Structural analysis of the carbohydrate moiety of arabinogalactan-proteins from stigmas and styles of Nicotiana alata

Arabinogalactan-proteins (AGPs) from the female reproductive tissues (stigmas and styles) of Nicotiana alata were isolated from the saturated ammonium sulfate supernatant of buffer-soluble extracts by precipitation with the β-glucosyl Yariv reagent, followed by gel-filtration chromatography under dissociating conditions. The AGPs had characteristics typical of other AGPs: a high proportion of carbohydrate (95%) with a high ratio of Galp to Araf (2:1), and a low protein content (5%) with high levels of alanine, serine, and hydroxyproline. The AGPs consisted of a major species which was almost neutral, and a minor species which was more negatively charged. Sedimentation equilibrium experiments showed that the purified AGPs had a weight-average molecular weight of 143 kD. Linkage analysis showed that the AGPs contained a highly branched backbone of 3-, 6-, and 3,6-linked Galp residues, bearing terminal Galp and terminal Araf residues. Analysis by one-dimensional and two-dimensional 1H and 13C NMR spectroscopy confirmed the presence of these glycosyl linkage types, and showed a high mobility of the terminal Araf residues consistent with their location on the periphery of the molecules. This analysis represents the most complete 1H assignment for AGP molecules in solution. No difference in the carbohydrate analyses was found between AGPs isolated separately from stigmatic or stylar tissue, or between AGPs isolated from stigmas and styles of plants of different self-incompatibility genotypes.