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Plant Physiol. (1999) 121: 135-146 Biochemical Characterization of the Suberization-Associated Anionic Peroxidase of Potato1
Program in Chemistry, University of Northern British Columbia, 3333 University Way, Prince George, British Columbia, Canada V2N 4Z9
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
Suberization is a tissue-specific process whereby cell walls
become impregnated with a poly(phenolic) matrix coincident with the
deposition of a poly(aliphatic) matrix between the plasmalemma and
carbohydrate cell wall (for review, see Bernards and Lewis, 1998
The macromolecular assembly process whereby monomeric hydroxycinnamic
acids (and/or their derivatives) are transported to and subsequently
incorporated (i.e. polymerized) into the carbohydrate cell wall matrix
remains undefined. By analogy to the oxidative cross-linking model
accepted for lignification, it has been hypothesized that the phenolic
component of suberized cell walls is polymerized via a
peroxidase/H2O2-mediated
process (Kolattukudy, 1980 While the suberin-associated anionic peroxidase has not been fully
characterized biochemically, it has been cloned and its molecular
biology studied. Thus, a cDNA clone of the wound-induced anionic
peroxidase of potato (Roberts et al., 1988 The specificity with which purified peroxidases oxidize different
phenolic substrates (e.g. Converso and Fernandez, 1995 General
Plant Material Potato (Solanum tuberosum cv Russet Burbank) tubers were obtained from Monashee Mountain Seed Potatoes (Lumby, British Columbia, Canada), a member of the British Columbia Seed Potato Growers Association, and propagated in the Prince George, British Columbia, Canada, area. Tubers were harvested each fall and stored at 5°C in the dark until used. Suberization was induced by slicing surface-sterilized tubers into 0.5- to 1-cm-thick cross-sectional pieces, and incubating them in sterile Magenta boxes as described previously (Bernards and Lewis, 1992 ). For purification of the anionic
peroxidase, acetone powders (Espelie and Kolattukudy, 1985) prepared
from the mechanically removed suberized layers of 7-d wound-healed tubers were used.
Isoform Analysis Total soluble protein extracts were prepared separately from 1 g each of suberized and nonsuberized (i.e. the tissue immediately underlying the suberized layer) tissues collected 7 d post wounding, for 30 min on ice in 10 mL of cold extraction buffer (50 mM potassium phosphate, pH 7.5, containing 300 mM Suc, 20 mM KCl, 10 mM DTT [added fresh at the time of extraction], 3 mM EDTA, and 0.1 mM MgCl2). After centrifugation at 13,500g, the supernatant was desalted (model P6-DG, Bio-Rad) into 25 mM Bis-Tris-iminodiacetate, pH 7.1, and chromatofocused on a Mono-P HR 5/5 column (Pharmacia) over a 7.1 to 3.5 pH range. The pH gradient was generated with buffer (Polybuffer 74, Pharmacia, pH adjusted to 3.5 with saturated iminodiacetate) at a flow rate of 0.5 mL min 1.
Fractions (0.5 mL) were assayed spectrophotometrically using both guaiacol/H2O2 (20 mM/10 mM; 470 nm) and
ferulic acid/H2O2 (0.15 mM/2 mM; 310 nm).
Purification of Anionic Peroxidase All purification steps were performed at 4°C or on ice. Column fractions were assayed for peroxidase activity spectrophotometrically using 20 mM guaiacol and 20 mM H2O2 in acetate buffer (20 mM, pH 5.0) by following the oxidation of guaiacol at 470 nm. Column eluants were monitored at 280 nm.
SDS-PAGE SDS-PAGE was carried out using 14% acrylamide gels essentially as originally described (Laemmli, 1970) and modified for the Bio-Rad Mini
Protean system according to the manufacturer's instructions but
without boiling. Gels were silver-stained using a modified protocol of
de Moreno et al. (1985). After successive fixing with 20% TCA (minimum
2 h) and MeOH:HOAc:H2O (4:1:5) (3 × 15 min), gels were rinsed with water (2 × 15 min) and treated with 0.1% AgNO3 (1 h). After rinsing (2 × 10 s),
protein bands were visualized using successive washes (3 × 100 mL) with a developer solution (3% [w/v]
Na2CO3 containing 0.0185%
[w/v] formaldehyde). Color development was stopped using 2.3 M citric acid (7.5 mL/100 mL developer solution).
Calibrated Molecular Sieving Chromatography A Bio-Prep SE 100/17 column (Bio-Rad, molecular mass range 5-100 kD) was calibrated using thyroglobulin A (670 kD void volume estimate), IgG (150 kD), BSA (67 kD), ovalbumin (43 kD), carbonic anhydrase (29 kD), myoglobin (17 kD), RNase A (13.7 kD), and vitamin B12 (1.3 kD total volume estimate). Standard solutions (5 mg mL1) of BSA, carbonic
anhydrase, and RNase A were prepared in elution buffer (20 mM Tris-HCl, pH 7.5, containing 150 mM KCl).
The remaining standards were part of a calibration kit (Bio-Rad) and
were prepared according to the manufacturer's instructions. Samples
were loaded individually (100 µL) and eluted with elution buffer at
0.25 mL min1. For purified anionic peroxidase,
100 µL of a 5 µM solution in elution buffer was used.
Fractions (0.25 mL) were collected and assayed for activity using
guaiacol/H2O2.
Chemical Deglycosylation Purified anionic peroxidase (35 µg) was deglycosylated according to the method of O'Donnell et al. (1992) and analyzed by SDS-PAGE
followed by silver staining.
Enzyme Assays Potato anionic peroxidase, potato cationic peroxidase, and horseradish type VIII (anionic) peroxidase were assayed at a final concentration of 0.5 nM. A molar extinction coefficient of 105 mM1 cm1
was used to adjust their concentrations. For ascorbate and guaiacol substrates, the method of Amako et al. (1994) was used essentially as
described, except guaiacol was substituted for pyrogallol. For phenolic
substrate specificity assays, solutions were preincubated at 40°C.
The following quasi-rapid mixing method, based on that described by
Rasmussen et al. (1995), was employed: anionic peroxidase (1 nM) and phenolic substrate (0-0.4 mM), in
assay buffer (50 mM citrate buffer, pH 4.5 or 6.5, containing 1 mM CaCl2) was placed in
one syringe (3 mL), while
H2O2 (4 mM) in
assay buffer (total 3 mL) was placed in another. Equal volumes (1 mL)
of each solution were mixed by simultaneous injection into a flow cell
(75 µL internal volume) and the initial rate of substrate
disappearance was monitored for 30 s. Triplicate reactions were
measured for each syringe filling and each substrate concentration,
with each repeated at least three times. Slopes (in absorbance units
min1) were measured for the initial, linear
phase of the reaction (usually over 5-10 s). The data were fitted to
straight lines using Wolfe-Hanes transformations, and apparent maximum
rates (Vmaxapp)
values were extrapolated from intercepts.
Synthesis of Phenolic Substrates N-(Hydroxycinnamoyl)tyramine derivatives 1b, 2b, 3b, and 4b and the 2-(phenyl)-ethylamine analog 3g were synthesized according to the method of Villegas and Brodelius (1990).
N-(Hydroxycinnamoyl)putrescine derivatives 1c and
3c were synthesized according to the method of Malmberg
(1984) and purified on a 1.5- × 30-cm polyamide column (model SC6,
Machery-Nagel, Duren, Germany, pre-equilibrated with water) eluted with
water. Hydroxycinnamyl
alcohol-4-O- -D-glucosides 5b, 6b, and 7b were synthesized via
reduction of the corresponding hydroxycinnamoyl-ethyl esters using
diisobutylaluminum hydride (Terashima et al., 1995).
Hydroxycinnamate-4-O--D-glucosides 1e, 3e, and 4e were synthesized using
the same basic procedure as for 5b, 6b, and
7b, but incorporating ester hydrolysis (10% [w/v] KOH in
MeOH for 1 h followed by acidification [HCl] and extraction into
ethylacetate) in place of the reduction with diisobutylaluminum
hydride. The identity of each product was verified by NMR spectroscopy
(1H and 13C) and comparison
with published spectral data.
E-[8-13C]Ferulic Acid Piperidine (50 µL) was added to a suspension of vanillin (97.3 mg, 0.64 mmol) and [2-13C]malonic acid (120.2 mg, 1.14 mmol, 1.8 equivalents) in freshly distilled pyridine (1 mL). The resulting yellow solution was stirred at 55°C for 17 h. The yellow pyridine solution was cooled to room temperature, poured into a 6 M solution of HCl (6 mL), and stirred vigorously for 15 min. The aqueous mixture was extracted with EtOAc (4 × 10 mL) and the organic solubles were combined, dried (MgSO4), concentrated in vacuo, and chromatographed on silica gel (EtOAc:CH2Cl2:MeOH, 5:5:1) to produce a yellow solid (94.6 mg, 76%). 1H-NMR (300 MHz, acetone-d6): 3.92 (3H,
s, Ar-OMe), 6.38 (1H, dd,
JH7-H8 = 15.9 Hz,
JC7-H8 = 160.8 Hz, H-8), 6.87 (1H, d,
JH5-H6 = 8.2 Hz, H-5), 7.14 (1H, dd,
JH2-H6 = 1.8 Hz, JH5-H6 = 8.2 Hz, H-6), 7.34 (1H, d, JH2-H6 = 1.8 Hz, H-2), 7.59 (1H, dd, JH7-H8 = 15.9 Hz, JC7-H7 = 2.7 Hz, H-7), 8.2 (1H, br
s, exchangeable with D2O, Ar-OH).
13C-NMR (75 MHz,
acetone-d6): 116.3 (C-8).
Isolation of Phenolic Substrates The 9-O--D-Glc esters
1d and 3d were isolated from young tomato leaves
after first feeding the appropriate hydroxycinnamate precursor (10 mM in water) for 2 to 3 d (Harborne and
Corner, 1961). For sinapoyl Glc 4d, 4-d-old radish seedlings
were used. In either case, a total phenolic extract was prepared (80%
aqueous MeOH) from 35 to 100 g fresh weight of plant material,
concentrated in vacuo (<40°C) until aqueous, filtered, and applied
to a 2.5- × 25-cm polyamide SC6 column (Machery-Nagel) pre-equilibrated with water. The hydroxycinnamoyl glucosides were eluted from the polyamide column with water and concentrated in vacuo
(<40°C). Free sugars were precipitated at 4°C by repeatedly adding
(four times) cold MeOH (to 80% [v/v]) to the aqueous fraction, followed by filtration and concentration in vacuo to remove the MeOH.
Crude, sugar-free samples were loaded onto a water-equilibrated 1.5- × 20-cm Sephadex LH20 column (Pharmacia), and the phenolic glucosides
eluted with a stepwise gradient of MeOH (100 mL each of 0%, 25%,
50%, 75%, and 100% [v/v] MeOH). Fractions containing hydroxycinnamoyl conjugates were selected using their distinctive UV spectra as a marker. Final purification was achieved using a semipreparative 25- × 100-mm HPLC C18 column
(NovaPak, Waters). Glucosides were eluted with an isocratic gradient
(3% [v/v] acetonitrile in water) at 9 mL
min1 and identified on the basis of their
1H-NMR spectra.
p-Coumaroylglucose 1d Isolated as an amorphous powder (5 mg). UV (MeOH, max) 330 nm.
1H-NMR (300 MHz,
MeOH-d4): 3.37-3.46 (4H,
m, Glc protons 2 , 3prime], 4, 5), 3.69 (1H,
dd, J = 12.0 Hz, 4.8 Hz, H-6B), 3.85 (1H,
dd, J = 1.0 Hz, 12.0 Hz, H-6A), 5.57 (1H,
d, JH1 -H2 = 7.6 Hz, H-1), 6.38 (1H,
d, JH7-H8 = 15.9 Hz, H-8), 6.38 (2H, d, JH5-H6 = 8.7 Hz, H-3, H-5) 7.49 (2H, d, JH2-H3 = 8.7 Hz,
H-2, H-6), 7.73 (1H, d, JH7-H8 = 16.0 Hz, H-7).
Feruloylglucose 3d Isolated as an amorphous powder (38 mg). UV (MeOH,max) 330 nm.
1H-NMR (300 MHz,
MeOH-d4): 3.31-3.43 (4H,
m, Glc protons 2, 3, 4, 5), 3.66 (1H, dd,
J = 12.1 Hz, 4.5 Hz, H-6B), 3.82 (1H, dd, J = 1.6 Hz, 12.0 Hz, H-6A), 3.86 (3H, s, Ar-OMe), 5.54 (1H, d, JH1-H2 = 7.6 Hz, H-1), 6.38 (1H,
d, JH7-H8 = 15.9 Hz, H-8), 6.79 (1H,
d, JH5-H6 = 8.2 Hz, H-5), 7.07 (1H,
dd, J H2-H6 = 1.6 Hz,
JH5-H6 = 8.2 Hz, H-6), 7.18 (1H,
d, JH2-H6 = 1.8 Hz, H-2), 7.70 (1H,
d, JH7-H8 = 16.1 Hz, H-7).
Sinapoylglucose 4d Isolated as pale yellow needles from water (28 mg). UV (MeOH,max) 330 nm. 1H-NMR (300 MHz,
MeOH-d4): 3.34-3.50 (4H, m, Glc
protons 2, 3, 4, 5), 3.70 (1H, dd, J = 8.0 Hz, 4.5 Hz, H-6B), 3.84 (1H, d, J = 1.8 Hz, H-6A), 3.88 (6H,
s, Ar-OMe), 5.59 (1H, d,
JH1-H2 = 7.9 Hz, H-1), 6.44 (1H, d,
JH7-H8 = 15.9 Hz, H-8), 6.93 (2H, s,
H-2, H-6), 7.72 (1H, d, JH7-H8 = 15.9 Hz, H-7).
Product Formation Polymeric products were prepared by the slow addition (0.8 mL h1) of
H2O2 (50 mM, 10 mL, in 10 mM phosphate buffer, pH 7) to a stirring solution
(10 mL, 10 mM phosphate buffer, pH 7) of pure potato
anionic peroxidase (0.14 mg) and either ferulic acid 3a (19.4 mg, 0.1 mmol) or
E-[8-13C]ferulic acid (19.5 mg, 0.1 mmol) in a 40°C water bath. All solutions were bubbled with
N2 gas prior to use. After 24 h, the
reaction mixture was deep red, and the product was precipitated with
the addition of a few drops of concentrated HCl, collected by
centrifugation (1250g, 10 min, room temperature), and washed
with water (two times), collecting the precipitate by centrifugation as
above. The final pellet was freeze-dried to yield a dark orange powder, reconstituted in 1 mL of 0.1 M NaOH, loaded onto
a 1.5- × 25-cm Sephadex G25-M column (Pharmacia) pre-equilibrated with
0.1 M NaOH, and eluted with 0.1 M NaOH at 1.8 mL min1.
The UV-absorbing eluant (A280) was
collected, acid precipitated with HCl, washed with water, and
freeze-dried as above to yield 10 mg (52%). BSA and ferulic acid were
used to estimate the void and total volumes, respectively, of the
column used. For NMR, equal amounts of either natural abundance or
13C-enriched reaction product were dissolved
separately in 1 mL of 0.1 M KOH in
D2O. A drop of
DMSO-d6 was added as an internal standard.
Isoform Analysis Wounding of potato tubers induced at least three groups of peroxidase isoforms, cationic, neutral, and anionic, in the suberizing tissue isolated from a 7-d-old wound site (Fig. 1). By contrast, the nonsuberized tissue underlying the suberized layer contained predominantly cationic and neutral forms, with only trace amounts of the anionic forms. All three groups of isoforms oxidized both ferulic acid 3a and guaiacol, albeit with different specific activity. For example, the cationic isoforms oxidized ferulic acid 3a approximately 1.5 times faster than guaiacol, while the anionic form oxidized ferulic acid 3a approximately 2.5 times faster than guaiacol. The neutral peroxidase oxidized both substrates equally well.
Purification of a Wound-Induced Anionic Peroxidase Potato anionic peroxidase was readily purified to apparent electrophoretic homogeneity (Fig. 2, lane b) from wound-induced tubers through a combination of size-exclusion and anion-exchange chromatography (Table I). Enzyme activity (using guaiacol as the substrate) was used as the basis for selection at each step. The final product, representing approximately 20% of the original (i.e. total) activity (measured using ferulic acid as substrate) was recovered in a total yield of 3.5 mg and had a Reinheitszahl value (ratio of heme A405 to protein A280) of 2.7. Typical values reported for purified peroxidases range from 1.6 (Kwak et al., 1995) to
4.1 (Converso and Fernandez, 1995), with most in the 2.6 to 3.3 range
(Zimmerlin et al., 1994; Padiglia et al., 1995; Rasmussen et al.,
1995). The relatively low pI of the protein (approximately 3.5) and its
small size (approximately 45 kD) facilitated purification. Remarkably,
the enzyme retained its
H2O2-dependent activity in
the SDS-PAGE gel (Fig. 2, lane d), confirming that the isolated protein
was a peroxidase.
Potato Anionic Peroxidase Characterization The purified potato anionic peroxidase has a molecular mass of 45.8 kD, based on SDS-PAGE (Fig. 2, lane b). Deglycosylation with trifluoromethane sulfonic acid (TFMS) yielded a 35.3-kD protein (Fig. 2, lane c). Calibrated molecular-sieving chromatography predicted a molecular mass of 44.9 kD for the purified protein (data not shown). The enzyme displayed a broad temperature optimum between 40°C and 60°C (data not shown) and a pH optimum of 4.5 for phenolic acids and 6.5 for monolignols (Fig. 3). In both cases, the pH optima were broad, and neutral conjugates (e.g. 3b) were equally good substrates at either pH (data not shown). For convenience, all substrates except the hydroxycinnamyl alcohols (5a, 5b, 6a, 6b, 7a, and 7b) were assayed at pH 4.5.
Substrate Specificity of Potato Peroxidases Anionic Peroxidase Twenty-five different phenolic compounds were tested as substrates (Table II; Scheme 1). The potato anionic peroxidase showed a strong preference for substrates with o-methoxyphenol-substituted aromatic ring systems. Thus the hydroxycinnamates 3a, 3b, 3c, 3d, 3f, and 3g were all excellent substrates, while (in decreasing order) the caffeoyl (2a, 2b, and 2h), p-coumaroyl (1a-1d), and sinapoyl (4a-4d) compounds were less effective. The hydroxycinnamyl alcohols (5a, 6a, and 7a) were poorer substrates than the corresponding hydroxycinnamates, but still showed the same pattern of maximal activity with the o-methoxyphenol-substituted coniferyl alcohol 6a. As expected, protection of the phenolic hydroxyl groups (i.e. the initial site of oxidation by peroxidase) with Glc moieties (e.g. 1e, 3e, 4e, 5b, 6b, and 7b) prevented their oxidation by the enzyme. The potato anionic peroxidase readily oxidized guaiacol, both in the presence and absence of p-chloromercuribenzoic acid (pCMB) (up to 200 µM), while ascorbate was a very poor substrate (data not shown).
Cationic Peroxidase A subset of the phenolics tested as substrates for the anionic peroxidase, including the hydroxycinnamates 1a, 2a, 3a, and 4a and coniferyl alcohol 6a, were also tested with the partially purified cationic peroxidase(s) of potato (Table III; Scheme 1). In contrast to the specificity apparent for the anionic peroxidase, the cationic peroxidase(s) oxidized ferulic acid 3a and coniferyl alcohol 6a equally well. A similar trend in preference for aromatic substitution patterns was observed. The (descending) order of substrate preference for the cationic isoform(s) was feruloyl > caffeoyl > syringyl > p-coumaryl.
Potato Anionic Peroxidase Reaction Products The acid-insoluble product(s) obtained from the slow addition of H2O2 to an enzyme/ferulic acid solution appeared to be polymeric (Mr > 5,000), on the basis of its elution in the void volume of a Sephadex G25-M column (Fig. 4). The natural abundance polymer had only a single weak resonance in its 13C-NMR spectrum, corresponding to the methoxyl carbon ( 56.8 ppm), owing to
the heterogeneous nature of the polymer as well as the low abundance of sample (data not shown). In the
13C-NMR spectrum obtained for the polymer
prepared from
E-[8-13C]ferulic acid
(Fig. 5), however, major resonances
wereapparent at 171.8, 124.2, 122.9, 118.0, 105.4, and 59.6 ppm,
with minor resonances observed at 136.3, 135.3, 130.4, and 55.3 ppm.
The Anionic Potato Peroxidase and Suberization Wounding of potato tubers results in a gradual increase in total soluble peroxidase activity over a period of 5 to 7 d (Borchert, 1978; Roberts et al., 1988). This involves the synthesis of new protein
and is preceded by the accumulation of mRNA (Roberts et al., 1988).
However, plants typically contain multiple peroxidase isoforms and it
is not surprising that several (i.e. cationic, neutral, and anionic)
are induced in potato under wound healing conditions (Borchert, 1978;
Borchert and Decedue, 1978; Fig. 1), with the cationic and anionic
forms predominating. Since suberization is restricted to the two to
three cell layers immediately below the wound site (Borchert, 1978;
Borchert and Decedue, 1978; Kolattukudy, 1980), and the anionic
isoform(s) is immunocytochemically (Espelie et al., 1986) and
biochemically (Fig. 1) localized to this region, it is strongly
implicated in the suberization process.
Potato Anionic Peroxidase Characterization Heme peroxidases are classified as either class I (intracellular), class II (fungal secretory), or class III (plant secretory), largely based on their structure (i.e. carbohydrate content, number of bound Ca2+ atoms, number of disulfide bridges, etc.) (Welinder, 1985; O'Donnell et al., 1992),
substrate preference (i.e. ascorbate versus guaiacol), and sensitivity
to pCMB (Amako et al., 1994). For example, class I
intracellular peroxidases, typified by ascorbate peroxidase, prefer
ascorbate as substrate and are inhibited by pCMB, while class III peroxidases (the so-called guaiacol or secretory peroxidases) prefer guaiacol as substrate and show no sensitivity to pCMB
(Amako et al., 1994). For potato anionic peroxidase, the large (10.7 kD) shift in molecular mass after treatment with TFMS, its preference for guaiacol over ascorbate as substrate, and its insensitivity to
pCMB clearly distinguish it as a class III peroxidase.
Substrate Specificity The reaction catalyzed by peroxidase is both complex (Scheme 2) and fast, and does not follow simple Michaelis-Menten kinetics (e.g. Nakajima et al., 1991). The initial
step involves the binding of
H2O2 by the Fe(III) heme,
followed by its oxidation, cleavage of the O-O bond, and the subsequent
formation of a ferryl (Fe[IV]=O)-porphorin  -cation radical
(referred to as compound I), accompanied by the release of water. Next,
the first of two reducing substrates (e.g. R-OH in Scheme 2) binds and
donates one electron to compound I, reducing the porphorin cation and
resulting in a ferryl (Fe[IV]=O) enzyme (referred to as compound II).
The reducing substrate is released as a radical (e.g.
R-O· in Scheme 2). In the last step of the
cycle, a second reducing substrate binds and donates an electron to
Fe(IV)=O, resulting in the reduction of the heme to Fe(III) and, with
the addition of two protons, the release of water. A second radical is
generated in the process. Thus, the stoichiometry of the reaction
involves 2 mol phenolic substrate oxidized for each mole of
H2O2 reduced, with
different binding affinities for the phenolics for compounds I and II.
Product Analysis Horseradish peroxidase has often been used to generate dehydrogenation polymers of coniferyl alcohol (e.g. Lewis et al., 1987; Lewis and Yamamoto, 1990). The type of product obtained depends on the
rate at which the substrates (i.e. coniferyl alcohol and H2O2) are added to the
enzyme (see Saake et al., 1996; Guan et al., 1997). In the present
study, the slow addition of
H2O2 to a stirring solution
of purified anionic peroxidase and ferulic acid 3a yielded a
polymer of Mr > 5,000, as judged by
chromatography on a Sephadex G25-M column (Fig. 4). More than half
(i.e. 52%) of the original monomer was recovered in this polymeric
fraction. Based on a monomer molecular mass of approximately 194 g
mol1, the recovered material represents a polymer with a
minimal degree of polymerization of 26. By contrast, either the rapid
addition of H2O2 or the
addition of both substrates at once to a stirring solution of purified
anionic peroxidase yielded mainly
low-Mr, acid-soluble products, not
unlike those reported by Zimmerlin et al. (1994) (data not shown).
The macromolecular assembly of the aromatic domain in suberized
tissues is hypothesized to involve a
peroxidase/H2O2-mediated free radical coupling process. One candidate peroxidase in potato tubers is the highly anionic isoform that is induced by wounding. The
biochemical evidence presented here supports this contention on two
counts. First, the anionic peroxidase is restricted to the suberizing
tissues in the immediate vicinity of the wound site. Second, the
anionic peroxidase of potato prefers
o-methoxyphenol-substituted hydroxycinnamates (typical of
those that accumulate in tubers during wound healing and incorporated
into the suberized cell wall) to other phenolic substrates (order of
substrate preference: guaiacyl > caffeoyl > p-coumaryl
2 Present address: Department of Plant Sciences, University of Western Ontario, London, ON, Canada N6A 5B7. 3 Present address: Department of Biology, University of Alberta, Edmonton, AB, Canada T6G 2E9. 4 Present address: Department of Chemistry, McGill University, Montreal, PQ, Canada H3A 1B1. * Corresponding author; e-mail bernards{at}julian.uwo.ca; fax 519-661-3935. Received January 12, 1999;
accepted May 22, 1999.
The authors gratefully acknowledge the assistance of Dr. David Dick (University of Northern British Columbia) in acquiring NMR spectra.
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Abstract
Introduction
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-[8-13C]ferulic acid,
H2O2, and the purified anionic enzyme contained a significant amount of cross-linking through C-8, albeit with retention of unsaturation.
4 and hydroxycinnamyl alcohols 5
-globulins as standards, according to
the manufacturer's instructions. The concentration of pure enzyme was
estimated using a molar extinction coefficient (
405) value of 105 mM
-lactalbumin (14.1 kD). Peroxidase activity was visualized
in gels without prior silver-staining using
guaiacol/H2O2 (50 mM each in 50 mM acetate buffer, pH 5), after
first rinsing the gels with water (2 × 15 min) to remove SDS. The
reaction was stopped by removing the substrates and rinsing the gels
with water.
), acetate (
, 
), His (
, 
), phosphate (
), and Tris
(
). Oxidation rates were measured in triplicate. Error bars
represent ±1 SD.
