Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method.
Airianah O. B., Vreeburg R. A. M., Fry S. C.
Author Affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
Annals of Botany 117 : 441-455
Abstract : Background and aims: Many fruits soften during ripening, which is important commercially and in rendering the fruit attractive to seed-dispersing animals. Cell-wall polysaccharide hydrolases may contribute to softening, but sometimes appear to be absent. An alternative hypothesis is that hydroxyl radicals (.OH) non-enzymically cleave wall polysaccharides. We evaluated this hypothesis by using a new fluorescent labelling procedure to 'fingerprint' .OH-attacked polysaccharides. Methods: We tagged fruit polysaccharides with 2-(isopropylamino)-acridone (pAMAC) groups to detect (a) any mid-chain glycosulose residues formed in vivo during .OH action and (b) the conventional reducing termini. The pAMAC-labelled pectins were digested with Driselase, and the products resolved by high-voltage electrophoresis and high-pressure liquid chromatography. Key Results: Strawberry, pear, mango, banana, apple, avocado, Arbutus unedo, plum and nectarine pectins all yielded several pAMAC-labelled products. GalA-pAMAC (monomeric galacturonate, labelled with pAMAC at carbon-1) was produced in all species, usually increasing during fruit softening. The six true fruits also gave pAMAC.UA-GalA disaccharides (where pAMAC.UA is an unspecified uronate, labelled at a position other than carbon-1), with yields increasing during softening. Among false fruits, apple and strawberry gave little pAMAC.UA-GalA; pear produced it transiently. Conclusions GalA-pAMAC arises from pectic reducing termini, formed by any of three proposed chain-cleaving agents (.OH, endopolygalacturonase and pectate lyase), any of which could cause its ripening-related increase. In contrast, pAMAC.UA-GalA conjugates are diagnostic of mid-chain oxidation of pectins by .OH. The evidence shows that .OH radicals do indeed attack fruit cell wall polysaccharides non-enzymically during softening in vivo. This applies much more prominently to drupes and berries (true fruits) than to false fruits (swollen receptacles). .OH radical attack on polysaccharides is thus predominantly a feature of ovary-wall tissue.