Hydroquinone peroxidase activity of maize root mitochondria

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Protoplasma (2007) 231: 137–144 DOI 10.1007/s00709-007-0260-0

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Hydroquinone peroxidase activity of maize root mitochondria Vesna Hadzˇi-Tasˇkovic´ Sˇukalovic´1, B. Kukavica 2, M. Vuletic´1 1 2

Laboratory of Plant Physiology, Maize Research Institute Zemun Polje, Belgrade Center for Multidisciplinary Studies, Belgrade University, Belgrade

Received 8 November 2006; Accepted 31 January 2007; Published online 10 October 2007 © Springer-Verlag 2007

Summary. The oxidation of hydroquinone with H2O2 in the presence of mitochondria isolated from maize (Zea mays L.) roots was studied. The results indicate that a reduced form of quinone may be a substrate of mitochondrial peroxidases. Specific activities in different mitochondrial isolates, the apparent Km for hydrogen peroxide and hydroquinone, and the influence of some known peroxidase inhibitors or effectors are presented. Zymographic assays revealed that all mitochondrial peroxidases, which were stained with 4-chloro-1-naphthol, were capable of oxidizing hydroquinone. A possible antioxidative role of hydroquinone peroxidase in H2O2 scavenging within the mitochondria, in cooperation with ascorbate or coupled with mitochondrial NAD(P)H dehydrogenases, is proposed. Keywords: Hydroquinone peroxidase; Mitochondria; Maize root; Zea mays. Abbreviations: BQ benzoquinone; HQ hydroquinone; HRP horseradish peroxidase; POD peroxidase; ROS reactive oxygen species.

Introduction Plant mitochondria generate reactive oxygen species (ROS), O2·– and H2O2, under normal conditions (Boveris 1984, Puntarulo et al. 1991, Møller 2001) and especially when the plants are subjected to biotic or abiotic stress (Hernández et al. 1993, Prasad et al. 1995, Dixit et al. 2002, Mittova et al. 2004). Also, increased ROS production has been observed during leaf and plant senescence (Droillard and Paulin 1990), aging, and fruit ripening (Purvis et al. 1995). When ROS production exceeds the capacity of the detoxification and repair pathways, oxidative damage to proteins, DNA, and phospholipids occurs, disrupting miCorrespondence: M. Vuletic´, Laboratory of Plant Physiology, Maize Research Zemun Polje, P.O. Box 89, 11185 Belgrade, Serbia. E-mail: [email protected]

tochondrial oxidative phosphorylation and leading to cell damage and death (Halliwell and Gutteridge 1989). Therefore, plant mitochondria have developed regulatory systems for the avoidance of ROS production, as well as antioxidative systems for ROS scavenging. ROS production can be avoided by using alternative oxidase and nonproton pumping NAD(P)H dehydrogenase activities (Purvis 1997, Maxwell et al. 1999, Vanlerberghe et al. 2002). The mitochondrial systems for ROS scavenging that have been identified are Mn-superoxide dismutase (Mn-SOD) (Scandalios 1993, Streller et al. 1994), nonspecific guaiacol peroxidases (POD) (Prasad et al. 1995, Iturbe-Ormaetxe et al. 2001, Hadzˇi-Tasˇkovic´ Sˇukalovic´ and Vuletic´ 2003, Mittova et al. 2004), and ascorbate and glutathione that act as POD substrates or as the components of the other antioxidative enzymatic systems (Jiménez et al. 1997, Mittova et al. 2004). In contrast to leaf mitochondria (Nakano and Asada 1981, Jiménez et al. 1997, Mittova et al. 2000), for roots the role of the mitochondrial antioxidant system in the protection against oxidative stress has rarely been investigated. Root mitochondria, like those in leaves, contain Mn-SOD for O2·– scavenging (Malecka et al. 2001, Mittova et al. 2004). However, information concerning the root mitochondrial H2O2 scavenging system(s) is scarce. Recently, guaiacol POD (EC activity has been identified in root mitochondria (Hadzˇi-Tasˇkovic´ Sˇukalovic´ and Vuletic´ 2003), in addition to that of ascorbate POD (EC (Mittova et al. 2004), and the role of PODs in the control of mitochondrial H2O2 content under salt stress has been documented (Mittova et al. 2004). Although a dual role of mitochondrial guaiacol POD(s) in H2O2 scavenging, involving oxidation of phenolics by H2O2


V. Hadzˇi-Tasˇkovic´ Sˇukalovic´ et al.: Hydroquinone peroxidase of maize root mitochondria

and the cooxidation of NAD(P)H, has been demonstrated in vitro (Hadzˇi-Tasˇkovic´ Sˇukalovic´ and Vuletic´ 2003), the natural physiological substrates have not been identified. The discovery of POD (EC associated to thylakoid membranes, which has a high affinity for hydroquinone (HQ) (Zapata et al. 1998, Casano et al. 2000), and the substratelike properties of the mitochondrial coenzyme Q (Estornell et al. 1992) prompted us to examine whether hydroquinones could also be natural substrates for mitochondrial POD(s). Oxidation of HQ with H2O2 in the presence of mitochondria isolated from maize roots and the identification of possible systems involved in HQ regeneration, such as enzymatic oxidation of NAD(P)H and nonenzymatic cooxidation of ascorbate, were investigated in vitro in the present study. Material and methods Plants and growth conditions Maize (Zea mays L.) inbred line VA35 seeds were germinated for three days and then transferred into plastic pots containing Knopp solution, with modified nitrogen content. For the first seven days, plants were grown on one-quarter strength nutrient solution and on full strength solution over the following four days. Nitrogen was supplied in the form of KNO3, Ca(NO3)2, and (NH4)2SO4. The concentrations of NO3– and NH4+ in the full-strength solution were 10.9 and 7.2 mM, respectively. The initial pH of the solution was adjusted to 5.6. Plants were kept in a growth chamber at a 12 h photoperiod at 22 °C at light and 18 °C at dark, with an irradiance of 40 W/m2 and a relative humidity of 70%. In addition, in some experiments, maize seedlings were grown for four days in the dark on moistened filter paper.

Unless otherwise noted, the spectrophotometric assay of POD was performed at 30 °C in a 1 ml assay volume containing 1 mM HQ and 5–10 g of mitochondrial protein in 50 mM phosphate buffer, pH 7.5. The reaction was initiated by adding 2.5 mM H2O2. The increase in absorbance at 250 nm was measured over a time period of 1 min. The oxidation rate was expressed as micromoles of BQ (benzoquinone) per minute with an extinction coefficient of 1.9  104 M1 cm1 (Zapata et al. 1998). NAD(P)H oxidation by the HQ POD system was measured at 340 nm in 1 ml of assay mixture containing the same concentrations of reactants as for the HQ POD reaction. NAD(P)H (0.2 mM) was added to the assay mixture after HQ oxidation by H2O2 or at the beginning of HQ oxidation. The oxidative activity of POD was determined in 50 mM phosphate buffer at pH 5.5 by monitoring the decrease in NADH absorbance at 340 nm in the assay mixture, which contained 0.2 mM NADH, 0.2 mM p-coumaric acid, 0.25 mM MnCl2, and 5 g of mitochondrial protein in a volume of 1 ml. The standard guaiacol test (Chance and Maehly 1955) for the determination of the peroxidative activity of POD was used. This involved measuring absorbance at 436 nm in the presence of H2O2 and 10 g of mitochondrial protein in 1 ml of 50 mM phosphate buffer at pH 6.5. The protein content was measured by the method of Lowry et al. (1951) with bovine serum albumin as standard.

Zymographic assays Mitochondria (about 40 g of protein) were disrupted with the addition of 1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-2-hydroxy-1-propanesulfonate. Isoelectric focusing of mitochondrial PODs was performed in a 7.5% polyacrylamide gel with 3% ampholite on a pH gradient running from 3.6 to 9.3. To determine the POD activity, the gel was incubated with 10% 4-chloro-1-naphthol and 0.03% H2O2 in 100 mM K-phosphate buffer (pH 6.5). The reaction of PODs with HQ was demonstrated using an incubation medium identical to that described in Zapata et al. (1992). The reaction was performed at 30 °C for 10 min. After removal of the HQ-containing media, the staining reaction was developed with 50 mM 4-aminoantipyrine in 0.1 M HCl until the color appeared. Unless otherwise noted, assays were performed with mitochondria isolated from the 2-week-old maize roots.

Isolation and purification of mitochondria Mitochondria were isolated from 2-week-old roots or seedling roots and shoots by the procedure of Schwitzguebel and Siegenthaler (1984) and purified by differential and Percoll gradient centrifugation. The discontinuous Percoll gradient was made of five layers prepared with 5 ml of 50%, 10 ml of 45%, 5 ml of 27%, 5 ml of 20%, and 5 ml of 13.5% Percoll, with polyvinylpyrrolidone added as described by Hadzˇi-Tasˇkovic´ Sˇukalovic´ and Vuletic´ (1998). The mitochondrial band was collected at the 27/45% interface. The activity of cytochrome c-dependent cytochrome oxidase (EC in the absence and presence of 0.025% (v/v) Triton X-100 was measured, in order to determine the intactness of mitochondria (Tolbert 1974). Possible contamination with microbodies and plastids was determined by analyzing the activities of the marker enzymes catalase (EC, according to Aebi (1974), and phosphogluconate dehydrogenase (EC, according to Journet and Douce (1985), as previously described (Hadzˇi-Tasˇkovic´ Sˇukalovic´ and Vuletic´ 1998). As in our previous study (Hadzˇi-Tasˇkovic´ Sˇukalovic´ and Vuletic´ 1998), the low specific activities of catalase and phosphogluconate dehydrogenase observed (0.091 and 0.062 mol per mg of protein per min, respectively) excluded the possibility of contamination of our isolates with peroxisomes and plastids. Enzyme assays and protein determination To determine enzymatic activity of POD (EC, the mitochondria were ruptured by three freeze-thaw cycles prior to the assay.

Results Characterization of the oxidation of HQ by mitochondrial POD Oxidation of HQ by a mitochondrial isolate from 2-weekold maize roots was monitored spectrophotometrically in the 200–400 nm region (Fig. 1). In the presence of H2O2, characteristic spectral changes for HQ oxidation (absorbance decrease at 290 nm) and BQ formation as the end-product (absorbance maximum at 250 nm) were obtained. The appearance of an isosbestic point at 268 nm in the consecutive spectra suggested that there was a constant transformation of HQ to BQ, the two species being kinetically correlated because very unstable semiquinone radicals are produced when HQ is oxidized by POD, which rapidly dismutate to give BQ and HQ (Beckman and Siedow 1985). The inability of the mitochondrial isolate to oxidize HQ in the absence of H2O2 (not shown)

V. Hadzˇi-Tasˇkovic´ Sˇukalovic´ et al.: Hydroquinone peroxidase of maize root mitochondria

Fig. 1. Spectrophotometric recording of the oxidation of HQ by mitochondria isolated from maize root (A) or horseradish peroxidase (B) induced by H2O2. Consecutive scans were recorded every 60 s after the addition of H2O2 (scan 1)

and the fact that the same spectral changes were observed with horseradish peroxidase (HRP) (Fig. 1) suggest that the oxidation of HQ to BQ might be attributed to POD activities. In addition, appropriate controls of chemical oxidation by H2O2 did not show significant changes. In order to characterize the peroxidative oxidation of HQ by mitochondrial isolates, the oxidation rate (estimated from the increases in absorbance at 250 nm) was determined at different pH values and H2O2 and HQ concentrations. Variation of the pH in the 5.0–8.0 range, using 50 mM phosphate buffer, did not significantly modify the HQ oxidation rate (data not shown). Since the pH within mitochondria is neutral, pH 7.5 was used in all subsequent kinetic studies. The dependence of the HQ oxidation rate on H2O2 and HQ concentrations showed


Fig. 2 A, B. Activity of mitochondrial HQ POD as a function of substrate concentrations. A HQ in the presence of 2.5 mM H2O2. B H2O2 in the presence of 1 mM HQ. Each data point is the mean from three experiments

Michaelis–Menten-type kinetics for mitochondrial POD activities in both cases (Fig. 2). Although valid Km values cannot be defined for oxidation catalyzed by PODs (Zapata et al. 1992), apparent Km values were determined. The activity of POD was determined in mitochondria isolated from seedling shoots and roots as well as in isolates from the 2-week-old roots with HQ and guaiacol as reductants. Significantly higher activities were obtained in the 2-week-old roots compared to seedling shoots and roots (Table 1) with both substrates. To further characterize HQ POD, its sensitivity to some inhibitors and effectors was investigated. The results presented in Table 2 show that a total inhibitory effect was obtained with 1 mM KCN, as well as with the peroxidase substrates guaiacol and 4-chloro-1-naphthol, when added

V. Hadzˇi-Tasˇkovic´ Sˇukalovic´ et al.: Hydroquinone peroxidase of maize root mitochondria


Table 1. Specific activity of POD from mitochondrial isolates Source of mitochondria

Seedling shoots Seedling roots 2-week-old roots

POD activity with substrate: Hydroquinone a

Guaiacol b

0.425  0 0.161  0.02 7.32  1.2

ND c 0.51  0.03 4.15  0.6


Values are given as micromoles of BQ per milligram of protein per minute; means with standard errors of duplicate assays from at least two isolations b Values are given as micromoles of tetraguaiacol per milligram of protein per minute; means with standard errors of duplicate assays from at least two isolations c ND, not detectable Table 2. Influence of inhibitors and effectors on HQ POD activity a Treatment

HQ POD activity (%)

None 1 mM KCN 0.02 mM cysteine 0.1 mM cysteine 0.1 mM glutathione (reduced) 0.05 mM NAD(P)H 4.5 mM guaiacol b 0.1 mM 4-chloro-1-naphthol b

100 0 34.3 19.5 11.2 100 0 0

Assays were performed with 5 g of mitochondrial protein isolated from 2-week-old maize roots in standard HQ POD assay conditions. The results are given as percentage of activity without treatment b Added during the oxidation of HQ a

during HQ oxidation. Thiol-type inhibitors induced partial inhibition of HQ POD. The presence of 0.02 mM cysteine induced approximately 66% inhibition, while 0.1 mM cysteine or 0.1 mM reduced glutathione induced approximately 80% and 89% inhibition of enzyme activity, respectively. No inhibitory effects of NADH were detected when it was present in a standard assay mixture. During HQ oxidation with H2O2 in the presence of mitochondrial isolates, simultaneous NADH oxidation was detected as absorbance changes at 340 nm (Fig. 3). The rate of NAD(P)H oxidation was dependent on the quantity of mitochondrial protein (data not presented) and was higher when NAD(P)H was added following the completion of HQ oxidation (NADH, 3.26  0.1; NADPH, 2.19  0.01 mol per mg of protein per min; means with standard errors, n  4) than when it was added at the start of the assay (NADH, 1.86  0.0; NADPH, 1.79  0.0 mol per mg of protein per min). Also, KCN exhibited no inhibitory effect on NADH oxidation when added after the completion of the POD reaction (NADH, 3.42 

Fig. 3. Spectral changes during POD-catalyzed oxidation of HQ in the presence of 0.2 mM NADH. Consecutive scans were recorded every 60 s after the addition of H2O2 (scan 1)

0.0 mol per mg of protein per min). Control assays without HQ or H2O2 did not show significant absorbance decreases. The oxidation of NAD(P)H by BQ in the presence of mitochondrial isolates was also detected (data not presented). The presence of ascorbate in the assay mixture delayed POD-dependent oxidation of HQ. The spectral changes due to HQ oxidation in a mixture of HQ and ascorbate, which was added to the mitochondrial isolate and H2O2, were completely suppressed during ascorbate oxidation (Fig. 4A). Oxidation of HQ started following total exhaustion of the reduced ascorbate pool (Fig. 4 inset), and spectral changes (Fig. 4B) were comparable to those obtained during oxidation of HQ without ascorbate (Fig. 1). The duration of ascorbate oxidation, preceding POD-dependent oxidation of HQ, increased with increasing ascorbate concentration without influencing the HQ oxidation rate (data not shown). The oxidation of ascorbate with H2O2 was not possible in the absence of HQ. A possible prooxidative role of mitochondrial HQ POD (generating H2O2 in the presence of NAD(P)H, and mediating the reduction of O2 to O2·–) was also investigated. When HQ was used as a phenolic cofactor in the oxidative cycle of POD, oxidation of NAD(P)H could not be detected. Furthermore, NADH oxidation in the assay with p-coumaric acid was inhibited with 1 mM HQ (0.75  0.01 mol of NADH per mg of protein per min; with p-coumaric acid as the only cofactor, 12.05  0.0 mol of NADH per mg of protein per min; means with standard errors, n  3).

V. Hadzˇi-Tasˇkovic´ Sˇukalovic´ et al.: Hydroquinone peroxidase of maize root mitochondria


Fig. 5. Comparative pattern of mitochondrial POD after protein separation by isoelectric focusing and staining with 4-chloro-1-naphthol (A) and HQ (B). Solid arrows indicate PODs and the dashed arrow indicates the well where sample was applied

the PODs located in the mitochondria were anionic and capable of oxidizing HQ to BQ. Discussion

Fig. 4 A, B. Spectral changes during POD-catalyzed oxidation of HQ in the presence of 0.1 mM ascorbate. A Continuous oxidation of ascorbate. Consecutive scans were recorded every 60 s after the addition of H2O2 (scan 1). B Oxidation of HQ after the total exhaustion of ascorbate. Consecutive scans were recorded every 60 s, the first scan being recorded 180 s after H2O2 addition. Inset Time course of ascorbate (265 nm) and HQ (250 nm) absorbance changes

Isoenzymes As PODs exist in multiple molecular forms with different affinities for substrates, we performed different staining techniques on PODs separated by isoelectric focusing on polyacrylamide gels. The comparative isoenzyme pattern of mitochondrial PODs obtained after staining with 4chloro-1-naphthol and HQ (Fig. 5) revealed that almost all

The results presented demonstrate that maize root mitochondrial POD can use HQ as a substrate in the peroxidative cycle of the enzyme reaction, in addition to the artificial compound guaiacol (for which this enzyme is named) and a variety of phenolic substrates recently reported by Hadzˇi-Tasˇkovic´ Sˇukalovic´ and Vuletic´ (2003). The identical spectral changes obtained when using the mitochondrial isolate or horseradish peroxidase (Fig. 1), which corresponded to published data (Zapata et al. 1998), identified BQ as the end-product of HQ oxidation with H2O2. The high specific activity of POD observed in mitochondria isolated from 2-week-old maize roots, as opposed to the low activity detected in seedling roots or shoots with either POD substrate (Table 1), as well as the inhibitory effect of guaiacol and 4-chloro-1-naphthol on HQ POD activity (Table 2), pointed to a similarity between the HQ POD and guaiacol POD enzymes. In addition, the identical zymograms obtained with 4-chloro1-naphthol (a specific substrate of PODs not oxidizable by other heme proteins) and with HQ showed that all mitochondrial POD isozymes can use HQ as a substrate. As guaiacol PODs have been found within the mitochondria (Hadzˇi-Tasˇkovic´ Sˇukalovic´ and Vuletic´ 2003), both membrane-bound (Prasad et al. 1995, Iturbe-Ormaetxe et al. 2001) and in the matrix (Iturbe-Ormaetxe et al. 2001), the possible contamination of this activity with soluble POD was excluded.


V. Hadzˇi-Tasˇkovic´ Sˇukalovic´ et al.: Hydroquinone peroxidase of maize root mitochondria

Mitochondrial PODs have been shown to act as bifunctional enzymes possessing peroxidative and oxidative activities. The oxidative activity, mediating the reduction of oxygen to O2·– and H2O2, with NAD(P)H as a substrate in the presence of salicylhydroxamic acid and p-coumaric acid as phenolic cofactors, was demonstrated by Hadzˇi-Tasˇkovic´ Sˇukalovic´ and Vuletic´ (2003). As peroxidases oxidize HQ in a way similar to that of a wide range of phenolics (Beckman and Siedow 1985), a possible prooxidative effect of a derived semiquinone radical (prior to its dismutation to p-BQ) could be envisaged. In addition, a dual role for natural mitochondrial quinone, ubiquinone, in ROS production and ROS protection has been proposed (Beyer 1992, Wagner and Purvis 1998). However, our results excluded an oxidative activity of mitochondrial POD using HQ as a cofactor. Moreover, HQ inhibited the oxidative cycle of POD when present together with p-coumaric acid as a cofactor. Thus, our results demonstrate that mitochondrial HQ POD possesses only the antioxidative activity. The kinetic analysis of HQ POD revealed high Km values for HQ (0.353  0.06 mM) and H2O2 (0.415  0.08 mM), raising the question of its physiological role in mitochondria. We propose that this enzyme controls the concentration of H2O2 produced in the older root tissue, or under stress conditions, by scavenging H2O2 in mitochondria that lack catalase. The radical-scavenging function of reduced ubiquinone, which protects mitochondria from damage when under stress, proposed by Wagner and Purvis (1998), could be extrapolated to HQ and/or other reduced quinones. For continuous H2O2 scavenging using POD, it would be important to maintain a pool of reduced quinone. Our in vitro experiments demonstrated that NAD(P)H could be an electron donor for the reduction of quinone in a HQ-POD-H2O2coupled reaction system. However, this system is different from the nonenzymatic cooxidation of NAD(P)H that delayed oxidation of phenolic substrates used in the peroxidative reaction (Hadzˇi-Tasˇkovic´ Sˇukalovic´ and Vuletic´ 2003). The continuous oxidation of NADH occurring simultaneously with HQ oxidation (Fig. 3), which is dependent on the amount of mitochondrial protein used, as well as the NADH oxidation by BQ in the presence of mitochondria, point to a possible enzymatic electron transfer from NAD(P)H to BQ as the end-product of the HQ POD reaction. The increased rate of NAD(P)H oxidation upon completion of the POD reaction, which was not inhibited by KCN, indicates that some enzyme other than POD is involved. We suggest that some of the mitochondrial dehydrogenases could catalyze this electron transfer from NAD(P)H to BQ. In addition, maintenance of the reduced-quinone pool during H2O2 scavenging could be achieved through an

H2O2-HQ-ascorbate system. Since ascorbate was oxidized in vitro by the mitochondrial isolate in the presence of exogenous H2O2 only upon addition of HQ, the oxidation of ascorbate by ascorbate POD was excluded. The oxidation of ascorbate in an H2O2-HQ-ascorbate system is similar to ascorbate cooxidation by the POD-phenolic system, which has been widely examined in many other cell compartments (Otter and Polle 1995, Hadzˇi-Tasˇkovic´ Sˇukalovic´ et al. 2003, Pérez et al. 2002), or by HRP (Galati et al. 2002), as well as cooxidation of ascorbate and HQ catalyzed by HRP (Rogozhin and Verkhoturov 1999). In this reaction, during delayed HQ oxidation, ascorbate is an electron donor to the semiquinone radical before its rapid dismutation to yield the stable product BQ. According to our findings, reductants other than ascorbate (e.g., NAD(P)H, cysteine, and reduced glutathione) were not able to constitute a similar system. However, thiol reagents induced strong HQ POD inhibition, similar to HRP (Sariri et al. 2006) or membrane-bound peroxidase (Onsa et al. 2004) inhibition. Proposed physiological significance of the HQ POD reaction The results presented above led us to propose a two-reaction model for H2O2 scavenging within root mitochondria that includes HQ (Fig. 6). Coupled reactions catalyzed by POD and NAD(P)H dehydrogenase(s), which, besides H2O2 scavenging, regulate the redox state of HQ and NAD(P)H pools, thus contributing to the overall mechanism protecting from oxidative damage. Cooxidation of ascorbate represents an effective regulatory system in H2O2 scavenging that recycles semiquinone radicals derived from the POD reaction. The primary oxidation product of ascorbate is a monodehydroascorbate

Fig. 6. Two-reaction model for H2O2 scavenging

V. Hadzˇi-Tasˇkovic´ Sˇukalovic´ et al.: Hydroquinone peroxidase of maize root mitochondria

(MDA) radical, which disproportionates spontaneously to dehydroascorbate (Takahama and Oniki 1997). This system is different from that of the ascorbate POD enzyme, also present in root mitochondria (Mittova et al. 2004), and can be considered to have a more important role than just its antioxidative one, since it consecutively regulates the redox state of ASC/DHA (and possibly glutathione), maintaining quinones in the reduced form. The fact that the last step of ascorbate biosynthesis (Bartoli et al. 2000) occurs in mitochondria, together with the system for regeneration of ascorbate (Jiménez et al. 2002), makes ascorbate cooxidation by HQ POD possible in vivo. It can be speculated that mitochondrial HQ POD plays a role in the protection of the root tissue from oxidative stress. Due to H2O2 diffusion across mitochondrial membranes, the same mitochondrion (or any part of it) may act alternatively as either a sink or a source of H2O2. Different roles of mitochondria in the recognition of the stress signal, H2O2, which are dependent on the stress severity, have been proposed by Vacca et al. (2004). In plants exposed to mild stress, mitochondria represent an important element in the stress-signaling network, but in cells affected by severe stress, the amplification of H2O2 production leads to programmed cell death. Our results, which demonstrate that HQ POD activity is consistent with guaiacol POD activities, and the lack of ascorbate POD, indicate a dominant role for the HQ POD system in H2O2 scavenging in mitochondria from older maize roots. Whether or not such a system functions in other mitochondria and under different stress conditions remains to be investigated. To conclude, the results presented in this paper show that mitochondrial PODs may use HQ as a reductant. Their role in H2O2 scavenging, but not in H2O2 production, has been demonstrated. Thus, in accordance with our in vitro experiments, an antioxidative role of mitochondrial HQ POD in the regulation of H2O2 content within root mitochondria is proposed.

Acknowledgments This work was supported by the Ministry of Science and Environmental Protection (Republic of Serbia), Project 143020.

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