Antioxidant response of Phragmites australis to Cu and Cd contamination

Ecotoxicology and Environmental Safety 109 (2014) 152–160

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Antioxidant response of Phragmites australis to Cu and Cd contamination A. Cristina S. Rocha a,n, C. Marisa R. Almeida b, M. Clara P. Basto a, M. Teresa S.D. Vasconcelos b a b

CIIMAR/CIMAR and Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Universidade do Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal

art ic l e i nf o

a b s t r a c t

Article history: Received 8 April 2014 Received in revised form 18 June 2014 Accepted 20 June 2014

Metals are known to induce oxidative stress in plant cells. Antioxidant thiolic compounds are known to play an important role in plants' defence mechanisms against metal toxicity but, regarding salt marsh plants, their role is still very poorly understood. In this work, the involvement of non-protein thiols (NPT), such as cysteine (Cys), reduced glutathione (GSH), oxidised glutathione (GSSG) and total acidsoluble SH compounds (total thiols), in the tolerance mechanisms of the marsh plant Phragmites australis against Cu and Cd toxicity was assessed. Specimens of this plant, freshly harvested in an estuarine salt marsh, were exposed, for 7 days, to rhizosediment soaked with the respective elutriate contaminated with Cu (0, 10 and 100 mg/L) or Cd (0, 1, 10 mg/L). In terms of NPT production, Cu and Cd contamination induced different responses in P. australis. The content of Cys increased in plant tissue after plant exposure to Cu, whereas Cd contamination led to a decrease in GSSG levels. In general, metal contamination did not cause a significant variation on GSH levels. Both metals influenced, to some extent, the production of other thiolic compounds. Despite the accumulation of considerable amounts of Cu and Cd in belowground tissues, no visible toxicity signs were observed. So, antioxidant thiolic compounds were probably involved in the mechanisms used by P. australis to alleviate metal toxicity. As P. australis is considered suitable for phytostabilising metalcontaminated sediments, understanding its tolerance mechanisms to toxic metals is important to optimise the conditions for applying this plant in phytoremediation procedures. & 2014 Elsevier Inc. All rights reserved.

Keywords: Reduced glutathione Oxidised glutathione Cysteine Phragmites australis

1. Introduction Copper is an essential metal for plants, playing key roles in several physiological processes (Yruela, 2005). On the other hand, Cd, without a known particular function in plant's metabolism (Påhlsson, 1989), is easily uptaken by plant roots and translocated to aboveground parts (Gill and Tuteja, 2011). Depending on their concentration in the medium, both metals have been proven to be toxic for plants (Påhlsson, 1989) due to not only a direct interference of metals in plant cells metabolism but also antagonistic effects between the metals and the uptake of essential metals, such as Fe, Mn, Zn (Ondo et al., 2012, El-Kafafi and Rizk, 2013). Evidence that these metals induce oxidative stress in plant cells is found in the literature (Anjum et al., 2012) and to cope with metal toxicity, plants rely on an efficient antioxidant defence mechanisms to repair the inhibitory effects of metal-induced oxidative stress (Gill and Tuteja, 2011).

n

Corresponding author. E-mail address: [email protected] (A.C.S. Rocha).

http://dx.doi.org/10.1016/j.ecoenv.2014.06.027 0147-6513/& 2014 Elsevier Inc. All rights reserved.

Non-protein thiols (NPT), such as glutathione (GSH) and cysteine (Cys), have been reported as important agents against oxidative stress. Cysteine is a hydrophilic α-amino acid involved in the chelation of trace metals (Jozefczak et al., 2012), in the synthesis of sulphur-organic compounds, proteins, vitamins and co-enzymes and in the biosynthesis of GSH and phytochelatins (PC) (Bonner et al., 2005). Glutathione presents several functions in plant metabolism and protection being considered as one of the most important NPT in cells (Noctor et al., 2012). This NPT functions as a substrate for several enzymes and as an important element in ascorbate regeneration (Arora et al., 2002). Moreover, it is also a reductant and scavenger of reactive oxygen species (ROS) and a substrate for PC synthesis (Anjum et al., 2012). Glutathione can also complex metal ions promoting their appropriate storage and reducing their toxicity (Guimarães et al., 2008). The consumption of GSH and its conversion into oxidised glutathione (GSSG) may be an important indicator of oxidative stress in cells (Anjum et al., 2012). Information on the response of soil plants, in terms of NPT production, to continuous Cd and Cu exposure regimes can be found in the literature (Ben Ammar et al., 2008; Bruns et al., 2001;

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De Vos et al., 1992). Nevertheless, for salt marsh plants, knowledge on the role of NPT on metal detoxification is still limited. Salt marsh plants face serious challenges to cope with the waterlogged, anoxic, reduced, saline and, sometimes polluted, environments in which they live. Nonetheless, salt marsh plants pose an advantage to plants inhabiting freshwater habitats since they had to develop efficient means to survive and reproduce in these salt-rich environments (Manousaki and Kalogerakis, 2010). Over the last decades, there has been an increasing interest in using halophytic plants for phytoremediation purposes, since these plants can remove and accumulate considerable amounts of metals (Almeida et al., 2011; Caçador et al., 2009), being therefore important to study the role of NPT on the phytoremediation processes. Phragmites australis is a macrophyte plant frequently found in wetlands throughout temperate and tropical regions of the world and has colonised many coastal salt marshes (Havens et al., 1997). In addition, it can accumulate several metals, including Cu and Cd (Fediuc and Erdei, 2002; Windham et al., 2003). The antioxidant mechanisms developed in P. australis specimens were studied in a few previous studies (Ederli et al., 2004; Fediuc and Erdei, 2002; Iannelli et al., 2002; Pietrini et al., 2003) and the results suggested that GSH, PC and antioxidant enzymes may play important protective roles in defending plant cells against Cd toxicity. These works used however synthetic hydroponic media (a non-natural medium) and specimens adapted to freshwater environments. In this work, specimens of P. australis, freshly collected from an estuarine salt marsh, were used to research the influence of Cu and Cd contamination on the content of NPT in plant roots and leaves in order to assess the involvement of NPT on the tolerance mechanisms of P. australis specimens against Cu and Cd toxicity. Information on this matter regarding salt marsh plants is still scarce. In fact, till now, only two studies using specimens adapted to salty environments were published: one regarding the presence of thiolic proteins in Halimione portucaloides tissues (Válega et al., 2009) and other regarding PC in Spartina maritima tissues (Padinha et al., 2000). Present experiments were carried out in vitro, rhizosediment soaked with the respective elutriate solution (prepared with estuarine water) being used as contaminated medium (after being spiked with Cu or Cd). This medium mimics the natural environment, minimising osmotic effects and other stress factors subsequent to the removal of the plants from their natural habitat. Copper and cadmium are commonly found in sediments of several Portuguese estuaries, sometimes at levels above the effect range – low (ERL) (Almeida et al., 2011, Long et al., 1995). In addition, in a previous field study (unpublished data), involving different salt marsh plants (including P. australis) from two Portuguese estuaries, significant correlations between the content of thiolic compounds and the levels of several metals found in plant roots were only found for Cu and Cd. For this reason, these two metals were chosen to pursue further research.

2. Material and methods 2.1. Sample collection Specimens of P. australis, rhizosediment and estuarine water were collected in Lima River estuary (41.6855 N; 8.8209 W), in spring of 2012, being transported to the laboratory within 1–2 h. Green plants without a senescent appearance and with similar size and age were selected. Rhizosediments (sediment in contact with plant roots) were retrieved by means of a plastic shovel, placed in polyethylene plastic bags and immediately stored in a portable refrigerator. Estuarine water was collected in 1.5 L plastic bottles (rinsed with water at the site). In the laboratory, plant roots were rinsed with deionised water to remove all sediment adhering to roots. A small portion of the plants was frozen at
20 1C for determining basal thiolic compounds, chlorophyll and carotenoids concentrations. Another small portion of the plants and of the rhizosediments was dried until

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constant weight for determining the basal metal concentrations. Remaining plants and rhizosediments were used in the experiments described below. 2.2. Experimental set Two independent sets of experiments were carried out in April. In experiment (1) P. australis (4 specimens per replicate) was exposed to Cu contamination and in experiment (2) identical number of specimens were exposed to Cd. P. australis specimens were placed in glass vessels (250 mL, wrapped in aluminium foil) containing rhizosediment soaked in elutriate enriched with a modified Hoagland nutrient solution and different concentrations of Cu or Cd (see below). Rhizosediment elutriate was obtained according to Environmental Protection Agency protocols (USEPA, 1991) and as described in Almeida et al. (2008). In order to guarantee that nutrient starvation would not be a factor contributing for oxidative stress, a modified Hoagland nutrient solution was added to the media in the day 1 of the experiment. Plant specimens were also exposed to media without nutrient solution addition or without metal contamination as “blank” test. In addition, rhizosediments soaked in elutriate enriched or not with metals, but without plants, were kept under the same conditions, for comparison purposes. Seven days experiments were carried out under 18–20 1C and subjected to natural day/night regime with natural sunlight. The used exposure period was selected in order to avoid oxidative stress caused by possible hypoxia. A longer exposure period (15 days) was also tested in a preliminary stage, but was eventually abandoned, since the obtained results (not shown) indicated that the stress induced by a longer exposure to the medium prevailed over the stress induced by metal contamination (even with regular agitation). All experiments were carried out in triplicate. For Cu treatment, the medium was contaminated with 10 mg/L and 100 mg/L Cu, in order to obtain Cu contaminations higher than the respective ERL (34 mg/g DW) or effect range – median (ERM) (270 mg/g DW), respectively (ERM being the sediment quality guideline that indicates the pollutant concentration above which adverse biological effects may frequently occur, and ERL the concentration below which adverse biological effects rarely occur, in marine and estuarine sediments (Long et al. 1995)). For the Cd treatment, elutriate solutions enriched with 1 mg/L and 10 mg/L Cd were mixed with rhizosediments in order to attain Cd contaminations similar to the respective ERM (9 mg/g DW), in the first case, or ten times higher than that parameter (90 mg/g DW). These concentrations may not normally occur in the environment, however, they may cause a marked effect on the plants and measurable results would be more easily attained in short term experiments, providing therefore important information on how plants can respond to metals toxicity. At the end of the experiments, plants were removed from the containers, washed with deionised water and part of the specimens, as well as rhizosediments, were dried at room temperature, until constant weight. Elutriate solutions were transferred to plastic tubes (15 mL), centrifuged and acidified. The contents of Cd and Cu were determined in initial estuarine water, elutriate solutions (at the end of the experiments), rhizosediments and in P. australis plant tissues. Part of fresh roots and leaves of P. australis were frozen and stored at
20 1C for thiolic compounds determination and for chlorophyll and carotenoids analysis. 2.3. Material, reagents and solutions To prevent contamination, all sampling and labware material were soaked in 20% (v/v) HNO3 solution for at least 24 h, being afterwards rinsed several times with deionised water. High performance liquid chromatography (HPLC) vials were washed with deionised water in an ultrasonic bath (Transsonic 460/H, Elmas). The sample manipulation was carried out in a clean room with Class ISO 5 filtered air. Concentrated stock solutions of Cys (100 mg/L, purity 499.0%), GSH (250 mg/L, purity 498.0%) and GSSG (750 mg/L, purity 498.0%), as well as, 6 mM dithiothreitol (DTT, purity 499.0%) and 6 mM N-ethylmaleimide (NEM, purity 499.0%) solutions, were daily prepared by dissolving an appropriate amount of each compound in deionised water. A 0.2 M aqueous solution of 2-(cyclohexylamino) ethanesulfonic acid (CHES, purity Z99.0%) was prepared weekly. The derivatization solution (30 mM monobromobimane (mBrB, purity 495.0%)) was prepared in methanol (HPLC grade). Solutions were kept in amber glass at 4 1C in a refrigerator (light protected from photo-degradation). For the measurement of total acidsoluble SH compounds (total thiols), 0.5 M K2HPO4 (pro analysis) and 10 mM 5,50 -dithiobis(2-nitrobenzoic acid) (purity 499.0%) solutions were prepared. The metal standards solutions used in the atomic absorption spectrometry (AAS) determinations were prepared by dilution of AAS standard solutions (BDH, Spectrosol grade) (1000 mg/L) in deionised water and acidified with 3% of concentrated HNO3 (suprapure). 2.4. Thiolic compounds analysis About 200 mg of frozen root or leaf material was reduced to fine powder, separately, with liquid nitrogen and a mortar and pestle. Thiolic compounds were extracted from the plant material with 2 mL of 0.1 M HCl solution, being the

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material homogenised on a vortex and centrifuged for 30 min at 2,500 rotations per minute. The supernatant was then filtered through a 0.45 mm cellulose nitrate membrane.

3. Results

2.4.1. Total acid-soluble SH compounds analysis For determining total thiols, 600 mL of the plant extract was mixed with 1.26 mL of 0.5 M K2HPO4 and 50 mL of 10 mM 5,50 -dithiobis(2-nitrobenzoic acid). The absorbance at 412 nm was read after 5 min (De Vos et al., 1992). Calibration was performed by using GSH standard solutions prepared in a 0.1 M HCl solution (0– 20 mg/L).

Copper and cadmium concentrations found in the below and aboveground tissues of P. australis at the end of the experiments are exhibited in Fig. 1 (Cu and Cd levels found in each plant tissue collected in the estuary (basal levels) are also included). P. australis roots were the main organ accumulating Cu and Cd, presenting significantly higher amounts of both metals than rhizomes, stems and leaves. In addition, the amounts of Cu and Cd found in rhizomes were also significantly higher in comparison to stems and leaves, denoting that both metals were mostly concentrated in the belowground organs of P. australis. Roots and rhizomes exhibited significantly higher amounts of Cu and Cd as the metal concentration increased in the medium. Therefore, metal accumulation in those structures was dependent on metal concentration in the medium. With regard to stems and leaves, Cu levels were statistically similar in both organs and no significant variations were observed after plant exposure to the contaminated media. On the contrary, the levels of Cd increased significantly as the Cd concentration increased in the medium. Nevertheless, [Cd]abovegournds/ [Cd]belowgrounds ratios were far smaller than one unit for both Cd concentrations tested (0.02 and 0.005 for 1 mg/L and 10 mg/L Cd, respectively), these results suggesting that P. australis has a low potential to translocate Cu and Cd. A significant decrease in the concentration of both metals in the elutriate solutions was observed after plant exposure to the media (Fig. 2), this decline being very pronounced in the contaminated elutriates. The elutriate solution from Cu-non-contaminated medium to which nutrient solution was added presented, without plant exposure, significantly higher Cu levels than the equivalent medium without nutrients addition (Fig. 2), probably resulting from the presence of copper sulphate in the nutrient solution. This explains the fact of the amount of Cu accumulated by the plant being higher in the presence of nutrients (Fig. 1). In rhizosediments, no marked differences were observed in media with and without plant (Fig. 2), the levels of Cu and Cd remaining statistically similar throughout the experiment. Nevertheless, in the case of Cd, the levels registered in non-contaminated rhizosediments, after the 7 days treatment, were tendentiously higher than the levels registered for media without plant (although differences were not significant, whether there was or not nutrient addition), suggesting that P. australis excreted some Cd.

2.4.2. Non protein thiols analysis Before proceeding to the determination of Cys, GSH and its oxidation product, GSSG, the pH of the extract was adjusted to 7.0 (by addition of 1 M NaOH solution). Then, two derivatization processes were carried out: a) to determine Cys and GSH, a 120 mL aliquot of the sample extract or of a standard solution was mixed with 180 mL of 0.2 M CHES buffer (pH 9.3) and 30 mL of 6 mM DTT solution. The solution was then incubated 1 h on ice. b) in the case of GSSG, a 200 mL aliquot of the extract or of a standard solution was mixed with 300 mL of CHES solution and 50 mL of 6 mM NEM. The reaction was carried out at room temperature for 10 min, being 30 mL of 3 mM DTT then added. The treated extracts were then incubated for 1 h on ice. At the end of the incubation period, 10 mL of 30 mM mBrB was added to the aliquots (for both determinations (a and b)). The derivatization reaction was stopped after 15 min by adding 250 mL of a 5% (v/v) solution of acetic acid (reaction must occur in the dark, therefore amber vials were used during the procedure). The analysis of the thiolic compounds was performed using a C8 Luna column (250 mm  4.60 mm, Phenomenex) in a Beckman Coulter System Gold HPLC with a 126 solvent module, provided with a fluorescence detector (JASCO, FP1520) and a 508 auto-sampler. The mobile phases were (A): 2% (v/v) methanol/98% (v/v) water/ 0.25% (v/v) acetic acid (pH 4.3) and (B): 90% (v/v) methanol/10% (v/v) water/0.25% (v/v) acetic acid (pH 3.9). The column operated at room temperature at a flow rate of 0.80 mL/min and each run took 30 min. The following gradient was used: 7% of eluent B (93% of eluent A), keeping isocratic conditions during 3 min, followed by 27 min of linear gradient to 100% of eluent B and, later, 3 min of a linear gradient to 3% of eluent B (97% eluent A). The derivatization product was detected by fluorescence (λexcitation ¼ 380 nm and λemission ¼ 480 nm). The used method was based on methods proposed for thiol analysis in biological samples (JaroszWilkołazka et al., 2006; Rother et al., 2006). Calibration was performed by means of standards solutions (five) prepared daily in a 0.1 M HCl solution (pH corrected to 7 with a 1 M NaOH solution) from more concentrated standard solutions (100 mg/L of Cys, 250 mg/L GSH and 750 mg/L GSSG). The thiol standard solutions concentration range used was: 0.1– 1.2 mg/L for Cys, 0.25–3 mg/L for GSH and 0.25–4.5 mg/L for GSSG. 2.5. Determination of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids in leaves of P. australis Chlorophyll a (Chl. a), chlorophyll b (Chl. b), total chlorophyll (total Chl.) and carotenoids in P. australis leaves were extracted and quantified according to a modified protocol of Abadía et al. (1984). For that, 12.5 mL of 40 mM calcium carbonate solution prepared in methanol were added to 0.5 g of P. australis leaves. After 48 h, the supernatant was collected and the absorbance signal was measured at 480 nm, 663 nm and 645 nm. 2.6. Metal determination Metals (Cd and Cu) were determined by AAS in rhizosediments, elutriate solutions and different tissues of P. australis (roots, rhizomes, stems and leaves) collected from their natural habitat and involved in the experiments performed. Aliquots of each sample were digested with suprapure concentrated HNO3 and with H2O2 (30% (v/v), only for plant tissues), using an advanced microwave digestion system (Ethos 1, Millestone). The procedure was adapted from and is fully described in (Almeida et al., 2004). 2.7. Statistical analysis For each treatment, mean values and respective standard deviation (n ¼3) were calculated. Data were analysed statistically using analysis of variance (ANOVA) and the Tukey pair wise comparisons were employed to determine the significance of the differences among treatments. Unpaired t-student test (p o 0.05) was performed to test for significant differences among mean concentrations. The statistical package used was GraphPad Prism 6 software and the confidence limit was 95%.

3.1. Metal accumulation in P. australis tissues

3.2. Assessment of toxicity signs The selected Cu and Cd concentrations are likely to cause adverse effects. However, P. australis specimens did not present any external visible deleterious effects after exposition to metal contaminated medium. Likewise, the photosynthetic activity was shown not to have been affected since the contents of Chl. a, Chl. b, Total Chl. and carotenoids were statistically similar in plants exposed and not exposed to Cu or Cd and also to basal levels (Fig. 3). 3.3. Levels of thiolic compounds Levels of Cys, GSH, GSSG and total thiols in roots and leaves of P. australis specimens subjected to experiment (1) and experiment (2) are presented in Table 1. 3.4. Exposure to non-contaminated medium After plant exposure to the non-contaminated medium with or without nutrient addition, it was observed in experiment (1) an increase in Cys and total thiols levels in roots in comparison to

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Fig. 1. Levels (mean and standard deviation, n ¼3) of Cu and Cd found in roots, rhizomes, stems and leaves of P. australis specimens exposed, for 7 days, to rhizosediment and the respective elutriate contaminated or not with Cu and Cd, in the absence or in the presence of nutrients. Basal levels are also shown. Different letters indicate statistical difference (po 0.05) between values (capital letter compares data regarding basal levels and non-contaminated media: small letter compares, for each plant tissue, data regarding media enriched with nutrients with our without metal addition).

basal levels (statistically significance only found in the case of no nutrients addition). The amounts of GSH remained statistically similar, whereas the amount of GSSG decreased significantly. In leaves, levels of Cys and GSH increased significantly regarding basal levels, although there were no significant differences between media with and without nutrient addition. The levels of GSSG and total thiols, on the other hand, remained statistically similar to basal ones. In experiment (2), a different behaviour was observed: P. australis roots and leaves maintained their levels of Cys, GSH, GSSG and total thiols statistically similar between both media (with or without nutrient addition) and in comparison to basal ones (a significant increase in total thiols was only observed for the non-contaminated medium with nutrient addition). In general, medium composition did not cause major stress in P. australis, the nutrients addition contributing in some cases for a reduction of experimental stress.

GSH contents in plants exposed and not exposed to Cu contamination. Conversely, GSSG levels were significantly lower than basal levels which might indicate some kind of stress. In leaves, no specific response to Cu contamination occurred, the amounts of Cys, GSH and GSSG measured in Cu-non-contaminated and Cucontaminated media being statistically similar, which is compatible with the absence of Cu translocation. Total thiols contents were generally significantly higher than the sum of all NPT measured in roots and leaves. Comparing to data registered for the nutrient-rich and non-contaminated medium, Cu contamination induced a decrease in total thiols levels: in roots, total thiols reached amounts statistically similar to the ∑NPT, and in leaves, for the medium containing the highest Cu concentration, there was a significant decrease. This suggests that Cu contamination affected majorly the production of other thiolic compounds.

3.4.1. Cu contamination Copper contamination triggered a significant increase in Cys levels in P. australis roots (Table 1). Nevertheless, P. australis response was only driven by the presence of the metal in detriment to Cu concentration in the medium and to the content of Cu retained in roots (statistically similar Cys levels were found for both concentrations tested). No significant variation was found in

3.4.2. Cd contamination P. australis roots and leaves exposed for 7 days to Cdcontaminated media (both concentrations tested) presented amounts of Cys and GSH statistically similar to those found in the absence of contamination. Nevertheless, Cd contamination induced a significant decrease in GSSG levels in both tissues: in roots, GSSG contents were below the limit of detection, whereas in

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Fig. 2. Levels (mean and standard deviation, n¼3) of Cu and Cd found, after 7 days, in elutriate solutions and rhizosediments of the experimental media enriched or not with Cu or Cd and/or nutrients and with or without plant exposure. Different letters indicate statistical difference (p o0.05) between values (capital letters compare data from before and after P. australis exposure; small letters compare data between the different tested variables).

leaves those levels were tendentiously lower than the equivalent non-contaminated medium (particularly for the most contaminated medium). Significantly higher contents of total thiols than those from the ∑NPT were also found in most cases. The content of total thiols in roots was generally similar between Cd-contaminated and noncontaminated media. On the contrary, in leaves, exposure to Cd led to a significant decrease on total thiols levels comparing with the nutrient-rich and non-contaminated medium. Nevertheless, this decrease was not proportional to the external Cd concentration, as statistically similar amounts of total thiols were measured in leaves exposed to both Cd concentrations tested (although there was a tendency for a lower amount of total thiols when the plant was exposed to the highest Cd concentration). Therefore, Cd contamination seems to influence significantly only the levels of GSSG in plant roots.

4. Discussion This work shows that P. australis is indeed a Cu and Cd accumulator, as indicated in other studies (Duman et al., 2007; Fediuc and Erdei, 2002). Both metals were mostly concentrated in

the belowground organs (roots and rhizomes), metal accumulation being dependent on metal medium concentration. P. australis specimens showed none or low potential for translocating Cu and Cd (to stems and leaves, respectively), functioning essentially as a phytostabiliser. Similar conclusions were reported by Iannelli et al. (2002) and Ait Ali et al. (2002) for P. australis specimens exposed to a hydroponic medium. Metal retention in the belowground parts and the prevention of metal translocation are known defence mechanisms of salt marsh plants against metal toxicity (Caçador et al., 2000; Reboreda and Caçador, 2007a). These mechanisms may protect the photosynthetic apparatus, considerably important for plant's vitality, from metal toxicity as suggested by Bragato et al. (2009). Exposure to metals has been shown to induce oxidative stress in plant cells due to interaction with functional groups, the displacement of essential elements and the enhancement in ROS production. Evidence that antioxidant thiolic compounds can be involved in plants' mechanisms to tolerate and cope with metal contaminated media can be found in literature especially regarding soil plants (Seth et al., 2012), being data on salt marsh plants very scarce. So, this study aimed at understanding the involvement of NPT on the tolerance mechanisms of P. australis specimens, adapted to estuarine environments, against metal toxicity. Metals

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Fig. 3. Levels (mean and standard deviation, n¼ 3) of chlorophyll a (Chl. a), chlorophyll b (Chl. b), total chlorophyll (Total Chl.) and carotenoids found in leaves of P. australis specimens exposed to rhizosediment and the respective elutriate, during 7 days, with or without addition of Cd or Cu and with or without addition of nutrients. Basal levels are also shown.

Table 1 Levels (mean and standard deviation, n¼3) of cysteine (Cys), reduced glutathione (GSH), oxidised glutathione (GSSG) and total acid-soluble SH compounds (Total Thiols), expressed in mg/g rootFW, found in roots and leaves of P. australis specimens exposed, during 7 days, to rhizosediment and the respective elutriate contaminated with Cd and Cu, with or without nutrient addition. Basal levels are also shown. ∑NPT is the sum of the three non protein thiols measured. Roots

Leaves

Cys

GSH a

57 1

∑NPT

GSSG a

9 72

a

16 73

Total Thiols A

26 75

a,B

Basal levels Cu experiment

2.17 0.6

0 mg/L Cu No nutrient 0 mg/L Cu nutrient 10 mg/L Cu nutrient 100 mg/L Cu nutrient

107 2b 4.17 0.4c,α 67 1β,γ 87 0.3γ

87 3a 4.4 7 0.4a,α 47 1α 47 1α

3 71b o1.8* 4 71 o1.8*

21 76A 8 72A 12 73A 11 72A

39 73b,B 32 78a,α,B 24710α,β,A 14 76β,A

1.7 7 0.3a 1.4 7 0.7a,α 1.717 0.06α 1.3 7 0.3α

67 2a 37 1a,α 47 1α 47 1α

7 73a 6 74a o1.8* o1.8*

12 77A 11 75A 5 71A 5 71A

26 78a,A 21 75a,α,A 39 710α,B 21 710α,B

Cys 2.17 0.2

GSH a

537 9

∑NPT

GSSG a

2007 50

a

255 7 59

Total thiols A

480 718a,B

57 1a,b 67 2b,α 57 2α 67 1α

1027 4b,c 867 9c,α 827 7α 827 22α

2727 41a 1747 71a,α 1527 15α 1827 70α

380 7 28A 2677 77A 2437 11A 2707 89A

530 726a,B 587 798a,α,B 496 7133α,A 429 76β,B

37 1a 37 1a,α 3.9 7 0.4α 47 1α

547 5a 577 9a,α 467 5α 427 5α

201 7 20a 1527 48a,α 1447 46α 1197 23α

258 7 24A 2127 53A 1947 51A 1617 24A

413 742a,b,B 719 724b,α,B 563 753β,γ,B 456 7104γ,B

Cd experiment 0 mg/L Cd No nutrient 0 mg/L Cd nutrient 1 mg/L Cd nutrient 10 mg/L Cd nutrient

Different letters indicate statistical difference (p o0.05) between values (small letters (a,b,c) compare data regarding basal levels and non-contaminated media; symbols (α,β, γ) compare data regarding media enriched with nutrients with our without metal addition; capital letters compare data regarding ∑NPT and Total Thiols). n

LOD

concentrations used were likely to cause adverse effects. However, results indicated that there was indeed a disturbance in the content of NPT of P. australis cells but no external visible deleterious effects, such as leaf chlorosis and wilting of tops (Yadav, 2010), were observed in the tested specimens and the photosynthetic activity of the plant was also not affected.

Copper and Cd, in particular, are known to cause oxidative stress in plants (Aygun et al., 2011; Razinger et al., 2008; Thounaojam et al., 2012). Cadmium, a non-essential metal for plant metabolism, can easily enter root cells probably by means of the same transmembrane carrier of essential micronutrient metal ions with similar chemical properties, namely, Zn (Benavides et al., 2005) and has a

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high affinity for sulfhydryl groups causing the denaturation of proteins and the deactivation of enzymes (Guimarães et al., 2008). Therefore, owing to its high mobility and high toxicity even at small concentrations, Cd contamination poses a considerable threat to plants and other living organisms (Gill and Tuteja, 2011; Påhlsson, 1989). On the contrary, Cu participates in several plant physiological processes (Yruela, 2005), being important for plant growth, seed production, disease resistance, etc. Nevertheless, in excess, Cu can induce several physiological, biochemical and cytological changes in plant (Påhlsson, 1989). Being a redox metal, Cu is likely to be involved in Haber–Weiss and/or Fenton reactions in which hydroxyl radicals (HO) are formed (Arora et al., 2002). This free radical is the most reactive ROS causing severe damage to cells due to lipid peroxidation and modification in DNA, proteins and many small molecules (Arora et al., 2002). In this study, P. australis specimens adapted to estuarine environments presented a different behaviour after exposure to Cu and Cd. Cu contamination triggered an increase in Cys levels in P. australis roots not affecting the contents of GSH and GSSG. In leaves, no specific response to Cu contamination was observed which is compatible with the absence of Cu translocation. On the contrary, previous studies using different plant species and different experimental conditions reported that Cu affected GSH and/or GSSG contents in plant cells (Aygun et al., 2011; De Vos et al., 1992; Thounaojam et al., 2012). With regard to Cd, no significant variation was observed in the contents of Cys and GSH either in roots or in leaves. Nevertheless, Cd contamination triggered a significant decrease in GSSG levels in roots and a tendentiously decrease in leaves particularly in the most contaminated medium. This is in accordance with fact that Cd was, contrary to Cu, translocated to some extent to the leaves. In addition, in shortterm experiments carried out in microcosms, also with P. australis stands, a similar behaviour pattern for GSSG was observed (submitted article). Therefore, the initial contact of plant roots with Cd contamination affects mainly GSSG levels, a compound generally associated to stressful conditions (namely oxidative stress). GSH is converted to GSSG by reacting with oxidising compounds produced inside the cell as consequence of oxidative stress (Szalai et al., 2009). In studies involving plant species adapted in nature to freshwater systems or soil, Cd was indeed shown to increase GSSG levels in cells of those plants. This increase has been related to the Cd concentration in the medium (Ben Ammar et al., 2008; Fediuc and Erdei, 2002; Iannelli et al., 2002). The medium selected for the experimental sets did not seem to cause major stress in P. australis roots, denoting that the changes in NPT levels were triggered by metal exposure. Similar experimental conditions were used in non-contaminated and contaminated media so the results attained in each variable were safe to compare. Some differences in plant response to the noncontaminated media were observed between experiment (1) and (2) fact explained by natural variability of plant response. Specimens with similar size and aspect were chosen but, it is impossible to guarantee that plants were all of the same age or in the same physiological state (experiments performed two weeks apart, this fact may explain the differences as reported by Katerova et al. (2009). The contents of total thiols in P. australis roots and leaves indicated that other thiolic compounds besides the NPT under evaluation incorporate plant cell cytoplasm. For instance, metallothioneins, DL-homocysteine, N-acetyl-L-cysteine, γ-glutamylcysteine, homoglutathione and PC have also been found in soil plant tissues (Alvarez-Legorreta et al., 2008; Cobbett and Goldsbrough, 2002; Diopan et al., 2010; Klapheck, 1988). P. australis leaves presented in general significantly higher amounts of NPT and total thiols than in roots. The ROS production is an usual phenomenon occurring in cells under normal or stressful physiological

conditions, being produced in the course of metabolic reactions taking place in different cellular compartments as chloroplasts, mitochondrias and peroxisomes (Arora et al., 2002). Leaf cells contain all three organelles so it is expected that leaves require larger pools of NPT than roots to scavenger ROS and maintain cell's equilibrium. In this work, both metals appeared to influence the production of other thiolic compounds in P. australis cells. Exposure to Cu contamination affected the content of total thiols in P. australis roots and leaves, reaching roots levels statistically similar amounts to those of ∑NPT measured. Cd mainly affected the content of total thiols in leaves, there being a significant decrease after plant exposure to the highest Cd concentration. Cu and Cd contamination seemed to inhibit the production of other thiolic compounds besides Cys and GSH, indicating that P. australis may have developed alternative defence mechanisms against Cu and Cd toxicity. Apart from NPT, several enzymes and other thiolic compounds have also a major role in the defence system of plants against oxidative stress (Bartosz, 1997). Phytochelatins, for instance, are well-known metal chelators involved in the complexation, transport and storage of metals in cell compartments aiming at diminishing metal toxicity (Cobbett and Goldsbrough, 2002). Glutathione has been reported to be involved in PC synthesis (De Vos et al., 1992) and Cd is considered an important inducer of PC in plant cells (Inouhe, 2005). Herein, no increment on GSH levels was registered as the amount of GSSG decreased. Considering the decline of total thiols concentrations, other thiolic compounds might have been used for metal sequestration. The low concentrations of GSSG may indicate that this compound could have been converted into GSH to be used in the production of those thiolic compounds to mitigate Cd and Cu toxicity. In roots and leaves of soil plants, the depletion of GSH was in fact associated to the increment on PC concentration after plant exposure to Cd (Ben Ammar et al., 2008). In contrast, Ederli et al. (2004) inferred that the biosynthesis of GSH was stimulated in consequence of PC production in P. australis roots. However, the experimental conditions (experiment in hydroponics and P. australis specimens adapted to freshwater systems) were different from those of the present work, differences that condition significantly the plant response to metal contamination. Herein, P. australis specimens were shown to tolerate the concentrations of Cu and Cd tested in the conditions used. To note that, despite results suggesting the induction of oxidative stress, no visible deleterious effects were registered. Salt marsh plants, like P. australis, which are well adapted to harsh environmental conditions, can be resistant to both metals toxicity, fact that corroborates the opinion of Manousaki and Kalogerakis (2010). Indeed, P. australis can easily adapt to very different environments (Ederli et al., 2004) and must possess efficient internal mechanisms to cope with the biogeochemical differences of the media. Data from this work indicate that thiolic compounds may play a role in such tolerance mechanisms. Nevertheless, other alternative defence mechanisms such as the increase of antioxidant enzymes activity (Fediuc and Erdei, 2002; Iannelli et al., 2002), production of other antioxidant compounds, like ascorbic acid and amino acids, (Lefèvre et al., 2009; Manousaki and Kalogerakis, 2010; Sharma and Dietz, 2006; Smirnoff, 1996) or exudation of ALMWOAs (Ryan et al., 2001; Tong et al., 2010), etc, may have also been triggered in the process and in unison configure the remarkable resistance of salt marsh plants. Estuarine ecosystems are very sensitive areas, their management and phytoremediation strategies facing severe challenges. For this reason, pursuing research on the dynamics behind salt marsh plants' success to cope with the harsh conditions of marsh areas and to remove pollutants from sediments is utterly important and will contribute for the planning of more efficient

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remediation projects of impacted marsh areas. This work has in fact given new insights regarding one of the tolerance mechanisms triggered in P. australis in response to metal contamination.

5. Conclusions P. australis exposure to Cu and Cd contamination did not cause any visual adverse effect in the specimens tested, indicating plant resistance to the metals concentrations used. Thiolic compounds seemed to participate in P. australis tolerance mechanisms against Cu and Cd toxicity. Cu contamination affected the content of Cys in the plant tissues whereas Cd contamination influenced the GSSG levels. Both metals had an effect on the content of other thiolic compounds, denoting that other thiolic compounds, apart from the Cys and GSH, were involved in the detoxification of Cu and Cd. Different responses were observed for Cu and Cd, suggesting that plant's response was dependent upon the nature of the metal. Taking into consideration the ability of the salt marsh plant to accumulate Cu and Cd in belowground tissues and the resistance of the plant to metal toxicity, P. australis is a suitable choice for phytostabilisation purposes so understanding metal tolerance mechanisms is imperative to optimise conditions for applying P. australis in phytoremediation.

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