Plasma membrane-bound phenolic peroxidase of maize roots: in vitro regulation of activity with NADH and ascorbate

Plant Science 165 (2003) 1429–1435

Plasma membrane-bound phenolic peroxidase of maize roots: in vitro regulation of activity with NADH and ascorbate Vesna Hadži-Taškovi´c Šukalovi´c a,∗ , Mirjana Vuleti´c a , Željko Vuˇcini´c b a

Laboratory of Plant Physiology, Maize Research Institute, Zemun Polje, P.O. Box 89, 11081 Beograd-Zemun, Yugoslavia b Center for Multidisciplinary Studies, Belgrade University, K. Višeslava 1a, 11000 Beograd, Yugoslavia Received 23 June 2003; received in revised form 8 August 2003; accepted 11 August 2003

Abstract Plasma membrane vesicles isolated and purified from 14-day-old maize roots (Zea mays L.) exhibited peroxidase activity detected as the oxidation of different phenolics with H2 O2. The enzyme activity, localized at the apoplastic side of the plasma membranes, with a maximum at pH 6.0, showed substrate preference toward phenolics in descending order: coniferyl alcohol > caffeic acid > ferulic acid > chlorogenic acid > p-coumaric acid. In vitro redox coupling of phenolics with ascorbate and NADH in a peroxidase system was established using caffeic acid as substrate. NADH oxidation in peroxidase/phenolic system with various phenolics was presented. A role for plasma membrane peroxidases in utilizing and scavenging H2 O2 is proposed. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Ascorbate; Peroxidase; Plasma membrane; Root; Zea mays

1. Introduction Peroxidases (EC 1.11.1.7.) constitute a group of N-glycosylated [1], heme and Ca2+ -containing [2] monomeric enzymes that are distributed in plant cells and catalyze the oxidation of diverse substrates by hydrogen peroxide [3]. The products of these oxidations have an important physiological role in plant growth, differentiation and development, as well as in pathogen defense responses and wound healing. In addition to this peroxidative activity, peroxidases exhibit an oxidase activity by which electrons can be transferred from reducing substrates to oxygen. Peroxidases can use a wide range of substrates as electron donors in their peroxidative activity. Since guaiacol has been widely used as an non-specific electron donor for the assay, peroxidases are frequently referred to as guaiacol peroxidases. Many isozymes of guaiacol peroxidase were demonstrated to be present in plant tissues, localized in the intra- and extracellular compartments. On the other hand, it was shown that different naturally occurring phenolic comAbbreviations: AA, ascorbic acid; HRP, horseradish peroxidase Corresponding author. Tel.: +381-11-3756704; fax: +381-11-3754994. E-mail address: [email protected] (V.H.-T. Šukalovi´c). ∗

pounds, such as flavonols, hydroxycinnamic acid esters and anthocyanin glycosides [4–7], could also be substrates for vacuolar peroxidases. Furthermore, cell wall-associated and soluble apoplastic peroxidases use hydroxycinnamic acid and hydroxycinnamic alcohol derivatives as substrates for the process of lignification of the cell walls [8–10] and suberization during wound healing [11]. Phenolics, like ferulic acid, were also reported to be the substrates for the peroxidase responsible for cross-linking of cell wall constituents [12–15]. It has been known for some time that ascorbic acid (AA) may be involved in the regulation of peroxidase-dependent oxidation of phenolics as secondary electron donor [16,17]. The peroxidase/phenolics/AA system, with different inhibitory effects of AA on the oxidation of phenolics by cell wall-bound and soluble phenolic peroxidases, has been observed in the apoplast [9,17,18], and in vacuoles [19]. The physiological role for the soluble apoplastic and vacuolar peroxidase/phenolics/AA system was proposed to be the scavenging of hydrogen peroxide [7]. Similar effects were supposed with NADH instead of AA in peroxidase/phenolic system with mitochondria isolated from maize roots [20]. Peroxidase activities associated with the plant plasma membrane have been mainly described as NAD(P)H oxidase activity, mediating the reduction of O2 to superoxide anion

0168-9452/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2003.08.006

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and H2 O2 by NAD(P)H when stimulated with MnCl2 and phenolics [21,22]. We have previously shown that in addition to the oxidase activity, plasma membranes from maize roots exibited guaiacol peroxidase activity, tightly bound to the apoplastic side of the plasma membrane [23]. Recently, there has been another report on the peroxidase activity of plasma membranes from maize roots [24]. This lack of detailed studies of possibly physiologically important peroxidative activity associated with the plasma membranes prompted us to investigate such activities catalyzing the oxidation of different phenolics as possible natural substrates by hydrogen peroxide, and the involvement of NADH and ascorbate in the regulation of this activity.

buffer without glycerol and centrifuged at 100,000 × g for 30 min. The resulting pellet, containing purified plasma membranes, was resuspended in the same washing buffer to give a final concentration of 3–4 mg of protein per ml and stored at −60 ◦ C. The purity and characterization of such membranes has been described previously [26]. 2.3. Determination of protein content The protein content was measured in the presence of 0.005% Triton X-100 by the method of Lowry et al. [27] with bovine serum albumin as a standard. 2.4. Determination of peroxidase activities

2. Material and methods 2.1. Plant material and growth conditions Maize (Zea mays L.) inbred line VA35 was used for the experiments. The seeds, germinated on water, were transferred after 3 days to plastic pots containing Knopp solution, modified in the nitrogen content. Nitrogen was supplied as KNO3 , Ca(NO3 )2 and (NH4 )2 SO4 , the concentrations of NO3 − and NH4 + in full strength solution being 10.9 and 7.2 mM, respectively. Plants were grown on 1/4 strength nutrient solution during the first 7 days, and on full strength solution during the following 4 days. The initial pH of the solution was adjusted to 5.6. Plants were kept in a growth chamber under a 12 h light/dark regime at 22/18 ◦ C, with light intensity of 190 ␮mol m−2 s−1 photosynthetically active radiation, and a relative humidity of 70%. 2.2. Isolation and purification of plasma membrane Cut roots were ground in cold grinding buffer (250 mM sucrose; 3 mM EDTA; 50 mM Tris–HCl, pH 7.5; 1 mM dithiotreitol; and 10% (w/v) glycerol) with a chilled mortar and pestle. The homogenate was filtered and centrifuged at 12,000 × g for 10 min. The supernatant was centrifuged at 100,000 × g for 30 min. The microsomal fraction in the pellet was washed with a washing buffer (2 mM Tris–HCl, pH 7.5; 250 mM sucrose; 10% (w/v) glycerol) by resuspending and pelleting by centrifugation at 100,000 × g for 30 min. The washed microsomes were suspended in phase buffer (5 mM K-phosphate buffer, pH 7.8; 330 mM sucrose; 3 mM KCl) for plasma membrane purification by partitioning in a two-phase system [25]. For phase partitioning microsomes (3 g) were added to make 12 g two-phase system that contained 6.5% (w/w) dextran T 500, 6.5% (w/v) polyethylene glycol 3,350,330 mM sucrose, 3 mM KCl, 5 mM K2 HPO4 , pH 7.8. The tubes were mixed by inversion and centrifuged in a swinging-bucket rotor for 5 min at 1,500 × g. The upper phase was collected and repartitioned on a fresh lower phase. This procedure was repeated three times and the final upper phase was diluted with 10 volumes of washing

Spectrophotometric measurements of peroxidase-dependent oxidation of phenolics were performed with singlebeam diode array spectrophotometer (Hewlett Packard 8451A). The assay mixture contained 0.1 mM of phenolic and about 15 ␮g of plasma membrane protein in 50 mM phosphate buffer (pH 6.0). The experiments were performed at 30 ◦ C. Spectra were recorded over the wavelength range of 190–500 nm, before and every 90 s after addition of 2.5 mM H2 O2 . Absorbance peaks were determined, and the extinction coefficient for each phenolic was estimated at an absorbance maximum wavelength in the same assay conditions. Phenolic peroxidase activities were determined by measuring the initial rate of the decrease of absorbance at corresponding maximum in 1 ml assay mixture containing 0.1 mM phenolic (except 0.05 mM of p-coumaric acid), 2.5 mM H2 O2 in 50 mM phosphate buffer pH 6.0. In some experiments, oxidation of caffeic acid was measured as an increase of the absorbance at 420 nm. The rate of phenolic oxidation without H2 O2 was measured as a control. NADH or ascorbate oxidation by the peroxidase/phenolic system was measured at 340 and 264 nm, respectively, in 1 ml assay mixtures containing NADH (or ascorbate), phenolic substrate and H2 O2 , in concentrations indicated in the legends to figures.

3. Results 3.1. Substrate specificity of plasma membrane peroxidases Five different natural phenolic compounds were tested as peroxidase substrates: coniferyl alcohol, caffeic, chlorogenic, p-coumaric and ferulic acid. Each of the tested phenolics showed absorption spectra changes in the presence of plasma membrane vesicles only upon H2 O2 addition to the assay mixture (Fig. 1). Oxidation of all investigated phenolics in the peroxidase reaction resulted in an absorbance decrease in UV range. Additionally, during oxidation of chlorogenic and caffeic acid, formation of brown components with absorption maxima at about 420 nm was

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Fig. 1. H2 O2 induced changes in absorption spectra of: coniferyl alcohol (A), caffeic acid (B), chlorogenic acid (C), p-coumaric acid (D) and ferulic acid (E) by plasma membrane vesicles isolated from maize roots and spectra without phenols added (F). Trace I in each panel shows the spectrum prior to the addition of H2 O2 . Numbers show peak wavelengths. Upward and downward arrows indicate increases and decreases in absorbance, respectively.

obtained. The extinction coefficients for each phenolic were determined at corresponding absorbance maxima in the UV range (Table 1). Specific activities of maize root plasma membrane-bound peroxidase with each of the tested phenolics, determined at same wavelengths, are presented in Table 1. The results showed that plasma membrane peroxidase more readily oxidized coniferyl alcohol than other investigated substrates and demonstrated descending order of substrate preference: coniferyl alcohol > caffeic acid > ferulic acid > chlorogenic acid > p-coumaric acid. However, lower p-coumaric acid peroxidase activity could be due to decreased concentration of p-coumaric acid in assay mixture in order to keep the initial absorbance at a measurable level. All of the assays were performed at pH 6.0, because that was determined to be the pH optimum for peroxidase activity with all of the investigated phenolics (data not presented). In order to investigate the localization of plasma membrane peroxidase activity, 0.025% Triton X-100 was added to the

assay mixture and absorbance changes at 420 nm during caffeic acid oxidation with H2 O2 were measured (data not presented). An increase in activity of about 15% was calculated. Subsequent experiments were performed without Triton X-100 in order to avoid its high absorbance in the UV range. 3.2. NADH oxidation in peroxidase/phenolic/NADH system We investigated NADH oxidation in the phenolic/ peroxidase system with different substrates: coniferyl alcohol, caffeic, fumaric and p-coumaric acid. The measurements were possible since these phenolics exhibited none or only insignificant absorbance changes at 340 nm during their oxidation with H2 O2 (Fig. 1). It was shown that plasma membrane preparations exhibited NADH oxidation in peroxidase/phenolics system, except in the presence of coniferyl alcohol (Table 2). In all other cases increased

Table 1 Substrate specificity of peroxidase activity of maize root plasma membrane preparations Substrate

Concentration (mM)

Wavelength assayed (nm)

Extinction coefficient (M−1 cm−1 )

Coniferyl alcohol Caffeic acid Chlorogenic acid Ferulic acid p-Coumaric acid

0.1 0.1 0.1 0.1 0.05

262 290 324 286 286

13400 15000 20200 16800 19700

The results are mean ± S.E. of at least three assays from one isolation.

± ± ± ± ±

683 559 1042 1402 1072

Specific activity (␮mol mg−1 protein min−1 ) 1.730 0.702 0.578 0.641 0.312

± ± ± ± ±

0.13 0.03 0.02 0.07 0.01

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Table 2 Rate of NADH oxidation by plasma membrane preparations in the presence of H2 O2 and various phenolic compounds Phenolic compound

Concentration (mM)

NADH (␮mol mg−1 protein min−1 )

None Coniferyl alcohol Coniferyl alcohol Coniferyl alcohol Caffeic acid Caffeic acid Caffeic acid Ferulic acid Ferulic acid p-Coumaric acid p-Coumaric acid p-Coumaric acid p-Coumaric acid

– 0.1 0.5 1.0 0.05 0.1 0.2 0.1 0.2 0.1 0.2 0.5 1.0

0 0 0 0 0.54 1.17 2.01 0.39 0.50 0.50 0.78 1.03 1.36

± ± ± ± ± ± ± ± ±

NADH and ascorbate oxidation were nearly linear until almost all of NADH or AA had been consumed and were independent of the concentration of NADH or AA in the presence of 0.1 mM caffeic acid (Fig. 2). The duration of NADH and AA oxidation increased with the increase of their concentrations (Fig. 2), and decreased with the increasing amount of caffeic acid in assay mixture (data not presented). In Fig. 3 parallel time courses of caffeic acid oxidation (Fig. 3A) and NADH (Fig. 3B, left) or AA (Fig. 3B, right) oxidations are presented. Oxidation of caffeic acid was measured in the absence and in the presence of NADH or AA. In the absence of NADH or AA absorbance at 420 nm increased rapidly due to caffeic acid oxidation immediately upon H2 O2 addition. However, in the presence of NADH or AA oxidation of caffeic acid was delayed and started after NADH or AA oxidation had been completed. Rates of NADH and AA oxidation were similar, about 1.1 ␮mol mg−1 protein min−1 . Also, the rate of caffeic acid oxidation, expressed as A420nm min−1 mg−1 protein, in the absence of NADH or AA (0.060 ± 0.001) was similar to caffeic acid oxidation rate in peroxidase/caffeic acid/NADH (0.055 ± 0.001) or peroxidase/caffeic acid/AA (0.054 ± 0.001) systems after NADH or ascorbate oxidation was completed. These results indicate that NADH is as effective an inhibitor of the oxidation of caffeic acid as AA. Similar time courses of oxidations were obtained when chlorogenic acid was used as phenolic compound (data not presented).

0.03 0.07 0.49 0 0.02 0.03 0.04 0.08 0.19

Assay mixtures contained 0.1 mM NADH, 5 mM H2 O2 and 15 ␮g plasma membrane protein. The results are mean ± S.E. of at least three assays.

NADH oxidation rate was demonstrated with increasing concentrations of phenolics. The most pronounced rate of NADH oxidation was observed in the presence of caffeic acid. 3.3. Interaction between caffeic acid and NADH or ascorbate In order to compare the reaction mechanism of NADH oxidation with that of ascorbate oxidation in plasma membrane phenolic/peroxidase system, we used caffeic acid as substrate. Caffeic acid proved itself to be the most suitable substrate due to its oxidation product absorbing at 420 nm and insignificant absorbance changes at 340 and 264 nm during the peroxidase reaction (Fig. 1B). Oxidation of NADH or AA by plasma membrane, induced by H2 O2 , was possible only in the presence of a phenolic substrate. Initial rates of

4. Discussion The phenolic substrates used in our experiments are widely distributed in plant cells as precursors for lignin biosynthesis and cross linking of structural wall polymers. Also, quinones, as their oxidation products, are responsible for the production of reactive oxygen species during

(A)

5

2.2

1.0

4

Absorbance

4 1.8 0.8

(B)

5

3

3 2

2

1.4

0.6

1

1 1.0

0.4 0

100

200

300

400

500

0

100

200

300

400

Time (s) Fig. 2. Effects of different NADH and AA concentrations on plasma membrane caffeic acid peroxidase-dependent oxidation of NADH (measured at 340 nm) or AA (measured at 264 nm). Reaction mixtures contained 2.5 mM H2 O2 , 0.1 mM caffeic acid and 15 ␮g of plasma membrane protein in 1 ml of 50 mM phosphate buffer (pH 6.0) and different concentrations of NADH (A) or AA (B) in ␮M: 25 (trace 1); 50 (trace 2); 75 (trace 3); 100 (trace 4); 125 (trace 5).

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0.25 0.20

A

A 0.20

0.15

-AA

A420

A420

-NADH +NADH

0.15

0.10

+AA

0.10

0.05

0.05 0

100

200

300

400

0

50

100

150

200

250

300

50

100

150

200

250

300

1.2

B

2.4

B

2.2

A264

A340

1.0

2.0

0.8 1.8

1.6

0.6 0

100

200

300

400

0

Time (s) Fig. 3. Time courses of the oxidation of caffeic acid, NADH and AA by plasma membrane vesicles. Reaction mixtures contained 0.2 mM caffeic acid, ±0.1 mM NADH or AA, 5 mM H2 O2 , 15 ␮g of plasma membrane protein in 1 ml of 50 mM phosphate buffer, at pH 6.0. (A) Oxidation of caffeic acid was detected by measuring A420 in the absence and in the presence of NADH or AA. (B) Oxidation of NADH and AA were detected by measuring A340 and A264 , respectively.

pathogen attack. Our results demonstrated that isolated maize root plasma membrane vesicles were able to oxidize different phenolics with H2 O2 . Since the oxidation of phenolics in the absence of H2 O2 was not observed, we can conclude that this reaction is catalyzed by a peroxidase. Coniferyl alcohol, known as the precursor for lignin synthesis, was shown in our experiments to be the preferentially oxidized substrate by plasma membrane peroxidase activity. The effectiveness with which plasma membrane-bound peroxidase oxidizes different phenolic substrates may provide clues to their in vivo role(s). Plasma membranes isolated by the two-phase partitioning consist mainly of right-side out vesicles as demonstrated previously [23,25]. Low stimulation of enzyme activity by Triton X-100 (15%) indicates that the peroxidase activity is localised on the apoplastic side of the plasma membrane. This is in agreement with our previous results, which demonstrated a tightly bound guaiacol peroxidase activity on the apoplastic side of the plasma membrane [23], resistant to osmotic shock and salt washing. Moreover, the acidic environment in apoplastic cell compartment is consistent with phenolic peroxidase activity having an optimum at pH 6.0.

The oxidation of NADH and AA by a plasma membrane peroxidase/phenolic system was shown in our experiments. Since AA and NADH were oxidized in vitro by plasma membrane bound peroxidase in the presence of exogenous H2 O2 only upon addition of a phenolic, the ascorbate peroxidase and NADH peroxidase reactions were excluded. The results obtained in the presence of caffeic acid demonstrated the oxidation of caffeic acid, measured as an increase of its oxidation product following complete exhaustion of NADH or AA. This indicates that AA or NADH reacts with phenoxy radical products of the reaction catalyzed by peroxidase and not with enzyme itself. Previous findings that ascorbate reacts rapidly with an oxidized intermediate generated from phenolics [7,17], and that oxidation of NADH in mitochondria is carried on in similar manner [20] are in agreement with these results. Takahama and Oniki [7] demonstrated that there was no cross-linking of phenoxy radicals during AA oxidation in peroxidase/phenolic/ascorbate system, suggesting roles for AA as a second electron donor, and the whole system in H2 O2 scavenging. We propose a similar reaction mechanism for NADH oxidation, and therefore both systems, peroxidase/phenolic/AA or NADH, could be considered as H2 O2 scavengers. Beside caffeic acid, NADH

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oxidation in the peroxidase/phenolic system was tested with other phenolics and demonstrated with p-coumaric and ferulic acid, but not with coniferyl alcohol. We can assume that oxidation of coniferyl alcohol by plasma membrane-bound coniferyl alcohol peroxidase can avoid NADH inhibitory effect similar to the cell wall-bound enzyme, being only partially inhibited by AA [14,18]. Unfortunately, the measurement of AA oxidation in the same system and comparison of AA and NADH oxidation rates in the presence of different phenolics was not possible using our method due to their absorption in the UV range, close to that of AA. These results argue in favor of the involvement of NADH and AA in the control of the phenolic peroxidase reaction. Peroxidase activity is under strict biological control, which comprises hormonally controlled synthesis and secretion of enzymes, the supply of substrates and the level of peroxidase-modulating agents. Ascorbate is a common constituent of the apoplast [9,17,18,28]. Its role as a secondary electron donor in phenolic peroxidase reactions was described in the case of apoplastic coniferyl alcohol [9,18], ferulic acid [14], and chlorogenic acid [29] peroxidase, and during oxidation of rutin and chlorogenic acid with horseradish peroxidase (HRP) [7]. Ascorbic acid acts as a strong inhibitor of guaiacol peroxidase in vitro, and its role in stimulation of root elongation by modulating in vivo peroxidase activity involved in the cross-linking, was also demonstrated [14]. The presence of the other possible modulating agent, NADH, in the apoplast has not been unequivocally demonstrated, although the presence of NAD+ in cell wall free space of cucumber hypocotyl [30], and reduction of NAD+ with cell wall malate dehydrogenase [31], were reported. Oxidation of NADH in the presence of phenolics and H2 O2 -mediating peroxidase has been shown with HRP [32], and with peroxidase ionically bound to the cell wall of lupin hypocotyls [33]. In addition, activity of apoplastic peroxidase, using NADH in its oxidation cycle, implies the presence of NADH in the extracellular compartment [9,33]. To conclude, we have shown that plasma membrane-bound peroxidase can reduce H2 O2 using phenolics as primary electron donors. Also, both NADH and AA can reduce phenoxy radicals and can be considered to act as secondary electron donors in the peroxidase/phenolic/NADH or AA system. Plasma membrane phenolic peroxidase in addition to apoplastic soluble and cell wall-bound peroxidase could be involved in some biosynthetic processes or to work as H2 O2 scavengers in extracellular space. Further experiments designed to examine the microenvironment in root tissue are required in order to understand the role of ascorbate and NADH as peroxidase modulating agents in vivo.

Acknowledgements This work was supported by the grants 1934 and BTR.5.02.0507.b of the Ministry for Science, Technologies and Development of R. Serbia.

References [1] C. Hu, R.B. van Huystee, Role of carbohydrate moieties in peanut peroxidases, Biochem. J. 263 (1989) 129–135. [2] M.J. Rodriguez-Mara´nón, R.B. van Huystee, Plant peroxidases: interaction between their prostetic groups, Phytochemistry 37 (1994) 1217–1225. [3] K.G. Welinder, Superfamily of plant, fungal and bacterial peroxidases, Curr. Opin. Struct. Biol. 2 (1992) 388–393. [4] P. Schloß, C. Walter, M. Mäder, Basic peroxidases in isolated vacuoles of Nicotiana tabacum L., Planta 170 (1987) 225–229. [5] A. Ros Barceló, M.A. Ferrer, E. Grac´ıa-Florenciano, R. Mu´noz, The tonoplast localization of two basic isoperoxidases of high pI in Lupinus, Bot. Acta 104 (1991) 272–278. [6] U. Takahama, T. Egashira, Peroxidase in vacuoles of Vicia faba leaves, Phytochemistry 30 (1991) 73–77. [7] U. Takahama, T. Oniki, A peroxidase/phenolics/ascorbate system can scavenge hydrogen peroxide in plant cells, Physiol. Plant. 101 (1997) 845–852. [8] U. Takahama, Redox state of ascorbic acid in the apoplast of stems of Kalanchoë daigremontiana, Physiol. Plant. 89 (1993) 791– 798. [9] T. Otter, A. Polle, The influence of apoplastic ascorbate on the activities of cell wall-associated peroxidase and NADH oxidase in needles of Norway spruce (Picea abies L.), Plant Cell Physiol. 35 (1994) 1231–1238. [10] A. Kärkönen, S. Koutaniemi, M. Mustonen, K. Syrjänen, G. Brunow, I. Kilpeläinen, T.H. Teeri, L.K. Simola, Lignification related enzymes in Picea abies suspension cultures, Physiol. Plant. 114 (2002) 343– 353. [11] M.A. Bernards, W.D. Fleming, D.B. Llewellyn, R. Priefer, X. Yang, A. Sabatino, G.L. Plourde, Biochemical characterization of the suberization-associated anionic peroxidase of potato, Plant. Physiol. 121 (1999) 135–145. [12] S.C. Fry, Feruloylated pectins from the primary cell wall: their structure and possible functions, Planta 157 (1983) 111–123. [13] K.S. Tan, T. Hoson, Y. Masuda, S. Kamisaka, Involvement of cell wall-bound diferulic acid in light-induced decrease in growth rate and cell wall extensibility of Oryza coleoptiles, Plant Cell Physiol. 33 (1992) 103–108. [14] M.C. Córdoba-Pedregosa, J.A. González-Reyes, M.S. Ca´nadillas, P. Navas, F. Córdoba, Role of apoplastic and cell-wall peroxidases on the stimulation of root elongation by ascorbate, Plant Physiol. 112 (1996) 1119–1125. [15] M. Sánchez, U.J. Pe´na, G. Revilla, I. Zarra, Changes in dehydrodiferulic acids and peroxidase activity against ferulic acid associated with cell walls during growth of Pinus Pinaster hypocotyl, Plant Physiol. 111 (1996) 941–946. [16] G.X. Chen, K. Asada, Hydroxyurea and p-aminophenol are suicide inhibitors of ascorbate peroxidase, J. Biol. Chem. 265 (1990) 2775– 2781. [17] U. Takahama, T. Oniki, Regulation of peroxidase-dependent oxidation of phenolics in the apoplast of spinach leaves by ascorbate, Plant Cell Physiol. 33 (1992) 379–387. [18] U. Takahama, Regulation of peroxidase-dependent oxidation of phenolics by ascorbic acid—different effects of ascorbic acid on the oxidation of coniferyl alcohol by the apoplastic soluble and cell wall-bound peroxidases from Vigna angularis, Plant Cell Physiol. 34 (1993) 809–817. [19] U. Takahama, Hydrogen peroxide scavenging systems in vacuoles of mesophyll cell of Vicia faba L., Phytochemistry 31 (1992) 1127– 1133. [20] V. Hadži-Taškovi´c Šukalovi´c, M. Vuleti´c, The characterization of peroxidases in mitochondria of maize roots, Plant Sci. 164 (2003) 999–1007.

V.H.-T. Šukalovi´c et al. / Plant Science 165 (2003) 1429–1435 [21] I.A. Møller, A. Bérczi, Oxygen consumption by purified plasmalemma vesicles from wheat roots, FEBS Lett. 193 (1985) 180– 184. [22] A. Viannelo, F. Macri, NAD(P)H oxidation elicts anion superoxide formation in radish plasmalemma vesicles, Biochim. Biophys. Acta 980 (1989) 202–208. [23] V. Hadži-Taškovi´c Šukalovi´c, M. Vuleti´c, Properties of peroxidase activities in plasma membrane and cell wall form maize roots, Jugoslav. Physiol. Pharmacol. Acta 34 (1998) 103–109. [24] A. Mika, S. Lüthje, Properties of guaiacol peroxidase activities isolated from corn root plasma membranes, Plant Physiol. 132 (2003) 1489–1498. [25] C. Larsson, Plasma membrane, in: H.F. Linskens, J.F. Jackson (Eds.), Cell Components, Modern Methods of Plant Analysis, New Series, vol. 1, Springer, Heidelberg, Berlin, New York, Tokyo, 1985, pp. 85–104. [26] V. Hadži-Taškovi´c Šukalovi´c, M. Vuleti´c, D. Ignjatovi´c-Mici´c, Ž. Vuˇcini´c, Plasma membrane-bound malate dehydrogenase activity in maize roots, Protoplasma 207 (1999) 203–212. [27] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275.

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[28] M.W.F. Luwe, U. Takahama, U. Heber, Role of ascorbate in detoxifying ozone in the apoplast of spinach (Spinacia oleracea L.) leaves, Plant Physiol. 101 (1993) 969–976. [29] U. Takahama, M. Hirotsu, T. Oniki, Age-dependent changes in levels of ascorbic acid and chlorogenic acid, and activities of peroxidase and superoxide dismutase in the apoplast of tobaco leaves: mechanism of the oxidation of chlorogenic acid in the apoplast, Plant Cell Physiol. 40 (1999) 716–724. [30] J.R. Shinkle, S.J. Swoap, P. Simon, R.L. Jones, Cell wall free space of Cucumis hypocotyls contains NAD and a blue light-regulated peroxidase activity, Plant Physiol. 98 (1992) 1336–1341. [31] G.G. Gross, C. Jance, E.F. Elstner, Involvement of malate, monophenols, and the superoxide radical in hydrogen peroxide formation by isolated cell walls from horseradish (Armoracia lapathifolia Gilib.), Planta 136 (1997) 271–276. [32] B. Halliwell, Lignin synthesis: the generation of hydrogen peroxide and superoxide by horseradich peroxidase and its stimulation by manganese(II) and phenols, Planta 140 (1978) 81–88. [33] M.A. Pedre´no, F. Sabater, R. Mu´noz, F. Garc´ıa-Carmona, Effect of different phenols on the NADH-oxidation catalysed by a peroxidase from lupin, Phytochemistry 26 (1987) 3133–3136.