Metabolites of 2,3-diketogulonate delay peroxidase action and induce non-enzymic H 2 O 2 generation: Potential roles in the plant cell wall

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.

Determination of cellular vitamin C dynamics by HPLC-DAD

“…The apparent loss in the total VC amount can be ascribed to those converted into other irreversible DHA metabolites. 17 Indeed, we have confirmed that these metabolites can be distinguished from the DHA-ASC pair on the same chromatograph (see the ESI - Fig. S3 †), further bolstering the specificity of the method.…”

Hydrogen peroxide metabolism and functions in plants

“…Arabidopsis has a family of germin‐like proteins, which do not have oxalate oxidase activity (Membré et al ., ), but which may have SOD activity. Oxalate is a product of ascorbate degradation in the cell wall, but it is also potentially relevant that oxidation products of ascorbate (dehydroascorbate (DHA) and 2,3‐diketogulonate) can be degraded under apoplastic conditions with non‐enzymatic H 2 O 2 production (Kärkönen et al ., ). Very interestingly, a currently unidentified oxidation product of ascorbate also inhibits peroxidase activity (Kärkönen et al ., ).…”

“…Oxalate is a product of ascorbate degradation in the cell wall, but it is also potentially relevant that oxidation products of ascorbate (dehydroascorbate (DHA) and 2,3‐diketogulonate) can be degraded under apoplastic conditions with non‐enzymatic H 2 O 2 production (Kärkönen et al ., ). Very interestingly, a currently unidentified oxidation product of ascorbate also inhibits peroxidase activity (Kärkönen et al ., ). Cell wall ascorbate oxidase maintains ascorbate in a relatively oxidized state, and its activity could therefore influence H 2 O 2 production.…”

Oxalyltransferase, a plant cell‐wall acyltransferase activity, transfers oxalate groups from ascorbate metabolites to carbohydrates

“…In preliminary work, we showed that radioactivity from [ 14 C]DHA was incorporated into the cellular polymeric fraction (AIR) (Figure S1). However, DHA has diverse metabolic fates, forming cOxT, OxT and OxA, as well as diketogulonate and its downstream products (Kärkönen et al ., ). We therefore focused on the incorporation of one specific naturally occurring DHA catabolite, OxT.…”