title: Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method.
abstract: OBJECTIVE
  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.
  
  
  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.
authors: 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 s.fry@ed.ac.uk.
authors: Othman B
authors: OB
authors: Airianah
authors: 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 s.fry@ed.ac.uk.
authors: Robert A M
authors: RA
authors: Vreeburg
authors: 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 s.fry@ed.ac.uk.
authors: Stephen C
authors: SC
authors: Fry
mesh_terms: D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D004586:Electrophoresis; D005456:Fluorescent Dyes / Q000378:metabolism*; D005638:Fruit / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D017665:Hydroxyl Radical / Q000378:metabolism*; D010368:Pectins / Q000737:chemistry / Q000378:metabolism*; D010944:Plants / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D005656:Fungal Proteins; D011134:Polysaccharides; D017665:Hydroxyl Radical; D010368:Pectins; D006026:Glycoside Hydrolases; C035212:driselase
keywords: Fruit; cell wall; fingerprint compounds; fluorescent labelling; hydroxyl radicals; non-enzymic scission; pectic polysaccharides; ripening