Feruloylated Arabinoxylans Are Oxidatively Cross-Linked by Extracellular Maize Peroxidase but Not by Horseradish Peroxidase  Burr Sally J. , Fry Stephen C.   Molecular Plant  Volume 2, Issue 5, Pages 883-892 (September 2009) DOI: 10.1093/mp/ssp044 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Effect of Hydrogen Peroxide or a Peroxide-Scavenger on the Cross-Linking of Soluble Extracellular Arabinoxylans in Maize Cell Cultures In Vivo. A preparation of [pentosyl-3H]arabinoxylans (0.75 kBq, 35 μl) was added to maize cell-suspension cultures (1 ml) of various ages containing no additives (○), or supplemented with 20 mM KI (•) or 7.3 μM H2O2 (▪). Incubation was then continued for 24 h, after which the cell-free culture filtrate was size-fractionated on Sepharose CL-2B. The y-axis shows the percentage of the 3H-labelled material that was eluted in the void volume. Similar experiments were performed four times, with essentially identical results. Molecular Plant 2009 2, 883-892DOI: (10.1093/mp/ssp044) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 Effect of H2O2 and Horseradish Peroxidase on Cross-Linking of Arabinoxylans in Dialysed Culture Filtrate In Vitro. Radioactive arabinoxylans in dialysed, cell-free, 8-d culture filtrate were incubated in the presence of acetate (Na+, pH 4.7, 100 mM) and the additives H2O2 and/or HRP for the durations indicated on the histograms, then size-fractionated on Sepharose CL-2B. (A) Time-course for effect of H2O2 alone on [pentosyl-3H]arabinoxylans. (B) Long-term effect of type-II HRP and/or H2O2 on [pentosyl-3H]arabinoxylans. (C) Dose–response curve for the long-term effect of HRP (in the presence of H2O2) on [pentosyl-3H]arabinoxylans. (D) Long-term effect of type-II HRP and/or H2O2 on [feruloyl-14C]arabinoxylans. Similar experiments were performed three times, with essentially identical results. Molecular Plant 2009 2, 883-892DOI: (10.1093/mp/ssp044) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 Heating Destroys the Ability of Horseradish Peroxidase to Oxidise o-Dianisidine But Not to Block the Cross-Linking of Arabinoxylans in Dialysed Culture Filtrate In Vitro. (A) HRP (type VI, 1 μg ml−1, 500 μl) was heated in a boiling water bath for 0–30 min (n  =  3), allowed to equilibrate to room temperature for 1–2 h, and then measured for initial rate in the o-dianisidine assay (mean ± SD; n  =  3). (B) Native [3H]arabinoxylans in dialysed culture filtrate (8 d after sub-culture) were treated with 7.3 mM H2O2 and 100 mM acetate (Na+, pH 4.7) plus type-VI HRP (5 μg ml−1) that had been boiled for 0–30 min. After 16 h, the products were size-fractionated on Sepharose CL-2B. Similar experiments were performed twice, with essentially identical results. Molecular Plant 2009 2, 883-892DOI: (10.1093/mp/ssp044) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Ability of Maize But Not Horseradish Peroxidase to Restore the H2O2-Dependent Cross-Linking of Arabinoxylans in Boiled, Dialysed Culture Filtrate. Native [3H]arabinoxylans (6 × 90 μl, 1.68 kBq ml−1) in dialysed 8-day-old cell-free culture filtrate were heated in a boiling water bath for 15 min, allowed to cool to room temperature, and supplemented with 100 mM acetate (Na+, pH 4.7). The solutions were incubated with various combinations of additives, summarized on the histograms: 7.3 mM H2O2, 67 μg ml−1 HRP type-II, and 20% (v/v) 8-day-old non-denatured culture filtrate (CF). After 16 h, the products were size-fractionated on Sepharose CL-2B. Similar experiments were performed three times, with essentially identical results. Molecular Plant 2009 2, 883-892DOI: (10.1093/mp/ssp044) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Heat-Labile But Not Heat-Stable Maize Peroxidase Catalyzes Cross-Linking of Arabinoxylans. Samples (90 μl) of dialysed culture filtrate containing [3H]arabinoxylans (1.68 kBq ml−1) from 8-day-old maize cultures were heated in a boiling water bath for 0–30 min (n  =  3), allowed to equilibrate to room temperature for 1–2 h and then either (A) measured for activity by the o-dianisidine assay (mean ± SD; n  =  3) or (B) incubated for 16 h with 7.3 mM H2O2 and 100 mM acetate (Na+, pH 4.7) and size-fractionated on Sepharose CL-2B. Similar experiments were performed twice, with essentially identical results. Molecular Plant 2009 2, 883-892DOI: (10.1093/mp/ssp044) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 6 Ionic Effects on the Ability of Maize Peroxidase to Cross-Link Arabinoxylans. (A) Native [3H]arabinoxylans in dialysed cell-free culture filtrate (8 d after sub-culture) were incubated for 24 h with 59 μM H2O2 and either no buffer or 100 mM formate (Na+, pH 3.7), 100 mM acetate (Na+, pH 4.7), or 100 mM phosphate (Na+, pH 7.2), then size-fractionated on Sepharose CL-2B. (B) As (A) but different concentrations of acetate (Na+, pH 4.7) were used. Similar experiments were performed twice, with essentially identical results. Molecular Plant 2009 2, 883-892DOI: (10.1093/mp/ssp044) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions