Metabolites of 2,3-diketogulonate delay peroxidase action and induce non-enzymic H2O2generation: Potential roles in the plant cell wall.
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 H2O2and delay peroxidase action on aromatic substrates. As DKG itself generates several by-products, we characterised these and their ability to generate H2O2and delay peroxidase action. DKG preparations rapidly produced a by-product, compound (1), with λmax271 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 (pKa≈ 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.
Department of Agricultural Sciences, Viikki Plant Science Center, University of Helsinki, Finland; The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh EH9 3BF, UK. Electronic address: anna.karkonen@luke.fi.
Anna
A
Kärkönen
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh EH9 3BF, UK.
Rebecca A
RA
Dewhirst
EastCHEM School of Chemistry, The University of Edinburgh, Edinburgh EH9 3FJ, UK.
C Logan
CL
Mackay
The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh EH9 3BF, UK.
Stephen C
SC
Fry
D015083:2,3-Diketogulonic Acid / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry*
D015083:2,3-Diketogulonic Acid; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase
Apoplast; Ascorbate; Dehydroascorbic acid; Diketogulonate; Hydrogen peroxide; Hydroxyl radical; Peroxidase; Plant cell wall