Tomato LeAGP-1 is a plasma membrane-bound, glycosylphosphatidylinositol-anchored arabinogalactan-protein.

Arabinogalactan-proteins (AGPs) are a class of highly glycosylated, hydroxyproline-rich glycoproteins that function in plant growth and development. Tomato LeAGP-1 represents a major AGP expressed in cultured cells and plants. Based on cDNA and amino acid sequence analyses along with carbohydrate and other biochemical analyses, tomato LeAGP-1 is hypothesized to be a classical AGP localized to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Here, this hypothesis was tested and supported with the following experiments. First, tomato (Lycopersicon esculentum, cv. UC82B) cotyledon protoplasts were isolated following cell wall digestion with cellulase and pectinase, and LeAGP-1 was immunolocalized to the plasma membrane with a LeAGP-1 antibody. Second, LeAGP-1 was shown to be a major AGP component in plasma membrane vesicles from tomato cv. Bonnie Best suspension-cultured cells by Western blot analysis with the LeAGP-1 antibody. Third, fluorescence microscopy of plasmolysed, transgenic tobacco (Nicotiana tabacum BY-2) suspension-cultured cells expressing a green fluorescent protein (GFP)-LeAGP-1 fusion product demonstrated localization to the plasma membrane and Hechtian threads. Fourth, the GFP-LeAGP-1 fusion protein was present in plasma membrane preparations from these transgenic tobacco cells by Western blot analysis with a GFP antibody. Fifth, GFP-LeAGP-1 secreted into the culture media contained ethanolamine, presumably attached to the C-terminal amino acid residue, consistent with its processing and release from the plasma membrane. Thus, these data support the hypothesis that LeAGP-1 is localized to the plasma membrane via a GPI anchor and suggest possible roles for LeAGP-1 in cellular signalling and matrix remodelling.

Alpha,2-, alpha,3-, and alpha,4-dehydrophenol radical anions: formation, reactivity, and energetics leading to the heats of formation of alpha,2-, alpha,3-, and alpha,4-oxocyclohexadienylidene.

We have regiospecifically generated the alpha,2-, alpha,3-, and alpha,4-dehydrophenoxide anions by collisional activation of o-, m-, and p-nitrobenzoate. The alpha,2 and alpha,4 isomers also were synthesized by reacting o-benzyne radical anion with carbon dioxide and electron ionization of p-diazophenol. All three dehydrophenol radical anions were differentiated from each other and identified by probing their chemical reactivity with several reagents. Each isomer was converted to phenoxide and its corresponding quinone as well. Thermochemical measurements were carried out on all three radical anions and their hydrogen-atom affinities, proton affinities, and electron binding energies are reported. These measured quantities are combined in thermodynamic cycles to derive the heats of formation of each of the radical anions and their corresponding carbenes (i.e., alpha,2-, alpha,3-, and alpha,4-dehydrophenol). These results are compared to MCQDPT2, G3, G2+(MP2), and B3LYP calculations and experimental data for appropriate reference compounds.