Arabinogalactan-proteins (AGPs) are hydroxyproline-rich glycoproteins that are expressed at plant cell surfaces. Two different approaches were employed to study AGPs, (1) perturbation studies in tobacco BY-2 cells to examine molecular nteractions of AGPs and (2) reverse genetics studies to elucidate functional roles of AGPs in Arabidopsis. In the first approach, role of a tomato AGP LeAGP-1, as a candidate linker protein between plasma membrane and cytoskeleton was examined. Fluorescent probes used to perform these studies were, green fluorescent protein-microtubule binding domain [(GFP-MBD) which labels cortical microtubules (MTs)], rhodamine-phalloidin (which labels F-actin) and GFP-LeAGP-1 (which labels LeAGP-1 on the cell surface and Hechtian strands). Treatment of BY-2 cells expressing GFP-MBD with â-Yariv reagent (which selectively binds AGPs) resulted in defects in terminal cell expansion accompanied with depolymerization/disorganization of MTs. Similarly, â-Yariv treatment of wild type BY-2 cells resulted in defects in organization of F-actin. Coversely, treating cells expressing GFP-LeAGP-1 with amiprophosmethyl (APM; promotes microtubule depolmerization) and cytochalasin-D (promotes F-actin depolymerization) resulted in relocalization of LeAGP-1 on Hechtian strands and cell surface. These studies indicate a likely role for glycosylphophatidylinositol (GPI)-anchored AGPs in cortical MT and F-actin organization and conversely roles for MTs and F-actin in organizing AGPs at cell surface.
In another approach, T-DNA insertion lines for four Arabidopsis classical AGPs, AtAGP3, AtAGP4, AtAGP7 and AtAGP9 were examined to identify their functions. TDNA homozygous mutants of AtAGP4, AtAGP7 and AtAGP9 were identified (a potential homozygous lethal mutant was identified for AtAGP3) and examined for phenotypic aberrations. Expression analyses were performed by northern blotting and reverse transcription-polymerase chain reaction (RT-PCR) studies. These studies indicate high expression for AtAGP4 and AtAGP9 in stem and young roots. Real-time quantitative-PCR studies demonstrate absence of RNA transcript in roots of atagp7, whereas, decreased transcript levels in atagp4 and atagp9(3) roots and increased transcript levels in atagp9(5) roots. Growth-based phenotypic analyses for atagp9 mutants indicate defects in lateral root development. Germination assays indicate atagp4, and atagp9 mutants show increased tolerance to osmotic and abscisic acid (ABA) stress, thereby enhancing germination whereas atagp7 shows sensitivity to ABA stress (decreasing germination). Collectively, these studies suggest roles of these classical AGPs in seed germination, stress signaling and root growth and development.