Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method

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.

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.

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.

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

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.