The O-Hyp glycosylation code in tobacco and Arabidopsis and a proposed role of Hyp-glycans in secretion Jianfeng Xu a , Li Tan a , Derek T.A. Lamport b , Allan M. Showalter c , Marcia J. Kieliszewski a, * a Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, United States b Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701, United States c School of Life Sciences, John Maynard Smith Building, University of Sussex, Falmer, Brighton BN1 9QG, UK Received 11 November 2007; received in revised form 30 January 2008 Available online 25 March 2008 Abstract Most aspects of plant growth involve cell surface hydroxyproline (Hyp)-rich glycoproteins (HRGPs) whose properties depend on arabinogalactan polysaccharides and arabinosides that define the molecular surface. Potential glycosylation sites are defined by an O-Hyp glycosylation code: contiguous Hyp directs arabinosylation. Clustered non-contiguous Hyp directs arabinogalactosylation. Elu- cidation of this code involved a single species, tobacco (Nicotiana tabacum) BY-2 cells. However, recent work suggests species variation, perhaps tissue specific Hyp glycosylation. Thus, the extent to which the Hyp glycosylation code is ‘global’ needs testing. We compared the ability of distantly related Arabidopsis cell cultures to process putative HRGP glycosylation motifs encoded by synthetic genes. The genes included: repetitive Ser-Pro, Ser-Pro 2 , Ser-Pro 4 and an analog of the tomato arabinogalactan-protein, LeAGP-1DGPI. All were expressed as enhanced green fluorescent protein (EGFP) fusion glycoproteins, designated: AtSO-EGFP (O = Hyp), AtSO 2 -EGFP, AtSO 4 -EGFP and AtEGFP-LeAGP-1DGPI, respectively. The Arabidopsis glycosylation patterns were essentially similar to those observed in Nicotiana: non-contiguous Hyp residues in AtSO-EGFP were glycosylated exclusively with arabinogalactan polysaccharides while contiguous Hyp in AtSO 2 -EGFP and AtSO 4 -EGFP was exclusively arabinosylated. Mixed contiguous and non-contiguous Hyp residues in AtEGFP-LeAGP-1DGPI were also arabinosylated and arabinogalactosylated consistent with the code. However, slightly more arabinogalactosylated Hyp and less non-glycosylated Hyp in AtEGFP-LeAGP-1DGPI than tobacco NtEGFP-LeAGP-1DGPI suggested Arabidopsis prolyl hydroxylases have a slightly broader specificity. Arabidopsis Hyp-arabinogalactans differed from tobacco in decreased glucuronic acid content and lack of rhamnose. Yields of the EGFP fusion glycoproteins were dramatically higher than tar- geted EGFP lacking Hyp-glycomodules. This validates earlier suggestions that the glycosylation of proteins facilitates their secretion. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Arabidopsis thaliana; Cruciferae; O-Glycosylation; Arabinogalactan-protein; Hydroxyproline-rich glycoprotein; (b-D-Galactosyl) 3 -Yariv reagent 1. Introduction Hydroxyproline-rich glycoproteins (HRGPs) expressed at the plant cell surface are of considerable current interest as they are involved in virtually all aspects of plant growth and development, from fertilization and cytokinesis (Hall and Cannon, 2002) to apoptosis and senescence (Nothna- gel, 1997; Showalter, 2001; Motose et al., 2004; Lamport et al., 2006). In the broadest sense, HRGPs comprise numerous cell surface proteins that contain glycosylated hydroxyproline. This includes extensins, arabinogalactan- proteins (AGPs), proline-rich proteins, and numerous other protein chimeras that contain HRGP glycomodules. HRGPs are dominated by O-Hyp linked arabinosides (Lamport, 1967) and arabinogalactan polysaccharides (Lamport, 1977; Pope, 1977) which define most of the interactive molecular surface. The ‘surface codes’ are there- fore of intrinsic interest. Recent work with tobacco BY-2 cells supports the Hyp contiguity hypothesis (Fong et al., 1992; Kieliszewski et al., 1992; Kieliszewski and Lamport, 1994) where a simple 0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2008.02.006 * Corresponding author. Tel.: +1 740 593 9466; fax: +1 740 597 1772. E-mail address: kielisze@helios.phy.ohiou.edu (M.J. Kieliszewski). www.elsevier.com/locate/phytochem Available online at www.sciencedirect.com Phytochemistry 69 (2008) 1631–1640 PHYTOCHEMISTRY