Environmental and Experimental Botany 66 (2009) 917

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Environmental and Experimental Botany

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Methyl jasmonate-induced antioxidant defence in root apoplast from sunower seedlings

Maria Carmen Parra-Lobato a , Nieves Fernandez-Garcia b , Enrique Olmos b, , Maria Carmen Alvarez-Tinaut a , Maria Carmen Gmez-Jimnez a
a b

Department of Plant Physiology, Faculty of Science, University of Extremadura, 06071-Badajoz, Spain CEBAS-CSIC, Campus de Espinardo, P.O. Box 164, 30100-Murcia, Spain

a r t i c l e

i n f o

a b s t r a c t
We have investigated the physiological functions of the rapid generation of reactive oxygen species (ROS) and the implication of the antioxidant enzymes in the apoplast and symplast of roots of sunower (Helianthus annuus L.) seedlings exposed to methyl jasmonate (MeJA, 50 M). MeJA-elicited roots showed a fast increase in ROS content, followed by a marked increase in the activity of H2 O2 scavenging enzymes, guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and catalase (CAT). The mechanisms responsible for MeJA-induced H2 O2 accumulation was investigated further by studying both the production and scavenging of H2 O2 in the extracellular matrix. Peroxidases active against (2,2 -azinobis-[3-ethylbenzthiazoline-6-sulfonic acid], ABTS) and guaiacol were found in the apoplastic uid, and proved to be ionically and covalently associated with sunower cell walls, although only the peroxidase activities of the soluble apoplastic fractions and those ionically linked to the cell wall were correlated with the accumulation of the H2 O2 detected. The results indicated that H2 O2 accumulation is a complex and highly regulated event requiring the time-dependent stimulation and down-regulation of differently located enzymes, some of which are involved in H2 O2 generation and degradation. It is concluded that exogenous MeJA may be involved in the oxidative stress processes by regulating antioxidant enzyme activities. 2009 Elsevier B.V. All rights reserved.

Article history: Received 6 May 2008 Received in revised form 11 December 2008 Accepted 13 January 2009 Keywords: Apoplast Cell wall Sunower Methyl jasmonate Peroxidases Reactive oxygen species Root

1. Introduction Methyl jasmonate (MeJA), a methyl ester of jasmonic acid (JA), is a phytohormone with ubiquitous distribution among plants and generally considered to modulate many physiological events in higher plants, such as defence responses, owering and senescence (Cheong and Choi, 2003). Plants respond to a variety of environmental stresses through the induction of antioxidant defence enzymes that protect against further damage (Mittler, 2002; Kawano, 2003; Hernandez et al., 2006). In recent years, MeJA was found not only to regulate a variety of plant-developmental responses, but also to be induced by pathogen attack or wounds that often lead to the generation of reactive oxygen species (ROS), including hydrogen peroxide

Abbreviations: ABTS, 2,2 -azino-bis-[3-ethylbenzthiazoline-6-sulphonic acid]; AF, apoplastic fraction; APX, ascorbate peroxidase; ASC, ascorbate; CAT, catalase; DMAB, 3-(dimethylamino)benzoic acid; GPX, peroxidase activity against guaiacol; G6PDH, glucose-6-phosphate dehydrogenase; H2 O2 , hydrogen peroxide; MBTH, 3-methyl-2-benzothiazoline hydrazone; MeJA, methyl jasmonate; ROS, reactive oxygen species; SSF, soluble symplastic fraction. Corresponding author. Tel.: +34 968 396336; fax: +34 968 39 6213. E-mail address: eolmos@cebas.csic.es (E. Olmos). 0098-8472/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2009.01.002

(H2 O2 ), superoxide anions (O2 ) and hydroxyl free radicals ( OH) (Devoto and Turner, 2003). These observations suggest that MeJA could be linked to oxidative stress. ROS have the potential to interact with many cellular components, leading to membrane damage, causing an immediate cellular response to trigger a plant-defence signal (Van Breusegem et al., 2001). Plants have evolved protective enzymatic and nonenzymatic mechanisms to scavenge ROS and minimize their harmful effects. These mechanisms consist of metabolites, including ascorbic acid and glutathione, and enzymes, including superoxide dismutases (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), and peroxidases such as ascorbate peroxidase (APX; EC 1.11.1.11) and guaiacol peroxidase (GPX; EC 1.11.1.7). Recently, several studies have also supported the idea that MeJA has a major role in modulating the plant response to several types of biotic and abiotic stresses (Salzman et al., 2005; Collen et al., 2006). Different effects of MeJA on protective enzymes activities could be associated with H2 O2 metabolism (Wang et al., 2005; Ali et al., 2006). The apoplast matrix contains many enzymatic and nonenzymatic components. The concentration and characteristics of these components are exhaustively monitored by the symplasm. In this regard, the apoplast and cell wall act as a reservoir of information on the biotic and abiotic environment surrounding the cell

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as well as a major conduit of information between cells (Pignocchi et al., 2006). Recently, several studies have focused on the roles of ROS as signals inuencing plant responses to environmental stimuli (Bolwell et al., 2002; Kwak et al., 2006), and on the redox-buffering capacity of the apoplast (Foyer and Noctor, 2005; Pignocchi et al., 2006). The release of O2 and H2 O2 in the apoplastic space is believed to contribute to several disease-resistance strategies in plants. Thus, several different enzyme activities may be responsible for the generation of apoplastic O2 in plants, depending on the particular physiological context and the particular plant species under investigation (Frahry and Schopfer, 2001). ROS and particularly H2 O2 have been considered to be signal molecules in the environmental stress response since they induce the expression of a variety of defence-related genes (Foyer and Noctor, 2005). The fact that APX has a high afnity for H2 O2 and is able to detoxify low concentrations of H2 O2 , whereas CAT has a high reaction rate but a low afnity for H2 O2 , renders the APX isoenzymes ideal candidates for a tight regulation of hydrogen peroxide as a signaling molecule (Noctor and Foyer, 1998). Therefore, it is crucial that plants should maintain the activities of these enzymes in order to accommodate these types of oxidative stress. Roots play a number of important roles during plant growth and development and are typically the rst part of the plant to encounter several types of environmental stress. Several papers have already been published about the increased ROS caused by MeJA in different plants tissues also including roots (Garrido et al., 2003). The rst evidence for direct interaction between the MeJAinduced ROS and peroxidase in vivo activity in the roots of sunower seedlings has already been shown (Garrido et al., 2003). In this study, attention is focused on the generation of ROS. However, the cellular antioxidant-defence systems and, in particular, antioxidant enzyme (APX, GPX and CAT) activities in response to MeJA addition have not been studied. The present work was carried out in an attempt to understand the MeJA-dependent defence response in root. Since MeJA could be linked to oxidative stress, it would be helpful to know the antioxidant response of sunower to elicitation with MeJA. For this, we investigated the role of MeJA on early production of H2 O2 and O2 , as well as temporal and spatial responses of antioxidant enzyme (CAT, APX and GPX) activities in the apoplastic and the symplastic fractions of sunower roots. In addition, its regulatory role on apoplastic and cell-wall-bound peroxidases was studied. 2. Materials and methods 2.1. Plant material Sunower (Helianthus annuus L. cv dwarf) seeds were germinated on wet lter paper in plastic trays (50 seeds and 175 mL distilled water) in darkness at 27 C and 70% relative humidity. Three-day-old seedlings were then treated by seedling immersion with distilled water (control) and MeJA (50 M in water) for 5, 15, 30, 60 and 120 min under light. Afterwards, the roots (35 mm) and cotyledons of treated seedlings were aseptically excised and immediately used. The excised roots were treated with MeJA before the excisions were done. 2.2. Hydrogen peroxide production Hydrogen peroxide production was determined according to Okuda et al. (1991). The reaction mixture contained 1 mL of distilled water (with or without MeJA at a nal concentration of 50 M), 400 L of 12.5 mM 3-(dimethylamino)benzoic acid (DMAB) in 0.375 M phosphate buffer (pH 6.5), 80 L of 1.3 mM 3-methyl-2benzothiazoline hydrazone (MBTH) and 20 L of peroxidase (0.25

unit) in a total volume of 1.5 mL. The reaction was started by carefully immersing the root of one intact seedling into the continuously aerated and stirred reaction mixture at 25 C, and the change in A590 nm was monitored every 5 min in a spectrophotometer (Shimadzu UV 1603). 2.3. Isolation of apoplastic uids and intracellular soluble fractions About 2 g of sunower seedlings (root + cotyledon) or excised roots were quickly washed in distilled water, placed in Petridishes in 10 mM sodium phosphate, pH 6.0, containing 1.5% (w/v) polyvinylpolypyrrolidone, 1 mM EDTA, and 0.5 mM phenylmethylsulfonyl uoride, and submitted to vacuum (60 kPa) for 5 min at 4 C. Afterwards, seedlings (root + cotyledon) or excised roots were carefully dried with lter paper and placed in syringes, which were placed in centrifugation tubes. Seedlings or excised roots were centrifuged at 150 g for 5 min, and the apoplastic uids (AF) recovered at the bottom of the tubes. With this procedure, we obtained 70110 L of AF for 1 g fresh weight of each material. The remaining seedlings or excised roots were used to obtain the soluble symplastic fraction (SSF) after homogenization in the same medium with an Ultraturrax T-25 (IKA Labortecnik, Staufen, Germany) and centrifugation at 15,000 g for 30 min. Cytosolic contamination of AF was monitored by assaying with glucose-6-phosphate dehydrogenase (G6PDH) activity as a marker. 2.4. Superoxide production Extracellular production of O2 radicals was estimated by the oxidation of epinephrine (Sigma, St. Louis, MO) to adrenochrome, which can be determined spectrophotometrically by measuring A480 (Takeshige and Minakami, 1979). To estimate changes in the production of O2 with time, we estimated O2 production in different samples shaken in 2 mL of 50 mM TrisHCl buffer (pH 7.8), to which 1 mM epinephrine was added after 0, 5, 15, 30, 60 and 120 min. 2.5. Antioxidant enzymatic activities Enzymatic activities were spectrophotometrically assayed for AF and SSF obtained from seedlings (cotyledon + root) or excised root. Except where noted, reactions were developed at 25 C for 5 min, with stirring, at a nal volume of 1 mL containing 2535 g of protein. GPX was determined according to Zheng and Van Huystee (1992). The reaction mixture contained 10 mM sodium phosphate (pH 6.0), 0.1 mL of 0.3% (v/v) H2 O2 , and 0.1 mL of 1% (v/v) tetraguaiacol. Reaction was initiated by the addition of H2 O2 and followed at 470 nm (extinction coefcient of tetraguaiacol = 26.6 mM1 cm1 ) at 30 C. For APX determination, seedlings or roots excised were vacuuminltrated in phosphate buffer containing 5 mM ASC. For SSF, homogenization buffer also contained 5 mM ASC. The activity was measured by the method of Nakano and Asada (1981). The reaction mixture contained 50 mM phosphate buffer (pH 7.0), 1 mM sodiumascorbate and 2.5 mM H2 O2 . After the addition of ascorbate to the mixture, the reaction was followed at 290 nm (extinction coefcient of ascorbate = 2.8 mM1 cm1 ). CAT activity was estimated using the method of Aebi (1983). The reaction mixture contained 50 mM potassium phosphate (pH 7.0) and 10 mM H2 O2 . After enzyme addition, the reaction was monitored by following decomposition of H2 O2 at 240 nm (extinction coefcient of H2 O2 = 43.6 mM1 cm1 ). G6PDH assay was made in 100 mM TrisHCl (pH 8.0) containing 1 mM MgCl2 , 0.2 mM NADP+ , and 1 mM glucose-6-phosphate.

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Generation of NADPH was measured at 340 nm, and the extinction coefcient was 6.2 mM1 cm1 (Weimar and Rothe, 1986). 2.6. Extraction and assay of cell-wall-bound peroxidase For root cell-wall isolation a modied procedure of Ros Barcelo et al. (1989) was employed. Samples (1 g) were homogenized in 11 volumes of 50 mM TrisHCl, pH 7.2 containing 250 mM sucrose. The homogenate was centrifuged at 4 C for 10 min at 1000 g, and the supernatant was discarded. The pellet, washed twice by resuspension in 10 mL of 50 mM TrisHCl, pH 7.2, containing 250 mM sucrose and 1% (w/v) Triton X-100 and four times in 30 mL of the same medium without Triton, was considered to be a puried cellwall fraction. As a means of obtaining ionically bound enzymes, the cell-wall fraction was incubated in 1 M NaCl for 30 min with continuous stirring at 4 C. The supernatant obtained after centrifugation at 1000 g for 10 min was used in experiments. This fraction was considered as the ionically bound protein fraction. The residue was exhaustively washed with water and treated with a mixture of 0.5% cellulose and 2.5% pectinase, both from Aspergillus niger (Sigma Chemical Co), in 0.1 M sodium acetate buffer pH 5.0 for 24 h at 25 C with shaking (Sanchez et al., 1995). The suspension was centrifuged at 10,000 g for 30 min, and the supernatant was dialysed against distilled water and concentrated with ultraltration cell (Amicon PM 10). This fraction was considered as the covalently bound protein fraction. Peroxidase activity was measured using two different substrates, 2,2 -azino-bis-[3-ethylbenzthiazoline-6-sulphonic acid] (ABTS) or guaiacol. The activity against ABTS was measured using the following reaction medium. Protein extract (10 g) was incubated with 0.5 mM ABTS, 0.2 mM H2 O2 in 50 mM glycineHCl buffer pH 2.57.0 in a reaction volume of 2.5 mL at 30 C for 30 s. The absorbance was measured at 414 nm (extinction coefcient was 31.1 mM1 cm1 , Arnao et al., 1990). Further experiments were carried out using a pH 3 buffer. The activity against guaiacol was determined following Zheng and Van Huystee (1992). Further experiments were carried out using a pH 5.0 buffer. The protein concentration was measured according to Bradford (1976) using bovine serum albumin as the standard. The data are the mean of six determinations in three different experiments. 3. Results 3.1. Early production of reactive oxygen species in sunower seedlings induced by MeJA Sunowers seedlings were incubated in a solution containing 50 M MeJA for increasing time ranging from 5 min to 2 h. The effect of MeJA on production of ROS in roots of sunower seedlings is shown in Fig. 1. The treatment resulted in higher production of hydrogen peroxide in sunower roots compared to the respective control (Fig. 1A). The elicitation also led to an induction of O2 accumulations (Fig. 1B). In sunower roots, production of H2 O2 and O2 accumulations increased 1.6 and 2.4 times, in comparison with control roots, at 5 min post-elicitation with 50 M MeJA (Fig. 1), respectively. 3.2. Enzymatic antioxidant response in cotyledons and roots of sunower seedlings In total homogenates of sunower seedlings, MeJA showed no effect on the antioxidant enzymes (GPX, APX, CAT) in the cotyledon tissues (Fig. 2, left panels). However, MeJA increased all enzyme activities in the roots (Fig. 2, right panels). In total homogenates of sunower root, GPX and CAT activities were much higher in the total

Fig. 1. Effects of methyl jasmonate on the levels of H2 O2 (A) and O2 production (B) of MeJA-treated sunower seedlings. Three-day-old sunower seedlings were treated with 50 M MeJA (solid line) or distilled water (broken line) from 0 to 2 h. In this and the following graphs, the error bars represent the standard deviation, n = 6, and overlapping error bars have been removed.

homogenates from roots than in cotyledons (Fig. 2A and C). However, the ascorbate peroxidase did not differ signicantly between untreated root and cotyledon tissues (Fig. 2B). The GPX activity in the total root homogenate signicantly increased during the rst 5 min of treatment, showing 39% more activity than in the non-treated roots (Fig. 2A). Meanwhile, the GPX activity in the cotyledons was similar during the 2 h of treatment, both in treated as well as in non-treated (Fig. 2A). The APX activity increased 2.8-fold at 5 min in the total homogenate of roots treated with MeJA with respect to the non-treated roots (Fig. 2B). In addition, in the total homogenate of the cotyledons, no differences were appreciated in the APX activity between treated and non-treated seedlings, both gradually increasing during the rst 30 min of treatment (Fig. 2B). CAT activity of cotyledon homogenates showed no changes between MeJA-treated and non-treated seedling. However, CAT activity of root homogenates was higher after 30 min in MeJAtreated roots (Fig. 2C). 3.3. Changes in intracellular and apoplastic enzymatic antioxidant response to MeJA in roots of sunower seedlings Sunower seedlings (root + cotyledon) or excised roots were treated with MeJA and the apoplastic (AF) and soluble symplastic fractions (SSF) were obtained as described previously. In both fractions G6PDH activity were measured. The results are displayed in Table 1. AF obtained in our experiments showed only slight contamination from the cytosol, as deduced from the controls made

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Fig. 2. Effects of methyl jasmonate on the activities of guaiacol peroxidase (A), ascorbate peroxidase (B) and catalase (C) of the total homogenates of the cotyledons and roots in sunower seedlings with 50 M MeJA (solid line) and control (broken line). Three-day-old sunower seedlings were treated with either distilled water or MeJA (U GPX, U APX and U CAT: mol min1 mg1 protein).

with G6PDH activity, a marker for cytosolic contamination. This activity was assayed for each apoplast extraction. All of the following data concerning apoplastic constituents have been corrected accordingly for cytosolic contamination.
Table 1 Glucose-6-Phosphate dehydrogenase (G6PDH) activity evaluated in soluble symplastic fractions (SSF) and apoplastic uids (AF) from the three-day-old sunower seedlings (cotyledon + root) or the excised roots of seedlings. SSF AF (nmol min1 g1 fresh weight) Sunower seedlings Excised root 516 41 340 29 0.8 0.23 (0.18) 1.6 0.51 (0.57)

Values are given standard deviation, n = 6. The percentage of apoplastic activity respect to the corresponding SSF is presented in parentheses.

Sunower root AF revealed different enzymatic activities (GPX, APX and CAT), depending on whether the seedling root had been excised or not before the MeJA treatment (Fig. 3). GPX and APX activities were signicantly lower in the AF than in the total homogenates. Thus activities calculated using the total homogenates were nearly identical to those from AF plus SSF. It seems that apoplastic APX was higher than the symplastic enzyme, and accordingly most of the total APX detected in roots seems to be apoplastic. The MeJA treatment increased GPX and APX in the apoplastic space from both excised and non-excised roots. Enzymatic activities assayed in SSF and AF of roots varied depending on the presence or absence of MeJA (Fig. 3). Peroxidase activity (GPX) was higher in MeJA-treated roots than in non-treated ones during the rst 30 min in both SSF and AF (Fig. 3A). In SSF, the APX activity was higher in excised roots and signicantly lower in

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Fig. 3. Effects of methyl jasmonate on the activities of guaiacol peroxidase (A), ascorbate peroxidase (B) and catalase (C) in the apoplastic (AF) and soluble symplastic fractions (SSF) from sunower root depending on whether the seedling root had been excised ( ) or not () before the MeJA treatment (solid line) or distilled water (broken line). Three-day-old sunower seedlings were treated with either distilled water or 50 M MeJA (U GPX, U APX and U CAT: mol min1 mg1 protein).

non-excised ones (Fig. 3B). However, in apoplast, this activity was higher in MeJA-treated roots during the rst 60 min in both, excised and non-excised roots (Fig. 3B). The pattern of CAT activity of SSF was unaltered by MeJA in both, excised and non-excised roots. However, the apoplast of MeJAtreated roots, excised and non-excised, showed a higher value of CAT activity during the rst minutes of treatment (Fig. 3C). 3.4. MeJA-induced changes in cell-wall peroxidase activity in sunower roots The effect of pH on the peroxidase activity against both substrates (ABTS and guaiacol) was tested using apoplastic, ionically and covalently bound fractions obtained from three-day-old roots when the activity was higher (Fig. 4). The pH dependence of peroxidase activity against ABTS showed a wide-shaped curve, with maximum values ranging between pH 3.0 and 4.0 (Fig. 4A). However, the maximum activity with guaiacol was found at pH 5.0 for

the three different fractions (Fig. 4B), this value being similar to the value found for pine peroxidase (Sanchez et al., 1995). The peroxidase activity of apoplastic, ionically and covalently bound fractions was higher when ABTS was used as substrate, the activity being lower when guaiacol was assayed as substrate for peroxidase. The changes in the cell-wall-bound peroxidase activity in response to MeJA are shown in Fig. 5. Peroxidase activity in the apoplastic uid against ABTS and guaiacol increased at 5 min in excised roots treated with MeJA, the increase being stronger in the presence of ABTS (Fig. 5A). Afterwards, peroxidase activity against both substrates dramatically declined. The peroxidase activity ionically bound to the cell wall against ABTS and guaiacol also increased with MeJA-treated roots, the activity of this POX isozyme being higher than the apoplastic and the covalently bound isozymes (Fig. 5B). Although the changes in peroxidase activity in the presence of MeJA showed some difference between ABTS and guaiacol substrates, in both cases, the activity was higher at 5 min with MeJA. The peroxidase activity covalently bound to the cell walls against

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Fig. 4. Effect of pH on peroxidase activity against ABTS (A) and guaiacol (B). Apoplastic ( ), ionically ( ) and covalently bound ( ) peroxidases (Unit of peroxidase (U PX): mol min1 mg1 protein).

ABTS was similar to the activity against guaicol, and only slightly increased with MeJA for longer incubation times (Fig. 5C). 4. Discussion MeJA and its free-acid jasmonic acid (collectively referred to as jasmonates) are important cellular regulators involved in diverse developmental processes, such as seed germination, root growth, fertility, fruit ripening, and senescence (Wasternack and Hause, 2002). In addition, jasmonates activate plant defence mechanisms in response to insect-driven wounding, various pathogens, and environmental stress (Cheong and Choi, 2003). In sunower roots, H2 O2 and O2 accumulation, observed after only 5 min of exposure to 50 M of MeJA, was one of the earliest detectable metabolic responses to the imposed stress. O2 is known to be harmful to all membrane constituents suggesting that MeJA leads to the production of O2 , resulting in increased lipid peroxidation. In accordance with these results, induction of oxidative stress has been observed in plants exposed to MeJA (Ali et al., 2006), as well as in suspension culture Panax ginseng roots (Ali et al., 2006) and in Brassica napus leaves (Sarosh and Meijer, 2007). Recently, in Arabidopsis thaliana, the expression of oxidative stress-tolerance genes (Mittler, 2002), including those for peroxidases and oxidases, was also up-regulated by MeJA treatment (Jung et al., 2007). The activities of antioxidant enzymes increased in sunower seedlings but the increase was signicant and consistent only in the root. The increases in H2 O2 and O2 concentrations are considered as an inducer of the responses of enzymatic systems linked to the ROS-scavenging process (Apel and Hirt, 2004). The results, in the present study, show that among the antioxidant enzymes assayed, GPX and APX activities augmented by around 0.4- to 2.8fold in total homogenates of sunower roots upon MeJA exposure (5 min) compared with controls; however, in the total homogenates of sunower cotyledons no differences were appreciated in the GPX and APX activities between treated and non-treated seedlings. The increased antioxidant enzyme activities were correlated with greater H2 O2 in the MeJA-treated roots, indicating rapid responses of antioxidant enzymes to oxidative stress in sunower roots. The activities of these scavenging enzymes increased signicantly in roots (5 min) upon MeJA exposure but the induction levels of activity of CAT were higher in roots after 30 min upon MeJA application. For instance, it is still necessary to conrm whether the CAT induc-

tion depends only on an increase in MeJA. However, it has been reported that other signaling molecules may be the compound causing CAT inhibition and induction of disease resistance by the signaling of H2 O2 and activation of the defence genes (Shim et al., 2003). Since enzymes involved in antioxidant metabolism are usually co-regulated and their activities increase upon exposure to stress, higher activity levels in roots indicate increased oxidative stress in sunower roots upon MeJA exposure. The observations also strongly imply a possibility that enzymatic antioxidant systems are also utilized in sunower to alleviate oxidative stress caused by MeJA, thus protecting the cells from oxidative damage. In addition, reports suggest that apoplastic compartment could be important in the plant-cell response to stress (Vanacker et al., 1999; Olmos et al., 2003; Diaz-Vivancos et al., 2006). However, most studies concerning oxidative stress caused by extreme environmental changes have not considered compartmentalization, and have not distinguished between ROS generated in the cytoplasm and those generated in the apoplast. Therefore, in the present study, the effects of MeJA on activities of apoplastic GPX, CAT and APX were investigated in sunower roots. It was hypothesized that MeJA can affect apoplastic antioxidant enzyme activities in the sunower roots. However, it is noteworthy that after MeJA treatment the GPX, CAT and APX activities detected in AF from the root were higher than in the control and, in some cases, less in terms of percentage from the corresponding SSF. GPX and APX activities increased signicantly in both SSF and AF from treated roots, whereas CAT increased in AF. When apoplastic CAT activity was determined in sunower roots the values of activity were similar to values of apoplastic CAT activity in onion roots (Cordoba-Pedregosa et al., 2003). Root apoplastic APX activity strongly increased (up to 95%) after a short-term MeJA treatment (5 min). Our results also suggest that the apoplastic CAT and APX activity could be involved in the regulation of H2 O2 in the extracellular spaces (Cordoba-Pedregosa et al., 2003; Patykowski and Urbanek, 2003). Under stress conditions, some soluble peroxidase isoforms can be easily released from the cell wall and circulated inside the apoplast of the intact plant (Minibaeva and Gordon, 2003). Thus, peroxidase protects cells against the damaging effects of H2 O2 during an oxidative-burst response (Bolwell et al., 2002). In the literature, only one work about the induction of peroxidase by

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Fig. 5. Changes in apoplastic (A), ionically bound (B) and covalently bound (C) cell-wall peroxidase activities in roots of sunower. Three-day-old sunower seedlings were treated with either distilled water (broken line) or MeJA (solid line) from 5 min to 2 h. ABTS or guaiacol were used as substrates (Unit of peroxidase (U PX): mol min1 mg1 protein).

exogenous MeJA was found (Garrido et al., 2003) stimulating the O2 synthesis by root cells. However, several reports concerning apoplastic CAT and peroxidase activities under salicylic acid treatment/cold stress, as well as under pathogen attack and ozone stress have been reported (Vanacker et al., 1999; Patykowski and Urbanek, 2003; Ranieri et al., 2003; Shim et al., 2003). Several functions have been postulated for soluble and cell-wall-bound extracellular peroxidases (Hiraga et al., 2001; Kawano, 2003). MeJA such as salicylic acid might function as a weak detergent and change the electric charge of the cell surface, facilitating secretion of soluble peroxidase isoforms into the apoplast (Minibaeva and Gordon, 2003). In addi-

tion to cellular CAT and peroxidase-activity studies under salicylic acid treatment and cold stress (Shim et al., 2003), several studies are available about the effect of pathogen attack and ozone stress on apoplastic CAT and peroxidase activities (Vanacker et al., 1999; Ranieri et al., 2003; Patykowski and Urbanek, 2003). The expression and activity of these enzymes may be regulated by different abiotic and biotic stress factors or other stimuli. For until now, no study has been published about the effect of MeJA treatment on cell-wall-bound peroxidase activity. In sunower roots, the origin of H2 O2 during MeJA exposure appears to be complex and highly regulated, whereby various sets

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of differently located enzymes undergo requisite time-dependent coordinated stimulation as well as down-regulation. Authors suggested that NADPH oxidase could be the main source of O2 generation that they observed. Superoxide can be dismutated by SOD to produce the H2 O2 detected in MeJA-treated roots. However, another mechanism can be considered for the apoplastic H2 O2 generation. A role for NADH-POX in the apoplastic H2 O2 generation could be considered (Patykowski and Urbanek, 2003; Diaz-Vivancos et al., 2006), although other enzymes such as oxalate oxidase and amine oxidase cannot be excluded (Bolwell and Wojtaszek, 1997). Extracellular peroxidase, which catalyzes lignication, needs an acidic environment, its optimal pH being between 4.5 and 6.0 (Otter and Polle, 1997). This agrees with results concerning the stimulation of peroxidase activity after ozone stress (Ranieri et al., 2003). The ionically bound fraction behaved differently, not being inuenced by ozone exposure in any time interval. Hence, it seems that the reaction to MeJA exposure by the different peroxidase isoforms depends on their subcellular location. Extracellular sunower peroxidases responded in a similar way to iron deciency, thus affecting peroxidase activity in the covalently (but not in the ionically) bound fraction (Ranieri et al., 2001). However, despite vigorous research, the different peroxidase isozymes have not until now been clearly correlated with their subcellular location in the cell wall (Passardi et al., 2006), although many different reactions catalyzed by peroxidases in the cell wall are assumed to require a ne control of microlocalization. It should be borne in mind that the nal response to MeJA depends on the crosstalk between the different signaling routes, involving not only ROS and MeJA, as well as perhaps ethylene and salicylic acid, which interact synergetically or antagonistically (Overmeyer et al., 2000). In this context, salicylic acid, by inhibiting catalase activity, reportedly induces H2 O2 accumulation through specic binding to the enzyme (Chen et al., 1993) or by triggering H2 O2 formation by peroxidases (Kawano and Muto, 2000). Meanwhile, jasmonates are thought to desensitize the oxidative burst induced by ozone and the salicylic acid-mediated amplication loop which produces excess ROS (Overmeyer et al., 2000; Ranieri et al., 2003). In conclusion, the early ROS accumulation observed in sunower seedlings, treated with MeJA, appears to result from a highly regulated time-dependent stimulation and down-regulation of differently located enzymes which produce or scavenge ROS. It seems that the increase in H2 O2 generation could induce specifically different H2 O2 -scavenging enzymes in the apoplastic space from sunower roots. However, there is a need for ongoing experiments to examine the behaviour of other extracellular enzymes and metabolites involved in H2 O2 turnover, together with studies to decipher the crosstalk among H2 O2 and other signaling molecules. Acknowledgments The authors wish to thank Dr. I. Zarra (University of Santiago de Compostela, Spain) for their help in the extraction and assay of cell-wall-bound peroxidase. References
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