalactan-proteins (AGPs) in Arabidopsis. They are distinguished from other AGPs by a short, C-terminal lysine-rich region. Organ-specific expression of these genes was revealed by Northern blot analysis. AtAGP17 was strongly expressed in leaves and stems, and weakly expressed in flowers and roots; AtAGP18 was strongly expressed in flowers, and moderately expressed in roots, stems, and young leaves, and AtAGP19 was strongly expressed in stems, moderately expressed in flowers and roots, and weakly expressed in young leaves. One of these genes, AtAGP17, was expressed and purified as a green fluorescent protein (GFP) fusion protein in transgenic tobacco cells using hydrophobic interaction chromatography, size exclusion chromatography, and reverse phase HPLC. The fusion (glyco)protein produced a characteristic AGP "smear" with a molecular mass of 80 to 150 kDa when detected by Western blot analysis. Glycosyl composition and linkage analyses of purified GFP-AtAGP17 showed that carbohydrate accounted for ~86% of the molecule with arabinose and galactose as major and rhamnose and glucuronic acid as minor glycosyl residues and with 1,3,6-Gal, 1,4-Glc A, 1,3-Gal, and t-Ara as major linkages. GFP-AtAGP17 was also precipitated by {beta}-Yariv reagent further confirming that AtAGP17 is a bona fide AGP. Confocal fluorescence microscopy of plasmolysed, transformed cells indicated that AtAGP17 is localized on the plasma membrane and in Hechtian strands. Hydroxyproline glycoside profiles of GFP-AtAGP17 in conjunction with the deduced protein sequence also served to corroborate the Hyp contiguity hypothesis, which predicts contiguous Hyp residues as attachment sites for arabinosides and clustered, non-contiguous Hyp residues as attachment sites for arabinogalactan polysaccharides.

Keywords: Arabinogalactan-proteins; Arabidopsis; Glycoprotein; Hydroxyproline.
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