Inactivation of genes, encoding tocopherol biosynthetic pathway enzymes, results in oxidative stress in outdoor grown Arabidopsis thaliana

Plant Physiology and Biochemistry 47 (2009) 384–390

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Research article

Inactivation of genes, encoding tocopherol biosynthetic pathway enzymes, results in oxidative stress in outdoor grown Arabidopsis thaliana Nadia M. Semchuk a, Oleh V. Lushchak a, Jon Falk b, Karin Krupinska b, Volodymyr I. Lushchak a, * a b

Department of Biochemistry, Precarpathian National University named after Vassyl Stefanyk, 57 Shevchenko Str., Ivano-Frankivsk 76025, Ukraine Institute of Botany, Christian-Albrechts University of Kiel, Olshausenstrasse 40, 24098 Kiel, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 June 2008 Accepted 30 January 2009 Available online 12 February 2009

Tocopherols (a-, b-, g- and d-tocopherols) represent a group of lipophilic antioxidants which are synthesized only by photosynthetic organisms. It is widely believed that protection of pigments and proteins of photosynthetic system and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species (ROS) is the main function of tocopherols. The wild type Columbia and two mutants of Arabidopsis thaliana with T-DNA insertions in tocopherol biosynthesis genes – tocopherol cyclase (vte1) and g-tocopherol methyltransferase (vte4) – were analyzed after long-term outdoor growth. The concentration of total tocopherol was up to 12-fold higher in outdoor growing wild type and vte4 plant lines than in plants grown under laboratory conditions. The vte4 mutant plants had a lower concentration of chlorophylls and carotenoids, whereas the mutant plants had a higher level of total glutathione than of wild type. The activities of antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate oxidase (AO, EC 1.10.3.3) were lower in both mutants, whereas activities of catalase (EC 1.11.1.6) and ascorbate peroxidase (APx, EC 1.11.1.11) were lower only in vte1 mutant plants in comparison to wild type plants. However, the activity of guaiacol peroxidase (GuPx, EC 1.11.1.7) was higher in vte1 and vte4 mutants than that in wild type. Additionally, both mutant plant lines had higher concentration of protein carbonyl groups and oxidized glutathione compared to the wild type, indicating the development of oxidative stress. These results demonstrate in plants that tocopherols play a crucial role for growth of plants under outdoor conditions by preventing oxidation of cellular components. Ó 2009 Elsevier Masson SAS. All rights reserved.

Keywords: Antioxidant enzymes Arabidopsis thaliana Chlorophylls Glutathione Oxidative stress Tocopherols

1. Introduction Reactive oxygen species (ROS), namely superoxide anion (O2),  lipid peroxides (ROO ), hydrogen peroxide (H2O2), hydroxyl radical  ( OH) and singlet oxygen (1O2) are continuously produced during metabolism in aerobic organisms. High levels of ROS are produced by photosynthetic organisms, because they accumulate in their green tissues high levels of oxygen by photosynthesis. Therefore, photosynthetic organisms have well-developed enzymatic and non-enzymatic defense systems, which both limit the formation of ROS and their harmful action [32]. The photosynthetic electron transport chain is one of the main sources of ROS in plants [10]. Tocopherols (a-, b-, g- and d-tocopherols) are lipophylic antioxidants synthesized only by photosynthetic organisms [1,27,44]. The 

Abbreviations: AO, ascorbate oxidase; APx, ascorbate peroxidase; CP, protein carbonyl groups; FW, fresh weight; GSSG, oxidized glutathione; tot GSH, total glutathione; GuPx, guaiacol peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase. * Corresponding author. Tel./fax: þ380 342714683. E-mail address: [email protected] (V.I. Lushchak). 0981-9428/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2009.01.009

biosynthesis of tocopherols takes place in chloroplasts where they do accumulate in all membranes. Therefore, it has been suggested that tocopherols are involved in the protection of pigments and proteins of the photosynthetic apparatus and polyunsaturated fatty acids against ROS generated during photosynthesis [1,24,34,45]. Tocopherols are synthesized from precursors derived from two metabolic pathways: homogentisic acid, an intermediate in aromatic amino acid degradation, and phytyl diphosphate, which arises from methylerythritol phosphate pathway (Fig. 1). d- and g-Tocopherols are produced from respective intermediates 2methyl-6-phytyl-1,4-benzoquinol (MPBQ) and 2,3-dimethyl-6phytyl-1,4-benzoquinol (DMPBQ) via reactions catalyzed by a tocopherol cyclase (TC, encoded by gene VTE1) [41]. A g-tocopherol methyltransferase (g-TMT, EC 2.1.1.95), encoded by gene VTE4, converts d- and g-tocopherols to a- and b- tocopherols, respectively [9]. All four types of tocopherols (a-, b-, g- and dtocopherols) consist of a chromanol head group attached to the phytyl tail and differ in the number and positions of methyl groups at the chromanol ring. The leaves of many higher plants contain predominately a-tocopherol, whereas g-tocopherol is

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Fig. 1. The tocopherol biosynthetic pathway in A. thaliana. Abbreviations: HGA, homogentisic acid; PDP, phytyl diphosphate; MPBQ, 2-methyl-6-phytyl-1,4-benzoquinol; DMPBQ, 2,3-dimethyl-6-phytyl-1,4-benzoquinol; MT, MPBQ methyltransferase; TC (VTE1), tocopherol cyclase; g-TMT (VTE4), g-tocopherol methyltransferase.

most abundant in seeds. The other two tocopherol derivatives band d-tocopherols are only present in minor amounts in seeds and leaves [9,27]. In this study we analyzed two tocopherol-deficient mutants of Arabidopsis thaliana, the tocopherol cyclase mutant vte1 and the g-tocopherol methyltransferase mutant vte4, derived from Columbia, which were grown outdoor. Both kinds of mutations were described previously [9,41]. So far, no data are available on the performance of such mutants during growth under outdoor conditions. In this study, oxidative damage to proteins as well as the levels and activities of different antioxidant systems was compared between the vte1 and vte4 mutants and the wild type after 7 months of growth in the field.

2.2. Concentrations of pigments The concentrations of total chlorophyll, carotenoids and anthocyanins are given in Table 2. The total chlorophyll concentration was found to be reduced significantly in the vte4 mutant plants and only slightly in vte1 plants compared to the wild type. The inactivation of the vte4 gene resulted in a lower chlorophyll a concentration of about 23% relative to the wild type, but the vte1 defect did not affect the concentration of the pigments (data not shown). The concentration of chlorophyll b was 24% lower in the vte1 mutant that hence had a higher chlorophyll a/b ratio than the other lines (Table 2). The concentration of another pigment, the vacuolar anthocyanins, was virtually the same in all three A. thaliana lines used.

2. Results 2.1. Tocopherol content Analyses of the concentrations of a- and g-tocopherols in wild type and the two knockout mutants showed that a-tocopherol is present only in wild type plants, while g-tocopherol is present both in wild type and vte4 plants (Table 1). The total amount of tocopherols, however, was virtually the same in wild type and vte4 plants as described before [9,41].

Table 1 The concentrations of tocopherols in wild type and vte4, vte1 mutant lines of A. thaliana. Data are means  SEM (n ¼ 6). FW, fresh weight. Plant lines

a-Tocopherol (mg/g FW)

g-Tocopherol (mg/g FW)

Wild type vte4 vte1

86.1  13.3 0 0

2.20  0.46 94.0  10.2 0

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Table 2 The concentrations of pigments in vte4, vte1 mutant lines in comparison to the wild type of A. thaliana. Data are means  SEM (n ¼ 6) and ‘‘wt’’, ‘‘vte1’’ indicates the significant difference from respective group of wt (P < 0.05) and vte1 type (P < 0.005). FW, fresh weight. Plant lines

Total chlorophyll (mmol/g FW)

Chl a/Chl b ratio

Carotenoids (mmol/g FW)

Anthocyanins (mmol/g FW)

Wild type vte4 vte1

0.89  0.07 0.69  0.04wt,vte1 0.79  0.03

3.2 3.2 3.9

0.29  0.03 0.22  0.01wt,vte1 0.29  0.01

0.43  0.04 0.40  0.03 0.36  0.02

to wild type (Fig. 5A, B). Activities of ascorbate peroxidase in vte4 plants were similar to wild type plants, while the activities of AO in vte4 plants were intermediate between wild type and the vte1 mutant. Tocopherol deficiency may not only cause oxidative stress in plants, but also induce adaptive response of the antioxidant system. In order to check this issue we measured the activities of one another hydrogen peroxide detoxifying enzyme, namely guaiacol peroxidase (GuPx). The activity of this enzyme in mutant lines vte4 and vte1 was 1.6- and 3.6-fold higher, respectively, than in wild type plants (Fig. 6). 3. Discussion

2.3. Concentration of protein carbonyl groups and glutathione The concentration of protein carbonyl groups (CP), a measure of oxidative modification of proteins, in vte1 and vte4 plants was nearly 1.4-fold higher than in wild type plants (Fig. 2). The concentrations of total (A) and oxidized (B) glutathione as well as their ratio (C) were measured in leaves of both mutants and the wild type. The inactivation of vte1 and vte4 genes resulted in 1.4fold and 1.7-fold higher concentrations of total and oxidized glutathione, respectively, in both mutant lines compared to the wild type. Consequently, both mutants had 1.2–1.3-fold higher [GSSG]/[tot GSH] ratio (Fig. 3C), which is indicating the development of oxidative stress in plants with inactivated vte1 and vte4 genes. 2.4. Activities of enzymes of the antioxidant system In addition to the levels of antioxidants the activities of the major enzymes of the antioxidant system of plants, namely superoxide dismutase (SOD) and catalase, were investigated in all three lines of A. thaliana (Fig. 4). The activities of SOD were 56 and 59% lower in vte4 and vte1 plants, respectively, as compared to those in wild type (Fig. 4A). However, catalase activity was virtually the same in wild type and vte4 plants, but in vte1 plants it was reduced by 17% (Fig. 4B). Because tocopherols operate in concert with glutathione and ascorbate, we further studied the activities of two enzymes of ascorbate metabolism – ascorbate peroxidase (APx) and ascorbate oxidase (AO) in all three lines of A. thaliana. Surprisingly, the activities of both enzymes were lower in vte1 mutant plants than those in wild type plants: for APx – 67% and for AO – 57% compared

Fig. 2. The concentration of protein carbonyl groups in wild type, vte1 and vte4 plants of A. thaliana. Data are means  SEM (n ¼ 6). aSignificantly different from respective group of wild type plants (P < 0.05).

Arabidopsis leaves normally accumulate only a-tocopherol like most plants [13]. The vte1 mutant that is deficient in tocopherol cyclase activity, lacks all tocopherols but accumulates the tocopherol biosynthetic pathway intermediate 2,3-dimethyl6-phytyl-1,4-benzoquinol (DMPBQ) at a level comparable to atocopherol in the wild type [25,33,41,44]. The vte4 mutant is defective in g-tocopherol methyltransferase activity and devoid of a-tocopherol, but accumulates instead g-tocopherol in the leaves [9,15,25,33]. The total tocopherol content in leaves of the vte4 mutant line did not differ from that of the wild type (Table 1). This is in the accordance with previous reports indicating that g-tocopherol could at least partially take over the function of a-tocopherol [9,25,33]. However, we found that in wild type and vte4 plant lines of A. thaliana, growing outdoor, the concentration of total tocopherol was up to 3- and 12-fold higher than those in tobacco and A. thaliana plants respectively, that were grown under laboratory conditions [1,9,29]. Tocopherol levels in plants were shown to change during the development and under different abiotic stresses [14,21,35]. Significantly increased a-tocopherol levels were observed in leaves during aging and senescence as well as in response to stress [13,14,25,29]. Accordingly, an enhanced tolerance towards drought stress was observed in transgenic tobacco plants overexpressing VTE1 [29]. The higher tocopherol concentration in outdoor grown plants versus indoor grown ones suggests that tocopherols play an important role in plant protection against stressful outdoor conditions. To explore whether tocopherols under outdoor conditions have a protective effect on the photosynthetic apparatus, we measured chlorophyll concentrations. The total chlorophyll concentrations in wild type and vte1 mutant lines were very similar (Table 2). This is in agreement with indoor studies [37,41], suggesting that the absence of all forms of tocopherols had no significant impact on photosynthesis. Moreover, in a recent study with the vte1 mutant of A. thaliana it has been suggested that the biosynthetic precursor of tocopherols, DMPBQ, could serve as an antioxidant and functionally replace tocopherols [22,24,25]. During outdoor growth, however, the total chlorophyll content in vte4 mutant plants was lower than in the wild type (Table 2), whereas under laboratory growth these parameters were similar in both lines of A. thaliana and tobacco plants [9]. It is likely that a higher ROS level is responsible for degradation of photosynthetic pigments in vte4 mutant plants. It is well known that a-tocopherol is a more active antioxidant than g-tocopherol [9,27]. It also plays the crucial role in the protection of the photosynthetic apparatus by scavenging singlet oxygen. ROS can react with different cellular components, such as nucleic acids, membrane lipids and proteins, and cause their oxidative modification. Such oxidative damages accumulate during the life cycle of many organisms and it can be one of the possible causes of aging [23]. Protein carbonylation is an irreversible oxidative process, which occurs as a result of a direct oxidative

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Fig. 3. The concentrations of total glutathione (A), oxidized glutathione (B) and [GSSG]/[tot GSH] ratio (C) in wild type, vte1 and vte4 plants of A. thaliana. Data are means  SEM (n ¼ 6). aSignificantly different from respective group of wild type plants (P < 0.05).

attack on certain amino acids residues, such as Cys, Lys, Arg, Pro or Thr and may lead to a loss of function of modified proteins [31,50]. During outdoor growth, the level of carbonylated proteins in mutant plants vte1 and vte4 was higher than in the wild type of A. thaliana. This was probably a consequence of the increase in the steady-state ROS level in mutant plants and intensification of free radical-induced protein oxidation. The most important low molecular mass antioxidant in chloroplasts is GSH. It is involved in scavenging of H2O2 and also participates in protection of membranes by regenerating tocopheroxyl radicals to its reduced form [39,45,46]. Together with its oxidized form (GSSG) glutathione maintains the intracellular redox balance. Similarly to tocopherol, the glutathione concentration may increase in response to oxidative stress [10,24]. The concentration of total GSH was higher in both the vte1 and vte4 mutants of A. thaliana compared to the wild type, as already shown before [22,24]. The results indicate that the absence of a-tocopherol in both mutant lines can be compensated by an increase in the total glutathione level. A higher ratio of oxidized glutathione to reduced glutathione [GSSG]/[tot GSH] in mutant plants vte1 and vte4 may be caused by an increased concentration of ROS in these plants. Further components supposed to have antioxidant effects in addition to its coloring function are vacuole localized anthocyanins [42] that are accumulating under different stress conditions [19,33]. We did not measure any difference in the levels of anthocyanins between the mutants impaired in tocopherol biosynthesis and the wild type. This suggests that they don’t play a role for protection of those processes where tocopherols are involved in as protectors. Besides low molecular weight antioxidants, the antioxidant system involves enzymes involved in detoxification of specific ROS  species. The superoxide anion O2 is an abundant ROS, produced mainly in chloroplasts, mitochondria and peroxisomes. Superoxide  dismutase (SOD) is a primary enzymatic scavenger of the O2 radical catalyzing its conversion to H2O2 [6]. Catalases and peroxidases such as ascorbate peroxidase (APx) and guaiacol peroxidase (GuPx) are also the primary H2O2 scavenging enzymes in plants [7]. Catalase converts H2O2 to H2O and O2 [49]. Superoxide dismutases and catalases in concert efficiently eliminate superoxide anions and hydrogen peroxide and indirectly protect plants against the more toxic hydroxyl radical [7]. In the present study, the activities of SOD in vte1 and vte4 mutant lines of A. thaliana were lower than those in wild type plants. Inhibition of SOD activity was observed in many experiments with plants exposed to different stress conditions [3,4]. It has been found that very high levels of H2O2 inhibit Cu,ZnSOD through Cu2þ to Cuþ reduction and in addition hydroxyl

radicals also inhibit Cu,Zn-SOD [7]. The activity of catalases in vte1 mutant plants was lower compared to wild type and vte4 ones. Similarly to SOD, the catalases are also sensitive to ROS, particularly  to O2 and can be inactivated by its increased levels [7]. In addition, other ways may be involved in plant defense against ROS, such as glutathione and ascorbate systems. Interestingly, the highest level of total glutathione was observed in vte1 mutant plants. Similarly, increased GSH concentrations were found in catalase-deficient mutants and plants with catalase activity reduced by antisense technology [38]. Ascorbate peroxidase, which utilizes ascorbate as a specific electron donor to reduce H2O2 to water, is the major enzyme responsible for the elimination of H2O2 in photosynthetic organisms [47]. The rapid inactivation of the enzyme under conditions where an electron donor is absent is one of the characteristic properties of APx, which distinguishes it from guaiacol peroxidase and glutathione peroxidase. In the absence of ascorbate, the heme of the two-electron-oxidized intermediate of APx is decomposed by hydrogen peroxide leading to inactivation of the enzyme [36]. We found that APx activity was lower in the tocopherol-deficient mutant vte1 than that in wild type plants. It is known that in the absence of membrane-associated antioxidant components, particularly tocopherol, ascorbate acts as a primary antioxidant scavenging lipid peroxides [8,45]. This can occur via direct oxidation of

Fig. 4. The activities of superoxide dismutase (A) and catalase (B) in wild type, vte1 and vte4 plants of A. thaliana. Data are means  SEM (n ¼ 6). aSignificantly different from respective group of wild type (P < 0.05) and bvte4 (P < 0.025).

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Fig. 7. Change in aaverage air temperature and bday length during the growth period of the plants used in this study. Fig. 5. The activities of ascorbate peroxidase (A) and ascorbate oxidase (B) in wild type, vte1 and vte4 plants of A. thaliana. Data are means  SEM (n ¼ 6). aSignificantly different from respective group of wild type (P < 0.05) and bvte4 (P < 0.005).

ascorbate. The significant decrease of a total ascorbate level can result in decreased APx activity [47]. Also the level of ascorbate oxidase (AO) that catalyzes the oxidation of ascorbate to monodehydroascorbate in the apoplast [48] was reduced in the mutant lines. However, previously, it was suggested that the AO activity is not connected to the level of ascorbic acid, because AO is located in the apoplast, whereas most of the ascorbic acid pool is in the cytoplasm [40]. Since activities of catalase and APx were reduced in mutants impaired in tocopherol biosynthesis, we searched for alternative enzymes which could be involved in protection against ROS. It is known that in plants also guaiacol peroxidases (GuPx) may participate in H2O2 detoxification. An enhanced activity of this enzyme was observed in response to different types of stresses [3,4]. Similarly to APx, GuPx scavenge H2O2 using plant phenolic compounds, in particular guaiacol (o-methoxyphenol) as an electron donor [18]. In our experiments, the mutation in the vte1 and vte4 genes resulted in an increased GuPx activity in both mutant lines. This result suggests that deficiency in a-tocopherol might result in increased steady-state levels of ROS that is responsible for inactivation of sensitive enzymes such as superoxide dismutase, catalase [7] and ascorbate peroxidase [47] in tocopherol-deficient lines. The reduced activity of catalase and ascorbate peroxidase

seemingly may be compensated by an enhanced guaiacol peroxidase activity. 4. Conclusion The results of this study suggest that under outdoor growth conditions tocopherol deficiency leads to oxidative stress in A. thaliana, as indicated by the increased concentrations of protein carbonyl groups and oxidized glutathione. It seems that tocopherols have an important function for photosynthetic performance and plants growth under outdoor conditions. Under such conditions the lack of a-tocopherol in vte1 and vte4 mutants may be compensated by other antioxidant mechanisms, particularly via increased level of glutathione and activity of GuPx. 5. Materials and methods 5.1. Plant material and growth conditions Seeds of A. thaliana wild type (Columbia) and mutant lines vte1 (GABI_11D07) and vte4 (SALK_03676), defective in vte1 and vte4 genes, respectively, were obtained from the GABI-Kat [43] and Salk Institute [5]. Homozygous plants were selected in the Institute of Botany (Kiel, Germany) and their seeds were used in the present study. All chemicals were obtained from ‘‘Sigma’’ (USA), ‘‘Fluka’’ (Germany) and ‘‘Reakhim’’ (Russia); chemicals were of the maximum purity available. All A. thaliana plants were seeded outdoor on 22nd September 2006 grow slowly during one month because of low temperature and overwintered outdoor. The average temperature in winter fluctuated between zero and þ5  C, although minimal and maximal average temperatures during December–February were 9.3 to þ14.1, 12.4 to þ13.2, 17.6 to þ10.6  C, respectively (Fig. 7). In March and April the average temperature was higher than in December–February and reached 6.4 and 9  C, respectively. Additionally, the day length also changed during growth (Fig. 7). In April the increase of temperature and light day duration induced fast growth and maturation of plants which entered reproductive stage. The biochemical analyses were carried out in April 2007. 5.2. Tocopherol analysis

Fig. 6. The activities of guaiacol peroxidase in wild type, vte1 and vte4 plants of A. thaliana. Data are means  SEM (n ¼ 6). aSignificantly different from respective group of wild type (P < 0.005) and bvte4 (P < 0.025).

For tocopherol analysis the leaves of A. thaliana were homogenized with ice-cold 96% ethanol 1/10 (w/v) and homogenates were centrifuged at 8000g for 10 min. The supernatants were used further. Ethanol was removed by evaporation in a boiling water

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bath during 1 h with nitrogen bubbling. Tocopherol species and concentrations were investigated as described by Falk et al. [17]. Briefly, samples were resolved in n-heptane and after centrifugation at 15,000g, the clear supernatants were taken for analysis by HPLC chromatography. 20 mL of the sample were chromatographically analyzed using a LiChrosphere Si 100 (5 mm) column (10  250 mm) with n-heptane/2-propanol (99.5 þ 0.5) as elutant and with a flow rate of 1.0 mL/min. Tocopherols were detected and quantified using a fluorescence detector (model RF10AXL, Shimadzu) set at lexcitation ¼ 290 nm and lemission ¼ 328 nm. To calibrate the system and verify the identity of individual peaks we used tocopherol standards purchased from Merck (Darmstadt, Germany). 5.3. Pigment measurements For pigment extraction leaves were homogenized in a Potter glass homogenizer with ice-cold 96% ethanol (1/10, w/v) in the presence of CaCO3 to prevent pheophytization. The homogenates were centrifuged at 8000g for 10 min (4  C), supernatants were collected and the pigments were repeatedly extracted twice from pellets with 1 mL ice-cold 96% ethanol. The concentrations of pigments were measured spectrophotometrically in the combined resulting extracts. Specific absorption coefficients for chlorophyll a, chlorophyll b, total chlorophyll and total carotenoids were used [28]. A molecular mass of 570 for carotenoids was used for calculation. Anthocyanin content was determined after extract acidification with concentrated HCl to a final concentration of 1% (v/v). The anthocyanin concentration was assayed spectrophotometrically at 530 nm wavelength and an absorption coefficient of 30 mM1 cm1 was used [19]. 5.4. Carbonyl derivatives of proteins and glutathione measurements The concentration of protein carbonyls was evaluated with 2,4dinitrophenylhydrazine (DNPH) as described earlier [26]. The leaves of A. thaliana were homogenized 1/5 (w/v) in the medium containing 50 mM potassium-phosphate (KPi) buffer (pH 7.0) and 0.5 mM ethylenediamine-tetraacetic acid (EDTA). The homogenates were centrifuged at 15,000g for 10 min at 4  C and protein carbonyls were measured in resulted supernatants. For measurement of glutathione content leaves were homogenized 1/3 (w/v) with ice-cold 5% (w/v) sulfosalicylic acid and then centrifuged at 5000g for 5 min. The total glutathione and oxidized glutathione (GSSG) concentrations were analyzed using glutathione reductase and vinylpyridine as described by Griffith [20]. 5.5. Enzyme assays For determination of enzyme activities the plant leaves were homogenized (1/5, w/v) with medium, containing 50 mM KPi buffer (pH 7.0), 0.5 mM EDTA and a few crystals of phenylmethylsulfonyl fluoride (PMSF), a protease inhibitor. The homogenates were centrifuged at 15,000g for 10 min at 4  C and the resulting supernatants were used for the assays. The activity of superoxide dismutase (SOD) (EC 1.15.1.1) was assayed as a function of its inhibitory action on quercetin oxidation [30]. The reaction mixture contained (final concentrations): 30 mM Tris–HCl buffer (pH 10.0), 0.5 mM EDTA, 0.8 mM N,N,N0 ,N0 -tetramethylethylenediamine (TEMED), 0.05 mM quercetin, and 1–50 mL of supernatant. The reaction was monitored at 406 nm wavelength for 8 different volumes of the supernatant. One unit of SOD activity is defined as the amount of enzyme (per protein milligram) that inhibits the quercetin oxidation reaction by 50% of the maximum inhibition.

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Catalase (EC 1.11.1.6) activity was measured spectrophotometrically at 240 nm wavelength. 2 mL of reaction medium contained (final concentrations): 50 mM KPi buffer (pH 7.0), 0.5 mM EDTA, 10 mM hydrogen peroxide and 25 mL supernatant. The extinction coefficient 39.4 M1 cm1 for hydrogen peroxide was used [2]. Ascorbate oxidase (AO) (EC 1.10.3.3) activity was determined according to Diallinas et al. [16]. The reaction medium contained 50 mM KPi buffer (pH 7.0) and 2.5 mM ascorbic acid. The reaction was initiated by the addition of 20 mL supernatant and followed for 3 min by measuring the decrease in absorbance at 265 nm wavelength due to ascorbate oxidation (3 ¼ 14,000 M1 cm1). The activity of ascorbate peroxidase (APx) (EC 1.11.1.11) was monitored by following the decrease of absorbance at 290 nm wavelength (3 ¼ 2800 M1 cm1) [12]. The reaction mixture contained 50 mM KPi buffer (pH 7.0), 0.5 mM ascorbic acid, 0.3 mM H2O2 and 20 mL supernatant. Guaiacol peroxidase (GuPx) (EC 1.11.1.7) activity was assayed spectrophotometrically following the increase in absorbance at 470 nm wavelength due to guaiacol oxidation (3 ¼ 26,600 M1 cm1) [3]. The 1.5 mL reaction mixture contained 50 mM KPi buffer (pH 7.0), 20 mM guaiacol, 10 mM hydrogen peroxide and 5 mL supernatant. 5.6. Protein measurements and statistics Protein concentration was determined with Coomassie brilliant blue G-250 according to the method of Bradford [11] with bovine serum albumin as a standard. Experimental data are expressed as mean  SEM, and statistical testing used Student’s t-test. Acknowledgements We are grateful to O. Kubrak, T. Nazarchuk, O. Lozinsky and M. Nykorak for technical assistance. References [1] A.R. Abbasi, M. Hajirezaei, D. Hofius, U. Sonnewald, L.M. Voll, Specific roles of a- and g-tocopherol in abiotic stress responses of transgenic tobacco, Plant Physiol. 143 (2007) 1720–1738. [2] H. Aebi, Catalases, in: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, vol. 2, Academic Press, New York, 1984, pp. 673–684. [3] M.B. Ali, E.J. Hahn, K.Y. Paek, Effect of temperature on oxidative stress defense systems, lipid peroxidation and lipoxygenase activity in Phalaenopsis, Plant Physiol. Biochem. 43 (2005) 213–223. [4] M.B. Ali, E.J. Hahn, K.Y. Paek, Effect of light intensities on antioxidant enzymes and malondialdehyde content during short-term acclimatization on micropropagated Phalaenopsis plantlet, Environ. Exp. Bot. 54 (2005) 109–120. [5] J.M. Alonso, A.N. Stepanova, T.J. Leisse, C.J. Kim, H. Chen, P. Shinn, D.K. Stevenson, J. Zimmerman, P. Barajas, R. Cheuk, C. Gadrinab, C. Heller, A. Jeske, E. Koesema, C.C. Meyers, H. Parker, L. Prednis, Y. Ansari, N. Choy, H. Deen, M. Geralt, N. Hazari, E. Hom, M. Karnes, C. Mulholland, R. Ndubaku, I. Schmidt, P. Guzman, L. Aguilar-Henonin, M. Schmid, D. Weigel, D.E. Carter, T. Marchand, E. Risseeuw, D. Brogden, A. Zeko, W.L. Crosby, C.C. Berry, J.R. Ecker, Genome-wide insertional mutagenesis of Arabidopsis thaliana, Science 301 (2003) 653–657. [6] R.G. Alscher, N. Erturk, L.S. Heath, Role of superoxide dismutases (SODs) in controlling oxidative stress in plants, J. Exp. Bot. 53 (2002) 1331–1341. [7] P. Aravind, M.N.V. Prasad, Zinc alleviates cadmium-induced oxidative stress in Ceratophyllum demersum L.: a free floating freshwater macrophyte, Plant Physiol. Biochem. 41 (2003) 391–397. [8] M. Baier, G. Noctor, C.H. Foyer, K. Dietz, Antisense suppression of 2-cysteine peroxiredoxin in Arabidopsis specifically enhances the activities and expression of enzymes associated with ascorbate metabolism but not glutathione metabolism, Plant Physiol. 124 (2000) 823–832. [9] E. Bergmu¨ller, S. Porfirova, P. Do¨rmann, Characterization of an Arabidopsis mutant deficient in g-tocopherol methyltransferase, Plant Mol. Biol. 52 (2003) 1181–1190. [10] O. Blokhina, E. Virolainen, K.V. Fagerstedt, Antioxidants, oxidative damage and oxygen deprivation stress: a review, Ann. Bot. 91 (2003) 179–194. [11] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 289–292.

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