Platinum uptake, distribution and toxicity in Arabidopsis thaliana L. plants

Ecotoxicology and Environmental Safety 147 (2018) 982–989

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Platinum uptake, distribution and toxicity in Arabidopsis thaliana L. plants a,⁎

a

a

MARK

b

Helena Gawrońska , Arkadiusz Przybysz , Elżbieta Szalacha , Katarzyna Pawlak , Katarzyna Bramab, Agata Miszczakb, Marta Stankiewicz-Kosyla, Stanisław W. Gawrońskia a Laboratory of Basic Research in Horticulture; Faculty of Horticulture, Biotechnology and Landscape Architecture; Warsaw University of Life Sciences-SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland b Chair of Analytical Chemistry; Faculty of Chemistry; Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland

A R T I C L E I N F O

A B S T R A C T

Keywords: Metal uptake and distribution Phytochelatins Photosynthetic apparatus efficiency Platinum Water status

Platinum (Pt) occurs at very low levels in parent rock and soils in unpolluted areas, however concentrations of this element in urban areas is steadily increasing. At the levels recorded in urban environments, Pt is not yet phytotoxic, but it already poses a threat to human health, particularly when present in airborne particulate matter. In this study an attempt was made to evaluate Pt(II) uptake, distribution and toxicity in Arabidopsis thaliana L. plants. Arabidopsis thaliana plants were hydroponically grown with increasing Pt(II) concentrations in the range of 0.025–100 µM. Pt(II) was taken up by the roots and translocated to the rosette. At lower Pt(II) concentrations (≤ 2.5 μM) hormesis was recorded, plant growth was stimulated, the efficiency of the photosynthetic apparatus improved and biomass accumulation increased. Higher Pt(II) concentrations were phytotoxic, causing growth inhibition, impairment of the photosynthetic apparatus, membrane injuries and a reduction in biomass accumulation. Exposure of A. thaliana to Pt(II) also resulted in an increased content of phytochelatins throughout the plant and glutathione in the rosette. Uptake and translocation of Pt(II) to harvestable organs of A. thaliana suggests that species of higher biomass accumulation from the Brassicaceae family can probably be used for the phytoextraction of Pt-polluted sites.

1. Introduction Platinum (Pt) is a rare noble element that occurs at concentrations of 0.14 µg kg−1 in unpolluted soils, 1.12 µg kg−1 in agricultural soils and 20.9 µg kg−1 in areas adjacent to roads (Zereini et al., 1997). Its chemical characteristics make it useful as a catalyst in a variety of chemical processes (Zereini et al., 2012), with the amount used in car catalysts, oncology, jewellery, the electrical and glass industries, polymer processing and pesticide production totalling around 200 t year−1 (Sobrova et al., 2012; Zereini and Wiseman, 2010). Today, the main source of Pt emission is catalytic converters in vehicles (50.4% of total emissions in Europe) (Pawlak et al., 2014). Each one can emit 38–146 ng Pt km−1 due to rapid changes in oxidation reduction conditions, high temperatures and mechanical abrasion (Limbeck et al., 2007), resulting in this element's diffuse contamination in environmental concentrations up to hundreds of µg kg−1 (Reith et al., 2014; Sobrova et al., 2012). Elevated levels of Pt are recorded in soils, airborne particulate matter, roadside dust and vegetation, rivers, and coastal and oceanic environments (Ravindra et al., 2004; Wiseman et al., 2016 and references therein).

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Environmental concentrations of Pt are already considered to pose a threat to human health (Ravindra et al., 2004). Occurring in the air and in street dust, Pt acts as a hypoallergenic compound and can be transported through the lungs and in the blood to other human organs (Zaray et al., 2004), while Pt from road dust can be soluble and consequently enters watercourses, sediments, soil and ultimately the food chain (Ravindra et al., 2004). Pt is principally emitted from catalysts in a metallic form or as oxides, however considerable quantities are converted in soil into bioavailable forms, mainly as chloro or organic complexes, and thus can be taken up by plants (Šebek et al., 2011; Pawlak et al., 2014). However, the solubility and mobility of Pt are dependent on the pH and the ability to form neutral or charged inorganic and organic complexes (Šebek et al., 2011). The presence of Pt has been reported in many cultivated and wild plant species, in concentrations ranging from a few µg kg−1 up to hundreds of µg kg−1, depending on the plant species and their environments (Reith et al., 2014). Concentrations of Pt in plants correspond well with levels of the element in urban dust (Orecchio and Amorello, 2010) and soils (Hooda et al., 2007), however a substantial proportion of accumulated Pt, especially in leaves, may come from the air in dust deposition, and

Corresponding author. E-mail address: [email protected] (A. Przybysz).

http://dx.doi.org/10.1016/j.ecoenv.2017.09.065 Received 25 May 2017; Received in revised form 19 September 2017; Accepted 26 September 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.

Ecotoxicology and Environmental Safety 147 (2018) 982–989

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2.2.1. Sample preparation Three samples of about 0.2 g dry matter representative for each harvested plant were mineralised in a Speedwave MWS-3+ microwave digestion system (Berghof) with 3 mL ultrapure HCl (37%) and 1 mL concentrated HNO3 (~65%, T) for 30 min at ~180 °C. After digestion, samples were cooled to ambient temperature and vented carefully. The solutions were evaporated in quartz crucibles until they were almost completely dry, and diluted to a volume of 10 mL with 0.5 M HCl. Samples were then subjected to sequential extraction using the procedure established and described by Połeć-Pawlak et al. (2005). The solutions obtained with different extracting solvents (A: 10 mM Tris-HCl, pH 7.4; B: 2% Driselase in solution A; C: 1% SDS in solution A; D: 10 mM ammonium acetate, pH 4.6) were centrifuged for 20 min at 10,000 rpm (600g) at 0 °C, and the supernatants filtered through 0.45 µm filters. The solutions obtained were diluted to a volume of 10 mL with 0.5 M HCl.

collection and adsorption on the external surface of plants (Hooda et al., 2007; Ravindra et al., 2004; Šebek et al., 2011). Plant species differ in the total amount of Pt taken up and in the proportion of Pt translocated to the aboveground parts, which is usually low (Kowalska et al., 2004; Mikulaskova et al., 2013; Nischkauer et al., 2013). The natural properties of plants can be used to remove Pt from the environment via an environmental biotechnology called phytoextraction. However, before such phytotechnology can be applied in practice, knowledge is required about the biological basis of plant response to Pt at environmental concentrations. To the best of the authors’ knowledge, complex data on the physiological basis of the plant response to Pt have not yet been presented in the literature. Therefore this study aimed to evaluate Pt(II) uptake, distribution and toxicity in model Arabidopsis thaliana L. plants. 2. Materials and methods 2.1. Plant material and growing conditions

2.2.2. Quantification conditions The total amount of Pt(II) was determined by inductively coupled plasma mass spectrometry (ICP-MS) (Model 7500a, Agilent Technologies, Tokyo, Japan) using 10 ng mL−1 indium as an internal standard, according to the modified procedure of Cyprien et al. (2008). Briefly, the calibration graphs to determine Pt(II) by ICP-MS were prepared in the range of 0.001–40 μg L−1. All standard solutions were prepared in 0.3 mol L−1 HCl. The detection limit (DL) was calculated for two Pt(II) isotopes (194 and 195) from the standard deviation of ten measurements of blanks and was found to be 0.001 μg L−1. ICP-MS measurement conditions (nebuliser gas flow 1.13 L min−1, RF power 1200 W and lenses voltage 40 – 120 V) were optimised daily using a standard built-in procedure. The limit of detection was established for samples of plants not exposed to Pt(II) (control group) and was found to be 0.008 and 0.006 μg L−1 for roots and leaves respectively. The recovery of Pt(II) from plant tissue was established for 0.2 g samples of plants from the control group and exposed to 25 µM Pt(II) in growth medium. Samples were spiked with 4 and 40 ng of Pt(II) in the form of [Pt(NH3)4](NO3)2] complex and the obtained recoveries were 90–92 and 102–105% respectively for the control group and 97–100 and 103–110% respectively for plants exposed to 25 µM Pt(II). The relative amount (%) of Pt(II) in each extract was established against the total amount of Pt(II) in the mineralised sample and post-extraction residue, in accordance with previous studies (Połeć-Pawlak et al., 2005). All the measurements with ICP MS were performed with three biological replicates derived from individual plants and each biological replication was the mean of five technical replications (n = 3).

Seeds of Arabidopsis thaliana L. Col 4, #N933 were purchased from the Nottingham Arabidopsis Stock Centre (NASC, UK). To ensure uniform germination, seeds were kept for three days in a refrigerator at 4 °C and sown onto multi-well plates (single well volume – 60 mL) filled with substrate (Universal Kronenerde) and mixed with sand in the proportion 2:1 v/v, with pH of 6.6. Plants were grown in a growth chamber (Simez Control s.r.o., Vsetin, Czech Republic) at a temperature of 20/18 °C (day/night) and relative humidity of 70%. The photosynthetic active radiation (PAR) at plant level was 200 µE m−2 s−1 (MASTER SON-T PIA Green Power, Philips) with a 8/16 h day/night regime. Five-week-old uniform plants at growth stage 1.12 (Boyes et al., 2001) were transferred to plastic containers. During transfer, the roots were gently rinsed with distilled water to remove substrate residues. Plants were then grown in hydroponic culture in 0.3 dm3 of Hoagland's nutrient solution, modified according to Siedlecka and Krupa (2002). The solution was continuously aerated and replaced weekly. During the first week the nutrient solution was used at 0.5 strength, and thereafter the complete composition of macro- and microelements was applied. The pH of the growing medium was 6.2 (Multilevel 1 m, WTW, Germany). On day 14, when the nutrient solution was changed, Pt in oxidation state II was added in the form of [Pt(NH3)4](NO3)2] at concentrations of 0.025, 0.25, 2.5, 5.0, 25, 50 and 100 μM. However, not all the concentrations were applied in each of the five experiments performed. The amount of N provided in Hoagland's solution was reduced by the amount of N added with Pt(II) salt. Control plants were grown in nutrient solution that did not contain Pt(II). During the experimental period the pH level was monitored daily (Multilevel 1 m, WTW, Germany) and an increase recorded from the initial 6.2–7.6 after 24 h and to around 8 at the time the nutrient solution was changed.

2.3. Effect of Pt(II) on selected morphological, physiological and biochemical processes In these experiments A. thaliana plants were treated with Pt(II) at concentrations of 0.025, 0.25, 2.5, 5, 25, 50 and 100 μM, and grown in the presence of Pt(II) in the conditions described in Section 2.1. Measurements were performed in vivo two weeks after application of Pt(II) or during harvest, which took place one week later. In all, three experiments were performed with five biological replications each. Since the results obtained in all showed similar trends, the data presented here are from the experiments with a wider range of Pt(II) concentrations and more parameters measured.

2.2. Pt(II) uptake from the growing medium and its concentration in the roots and rosette To determine the uptake and accumulation of Pt(II) by A. thaliana plants, two experiments were performed with five biological replications each. Since the results obtained in both showed similar trends, the data presented here are from the experiment with a wider range of Pt (II) concentrations. Plants were exposed to Pt(II) at concentrations of 0, 2.5, 5.0 and 25 μM and grown in the conditions described in Section 2.1. Two weeks after the treatment, three uniform plants (biological replications) from each Pt(II) concentration and the control were harvested separately and divided into the rosette and roots. The roots were gently rinsed twice with distilled water, followed by rinsing with redistilled water to remove Pt(II). The biological material was immediately frozen in liquid nitrogen, freeze dried, ground in a mortar and stored at −80 °C until analysis.

2.3.1. Parameters/processes measured during plant growth During plant growth the efficiency of the photosynthetic apparatus and transpiration rate were measured. The efficiency of the photosynthetic apparatus was assessed on fully expanded, undamaged leaves from the middle part of the rosette. Plant gas exchange was measured using the LI-6200 Photosynthesis System (LI-COR, Inc., USA). Measurements of net photosynthesis and stomatal resistance were taken under ambient temperature (20 °C), humidity (70%) and irradiance 983

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(200 µE m−2 s−1), with a CO2 level in a LI-6200-leaf chamber of approximately 400 μmol mol−1. The chlorophyll content was evaluated using chlorophyll meter CCM-200 (Opti-Sciences, USA). Chlorophyll a fluorescence was determined by a continuous excitation fluorimeter (Handy PEA, Hansatech Instruments, UK) in leaves adapted to darkness for 60 min. The saturating light pulse intensity was 1500 µmol m−2 s−1 and its duration was 1 s. The basic fluorescence parameters – minimal fluorescence (Fo), maximum fluorescence (Fm) and variable fluorescence (Fv = Fm-Fo) – were recorded automatically and used to calculate the maximum quantum efficiency of PSII (Fv/Fm) and the potential photochemical efficiency of PSII (Fv/Fo). Furthermore the performance index (PI = Abs/CS*TRo/CS*ETo/CS, where Abs/CS = absorption flux per cross-section, TRo/CS = trapped energy flux per cross-section and ETo/CS = electron transport flux per cross-section) was determined. Simultaneously the transpiration rate was measured with the LI-6200 Photosynthesis System (LI-COR, Inc., USA). All the measurements were performed with five biological replicates, with three (gas exchange and chlorophyll a fluorescence) or two (chlorophyll content) independent measurements per biological replication (n = 5).

followed by 24 h at 70 °C, and the dry matter recorded (VIBRA AJ-CE Series). All the measurements were performed in three biological replications (n = 3). In the cases of relative water content and membrane injuries, each biological replication was the mean of two independent measurements. 2.4. Statistical data analysis All the data were analysed using Statgraphics Plus 4.1. (StatPoint Technologies, Inc., USA). The Shapiro-Wilk test was used to examine the normality of distribution, while Bartlett's test verified the homogeneity of variances. One-way ANOVA was carried out to investigate plant responses to different concentrations of Pt(II) in growing media, followed by post-hoc Fisher's least significant difference (LSD) verifying the differences between treatment groups. Means were considered to be significantly different at P < 0.05. The results presented are means ± SE. In order to investigate the relationship between 20 different parameters/processes studied in this work, the correlation coefficients (r) were calculated using Microsoft Excel Data Analysis Tool Pak for each combination of two variables. The obtained matrix of r was used to estimate the similarity between each pair of random vectors using the equation for the calculation of tangent distance:

2.3.2. Parameters/processes measured at harvest At harvest plants were harvested individually and samples collected from each individual to determine (i) the content of glutathione (GSH) and phytochelatins (PCs), (ii) relative water content (RWC) and (iii) membrane injuries, and data recorded on (iv) the number and area of leaves, (v) the length of the longest roots and (vi) the biomass accumulated by the whole plant and particular organs. The effect of Pt(II) on changes in GSH and PCs levels was monitored by means of reversed-phase liquid chromatography with electrospray ionisation mass spectrometry (RPLC-ESI-MS). The extraction and measurement conditions have been reported by Połeć-Pawlak et al. (2005). In brief, selected ions were monitored by ESI MS (SIM) in positive ion mode. In the case of GSH, the ions at m/z 308 were recorded. The other ions were quasi-molecular ions of PCs and their isomers followed the pattern (G+ n232) corresponding to (Cys-Glu)n linkage, where n = 0–5. G means GSH or its isomer quasi-molecular ion at m/z 308 for PCs, 322 for homo-PCs, 380 for iso-PCs, 338 for hydroxymethyl-PCs, 251 for desGly-PCs, 411 for PCs-Glu and 379 for PC-related peptides with Cterminal glutamine. The identity of the peptides was confirmed by a comparison of retention times (percentage relative standard deviation < 2.0%) corresponding to ions of peptides, with the same m/z recorded for extracts of A. thaliana treated with Cd at concentrations 25 and 50 μM. RWC is an indicator of plant water status and expresses water content as a percentage at a given time as related to the water content at full turgor (gravimetrically, VIBRA AJ-CE Series). It was calculated as follows: RWC (%) = [(FW-DM) / (TW-DM)] × 100, where FW is actual fresh weight of leaf, DM is dry matter of leaf and TW is turgid fresh weight of leaf. Membrane injuries in roots and the rosette were measured using an electrical conductivity meter (Multilevel 1 m, WTW, Germany). Each sample (0.5 g) was cut into 0.5 cm-wide pieces, rinsed with distilled water and placed in beakers filled with 50 cm3 distilled water. After 1 h, the electroconductivity of the solutions was measured and the beakers placed in a water bath (95 °C) for 20 min, then cooled to room temperature and electroconductivity measured again. The membrane injuries were calculated using the following formula: % injury = 1-[1((T1/T2)/1)-C1/C2]*100, where electro conductivity with the symbol T means treated with Pt, C means the control, 1 means before boiling and 2 means after boiling. The number of leaves, total area of the leaves and length of the longest root were determined using the Leaf Area and Root Length Image Analysis System and Skye Leaf software (Skye Instruments Ltd, UK). The fresh weight of the leaves and roots was recorded (VIBRA AJ-CE Series) and the plant material then oven-dried for 2 h at 105 °C,

d=

1 − r2 . r2

The method for hierarchical clustering following the shortest distance was not used to establish the relationship between samples, as is typically carried out, but to establish the relationship between measured parameters. The results of these analyses are presented as an Online Resource. 3. Results 3.1. Pt(II) concentration in plant parts The concentration of Pt(II) in A. thaliana plant parts increased significantly with increasing concentrations of the element in the nutrient solution (Table 1). After uptake, part of the Pt(II) was translocated from the roots to the rosette. The root concentrations of Pt(II) ranged from 100.2 to 425.4 ng g−1 DM, while in the rosette concentrations were always significantly lower and varied between 17.9 and 210.5 ng g−1 DM. The concentration of Pt(II) was therefore 51–86% greater in the roots. However, accounting for the DM of the roots and the rosette, the calculated Pt(II) amount was either similar in both organs or even higher in the rosette, which was especially evident when plants were treated with the highest Pt(II) concentration (76% of Pt(II) yield was accumulated in the rosette) (Table 1). 3.2. Effect of Pt(II) on morphological, physiological and biochemical parameters/processes The effect of Pt(II) on A. thaliana plants depended on the Pt(II) concentration in the growing medium, and ranged from stimulating growth to being toxic. Plants grown in the concentration of 2.5 μM Pt (II) were more vigorous than the control, while in higher Pt(II) concentrations the opposite was found. Leaves of plants exposed to Pt(II) at concentrations of 25 μM and above were light green or nearly yellowish in colour, with clear chlorosis and/or necrosis developing in the older leaves and withering symptoms in the oldest ones. After treatment with Pt(II) at concentrations of 25 μM and above, roots changed colour from creamy white to light brown and became easy to smear. Pt(II) applied at 2.5 μM had a stimulatory effect on total leaf area (23% increase), length of the longest root (3.5% increase), fresh weight 984

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Table 1 Concentration and estimated content of Pt(II) in different organs of A. thaliana plants grown in growing medium with increasing Pt(II) levels. Data are mean ± SE, n = 3 (3 biological replicates, each biological replication is the mean of 5 technical replications). Pt(II) concentration in growing medium µM

Plant part

Pt(II) concentration ng g−1 DM

Control (0)

Rosette Roots Rosette Roots Rosette Roots Rosette Roots

0.31 ± 0.41 A* a** < 0.001 0.40 ± 0.61 a a 17.9 ± 0.41 B a < 0.001 100 ± 2.26 b b 23.6 ± 0.73 B a < 0.001 168 ± 7.54 c b 210 ± 4.40 C a < 0.001 425 ± 5.86 d b rosette < 0.001; roots < 0.001

2.5 5 25 P values for organs:

P values for concentrations

Dry matter g organ−1

Pt(II) content µg organ−1

Pt(II) content ratio roots:rosette

0.75 ± 0.02 0.13 ± 0.01 0.81 ± 0.02 0.13 ± 0.00 0.68 ± 0.03 0.11 ± 0.00 0.60 ± 0.02 0.09 ± 0.00

nc*** nc 0.014 0.013 0.016 0.019 0.127 0.039

nc 0.93 1.19 0.31

Data with the same letter do not differ significantly as determined by one-way ANOVA, Fisher's LSD test, p < 0.05. * Comparisons between different Pt(II) concentrations within the same plant organ (capital letters: rosette; lowercase letters: roots) ** Comparisons of different organs within the same Pt(II) concentration. *** Not calculated, since Pt(II) was not added. Table 2 Leaf area, number of leaves, length of the longest root, fresh weight and dry matter in A. thaliana plants grown in growing medium with increasing Pt(II) levels. Data are mean ± SE, n = 3. Pt(II) concentration in growing medium

Control (0) 2.5 25 50 P value

Measured parameters Leaf area (cm2 plant−1)

223 ± 1.48 274 ± 15.7 217 ± 6.07 219 ± 0.67 0.004

*

a b a a

Number of leaves (plant−1)

143 ± 3.21 a 145 ± 6.43 a 161 ± 17.15 a 193 ± 4.58 b 0.021

Length of the longest root (cm plant−1)

20.1 ± 0.38 20.8 ± 0.28 18.0 ± 0.56 17.8 ± 0.62 0.005

b b a a

Fresh weight (g plant−1)

Dry matter (g plant−1)

Roots

Roots

Rosette

2.21 ± 0.12 3.19 ± 0.72 2.15 ± 0.27 1.90 ± 0.28 0.211

a a a a

15.2 ± 1.84 17.1 ± 2.19 12.9 ± 0.47 8.72 ± 1.00 0.024

b b a a

0.16 ± 0.01 0.23 ± 0.02 0.15 ± 0.01 0.14 ± 0.00 0.003

Rosette DM/roots DM ratio

Rosette a b a a

1.60 ± 0.06 1.74 ± 0.21 1.33 ± 0.12 1.02 ± 0.11 0.025

b b a a

10 7.56 8.87 7.29

Membrane injuries (% of control) Roots

Rosette

0 21.2 24.4 32.5

0 4.74 4.95 8.68

* Values with the same letter in the columns do not differ significantly as determined by one-way ANOVA, Fisher's LSD test, p < 0.05.

(12.5%, 44% and 16.5% increase for rosette, roots and whole plants respectively) and dry matter (9%, 44% and 12% increase for rosette, roots and whole plants respectively), significantly so in case of leaf area and dry matter of roots (Table 2). The plants grown at 5 μM Pt(II) or below also had significant increases in net photosynthesis (by 11–20%, Fig. 1a) and total chlorophyll content (by 4–13%, Table 3) compared to the controls. Higher concentrations of Pt(II) adversely affected nearly all the parameters/processes examined, with the negative impact being most evident in plants treated with 25–100 μM Pt(II). Root length was significantly reduced in plants at 25 and 50 μM Pt(II) by 10.5% and 11.5% respectively (Table 2). The high concentrations of Pt(II) in the growing medium also negatively affected biomass accumulation, which decreased in both the rosette (by 15–43% and 17–36% for fresh weight and dry matter respectively) and the roots (by 3–14% and 6–12.5% for fresh weight and dry matter respectively), albeit significantly only for the rosette. Biomass distribution in Pt-treated plants was also affected, with a decrease in the rosette-to-roots ratio. Of the morphological parameters, only total leaf area was unchanged by Pt(II) exposure. Developed leaves were smaller, but their number grew with increasing Pt(II) concentration in the growing medium, reaching a significant 35% increase after treatment with 50 μM Pt(II) (Table 2). Net photosynthesis in plants grown at 25, 50 and 100 μM Pt(II) was 3%, 11% and 39% lower, respectively, than in the control plants (Fig. 1a). At each Pt(II) concentration except the lowest, stomatal resistance increased, ranging from 120% to 293% of the values recorded for the control plants. However, negative changes recorded in the gas exchange were only significant with the highest Pt(II) concentration (Fig. 1a). The chlorophyll content significantly decreased in plants grown at 50 μM Pt(II) and higher concentrations, reducing to 47% lower than the control at 100 μM Pt(II) (Table 3).

Fig. 1. Net photosynthesis and stomatal resistance (A) and rate of transpiration and relative water content (RWC) (B) in leaves of A. thaliana plants grown in growing medium with increasing Pt(II) levels. Data are mean ± SE, n = 5 (in case of gas exchange, each biological replication is mean of 3 independent measurements)* Values with the same letter do not differ significantly as determined by one-way ANOVA, Fisher's LSD test, p < 0.05.

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Table 3 The fluorescence of chlorophyll a (Fv/Fm, Fv/Fo and Performance Index (PI)) and chlorophyll content in A. thaliana plants grown in growing medium with increasing Pt(II) levels. Data are mean ± SE, n = 5 with 3 (chlorophyll a fluorescence) or 2 (chlorophyll content) independent measurements per each biological replication. Pt(II) concentration in growing medium

Measured parameters Fv/Fm

Control (0) 0.025 0.25 2.5 5 25 50 100 P value

0.83 ± 0.002 0.84 ± 0.003 0.84 ± 0.002 0.84 ± 0.002 0.82 ± 0.002 0.83 ± 0.001 0.82 ± 0.002 0.79 ± 0.004 < 0.001

PI

Fv/Fo bc c c c b bc b a

*

5.05 ± 0.08 5.06 ± 0.09 5.11 ± 0.08 5.09 ± 0.08 5.07 ± 0.03 4.79 ± 0.04 4.42 ± 0.06 3.93 ± 0.16 < 0.001

cd cd d d cd bc b a

27.9 ± 0.69 26.8 ± 1.87 28.6 ± 1.24 30.2 ± 2.12 29.0 ± 0.97 24.0 ± 0.71 19.6 ± 1.05 12.9 ± 0.65 < 0.001

Chlorophyll content (relative value) cd c cd d d c b a

11.5 ± 1.01 10.6 ± 0.64 12.0 ± 0.64 13.0 ± 0.72 12.6 ± 0.53 11.1 ± 0.87 7.62 ± 0.74 6.06 ± 0.54 < 0.001

bc b bc c bc bc a a

* Values with the same letter in the columns do not differ significantly as determined by one-way ANOVA, Fisher's LSD test, p < 0.05.

4. Discussion

The values of the Fv/Fm parameter significantly decreased only at the highest Pt(II) concentration (by 5%), while in the case of Fv/Fo a reduction was already recorded from 25 μM Pt(II) (by 5–22%) (Table 3). The highest value of PI was recorded in plants grown at 2.5 μM Pt(II), where values of this parameter were 8% higher than in the control, while they were significantly the lowest (by 54%) at 100 μM compared to the untreated plants (Table 3). Plants exposed to Pt(II), irrespective of its concentration, lost significantly less water via transpiration, with the rate being 7–63% lower in Pt(II)-treated plants than in the control. The reduction peaked at the highest Pt(II) concentration (Fig. 1b). RWC was almost unaffected by the presence of Pt(II) in the growing medium, although insignificantly lower values of this parameter (by 1–2%) were recorded for plants treated with the four highest Pt(II) concentrations (5–100 μM) (Fig. 1b). Pt(II) uptake by plants resulted in plasma membrane damage (Table 2). At a Pt(II) concentration of 50 μM, membrane injuries in roots and rosette were 32.5% and 9% higher respectively than the control (Table 2). The presence of Pt(II) in growing medium affected GSH and PCs homeostasis in the roots and rosette of A. thaliana plants (Fig. 2). Peak areas for GSH in roots significantly decreased with increasing Pt(II) concentrations, while for PCs contrasting results were recorded. In the rosette, peak areas for both GSH and PCs significantly reached a maximum value at the highest Pt(II) concentration (Fig. 2).

4.1. Pt(II) uptake, translocation to rosette and predictions of its phytoextraction Arabidopsis thaliana plants have the ability to take up and accumulate Pt(II). The Pt(II) concentration was always higher in the roots than in the rosette, confirming the previous findings of Leśniewska et al. (2004), Mikulaskova et al. (2013) and Nischkauer et al. (2013). Nischkauer et al. (2013) showed that the concentration of Pt is in the decreasing order: roots > stem > old leaves > young leaves. However, when the total amount of Pt(II) in particular organs was calculated in the present study, the highest value occurred in the rosette (52% and 76% for 2.5 and 25 µM Pt(II) respectively). Due to its limited size, Arabidopsis is not a species of practical importance, but its ability to translocate metals to harvestable organs, as shown in this study, is one of the critical conditions that must be met by plants used in phytoextraction. Besides being an important model plant, A. thaliana belongs to the Brassicaceae family, which contains many metal-accumulating species, including the best-known metal hyperaccumulators Noccaea caerulescens and Alyssum bertolonii (Mourato et al., 2015). The present study demonstrated the uptake and easy translocation of Pt(II) to the rosette in A. thaliana plants, which suggests that other species of the Brassicaceae and/or other families suited to phytoextraction (e.g.

Fig. 2. Changes in the peak areas of glutathione (GSH) and isoforms and homologues of phytochelatins (PCs) registered by LC-MS for the roots (A) and rosette (B) of A. thaliana plants grown in growing medium with increasing Pt(II) levels. Data are mean ± SE, n = 3 * Values with the same letter do not differ significantly as determined by one-way ANOVA, Fisher's LSD test, p < 0.05.

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at 2.5 µM Pt(II), two genes controlling photosynthesis were up-regulated (At1g29930 – involved in light harvesting and At5g38420 – encoding a member of the Rubisco small subunit) (data not published). So far, the hormetic effects of metals have been shown for Cd, Cr, Al and Pb and non-metallic ions such as As and Se (Poschenrieder et al., 2013), but not yet for Pt. Therefore these results are the first to show the hormetic effect of Pt(II). The lack of Pt(II) toxicity in plants treated with 2.5 µM Pt(II) was also partly proved by the low content of PCs. PCs together with metallothioneins (MTs) are metal-binding ligands that play a crucial role in heavy metal detoxification (Cobbett and Goldsbrough, 2002). Their low level may indicate that, in the conditions described, Pt(II) was more of a stimulating and non-toxic factor or other mechanisms were able to mitigate the stress caused by Pt(II) efficiently. However a high content of GSH was recorded in the roots of these plants. GSH is the substrate for PCs biosynthesis (Cobbett and Goldsbrough, 2002), but is also an antioxidative compound and element in signal transduction (Jozefczak et al., 2012). It seems that at 2.5 µM Pt(II) GSH was used for processes other than metal detoxification, e.g. to reduce ascorbate or maintain protein integrity (Noctor et al., 2011).

Salicaceae), characterised by more rapid growth and higher biomass production, could be used for the efficient phytoextraction of Pt from polluted urban soils. According to Reith et al. (2014), transfer coefficients of Pt from soils into plants are in the same range as other moderately mobile elements, and dicotyledons have a greater ability to accumulate Pt than monocotyledons. High biomass producers from Brassicaceae, e.g. Brassica juncea, have already been used successfully in the phytomining of gold, which is also a noble metal (Anderson et al., 1998). Bearing in mind the growing concentrations of Pt in the environment (Reith et al., 2014), phytoextraction could be considered as a phytotechnology option for remediating sites with elevated levels of this metal. The amount of Pt(II) taken up by plants is affected by many factors, including its concentration in the growing medium (Hooda et al., 2007; Mikulaskova et al., 2013) and pH, which is greater in higher metal concentrations and usually under more acidic conditions (Dahlheimer et al., 2007). In this study, uptake of Pt(II) by A. thaliana plants rose with the increase in metal concentration in the nutrient solution, but was less pH dependent. During growth, plants took up NO3- from the growing medium, which resulted in an increase in pH from the initial 6.2–7.6 after 24 h and to around 8 at the weekly change of growing medium. Increased pH could reduce Pt(II) bioavailability, therefore it can be assumed that if pH were kept close to the outset value, the recorded uptake of Pt(II) could be higher. However, this may depend on Pt(II) speciation and in some cases might be the same. In the urban environment, however, the highest concentrations of Pt(II) are found alongside busy roads where the soil pH is even higher (8–10) due to road de-icing. It should be noted that the present study was conducted in a hydroponic system and therefore cannot be directly transferred to soil conditions, but the results obtained here indicate that well-selected plants species will most probably have the ability to take up bioavailable Pt from soil solution, even in neutral or slightly alkaline solutions. Nevertheless, this is obviously an area that requires further investigation. In the near future it may be important to select species that are able to accumulate Pt present not only in the soil, but in the air as well, as there is increasing evidence that this metal is deposited on the breathable (PM10) and respirable (PM2.5) fractions of air particulates (Diong et al., 2016). Šebek et al. (2011) have shown a correlation between the number of vehicles equipped with catalysts and increased levels of Pt in plant material samples. Presumably, a considerable proportion of Pt in selected samples was recorded due to the ability of plants to take up metals from the soil and water through their root systems, but it could also be linked to Pt deposition on external plant surfaces (Hooda et al., 2007; Ravindra et al., 2004; Šebek et al., 2011).

4.3. Toxic effect of high concentrations of Pt(II) At concentrations above 2.5 μM, Pt(II) had a negative effect on A. thaliana plants. The rosette was less vigorous and had smaller leaves, but there were more of them. The development of additional leaves was probably a response to lethal chlorosis, with necrosis appearing in older leaves that were no longer functional. The root system was smaller, shorter and became brownish in colour. Similar changes in the morphology and development of plants have been reported in duckweed treated with Pt(IV) (Bednarova et al., 2014), but they are also typical for other metals (Poschenrieder and Barceló, 1999). The disturbances in the growth and development of Pt(II)-treated plants led to a decreased biomass accumulation and affected its distribution, which is in agreement with the findings of Gagnon et al. (2006) and Mikulaskova et al. (2013) in peat moss, pea and maize. Biomass accumulation in Pt(II)-treated plants was most probably reduced due to the impairment of the photosynthetic apparatus, as manifested by (i) the lower chlorophyll content, (ii) reduced net photosynthesis and (iii) decreased values of the examined parameters of chlorophyll a fluorescence. The chlorophyll content decreased with increasing Pt(II) concentrations, as has previously been recorded for many species treated with other metals (Huang and Wang, 2010; Li et al., 2010; Pandey and Sharma, 2002). Although the concentration of Pt(II) in mesophyll cells/ chloroplasts was not measured in this study, it can be assumed that Pt (II), when present in the rosette, will reach these organelles, as has been previously noted for Cd, Cu, Co, Hg, Ni, Pb and Zn (Prasad and Strzałka, 1999). If so, it is possible that Pt(II) also has an inhibitory effect on the biosynthesis of photosynthetic pigments, as reported for cabbage exposed to Co, Ni and Cd (Pandey and Sharma, 2002). The above may partly explain the reduction in the chlorophyll content reported in this study. A common symptom of metal toxicity is a decreased intensity of photosynthesis (Nagajyoti et al., 2010). In the present study, the lower net photosynthesis corresponded well with increased stomatal resistance. The analysis of tangent distance between vectors of the studied parameters showed that stomatal resistance was strongly related to the Pt(II) amount in roots (d 0.5, Online Resource) and the rosette (d 0.4, Online Resource), which may indicate that Pt(II) applied in high concentrations affects the homeostasis of K+, Ca2+, Cl- and abscisic acid (ABA) in stomata cells, leading to the stomata closing. A reduction in stomatal conductance during heavy metal stress has been reported in many species treated with several metals (Nagajyoti et al., 2010). Increased stomatal resistance usually results in a decrease in transpiration rate, as noted in this study and in many other species, even including a

4.2. Stimulatory effect of low concentrations of Pt(II) Treatment with Pt(II) at concentrations lower than or equal to 2.5 μM had a positive effect on nearly all the measured parameters and processes in A. thaliana plants. This can be explained by the fact that Pt, as a noble metal and recognised catalyst (Zereini et al., 2012), might play a catalytic role in plants when it is present in cells at low concentrations. Moreover, the very likely induction of ROS production by mild Pt(II) stress may lead to the activation of antioxidant defence, stress-signalling hormones or adaptive growth responses, which are the most probable pathways for hormetic responses (Poschenrieder et al., 2013). Hormesis is quite common in nature and plays an important role in plant fitness and the ability of plants to survive in adverse growing conditions that are not yet generating stress (Poschenrieder et al., 2013). It is believed that in response to low concentrations of a presumably dangerous substance, alternative metabolic pathways are activated, leading to higher biomass accumulation and greater intensity of photosynthesis. In preliminary studies with Pt(II) using the cDNA-AFLP technique (amplified fragment length polymorphism), it was found that 987

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concentrations. Stimulation of most of the measured parameters/processes at 2.5 µM Pt(II) could be attributed to the phenomenon of hormesis. The negative impact of Pt(II) on plants involved disturbances to plant morphology, biomass accumulation, the functioning of photosynthetic apparatus and membrane integrity. In A. thaliana plants treated with Pt(II), the concentration of PCs was higher than in the control. In view of steadily increasing concentrations of Pt in the urban environment, the high proportion of total Pt(II) found in the rosette and the relatively high tolerance of plants to Pt(II) toxicity mean that phytoextraction using Brassicaceae plants, with the potential of biomass accumulation, may be a promising future biotechnology to reduce the labile Pt pool in the soil.

heavy metal-tolerant variety of Indian mustard treated with Cd (HaagKerwer et al., 1999). Here the statistical relationship between the Pt(II) amount in roots and rosettes and transpiration was surprisingly low (d 1.5, Online Resource). It can be assumed that the improved antioxidant defence indicated by changes in non-oxidised GSH most probably protected the gas exchange from being significantly reduced in concentrations up to 25 µM Pt(II). Increased stomatal resistance and reduced transpiration caused by Pt(II) resulted in limited changes in RWC. The parameter Fv/Fm, which provides information on the maximum efficiency of PSII, decreased only slightly after treatment with Pt (II). This may indicate that Pt(II) reduced the efficiency of the carbon assimilation reaction, but without any significant effects on primary photochemical reactions and photophosphorylation potential. Some earlier reports have also demonstrated that the impact of metals on the inhibition of PSII may be small (Burzyński and Kłobus, 2004). Of the measured parameters of chlorophyll a fluorescence, PI (performance index) decreased most and reached its lowest value with the highest Pt (II) concentration, proving the previously discussed negative impact of Pt(II) on the vitality of A. thaliana plants. If accumulated in excessive amounts, metals lead to membrane damage (Morsy et al., 2012). This was also found in the present study, particularly in roots. It is worth mentioning that membrane injuries occurred even at the lowest Pt(II) concentration used, i.e. where hormesis was noted. This may suggest the induction of oxidative stress, peroxidation of membranes and some changes in the root ionome and ultrastructure (Sobrova et al., 2012). The statistically described similarity of the membrane injury to Pt(II) concentration in the growing medium was high (d < 0.2, Online Resource), indicating that membrane dysfunction can be recognised as one of the primary symptoms of Pt(II) stress and a possible reason for the greater allocation of biomass in the rosette rather than in dysfunctional roots. Pt(II) excess forced the induction of the plant's defence mechanisms. As previously mentioned, one of the most effective ways to detoxify metals is to produce PCs and MTs (Cobbett and Goldsbrough, 2002). Here the content of PCs was significantly higher in plants at 5 and 25 µM Pt(II) than in those exposed to 2.5 µM Pt(II). A high content of PCs was recorded in both the roots and the rosette, which is associated with efficient Pt(II) translocation to the rosette (the similarity between the amount of PCs and Pt(II) translocation to the rosette was about 1, Online Resource). Mikulaskova et al. (2013) also noted that PCs concentration increases markedly with an increasing concentration of Pt (IV) in maize and pea plants. The effectiveness of PCs in Pt detoxification has been proven by Klueppel et al. (1998), who showed that the majority of Pt(IV) in plants cells is in the bound form with sulfhydryl-group-containing compounds such as PCs. The effect of Pt(II) on GSH was less evident. In roots its content decreased in a concentration-dependent manner, while in the rosette the opposite results were recorded. It can be assumed that in roots through which Pt(II) was taken up, most GSH was transformed to PCs. In the rosette the utilisation of GSH was more diversified, as GSH for instance could be used as an antioxidant compound against hydrogen peroxide in the glutathione-ascorbate cycle, what may result in the stabilisation of gas exchange and membranes (Noctor et al., 2011). Mikulaskova et al. (2013) showed that the content of GSH in plants treated with Pt(IV) is not only organ dependent, but species dependent as well.

Acknowledgements This study was supported by an EEA grant from Norway through the Norwegian Financial Mechanism, PNRF - 193 - AI - 1/07 granted to S.W. Gawroński and A. Sæbø, and Warsaw Plant Health Initiative FP7REGPOT-2011-1-286093. The authors would like to thank the anonymous reviewers and editor for their time and effort in improving the quality of the paper. We are grateful to Monika Małecka-Przybysz for her laboratory support and for her technical editing of the entire manuscript. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2017.09.065. References Anderson, C.W.N., Brooks, R.R., Stewart, R.B., Simcock, R., 1998. Harvesting a crop of gold in plants. Nature 395, 553–554. Bednarova, I., Mikulaskova, H., Havelkova, B., Strakova, L., Beklova, M., Sochor, J., Hynek, D., Adam, V., Kizek, R., 2014. Study of the influence of platinum, palladium and rhodium on duckweed (Lemna minor). Neuroendocrinol. Lett. 35 (Suppl. 2), 35–42. Boyes, D.C., Zayed, A.M., Ascenzi, R., McCaskill, A.J., Hoffman, N.E., Davies, K.R., Görlach, J., 2001. Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13, 1499–1510. Burzyński, M., Kłobus, G., 2004. Changes of photosynthetic parameters in cucumber leaves under Cu, Cd and Pb stress. Photosynthetica 42, 505–510. Cobbett, Ch, Goldsbrough, P., 2002. Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol. 53, 159–182. Cyprien, M., Barbaste, M., Masson, P., 2008. Comparison of open digestion methods and selection of internal standards for the determination of Rh, Pd and Pt in plant samples by ICP-MS. Int. J. Environ. Anal. Chem. 88, 525–537. Dahlheimer, S.R., Neal, C.R., Fein, J.B., 2007. Potential mobilization of platinum-group elements by siderophores in surface environments. Environ. Sci. Technol. 41, 870–875. Diong, H.T., Das, R., Khezri, B., Srivastava, B., Wang, X., Sikdar, P.K., Webster, R.D., 2016. Anthropogenic platinum group element (Pt, Pd, Rh) concentrations in PM10 and PM2.5 from Kolkata, India. SpringerPlus 5, 1242. Gagnon, Z.E., Newkirk, C., Hicks, S., 2006. Impact of platinum group metals on the environment: a toxicological, genotoxic and analytical chemistry study. J. Environ. Sci. Health A Toxicol. Hazard Subst. Environ. Eng. 41 (3), 397–414. Haag-Kerwer, A., Schäfer, H.J., Heiss, S., Walter, C., Rausch, T., 1999. Cadmium exposure in Brassica juncea causes a decline in transpiration rate and leaf expansion without effect on photosynthesis. J. Exp. Bot. 50, 1827–1835. Hooda, P.S., Miller, A., Edwards, A.C., 2007. The distribution of automobile catalysts-cast platinum, palladium and rhodium in soils adjacent to roads and their uptake by grass. Sci. Total Environ. 384, 384–392. Huang, G., Wang, Y., 2010. Physiological and biochemical responses in the leaves of two mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza) exposed to multiple heavy metals. J. Hazard. Mater. 182, 848–854. Jozefczak, M., Remans, T., Vangrosveld, J., Cuypers, A., 2012. Glutathione is a key player in metal-induced oxidative stress defenses. Int. J. Mol. Sci. 13 (3), 3145–3175. Klueppel, D., Jakubowski, N., Messerschmidt, J., Stuewer, D., Klockow, D., 1998. Speciation of platinum metabolites in plants by size-exclusion chromatography and inductively coupled plasma mass spectrometry. J. Anal. At. Spectrom. 13, 255–262. Kowalska, J., Huszal, S., Rawicki, M., Asztemborska, M., Stryjowska, E., Szalacha, E., Golimowski, J., Gawroński, S.W., 2004. Voltammetric determination of platinum in plant material. Electroanalysis 15, 1266–1270. Leśniewska, B.A., Messerschmidt, J., Jakubowski, N., Hulanicki, A., 2004. Bioaccumulation of platinum group elements and characterization of their species in

5. Conclusions This study showed that Pt(II) was taken up by A. thaliana roots and then translocated relatively easily to the shoots. Consequently, the amount of Pt(II) accumulated in the rosette was higher than in the roots. The accumulation of Pt(II) in plant tissues affected vital processes, with stimulatory effects at low concentrations and toxic effects at high 988

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Prasad, M.N.V., Hagemeyer, J. (Eds.), Heavy Metal Stress in Plants. From Molecules to Ecosystems. Springer-Verlag, Berlin, pp. 207–230. Poschenrieder, Ch, Cabot, C., Martos, S., Gallego, B., Barceló, J., 2013. Do toxic ions induce hormesis in plant? Plant Sci. 212, 15–25. Prasad, M.N.V., Strzałka, K., 1999. Impact of heavy metals on photosynthesis. In: Prasad, M.N.V., Hagemeyer, J. (Eds.), Heavy Metal Stress in Plants. From Molecules to Ecosystems. Springer-Verlag, Berlin, pp. 117–138. Reith, F., Campbell, S.G., Ball, A.S., Pring, A., Southam, G., 2014. Platinum in Earth surface environments. Earth-Sci. Rev. 131, 1–21. Ravindra, K., Bencs, L., Van Grieken, R., 2004. Platinum group elements in the environment and their health risk. Sci. Total Environ. 318 (1–3), 1–43. Šebek, O., Mihaljevič, M., Strnad, L., Ettler, V., Ježek, J., Štědrý, R., Drahota, P., Ackerman, L., Adamec, V., 2011. Dissolution kinetics of Pd and Pt from automobile catalysts by naturally occurring complexing agents. J. Hazard Mater. 198, 331–339. Siedlecka, A., Krupa, Z., 2002. Simple method of Arabidopsis thaliana cultivation in liquid nutrients medium. Acta Physiol. Plant. 24, 163–166. Sobrova, P., Zehnalek, J., Adam, V., Beklova, M., Kizek, R., 2012. The effects on soil/ water/plant/animal systems by platinum group elements. Eur. J. Chem. 10 (5), 1369–1382. Wiseman, C.L.S., Pourc, Z.H., Zereini, F., 2016. Platinum group element and cerium concentrations in roadside environments in Toronto, Canada. Chemosphere 145, 61–67. Zaray, G., Ovari, M., Salma, I., Steffan, I., Zeiner, M., Caroli, S., 2004. Determination of platinum in urine and airborne particulate matter from Budapest and Vienna. Microchem. J. 76, 31–34. Zereini, F., Alsenz, H., Wiseman, C.L.S., Püttmann, W., Reimer, E., Schleyer, R., Bieber, E., Wallasch, M., 2012. Platinum group elements (Pt, Pd, Rh) in airborne particulate matter in rural vs. urban areas of Germany: concentrations and spatial patterns of distribution. Sci. Total Environ. 416, 261–268. Zereini, F., Skerstupp, B., Alt, F., Helmers, E., Urban, H., 1997. Geochemical behaviorof platinum-group elements (PGE) in particulate emissions by automobileexhaust catalysts: experimental results and environmental investigations. Sci. Total Environ. 206, 137–146. Zereini, F., Wiseman, C.L.S., 2010. Platinum group elements. In: Hooda, P.S. (Ed.), Trace Elements in Soils. Wiley Publication, Chichester, pp. 567–577.

Lolium multiflorum by size-exclusion chromatography coupled with ICP-MS. Sci. Total Environ. 322, 95–108. Li, Q., Cai, S., Mo, C., Chu, B., Peng, L., Yang, F., 2010. Toxic effects of heavy metals and their accumulation in vegetables grown in a saline soil. Ecotoxicol. Environ. Saf. 73, 84–88. Limbeck, A., Puls, Ch, Handler, M., 2007. Platinum and palladium emissions from on-road vehicles in the Kaisermühlen tunnel (Vienna, Austria). Environ. Sci. Technol. 41, 4938–4945. Mikulaskova, H., Merlos, M.A.R., Zitka, O., Kominkova, M., Hynek, D., Adam, V., Beklova, M., Kizek, R., 2013. Employment of electrochemical methods for assessment of the maize (Zea mays L.) and pea (Pisum sativum L.) response to treatment with platinum (IV). Int. J. Electrochem. Sci. 8, 4505–4519. Morsy, A.A., Salama, K.H.A., Kamel, H.A., Mansour, M.M.F., 2012. Effect of heavy metals on plasma membrane lipids and antioxidant enzymes of Zygophyllum species. Eurasia J. Biosci. 6, 1–10. Mourato, M.P., Moreira, I.N., Leitão, I., Pinto, F.R., Sales, J.R., Martins, L.L., 2015. Effect of heavy metals in plants of the genus brassica. Int. J. Mol. Sci. 16 (8), 17975–17998. Nagajyoti, P.C., Lee, K.D., Sreekanth, T.V.M., 2010. Heavy metals, occurrence and toxicity for plants: a review. Environ. Chem. Lett. 8, 199–216. Nischkauer, W., Herincs, E., Puschenreiter, M., Wenzel, W., Limbeck, A., 2013. Determination of Pt, Pd and Rh in Brassica napus using solid sampling electrothermal vaporization inductively coupled plasma optical emission spectrometry. Spectrochim. Acta B 89, 60–65. Noctor, G., Queval, G., Mhamdi, A., Chaouch, S., Foyer, Ch.H., 2011. Glutathione. The Arabidopsis Book. 9. pp. e0142. Orecchio, S., Amorello, D., 2010. Platinum and rhodium associated with the leaves of Nerium oleander L.; analytical method using voltammetry; assessment of air quality in the Palermo (Italy) area. J. Hazard. Mater. 174, 720–727. Pandey, N., Sharma, C.P., 2002. Effect of heavy metals Co2+, Ni2+ and Cd2+ on growth and metabolism of cabbage. Plant Sci. 163, 753–758. Pawlak, J., Łodyga-Chruścińska, E., Chrustowicz, J., 2014. Fate of platinum metals in the environment. J. Trace Elem. Med. Biol. 28, 247–254. Połeć-Pawlak, K., Ruzik, R., Abramski, K., Ciurzyńska, M., Gawrońska, H., 2005. Cadmium speciation in Arabidopsis thaliana as a strategy to study metal accumulation system in plants. Anal. Chim. Acta 540, 61–70. Poschenrieder, Ch, Barceló, J., 1999. Water relations in heavy metal stressed plants. In:

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