Abstract
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 λmax 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 (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.
Highlights
L-Ascorbic acid (C6H8O6; vitamin C) is an important redox compound in all plants and animals. It is synthesised in the protoplast, and a proportion of it is released into the apoplast [11,37], where some of it is enzymically and non-enzymically oxidised by O2 to form monodehydroascorbate, which rapidly disproportionates into ascorbate and dehydro-L-ascorbic acid (DHA; C6H6O6)
Ascorbate oxidase (AAO), 2,20-azino-bis(3-ethylbenzthiazoline6-sulphonic acid) (ABTS), catalase, dehydroascorbic acid and dehydro-L-ascorbic acid dimer, o-dianisidine dihydrochloride and horseradish peroxidase type II were obtained from Sigma-Aldrich
The DHA had negligible absorbance at this wavelength or at 265 nm
Summary
L-Ascorbic acid (C6H8O6; vitamin C) is an important redox compound in all plants and animals. Apoplastic ascorbate and its downstream metabolites have been widely discussed as important players in protecting the plant against environmental oxidative stresses, especially ultraviolet irradiation, atmospheric ozone pollution and pathogen challenge [4,5,10,40,41,44,47,53]. Apoplastic ascorbate metabolites have been proposed to serve roles in modulating the cell wall's biophysical properties, especially influencing the softening of ripening fruits and the extensibility of the primary wall [1,9,12,15,25]. Ka€rko€nen et al / Archives of Biochemistry and Biophysics 620 (2017) 12e22 ascorbate degradation products are of interest in cell-wall physiology and in pathogen defence because of their unique redox properties
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