Metabolites of 2,3-diketogulonate delay peroxidase action and induce non-enzymic H2O2 generation: Potential roles in the plant cell wall

Metabolites of 2,3-diketogulonate delay peroxidase action and induce non-enzymic H 2 O 2 generation: Potential roles in the plant cell wall Anna K€ ark€ onen a , b , * , Rebecca A. Dewhirst b , 1 , C. Logan Mackay c , Stephen C. Fry b a Department of Agricultural Sciences, Viikki Plant Science Center, University of Helsinki, Finland b The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh EH9 3BF, UK c EastCHEM School of Chemistry, The University of Edinburgh, Edinburgh EH9 3FJ, UK article info Article history: Received 30 September 2016 Received in revised form 22 February 2017 Accepted 12 March 2017 Available online 14 March 2017 Keywords: Ascorbate Dehydroascorbic acid Diketogulonate Apoplast Peroxidase Hydrogen peroxide Hydroxyl radical 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 H 2 O 2 and delay peroxidase action on aromatic substrates. As DKG itself generates several by-products, we characterised these and their ability to generate H 2 O 2 and delay peroxidase action. DKG preparations rapidly produced a by-product, compound (1), with l max 271 and 251 nm at neutral and acidic pH respectively. On HPLC, (1) co-eluted with the major H 2 O 2 -generating and peroxidasedelaying 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 z2.3) compound. Mass spectrometry suggested that un-ionised (1) has the formula C 6 H 6 O 5 , i.e. it is a reduction product of DKG (C 6 H 8 O 7 ). In conclusion, compound (1) is the major H 2 O 2 -generating, peroxidase-delaying principle formed nonenzymically from DKG in the pathway ascorbate /dehydroascorbic acid /DKG /(1). We hypothesise that (1) generates apoplastic H 2 O 2 (and consequently hydroxyl radicals) and delays cell-wall crosslinking dboth these effects favouring wall loosening, and possibly playing a role in pathogen defence. ©2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction L -Ascorbic acid (C 6 H 8 O 6 ; vitamin C) is an important redox compound in all plants and animals. In plants, it is synthesised in the protoplast, and a proportion of it is released into the apoplast (aqueous solution that permeates the cell wall) [11,37], where some of it is enzymically and non-enzymically oxidised by O 2 to form monodehydroascorbate, which rapidly disproportionates into ascorbate and dehydroL -ascorbic acid (DHA; C 6 H 6 O 6 ). Ascorbate oxidase is well established to be a wall-localised enzyme capable of modulating the ascorbate:DHA ratio [43]. 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 also 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]. DHA is unstable in neutral aqueous solutions and is easily delactonised to 2,3-diketoL -gulonic acid (DKG; C 6 H 8 O 7 ) which itself converts non-enzymically to several further degradation products depending on the incubation conditions [8,26,35,46,51]. In the apoplast of cultured rose cells, a portion of the DHA is oxidised to oxalate and L -threonate (and esters thereof), some of these reactions being proposed to generate H 2 O 2 , and a further portion of the DHA is hydrolysed to DKG [17,18]. The balance between these two pathways (oxidation:hydrolysis ratio of DHA) is dictated by the severity of the ambient oxidising conditions [38,39]. Some Abbreviations used: AAO, ascorbate oxidase; ABTS, 2,2 0 -azino-bis(3ethylbenzthiazoline-6-sulphonic acid; DHA, dehydroascorbate; DKG, 2,3-diketoL - gulonic acid; 2,3-enediol-DKGL, the 2,3-enediol form of 2,3-diketogulonod - lactone; 3,4-enediol-DKGL, the 3,4-enediol form of 2,3-diketogulonod -lactone; ROS, reactive oxygen species; XO, xylenol orange. *Corresponding author. Current address: Natural Resources Institute Finland (Luke), Green Technology, Latokartanonkaari 9, Helsinki, Finland. E-mail address: anna.karkonen@luke.fi(A. K€ ark€ onen). 1 Current address: wildFIRE Lab, Hatherly Laboratories, University of Exeter, Prince of Wales Road, Exeter EX4 4PS, UK. Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi http://dx.doi.org/10.1016/j.abb.2017.03.006 0003-9861/©2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 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. DKG is a highly unstable compound, readily forming a wide range of by-products. Some of the many reported ascorbate degradation products, probably formed via DKG, include 2,3enediol-DKGL (C 6 H 6 O 6 ; the 2,3-enediol form of 2,3-diketogulonod -lactone), 3,4-enediol-DKGL (also C 6 H 6 O 6 ; the 3,4-enediol form of 2,3-diketo-gulonod -lactone) and L -erythroascorbic acid (C 5 H 6 O 5 ), have reducing activity and might function as reducing agents in a similar way to ascorbate in vivo [26,27,35,48]. The pathway from DKG to erythroascorbate was suggested to proceed via 2,3-enediolL -lyxose, with O 2 (or DHA if present) serving as oxidant in the conversion of 2,3-enediolL -lyxose to erythroascorbate [19]. However, the physiological significance of erythroascorbate formation from DKG may be limited, as the process was only rapid in the presence of cyanide, 0.5 M phosphate, and a pH of 8. De-lactonisation of DHA to DKG is often considered to be irreversible [35]. However, some formation of DHA by re-lactonisation of DKG has been demonstrated, especially at lower pH values [31]. DHA can be reduced to ascorbate when a suitable reductant is present; 3,4-enediol-DKGL formed from DKG was considered to be this reductant [48]. Indeed, the concentration of 3,4-enediol-DKGL rose to 10% of that of DKG after 30 min incubation of DKG at neutral pH in a nitrogen atmosphere [48]. DKG has also been reported to undergo decarboxylation to L - xylosulose (¼ L -xylosone) [19,32,55], from which erythroascorbate may be formed [21]. L -Xylosulose may also give rise to several strongly acidic redox compounds e.g. 2-furoic acid and the delactonised form of 5-methyl-3,4-dihydroxytetrone, although most of these were only formed under highly unphysiological conditions such as 0.5 M H 2 SO 4 at 90  C[26]. Non-acidic dioxo products may be formed from DKG after the loss of oxalate by hydrolysis to form L -erythrulose and subsequently 3-deoxyL -threosulose (¼3-deoxyL -threosone) or after the loss of oxalate by an oxidative pathway to form L -threosulose dall of which are compounds discussed as being of relevance to the ageing of animal lens proteins [34]. However, we are not aware of any of these particular dioxo products being reported to delay peroxidase action or to reduce O 2 to H 2 O 2 . Additional products of DKG degradation, formed in the plant apoplast, include compounds ‘C’and ‘E’[17], provisionally identified [39] as C¼2-carboxyL -xylonolactone plus 2-carboxyL -lyxonolactone and E¼their de-lactonised product; Cand Eare interconvertible, but otherwise relatively stable both in vivo and in vitro. ‘DKG’preparations have biologically interesting redox properties, probably due to the formation of DKG degradation products. An eneediol group or some other functional group that is as easily oxidisable as an eneediol is thought to be a common feature of the ascorbate degradation products having reducing properties [51]. For example, ‘DKG’(120 m M) has been reported to be an antioxidant against the oxidative modification of yolk lipoprotein in a copper-containing solution, whereas it has a pro-oxidative effect at lower concentrations (75 m M; [29]). Furthermore, ‘DKG’at micromolar concentrations delays copper-induced oxidative formation of conjugated dienes in yolk lipoprotein, the lag time lengthening with increasing ‘DKG’concentrations [29]. Since DKG itself has no reducing activity, it was suggested that 3,4-enediolDKGL, the most prevalent breakdown product detected, was responsible for the anti-oxidative effect [29]. 2,3-Enediol-DKGL was also present and possibly contributed to the anti-oxidative function. Likewise, [42] hypothesised that the protective effect of DHA on copper-induced oxidative modification of human low-density lipoprotein was due to stable modification of the protein by DHA or its breakdown product(s). ‘DKG’has previously been observed to accelerate the peroxidation of linoleic acid in neutral but not in slightly acidic solutions [49]. A superoxide-scavenging agent, Tiron, suppressed linoleate peroxidation whereas catalase had no inhibitory effect, suggesting that superoxide was the reactive oxygen species (ROS) generated during incubation with DKG. Although H 2 O 2 was the ROS detected in the present study, the possibility remains that superoxide was the original ROS generated, forming H 2 O 2 by dismutation. Ascorbate induces clear non-enzymic H 2 O 2 generation when added into a solution containing a transition metal [7,12]. Also DHA, and especially a DKG preparation prepared from commercial DHA, led to H 2 O 2 generation when added into a solution containing a trace of copper ions [22]. To detect the H 2 O 2 generated after DKG addition we used two separate assays: the xylenol orange (XO) assay [3,16] and an indirect peroxidase activity assay in which odianisidine was used as a peroxidase substrate. Differences in the results obtained by these two methods led to the discovery that the DKG preparation contained a compound that inhibits peroxidase activity. Since the DKG preparation contained several breakdown products, as observed by paper electrophoresis followed by silver staining (Fig. 9 in Ref. [22], we wanted to resolve which of these was the major active component inducing non-enzymic H 2 O 2 generation and delaying peroxidase action. 2. Materials and methods 2.1. Chemicals Ascorbate oxidase (AAO), 2,2 0 -azino-bis(3-ethylbenzthiazoline6-sulphonic acid) (ABTS), catalase, dehydroascorbic acid and dehydroL -ascorbic acid dimer, o-dianisidine dihydrochloride and horseradish peroxidase type II were obtained from Sigma-Aldrich. AAO was dissolved as a stock at 1000 U ml 1 in 50 mM succinate (Na þ ) buffer, pH 5.6, supplemented with 0.05% bovine serum albumin. Peroxidase was dissolved (1 m g m l 1 ) and further diluted in the same buffer. DKG was prepared from the commercial DHA by alkali treatment [56]. A stock of DHA (50 mM) was prepared in water (it took at least 30 min to dissolve DHA). A slight molar excess of NaOH (1.3 ) was added and the mixture incubated at 20  C for 6 min. Routinely, the hydrolysis was then stopped with 1 M L -tartaric acid and the pH was checked by pH paper (~3.5e4.0). However, for samples to be fractionated by HPLC, hydrolysis was stopped with 1MH 2 SO 4 to a final pH of ~1 or ~6. Freshly-made DHA and DKG solutions were stored on ice before the assays. DKG was prepared also by an iodate method [20]. A solution of ascorbic acid (0.12 M) was incubated with potassium iodate (0.36 M) for 5 min. KOH (1 M) was then added dropwise until the solution became colourless. Cold ethanol (8 vol, 20  C) was added, and the precipitated DKG was vacuum filtered, rinsed in 70% ethanol, dried and stored at 80  C. 2.2. In-vitro peroxidase activity assays The effects of various ascorbate breakdown products on peroxidase activity were tested in vitro. DHA and DKG stock solutions were freshly prepared and added at various concentrations to a reaction mixture (total volume 1.0 ml) that contained either 550 m M ABTS or 800 m Mo-dianisidine, and 250 or 500 m MH 2 O 2 , 3.13 or 6.25 ng/ml horseradish peroxidase type II and the compound of interest, in 44 mM succinate (Na þ ) buffer, pH 5.6. [All concentrations quoted are final, in the complete reaction mixture, unless otherwise stated.] The reaction was initiated by the addition A. K€ ark€ onen et al. / Archives of Biochemistry and Biophysics 620 (2017) 12e22 13 of the enzyme and followed at 420 and 405 nm for ABTS and odianisidine respectively. When an ascorbate oxidase (AAO) pretreatment was included, the compound of interest (~0.5 mM) was pre-incubated in 4 U/ml AAO and 44 mM succinate (Na þ ) buffer, pH 5.6, for 10, 15 or 60 min at 20  C before addition of the other assay components. 2.3. Search for the active component(s) in the DKG preparation that stimulates H 2 O 2 production and inhibits peroxidases As the DKG preparation contained several compounds in addition to DKG (Fig. 9 in Ref. [22], these ‘metabolites’were separated by preparative high-voltage paper electrophoresis at pH 2.0, 3.5 and 6.5 according to [14]. Each electrophoretogram was cut into strips, and the compounds were eluted from the paper in water, concentrated in vacuo (SpeedVac, Savant) and stored at 75  C. The effect of eluted compounds on non-enzymic H 2 O 2 production was tested in vitro:10 m l of each fraction, supplemented with 1 m M CuSO 4 , was tested for H 2 O 2 formation by the xylenol orange (XO) method [3]. The XO method detects hydroperoxides that oxidise Fe 2þ in an acidic solution, and the amount of ferric product is measured as a XO complex [16]. Also the effect of each fraction on peroxidase activity in vitro was tested. To confirm the identity and stability of compounds used in the assays, we re-electrophoresed each fraction at the original pH, and stained the solutes with AgNO 3 [13]. 2.4. Search for the AAO-responsive ‘metabolite’in the DKG preparation by HPLC DKG [4.7 mM, in 45 mM succinate (Na þ ) buffer, pH 5.6] was treated with AAO (12 U/ml) or denatured AAO (10 min boiling) at 25  C for 15 min with gentle mixing, then the enzymic reaction was terminated by addition of H 2 SO 4 to pH ~1. Reaction products, Table 1 Ultraviolet absorption properties of ascorbate and some of its degradation products. Compound l max at acidic pH (nm) l max at neutral pH (nm) References Cmpd (1) 251 271 present work L -Ascorbate 245 265 [19] DehydroL -ascorbic acid a <195 [54] 223 [2,54,59] 225 w 2,3-DiketoL -gulonate <195 <225 [36,54] L -Erythroascorbate 245 265 [19] 2,3-Enediol-DKGL 210, 300 225, 345 [29,51] 3,4-Enediol-DKGL 245 265 [29,36] 2-Furoic acid 255 245 [26,60] 252 5-Methyl-3,4-dihydroxytetrone 245 265 [26] L -Erythrulose 279 279 [33] Oxalate <205 <205 Succinate b <205 <205 w¼Weak absorbance maximum. a Fig. 3aof[21] shows that fresh dehydroascorbic acid has almost no absorbance at ~300 nm, but acquires absorbance at or near that wavelength after lengthy storage of the solution. b Not a product of ascorbate catabolism, but used in the present work as a buffer. Fig. 1. Effect of dehydroascorbate and a diketogulonate preparation on the peroxidase reaction with o-dianisidine as substrate. The effect of the DKG preparation pre-treated with AAO is also shown (þAAO). DHA, dehydroascorbic acid; DKG, diketogulonate. A. K€ ark€ onen et al. / Archives of Biochemistry and Biophysics 620 (2017) 12e2214 analysed by HPLC, were compared with those in an untreated DKG aliquot. 2.5. Semi-purification of the active compound(s) in DKG preparation by HPLC HPLC was used to purify the compound(s) that inhibits peroxidases and generates H 2 O 2 when added into 1 m MCu 2þ .DKG preparations (~46 mM, pH ~1 and ~6) were filtered (0.4 m m, Chromacol), and 40 m l was fractionated on a Phenomenex Rezex ROA column, run (0.5 ml min 1 )at35  C, routinely with 47 mM H 2 SO 4 as mobile phase. In some experiments, 13 mM TFA [0.1% (v/v)] was used when a volatile mobile phase was required. Degradation products were detected by UV absorbance at various wavelengths. The major peak of cmpd (1) was collected and stored frozen prior to analysis. Mass spectrometry measurements were performed by electrospray on a 12T SolariX Fourier transform mass spectrometer (Bruker Daltonics) equipped with an infinity cell and operating in positive mode. Spectra were the sum of 20 mass analyses and collected with a data size of 4 Mword. Agilent tune mix was used for external calibration. Analysis was achieved with Data Analysis version 4.4 (Bruker Daltonics). 2.6. Analysis of redox properties of HPLC-fractionated metabolites For preparative purposes, HPLC fractions (0.5 ml) were collected and used in peroxidase or H 2 O 2 assays either immediately or after storage. In the latter case the fractions were frozen in liquid nitrogen before storage at 75  C. Since ascorbate degradation products were eluted from the HPLC column in 47 mM H 2 SO 4 (pH ~1), the assays were modified as follows. The peroxidase activity assay mixture (1.0 ml) contained (added in the following order; final concentrations are given): 25 mM Na 2 -succinate, 37 mM succinate (Na þ ) buffer (pH 5.6), 225 m l of the HPLC fraction (if <225 m l, the remaining volume was added as 47 mM H 2 SO 4 ), 550 m M ABTS, 250 m MH 2 O 2 , and 3.13 ng/ml peroxidase. When an AAO treatment was included, AAO (1 U/ml; active or boiled) was added after the HPLC fraction had been mixed with the succinate; then, after 10 min incubation at 20  C, ABTS and H 2 O 2 were added and the assay was started by addition of peroxidase. The assay mixture for non-enzymic H 2 O 2 generation contained (final volume 3.0 ml): 8.3 mM Na 2 -succinate and 225 m l of HPLC fraction (if <225 m l, the remaining volume was added as 47 mM H 2 SO 4 ) and 1 m M CuSO 4 (added last). When an AAO pretreatment was included, the enzyme (0.7 U/ml) was added after the HPLC fraction had been mixed with the succinate, and the vials were incubated for 10 min on a shaker (100 rpm) at 20  C before addition of CuSO 4 (to 1 m M). As a control for the AAO treatment, the HPLC fraction was treated for 10 min with denatured AAO (10 min boiling). This treatment was also important to show whether incubation at an increased pH was enough to alleviate the effect of the compound, i.e. whether the compound was more labile at pH~5 than at pH~1. H 2 O 2 generated was measured by the XO assay [3,16,24]. Aliquots (100 m l) of the reaction mixture were sampled at time points and immediately added to 1 ml of XO mixture (containing 125 m M XO, 100 mM D -sorbitol, 250 m M FeSO 4 , 250 m M (NH 4 ) 2 SO 4 and 25 mM H 2 SO 4 ). All sample-XO mixtures were incubated for 40 min at room temperature before measurement of A 560 against a Fig. 2. HPLC of diketogulonate and its by-products. (a) The DKG preparation was fractionated by HPLC with 47 mM H 2 SO 4 as eluent, revealing several metabolites. The eluate was monitored simultaneously for absorbance at 210, 250 and 300 nm, and fractions (labelled fr1 to fr13) were collected. A possible structure for cmpd (1), based on its mass spectrum (Supplemental Fig. 5), is shown. (b) UV spectrum of the peak containing cmpd (1), eluting at 11.01 min. (c) Ascorbate oxidase (AAO; 12 U/ml), or denatured enzyme as a control,was applied to a new preparation of DKG for 15 min, then the products were fractionated as in (a). Absorbance at 250 nm is shown. Solid arrows, nomenclature of significant peaks; open arrows with names in brackets, expected elution positions of the named compounds. A. K€ ark€ onen et al. / Archives of Biochemistry and Biophysics 620 (2017) 12e22 15 blank prepared with 100 m l solution containing 1 m M CuSO 4 and 8.3 mM Na 2 -succinate þ1 ml of XO mixture. CuSO 4 was observed not to interfere with the XO assay. A standard curve was prepared with different concentrations of H 2 O 2 in 1 m M CuSO 4 . A dilution series of H 2 O 2 was prepared in water and a portion of each solution was adjusted to 1 m M CuSO 4 immediately before addition to XO reagent. 3. Results 3.1. Diketogulonate generates a by-product that reduces O 2 to H 2 O 2 and delays peroxidase action on model substrates DKG, prepared by hydrolysis of DHA, exhibited a prominent peak of UV absorbance at pH 5.6 ( l max 271e272 nm; [22].In contrast, the DHA had negligible absorbance at this wavelength or at 265 nm (which is the l max of ascorbate, erythroascorbate and 3,4-enediol-DKGL in neutral solution; Table 1). Since freshly-made aqueous DKG is stated to have no strong absorption above 225 nm [36], the observed absorbance at 271e272 nm was probably attributable to unidentified DKG degradation products, the major one of which is here termed cmpd (1). Similar DKG solutions had been shown to reduce O 2 to H 2 O 2 non-enzymically in the presence of a trace of Cu 2þ [22]. We now give evidence that this is due to the presence of cmpd (1). In addition, we have found that in in-vitro peroxidase assays, the DKG preparation caused a concentration-dependent lag, presumably also due to cmpd (1), before the oxidation of a model substrate, odianisidine, became visible (Fig. 1). After this lag, the reaction velocity was similar to (or, in the case of high ‘DKG’concentrations, slightly slower than) that of the non-inhibited peroxidase, and the duration of the lag correlated with the amount of ‘DKG’added. These effects, which seem unlikely to be due to DKG itself, are similar to those exerted by ascorbate [50]. DHA, on the contrary, showed little inhibition of peroxidase activity. 3.2. Ascorbate oxidase partially inactivates cmpd (1) Since the UV spectrum of the DKG preparation ( l max 271 nm at pH 5.6; Fig. 8 of [22] was reminiscent of that of ascorbate ( l max 265 nm at pH 5.6), we explored the possible presence of ascorbatelike substances. Pre-treatment of the DKG preparation with ascorbate oxidase (AAO) diminished but did not abolish the lag period (Fig. 1). The A 271 of the DKG preparation decreased slowly without any enzyme addition; addition of AAO accelerated this reaction (Fig. 8 of [22], but it took several minutes before the absorbance value reached a minimum, and even then some A 271 persisted. In contrast, the A 265 of a solution of pure ascorbate reaches zero a few seconds after AAO addition (Supplemental Fig. 1), confirming that cmpd (1) is not ascorbate. In a mixture of the DKG preparation and pure ascorbate, AAO caused a rapid decrease in A 265 (due to ascorbate oxidation) followed by a slower decrease due to cmpd (1) oxidation (Supplemental Fig. 1); thus it cannot be argued that the DKG was inhibiting the AAO. Nevertheless, cmpd (1) appears to be ascorbate-related since AAO has a high specificity towards L - ascorbate and related compounds that have a lactone ring with an adjacent eneediol group such as erythroascorbate [6]. 3.3. High-voltage electrophoresis of the crude DKG preparation As the alkali-generated DKG preparation contained several byproducts [22], we attempted to separate these by electrophoresis and test them individually for peroxidase action delay (Supplemental Fig. 2) and non-enzymic H 2 O 2 generation (data not shown). After electrophoresis at pH 2.0 (Supplemental Fig. 2a), only fraction 2 (containing neutral and weakly acidic material) exerted these effects, and only to a low degree. Thus the only active principle detected had clearly separated from DKG itself (a relatively strong acid, found in fractions 3 and 4). However, the total recovered zones had far less H 2 O 2 -generating and peroxidase-delaying capacity than the crude DKG that had been applied to the electrophoretogram. After electrophoresis at pH 3.5 (Supplemental Fig. 2b) or 6.5 (Supplemental Fig. 2c), the only active principle detected was found to have co-migrated with DKG. This could indicate that (i) cmpd (1) co-migrated with DKG at these pH values, and/or (ii) the cmpd (1) originally present was degraded during the electrophoresis and subsequent elution but new cmpd (1) was formed from the eluted DKG itself. 3.4. HPLC of the DKG preparation On HPLC, freshly prepared crude DKG revealed several peaks of absorbance at 210 nm (A 210 peaks), suggesting carboxylic acids, esters or lactones, and at least three A 250 peaks (suggesting conjugated double-bonds; labelled 1,2and 3in Fig. 2). Cmpd (1)was eluted shortly after the DKG peak, only partially separated from it (retention times 11.02 and 10.64 min respectively; Fig. 2). We propose that cmpd (1) showed a pH-dependent absorbance shift, Fig. 3. Selected HPLC fractions from a diketogulonate preparation delay peroxidase action. ABTS was used as peroxidase substrate. Volume of HPLC fraction added to the assay: 150 m l. The effect of pretreatment of the fractions with AAO (1 U/assay; þAAO) or with boiled AAO (þboiled enz) at pH ~5.2e5.6 for 10 min before the peroxidase activity assay is also shown. A. K€ ark€ onen et al. / Archives of Biochemistry and Biophysics 620 (2017) 12e2216 with l max 251 (Fig. 2b) and 271 nm at acidic and neutral pH respectively (the HPLC eluent was 47 mM H 2 SO 4 ). This shift would mirror the behaviour of ascorbate and 3,4-enediol-DKGL, which have l max 245 and 265 nm in acidic and neutral solutions respectively [36,48]. Cmpd (1) was again confirmed not to be ascorbate itself, as this elutes at 13.25 min in this system. 3.5. Ability of HPLC fractions to generate H 2 O 2 and delay peroxidase action Fraction 5 (Fig. 2a), which contained the majority of cmpd (1), was the most effective fraction at delaying peroxidase action on a model substrate (ABTS; Fig. 3). Other delaying agents were also detected, e.g. in fractions 8 and 9. A moderate delaying effect was also observed in fraction 4, which contained most of the DKG (Figs. 2 and 3). However, since 24% of cmpd (1) eluted in fraction 4, and because some of the DKG may be further degraded to cmpd (1) after elution from the column, we conclude that the peroxidase delaying agent in fraction 4 was cmpd (1), not the DKG itself. Fractions 4 and 5 also caused non-enzymic H 2 O 2 production in the presence of O 2 and a trace of Cu 2þ , fraction 5 again being more effective (Supplemental Fig. 3). The other HPLC fractions tested, even those that caused a slight delay in peroxidase activity assays, did not generate H 2 O 2 . 3.6. Ascorbate oxidase diminishes the ability of HPLC fractions to delay peroxidase action and generate H 2 O 2 AAO pre-treatment of the peroxidase-retarding HPLC fractions (4, 5, 8 and 9; Fig. 2a) diminished their ability to delay peroxidase action (Fig. 3). Treatment with heat-denatured AAO (i.e., ‘ageing’ the fractions at elevated pH (5.2e5.6) in the absence of active AAO) also slightly reduced the lag caused by fractions 4, 5, 8 and 9. However, AAO did not completely destroy the peroxidase delaying effects of any of these fractions, suggesting either that several agents were present in each fraction, only some of them being AAOoxidisable, or that the AAO generated new products whose peroxidase-delaying properties were weaker than those of the initial compounds. AAO-pretreatment of fractions 4 and 5 also diminished their ability to non-enzymically generate H 2 O 2 (Supplemental Fig. 3). 3.7. UV-detectable compounds (1), (2) and (3) can be oxidised by ascorbate oxidase Since AAO affected the HPLC fractions' effects on H 2 O 2 Fig. 4. Effect of treatment at pH 1 or 6 on the HPLC profile of diketogulonate and its by-products. The DKG, prepared by NaOH treatment of DHA, was adjusted to pH 1 or pH 6 with H 2 SO 4 and, after storage for 0.5e3.0 h at 0 C, fractioned by HPLC. During each run, absorbances at (a) 210, (b) 250 and (c) 300 nm were simultaneously monitored. Solid arrows, nomenclature of significant peaks; open arrows with names in brackets, expected elution positions of the named compounds. A. K€ ark€ onen et al. / Archives of Biochemistry and Biophysics 620 (2017) 12e22 17 generation and peroxidase action, we attempted to determine which UV-detectable compounds were affected by the AAO treatment. The crude DKG preparation was treated with AAO (active or denatured) for 15 min and re-run by HPLC. The most remarkable changes were observed in compounds absorbing at 250 nm (Fig. 2c). In particular, active AAO strongly diminished the cmpd (1) peak. A slight decrease was also noticed in the A 210 of this peak (data not shown), suggesting that cmpd (1) is a carboxylic acid or ester as well as possessing conjugated double bonds. Compounds 2 and 3also diminished (Fig. 2c; the peak eluting at 15.8 min is probably the succinate buffer). 3.8. Cmpd (1) is less stable at pH 6 than at pH 1 Since treatment even with denatured AAO (at pH ~5.6) modified the HPLC profile (Fig. 2c) and the fractions' ability to delay peroxidase action (Fig. 3) and to promote H 2 O 2 production (Supplemental Fig. 3), we tested the effect of pH on the stability of DKG and its by-products. For this work, DKG was prepared by alkaline hydrolysis of DHA and the reaction was stopped with H 2 SO 4 either to pH 6 or to pH 1 (Fig. 4). DKG itself, detected at 210 nm, was almost unaffected by storage on ice for 0.5e3 h at pH 1 or 6 (Fig. 4a). When pre-treated at pH 6, instead of pH 1, compounds (1), (2) and (3) were diminished, little affected and increased respectively (Fig. 4b). Therefore, if cmpd (1) was the reductant that delays peroxidase action, then its effect should be weakened by storage at pH 6. This prediction was tested on samples stored at pH 1 or 6 before HPLC. Pre-treatment of the unfractionated preparation only slightly diminished its ability to delay peroxidase action regardless of the pH to which they were adjusted (Fig. 5a), possibly because other reductants e.g. compound (3) increased after treatment at pH 6 (Fig. 4). However, in HPLC-purified preparations (fractions 4 and 5), storage at pH 6 did weaken the effect compared with storage at pH 1, approximately halving the lag period observed before peroxidase action began (Fig. 5b) [the pH of the peroxidase reactions was adjusted to >5 with the addition of Na 2 -succinate to HPLC fractions]. These data strongly support the idea that cmpd (1), the main 250-nm-absorbing compound in fractions 4 and 5, was the major reductant that delayed peroxidase action on its model substrate. 3.9. Analysis of cmpd (1) by high-voltage paper electrophoresis HVPE has proved very useful for resolving ascorbate metabolites [17,18,38,39]. Partially purified cmpd (1) obtained by preparative HPLC with 13 mM TFA as eluent (chosen because it is readily volatile) gave stainable spots on analytical electrophoretograms (Fig. 6). The HPLC profile with TFA as eluent (Supplemental Fig. 4a) was broadly similar to that with H 2 SO 4 (Fig. 2a), DKG eluting slightly before cmpd (1). The greatest amounts of cmpd (1) were found between 10.5 and 11.25 min (Supplemental Fig. 4b and c). This 0.75-min window of fractions was pooled, dried in vacuo and re-dissolved in water; portions were electrophoresed at pH 2.0 and 6.5 and stained with AgNO 3 (Fig. 6), revealing: DKG, which is only partially resolved from cmpd (1) by the HPLC system used; compounds Cand E(a trace), which are proposed [39] to be C¼2-carboxyL -xylonolactone plus 2-carboxyL -lyxonolactone; E¼their de-lactonised product (a dianionic carboxypentonate); and a spot whose m DKG values were 1.22 at pH 2.0 and 1.05 at pH 6.5, which stained a slightly yellowish brown rather than the greyish brown usually produced by AgNO 3 . Of these possible identities, we have already shown that cmpd (1) is not DKG itself. Furthermore, we found that cmpd (1) is not C or Esince purified Cand Egave peaks clearly resolved from cmpd (1) on HPLC (Fig. 7). This was demonstrated when samples of Cand E(eluted from paper after preparative electrophoresis [17];were analysed by HPLC with 13 mM TFA as eluent (Fig. 7b), and a sample containing cmpd (1) was run immediately thereafter (Fig. 7a). Cand Eshowed major peaks of A 210 at 10.1 and 9.2 min respectively, and only small peaks of A 250 (Fig. 7b); in contrast, cmpd (1) showed greater absorbance at 250 nm and eluted at 10.5 min (Fig. 7a). Thus cmpd (1) is clearly distinguished from cmpds Cand E. Compounds Cand Eform from DKG in aqueous conditions [17,39], and the spots of them seen in Fig. 6 would have formed from the DKG after being eluted from the column. We therefore suggest that, of the spots seen in Fig. 6, one with m DKG values 1.22 and 1.05 at pH 2.0 and 6.5 respectively is likely to be cmpd (1). Although its structure remains unknown, some of its ionic properties can be deduced from the electrophoretic mobilities. At pH 6.5, all eCOOH groups are almost fully ionised, so the Fig. 5. Effect of pH on the ability of diketogulonate and/or its by-products to delay peroxidase action. The samples tested were (a) the whole DKG preparation was stored at 0 C for 0.5e3.0 h at pH ~1 or at pH ~6; and (b) HPLC fractions thereof that had been collected in 47 mM H 2 SO 4 (pH ~1) as in Fig. 2a. In each case, the samples were then tested for effect on in-vitro peroxidase action with ABTS as substrate. In (a), the lag times caused by the whole preparation were very long, so the samples were diluted for the assay (to ~23 and 46 m Mfinal). In (b), with HPLC fractions 4 and 5, undiluted 100m l portions were included in 1-ml peroxidase reaction mixtures with Na 2 -succinate addition to buffer the pH above 5. A. K€ ark€ onen et al. / Archives of Biochemistry and Biophysics 620 (2017) 12e2218 compound's proximity to DKG and Cat that pH (Fig. 6b) indicates that it has a charge:mass ratio similar to theirs, i.e. 1 negative charge per ~6 carbon atoms. Furthermore, since the compound appears to be anionic even at pH 2.0, with a mobility exceeding that of DKG (m DKG ¼1.22; Fig. 6a), it is probably a strong acid with a pK a even lower than that of DKG (predicted pK a z2.38; http://www. hmdb.ca/metabolites/HMDB05971). 3.10. Mass spectrometry of cmpd (1) A further sample of cmpd (1), partially purified by HPLC as in Supplemental Fig. 4, was analysed by MS in positive mode (Supplemental Fig. 5). Ion peaks at m/z159.03164 and 181.01351 were observed, which were absent from the blank. These ions are interpreted as C 6 H 6 O 5 $H þ and C 6 H 6 O 5 $Na þ (m/zvalues respectively 17 and 12 ppm deviation from theoretical, which is acceptable since the nearest calibration point was at m/zz332). 4. Discussion Freshly prepared DKG is reported to have little UV absorbance at wavelengths above about 225 nm [36]. However, our DKG preparations rapidly produced a proportion of compound (1), with l max 271 and 251 nm at neutral and acidic pH respectively. These l max values may be compared with those of ascorbate and some of its previously reported degradation products (Table 1). Cmpd (1)’s l max values, and its bathochromic shift when the pH is adjusted from acidic to neutral, suggest some chemical similarities to ascorbate. Furthermore, like ascorbate, DHA and 3,4-enediol-DKGL [48], cmpd (1) was more stable at acidic than neutral pH. Fig. 6. Analysis of HPLC-enriched cmpd (1) by high-voltage electrophoresis. Cmpd (1) was partially purified by HPLC with 13 mM TFA as eluent (see Supplemental Fig. 4), then analysed by electrophoresis at pH 2.0 (a) or 6.5 (b). Each sample, and the markers, contained a trace of Orange G, which was circled in pencil before the other compounds were stained in AgNO 3 . Abbreviations used: EryR, erythrarate (¼meso-tartrate); ThrR, L-threarate (¼L-tartrate); ThrO, threonate; OxT, oxalyl threonate; cOxT, cyclic oxalyl threonate; C,2carboxy-L-xylonolactone and/or 2-carboxy-L-lyxonolactone; E, de-lactonised C; DHA, dehydroascorbic acid; DKG, diketogulonate (prepared by the iodate method). Spots present in the cmpd (1) preparation are labelled to the right, markers to the left of each electrophoretogram. Spots labelled () are contaminants as they are not precisely in line with the other spots in the lane. A. K€ ark€ onen et al. / Archives of Biochemistry and Biophysics 620 (2017) 12e22 19 In agreement with the noted resemblance between cmpd (1) and ascorbate, the delaying effect of cmpd (1) on peroxidase action mimicked that of ascorbate. Ascorbate serves as an anti-oxidant, scavenging peroxidase-generated phenolic radicals, so that the oxidation of aromatic substrates becomes visible only when all ascorbate has been oxidised [50]. Thus, cmpd (1) probably affects peroxidase action in a similar manner. In the present work, several metabolites obtained from a DKG preparation induced the non-enzymic production of H 2 O 2 and delayed the onset of substrate oxidation in in-vitro peroxidase assays. A scheme for ascorbate degradation has been proposed [17,18,38,39] in which the initial oxidation product, DHA, is either further oxidised (to oxalyl threonate, cyclic oxalyl threonate and oxalate þthreonate) or hydrolysed (to DKG and its own downstream products Cand E). The oxidising branch was proposed to include, or lead to, three steps that might generate H 2 O 2 . On the other hand, the hydrolytic pathway was not proposed to yield H 2 O 2 ; therefore the discovery reported here that DKG by-products, principally cmpd (1), do generate ROS, probably mainly H 2 O 2 ,was of great interest. Cmpd (1) has two effects which superficially seem contradictory: (a) when present in peroxidase assays it appears to serve as an anti-oxidant, scavenging phenolic radicals such that the oxidation of aromatic substrates (o-dianisidine and ABTS) becomes visible only when all the cmpd (1) has been oxidised, and (b) it nonenzymically reduces O 2 to H 2 O 2 , the oxidising substrate of peroxidase. Effect (a) would delay peroxidase action, whereas effect (b) would promote it. Both these effects could have biological significance in the plant cell wall. Effect (a) would delay the peroxidasecatalysed cross-linking of cell-wall phenolics [50], e.g. of ferulate to diferulates and tyrosine to isodityrosine, thus potentially preventing wall tightening. Conversely, the H 2 O 2 generated in effect (b) can non-enzymically lead to the formation of other ROS. In particular, the hydroxyl radical (  OH) is readily formed from H 2 O 2 , especially in the presence of some remaining ascorbate [12], the precursor of cmpd (1). It is known that  OH causes non-enzymic scission of cellwall polysaccharides [12,45,52], potentially loosening the primary cell wall. Preventing wall tightening and promoting wall loosening, caused by (a) and (b) respectively, are both expected to lead to a more readily extensible or softer wall. Therefore the two apparently divergent effects of cmpd (1) may act in an equivalent direction, both of them facilitating biological processes that depend on a ‘loose’cell wall such as cell expansion, fruit softening and abscission. Additionally, similarly to ascorbate, cmpd (1) may influence the oxidative burst occurring during pathogen attack, and hence plant defence responses [41]. The only known source of cmpd (1), potentially exerting such wall-loosening effects, is DKG dwhich is formed by the nonenzymic hydrolysis of apoplastic DHA. DHA can itself be formed from apoplastic ascorbate, about 50% by endogenous AAO action Fig. 7. Cmpd (1) is not a carboxypentonate. (a) Cmpd (1), purified by HPLC eluted in 13 mM TFA, was re-run by HPLC in water and the products were detected by A 210 and A 250 . (b) A sample containing compounds Cand E(carboxypentonates, prepared by alkali treatment of DHA and subsequent elution from a preparative electrophoretogram) was also run by HPLC in water and monitored by A 210 and A 250 . A. K€ ark€ onen et al. / Archives of Biochemistry and Biophysics 620 (2017) 12e2220