Efficiency of antioxidant response in Spartina densiflora: An adaptative success in a polluted environment

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Environmental and Experimental Botany 62 (2008) 69–77

Efficiency of antioxidant response in Spartina densiflora: An adaptative success in a polluted environment夽 D. Mart´ınez-Dom´ınguez, M.A. de las Heras, F. Navarro, R. Torronteras, F. C´ordoba ∗ Department of Environmental Biology and Public Health, Faculty of Experimental Sciences, University of Huelva, E-21071 Huelva, Spain Received 10 July 2007; accepted 25 July 2007

Abstract The effects of different culture conditions, unpolluted and polluted substrates, on an antioxidative system – antioxidant enzymes, such as catalase, ascorbate peroxidase and guaiacol peroxidase, and ascorbic acid – were investigated to establish its relationship with the acclimatization success of Spartina densiflora. Plants of this species growing in the polluted Odiel marshes (Huelva, Spain) showed high levels of catalase, ascorbate and guaiacol peroxidase activities and ascorbate concentration (reduced and oxidized ascorbate). In addition, we found significant oxidation of the ascorbate pool, since only 40% of ascorbate was reduced, and low levels of photosynthetic pigments, suggesting that an oxidative stress was impairing S. densiflora. Transplantation to an unpolluted substrate in the laboratory led to a gradual change in all tested parameters: antioxidative activities and total ascorbate concentration decreased while the percentage of reduced ascorbate and pigment concentrations increased; these data agreed with the hypothesis that oxidative stress conditions in S. densiflora habitat were due to a polluted substrate. After 28 days, the plants were transplanted for a second time to polluted conditions, equivalent to those in their habitats, and a rapid alteration of the antioxidative system was observed. In the first 24 h, catalase and guaiacol peroxidase activities and ascorbate concentration increased greatly and the percentage of reduced ascorbate fell drastically. Regardless of this fact, ascorbate peroxidase activity did not change until the end of the first week, while photosynthetic pigments declined at a constant rate during the whole culture period. Subsequently, we found that the antioxidative system improved its reductive capacity gradually and slowly – over weeks – but this reductive power was rapidly lost within days or even hours. It may be concluded that S. densiflora undergoes oxidative stress in its natural environment and is able to modulate its antioxidative system, based on the degree of pollution, in order to acclimatize successfully to its fluctuating environment. © 2007 Elsevier B.V. All rights reserved. Keywords: Spartina densiflora; Metal pollution; Ascorbate; Oxidative stress; Environmental adaptation

1. Introduction High levels of metal pollution occur in the Tinto-Odiel estuary (Huelva, SW Spain), due to the wastes from nearby petrochemical industries and natural biolixiviation, together with effluents from the ancient mining activity in the Iberian Pyrite Belt located in the river basin. Metals such as Fe, Ni, Cu, Pb, Zn, As, Cd, etc. are abundant in the water and sediments

夽 This work was supported by Grants AGL2003-06555 (Ministerio Educaci´ on y Ciencia) and CVI 282 (Plan Andaluz de Investigaci´on, Junta de Andaluc´ıa, Spain). ∗ Corresponding author at: Department of Environmental Biology and Public Health, Faculty of Experimental Sciences, Bulevar de las Artes y las Ciencias, s/n, University of Huelva, 21071 Huelva, Spain. Tel.: +34 959219875; fax: +34 959219876. E-mail address: [email protected] (F. C´ordoba).

0098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2007.07.005

(e.g. Luque et al., 1998; Santos-Bermejo et al., 2003; Nieto et al., 2007; personal data). At present, the Tinto-Odiel estuary is considered as one of the most contaminated estuaries in the world (Environmental Agency of Andalusia, 1994). However, a very high biodiversity is present in the estuary, mainly at the Odiel marshes, thus the varied marsh organisms are exposed to toxic levels of contamination. Moreover, the marsh plants are exposed to changeable salinity levels, under the influence of the sea. This environment is unstable due to the discontinuous contribution of metals, variable salinity and fluctuations in fresh water availability, depending on the tide regime, sunshine hours and industrial activities. Under these conditions, marsh plants must be able to respond rapidly to unpredictable changes in the substrate, which probably leads to a variable degree of oxidative stress. It is well-known that reactive oxygen species (ROS) are produced in normal metabolic processes in all aerobic organisms

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(Asada and Takahashi, 1987; Mittler, 2002) and also wellestablished is their implication as key molecules in pathogen defense, programmed cell death, abiotic stress responses and systemic signaling (Desikan et al., 2001; Mittler, 2002). However, several stress conditions (metal pollution, salt stress, chilling, UV radiation, pathogen attack, etc.) can unbalance the steady-state level of ROS production (Foyer et al., 1997). These ROS include the superoxide radical (O2 •− ), hydroxyl radical (• OH) and hydrogen peroxide (H2 O2 ), which are produced during electron transport activities in the cell membrane as well as by a number of metabolic pathways (Shi et al., 2006). ROS accumulation induces oxidative processes such as membrane lipid peroxidation, protein oxidation, enzyme inhibition and DNA and RNA damage, resulting in cell damage and, eventually, cell death (Dat et al., 2000; Hammond-Kosack and Jones, 1996). To avoid the deleterious effect of ROS, plant cells possess efficient antioxidant defense mechanisms, comprising both enzymatic components, such as ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD) or guaiacol peroxidase (POD); as well as non-enzymatic components, such as ascorbic acid (ASC) or glutathione (Arrigoni and De Tullio, 2002). The capacity of ascorbic acid to eliminate directly or indirectly – via APX activity – different ROS including singlet oxygen, superoxide, hydrogen peroxide and hydroxyl radicals is widely reported (Foyer and Halliwell, 1976; Asada and Takahashi, 1987; Padh, 1990; Asada, 1992). These defensive mechanisms against oxidative damage have been specifically observed in plants subjected to saline stress (Gossett et al., 1994, 1996; Lee et al., 2001) or metal pollution, in the form of excessive iron (Kampfenkel et al., 1995), similar environmental conditions applying to the plants growing in the Odiel marshes. Marsh plants may be suitable to investigate defensive responses against environmental stress from multiple and variable sources. In our study, we chose Spartina densiflora Brong, an invasive cordgrass from South America (Mobberley, 1956), because of its wide distribution in the Odiel marshes, salinity tolerance and metal accumulating properties (Luque et al., 1999). The successful adaptation of this species to stressing conditions in the Odiel marsh could be related to an efficient defensive antioxidant mechanism. If so, we can expect that Spartina would rapidly modulate its antioxidant system when environmental stressors, mainly metals in soils, fluctuate from low to high levels (and vice versa). In order to validate this hypothesis, we have developed a new experimental approach with Spartina individuals exposed to different soil conditions (polluted versus unpolluted) in a continuously controlled culture. The present study reveals that S. densiflora undergoes oxidative stress in its polluted location but is able to rapidly modulate its redox status when cultured under the presence or absence of polluting agents. 2. Materials and methods 2.1. Plant material and growth conditions S. densiflora specimens were collected in the Odiel River salt marshes (Huelva, SW, Spain). Ten plants of S. densiflora were transplanted from their polluted habitat to individual pots in our

Fig. 1. Scheme of the experimental design. Ten plants of Spartina densiflora were collected in situ and transplanted (T) to individual pots in the laboratory: five pots contained polluted substrate as in situ (Control plants) and the other five pots contained unpolluted substrate (Experimental plants). After 28 days in these conditions (Period A), all plants were again transplanted to individual pots containing polluted substrate in both Control and Experimental groups until 43 days after collection (Period B). Periodically, leaf samples were removed to analyse metabolites and enzymes as indicated in Section 2.

laboratory and divided into two groups. A group of five plants, denominated Control plants, was cultivated using their natural, metal-polluted sandy substrate. Another group of five plants, named Experimental plants, was cultivated in non-stressing conditions with clean, metal-unpolluted substrate (marine sand). After 28 days of culture (period A), both Control and Experimental plants were again transplanted to a polluted substrate obtained from the marshes, and their growth was followed for 15 days (period B). A schematic drawing of the experimental design is shown in Fig. 1. From the onset of the experimental periods, all plants were cultured in individual pots in a greenhouse, with humidity, temperature, light intensity and photoperiod controlled similar to conditions in the Odiel River marshes. Plants were watered with Hoagland’s nutritious solution (Hoagland and Arnon, 1950) and, since S. densiflora is a halophyte, this nutritious solution was enriched with 4 g/l of NaCl. Periodically, leaf samples, similar in aspect and size, were removed and conserved at −80 ◦ C until homogenization and biochemical analysis was performed. 2.2. Determination of metals concentrations The determination of metals concentrations was carried out according to Chaoui et al. (1997) with minor modifications. At harvest, leaves of S. densiflora were washed in distilled water and then desiccated for 48 h at 70 ◦ C. Then, oven-dried plant material was wet-ashed with an acid mixture (HNO3 :HClO4 , 4:1) and analysed for Fe, Cu, Ni, Pb, Zn, As, Cd by atomic absorption spectrophotometry. Soil samples from the Odiel marshes were analysed in the same way, but without being washed in distilled water. 2.3. Photosynthetic pigments determination For the determination of carotenes, chlorophylls a and b, and total chlorophyll, we used approximately 100 mg fresh weight of leaves. Plant material from S. densiflora was homogenized using 50% ice-cold acetone in a mortar, with washed sea sand, and later on, the extract was centrifuged (Arnon, 1949). Finally, the parameters previously mentioned were determined by means of spectrophotometry from the supernatant (Lichtenthaler, 1987).

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2.4. Growth rate

2.7. Samples and statistical analysis

The growth rate in leaves of S. densiflora was measured throughout the whole experiment. The measurements were done by marking at least three leaves per plant, and following their growth with a metric scale. Growth rate was calculated by observing the length (mm) of leaf blades as (L2 − L1 )/d, where L1 and L2 are the lengths on the first day of the culture periods (A or B) and on the last days of the culture periods (A or B), respectively, and d is the number of days (28 days for period A and 15 days for period B).

During the experimental period, three leaf samples were removed to measure enzymes and metabolites from each plant cultured (i.e. the five Control and five Experimental plants) at the time indicated in the figures. All spectrophotometric analyses were performed twice for each leaf extract. We show n = 5 in the figure legends to indicate the number of individuals analysed in both experimental groups. The metal concentrations in the marsh soils and leaves from in situ plants were determined in three soil samples and in three leaves removed, respectively, from three randomly selected, healthy representative plants. The mean values ± S.D. are given in figures and tables. Results were firstly analysed by a two way ANOVA, followed by a post hoc Duncan’s Multiple Range Test to compare changes within a group during the study period and between both groups at the same selected time. For comparative purposes, data obtained after analysis from leaf samples removed from in situ plants are shown in all figures. When a time course is followed, these data are included at time 0 to indicate the onset of the time course in our experimental design.

2.5. Enzymatic activities determination Leaves (0.2–0.3 g) frozen using liquid N2 were macerated in porcelain mortars. The extraction was made in 1 ml of 50 mM phosphate buffer (pH 7.0) with 1 mM PMSF, 0.1 mM EDTA, 1% (w/v) polivinylpirrolidone (PVP) and 5 mM ASC (only for ascorbate peroxidase). The homogenized leaf material was centrifuged for 20 min at 15,000 × g, using the supernatant for determining the enzymatic activities. The oxidorreductase activity, which uses H2 O2 as a donor, measured in these assays includes a group of non-specific enzymes, from multiple cellular sources, and generally referred to as guaiacol peroxidase activity (POD) (EC 1.11.1.7). The method followed for its determination was the one described by Putter (1974), with some modifications. The total peroxidase activity was determined by spectrophotometry following the rate of formation of tetraguaiacol at 470 nm (EmM = 26.6 cm−1 ). The ascorbate peroxidase activity (APX) (EC 1.11.1.11), which uses ascorbic acid as a substrate, was determined according to the method described by Asada (1984), with modifications (Amako et al., 1994). The reaction was started with the addition of hydrogen peroxide (1 mM) and the rate of oxidation of ascorbate at 290 nm (EmM = 2.80 cm−1 ) was followed. The catalase activity (CAT) (EC 1.11.1.6) catalyses the dismutation of hydrogen peroxide into oxygen and water (Inz´e and Van Montagu, 1995) without using an electron-donor substrate. This activity was determined using the method described by C´ordoba-Pedregosa et al. (2003), in 50 mM phosphate buffer (pH 7.0) with 20 mM of hydrogen peroxide. The reaction was started with the addition of the sample, and the decomposition of H2 O2 at 240 nm (EmM = 0.043 cm−1 ) was followed. 2.6. Total and reduced ascorbic acid, and dehidroascorbate determination The levels of ascorbate and dehidroascorbate in leaves were quantified by means of the bypiridyl method (Kn¨ozer et al., 1996). In the extractions, the frozen material was macerated with liquid N2 in porcelain mortars using a buffer solution with metaphosphoric acid at 5%. The homogenate was centrifuged at 19,000 × g and the absorbance was measured at 525 nm (EmM = 12.3 cm−1 ). The oxidized ascorbate (DHA) was calculated as the difference between total and reduced levels of ascorbate.

3. Results Highly significant changes in the parameters tested were observed during all the study periods in the Experimental plants in response to the presence of unpolluted and polluted substrates, with heavy metals as stressing agents. On the other hand, Control plants did not show significant changes during the study period compared to the values of the same parameters observed in their habitat. In Experimental plants, the levels of ascorbate and enzymatic activities reached at the end of period A (28 days with unpolluted substrate) stayed constant during at least 60 days in the same culture conditions (data not shown), and were considered as basal levels in our experimental conditions. 3.1. Metals concentrations The data on metal concentration in the leaves and soil of S. densiflora in the Odiel Marshes shows high levels of these metals in both materials. The amount of Fe found was the greatest among all the metals, both in leaves as in soil (Table 1). Moreover, we also found that the concentrations of all metals were higher in the soil than in the leaves (Table 1). 3.2. Ascorbate measurements Total ascorbate concentration (ASC + DHA) decreased about 37% in Experimental plants during period A (Fig. 2). However, in period B, ASC concentration increased rapidly again (50% in 24 h). During the following 7 days, this increase continued but at a slower rate; finally, in the last week of this period, the level of ASC tended to stabilise at similar values to those in the Control plants.

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Table 1 Concentration of metals in S. densiflora leaves and in its natural substrate Metal concentration (ppm)

Leaves Soil

Fe

Ni

Cu

Zn

As

Cd

Pb

3378 ± 78 15,752 ± 102

5.4 ± 0.6 36 ± 1.1

368 ± 20 702 ± 53

322 ± 31 573 ± 77

28 ± 3 159 ± 11

0.29 ± 0.03 2.1 ± 0.9

54 ± 7 176 ± 26

Samples of the Odiel marsh soil and leaves of Spartina densiflora growing in such a location were collected and assayed for metal concentration as indicated in Section 2. Means ± standard deviation (S.D.) (n = 3) are shown.

In Fig. 3, the change in the percentage of reduced ASC is shown during the whole study period. The use of unpolluted substrate (period A) provoked a great increase in the values of reduced ASC (ca. 130%) for up to 28 days of culture and then a rapid decrease (45% in 24 h) when these plants were transplanted to the polluted medium (period B). In Fig. 3A, the concentration of reduced ASC and DHA (oxidized ASC) is shown in Experimental plants. These plants showed high levels of DHA at the beginning of study (i.e. the plants were stressed), the ratio DHA/ASC being 1.5. Experimental plants during period A showed an increase in reduced ASC concentration of about 32%, whereas oxidized ASC (DHA) decreased more than 90% from initial levels. Thus, the ratio DHA/ASC dropped to 0.1 in this period, only 7% of the starting value. On the other hand, the Experimental plants during period B showed a dramatic nine-fold increase in the oxidized form of ASC (DHA) and a small, non-significant decrease in reduced ASC. The levels of Total ASC concentration (Fig. 2), percentages of reduced ASC (Fig. 3), and reduced and oxidized ASC concentrations (Fig. 3B) in Control plants showed no significant variation during the whole study period.

Fig. 2. Concentration of total ascorbate. The concentration of the total ascorbate pool (reduced ASC + DHA) was analysed during the whole study period. Means ± S.D. (n = 5) for Control and Experimental plants are shown on dark and clear gray bars, respectively. The dotted line marks the change from period A to period B. Letters (a and b) indicate the level of significance (p < 0.05) over the time course within the Experimental group in such a way that the means followed by the same letter were not significantly different. An asterisk symbol (*) denotes significance at p < 0.05 between Experimental vs. Control plants within a particular day. No significant differences were found for values of Control plants on the different days of the study. For comparative purposes, mean values from in situ plants are shown (white bar).

3.3. Antioxidant enzymatic activities When S. densiflora plants were transplanted from the in situ polluted environment (Odiel River marshes) to the unpolluted controlled cultures in the laboratory (Experimental plants), all enzymatic activities decreased dramatically (Fig. 4 A–C). After that, during period B, these plants showed a great recovery of their initial enzymatic activity values, reaching the levels found in Control plants (Fig. 4A–C). On the other hand, Control plants, kept in their polluted substrate, maintained high levels of these enzymatic activities during the whole study period. Firstly, POD activity (Fig. 4A) decreased from 3.4 mU/mg fw in in situ, polluted plants, to 0.5 mU/mg fw (ca. 85% of reduction) after 28 days in unpolluted cultures; but after transplantation to a polluted substrate this activity showed a recovery, reaching levels similar to Control plants in 7 days, a six-fold increase. Secondly, CAT activity (Fig. 4B) at time zero was about 0.77 mU/mg fw, but reached only 0.23 mU/mg fw (ca. 75% of reduction) at the end of period A in Experimental plants. After transplanting of plants to a polluted medium, this activity showed a great and rapid increase; in only 24 h its activity exceeded the activity of the Control plants. In the following week, CAT activ-

Fig. 3. Redox state of ascorbate. The effects of different culture conditions on the redox state of ASC (% reduced ASC vs. Total ASC) were analysed. Data for reduced ASC and DHA for Experimental (inset A) and Control (inset B) plants are also shown. In situ values are used as time 0 to indicate the onset of the time course in our experiment. The number of samples, statistical symbols and significance are the same as in Fig. 2. More details are in Section 2.

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Fig. 5. Photosynthetic pigments and chlorophyll a/b ratio. Effects of different culture conditions on photosynthetic pigments in leaves of S. densiflora. The ratio of chlorophylls a/b (A), total chlorophyll concentrations (B) and total carotenes (C) are shown. The number of samples, statistical symbols and significance are as in Fig. 2. More details are in Section 2.

Fig. 4. Antioxidant enzymatic activities. Effects of different culture conditions on the activities of POD (A), CAT (B) and APX (C) in leaves of S. densiflora. The number of samples, statistical symbols and significance are as in Fig. 2. More details are in Section 2.

ity diminished to Control levels and continued at this level for the rest of the experiment. Finally, APX activity (Fig. 4C) was 5.96 mU/mf fw at the beginning, but it dropped continuously to a value of 2.61 after 28 days of unpolluted, controlled culture. After transplantation, plants did not show any significant changes in the first 24 h; afterwards, APX activity increased until it reached the Control levels at the end of the experiment (period B).

3.4. Photosynthetic pigments During the period of culture in the absence of toxic compounds and stressing conditions (period A), in Experimental plants the concentration of photosynthetic pigments increased continuously (Fig. 5). When compared to Control plants, total chlorophyll increased ca. 70% by the 28th day in plants growing in unpolluted substrate. Interesting, the ratio chlorophyll a/b remained stable during the whole experimental period and no significant differences were found between both plant groups. Carotene values also increased, by more than 40%, in the Experimental plants during period A, in contrast to Control plant levels, similar to the behaviour of the chlorophylls.

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Fig. 6. Growth rate of S. densiflora leaves during the study period. Effects of different culture conditions on the mean growth rate of S. densiflora leaves. As indicated in Section 2, leaf growth was calculated as an average of the length variation within both periods A and B in five plants from the each of the Experimental and Control groups. No statistical differences were observed between Experimental and Control plants over the time course of the whole experiment.

After 28 days of culture in the laboratory, when Experimental plants were transplanted to a new contaminated substrate (period B), they underwent a constant diminution of their pigment concentration, including total chlorophylls and carotenes, reaching levels comparable to those in Control plants at the end of this period. 3.5. Growth rate of S. densiflora The growth rate of Spartina leaves (Fig. 6) was chosen as a parameter to give us information about the physiology and development of the whole plant. The growth rate was very stable, for both groups of plants and during both periods of culture, fluctuating nearly 1 mm/day. No significant difference was found for any culture period or group at p ≤ 0.05. 4. Discussion The habitat of S. densiflora in the Odiel marshes has high levels of metal pollution, high but variable salinity, and partially anoxic sediments. These conditions have been widely described as inducing oxidative stress in plants (Kampfenkel et al., 1995; Gossett et al., 1996; Melonia et al., 2003). However, S. densiflora grows successfully in this environment, taking the place of indigenous species and colonising new areas. Our findings confirm that its ability to grow in fluctuating environments with prooxidant characteristics seems to be related to the rapid and efficient modulation of a series of antioxidant defence mechanisms, both enzymatic and non-enzymatic. Our results for the concentration of heavy metals in leaves of S. densiflora agreed with previous reports in the same species (Luque et al., 1999). Likewise, the comparison of heavy metal concentrations found in leaves of S. densiflora and in its natural polluted substrate with the Toxicity Reference Values (TRV)

published by EPA (U.S. Environmental Protection Agency, 1999), revealed concentrations in leaves and substrates higher than cytotoxic TRV values in all cases, except for Ni. Not only have EPA data showed the toxic effects induced by these metals when their concentrations are higher than determined values, but also abundant studies have reported oxidative stress induced in plants exposed to concentrations, even lower than in our study, of these same metals (Kampfenkel et al., 1995; Kukkola et al., 2000; MacFarlane and Burchett, 2001; Sandalio et al., 2001; Sch¨utzend¨ubel et al., 2001; Singh et al., 2006). Our experimental design focused on comparing the original and final state of the health of Spartina with the aim of assessing Spartina’s ability to adapt to new situations due to the efficient modulation of antioxidative mechanisms such as ascorbic acid (ASC) and associated enzymes. In our study, we observed a decrease in total ASC in leaves during exposure to an unpolluted substrate and an increase during exposure to a polluted substrate; which seems to be related to an enhancement of ASC-dependent detoxification processes (Horemans et al., 2000). Similarly, these high levels of total ASC have been previously observed in plants growing in polluted substrates where reactive oxygen species (ROS) were increased (Smirnoff, 1995, 1996; Horemans et al., 2000). Therefore, we can assume that our plants experienced the effect of ROS in response to the presence of high levels of heavy metals, among other toxic agents, and presumably to a decreased aeration in the original Odiel marshes substrate. We can also assume that the ASC is, at least in part, an important defence system used by this plant, since Control plants showed extremely high levels of total ASC in leaves, 10 times higher than observed in other species (Takahama, 1993). In stressing conditions, as experienced by the in situ Spartina plants, ASC oxidation is enhanced and results in an increase of dehidroascorbate (DHA) (Paolacci et al., 1997; Savoure et al., 1999), as was also observed in our Experimental plants in the presence of pollutants (period B), where the DHA levels were higher than in the absence of pollutants (period A). This correlation between the presence of toxic agents and a great increase in DHA levels has frequently been implied as a biochemical indicator of oxidative stress in plant metabolism (Wise, 1995). So, we observed significant changes in the redox state of the ASC pool when the plants were transplanted from a polluted substrate (Odiel marshes) to an unpolluted one (period A in the laboratory) and vice versa, from an unpolluted substrate (period A in the laboratory) to a polluted one (period B in the laboratory). However, this process of a reverse in the ASC redox state did not follow the same time course: in the absence of pollutants (period A), the ASC reduction took 28 days (ca. 1 month) to change from 40% of reduced ASC to 91.7%; however, when we put the plants in the presence of pollutants again (period B), within only 7 days the values of reduced ASC reverted to become similar to those of the Control plants (54%). Therefore, the recovery of reduced ASC levels in the absence of stress seemed to be slower than its oxidation in stress conditions. This could be possible because the continuous presence of ROS – due to an extremely polluted habitat – avoids the maintenance of high levels of reduced ASC resulting from synthesis de novo or recycling (Conklin, 2001).

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Tolerance of plants to oxidative stressing conditions may be explained by the enhanced activity of one or more antioxidative enzymes preventing cell and tissue damage (Shi et al., 2006). When Experimental plants were grown in the presence of unpolluted substrate (period A), a significant decrease in POD, CAT and APX activity was observed, probably related to a fall in ROS concentration (Hern´andez et al., 1993; Gonz´alez et al., 1998; Noctor and Foyer, 1998; Smirnoff, 1998; Dietz et al., 1999). Interesting results were observed when Experimental plants were re-transplanted again to their natural and polluted substrate (period B) from unpolluted substrate (period A): the levels of antioxidative enzymes significantly increased again, representing a new defensive response against oxidative stress probably triggered, directly or indirectly, by metal-catalysing ROS production. However, levels of enzymatic activities in the presence of unpolluted or polluted substrates also showed different time-courses: CAT and POD activities increased greatly during the first 24 h after exposure to polluted substrates, reaching levels even higher than the those found in Control plants. By contrast, APX activity did not alter significantly in the first 24 h, but needed 1 or 2 weeks to reach similar levels to Control plants. Our findings agree with those of Willekens et al. (1994) and L´opez et al. (1996), who found that induction of APX by exposure to pollutants requires days or even weeks to be effective, while CAT expression was induced in a few hours. So, the induced APX expression could be then considered as a late stress response and the induced CAT and POD expression an early response. Likewise, greater APX activity was observed at the end of period B rather than during the stress period itself (period B), which could indicate a possible post-transcriptional regulation for this enzyme, as it was also observed in pea APX transcripts induced by drought stress (Mittler and Zilinskas, 1994). The data on photosynthetic pigments showed that Control plants had significantly lower levels than measured in Experimental plants transplanted to an unpolluted culture (period A). It has been reported that plant exposure to heavy metals, salinity, anoxia, etc. leads to damage in the photosynthetic machinery and then the efficiency of photosynthesis declines (Sinha et al., 1997; MacFarlane and Burchett, 2001; Mascher et al., 2002; P¨atsikk´a et al., 2002). Therefore, our data indicate that a stressing condition, due to substrate composition, probably impaired the photosynthetic machinery. In fact, a decline in the photosynthetic pigments has been described as an indicator of stress due to metals (Dietz et al., 1999). At the same time, in our experimental design, the observed decrease in the photosynthetic pigments in the presence of polluted substrate did not affect the ratio of chlorophylls a and b, although it has been reported that chlorophyll b is more sensitive and suffers greater depletions in response to exposure to metals (MacFarlane and Burchett, 2001). If so, our results for the chlorophyll a/b ratio could mean that the toxic effect of pollutants acts mainly on the chlorophyll biosynthetic machinery, affecting either types of chlorophyll. In support of this hypothesis, it has been reported that heavy metals inhibit ␦-aminolaevulinic acid (ALA)-dehydratase (EC 4.2.1.24) and protochlorophyllide reductase (MacFarlane and Burchett, 2001); both enzymes involved in chlorophyll biosynthesis (Van Assche and Clijsters, 1990; De Filippis and Pallaghy,

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1994). However, in spite of the damage observed to chlorophyll pigments, Spartina did not show chlorosis symptoms nor impaired leaf growth rates in plants growing in polluted soils. These results, together with the observed data on the antioxidative response of Spartina, seem to indicate that Spartina has developed an efficient antioxidative machinary able to counteract the harmful effect of prooxidant compounds in soils, thus preventing major physiological damages. Similarly, Gossett et al. (1996) observed no reduction in the growth of a cotton cell line in the presence of high NaCl levels, and suggested that cotton cells achieved tolerance to NaCl stress through a significant increase in their antioxidant activity. Thus, the expected Spartina impairment from chronic exposure to environmental pollutants is apparently controlled by an efficient defensive mechanism that maintains an adequate state of health, enabling Spartina to become successfully acclimated to the metal polluted environment of the Odiel marshes. 5. Conclusions In summary, our experimental design has proved useful to test defence mechanisms in plants subjected to pollutants in their natural environments, in situations where it is impossible to conduct comparative studies between polluted and non-polluted individuals, as in the S. densiflora population growing in the Odiel marshes, where all plants are subjected to a variety of pollutants from multiple and undefined sources. In our experimental design, the only difference between Control and Experimental plants transplanted from the marsh is that Control plants were permanently exposured to natural, polluted soil, while the Experimental plants underwent transient exposure to an unpolluted substrate, followed by exposure to the original polluted substrate. This experimental approach allowed us to evaluate the ability of Spartina to adapt to different soil conditions over a short, continuous period of time. We used oxidative stress markers to assess its adaptative responses to the high content of metals in marsh soils. However, although the presence/absence of toxic metals, triggering oxidative stress, is the most conspicuous difference between the polluted and unpolluted substrates used in our experimental design, parameters other than metals may modify the redox status in plants, particularly the possible differences in aeration around the roots due to the different particle sizes of the original sandy substrate obtained from the marsh and the sea sand used in the laboratory. To investigate this, we are currently analysing the effects of exposure to a single metal using a single substrate (sea sand) in the laboratory to elucidate the contribution of oxidative stress triggered by metals to the adaptability of Spartina. Our data indicate that S. densiflora is endowed with an efficient antioxidative system that is rapidly modulated in response to variable stressor levels in soil. Thus, the capacity of Spartina to invade and compete successfully in polluted marshes may be explained, at least in part, by the ability of the plant to up-regulate the ascorbate cycle and related enzymes. Our data also show that “relaxing” of antioxidative defence mechanisms, after removing pollutants, is a slow and gradual process (taking weeks), whereas its “activation”, after exposure to pollutants,

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is a rapid process (hours or days), and it could be related to different rates of de novo ASC biosynthesis, ASC depletion by ROS scavenging and its recycling from oxidized forms of ASC, such dehydroascorbate and semidehydroascorbate. Thus, further research will be necessary to elucidate the contribution of the biosynthetic and recycling ASC pathways in S. densiflora subjected to changing environments, and to test the role of different antioxidative enzymes, mainly those related to glutathione metabolism, probably involved in the defensive response of Spartina to enviromental pollutants and related to ascorbate metabolism by the Halliwell–Asada cycle. Acknowledgements The authors are grateful to Junta de Andalucia (Spain) for providing a fellowship (FPDI2003-48906692K) to David Mart´ınez Dom´ınguez. Dr F. C´ordoba and Dr. R. Torronteras are codirectors of this project. The authors want to express sincere thanks to Dr. Jim´enez-Nieva (Assistant Professor of Ecology) for technical advice on Spartina sampling in the Odiel marshes and Dr. Canalejo (Associate Professor of Cell Biology) for his critical analysis and constructive suggestions for this manuscript. References Amako, K., Chen, G.X., Asada, K., 1994. Separate assays specific for ascorbate peroxidase and guaiacol peroxidase and for the chloroplastic and cytosolic isozymes of ascorbate peroxidase in plants. Plant Cell Physiol. 35, 497–504. Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Arrigoni, O., De Tullio, M.C., 2002. Ascorbic acid: much more than just an antioxidant. Biochem. Biophys. Acta: Gen. Subjects 1569, 1–9. Asada, K., 1984. Chloroplasts: formation of active oxygen and its scavenging. Met. Enzymol. 105, 422–429. Asada, K., 1992. Ascorbate peroxidase—a hydrogen peroxide-scavenging enzyme in plants. Physiol. Plant. 85, 235–241. Asada, K., Takahashi, M., 1987. Production and scavenging of active oxygen in photosynthesis. In: Kyle, D.J., et, al. (Eds.), Photoinhibition. Elsevier Science Publisher, Amsterdam, pp. 227–288. Chaoui, A., Ghorbal, M.H., El Ferjani, E., 1997. Effects of cadmium–zinc interaction on hydroponically grown bean (Phaseolus vulgaris). Plant Sci. 126, 21–28. Conklin, P.L., 2001. Recent advances in the role and biosynthesis of ascorbic acid in plants. Plant Cell Environ. 24, 383–394. C´ordoba-Pedregosa, M.C., C´ordoba, F., Villalba, J.M., Gonz´alez-Reyes, J.A., 2003. Zonal changes in ascorbate and hydrogen peroxide contents, peroxidase, and ascorbate-related enzyme activities in onion roots. Plant Physiol. 131, 697–706. Dat, J., Vandenabeele, S., Vranova, E., Van Montagu, M., Inz´e, D., Van Breusegem, F., 2000. Dual action of the active oxygen species during plant stress responses. Cell. Mol. Life Sci. 57, 779–795. De Filippis, L.F., Pallaghy, C.K., 1994. Heavy metals: sources and biological effects. In: Rai, L.C., Gaur, J.P., Soeder, C.J. (Eds.), Algae and Water Pollution. Schweizerbart’sche Verlagsbuchhandlung, E. Stuttgart, Germany, pp. 31–77. Desikan, R., Mackerness, A.H., Hancock, J.T., Neill, S.J., 2001. Regulation of the Arabidopsis transcriptosome by oxidative stress. Plant Physiol. 127, 159–172. Dietz, K.J., Baier, M., Kr¨amer, U., 1999. Impact of heavy metals in photosynthesis. In: Prasad, M.N.V., Hagemeyer, J. (Eds.), Heavy Metal Stress in Plants: Molecules to Ecosystem. Springer-Verlag, Berlin, pp. 73–97. Environmental Agency of Andalusia, 1994. Environment in Andalusia: 1994 file. Andalusia Regional Government, Seville, Spain.

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