Lawrence Berkeley National Laboratory

A proportion of the plant's l-ascorbate (vitamin C) occurs in the apoplast, where it and its metabolites may act as pro-oxidants and anti-oxidants. One ascorbate metabolite is 2,3-diketogulonate (DKG), preparations of which can non-enzymically generate H₂O₂ and delay peroxidase action on aromatic substrates. As DKG itself generates several by-products, we characterised these and their ability to generate H₂O₂ and delay peroxidase action. DKG preparations rapidly produced a by-product, compound (1), with λ_max 271 and 251 nm at neutral and acidic pH respectively. On HPLC, (1) co-eluted with the major H₂O₂-generating and peroxidase-delaying principle. Compound (1) was slowly destroyed by ascorbate oxidase, and was less stable at pH 6 than at pH 1. Electrophoresis of an HPLC-enriched preparation of (1) suggested a strongly acidic (pK_a ≈ 2.3) compound. Mass spectrometry suggested that un-ionised (1) has the formula C₆H₆O₅, i.e. it is a reduction product of DKG (C₆H₈O₇). In conclusion, compound (1) is the major H₂O₂-generating, peroxidase-delaying principle formed non-enzymically from DKG in the pathway ascorbate → dehydroascorbic acid → DKG → (1). We hypothesise that (1) generates apoplastic H₂O₂ (and consequently hydroxyl radicals) and delays cell-wall crosslinking - both these effects favouring wall loosening, and possibly playing a role in pathogen defence.