Abstract

The hydroxyl radical produced in the apoplast has been demonstrated to facilitate cell wall loosening during cell elongation. Cell wall-bound peroxidases (PODs) have been implicated in hydroxyl radical formation. For this mechanism, the apoplast or cell walls should contain the electron donors for (i) H2O2 formation from dioxygen; and (ii) the POD-catalyzed reduction of H2O2 to the hydroxyl radical. The aim of the work was to identify the electron donors in these reactions. In this report, hydroxyl radical (·OH) generation in the cell wall isolated from pea roots was detected in the absence of any exogenous reductants, suggesting that the plant cell wall possesses the capacity to generate ·OH in situ. Distinct POD and Mn-superoxide dismutase (Mn-SOD) isoforms different from other cellular isoforms were shown by native gel electropho‑resis to be preferably bound to the cell walls. Electron paramagnetic resonance (EPR) spectroscopy of cell wall isolates containing the spin-trapping reagent, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO), was used for detection of and differentiation between ·OH and the superoxide radical (O2·). The data obtained using POD inhibitors confirmed that tightly bound cell wall PODs are involved in DEPMPO/OH adduct formation. A decrease in DEPMPO/OH adduct formation in the presence of H2O2 scavengers demonstrated that this hydroxyl radical was derived from H2O2. During the generation of ·OH, the concentration of quinhydrone structures (as detected by EPR spectroscopy) increased, suggesting that the H2O2 required for the formation of ·OH in isolated cell walls is produced during the reduction of O2 by hydroxycinnamic acids. Cell wall isolates in which the proteins have been denaturated (including the endogenous POD and SOD) did not produce ·OH. Addition of exogenous H2O2 again induced the production of ·OH, and these were shown to originate from the Fenton reaction with tightly bound metal ions. However, the appearance of the DEPMPO/OOH adduct could also be observed, due to the production of O2· when endogenous SOD has been inactivated. Also, O2· was converted to ·OH in an in vitro horseradish peroxidase (HRP)/H2O2 system to which exogenous SOD has been added. Taken together with the discovery of the cell wall-bound Mn-SOD isoform, these results support the role of such a cell wall-bound SOD in the formation of ·OH jointly with the cell wall-bound POD. According to the above findings, it seems that the hydroxycinnamic acids from the cell wall, acting as reductants, contribute to the formation of H2O2 in the presence of O2 in an autocatalytic manner, and that POD and Mn-SOD coupled together generate ·OH from such H2O2.

Introduction

It has been shown that intact plant tissues and cells can produce reactive oxygen species, such as H2O2, O2· and ·OH (Aver’yanov 1985, Kuchitsu et al. 1995, Schopfer et al. 2001, Rodriguez-Serrano et al. 2006), which can have important physiological functions, especially in the oxidative metabolism of the cell wall. These are closely linked with the two enzymes utilizing and producing these reactive oxygen species, the peroxidases (PODs) and superoxide dismutases (SODs). It is well established that plant cell walls contain class III POD (EC 1.11.1.7), and various POD isoforms are present in the cell walls. Their physiological roles are related to polymerization reactions such as lignification, suberization and cross-linking, consuming H2O2 (Kolattukudy 1980, Lewis and Yamamoto 1990, Iiyama et al. 1994). It has been shown, however, that PODs also possess the capacity to produce H2O2 while oxidizing different types of reductants including phenolics in the presence of trace amounts of metal ions (Vianello and Macri 1991, Pichorner et al. 1992, Jiang and Miels 1993, Hadži-Tašković Šukalović et al. 2005).

CuZn-SOD has been found in apoplastic fluid (Streller and Wingsle 1994, Ogawa et al. 1997, Schinkel et al. 1998, Karpinska et al. 2001, Bogdanović et al. 2006) and the cell wall (Karlsson et al. 2005). In addition, the presence of Mn-SOD has been demonstrated in the cell wall of moss (Yamahara et al. 1999). SOD has also been shown to be capable of catalyzing the formation of ·OH in the presence of H2O2 (Yim et al. 1990, Yim et al. 1993).

A physiological role for ·OH has been proposed in loosening of the cell wall and cleavage of polysaccharide polymers (Fry 1998, Schweikert et al. 2000). Schopfer and associates (Chen and Schopfer 1999, Schopfer et al. 2002, Liszkay et al. 2003) have demonstrated on whole roots and coleoptiles a POD-associated production of ·OH, in the presence of externally added reductants such as NADH or dihydroxyfumarate, using spectrofluorimetry and electron parametric resonance (EPR) spectroscopy.

The question of the naturally occurring reductant in the apoplastic space, participating in the production of reactive oxygen species, however, remains unclear. Involvement of the superoxide anion radical in ·OH production by the cell wall has been proposed in the literature (Chen and Schopfer 1999, Liszkay et al. 2004, Karkonen and Fry 2006). Various cellular components including plasma membranes were shown to be capable of generating O2· (Vuletić et al. 2003, Mojović et al. 2004). Plasma membrane-bound NADH oxidase has been implicated, based on an inhibitory effect of iodonium compounds, as a crucial enzyme responsible for the generation of O2· in the apoplast (Doke 1985, Murphy and Auh 1996, van Gestelen et al. 1997). The problem with the membrane-associated sources of O2· is the extreme reactivity of this radical species, the half-lives of O2· in water being 0.2 and 20 ms at concentrations of 10 and 1 μM, respectively (Bielski et al. 1985), which can be assumed to be similar to that in the apoplastic fluid. SOD accelerates the dismutation by 400-fold (rate constant, 2.4×109 M–1 s–1) (Scandalios 1997). Thus, it is very hard to envisage that plasma membrane-generated O2· can participate in the apoplastic space reactions in a controlled manner, and there is no evidence in the available literature for the presence of NAD(P)H in the apoplastic compartment.

In contrast to the adenylates, phenolics are ubiquitous apoplastic components (Takahama 2004). Since some phenolics are auto-oxidizable and can function as reductants, phenolics in the apoplast including the cell wall may contribute to the generation of H2O2 by reducing O2 (Takahama 2004). If H2O2 is generated in the cell walls by the auto-oxidation of phenolics, PODs and SODs in the walls may also be able to catalyze the formation of ·OH in addition to its formation by the Fenton reaction (Fry 1998, Karkonen and Fry 2006).

The aim of this work was to elucidate the mechanism of production of ·OH using cell wall isolated and purified from pea roots and to determine whether endogenous phenolics from the cell wall can act as reductants. To discuss the mechanism of ·OH production, mechanisms of production of O2· and H2O2 were also studied, taking O2-dependent oxidation of hydroxycinnamic acids into consideration. ·OH and O2· generated in cell wall isolates were detected using a spin-trapping reagent, 5-(diethoxyphosphoryl)-5-methyl-1-pyroline N-oxide (DEPMPO), due to its capacity to differentiate between O2· and ·OH (Frejaville et al. 1995, Mojović et al. 2004). The results obtained in this study suggest that cell wall isolates could reduce O2 to O2· by auto-oxidation of hydroxycinnamic acids bound to the cell wall, which in turn can be transformed by cell wall-bound SOD to H2O2. Subsequently, the cell wall-bound PODs can produce ·OH using the H2O2 generated.

Results

Components of cell wall isolates

First, we examined the purity of cell wall isolates prepared in this study. The total activity of glucose-6-phosphate dehydrogenase (G6PD), a cytosol marker enzyme, in the soluble pea root fraction was 0.53 ± 0.072 μmol gFW–1 min–1 (means ± SD) and in the cell wall isolate was 0.0027 ± 0.0011 μmol gFW–1 min–1 (means ± SD). The percentage of total activities of G6PD in cell wall isolates relative to that in the soluble root fraction was calculated to be 0.5 ± 0.06%, confirming that cell wall isolates were not contaminated with intracellular components. In addition, we could not detect NAD(P)H oxidase in the cell wall isolates; only the activity of ascorbate oxidase was observed (data not shown).

The content of metals and hydroxycinnamic acids and the activities of SODs and PODs were also measured using cell wall isolates. Table 1 gives the content of metals found in cell wall isolates. The quantity of the redox active metals (Fe and Cu) did not change significantly by treatment of cell wall isolates with SDS/heat (see Materials and Methods), suggesting that the metals were bound to the cell wall tightly. Table 2 shows the content of hydroxycinnamic acid in three root fractions. In cell wall isolates, ferulic, chlorogenic, caffeic and p-coumaric acids were found. By comparing the amount of each hydroxycinnamic acid in different fractions, it was clear that isoferulic acid was present in root extract and apoplastic fluid but not in cell wall isolates. This result additionally suggests that cell wall isolates were not contaminated with the soluble hydroxycinnamic acid contained in cytoplasm and apoplastic fluid. Fig. 1 shows SOD isoforms found in root extracts and cell wall isolates. In root extracts, three SOD isoforms were detected (Fig. 1A). H2O2 and KCN inhibited the activity of two isoforms, suggesting the presence of two isoforms of Cu,Zn-SOD and one isoform of Mn-SOD. After treatment with KCN, faint bands were detected, suggesting the presence of Fe-SOD. In cell wall isolates, several isoforms of SOD were also separated and their activities were not affected by either H2O2 or KCN (Fig. 1B). The latter indicates that the cell wall isolates contain several Mn-SOD isoforms (CWSODs), as opposed to the soluble root fraction which contains Cu,Zn-SOD.

Fig. 1

Separation of SOD isoforms in root extracts and cell wall isolates by native PAGE. (A) root extract; (B) cell wall isolate. Arrows indicate the presence of SOD isoforms. The amount of protein applied to each well was equivalent to 50 μg of bovine serum albumin. H2O2, H2O2-treated gel; KCN, KCN-treated gel.

Fig. 1

Separation of SOD isoforms in root extracts and cell wall isolates by native PAGE. (A) root extract; (B) cell wall isolate. Arrows indicate the presence of SOD isoforms. The amount of protein applied to each well was equivalent to 50 μg of bovine serum albumin. H2O2, H2O2-treated gel; KCN, KCN-treated gel.

Table 1

The metal content (μg gDW–1) in cell wall isolates of pea root (with intact and denatured proteins)

 Fe Mn Zn Cu 
Cell wall isolate 165 ± 45 3.8 ± 1.4 30 ± 3 23 ± 6 
Denatured cell wall isolate 188 ± 37 3.4 ± 1.6 14.1 ± 5 17 ± 5.4 
 Fe Mn Zn Cu 
Cell wall isolate 165 ± 45 3.8 ± 1.4 30 ± 3 23 ± 6 
Denatured cell wall isolate 188 ± 37 3.4 ± 1.6 14.1 ± 5 17 ± 5.4 

Cell wall isolates with denatured proteins were prepared by boiling the isolates in 2% SDS. Data are means ± SD (n = 3).

Table 2

The content of hydroxycinnamic acids (nmol gFW–1) in roots, cell wall isolates and apoplastic fluid

Hydroxycinnamic acids Roots Cell wall isolates Apoplast 
Ferulic acid 9.8 ± 1 14 ± 3 20 ± 10 
Isoferulic acid 26 ± 3 n.d. 2210 ± 220 
Chlorogenic acid n.d.a 20 ± 1.1 26.1 ± 1.4 
Caffeic acid n.d. 50 ± 2.3 n.d 
p-Coumaric acid n.d. 60 ± 0.5 n.d. 
Hydroxycinnamic acids Roots Cell wall isolates Apoplast 
Ferulic acid 9.8 ± 1 14 ± 3 20 ± 10 
Isoferulic acid 26 ± 3 n.d. 2210 ± 220 
Chlorogenic acid n.d.a 20 ± 1.1 26.1 ± 1.4 
Caffeic acid n.d. 50 ± 2.3 n.d 
p-Coumaric acid n.d. 60 ± 0.5 n.d. 

Hydroxycinnamic acids were identified by comparing their retention times with standard compounds. The retention times of chlorogenic, caffeic, p-coumaric, ferulic and isoferulic acid were 6.32, 8.17, 9.58, 11.82 and 12.65 min, respectively. Data are means ± SDs (n = 3)

aCould not be detected.

POD isoforms were also found in root extracts and cell wall isolates (Fig. 2). Three isoforms were detected in soluble root extracts (lane 1). In cell wall isolates that were not digested with cellulase and pectinase, POD activity was bound to the cell wall isolates (lane 2). When cell wall isolates were pre-digested with cellulase and pectinase, only one POD isoform could be detected, and this isoform was different from those observed in the cytoplasm (lane 3). These results suggest that the cell wall isolate posseses a distinct POD isoform (CWPOD), and that such cell wall isolates were not contaminated with cytoplasmic POD.

Fig. 2

Separation of POD isoforms in root extracts and cell wall isolates by native PAGE. 1, root extract; 2, cell wall isolate; 3, cell wall isolate after treatment with cellulase and pectinase. Arrows indicate the presence of POD isoforms.

Fig. 2

Separation of POD isoforms in root extracts and cell wall isolates by native PAGE. 1, root extract; 2, cell wall isolate; 3, cell wall isolate after treatment with cellulase and pectinase. Arrows indicate the presence of POD isoforms.

In addition to 4-chloro-α-naphtol used as a substrate for the POD reaction, we also studied whether cell wall isolates are capable of oxidizing hydroxycinnamic acids present normally in plants. Chlorogenic, caffeic and ferulic acids were oxidized at rates of 571 ± 131 ΔA410 mg protein–1 min–1, 532 ± 272 ΔA450 mg protein–1 min–1 and 227 ± 31 ΔA356 mg protein–1 min–1 (means ± SD), respectively, when H2O2 was added.

The results presented in Table 2 and Figs. 1 and 2 demonstrate that the cell wall isolates were not contaminated with either cytoplasmic phenolics, cytoplasmic isoforms of SOD or POD, and that the cell wall-bound POD could oxidize endogenous hydroxycinnamic acids (Table 2).

Formation of ·OH in cell wall isolates

When cell walls isolated from pea roots were suspended in buffer solution in the presence of DEPMPO, an EPR spectrum was observed, as shown in Fig. 3 (spectrum 3A). By comparing the EPR spectra of DEPMPO/OH and DEPMPO/OOH adducts, which were formed in chemical systems generating ·OH and O2·, respectively, with spectrum 3A (Mojović et al., 2004), we could conclude that the spectrum of the DEPMPO/OH adduct was contained in spectrum 3A. Computer simulation of the spectra (dashed line in spectrum 3A) supported the presence of EPR signals of the DEPMPO/OH adduct in spectrum 3A. In addition, signals of DEPMPO/H and DEPMPO/CH3 adducts were included in the spectrum. In Fig. 3G, the simulated spectra of each of the possible DEPMPO adducts are given. Protein denaturation of cell wall isolates by SDS/heat treatment abolished the generation of not only the DEPMPO/OH adduct but also other adducts (Fig. 3D). This finding suggests the contribution of proteins to the generation of spectrum 3A by cell wall isolates. The addition of H2O2 to the denatured cell wall resulted in the appearance of signal of DEPMPO/OOH in addition to DEPMPO/OH, DEPMPO/H and DEPMPO/CH3 adducts (spectra 3A and 3E). The signal of the DEPMPO/OOH adduct was observed transiently during the initial period after the addition of H2O2. The similarity of spectrum 3A to spectrum 3E except for the DEPMPO/OOH adduct suggests that H2O2 was generated during incubation of cell wall isolates. The addition of H2O2 to native cell wall isolates resulted in an increase in signal intensities from DEPMPO/OH and DEPMPO/H adducts. No signal due to the DEPMPO/OOH adduct could be observed in native cell wall isolates (spectrum 3B).

Fig. 3

EPR spectra of DEPMPO/OH adducts and other adducts in cell wall isolates. (A–C) Cell wall isolates; (D–F) denatured cell wall isolates. A and D, incubated for 5 min at room temperature; B and E, incubated for 5 min after the addition of 4 mM H2O2; C incubated for 5 min in the presence of 3 mM DETAPAC and 4 mM H2O2; F, incubated for 5 min in the presence of 3 mM DETAPAC and 4 mM H2O2. Filled circles, a characteristic peak of the DEPMPO/OH adduct; filled inverted triangles, a characteristic peak of the DEPMPO/H adduct; open inverted triangles, a characteristic peak of the DEPMPO/CH3 adduct; open circles, a characteristic peak of the DEPMPO/OOH adduct. Spectral simulations of spectra A, B and E were performed using the parameters given in Materials and Methods. (A) 50% DEPMPO/OH, 25% DEPMPO/CH3, 25% DEPMPO/H. (B) 25% DEPMPO/OH, 50% DEPMPO/CH3, 25% DEPMPO/H. (E) 38.5% DEPMPO/OOH, 38.5% DEPMPO/OH, 11.5% DEPMPO/CH3, 11.5% DEPMPO/H. (G) Spectral simulations of the four possible participating DEPMPO adducts (OH, OOH, CH3 and H).

Fig. 3

EPR spectra of DEPMPO/OH adducts and other adducts in cell wall isolates. (A–C) Cell wall isolates; (D–F) denatured cell wall isolates. A and D, incubated for 5 min at room temperature; B and E, incubated for 5 min after the addition of 4 mM H2O2; C incubated for 5 min in the presence of 3 mM DETAPAC and 4 mM H2O2; F, incubated for 5 min in the presence of 3 mM DETAPAC and 4 mM H2O2. Filled circles, a characteristic peak of the DEPMPO/OH adduct; filled inverted triangles, a characteristic peak of the DEPMPO/H adduct; open inverted triangles, a characteristic peak of the DEPMPO/CH3 adduct; open circles, a characteristic peak of the DEPMPO/OOH adduct. Spectral simulations of spectra A, B and E were performed using the parameters given in Materials and Methods. (A) 50% DEPMPO/OH, 25% DEPMPO/CH3, 25% DEPMPO/H. (B) 25% DEPMPO/OH, 50% DEPMPO/CH3, 25% DEPMPO/H. (E) 38.5% DEPMPO/OOH, 38.5% DEPMPO/OH, 11.5% DEPMPO/CH3, 11.5% DEPMPO/H. (G) Spectral simulations of the four possible participating DEPMPO adducts (OH, OOH, CH3 and H).

In the case of SDS/heat-treated cell wall isolates, no EPR signals could be observed in the absence of H2O2 (spectrum 3D). When H2O2 was added to such denaturated cell wall isolates, the DEPMPO/OOH adduct could be observed, in addition to the DEPMPO/OH and DEPMPO/H adducts (spectrum 3E). When the chelator DETAPAC (diethyl-enetriamine-N,N,N′,N′′,N′′-pentaacetic acid) was added to SDS/heat-treated and intact cell wall isolates and H2O2 subsequently added, no EPR signal was observed in the case of SDS/heat-treated isolates (spectrum 3F), as opposed to intact isolates where DETAPAC partially suppressed the generation of EPR signals due to the DEPMPO/OH adduct (spectrum 3C). As DETAPAC can chelate transition metal ions, the result suggests the formation of ·OH and O2· by metal ion-catalyzed reactions. The presence of transition metals in cell wall isolates has been shown (Table 1). Thus, the Fenton reaction might contribute to the formation of ·OH in cell wall isolates with denaturated proteins.

Participation of POD/H2O2 in ·OH formation by cell wall isolates

It was demonstrated in Fig. 3 that ·OH was formed in cell wall isolates. To elucidate the mechanism of ·OH formation, we studied the effects of POD inhibitors [KCN, NaN3 and salicylhydroxyamic acid (SHAM)] and scavengers of H2O2 (pyruvate and catalase) on the formation of the DEPMPO/OH adduct in cell wall isolates. Not only the inhibitors of POD but also the scavengers of H2O2 suppressed the formation of the DEPMPO/OH adduct (Fig. 4). The result suggests that H2O2 was generated in cell wall isolates and that the H2O2 contributed to the formation of ·OH. Cell wall isolates oxidized NADH in the presence of Mn2+ and SHAM, p-coumaric acid or ferulic acid, and rates of the oxidation were 0.90 ± 0.03, 0.77 ± 0.02 and 0.32 ± 0.09 mM mg protein–1 min–1 (means ± SD). As it has been reported that H2O2 is generated in a mixture of POD, Mn2+ and phenolics (Halliwell 1978), the reduction of cell wall isolates by exogenously added reductant (in this experiment NADH) and the presence of phenolics supports the concept of H2O2 generation by cell wall isolates. As described above, POD and H2O2 might participate in the formation of ·OH in cell walls as proposed previously (Schopfer 2002) and, since we found a SOD isoform tightly bound to the cell wall, we studied the formation of ·OH in an in vitro horseradish peroxidase (HRP)/H2O2 system in the presence and absence of bovine CuZn-SOD (Fig. 5). The addition of H2O2 to HRP resulted in the appearance of not only the DEPMPO/OH adduct but also the DEPMPO/OOH adduct (spectrum 5B). Computer simulation also suggested the formation of the two products, although the concentration of the DEPMPO/OH adduct seemed to be much lower than that of DEPMPO/OOH. The result indicates that O2· as well as ·OH was produced in the HRP/H2O2 system. When both H2O2 and SOD were added to HRP, EPR signals due to the DEPMPO/OH adduct increased (spectrum 5C), suggesting that scavenging of O2· resulted in an increase of the formation of ·OH. Control experiments with the spin trap and HRP without H2O2 (spectrum 5A) or spin trap and H2O2 without HRP (data not shown) did not generate any adducts.

Fig. 4

Effect of inhibitors of POD and scavengers of H2O2 on the generation of the DEPMPO/OH adduct in cell wall isolates. Cell wall isolates were incubated with DEPMPO for 5 min in the presence and absence of the inhibitors or the scavengers: (A) no addition; (B) 3 mM KCN; (C) 3 mM NaN3; (D) 3 mM SHAM; (E) 1,500 U of catalase ml–1; F, 3 mM pyruvate. Filled cirles, DEPMPO/OH adduct; filled inverted triangles, DEPMPO/H adduct; filled squares, quinhydrone structures (see Fig. 6).

Fig. 4

Effect of inhibitors of POD and scavengers of H2O2 on the generation of the DEPMPO/OH adduct in cell wall isolates. Cell wall isolates were incubated with DEPMPO for 5 min in the presence and absence of the inhibitors or the scavengers: (A) no addition; (B) 3 mM KCN; (C) 3 mM NaN3; (D) 3 mM SHAM; (E) 1,500 U of catalase ml–1; F, 3 mM pyruvate. Filled cirles, DEPMPO/OH adduct; filled inverted triangles, DEPMPO/H adduct; filled squares, quinhydrone structures (see Fig. 6).

Fig. 5

H2O2-dependent generation of ·OH and O2· by HRP. The reaction mixture contained 2.63 U of HRP (type II) and 42.5 mM DEPMPO in 100 mM potassium phosphate buffer (pH 7.0). (A) No addition; (B) the reaction mixture contained 2.63 U of HRP (type II) and 42.5 mM DEPMPO in 100 mM potassium phosphate buffer (pH 7.0) and 10 mM H2O2; (C) the reaction mixture contained 2.63 U of HRP (type II), 3.7 U of SOD and 42.5 mM DEPMPO in 100 mM potassium phosphate buffer (pH 7.0); the spectrum was recorded 5 min after the addition of 10 mM H2O2. Filled circles, DEPMPO/OH adduct; open circles, DEPMPO/OOH adduct. Spectral simulations of spectra B and C were performed using the parameters given in Materials and Methods: (B) 97% DEPMPO/OOH, 3% DEPMPO/OH. (C) 35.5% DEPMPO/OOH, 64.5% DEPMPO/OH.

Fig. 5

H2O2-dependent generation of ·OH and O2· by HRP. The reaction mixture contained 2.63 U of HRP (type II) and 42.5 mM DEPMPO in 100 mM potassium phosphate buffer (pH 7.0). (A) No addition; (B) the reaction mixture contained 2.63 U of HRP (type II) and 42.5 mM DEPMPO in 100 mM potassium phosphate buffer (pH 7.0) and 10 mM H2O2; (C) the reaction mixture contained 2.63 U of HRP (type II), 3.7 U of SOD and 42.5 mM DEPMPO in 100 mM potassium phosphate buffer (pH 7.0); the spectrum was recorded 5 min after the addition of 10 mM H2O2. Filled circles, DEPMPO/OH adduct; open circles, DEPMPO/OOH adduct. Spectral simulations of spectra B and C were performed using the parameters given in Materials and Methods: (B) 97% DEPMPO/OOH, 3% DEPMPO/OH. (C) 35.5% DEPMPO/OOH, 64.5% DEPMPO/OH.

H2O2-induced formation of quinhydrone structures in cell wall isolates

To clarify the cause of the differences between the HRP/H2O2/SOD system (Fig. 5B) and cell wall-derived EPR spectra (Fig. 3A), we analyzed the kinetics of the observed changes after the addition of H2O2 to the cell wall (Fig. 6). On the addition of DEPMPO to cell wall isolates, the signal of the DEPMPO/OH adduct was detected (spectrum 6A). Addition of 20 mM H2O2 to cell wall isolates resulted in the increase in signals of DEPMPO/OH and DEPMPO/H adducts and a quinhydrone-derived radical (spectrum 6B). The EPR spectrum in Fig. 6B changed as a function of time. The signal intensity of DEPMPO/OH and DEPMPO/H adducts decreased to nearly zero, and then the signal of the DEPMPO/OH adduct reappeared gradually as a function of incubation time. As the signals due to DEPMPO/OH and DEPMPO/H adducts decreased, the EPR spectrum of the quinhydrone-derived radical became clear. The signal intensity of the quinhydrone-derived radical increased 10- to 20-fold by increasing the pH of the mixture to 12 after H2O2 treatment (data not shown). The line shape was asymmetric and the line width was about 0.48 mT. These findings suggest that the signal was due to quinhydrone structures (Chio et al. 1982, Arnaud et al. 1983, Takahama et al. 2001). If the signal was due to free phenoxyl radicals, the line width would be smaller and hyperfine structures would be observed. A similar EPR signal, the intensity of which increased under alkaline conditions, was also observed in intact roots (data not shown). It has been reported that the presence of quinhydrone structures may be related to the reduction of O2 by phenolics to O2· and H2O2 (Furman 1986, Takahama et al. 2001). After removing phenolic compounds by acid and alkali hydrolysis of cell wall isolates, no EPR signal due to quinhydrone structures could be detected (data not shown). Oxidation of cell wall components by incubation of cell wall isolates with a high concentration of H2O2 (0.1 M), followed by thorough washing of the cell walls, resulted in a significant decrease in the intensity of signals due to the DEPMPO/OH adduct and quinhydrone structures (Fig. 7, lower spectrum in box A). Such oxidized cell walls had greatly reduced, or sometimes completely abolished, quantities of hydroxycinnamic acids (Fig. 7, box B). However, reduction of cell wall isolates by a high concentration of ascorbic acid (20 mM) resulted in the disappearance of quinhydrone structures as well as the DEPMPO/OH adduct, and the appearance of the signal of the ascorbyl radical (data not shown). Pea roots and the apoplastic fluid contained ascorbic and dehydroascorbic acids, and about 69 and 16% of ascorbic acid plus dehydroascorbic acid were present as the reduced form in the roots and the apoplastic fluid, respectively (Table 3).

Fig. 6

Changes in EPR spectra after the addition of H2O2 to cell wall isolates. (A) At 5 min after the addition of DEPMPO to cell wall isolates; (B) 5 min after the addition of 20 mM H2O2 to A; (C–J) EPR spectra measured every 2 min following the registration of spectrum B. Filled circles, DEPMPO/OH adduct; filled inverted triangles, DEPMPO/H adduct; filled squares, quinhydrone structures. Before addition of DEPMPO to cell wall isolates, the EPR signal marked with a square was also detected.

Fig. 6

Changes in EPR spectra after the addition of H2O2 to cell wall isolates. (A) At 5 min after the addition of DEPMPO to cell wall isolates; (B) 5 min after the addition of 20 mM H2O2 to A; (C–J) EPR spectra measured every 2 min following the registration of spectrum B. Filled circles, DEPMPO/OH adduct; filled inverted triangles, DEPMPO/H adduct; filled squares, quinhydrone structures. Before addition of DEPMPO to cell wall isolates, the EPR signal marked with a square was also detected.

Fig. 7

Effects of pre-oxidation of cell wall-associated phenolics on the formation of the DEPMPO/OH adduct in cell wall isolates. Box A: upper EPR spectrum, control cell wall isolates plus DEPMPO; lower EPR spectrum, oxidized cell wall isolates previously incubated with 0.1 M H2O2 for 30 min at 4°C and thoroughly washed before addition of DEPMPO. Box B: HPLC analysis of the hydroxycinnamic acids present in control and pre-oxidized cell wall isolates.

Fig. 7

Effects of pre-oxidation of cell wall-associated phenolics on the formation of the DEPMPO/OH adduct in cell wall isolates. Box A: upper EPR spectrum, control cell wall isolates plus DEPMPO; lower EPR spectrum, oxidized cell wall isolates previously incubated with 0.1 M H2O2 for 30 min at 4°C and thoroughly washed before addition of DEPMPO. Box B: HPLC analysis of the hydroxycinnamic acids present in control and pre-oxidized cell wall isolates.

Table 3

The content of ascorbic and dehydroascorbic acid (μmol gFW–1) in whole pea root extract and apoplastic fluid

 Ascorbic acid + dehydroascorbic acid Ascorbic acid (% of total) 
Root extract 0.98 ± 013 69 ± 5 
Apoplastic fluid 0.240 ± 0.025 16 ± 4 
 Ascorbic acid + dehydroascorbic acid Ascorbic acid (% of total) 
Root extract 0.98 ± 013 69 ± 5 
Apoplastic fluid 0.240 ± 0.025 16 ± 4 

Whole pea root extract and apoplastic fluid were prepared as described in Materials and Methods. Data are means ± SDs (n = 4).

These results indicate that quinhydrone structures observed in this study consisted of hydroquinone–quinone couples in macromolecules. Caffeic and chlorogenic acids and their quinone forms in the cell wall isolates may contribute to the formation of quinhydrone structures as quinhydrone structures are easily formed when quinones and hydroquinones are close to each other (Rex 1960, Steelink 1964, Furman 1986, Oniki 1998).

The effects of pH on the generation of the DEPMPO/OH adduct and formation of quinhydrone structures were studied using cell wall isolates (Fig. 8). The optimum pH for the generation of the DEPMPO/OH adduct was observed at about pH 7 and that for the formation of quinhydrone structures was also at pH 7. The result suggests that the production of ·OH was related to the oxidation of hydroxycinnamic acids to their quinone forms, which were included in the formation of quinhydrone structures.

Fig. 8

Effects of pH on the formation of the DEPMPO/OH adduct and quinhydrone structures in cell wall isolates. Cell wall isolates were incubated for 5 min in buffer solutions (pH 3–8). Upper panel, cell wall isolates with DEPMPO; lower panel, cell wall isolates without DEPMPO. Signal intensities marked with closed circles (A) and closed squares (B) were plotted as a function of pH.

Fig. 8

Effects of pH on the formation of the DEPMPO/OH adduct and quinhydrone structures in cell wall isolates. Cell wall isolates were incubated for 5 min in buffer solutions (pH 3–8). Upper panel, cell wall isolates with DEPMPO; lower panel, cell wall isolates without DEPMPO. Signal intensities marked with closed circles (A) and closed squares (B) were plotted as a function of pH.

Discussion

To study biochemical reactions taking place in cell walls, it is necessary to know how much the cell wall preparations were contaminated with symplastic components. The cell wall isolates used in this study seemed not to be contaminated with hydroxycinnamic acids, SOD- and POD-derived symplast, or NAD(P)H oxidase and G6PD (< 1% of symplast). Thus, the cell wall isolates prepared in this study can be used as a model system to simulate the reactions occurring between POD, SOD, metals, hydroxycinnamic acids and O2 in cell walls.

A HRP/H2O2 in vitro system generated ·OH and O2·, and the generation of ·OH was enhanced by SOD (Fig. 5). The generation of ·OH and O2· might be explained if the following reactions were taken into consideration (Adediran and Lambeir 1989, Wariishi and Gold 1990, Chen and Schopfer 1999, Hernandez-Ruiz et al. 2001, Liszkay et al. 2003, Furtmüller et al. 2004, Liszkay et al. 2004):  

(1)
formula
 
(2)
formula
 
(3)
formula
 
(4)
formula
 
(5)
formula

SOD-dependent enhancement of the formation of the DEPMPO/OH adduct might be explained by the inhibition of reaction 3 and enhancement of reactions 2 and 4 producing compound III and of reaction 5 producing hydroxyl radical. Besides the above-mentioned chain of reactions, the metal-catalyzed Fenton reaction can be also a source of hydroxyl radical production in the cell wall preparations (Fig. 3) via the following reaction:  

formula

The DEPMPO/OH adduct was detected in cell wall isolates, (Fig. 3). As cell wall isolates could produce H2O2 by auto-oxidation of hydroxycinnamic acids, the formation of ·OH in cell wall isolates could also be explained by the reaction between compound III and H2O2 (see also Fig. 9). Cell wall-bound SOD in the isolates might facilitate the production of ·OH similarly to SOD in the HRP/H2O2 in vitro system. If SOD contributed to the formation of ·OH from H2O2, the presence of CWSOD would be advantageous for the production of ·OH as this Mn-SOD isoform is resistant to inactivation with H2O2 (Beauchamp and Fridovich 1971, Weisiger and Fridovich 1973). DEPMPO/OOH was not observed in cell wall isolates with inactivated proteins unless H2O2 was added. Inactivation of enzymes (including POD and SOD) in the cell wall isolates by SDS/heat treatment demonstrated that besides the POD-associated production of ·OH, tightly bound metal ions can also, in the presence of H2O2, produce these radical species through the activity of the Fenton reaction. This potential source of hydroxyl radical species was abolished by the chelator DETAPAC. What is interesting, however, is that in the case of cell walls with inactivated proteins, one could also observe the production of the superoxide radical (Fig. 3E). This argues in favor of the participation of the CWSOD in dismutation of O2· and the contribution to ·OH production in the cell wall. Indeed, our in vitro experiment with purified HRP and SOD, which in the presence of O2· produced ·OH, supports such a mechanism.

Fig. 9

Schematic diagram showing the possible reactions occurring in the cell wall isolates. The scheme is a modification of the model proposed by Chen and Schopfer (1999), based on the results obtained in this study. MX+ and M(X–1)+, oxidized and reduced forms of a metal ion, respectively; -PhOH, -PhO· and -Ph = O, cell wall-bound o-dihydroxyphenolic, phenoxyl radical and quinone, respectively.

Fig. 9

Schematic diagram showing the possible reactions occurring in the cell wall isolates. The scheme is a modification of the model proposed by Chen and Schopfer (1999), based on the results obtained in this study. MX+ and M(X–1)+, oxidized and reduced forms of a metal ion, respectively; -PhOH, -PhO· and -Ph = O, cell wall-bound o-dihydroxyphenolic, phenoxyl radical and quinone, respectively.

The observed spectra generated by cell wall isolates are more complex than simple in vitro reactions. Simulations performed are best fitted if one assumes the participation of DEPMPO/H and/CH3 adducts. This could be due to ·H formation (marked by inverted triangles in Figs. 1, 2 and 7) (Bacčić et al. 2008). This would be in line with the proposal of Ward et al. (2003) who studied the lignin peroxidase-catalyzed oxidation of a series of phenolic compounds in detail and suggested the participation of phenoxyl radicals (probably DEPMPO/CH3 adducts) in the formation of ·H under certain pH and redox conditions.

As described above, H2O2 was generated in cell wall isolates. We propose that the mechanism of its formation in cell wall isolates is as follows. The initial step of the generation of H2O2 may be the reduction of O2 to O2· that can be transformed into H2O2. As the activity of NAD(P)H oxidase, which reduces O2 to O2·, was not detected in cell wall isolates used in this study, other reductants for the formation of O2· should be considered. The phenolics contained in cell walls are likely candidates because hydroxycinnamic acids can be oxidized by Fe3+ and Cu2+ to the radicals, which can reduce O2 to O2· (Takahama 2004). The occurrence of metals (Table 1) and o-dihydroxycinnamic acids such as caffeic and chlorogenic acids (Table 2) in cell walls satisfies the requirements for this reaction. No formation of the DEPMPO/OH adduct in the cell wall isolates after extensive oxidation of their bound hydroxycinnamic acids (Fig. 7) supports the necessity of hydroxycinnamic acids for the production of H2O2, the source of the hydroxyl radical. Once H2O2 is formed, the CWPOD/H2O2 system in the cell wall can oxidize caffeic, chlorogenic and ferulic acids in the isolates, enhancing the production of radicals of the hydroxycinnamic acid, which can react with O2, producing O2· (Fig. 9). It has been reported that o-dihydroxycinnamic acids found in cell wall isolates are auto-oxidizable and that the oxidation products (polymers with quinhydrone structures) are also auto-oxidizable (Takahama et al. 1999). The detection of an EPR signal of quinhydrone structures (Fig. 6) not only in cell wall isolates but also in intact roots (data not shown) suggests the presence of auto-oxidizable polymers in intact cell walls in addition to hydroxycinnamic acids. It has been reported that ascorbate added to the cell wall isolates suppressed the formation of ·OH (Veljović-Jovanović et al. 2005) and that cell wall POD-catalyzed oxidation of phenolics is inhibited by ascorbate (Takahama and Oniki 1992, Takahama 1993, Takahama and Oniki 1994). In this study, EPR signals of the DEPMPO/OH adduct and quinhydrone structures disappeared by adding ascorbate to the cell wall isolates (data not shown). The result suggests that ascorbate could inhibit the formation of ·OH, scavenge the DEPMPO/OH adduct and reduce quinones in quinhydrone structures. Ascorbate in the apoplast might be included in scavenging of ·OH generated in cell walls, resulting in the inhibition of elongation growth if ·OH participated in the loosening of cell walls on elongation growth. This idea is supported by reports that the concentration of apoplastic ascorbate along hypocotyls shows a significant negative correlation with the growth rate (Cordoba-Pedregosa et al. 2003, Rodriguez-Serrano et al. 2006) and that the concentration of ascorbate plus dehydroascorbate in IAA-treated epicotyls of Vigna angularis was similar to that in untreated hypocotyls, whereas the ratio of ascorbate to ascorbate plus dehydro-ascorbate is somewhat decreased by IAA (Takahama and Oniki 1994).

Conclusions

The data presented in this report demonstrate that hydroxycinnamic acids in cell walls can be electron donors of metal-catalyzed reduction of dioxygen to form superoxide, and that cell wall-bound POD mediates the production of hydroxyl radical from H2O2, which is formed from the superoxide. Mn-superoxide dismutase isoforms (CWSOD) were found in cell wall isolates, indicating their contribution to the facilitated formation of H2O2 within the cell wall. The presence of quinhydrone structures in cell wall isolates as well as in excised root was also demonstrated. The quinhydrone structures disappeared by oxidation and reduction of cell wall isolates, and the pH dependency of their formation was similar to that of the formation of the DEPMPO/OH adduct. The latter indicates that the reduction of O2 to H2O2 in the cell walls might be related to the transformation of a dihydroxycinnamic acid to its quinone form. Ascorbate seems to have an important role in regulating the production of ·OH and quinhydrone structures, which in turn may result in the regulation of root growth.

Materials and Methods

Reagents

DEPMPO was obtained from Alexis Biochemicals (Lausen, Switzerland). DETAPAC, SHAM, CuZn-SOD from bovine erythrocytes, HRP (type II), ascorbate oxidase and cellulase were from Sigma (St Louis, MO, USA). Pectinase was obtained from Serva (New York, USA). HPLC-grade acetonitrile and methanol were from J. T. Baker (Deventer, The Netherlands) and Carbo Reagenti (Milano, Italy), respectively. All solutions were prepared daily by dissolving in 18 M redistilled and deionized water (Millipore, Bedford, MA, USA).

Isolation of cell walls

Pea plants (Pisum sativum L.) were grown in hydroponic culture under an 8/16 h day/night regime. Fourteen-day-old plants were used for analysis and cell wall isolation. Cell walls were isolated from pea roots following the method described by Carpita (1984) with some minor modifications. Roots (60 g) were powdered in liquid N2 and homogenized in 120 ml of buffer [50 mM Tris–HCl (pH 7.2), 50 mM NaCl; 0.05% Tween-80; 1 mM phenylmethylsulfonyl fluoride (PMSF)]. The homogenate was filtered through two layers of cloth. The filtrate was sonicated for 1 min and centrifuged at 1,000×g for 20 min. The cell wall pellet was washed four times in the above buffer without detergent and salt and then suspended in 10 ml of 1 M NaCl, followed by incubation for 30 min at 4°C and centrifugation at 1,000 × g for 15 min. The pellet (2 g) (in the following, cell wall isolates) was washed and centrifuged with 5 ml of 50 mM Tris–HCl (pH 7.2) several times and finally suspended in 5 ml of 50 mM Tris–HCl (pH 7.2) for measurement of EPR spectra in the presence and absence of DEPMPO. When the effects of pH on the formation of radicals was determined, the pellet was suspended in buffers with various pH values (see below). The suspension was kept at 0°C until used for experiments.

Cell wall isolate (2 g) obtained as described above was digested for 24 h at 4°C in 5 ml of a mixture containing 0.5% cellulase and 2.5% pectinase in 50 mM Tris–HCl (pH 7.2), and centrifuged at 10,000 × g for 10 min (Lin and Kao 2001). POD and SOD isoforms bound to the cell wall isolates were released by this procedure, and POD and SOD in the supernatant analyzed by native PAGE (see below).

Cell wall isolates with denatured proteins were prepared by incubation of the isolates in 2% SDS for 30 min at 100°C, which were then washed four times by suspension and centrifugation at 1,000 × g for 15 min in 50 mM Tris–HCl (pH 7.2).

Extraction of soluble proteins and metabolites from roots

For extraction of soluble enzymes from roots, 0.5 g of frozen roots was powdered in a mortar containing liquid N2 and suspended in 100 mM potassium phosphate buffer (pH 6.5) and 1 mM PMSF. The homogenate was centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was used for the separation of SOD isoforms by native PAGE (see below) and measurement of the activity of G6DP. For determination of total ascorbate (ascorbate plus dehydroascorbate), roots (0.5 g) were homogenized in 4 ml of 5% (v/v) HClO4 and the homogenate was centrifuged at 12,000 × g for 10 min at 4°C to obtain the supernatant.

Extraction of apoplastic fluid

Freshly cut roots (20 root sections of length 3 mm) were vacuum infiltrated with distilled water for 10 min, and, after water on the surface was removed by filter paper, placed in 5 ml Eppendorf tips, with the anterior ends down. The Eppendorf tip was placed in a centrifugation tube containing 100 μl of extraction medium for ascorbate [5% (v/v) HClO4] or for proteins [100 mM phosphate buffer (pH 6.5)]. Upon centrifugation at 500 × g for 10 min, apoplastic fluid eluting from the cut roots was instantly mixed with the extraction medium. Protein extract was used for determination of the G6PD activity.

EPR spectroscopy

EPR measurements were made using a custom-constructed Teflon flat cell (50 μl) with one side formed from oxygen-permeable thin Teflon foil. EPR spectra were recorded at room temperature using a Varian E104-A EPR spectrometer operating at the X-band (9.51 GHz) under the following settings: modulation amplitude, 0.2 mT; modulation frequency, 100 kHz; microwave power, 10 mW; centre of magnetic field, 341 mT; scan range, 20 mT; scan speed, 4 mT min–1. Spectra were recorded and analyzed using EW software (Scientific Software). DEPMPO (final concentration, 42.5 mM) was added to cell wall isolates, apoplastic fluid or HRP. EPR signals of quinhydrone structures and ascorbyl radicals were also measured using the settings for the measurement of DEPMPO/OH and DEPMPO/OOH adducts. The pH dependence of the formation of the DEPMPO/OH adduct and quinhydrone structures was measured in the pH range from 3 to 8. The buffer solutions used were 50 mM citrate/phosphate buffer (pH range from 3 to 7) and 50 mM potassium phosphate buffer (pH range from 7 to 8). Computer simulations of the EPR spectra were performed using the WINEPR SinFonia program. Parameters indicated in parentheses were used for the simulation of the DEPMPO/OH adduct (aP = 46.70; aN = 13.64; aHβ = 12.78) and the DEPMPO/OOH adduct [isomer I (55%): aP = 50.15, aN = 13, aHβ = 11.3, aH_ = 0.85, aH_ = 0.35, aH_(3) = 0.53; isomer II (37%): aP = 48.68, aN = 13.8, aHβ = 0.88G, aH_ = 10.2, aH_ = 0.41, aH_ = 0.34; isomer III (8.5%): aP = 40.8, aN = 13.3, aHβ = 1.5, aH_ = 10] (Vasquez-Vivar et al. 2000; Mojović et al. 2004). The DEPMPO/H adduct was simulated using the following parameters (aP = 45.32; aN = 13.97; aHβ = 17.47; aHβ = 3.36) from Bacčić et al. (2008) and the DEPMPO/CH3 adduct was simulated using the following parameters (aP = 46.26; aN = 14.35; aHβ = 21.48) from Berliner et al (2002).

Analysis of phenolics by HPLC

Aliquots of root extract, apoplastic fluid and cell wall isolates were boiled in methanol for 30 min to extract phenolics. After cooling, the extracts were centrifuged at 10,000 × g for 15 min to remove methanol-insoluble components. For acid hydrolysis, the pellet with cell wall isolates was suspended in 1 mol l–1 HCl and incubated at 37°C for 3 h in a water bath. The rest of the cell wall isolates, after removing methanol and acid-solubilized phenolics, were hydrolyzed in a similar way using 100 g l–1 KOH in methanol (incubated at 60°C for 1 h in a water bath). Aliquots of samples (supernatants) prepared as above were injected in a Breeze HPLC system with a Waters 2465 electrochemical detector equipped with 3 mm gold working and hydrogen reference electrodes (Waters, Milford, MA, USA). Signals were detected in the direct scan mode at the constant potential of + 0.6 V. Phenolics were separated on a Waters Symmetry C-18 RP column (125×4 mm) with 5 μm particle size. The mobile phases were 0.1% phosphoric acid (adjusted to pH 2.4 in K2HPO4) (mobile phase A) and acetonitrile (mobile phase B) with the following gradient profile: in the first 10 min from 10 to 22% of mobile phase B, followed by a 10 min linear rise up to 30% of mobile phase B, ending with 5 min reversion to 10% of mobile phase B. The flow rate was 1 ml min–1.

Native PAGE

Native PAGE was performed in a 10% polyacrylamide gel with a reservoir buffer consisting of 0.025 M Tris and 0.192 M glycine (pH 8.3) at 24 mA for 120 min. To detect POD activity after electrophoresis, the gel was incubated with 10% 4-chloro-α-naphthol and 0.03% H2O2 in 100 mM potassium phosphate buffer (pH 6.5). SOD separated by electrophoresis was detected according to Beauchamp and Fridovich (1971) by incubating the gel in a reaction mixture containing 0.01 M EDTA, 0.098 mM nitroblue tetrazolium, 0.030 mM riboflavin and 2 mM TEMED in 50 mM potassium phosphate (pH 7.8) for 30 min in the dark, followed by washing with distilled water and illumination by a fluorescent lamp (30 μEm–2 s–1 for 15 min). CuZn-, Mn-and Fe-SODs were distinguished from each other by incubating the gel with 5 mM KCN and/or 5 mM H2O2 before staining (Weisiger and Fridovich 1973. Yamahara et al. 1999). Protein content was measured according to Bradford (1976).

Enzyme assays

POD activities were measured in a reaction mixture (3 ml) containing a 50 μl suspension of cell wall isolates, 3.3 mM H2O2 and 4 mM chlorogenic, caffeic or ferulic acids in 100 mM potassium phosphate buffer (pH 6.5). Oxidation of the above hydroxycinnamic acids was measured by the increase in absorbance at 410, 450 and 356 nm, respectively, according to Bestwick et al. (1998). The oxidation of NADH by cell wall isolates was determined by measuring the absorbance decrease at 340 nm (ε = 6.22 mM–1 cm–1) (Halliwell 1978). The reaction mixture (3 ml) contained 50 μl of cell wall isolates, 0.2 mM NADH, 0.25 mM MnCl2 and 0.2 mM SHAM, p-coumaric acid or ferulic acid in 50 mM potassium phosphate buffer (pH 5.5). Cytosolic contamination of apoplastic fluid and cell wall isolate was monitored by assaying G6PD activity as marker enzyme. The activity of G6PD was determined in a reaction mixture (3 ml) consisting of 5 mM MgSO4, 10 mM glucose-6-phosphate and 0.1 mM NADPH in 100 mM potassium phosphate buffer (pH 8). The rate of oxidation of NADPH was estimated by the absorbance decrease at 340 nm.

Determination of the concentration of ascorbic and dehydroxyascorbic acids

The pH of the apoplastic fluid centrifuged down into 5% HClO4 and roots extracted with 5% HClO4 was adjusted to pH 4 by adding 5 M K2CO3, and insoluble components were removed by centrifugation at 12,000 × g for 1 min at 4°C. The reduced form of ascorbic acid was assayed in a reaction mixture (3.5 ml) containing 100 μl of the root homogenate or apoplastic fluid in 300 mM potassium phosphate buffer (pH 5.6). The decrease in absorbance at 262 nm after addition of 1 U of ascorbate oxidase was taken for calculation of the amount of ascorbate (ε = 14.3 mM–1 cm–1). The concentration of dehydroascorbate was estimated in a reaction mixture (3.5 ml) containing 100 μl of root homogenate or apoplastic fluid and 20 μl of 100 mM dithiothreitol in 300 mM potassium phosphate buffer (pH 7.6). The absorbance increase at 262 nm after the addition of dithiothreitol was used to calculate the concentration of dehydroascorbate.

Determination of metal contents

Cell wall isolates were dried at 60°C for 24 h and then milled. Samples were digested in 1 M HNO3, and metal content was determined by flame atomic absorption spectrometry (Varian Spectra 220).

Funding

The Ministry of Science of Republic of Serbia (Project Nos. 143020B and 143016B).

Acknowledgments

The authors would like to thank the reviewers for their helpful suggestions.

References

Adediran
SA
Lambeir
A
Kinetics of the reaction of compound II of horseradish peroxidase with hydrogen peroxide to form compound III
Eur. J. Biochem
 , 
1989
, vol. 
186
 (pg. 
571
-
576
)
Arnaud
R
Perbet
G
Defladre
A
Lang
G
Electron spin resonance of melanin from hair. Effects of temperature, pH and light irradiation
Photochem. Photobiol.
 , 
1983
, vol. 
38
 (pg. 
161
-
168
)
Aver’yanov
AA
Superoxide radical generation by intact pea roots
Fiziol. Rast.
 , 
1985
, vol. 
32
 (pg. 
268
-
273
)
Bacčić
GG
Spasojević
I
Šećerov
B
Mojović
M
Spin-trapping of oxygen free radicals in chemical and biological systems: new traps, radicals and possibilities
Spectrochim. Acta A
 , 
2008
, vol. 
69
 (pg. 
1354
-
1366
)
Beauchamp
C
Fridovich
I
Superoxide dismutase: improved assay and assay applicable to acrylamide gels
Anal. Biochem.
 , 
1971
, vol. 
44
 (pg. 
276
-
287
)
Berliner
LJ
Kharamatsov
V
Clanton
TL
Fujii
H
NMR and MRI spin trapping: using NMR to learn about free radical reaction
Curr. Top. Biophys.
 , 
2002
, vol. 
26
 (pg. 
21
-
27
)
Bestwick
CS
Brown
IR
Mansfield
JW
Localized changes in peroxidases activity accompany hydrogen peroxide generation during the development of a nonhost hypersensitive reaction in lettuce
Plant Physiol.
 , 
1998
, vol. 
118
 (pg. 
1067
-
1078
)
Bielski
BHJ
Cabelli
DE
Arudi
RL
Reactivity of HO2/O2· radicals in aqueous solution
J. Phys. Chem. Ref. Data
 , 
1985
, vol. 
14
 (pg. 
1041
-
1100
)
Bogdanović
J
Prodanović
R
Milosavić
N
Prodanović
O
Radotić
K
Multiple forms of superoxide dismutase in the apoplast and whole-needle extract of Serbian spruce (Picea omorika (Pancč.) Purkyne)
Arch. Biol. Sci.
 , 
2006
, vol. 
58
 (pg. 
211
-
214
)
Bradford
MM
A rapid and sensitive method for quantitation of microgram of protein utilizing the principle of protein–dye binding
Anal. Biochem.
 , 
1976
, vol. 
72
 (pg. 
248
-
254
)
Carpita
NC
Cell wall development in maize coleoptiles
Plant Physiol.
 , 
1984
, vol. 
76
 (pg. 
205
-
212
)
Chen
S
Schopfer
P
Hydroxyl-radical production in physiological reactions. A novel function of peroxidase
Eur. J. Biochem.
 , 
1999
, vol. 
260
 (pg. 
726
-
773
)
Chio
S-S
Hyde
JS
Sealy
RC
Paramagnetism in melanins: pH dependence
Arch. Biochem. Biophys.
 , 
1982
, vol. 
215
 (pg. 
100
-
106
)
Córdoba-Pedregosa
MC
Córdoba
F
Villalba
JM
González-Reyes
JA
Zonal changes in ascorbate and hydrogen peroxide contents, peroxidase, and ascorbate-related enzyme activities in onion roots
Plant Physiol.
 , 
2003
, vol. 
131
 (pg. 
697
-
706
)
Doke
N
NADPH-dependent O2· generation in membrane fractions isolated from wounded potato tubers inoculated with Phytophtora infestans
Physiol. Plant Pathol.
 , 
1985
, vol. 
27
 (pg. 
311
-
322
)
Frejaville
C
Karoui
H
Tuccio
B
Le Moigne
F
Culcasi
M
Pietri
S
, et al.  . 
5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide: a new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals
J. Med. Chem.
 , 
1995
, vol. 
38
 (pg. 
258
-
265
)
Fry
SC
Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals
Biochem. J.
 , 
1998
, vol. 
332
 (pg. 
507
-
515
)
Furman
GS
The contribution of charge-transfer complexes of the color of kraft lignin
 , 
1986
Appleton, Wisconsin
Lawrence University
Furtmüller
PG
Jantschko
W
Zederbauer
M
Jakopisch
C
Arnhold
J
Oblinger
C
Kinetics of interconversion of redox intermediates of lactoperoxidase, eosinophil peroxidase and myeloperoxidase
Jpn. J. Infect. Dis.
 , 
2004
, vol. 
57
 (pg. 
S30
-
S31
)
Hadži-Tašković Šukalović
V
Vuletić
M
Vucčinić
Ž.
The role of p-coumaric acid in oxidative and peroxidative cycle of the ionically bound peroxidase of the maize root cell wall
Plant Sci.
 , 
2005
, vol. 
168
 (pg. 
931
-
938
)
Halliwell
B
Lignin synthesis: the generation of hydrogen peroxidase and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols
Planta
 , 
1978
, vol. 
140
 (pg. 
81
-
88
)
Hernandez-Ruiz
J
Arno
MB
Hiner
A.NP
Garcia-Canovas
F
Acosta
M
Catalase-like activity of horseradish peroxidase: relationship to enzyme inactivation by H2O2
Biochem. J.
 , 
2001
, vol. 
354
 (pg. 
107
-
114
)
Iiyama
K
Lam
T.BT
Setne
B
Covalent cross-links in the cell wall
Plant Physiol.
 , 
1994
, vol. 
104
 (pg. 
315
-
320
)
Jiang
Y
Miles
PW
Generation of H2O2 during enzymic oxidation of catechin
Phytochemistry
 , 
1993
, vol. 
33
 (pg. 
29
-
34
)
Karlsson
M
Melzer
M
Prokhorenko
I
Johansson
T
Wingsle
G
Hydrogen peroxide and expression of hipI-superoxide dismutase are associated with the development of secondary cell walls in
Zinnia elegans. J. Exp. Bot.
 , 
2005
, vol. 
56
 (pg. 
2085
-
2093
)
Karkonen
A
Fry
SC
Effect of ascorbate and its oxidation products on H2O2 production in cell-suspension cultures of Picea abies and in the absence of cells
J. Exp. Bot.
 , 
2006
, vol. 
57
 (pg. 
1633
-
1644
)
Karpinska
B
Karlsson
M
Schinkel
H
Streller
S
Suss
KH
Melzer
M
, et al.  . 
A novel superoxide dismutase with a high isoeletric point in higher plant. Expression, regulation, and protein localization
Plant Physiol.
 , 
2001
, vol. 
126
 (pg. 
1668
-
1677
)
Kolattukudy
PE
Biopolyester membranes of plants: cutin and suberin
Science
 , 
1980
, vol. 
208
 (pg. 
990
-
1000
)
Kuchitsu
K
Kosaka
H
Shiga
T
Shibuya
N
EPR evidence for generation of hydroxyl radical triggered by N-acetylchitooligosaccharide elicitor and a protein phosphatase inhibitor in supension-cultured rice cells
Protoplasma
 , 
1995
, vol. 
188
 (pg. 
138
-
142
)
Lewis
NG
Yamamoto
E
Lignin: occurrence, biogenesis and biodegradation
Annu. Rev. Plant Physiol. Plant Mol. Biol.
 , 
1990
, vol. 
41
 (pg. 
55
-
496
)
Lin
CC
Kao,
CH
Cell wall peroxidase activity, hydrogen peroxide level and NaCl-inhibited root growth of rice seedlings
Plant Soil
 , 
2001
, vol. 
230
 (pg. 
135
-
143
)
Liszkay
A
Kenk
B
Schopfer
P
Evidence for involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth
Planta
 , 
2003
, vol. 
217
 (pg. 
658
-
667
)
Liszkay
A
van der Zalm
A
Schopfer
P
Production of reactive oxygen intermediates (O2· H2O2 and ·OH) by maize roots and their role in wall loosening and elongation growth
Plant Physiol.
 , 
2004
, vol. 
136
 (pg. 
3114
-
3123
)
Mojović
M
Bacčić
G
Vucčinić
Ž.
Vuletić
M
Oxygen radicals produced by plant plasma membranes: an EPR spin-trap study
J. Exp. Bot.
 , 
2004
, vol. 
55
 (pg. 
2523
-
2531
)
Murphy
TM
Auh
CH
The superoxide synthases of plasma membrane preparations from cultured rose cells
Plant Physiol.
 , 
1996
, vol. 
110
 (pg. 
621
-
629
)
Ogawa
K
Kanematsu
S
Asada
K
Generation of superoxide anion and localization of CuZn-superoxide dismutase in the vascular tissue of spinach hypocotyls: their association with lignification
Plant Cell Physiol.
 , 
1997
, vol. 
38
 (pg. 
1118
-
1126
)
Oniki
T
Origin of free radicals produced from the syringyl end groups in lignins
J. Wood Sci
 , 
1998
, vol. 
44
 (pg. 
314
-
319
)
Pichorner
H
Couperus
A
Korori
S.AA
Ebermann
R
Plant peroxidase has a thiol oxidase function
Phytochemistry
 , 
1992
, vol. 
31
 (pg. 
3371
-
3376
)
Rex
RW
Electron paramagnetic resonance studies of stable free radicals in lignin and humic acid
Nature
 , 
1960
, vol. 
188
 (pg. 
1185
-
1186
)
Rodriguez-Serano
M
Romero-Puertas
M
Zabalza
A
Corpas
FJ
Gomez
M
Del Rio
LA
, et al.  . 
Cadmium effect on oxidative metabolism of pea (Pisum sativum L.) roots. Imaging of reactive oxygen species and nitric oxide accumulation in vivo
Plant Cell Environ.
 , 
2006
, vol. 
29
 (pg. 
1532
-
1544
)
Scandalios
JG
Scandalios
JG
Molecular genetics of superoxide dismutases in plants
Oxidative Stress and the Molecular Biology of Antioxidative Defenses
 , 
1997
NY
Cold Spring Harbor Laboratory Press, Cold Spring Harbor
(pg. 
527
-
568
)
Schinkel
H
Streller
S
Wingsle
G
Multiple forms of extracellular superoxide dismutase in needles, stem tissues and seedlings of Scots pine
J. Exp. Bot.
 , 
1998
, vol. 
49
 (pg. 
931
-
936
)
Schopfer
P
Liszkay
A
Bechtold
M
Frahy
G
Wagner
A
Evidence that hydroxyl radicals mediate auxin-induced extension growth
Planta
 , 
2002
, vol. 
214
 (pg. 
821
-
828
)
Schopfer
P
Plachy
C
Frahry
G
Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid
Plant Physiol.
 , 
2001
, vol. 
125
 (pg. 
1591
-
1602
)
Steelink
C
Free radical studies of lignin, lignin degradation products and soil humic acids
Geochim. Cosmochim. Acta
 , 
1964
, vol. 
28
 (pg. 
1615
-
1622
)
Streller
S
Wingsle
G
Pinus sylvestris L. needles contain extracellular CuZn superoxide dismutase
Planta
 , 
1994
, vol. 
192
 (pg. 
195
-
201
)
Schweikert
C
Liszkay
A
Schopfer
P
Scission of polysaccharides and peroxidase-generated hydroxyl radicals
Phytochemistry
 , 
2000
, vol. 
53
 (pg. 
565
-
570
)
Takahama
U
Oniki
T
Regulation of peroxidase-dependent oxidation of phenolics in the apoplast of spinach leaves by ascorbate
Plant Cell Physiol.
 , 
1992
, vol. 
33
 (pg. 
379
-
387
)
Takahama
U
Redox state of ascorbic acid in the apoplast of stems of
Kalanchoe daigremontiana. Physiol. Plant.
 , 
1993
, vol. 
89
 (pg. 
791
-
798
)
Takahama
U
Oxidation of vacuolar and apoplastic phenolic substrates. Physiological significance of the oxidation reactions
Phytochem. Rev.
 , 
2004
, vol. 
3
 (pg. 
207
-
219
)
Takahama
U
Hirotsu
M
Oniki
T
Age-dependent changes in levels of ascorbic acid and chlorogenic acid, and activities of peroxidase and superoxide dismutase in the apoplast of tobacco leaves: mechanism of the oxidation of chlorogenic acid in the apoplast
Plant Cell Physiol.
 , 
1999
, vol. 
40
 (pg. 
716
-
724
)
Takahama
U
Oniki
T
The association of ascorbate and ascorbate oxidase in the apoplast with IAA-enhanced elongation of epicotyls from Vigna angularis
Plant Cell Physiol.
 , 
1994
, vol. 
35
 (pg. 
257
-
266
)
Takahama
U
Oniki
T
Hirota
S
Phenolic compounds of brown scales of onion bulbs produce hydrogen peroxide by autooxidation
J. Plant Res.
 , 
2001
, vol. 
114
 (pg. 
395
-
402
)
van Gestelen
P
Asard
H
Caubergs
RJ
Solubilization and separation of plant plasma membrane NADPH-O2-synthase from other NAD(P)H oxidoreductases
Plant Physiol.
 , 
1997
, vol. 
115
 (pg. 
543
-
550
)
Vásquez-Vivar
J
Kalyanaraman
B
Kennedy
MC
Mitochondrial aconitase is a source of hydroxyl radical. An electron spin resonance investigation
J. Biol. Chem.
 , 
2000
, vol. 
275
 (pg. 
14064
-
14069
)
Veljović-Jovanović
S
Kukavica
B
Cvetić
T
Mojović
M
Vucčinić
Ž.
Ascorbic acid and the oxidative processes in pea root cell wall isolates. Characterization by fluorescence and EPR spectroscopy
Ann. NY Acad. Sci.
 , 
2005
, vol. 
1048
 (pg. 
501
-
505
)
Vianello
A
Macri
F
Generation of superoxide anion and hydrogen peroxide at the surface of plant cells
J. Bioenerg. Biomembr.
 , 
1991
, vol. 
23
 (pg. 
409
-
423
)
Vuletić
M
Hadži-Tašković
V
Vucčinić
Ž
Superoxide synthase and dismutase activity of plasma membranes from maize roots
Protoplasma
 , 
2003
, vol. 
221
 (pg. 
73
-
77
)
Ward
G
Hadar
Y
Bilkis
I
Dosoretz
CG
Mechanistic features of lignin peroxidase-catalyzed oxidation of substituted phenols and 1,2-dimethoxyarenes
J. Biol. Chem.
 , 
2003
, vol. 
278
 (pg. 
39726
-
39734
)
Wariishi
H
Gold
MH
Lignin peroxidase compound III. Mechanism of formation and decomposition
J. Biol. Chem.
 , 
1990
, vol. 
265
 (pg. 
2070
-
2077
)
Weisiger
R
Fridovich
I
Superoxide dsmutase. Organelle specificity
J. Biol. Chem.
 , 
1973
, vol. 
248
 (pg. 
3582
-
3592
)
Yamahara
T
Shiono
T
Suzuki
T
Tanaka
K
Takio
S
Sato
K
, et al.  . 
Isolation of a germin-like protein with manganese superoxide dismutase activity from cells of a moss
Barbula unguiculata. J. Biol. Chem.
 , 
1999
, vol. 
274
 (pg. 
33274
-
33278
)
Yim
MB
Chock
PB
Stadtman
ER
Copper, zinc superoxide dismutase catalyzes hydroxyl radical production from hydrogen peroxide (electron paramagnetic resonance/spin trapping/oxidative damage)
Proc. Natl Acad. Sci. USA
 , 
1990
, vol. 
87
 (pg. 
5006
-
5010
)
Yim
MB
Chock
PB
Stadtman
ER
Enzyme function of copper, zinc superoxide dismutase as a free radical generator
J. Biol. Chem.
 , 
1993
, vol. 
268
 (pg. 
4099
-
4105
)

Abbreviations:

    Abbreviations:
  • CWPOD

    cell wall peroxidase

  • CWSOD

    cell wall superoxide dismutase

  • DEPMPO

    5-(diethoxypho-sphoryl)-5-methyl-1-pyrroline-N-oxide

  • DETAPAC

    diethyl-enetriamine-N,N,N′,N′′,N′′-pentaacetic acid

  • EPR

    electro-paramagnetic resonance

  • G6PD

    glucose-6-phosphate dehydrogenase

  • HRP

    horseradish peroxidase

  • PMSF

    phenylmethylsulfonyl fluoride

  • POD

    peroxidase

  • SHAM

    salicylhydroxyamic acid

  • SOD

    superoxide dismutase.