Minisymposium 5: Cell Walls

Abs # 15002: Structure, Expression and Function of the Tomato LeAGP-1 Arabinogalactan-Protein with its Distinctive Lys-rich Subdomain: GPI Anchoring and LeAGP-1 Overexpression Studies

Presenter:	Sun, Wenxian , ws341890@ohiou.edu
Authors	Sun, Wenxian  (A) (C)  Zhao, Zhangdong  (B)   Hare, Michael C (B)   Kieliszewski, Marcia J (B) (C)  Showalter, Allan M (A) (C)
Affiliations:	(A): Department of Environmental and Plant Biology (B): Department of Chemistry and Biochemistry (C): Molecular and Cellular Biology Program, Ohio University

Arabinogalactan-proteins (AGPs) are a family of hyperglycosylated, hydroxyproline-rich proteins found at plant cell surfaces (plasma membranes-PM, cell walls-CW, extracellular secretions). In order to elucidate the structure, expression and function of AGPs, we have focused our attention on a major tomato AGP called LeAGP-1. We have generated a great deal of information on and research tools for LeAGP-1 including the following: full-length cDNA and gene sequences, a specific antibody against the Lys-rich subdomain of LeAGP-1, performed extensive northern blotting and immunolocalization experiments, purified and characterized deglycosylated LeAGP-1, and purified and characterized native LeAGP-1 as a fusion protein with green fluorescent protein (GFP). It is now clear that LeAGP-1 is a PM-bound AGP that contains a glycosylphosphatidylinositol (GPI)–membrane anchor. This anchor is cleaved to release LeAGP-1 to the CW and extracellular environment. Expression of GFP-LeAGP-1 fusion protein in tobacco cells shows LeAGP-1 occurs in Hechtian strands, adhesion sites between the PM and CW. Transgenic strategies were employed to elucidate LeAGP-1 function(s). Transgenic tomato plants expressing antisense LeAGP-1 cDNA showed no significantly different phenotypes from control plants. Meanwhile, transgenic tomato plants expressing GFP fused to LeAGP-1 [GFP-LeAGP-1] or an LeAGP-1 variant, lacking the GPI-anchor domain [GFP-LeAGP-1DC], under the control of the CaMV35S promoter were produced. Transgenic lines with high levels of both GFP-LeAGP-1 mRNA and protein: 1) were significantly shorter (some were only one-third as tall as control plants), 2) were highly branched (1.5-3 times as much branching as control plants), 3) produced more flower buds, but most flowers did not mature, resulting in less fruit production, and 4) produced seeds that were significantly smaller than normal seeds. Transgenic plants overexpressing GFP-LeAGP-1DC showed similar phenotypes. Analysis of the second generation of GFP-LeAGP-1 transgenic plants confirmed these observations. Since the phenotype of GFP-LeAGP-1 overexpressing transgenic plants is similar to that of cytokinin-overproducing plants, we examined LeAGP-1 mRNA expression patterns following hormone treatments. Cytokinins up-regulate LeAGP-1 mRNA expression, while auxins and ABA down-regulate LeAGP-1 mRNA expression. Based on these results, we speculate that GPI-anchored LeAGP-1 might be an extracellular mediator of cytokinin action.