Identifying the polysaccharides to which ferulic acid is esterified in the cell walls of purslane (Portulaca oleracea)

Abstract

Among angiosperms, the hydroxycinnamic acid ferulic acid is known to occur ester-linked to the primary cell walls of species in the 'core' Caryophyllales of eudicotyledons. Purslane (Portulaca oleracea) in the family Portulacaceae is also in the 'core' Caryophyllales. The ester-linkage of ferulic acid to polysaccharides in the primary cell walls of purslane internodes was investigated by treating cell-wall preparations with a purified 'Driselase' enzyme preparation. Fractionation of the solubilised products using solid phase extraction cartridges packed with C-18 reverse phase beads was followed by separation of the feruloylated oligosaccharides by descending paper chromatography. Several spots fluoresced in UV radiation, changing from blue to green after exposure to ammonia vapour, indicating the presence of ester-linked ferulic acid. The largest fluorescing spot which is referred to as spot A, was at Rf 0.60. Two weaker spots at Rf 0.44 and 0.24 are referred to as spots B and C, respectively, with an extremely weak spot D at an Rf of 0.13. The compounds were eluted and analyzed by HPLC ESI-IT MS. HPLC of spot A revealed the presence of two large peaks. Their mass fragmentation pattern along with their neutral monosaccharide analysis showed them to be O-(2-O-trans-feruloyl-Larabinofuranosyl)-( 1--5)-L-arabinofuranose. Spot B was identified as O-(6-O-trans-feruloyl)-β-Dgalactopyranosyl)-( 1--4)-D-galactopyranose. Another feruloylated oligosaccharide with an m/z 913 was also discovered in spot B although its structure could not be identified. The structure of the feruloylated pseudomolecular ion at m/z 985 seen in spot C was also unable to be identified. The feruloylated oligosaccharides identified from purslane cell-walls are likely to originate from arabinans or galactan side chains of rhamnogalacturonan I, as have previously been found in the Amaranthaceae. The neutral monosaccharide composition of the cell walls of 4-6-week-old purslane internodes showed the walls contained, in order of abundance: arabinose, xylose, galactose, rhamnose, glucose and fucose. Moreover, the major neutral monosaccharide in hydrolysates of spot A was arabinose. The uronic acid content of the whole cell walls was found to be 15.24%. Saponification of the whole cell walls of purslane revealed the presence of ferulic acid, predominantly in the trans- configuration, but no p-coumaric acid. This study shows that the walls of Portulaca oleraceae in the family Portulacaceae, which is phylogenetically distinct from that of Amaranthaceae, contain ferulic acid ester-linked to similar polysaccharides to those in the walls of amaranth, spinach and sugar beet. It is therefore possible that walls of species in other families within the 'core' Caryophyllales will have ferulic acid present, ester-linked in a similar manner to those in Amaranthaceae and Portulacaceae