Biochemical characterization of the suberization-associated anionic peroxidase of potato

Abstract

The anionic peroxidase associated with the suberization response in potato (Solanum tuberosum L.) tubers during wound healing has been purified and partially characterized at the biochemical level. It is a 45-kD, class III (plant secretory) peroxidase that is localized to suberizing tissues and shows a preference for feruloyl (o-methoxyphenol)-substituted substrates (order of substrate preference: feruloyl > caffeoyl > p-coumaryl approximately syringyl) such as those that accumulate in tubers during wound healing. There was little influence on oxidation by side chain derivatization, although hydroxycinnamates were preferred over the corresponding hydroxycinnamyl alcohols. The substrate specificity pattern is consistent with the natural substrate incorporation into potato wound suberin. In contrast, the cationic peroxidase(s) induced in response to wound healing in potato tubers is present in both suberizing and nonsuberizing tissues and does not discriminate between hydroxycinnamates and hydroxycinnamyl alcohols. A synthetic polymer prepared using E-[8-(13)C]ferulic acid, H(2)O(2), and the purified anionic enzyme contained a significant amount of cross-linking through C-8, albeit with retention of unsaturation.

Scheme 1

Hydroxycinnamic acid and hydroxycinnamyl alcohol derivatives used as substrates in this study. The aromatic ring substitution pattern is denoted by a number (i.e. hydroxycinnamates 1−4 and hydroxycinnamyl alcohols 5−7), while derivatives are denoted by lowercase letters. Not every possible derivative was used; refer to Table II for a complete listing of the 25 phenolics used in the substrate specificity study.

Figure 1

Isoform analysis of wound-induced peroxidases of potato tubers. Total soluble proteins were extracted from either the (mechanically removed) suberized layer or the unsuberized tissue immediately below the suberized layer of 7-d wound-healed potato tubers, and chromatofocused on a Mono-P HR 5/5 column (Pharmacia). Proteins were loaded at pH 7.1, and the pH gradient (7.1–3.5 over approximately 15 min) started after all of the unbound protein had been washed through (indicated by an arrow). Fractions were assayed separately for activity using both ferulic acid and guaiacol.

Figure 2

SDS-PAGE analysis of purified anionic peroxidase from potato electrophoresed in a 14% acrylamide gel under denaturing conditions, before (lane b) and after (lane c) treatment with TFMS. Lane a, Molecular mass markers, including BSA (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), soybean trypsin inhibitor (20.1 kD), and α-lactalbumin (14.1 kD). Lane d, Activity stain using guaiacol/H₂O₂. Proteins in lanes a to c were visualized by silver staining. After staining, gels were dried onto cellulose acetate sheets (Bio-Rad) and scanned to generate digital images.

Figure 3

Dependence of phenolic oxidation rate by potato anionic peroxidase on pH. The rate of oxidation of ferulic acid 3a (black symbols) and coniferyl alcohol 6a (white symbols) was measured spectrophotometrically in buffers of differing pH. The buffers used (all at 50 mm) were citrate (•), acetate (▪, □), His (▴, ▵), phosphate (▿), and Tris (⋄). Oxidation rates were measured in triplicate. Error bars represent ±1 sd.

Figure 4

M_r characterization of the polymeric product prepared by the incubation of ferulic acid/H₂O₂ with purified anionic peroxidase. The acid-insoluble precipitate collected after incubation of anionic peroxidase with ferulic acid/H₂O₂ was dissolved in 0.1 m NaOH and eluted from a Sephadex G25-M column. BSA was used to mark the void volume of the column. The ferulic acid monomer was used to mark the total volume of the column.

Figure 5

Solution-state ¹³C-NMR spectroscopic analysis of the polymeric product prepared by the incubation of E-[8-¹³C]ferulic acid/H₂O₂ with purified anionic peroxidase. Ten milligrams of the polymeric product collected from a Sephadex G25-M column was dissolved in 0.1 m KOH prepared in deuterated water. A drop of DMSO was added as an internal standard. With the exception of the resonance at 56.79 ppm, all resonances are due to the enhanced C-8 of the initial substrate.

Scheme 2

General reaction mechanism for peroxidase. See text for detailed description of each step. R-OH, Phenolic substrate; R-O^·, phenolic radical.