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Ascorbic acid and flavonoid-peroxidase reaction as a detoxifying system of H2O2 in grapevine leaves
Phytochemistry 60 (2002) 573–580 www.elsevier.com/locate/phytochem
Ascorbic acid and flavonoid-peroxidase reaction as a detoxifying system of H2O2 in grapevine leaves Francisco J. Pe´rez*, Daniel Villegas, Nilo Mejia Universidad de Chile, Facultad de Ciencias, Lab. de Bioquı´mica Vegetal, Casilla 653, Santiago, Chile Received in revised form 10 April 2002
Abstract Biosynthesis of both ascorbic acid (AsA) and peroxidase activity were induced by light in cv. Sultana grapevine leaves. Induced peroxidase activity mainly involved basic isoenzymes of pI 9.8 and 9.6 and catalyzed the oxidation of flavonoids like quercetin and kaempferol and derivatives of hydroxycinnamic acids such as ferulic and p-coumaric acids, but not AsA. However, the peroxidasedependent oxidation of ferulic acid and quercetin was temporarily suppressed by AsA as long as it remained in the reaction medium. Kinetics and spectroscopic results indicated that AsA was oxidized to dehydroascorbic acid only in the presence of phenols or flavonoids, and did not interfere with the catalytic activity of the peroxidase. Ascorbate peroxidase isoenzymes (APx), whose activities are widely considered central for detoxification of H2O2 in most plant cells, were not detected in grape leaves extracts. The significance of light stimulus on peroxidase activity and leaf AsA content is discussed in terms of a flavonoid-redox cycle proposed as an alternative system to detoxify H2O2 in grapevine leaves. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Vitis vinifera; Vitaceae; Ascorbic acid; Plant peroxidases; Phenols; Flavonoids
1. Introduction H2O2 scavenging in photosynthetically active tissues is critical in preventing oxidative damage. Green tissues are prone to produce H2O2 especially when exposed to strong light, since under such conditions, increased photoreduction of O2 in PSI of chloroplasts leads to increased production of H2O2 (Asada, 1999). Moreover, H2O2 is also stimulated by strong light through increases in photorespiration (Baker, 1991) and probably during respiration (Moller, 2001). Therefore, different cellular compartments such as chloroplast, peroxisomes and mitochondria are well equipped with enzymes capable of detoxifying H2O2. In most plants, peroxidases use different electron donors to remove H2O2. Catalase (EC. 1.11.16) is the only detoxifying enzyme that disproportionates H2O2 to H2O and O2 without electron donors. It is mainly localized in peroxisomes and has low substrate affinities, since the reaction requires simultaneous access to the active site of two H2O2 molecules (Willeckens et al., 1995). Ascorbate peroxidases (APx) (EC 1.11.1.11) uti* Corresponding author. E-mail address: [email protected] (F.J. Pe´rez).
lize ascorbic acid (AsA) as electron donor, and according to their amino acid sequence belong to the superfamily of heme-peroxidase Class I (Wellinder, 1992). They are found in chloroplasts and also in the cytosol and play an important role in scavenging H2O2 (Asada, 1992; Miyake and Asada, 1992; Ishikawa et al., 1998; Yoshimura et al. 2000). Recent studies in spinach, demonstrated that cytosolic APx (cAPx) activity is induced under oxidative stress, while the activities of the other APx isoforms remained unaltered. Similar observations of cAPx induction by oxidative stress were reported in peas, maize, rice and Arabidopsis (Mitler and Zilinskas, 1992; Donahue et al., 1997; Kaprinski et al., 1997; Storozhenko et al., 1998; Morita et al., 1999). Classical guaiacol peroxidases of plants belonging to Class III (GPx) (EC 1.11.1.7) are distinguishable from APx in amino acid sequence, substrate specificity and physiological function (Wellinder, 1992). GPx can use a wide range of aromatic compounds as reducing substrates and has been implicated in several metabolic reactions such as the biosynthesis of lignin (Gaspar et al., 1991; Christensen et al., 1993), decomposition of IAA (Pedren˜o et al. 1988) and defense against pathogens (Flott et al., 1989). It is still not clear however, whether GPx’s participate in scavenging H2O2 in plant cells. It
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(02)00146-2
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Table 1 Effect of PPFDs on ascorbic acid (AsA) content and peroxidase activity in extracts from leaves of grapevine exposed to different levels of light for different intervals of time Light PPFDs (mmol photons m2 s1)
50 700 1500
AsA (mmol g1 fr.wt)
Perox. Act. (mmolmin1 g1 fr.wt)
Time of exposure (h)
Time of exposure (h)
2
4
6
2
4
6
23.61.9 37.11.9 38.34.1
18.6 6.5 35.2 4.3 36 4
22.05.3 31.22.9 35.55.6
0.21 0.06 0.64 0.17 0.90 0.22
0.2 0.07 0.6 0.13 0.7 0.2
0.240.08 0.580.17 0.850.23
Peroxidase activity and AsA concentration in dark adapted leaves were 0.18 (mmolmin1g1 fr. wt) and 23 (mmol g1 fr. wt), respectively. Values correspond to the average of three independent measurements with their respective S.D.
has been suggested that AsA may indirectly be the electron donor even for non-specific peroxidases mediated by low molecular weight components in the cells (Mehlborn et al., 1996). Here we report the stimulatory effects of light on peroxidase activity and on AsA content in leaves of grapevine cv. Sultana. The inhibition of either the phenol or flavonoid peroxidase catalyzed reaction by AsA, and its potential as a possible flavonoid redox system capable of detoxifying H2O2 in grape leaves is analyzed.
2 h of exposure of the leaves to full-sunlight (1500 mmol photons m2 s1) peroxidase activity increased 4-fold with respect to deep shaded leaves (50 mmol photons m2 s1) and 5-fold with respect to dark adapted leaves. Exposure of leaves to longer periods of light ca. 4 or 6 h Table 2 Apparent kinetic parameters in the oxidation of ferulic acid (FA) and quercetin by partially purified basic peroxidases. The rates for FA oxidation were determined at a concentration of 0.6 mM H2O2 and for quercetin oxidation 0.44 mM H2O2. Kinetic parameters for H2O2 consumption were determined at fixed concentrations of 0.05 mM FA and 0.15 mM quercetin
2. Results 2.1. Light effect on peroxidase activity in grapevine leaves Exposure to light increased peroxidase activity in the leaves of grapevine cv. Sultana according to the photosynthetic photon flux densities (PPFDs) (Table 1). After
Compounds
Vmaxa (mM s1)
Km (mM)
Vmax/Km (m1 s1)
Ferulic acid H2O2 Quercetin H2O2
18.8 12.6 161 184
94 149 39 200
0.2 0.08 4.2 0.9
a
Rate values are normalized per gram of fr. wt.
Fig. 1. Isoelectrofocusing (IEF) analysis in a 3–10 pH gradient of peroxidases from extracts (10 ml) corresponding to equivalent amounts of grapevine leaves (7 mg fr. wt.) exposed to low (50 mmol photons m2 s1), lane (2) ; medium (700 mmol photons m2 s1), lane (3) and high (1500 mmol photons m2s1), lane (4) PPFDs. Lane (1) correspond to pH-markers, and the peroxidase activity bands were localized with O-methoxy-naphthol and H2O2.
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Fig. 2. Effect of ascorbic acid (AsA) on the peroxidase-dependent oxidation of ferulic acid (A) and quercetine (B). The oxidation of ferulic acid was monitored at 318 nm and AsA concentrations were 0 (—); 10 (- - -); 20 (-.-.-) and 40 (. . ..) mM. The oxidation of quercetine was monitored at 380 nm and the concentration of AsA were 0 (—); 10 (- - -); 20 (–); 30 (. . .) and 40 (-.-.-) mM.
did not increase enzyme activity further. The main peroxidases induced by light were basic in nature, with isoelectric points of 9.8 and 9.6 as shown by isoelectrofocusing analysis (IEF) of the leaf extracts (Fig. 1). Partially purified basic peroxidase oxidized hydroxycinnamic acid derivatives like ferulic and p-coumaric acids and flavonoids like quercetin and kaempferol (Table 2). However, it did not oxidize either ascorbic acid (AsA) or syringaldazine, the latter being a substrate widely used for measurements of peroxidase activity associated with lignification (Lewis and Yamamoto, 1990). Moreover, peroxidase activity was not suppressed by hydroxyurea and P-aminophenol (results not shown) which are typical suicide inhibitors of APx (Chen and Asada, 1990).
2.2. Light effect on ascorbic acid (AsA) content in grapevine leaves Leaf AsA content was also stimulated by light though the response was less pronounced and less dependent on the PPFD than peroxidase activity. After 2 h exposure to PPFDs of 700 and 1500 mmol photons m2 s1, AsA content increased only 57 and 63%, respectively, in relation to dark adapted and deep shaded leaves (Table 1). 2.3. Flavonoid determinations Total flavonoids estimated from the A320 of extracts from full-sunlight exposed leaves was 0.2 mmol per gram of fr. wt, which is about 10 times the concentration of AsA found in leaves. 2.4. Inhibition of peroxidase-dependent oxidation of phenols and flavonoids by AsA
Fig. 3. Activation of ascorbic acid (AsA) oxidation by gentisic acid (GA); p-coumaric acid (p-C); ferulic acid (FA) and quercetine (Q) (100 mM) in the presence of basic peroxidases. Controls ( c ) were carriedout in the absence of phenols. The reactions were monitored at 265 nm and the initial concentrations of AsA and H2O2 were 0.1 and 0.5 mM respectively.
Ascorbic acid (AsA) at low concentrations temporarily suppressed the peroxidase-dependent oxidation of quercetin and ferulic acid. AsA delayed the initiation of the reaction in proportion to its concentration in the reaction medium (Fig. 2A and B). However, once the oxidation of phenolic started, the rates were similar to those in the absence of AsA. This suggests that AsA does not interfere with peroxidase catalytic activity (Fig. 2A and B). Moreover, in the absence of phenols, basic peroxidase did not catalyze the oxidation of AsA (Fig. 3), however, in their presence, AsA was readily, oxidized. Fig. 3 shows the activation of AsA oxidation by different phenols. The results revealed that quercetin was the strongest activator followed by ferulic, p-coumaric and gentisic acids. The rate of AsA oxidation in the presence of quercetin and ferulic acid was directly related to the latter’s capacity to act as electron donors for
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Fig. 4. Overlay plot of ferulic acid (FA) oxidation in the presence of 40 mM AsA and AsA oxidation in the presence of 0.1 mM (FA).
the induced basic peroxidases (Table 2), suggesting that AsA is oxidized by the phenoxy radicals. An overlay plot of ferulic acid (FA) oxidation in the presence of AsA, and AsA oxidation in the presence of FA measured under identical experimental conditions (Fig. 4) showed that the onset of FA oxidation is preceded by the oxidation of AsA. It is well known that the
dithiotreitol (DTT) induced reduction of dehydroascorbic acid (DHA) to AsA can be followed spectroscopically by the increase in the absorption at 265 nm, since DHA does not absorb in the UV region (Morris and Redfearn, 1969), while AsA absorbs strongly at 265 nm. The spectra shown in Fig. 5 correspond to a mixture of FA and AsA. The maximum absorption (275 nm) as expected was centered within the maximam of AsA (265 nm) and FA (285 nm), respectively. After full AsA oxidation, the maximum shifted to 285 nm due to the predominance of FA, and the absorption at 265 nm decreased due to AsA oxidation. Addition of DTT recovered mostly the absorption at 265 nm and shifted again the maximum to 285 nm suggesting AsA regeneration. However, the initial spectral characteristics of the reaction mixture was not fully recovered, since after AsA oxidation FA was also oxidized as is shown by the decrease in the absorption at 310 nm.
3. Discussion
Fig. 5. Effect of DTT (10 mM) addition after full AsA oxidation on phenol peroxidase-dependent AsA oxidation. Line (1) represents the spectrum of the mixture FA and AsA at the beginning of the reaction, Line (2) represents the same spectrum after full AsA oxidation and line (3) represents the spectrum after DTT addition.
Increases in ascorbic acid (AsA) content with increased PPFDs in grapevine leaves is expected, since AsA is a key metabolite of the ascorbate glutathione cycle (Noctor and Foyer, 1998) generally considered as one of the main ROS detoxification systems in plant cells. Moreover, AsA is also required for the reductive de-epoxidation of violaxanthine to antheraxanthine and
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zeaxanthine (Yamamoto, 1979), an essential mechanism for thermal dissipation of excess energy in PSII (Demmig-Adams and Adams, 1992). However, the strong induction of basic peroxidases by light is surprising, especially considering that these isoenzymes belong to the guaiacol type peroxidase (GPx, Class III) which are unrelated to H2O2 scavenging (Asada, 1999). Class I ascorbate peroxidase isoenzymes (APx) are considered to be the main peroxidase involved in H2O2 detoxification, and has been reported to be present in very low amounts in extracts of grapevine leaves (Papadakis et al., 2001). Nevertheless, we were unsuccessful in detecting APx activity in leaf extracts of grapevine. The difference in our results and those obtained by Papadakis et al. (2001), could be due to differences in the grape varieties or methods used. AsA oxidation in leaf extracts due to the presence of phenols or flavonoids and basic peroxidase can be easily confused with the activity of native APx. However, if the extract is desalted to eliminate low molecular weight compounds, residual activity will reflect the presence of native Apx, which is generally considered the main peroxidase involved in the detoxification of H2O2 in plant cells. Since APx activity in extracts of grape leaves was not detected despite the precaution taken of using AsA in the extraction buffer (Amako et al., 1994), it would seem reasonable that instead of Apx, the light-induced basic peroxidase works together with AsA and phenols or flavonoids, which are also stimulated by light (Kolb et al., 2001), in the scavenging of H2O2 in grape leaves through a flavonoid redox cycle similar to that proposed by Yamasaki et al. (1997). In this system (Scheme 1) phenoxy or flavonoxy radicals are generated during the peroxidase reaction (Monties, 1989) and can accept electrons from AsA more readily than from other radicals (Nijs and Kelly, 1991) avoiding the formation of dimers (Takahama, 1993; Takahama and Oniki, 1994). The primary oxidation product of AsA in the proposed scheme is the monodehydroascorbate radical (MDA ) which disproportionates spontaneously to DHA. The recovered absorption at 265 nm after DTT addition to the reaction mixture indicates that DHA is the final oxidised product of AsA in the phenol peroxidase catalyzed reaction (Fig. 5). However, in vivo MDA radical can also be reduced to AsA in a reaction catalyzed by the monodehydroascorbate reductase (Hossain et al., 1984; Sano et al., 1995). The functionality of the system will depend on the capacity of
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MDA radical and DHA to regenerate AsA (Fig. 6), and on the cellular compartmentalization of all participating enzymes and metabolites in the same site. Therefore the subcellular localization of basic peroxidases, the detection of MDA-reductase and DHAreductase activities and their subcellular localization become interesting objectives for future research on the scavenging of H2O2 in grapevine leaves.
4. Experimental 4.1. Chemicals and plant material Ferulic acid (FA) (3-methoxy-4-hydroxycinnamic acid); p-coumaric acid (4-hydroxycinnamic acid); gentisic acid (2, 5 dihydroxybenzoic acid), quercetine (3, 30 , 40 , 5, 7 pentahydroxyflavone), syringaldazine (4-hydroxy3,5-dimethoxybenzaldehydeazine), ascorbate oxidase from Curcubita sp., and H2O2 were purchased from Sigma (USA). Eight year-old plants of Vitis vinifera cv. Sultana were used in this study. The plantation is situated in the central valley of Chile 32 SL 78 W. For light treatments, plants were covered with plastic nets to control the PPFDs. Before treatment, and during the night, plants were kept in darkness for 14 h covered with a black plastic. Afterwards, at 9 a.m. plants were exposed in average to PPFDs of 1500, 700 and 50 mmol photons m2 s1 for 2, 4 and 6 h. All the leaf material collected was immediately frozen in liquid N2 and kept frozen (80 ) until analyzed. 4.2. Partial purification of basic peroxidases Leaves (5 g fr. wt) were ground in liquid nitrogen and homogenized with a 3-fold volume of 0.25 M Tris–HCl adjusted to pH 7.5 containing 3 mM EDTA, 20 mM DTT, 0.1 mM PMSF, 2% insoluble PVP and 1% Tween 20. The homogenate was filtered through four layers of gauze and centrifuged at 10,000g for 10 min. The supernatant (crude extract) was desalted by filtering through a Sephadex G-25 column, and applied to a DEAE-Sephadex column previously equilibrated with 5 mM Tris–HCl pH 8.2. The column was washed with the equilibration buffer and activity of basic peroxidases was recovered with the washing buffer. Peroxidases of pI 9.8 and 9.6 were the main isoenzymes recovered as shown by IEF analysis. 4.3. Peroxidase assays
Scheme 1.
Peroxidase (donor: hydrogen peroxide oxidoreductases, EC1.11.1.7) activity was routinely assayed by measuring the H2O2 dependent oxidation of o-phenylendiamine (o-PDA) spectrophotometrically at 450 nm ("=1.1 mM1 cm1) or guaiacol (2-methoxyphenol) at
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Fig. 6. A proposed scheme for the scavenging of H2O2 by the flavonoid-redox cycle in grape leaves. GPx (guaiacol type peroxidase) light-induced, catalyze the oxidation of Flav (Flavonoids) or phenols (Phe), which are also light-induced, in the presence of H2O2 giving the corresponding radical Flav (Flavonoid radical) and H2O. In the presence of AsA (ascorbic acid) the Flav radical is reduced and AsA oxidized to MDA (monodehydroascorbic radical) which can regenerate AsA by the action of the MDA-reductase or disproportionate to DHA (dehydroascorbic acid). DHA can be reduced to AsA by the action of the cDHA-R (cytosolic dehydroascorbic acid reductase). In the absence of AsA, the Flav or Phe radicals reacts between them giving rise to the corresponding polymers.
470 nm ("=26.6 mM1 cm1). The reaction mixture contained 0.1 M sodium citrate pH 4.5, 44 mM o-PDA, 1 mM H2O2 and 5–10 ml of extract. For staining peroxidase activity in polyacrylamide gels 4-methoxy-1naphthol (4.5 mM) and H2O2 (1 mM) were used as substrates. 4.4. Isoelectric focusing Bio-Lyte ampholytes (Bio-Rad) in a pH range of 3–10 were utilized in polyacrylamide gels using a Bio-Rad 111 mini-IEF chamber according to the manufacturer‘s specifications. IEF standards (Bio-Rad) were used to assigned pH values in the gel. Equivalent amounts of fr. wt (7 mg) corresponding approximately to 10 ml of extracts of leaves exposed to low, medium and high radiation were loaded on the gel. The units of peroxidase activity loaded in the gel were 0.6 (L); 1.90 (M) and 4.3 (H). One unit was defined as 1 mmol of o-PDA oxidized min1 (ml of extract)1.
nected to a PC. The oxidation of ferulic acid (FA) and quercetin were monitored by the changes in absorbance at 318 nm ("=15.2 mM1 cm1 ) and 380 nm ("=13.6 mM1 cm1 ), respectively. The reactions were carried out in 1 ml cuvette containing 0.1 M citrate buffer pH 4.5, 0.6 mM H2O2 and FA and quercetin at variable concentrations. In these studies partially purified basic isoenzymes were used. Rates were determined from the initial slopes of the progress curves, and the dependence on substrate concentration followed the hyperbolic function in all cases. Michaelis–Menten parameters Vmax and Km were determined using a non linear regression program. Inhibition studies by ascorbic acid (AsA) were performed under the same conditions using fixed concentrations (100 mM) of both substrates and variable concentrations of AsA ranging between 10 and 40 mM. The oxidation of AsA was monitored by the changes in absorbance at 265 nm ("=9.8 mM1 cm1) (Yamasaki et al., 1997). 4.6. Ascorbate and total flavonoid determinations
4.5. Kinetic studies Spectrophotometric measurements were performed with a Jenway 6405 UV-Vis spectrophotometer con-
Ascorbate was measured as described by Wise and Naylor (1987). Leaves (0.5 g fr. wt) frozen in liquid N2 were ground in a mortar with 5 ml of 6% (v/v) HClO4
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and centrifuged at 10,000g for 5 min. The supernatant was neutralized to pH 6 with 1.25 M K2CO3. A 100 ml aliquot of the neutralized extract was added to 900 ml of 0.1 M phosphate buffer pH 7.0 and recorded immediately (A265) and again 5 min after the addition of 5 units of ascorbate oxidase. The difference between both measurements represents the ascorbate concentration. MeOH extracts of 0.5 g leaves were used for determining total flavonoids. The extract was centrifuged at 10,000g for 5 min, and filtered through a Sep-pack C18 cartridge to exclude chlorophyll and carotenoid pigments. Spectrophotometric measurements at 360 nm ("=13.6 mM1 cm1) were used to determine total flavonoids (Markham, 1993).
Acknowledgements The financial support of FONDECYT project 1000839 is gratefully acknowledged. We thank Alex Crawford for stimulating discussion and skillful proof reading. References Amako, K., Chen, G.X., Asada, K., 1994. Separate assays for ascorbate peroxidase and guaiacol peroxidase and for the chloroplastic and cytosolic isozymes of ascorbate peroxidases in plants. Plant Cell Physiology 35, 497–504. Asada, K., 1992. Ascorbate peroxidase a hydrogen-peroxide-scavenging enzyme in plants. Physiologia Plantarum 85, 235–241. Asada, K., 1999. The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annual Review of Plant Physiology and Plant Molecular Biology 50, 601–639. Baker, N.R., 1991. A possible role for photosystem II in environmental perturbation of photosynthesis. Physiologia Plantarum 81, 563–570. Chen, G.X., Asada, K., 1990. Hydroxyurea and p-aminophenol are the suicide inhibitors of ascorbate peroxidase. Journal of Biological Chemistry 26, 2775–2781. Christensen, J.H., Bauw, G., Wellinder, K.G., Van Montagu, M.V., Boerjan, W., 1993. Purification and characterization of peroxidases correlated with lignification in poplar xylem. Plant Physiology 118, 125–135. Donahue, J.L., Okpodu, C.M., Cramer, C.L., Grabau, E.A., Alscher, R.G., 1997. Response of antioxidants to paraquat in pea leaves. Plant Physiology 113, 249–257. Demmig-Adams, B., Adams III., W.W., 1992. Photoprotection and other responses of plants to high light stress. Annual Review of Plant Physiology and Plant Molecular Biology 43, 599–626. Flott, B.E., Moerschbacher, B.M., Reisner, H.J., 1989. Peroxidase isoenzyme patterns of resistant and susceptible wheat leaves following stem rust infection. New Phytologist 111, 413–421. Gaspar, Th., Penel, C., Hagege, D., Greppin, H., 1991. Peroxidases in plant growth, differentiation and development processes. In: Lobarzewiski, J., Greppin, H., Penel, C., Gaspar, Th. (Eds.), Biochemical, Molecular and Physiological Aspects of Plant Peroxidases. Universite´ de Geneve Press, Geneva, pp. 249–281. Hossain, M.A., Nakano, Y., Asada, K., 1984. Monodehydroascorbate reductase in spinach chloroplasts and its participation in regeneration of ascorbate for scavenging hydrogen peroxide. Plant Cell Physiology 25, 385–391.
579
Ishikawa, T., Yoshimura, K., Sakai, K., Tamoi, M., Takeda, T., Shigeoka, S., 1998. Molecular characterization and physiological role of a glyoxysome-bound ascorbate peroxidase from spinach. Plant Cell Physiology 39, 23–31. Kaprinski, S., Reynolds, H., Karpinska, B., Wingsle, G., Creissen, G., Mullineaux, P., 1997. Systemic signalling and acclimation in response to excess excitation energy in Arabidopsis. Science 284, 654–658. Kolb, C.A., Ka¨ser, M.A., Kopecky, J., Zotz, G., Riederer, M., Pfu¨ndel, E.E., 2001. Effects of natural intensities of visible and ultraviolet radiation on epidermal screening and photosynthesis in grape leaves. Plant Physiology 127, 863–875. Lewis, N.G., Yamamoto, E., 1990. Lignin: occurrence, biogenesis and biodegradation. Annual Review of Plant Physiology and Plant Molecular Biology 41, 455–496. Markham, K.R., 1993. Flavones, flavonols and their glycosides. In: Harbone, J.B. (Ed.), Methods in Plant Biochemistry. Academic Press, London, pp. 197–235. Mehlborn, H., Lelandais, M., Korth, H.G., Foyer, C.H., 1996. Ascorbate is the natural substrate for plant peroxidases. FEBS Letters 378, 203–208. Mitler, R., Zilinskas, B.A., 1992. Molecular cloning and characterization of a gene encoding pea cytosolic ascorbate peroxidase. Journal of Biological Chemistry 267, 21802–21809. Miyake, C., Asada, K., 1992. Thylakoid-bound ascorbate peroxidase in spinach chloroplasts and photoreduction of its primary oxidation product monodehydroascorbate radicals in thylakoids. Plant Cell Physiology 33, 541–547. Moller, M., 2001. Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Annual Review of Plant Physiology and Molecular Biology 52, 561–591. Monties, B., 1989. Lignins. In: Harborne, J.B. (Ed.), Methods in Plant Biochemistry. Academic Press, London, pp. 113–131. Morita, S., Kaminaka, H., Masumura, T., Tanaka, K., 1999. Induction of rice cytosolic ascorbate peroxidase mRNA by oxidative stress: the involvement of hydrogen peroxide in oxidative stress signalling. Plant Cell Physiology 40, 417–423. Morries, J.G., Redfearn, E.R., 1969. Vitamins and coenzymes. In: Dawson, R.M.C., Elliot, D.C., Jones, K.M. (Eds.), Data for Biochemical Research. Clarendon Press, Oxford, pp. 123–167. Nijs, D., Kelly, P.M., 1991. Vitamin C and E donate single hydrogen in vivo. FEBS Letters 284, 147–150. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione keeping active oxygen under control. Annual Review of Plant Physiology and Plant Molecular Biology 49, 249–279. Papadakis, A.K., Siminis, C.I., Roubelakis-Angelakis, K.A., 2001. Reduced activity of antioxidant machinery is correlated with suppression of totipotency in plant protoplasts. Plant Physiology 126, 434–441. Pedren˜o, M.A., Mun˜oz, R.A., Sabater, F., 1988. Oxidase and peroxidase activities of a cell-wall bound peroxidase acting on indoleacetic acid: control by p-coumaric acid. Biochemistry International 16, 452–457. Sano, S., Miyake, C., Mikami, B., Asada, K., 1995. Molecular characterization of monodehydroascorbate radical reductase from cucumber highly expressed in E. Coli. Journal of Biological Chemistry 270, 21354–21361. Storozhenko, S., Pauw, P.D., Montagu, M.V., Inze´, D., Kushnir, S., 1998. The heat shock element is functional component of the Arabidopsis APX1 gene promoter. Plant Physiology 118, 1005– 1013. Takahama, U., 1993. Redox state of ascorbic acid in the apoplast of stems of Kalanchoe daigremontiana. Physiologia Plantarum 89, 791– 799. Takahama, U., Oniki, T., 1994. Effects of ascorbate on the oxidation of derivatives of hydroxycinnamic acid and the mechanism of
580
F.J. Pe´rez et al. / Phytochemistry 60 (2002) 573–580
oxidation of sinapic acid by cell wall-bound peroxidases. Plant Cell Physiology 35, 593–600. Wellinder, K.G., 1992. Plant peroxidase: structure, functions, relationships. In: Pennel, C., Gaspar, T., Greppin, H. (Eds.), Plant Peroxidases 1980–1990, Topics and Detailed Literature on Molecular, Biochemical and Physiological Aspects. Geneva, Switzerland, pp. 1–24. Willeckens, H., Inze´, D., Van Montagu, M.V., Van Camp, W., 1995. Catalase in plants. Molecular Breeding 1, 207–215. Wise, R.R., Naylor, A.W., 1987. Chilling-enhanced photooxidation: evidence for the role of singlet oxygen and superoxide in the break-
down of pigments and endogenous antioxidants. Plant Physiology 83, 278–285. Yamamoto, H.Y., 1979. Biochemistry of the violaxanthine cycle in higher plants. Pure and Applied Chemistry 51, 639–645. Yamasaki, H., Sakihama, Y., Ikehara, N., 1997. Flavonoid-peroxidase reactions as a detoxification mechanism of plant cells against H2O2. Plant Physiology 115, 1405–1412. Yoshimura, K., Yabuta, Y., Ishikawa, T., Shigeoka, S., 2000. Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stress. Plant Physiology 123, 223–233.
Ascorbic acid and flavonoid-peroxidase reaction as a detoxifying system of H2O2 in grapevine leaves Francisco J. Pe´rez*, Daniel Villegas, Nilo Mejia Universidad de Chile, Facultad de Ciencias, Lab. de Bioquı´mica Vegetal, Casilla 653, Santiago, Chile Received in revised form 10 April 2002
Abstract Biosynthesis of both ascorbic acid (AsA) and peroxidase activity were induced by light in cv. Sultana grapevine leaves. Induced peroxidase activity mainly involved basic isoenzymes of pI 9.8 and 9.6 and catalyzed the oxidation of flavonoids like quercetin and kaempferol and derivatives of hydroxycinnamic acids such as ferulic and p-coumaric acids, but not AsA. However, the peroxidasedependent oxidation of ferulic acid and quercetin was temporarily suppressed by AsA as long as it remained in the reaction medium. Kinetics and spectroscopic results indicated that AsA was oxidized to dehydroascorbic acid only in the presence of phenols or flavonoids, and did not interfere with the catalytic activity of the peroxidase. Ascorbate peroxidase isoenzymes (APx), whose activities are widely considered central for detoxification of H2O2 in most plant cells, were not detected in grape leaves extracts. The significance of light stimulus on peroxidase activity and leaf AsA content is discussed in terms of a flavonoid-redox cycle proposed as an alternative system to detoxify H2O2 in grapevine leaves. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Vitis vinifera; Vitaceae; Ascorbic acid; Plant peroxidases; Phenols; Flavonoids
1. Introduction H2O2 scavenging in photosynthetically active tissues is critical in preventing oxidative damage. Green tissues are prone to produce H2O2 especially when exposed to strong light, since under such conditions, increased photoreduction of O2 in PSI of chloroplasts leads to increased production of H2O2 (Asada, 1999). Moreover, H2O2 is also stimulated by strong light through increases in photorespiration (Baker, 1991) and probably during respiration (Moller, 2001). Therefore, different cellular compartments such as chloroplast, peroxisomes and mitochondria are well equipped with enzymes capable of detoxifying H2O2. In most plants, peroxidases use different electron donors to remove H2O2. Catalase (EC. 1.11.16) is the only detoxifying enzyme that disproportionates H2O2 to H2O and O2 without electron donors. It is mainly localized in peroxisomes and has low substrate affinities, since the reaction requires simultaneous access to the active site of two H2O2 molecules (Willeckens et al., 1995). Ascorbate peroxidases (APx) (EC 1.11.1.11) uti* Corresponding author. E-mail address: [email protected] (F.J. Pe´rez).
lize ascorbic acid (AsA) as electron donor, and according to their amino acid sequence belong to the superfamily of heme-peroxidase Class I (Wellinder, 1992). They are found in chloroplasts and also in the cytosol and play an important role in scavenging H2O2 (Asada, 1992; Miyake and Asada, 1992; Ishikawa et al., 1998; Yoshimura et al. 2000). Recent studies in spinach, demonstrated that cytosolic APx (cAPx) activity is induced under oxidative stress, while the activities of the other APx isoforms remained unaltered. Similar observations of cAPx induction by oxidative stress were reported in peas, maize, rice and Arabidopsis (Mitler and Zilinskas, 1992; Donahue et al., 1997; Kaprinski et al., 1997; Storozhenko et al., 1998; Morita et al., 1999). Classical guaiacol peroxidases of plants belonging to Class III (GPx) (EC 1.11.1.7) are distinguishable from APx in amino acid sequence, substrate specificity and physiological function (Wellinder, 1992). GPx can use a wide range of aromatic compounds as reducing substrates and has been implicated in several metabolic reactions such as the biosynthesis of lignin (Gaspar et al., 1991; Christensen et al., 1993), decomposition of IAA (Pedren˜o et al. 1988) and defense against pathogens (Flott et al., 1989). It is still not clear however, whether GPx’s participate in scavenging H2O2 in plant cells. It
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(02)00146-2
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Table 1 Effect of PPFDs on ascorbic acid (AsA) content and peroxidase activity in extracts from leaves of grapevine exposed to different levels of light for different intervals of time Light PPFDs (mmol photons m2 s1)
50 700 1500
AsA (mmol g1 fr.wt)
Perox. Act. (mmolmin1 g1 fr.wt)
Time of exposure (h)
Time of exposure (h)
2
4
6
2
4
6
23.61.9 37.11.9 38.34.1
18.6 6.5 35.2 4.3 36 4
22.05.3 31.22.9 35.55.6
0.21 0.06 0.64 0.17 0.90 0.22
0.2 0.07 0.6 0.13 0.7 0.2
0.240.08 0.580.17 0.850.23
Peroxidase activity and AsA concentration in dark adapted leaves were 0.18 (mmolmin1g1 fr. wt) and 23 (mmol g1 fr. wt), respectively. Values correspond to the average of three independent measurements with their respective S.D.
has been suggested that AsA may indirectly be the electron donor even for non-specific peroxidases mediated by low molecular weight components in the cells (Mehlborn et al., 1996). Here we report the stimulatory effects of light on peroxidase activity and on AsA content in leaves of grapevine cv. Sultana. The inhibition of either the phenol or flavonoid peroxidase catalyzed reaction by AsA, and its potential as a possible flavonoid redox system capable of detoxifying H2O2 in grape leaves is analyzed.
2 h of exposure of the leaves to full-sunlight (1500 mmol photons m2 s1) peroxidase activity increased 4-fold with respect to deep shaded leaves (50 mmol photons m2 s1) and 5-fold with respect to dark adapted leaves. Exposure of leaves to longer periods of light ca. 4 or 6 h Table 2 Apparent kinetic parameters in the oxidation of ferulic acid (FA) and quercetin by partially purified basic peroxidases. The rates for FA oxidation were determined at a concentration of 0.6 mM H2O2 and for quercetin oxidation 0.44 mM H2O2. Kinetic parameters for H2O2 consumption were determined at fixed concentrations of 0.05 mM FA and 0.15 mM quercetin
2. Results 2.1. Light effect on peroxidase activity in grapevine leaves Exposure to light increased peroxidase activity in the leaves of grapevine cv. Sultana according to the photosynthetic photon flux densities (PPFDs) (Table 1). After
Compounds
Vmaxa (mM s1)
Km (mM)
Vmax/Km (m1 s1)
Ferulic acid H2O2 Quercetin H2O2
18.8 12.6 161 184
94 149 39 200
0.2 0.08 4.2 0.9
a
Rate values are normalized per gram of fr. wt.
Fig. 1. Isoelectrofocusing (IEF) analysis in a 3–10 pH gradient of peroxidases from extracts (10 ml) corresponding to equivalent amounts of grapevine leaves (7 mg fr. wt.) exposed to low (50 mmol photons m2 s1), lane (2) ; medium (700 mmol photons m2 s1), lane (3) and high (1500 mmol photons m2s1), lane (4) PPFDs. Lane (1) correspond to pH-markers, and the peroxidase activity bands were localized with O-methoxy-naphthol and H2O2.
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Fig. 2. Effect of ascorbic acid (AsA) on the peroxidase-dependent oxidation of ferulic acid (A) and quercetine (B). The oxidation of ferulic acid was monitored at 318 nm and AsA concentrations were 0 (—); 10 (- - -); 20 (-.-.-) and 40 (. . ..) mM. The oxidation of quercetine was monitored at 380 nm and the concentration of AsA were 0 (—); 10 (- - -); 20 (–); 30 (. . .) and 40 (-.-.-) mM.
did not increase enzyme activity further. The main peroxidases induced by light were basic in nature, with isoelectric points of 9.8 and 9.6 as shown by isoelectrofocusing analysis (IEF) of the leaf extracts (Fig. 1). Partially purified basic peroxidase oxidized hydroxycinnamic acid derivatives like ferulic and p-coumaric acids and flavonoids like quercetin and kaempferol (Table 2). However, it did not oxidize either ascorbic acid (AsA) or syringaldazine, the latter being a substrate widely used for measurements of peroxidase activity associated with lignification (Lewis and Yamamoto, 1990). Moreover, peroxidase activity was not suppressed by hydroxyurea and P-aminophenol (results not shown) which are typical suicide inhibitors of APx (Chen and Asada, 1990).
2.2. Light effect on ascorbic acid (AsA) content in grapevine leaves Leaf AsA content was also stimulated by light though the response was less pronounced and less dependent on the PPFD than peroxidase activity. After 2 h exposure to PPFDs of 700 and 1500 mmol photons m2 s1, AsA content increased only 57 and 63%, respectively, in relation to dark adapted and deep shaded leaves (Table 1). 2.3. Flavonoid determinations Total flavonoids estimated from the A320 of extracts from full-sunlight exposed leaves was 0.2 mmol per gram of fr. wt, which is about 10 times the concentration of AsA found in leaves. 2.4. Inhibition of peroxidase-dependent oxidation of phenols and flavonoids by AsA
Fig. 3. Activation of ascorbic acid (AsA) oxidation by gentisic acid (GA); p-coumaric acid (p-C); ferulic acid (FA) and quercetine (Q) (100 mM) in the presence of basic peroxidases. Controls ( c ) were carriedout in the absence of phenols. The reactions were monitored at 265 nm and the initial concentrations of AsA and H2O2 were 0.1 and 0.5 mM respectively.
Ascorbic acid (AsA) at low concentrations temporarily suppressed the peroxidase-dependent oxidation of quercetin and ferulic acid. AsA delayed the initiation of the reaction in proportion to its concentration in the reaction medium (Fig. 2A and B). However, once the oxidation of phenolic started, the rates were similar to those in the absence of AsA. This suggests that AsA does not interfere with peroxidase catalytic activity (Fig. 2A and B). Moreover, in the absence of phenols, basic peroxidase did not catalyze the oxidation of AsA (Fig. 3), however, in their presence, AsA was readily, oxidized. Fig. 3 shows the activation of AsA oxidation by different phenols. The results revealed that quercetin was the strongest activator followed by ferulic, p-coumaric and gentisic acids. The rate of AsA oxidation in the presence of quercetin and ferulic acid was directly related to the latter’s capacity to act as electron donors for
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Fig. 4. Overlay plot of ferulic acid (FA) oxidation in the presence of 40 mM AsA and AsA oxidation in the presence of 0.1 mM (FA).
the induced basic peroxidases (Table 2), suggesting that AsA is oxidized by the phenoxy radicals. An overlay plot of ferulic acid (FA) oxidation in the presence of AsA, and AsA oxidation in the presence of FA measured under identical experimental conditions (Fig. 4) showed that the onset of FA oxidation is preceded by the oxidation of AsA. It is well known that the
dithiotreitol (DTT) induced reduction of dehydroascorbic acid (DHA) to AsA can be followed spectroscopically by the increase in the absorption at 265 nm, since DHA does not absorb in the UV region (Morris and Redfearn, 1969), while AsA absorbs strongly at 265 nm. The spectra shown in Fig. 5 correspond to a mixture of FA and AsA. The maximum absorption (275 nm) as expected was centered within the maximam of AsA (265 nm) and FA (285 nm), respectively. After full AsA oxidation, the maximum shifted to 285 nm due to the predominance of FA, and the absorption at 265 nm decreased due to AsA oxidation. Addition of DTT recovered mostly the absorption at 265 nm and shifted again the maximum to 285 nm suggesting AsA regeneration. However, the initial spectral characteristics of the reaction mixture was not fully recovered, since after AsA oxidation FA was also oxidized as is shown by the decrease in the absorption at 310 nm.
3. Discussion
Fig. 5. Effect of DTT (10 mM) addition after full AsA oxidation on phenol peroxidase-dependent AsA oxidation. Line (1) represents the spectrum of the mixture FA and AsA at the beginning of the reaction, Line (2) represents the same spectrum after full AsA oxidation and line (3) represents the spectrum after DTT addition.
Increases in ascorbic acid (AsA) content with increased PPFDs in grapevine leaves is expected, since AsA is a key metabolite of the ascorbate glutathione cycle (Noctor and Foyer, 1998) generally considered as one of the main ROS detoxification systems in plant cells. Moreover, AsA is also required for the reductive de-epoxidation of violaxanthine to antheraxanthine and
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zeaxanthine (Yamamoto, 1979), an essential mechanism for thermal dissipation of excess energy in PSII (Demmig-Adams and Adams, 1992). However, the strong induction of basic peroxidases by light is surprising, especially considering that these isoenzymes belong to the guaiacol type peroxidase (GPx, Class III) which are unrelated to H2O2 scavenging (Asada, 1999). Class I ascorbate peroxidase isoenzymes (APx) are considered to be the main peroxidase involved in H2O2 detoxification, and has been reported to be present in very low amounts in extracts of grapevine leaves (Papadakis et al., 2001). Nevertheless, we were unsuccessful in detecting APx activity in leaf extracts of grapevine. The difference in our results and those obtained by Papadakis et al. (2001), could be due to differences in the grape varieties or methods used. AsA oxidation in leaf extracts due to the presence of phenols or flavonoids and basic peroxidase can be easily confused with the activity of native APx. However, if the extract is desalted to eliminate low molecular weight compounds, residual activity will reflect the presence of native Apx, which is generally considered the main peroxidase involved in the detoxification of H2O2 in plant cells. Since APx activity in extracts of grape leaves was not detected despite the precaution taken of using AsA in the extraction buffer (Amako et al., 1994), it would seem reasonable that instead of Apx, the light-induced basic peroxidase works together with AsA and phenols or flavonoids, which are also stimulated by light (Kolb et al., 2001), in the scavenging of H2O2 in grape leaves through a flavonoid redox cycle similar to that proposed by Yamasaki et al. (1997). In this system (Scheme 1) phenoxy or flavonoxy radicals are generated during the peroxidase reaction (Monties, 1989) and can accept electrons from AsA more readily than from other radicals (Nijs and Kelly, 1991) avoiding the formation of dimers (Takahama, 1993; Takahama and Oniki, 1994). The primary oxidation product of AsA in the proposed scheme is the monodehydroascorbate radical (MDA ) which disproportionates spontaneously to DHA. The recovered absorption at 265 nm after DTT addition to the reaction mixture indicates that DHA is the final oxidised product of AsA in the phenol peroxidase catalyzed reaction (Fig. 5). However, in vivo MDA radical can also be reduced to AsA in a reaction catalyzed by the monodehydroascorbate reductase (Hossain et al., 1984; Sano et al., 1995). The functionality of the system will depend on the capacity of
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MDA radical and DHA to regenerate AsA (Fig. 6), and on the cellular compartmentalization of all participating enzymes and metabolites in the same site. Therefore the subcellular localization of basic peroxidases, the detection of MDA-reductase and DHAreductase activities and their subcellular localization become interesting objectives for future research on the scavenging of H2O2 in grapevine leaves.
4. Experimental 4.1. Chemicals and plant material Ferulic acid (FA) (3-methoxy-4-hydroxycinnamic acid); p-coumaric acid (4-hydroxycinnamic acid); gentisic acid (2, 5 dihydroxybenzoic acid), quercetine (3, 30 , 40 , 5, 7 pentahydroxyflavone), syringaldazine (4-hydroxy3,5-dimethoxybenzaldehydeazine), ascorbate oxidase from Curcubita sp., and H2O2 were purchased from Sigma (USA). Eight year-old plants of Vitis vinifera cv. Sultana were used in this study. The plantation is situated in the central valley of Chile 32 SL 78 W. For light treatments, plants were covered with plastic nets to control the PPFDs. Before treatment, and during the night, plants were kept in darkness for 14 h covered with a black plastic. Afterwards, at 9 a.m. plants were exposed in average to PPFDs of 1500, 700 and 50 mmol photons m2 s1 for 2, 4 and 6 h. All the leaf material collected was immediately frozen in liquid N2 and kept frozen (80 ) until analyzed. 4.2. Partial purification of basic peroxidases Leaves (5 g fr. wt) were ground in liquid nitrogen and homogenized with a 3-fold volume of 0.25 M Tris–HCl adjusted to pH 7.5 containing 3 mM EDTA, 20 mM DTT, 0.1 mM PMSF, 2% insoluble PVP and 1% Tween 20. The homogenate was filtered through four layers of gauze and centrifuged at 10,000g for 10 min. The supernatant (crude extract) was desalted by filtering through a Sephadex G-25 column, and applied to a DEAE-Sephadex column previously equilibrated with 5 mM Tris–HCl pH 8.2. The column was washed with the equilibration buffer and activity of basic peroxidases was recovered with the washing buffer. Peroxidases of pI 9.8 and 9.6 were the main isoenzymes recovered as shown by IEF analysis. 4.3. Peroxidase assays
Scheme 1.
Peroxidase (donor: hydrogen peroxide oxidoreductases, EC1.11.1.7) activity was routinely assayed by measuring the H2O2 dependent oxidation of o-phenylendiamine (o-PDA) spectrophotometrically at 450 nm ("=1.1 mM1 cm1) or guaiacol (2-methoxyphenol) at
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Fig. 6. A proposed scheme for the scavenging of H2O2 by the flavonoid-redox cycle in grape leaves. GPx (guaiacol type peroxidase) light-induced, catalyze the oxidation of Flav (Flavonoids) or phenols (Phe), which are also light-induced, in the presence of H2O2 giving the corresponding radical Flav (Flavonoid radical) and H2O. In the presence of AsA (ascorbic acid) the Flav radical is reduced and AsA oxidized to MDA (monodehydroascorbic radical) which can regenerate AsA by the action of the MDA-reductase or disproportionate to DHA (dehydroascorbic acid). DHA can be reduced to AsA by the action of the cDHA-R (cytosolic dehydroascorbic acid reductase). In the absence of AsA, the Flav or Phe radicals reacts between them giving rise to the corresponding polymers.
470 nm ("=26.6 mM1 cm1). The reaction mixture contained 0.1 M sodium citrate pH 4.5, 44 mM o-PDA, 1 mM H2O2 and 5–10 ml of extract. For staining peroxidase activity in polyacrylamide gels 4-methoxy-1naphthol (4.5 mM) and H2O2 (1 mM) were used as substrates. 4.4. Isoelectric focusing Bio-Lyte ampholytes (Bio-Rad) in a pH range of 3–10 were utilized in polyacrylamide gels using a Bio-Rad 111 mini-IEF chamber according to the manufacturer‘s specifications. IEF standards (Bio-Rad) were used to assigned pH values in the gel. Equivalent amounts of fr. wt (7 mg) corresponding approximately to 10 ml of extracts of leaves exposed to low, medium and high radiation were loaded on the gel. The units of peroxidase activity loaded in the gel were 0.6 (L); 1.90 (M) and 4.3 (H). One unit was defined as 1 mmol of o-PDA oxidized min1 (ml of extract)1.
nected to a PC. The oxidation of ferulic acid (FA) and quercetin were monitored by the changes in absorbance at 318 nm ("=15.2 mM1 cm1 ) and 380 nm ("=13.6 mM1 cm1 ), respectively. The reactions were carried out in 1 ml cuvette containing 0.1 M citrate buffer pH 4.5, 0.6 mM H2O2 and FA and quercetin at variable concentrations. In these studies partially purified basic isoenzymes were used. Rates were determined from the initial slopes of the progress curves, and the dependence on substrate concentration followed the hyperbolic function in all cases. Michaelis–Menten parameters Vmax and Km were determined using a non linear regression program. Inhibition studies by ascorbic acid (AsA) were performed under the same conditions using fixed concentrations (100 mM) of both substrates and variable concentrations of AsA ranging between 10 and 40 mM. The oxidation of AsA was monitored by the changes in absorbance at 265 nm ("=9.8 mM1 cm1) (Yamasaki et al., 1997). 4.6. Ascorbate and total flavonoid determinations
4.5. Kinetic studies Spectrophotometric measurements were performed with a Jenway 6405 UV-Vis spectrophotometer con-
Ascorbate was measured as described by Wise and Naylor (1987). Leaves (0.5 g fr. wt) frozen in liquid N2 were ground in a mortar with 5 ml of 6% (v/v) HClO4
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and centrifuged at 10,000g for 5 min. The supernatant was neutralized to pH 6 with 1.25 M K2CO3. A 100 ml aliquot of the neutralized extract was added to 900 ml of 0.1 M phosphate buffer pH 7.0 and recorded immediately (A265) and again 5 min after the addition of 5 units of ascorbate oxidase. The difference between both measurements represents the ascorbate concentration. MeOH extracts of 0.5 g leaves were used for determining total flavonoids. The extract was centrifuged at 10,000g for 5 min, and filtered through a Sep-pack C18 cartridge to exclude chlorophyll and carotenoid pigments. Spectrophotometric measurements at 360 nm ("=13.6 mM1 cm1) were used to determine total flavonoids (Markham, 1993).
Acknowledgements The financial support of FONDECYT project 1000839 is gratefully acknowledged. We thank Alex Crawford for stimulating discussion and skillful proof reading. References Amako, K., Chen, G.X., Asada, K., 1994. Separate assays for ascorbate peroxidase and guaiacol peroxidase and for the chloroplastic and cytosolic isozymes of ascorbate peroxidases in plants. Plant Cell Physiology 35, 497–504. Asada, K., 1992. Ascorbate peroxidase a hydrogen-peroxide-scavenging enzyme in plants. Physiologia Plantarum 85, 235–241. Asada, K., 1999. The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annual Review of Plant Physiology and Plant Molecular Biology 50, 601–639. Baker, N.R., 1991. A possible role for photosystem II in environmental perturbation of photosynthesis. Physiologia Plantarum 81, 563–570. Chen, G.X., Asada, K., 1990. Hydroxyurea and p-aminophenol are the suicide inhibitors of ascorbate peroxidase. Journal of Biological Chemistry 26, 2775–2781. Christensen, J.H., Bauw, G., Wellinder, K.G., Van Montagu, M.V., Boerjan, W., 1993. Purification and characterization of peroxidases correlated with lignification in poplar xylem. Plant Physiology 118, 125–135. Donahue, J.L., Okpodu, C.M., Cramer, C.L., Grabau, E.A., Alscher, R.G., 1997. Response of antioxidants to paraquat in pea leaves. Plant Physiology 113, 249–257. Demmig-Adams, B., Adams III., W.W., 1992. Photoprotection and other responses of plants to high light stress. Annual Review of Plant Physiology and Plant Molecular Biology 43, 599–626. Flott, B.E., Moerschbacher, B.M., Reisner, H.J., 1989. Peroxidase isoenzyme patterns of resistant and susceptible wheat leaves following stem rust infection. New Phytologist 111, 413–421. Gaspar, Th., Penel, C., Hagege, D., Greppin, H., 1991. Peroxidases in plant growth, differentiation and development processes. In: Lobarzewiski, J., Greppin, H., Penel, C., Gaspar, Th. (Eds.), Biochemical, Molecular and Physiological Aspects of Plant Peroxidases. Universite´ de Geneve Press, Geneva, pp. 249–281. Hossain, M.A., Nakano, Y., Asada, K., 1984. Monodehydroascorbate reductase in spinach chloroplasts and its participation in regeneration of ascorbate for scavenging hydrogen peroxide. Plant Cell Physiology 25, 385–391.
579
Ishikawa, T., Yoshimura, K., Sakai, K., Tamoi, M., Takeda, T., Shigeoka, S., 1998. Molecular characterization and physiological role of a glyoxysome-bound ascorbate peroxidase from spinach. Plant Cell Physiology 39, 23–31. Kaprinski, S., Reynolds, H., Karpinska, B., Wingsle, G., Creissen, G., Mullineaux, P., 1997. Systemic signalling and acclimation in response to excess excitation energy in Arabidopsis. Science 284, 654–658. Kolb, C.A., Ka¨ser, M.A., Kopecky, J., Zotz, G., Riederer, M., Pfu¨ndel, E.E., 2001. Effects of natural intensities of visible and ultraviolet radiation on epidermal screening and photosynthesis in grape leaves. Plant Physiology 127, 863–875. Lewis, N.G., Yamamoto, E., 1990. Lignin: occurrence, biogenesis and biodegradation. Annual Review of Plant Physiology and Plant Molecular Biology 41, 455–496. Markham, K.R., 1993. Flavones, flavonols and their glycosides. In: Harbone, J.B. (Ed.), Methods in Plant Biochemistry. Academic Press, London, pp. 197–235. Mehlborn, H., Lelandais, M., Korth, H.G., Foyer, C.H., 1996. Ascorbate is the natural substrate for plant peroxidases. FEBS Letters 378, 203–208. Mitler, R., Zilinskas, B.A., 1992. Molecular cloning and characterization of a gene encoding pea cytosolic ascorbate peroxidase. Journal of Biological Chemistry 267, 21802–21809. Miyake, C., Asada, K., 1992. Thylakoid-bound ascorbate peroxidase in spinach chloroplasts and photoreduction of its primary oxidation product monodehydroascorbate radicals in thylakoids. Plant Cell Physiology 33, 541–547. Moller, M., 2001. Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Annual Review of Plant Physiology and Molecular Biology 52, 561–591. Monties, B., 1989. Lignins. In: Harborne, J.B. (Ed.), Methods in Plant Biochemistry. Academic Press, London, pp. 113–131. Morita, S., Kaminaka, H., Masumura, T., Tanaka, K., 1999. Induction of rice cytosolic ascorbate peroxidase mRNA by oxidative stress: the involvement of hydrogen peroxide in oxidative stress signalling. Plant Cell Physiology 40, 417–423. Morries, J.G., Redfearn, E.R., 1969. Vitamins and coenzymes. In: Dawson, R.M.C., Elliot, D.C., Jones, K.M. (Eds.), Data for Biochemical Research. Clarendon Press, Oxford, pp. 123–167. Nijs, D., Kelly, P.M., 1991. Vitamin C and E donate single hydrogen in vivo. FEBS Letters 284, 147–150. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione keeping active oxygen under control. Annual Review of Plant Physiology and Plant Molecular Biology 49, 249–279. Papadakis, A.K., Siminis, C.I., Roubelakis-Angelakis, K.A., 2001. Reduced activity of antioxidant machinery is correlated with suppression of totipotency in plant protoplasts. Plant Physiology 126, 434–441. Pedren˜o, M.A., Mun˜oz, R.A., Sabater, F., 1988. Oxidase and peroxidase activities of a cell-wall bound peroxidase acting on indoleacetic acid: control by p-coumaric acid. Biochemistry International 16, 452–457. Sano, S., Miyake, C., Mikami, B., Asada, K., 1995. Molecular characterization of monodehydroascorbate radical reductase from cucumber highly expressed in E. Coli. Journal of Biological Chemistry 270, 21354–21361. Storozhenko, S., Pauw, P.D., Montagu, M.V., Inze´, D., Kushnir, S., 1998. The heat shock element is functional component of the Arabidopsis APX1 gene promoter. Plant Physiology 118, 1005– 1013. Takahama, U., 1993. Redox state of ascorbic acid in the apoplast of stems of Kalanchoe daigremontiana. Physiologia Plantarum 89, 791– 799. Takahama, U., Oniki, T., 1994. Effects of ascorbate on the oxidation of derivatives of hydroxycinnamic acid and the mechanism of
580
F.J. Pe´rez et al. / Phytochemistry 60 (2002) 573–580
oxidation of sinapic acid by cell wall-bound peroxidases. Plant Cell Physiology 35, 593–600. Wellinder, K.G., 1992. Plant peroxidase: structure, functions, relationships. In: Pennel, C., Gaspar, T., Greppin, H. (Eds.), Plant Peroxidases 1980–1990, Topics and Detailed Literature on Molecular, Biochemical and Physiological Aspects. Geneva, Switzerland, pp. 1–24. Willeckens, H., Inze´, D., Van Montagu, M.V., Van Camp, W., 1995. Catalase in plants. Molecular Breeding 1, 207–215. Wise, R.R., Naylor, A.W., 1987. Chilling-enhanced photooxidation: evidence for the role of singlet oxygen and superoxide in the break-
down of pigments and endogenous antioxidants. Plant Physiology 83, 278–285. Yamamoto, H.Y., 1979. Biochemistry of the violaxanthine cycle in higher plants. Pure and Applied Chemistry 51, 639–645. Yamasaki, H., Sakihama, Y., Ikehara, N., 1997. Flavonoid-peroxidase reactions as a detoxification mechanism of plant cells against H2O2. Plant Physiology 115, 1405–1412. Yoshimura, K., Yabuta, Y., Ishikawa, T., Shigeoka, S., 2000. Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stress. Plant Physiology 123, 223–233.