Arabinogalactan-proteins in roots of Echinacea purpurea: Structural investigations and microscopic localization by immunofluorescent labelling

Abstract

Arabinogalactan-proteins (AGPs) are glycoproteins which are supposed to be cell wall associated and belong to the putative active compounds of Echinacea preparations (1). (β-D-Glc)3Yariv phenylglycoside (Yariv) specifically binds to most plant-AGPs and was used to isolate AGPs from the high molecular weight fraction (>30.000Da) of an aqueous extract of root material from Echinacea purpurea (L.) Moench (Asteraceae). These AGPs with a carbohydrate-moiety consisting predominantly of Gal and Ara (2:1, >90%) have been quantitatively and structurally characterized. Methylation analysis detected as main components 1,6-Galp, 1,3-Galp, 1,3,6-Galp and in the side chains 1,5-Araf and terminal 1-Araf. Determination of the acidic compounds showed the presence of 1-GlcAp. For microscopic localization, a polyclonal antibody (generated in rabbit directed against Echinacea AGP) (2) in combination with a secondary FITC-labelled anti-rabbit antibody was used for an immunofluorescent labelling of AGPs on thin sections (16µm) of root material of Echinacea purpurea. Furthermore, a Yariv-antibody (generated in rabbit directed against albumin-coupled Yariv) was used to label a Yariv-treated thin section of the same roots. After addition of the FITC-conjugated secondary antibody, the sections were observed with a Confocal Laser Scanning Microscope (CLSM). In both cases the labelled AGP was located in the cell walls, often in contact with the cytoplasma membrane. Confirming results on tomato roots (3), especially xylem tracheary elements showed very strong immunolabelling of the secondary wall.