Effects of binary metal combinations on zinc, copper, cadmium and lead uptake and distribution in Brassica juncea

Journal of Trace Elements in Medicine and Biology 44 (2017) 32–39

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Biochemistry

Effects of binary metal combinations on zinc, copper, cadmium and lead uptake and distribution in Brassica juncea

MARK

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Agnieszka Kutrowskaa, , Arleta Małeckaa, Aneta Piechalaka, Wacław Masiakowskia, Anetta Hanćb, Danuta Barałkiewiczb, Barbara Andrzejewskac, Janina Zbierskac, Barbara Tomaszewskaa a Department of Biochemistry, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznań, Umultowska 89, 61-614 Poznan, Poland b Department of Trace Element Analysis by Spectroscopy Method, Faculty of Chemistry, Adam Mickiewicz University in Poznań, Umultowska 89b, 61-614 Poznan, Poland c Department of Ecology and Environmental Protection, Poznan University of Life Sciences, Wojska Polskiego 28, 60-637 Poznan, Poland

A R T I C L E I N F O

A B S T R A C T

Keywords: Trace elements Phytoremediation Phytoextraction Metal uptake Metal crosstalk

The interaction between lead, copper, cadmium and zinc in their binary combinations was investigated in Indian mustard seedlings (Brassica juncea L. var. Malopolska). Fourteen-days-old seedlings were treated with Pb(NO3)2, CuSO4, CdCl2, ZnSO4 at 50 μmol of metal ion concentration and at 25 μmol of each metal ion in combinations. Metal combinations were generally more inhibiting in terms of biomass production. This inhibiting effect followed an order: Cu + Cd > Cu + Zn, Cd + Pb > Cu + Pb > Zn + Pb, Cu > Cd > Zn > Zn + Cd > Pb. We observed synergistic and antagonistic effects of metal uptake in binary metal treatments, suggesting metal crosstalk at the plant uptake site. Metal content in plant tissues varied among different combinations. The metal concentrations followed an order of Pb > Cu > Zn > Cd in roots, Zn > Cu > Pb > Cd in the stem and Zn > Cu > Cd > Pb in leaves. Presence of metals altered the distribution of micronutrients (Cu, Zn) in plants: Cu concentration was lowered in roots and leaves and increased in stems; Zn content was increased in plants, with stems having up to 4 or 5 times more Zn than in control plants.

1. Introduction Trace metals affect growth, development, flowering and plant yield, because they alter many basic physiological processes, including photosynthesis and respiration [1]. Level of toxicity of non-essential metals (e.g. Cd, Pb, Hg) depends on their concentration; but even essential metals like copper (Cu) and zinc (Zn) in excess can be harmful to organisms [2]. Several methods are available to remediate soils contaminated with metals, though most of them are expensive and laborious (e.g. excavation of contaminated material and an offsite treatment) [3]. Phytoextraction, the use of plants to remove trace metals, has become a tangible alternative, because it is environmentally friendly and costeffective. Hyperaccumulators are extreme examples of plants accumulating trace elements: able to grow on metalliferous soils and tolerating high amounts of metals in their aboveground organs at concentrations 100–1000-fold higher than in most species [4,5]. Indian mustard (Brassica juncea) exhibits some traits of a hyperaccumulator – it can

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

http://dx.doi.org/10.1016/j.jtemb.2017.05.007 Received 28 November 2016; Received in revised form 19 May 2017; Accepted 19 May 2017 0946-672X/ © 2017 Elsevier GmbH. All rights reserved.

take up significant quantities of Pb, Cd [6,7] and Cr, Cu, Ni, Pb and Zn [8,9], though its translocating ability is not as efficient. However, when considering metal uptake and translocation, not only plant features impacts the phytoextraction, but also other factors, such as: soil structure, pH, water retention capacity, ion sorption, organic compound content, microorganisms (especially rhizobacteria), and presence of other metal ions. Generally, metal cross-talk is one of the least studied aspects of metal removal. The antagonistic and synergistic interactions between metal ions remain to be employed for an efficient phytoextraction or reduction of non-essential metal uptake in crops. This study aimed to characterise influence of metal cross-talk in binary metal combinations on metal uptake, translocation and physiological aspects of response to metals in Indian mustard seedlings. 2. Material and methods 2.1. Plant material Seeds of Indian mustard (B. juncea L. var. Malopolska) were

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germinated on Petri dishes for 7 days. Seedlings were transferred to a hydroponic culture on the Hoagland medium (diluted 10×) and grown for 7 days in a growth chamber with a 16/8 h day/night photoperiod, at room temperature and light intensity of 82 μmol m2 s−1. The modified Hoagland’s medium used by us consisted of: 28 mM NH4H2PO4, 6 mM KNO3, 4 mM Ca(NO3)2·4H2O, 2 mM MgSO4·7H2O, 46 μM H3BO3, 9 μM MnCl2·4H2O, 3.7 μM ZnSO4·7H2O, 3.2 μM CuSO4·5H2O, 0.5 μM Na2MoO4, 70 μM FeC6H5O7·5H2O. Next, medium was diluted 100× and metals were applied in the following concentrations: 50 μM of Cu, Pb, Cd and Zn in separate treatment and 25 μM of each ion in binary combinations of Cu + Pb, Cu + Cd, Cu + Zn, Pb + Cd, Zn + Pb, Zn + Cd. Pb(NO3)2, CuSO4, CdCl2 and ZnSO4 solutions were used. The metal salts were chosen for plant treatment due to their solubility, taking under account the final concentration of essential ions (nitrate and sulphate) not exceeding the physiological range for plants. Seedlings were harvested and weighed after 0, 24, 48, 72 and 96 h of exposition to metals; samples after 96 h of exposition were analysed for metals, chlorophyll and glutathione content. Root samples were rinsed with 10 mM of CaCl2 and 10 mM EDTA to remove metals adsorbed on surface.

single variable method. Table S1 shows the operating parameters used for the ICP-MS and LA-ICP-MS measurements. More information about ICP-MS procedures used in this research can be found on page S2.

2.2. Estimation of glutathione content

Each experiment was performed in three biological and technical replicates. The mean values ( ± S.D or ± S.E) are given in the tables and figures. The data were analysed statistically using IBM SPSS Statistics (Version 22 for Windows). Significant differences among treatments were analysed by one-way ANOVA, taking p < 0.05 as the significant level, and the b-Tukey post hoc test was conducted to pairwise comparisons between treatments.

2.5. Calculation of accumulation and translocation factor Using results obtained with ICP-MS method, accumulation and translocation factors were calculated. Accumulation factor (AF) is presented as the ratio of metal content accumulated in the seedlings (in their roots, stems and leaves) divided by a metal content available for the plant to uptake from the medium. This factor represents the plant's ability to uptake metals. Translocation factor (TF) is expressed as the ratio of metal content accumulated in the shoots of seedlings (in their stems and leaves) divided by metal content in the whole plant. This factor represents the plant's ability to transport metals to the aboveground tissues. Product of TF and AF (TF*AF) is a combined factor representing the plant's ability to uptake and translocation metals to the aboveground tissues. 2.6. Statistical analysis

Total and oxidized levels of non-protein thiols were measured spectrophotometrically by monitoring the reduction of 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) at 412 nm, according to [10]. The extract was obtained using 5% sulfosalicylic acid (SSA). For the measurements of total glutathione (reduced and oxidized: GSH + GSSG), 150 μL neutralized extract was added to 1.2 mL of 0.3 mM NADPH, 150 μl of 6 mM DTNB and 1 unit of glutathione reductase (GR). For oxidized glutathione determination, 150 μL of neutralized root extract was incubated with 2 μL of 2-vinylpyridine for 1 h at 25 μC, and then added to 1.2 mL of 0.3 mM NADPH, 100 μL of 6 mM DTNB, and 1 unit of GR. The concentration of GSH was calculated as the difference between total and oxidized glutathione.

3. Results 3.1. Plant condition and biomass production The appearance and shape of seedlings treated with metals was altered in all research variants, especially in the roots after treatment with Cu + Pb, Cu + Cd, Cu + Zn and Cd + Pb. We observed inhibition of root elongation, a decrease in dry and fresh weight, root sliming and changes in the root colour (from a creamy white to the dark brown), probably caused by suberification or overproduction of the phenol derivatives. The magnitude of inhibition effects (Table S3) followed an order: Cu + Cd > Cu + Zn, Cd + Pb > Cu + Pb > Zn + Pb, Cu > Cd > Zn > Zn + Cd > Pb, with the combinations of first four combinations not only inhibited biomass production, but also decreased seedlings primary biomass by 38–54%. Cu and its combinations were most inhibiting, while Pb had lowest effect on biomass production. Binary combination of Zn + Cd was 1,5-fold less inhibiting than the exposure to individual metals, in terms of biomass production.

2.3. Chlorophyll content measurement The level of chlorophyll a and b was measured using DMSO according to a modified method described by Ronen and Galun [26]. Leaf tissue (200 mg) from pea plants was cut into small (4–16 mm2) pieces and placed in a vial with 5 mL DMSO. Three replicates of samples were incubated in a water bath at 65 °C during 120 min. Chlorophyll extract was transferred to a cuvette and spectrophotometric readings were made at 649 and 665 nm using UV–VIS spectrophotometer (Shimadzu Scientific Instruments, Japan). The reference probe was DMSO. 2.4. Elemental analysis by ICP-MS and LA-ICP-MS

3.2. Chlorophyll level Plant material (roots, stems and leaves) was washed with distilled water, gently dried on a blotting paper, weighed and dried at 70 ± 2 °C. Weighed dried samples were mineralised with microwaves in an MDS-2000 (CEM Corporation Matthews, NC, USA). Three-stage dilution was conducted in a closed system using 5 mL of 65% HNO3. After mineralization, samples were put in 10 mL measuring flasks filled with deionised water. An inductively coupled plasma mass spectrometry (ICP-MS) model Elan DRC II, (Perkin Elmer Sciex, Canada) was used to determine the concentration of elements in mineralized samples of the plants tissues. Plant roots, stems and leaves were taken after 72 h of treatment for the analysis of metal distribution. Samples were cut into 3 mm wide pieces and ablated along the pre-defined line across the cross-sections. Plant tissues were analysed in vivousing an ICP-MS spectrometer equipped with a laser ablation system (LA; model LSX-500, CETAC Technologies, USA) operating at a wavelength of 266 nm. Laser performance was optimized according to a detailed scheme [11] using a

Chlorophyll a level was steady in all separate and binary metal combinations, while chlorophyll b level decreased significantly (Fig. 1). Highest inhibition was observed in plants treated with Cu, Cd, Cu + Zn, Cu + Cd, Zn + Pb, Cd + Pb. 3.3. Glutathione Glutathione level in the aboveground plant parts was significantly increased in all tested variants, compared to the control plants (Fig. 2). Binary metal treatments caused greater changes to the GSSG to GSH ratio, with the higher increase of the oxidized glutathione content, than observed in separate treatments (with exception of copper). 3.4. Metal accumulation and distribution Metal content accumulated in the roots, stems and leaves of B. 33

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Fig. 1. Chlorophyll a and b level in shoots of Brassica juncea L. grown hydroponically, after treatment for 96 h with metal combinations. Values represent mean ± S.D. (n = 3). Different letters on bars (a,b,c… for chlorophyll a; a,b,c… for chlorophyll b) indicate significant differences (p < 0.05) between means according to Tukey test.

4. Discussion

juncea seedlings grown at different treatments of heavy metals (Zn, Cu, Pb, Cd, Cu + Zn, Zn + Cd, Pb + Cd, Cu + Cd, Pb + Zn, Cu + Pb) is presented in Table S4. Metal content in plant tissues varied among different combinations and followed the order Pb > Cu > Zn > Cd in roots, Zn > Cu > Pb > Cd in stems and Zn > Cu > Cd > Pb in leaves. Fig. 3A shows distribution of lead in roots and stems of plants treated with Pb, Cu + Pb, Cd + Pb, Zn + Pb, studied with the use of LA-ICP-MS. In roots of plants treated with binary combinations of Pb and in stems of plants from variants Zn + Pb and Cu + Pb highest peak is observed at the center of vascular tissue. In case of roots treated only with Pb and stems treated with Pb + Cd no peak at the center was observed. Fig. 3B shows distribution of Cd in cross-sections of roots and stems of plants treated with Cd, Cu + Cd, Zn + Cd, Cd + Pb. In roots treated with Cu + Cd and Zn + Cd we observed highest signal intensity at the center of vascular tissue. In stems, Cd distribution pattern is similar in most cross-sections, with highest relative intensity of Cd:C in plants treated with Zn + Cd.

There has been a continuing interest in studying metal uptake in plants, in order to increase phytoextraction efficiency to an economically feasible level. However, as the process is complex, many factors alter metal acquisition and translocation. Our study intended to show how presence of Cu, Cd, Zn and Pb in binary combinations affects metal uptake and transport. Here we show that B. juncea L. accumulated and translocated to shoots high amounts of metals. Our results suggest that the transport of Pb and Cd throughout the seedlings can be increased in the presence of other metal ions, especially in combinations with Zn. Indian mustard has been proven to take up significant quantities of Pb and Cd under a variety of experimental conditions [7] and to accumulate metals in shoots [12]. In our experimental setup, Indian mustard shoots never passed the threshold level for Pb hyperaccumulator (> 1000 mg kg−1 DW) [4], though reaching as high as 695.11 mg of Pb kg−1 DW in treatment with Pb and 624.37 mg of Pb kg−1 DW in Zn + Pb combination (Table S4). However, we have used a relatively low Pb concentration (25 μM in binary combination and

Fig. 2. Glutathione content in shoots of Brassica juncea L. grown hydroponically, after treatment for 96 h with metal combinations. Values represent mean ± S.D. (n = 3). GSH – reduced glutathione, GSSG – glutathione disulfide. Different letters on bars (a,b,c… for GSH; a,b,c… for GSSG) indicate significant differences (p < 0.05) between means according to Tukey test.

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Fig. 3. Distribution of Pb (part A) and Cd (part B) in cross-sections of the roots and stems of B. juncea L. seedlings treated with separate and binary combinations of metals (Cu, Zn, Cd, Pb). The distribution is presented as a ratio of analytical signals intensity of metals (Pb or Cd) to C as internal standard (i.e. Pb/C, Cd/C). The results were obtained using LA-ICP-MS technique.

50 μM in separate treatment) for only 5 days. In a similar study [13], Sesbania drummondii seedlings were treated with Pb at around 1.20 mM for 10 days and crossed the threshold. Our results show that B. juncea exhibits higher translocation rate for Pb than S. drummondii, expressed as a ratio of metal content in shoots to metal content in the whole plant – 0.18 for Pb and 0.26 for Zn + Pb in B. juncea as opposed to 0.021 and 0.025, respectively, in S. drummondii. When we consider a very low Pb mobility in soil and low translocation rate to shoots, Indian mustard may be very useful for Pb phytoextraction, contrarily to most studied plants [14,15,13]. Indian mustard has not met the criteria for a Zn and Cd hyperaccumulator, but when treated with Cu it passed the threshold (> 1000 mg kg−1). An ideal plant for metal removal is envisioned as one with high biomass production, easily harvested and with high capacity and tolerance for metal accumulation [16]. Thus, the product of TF and AF enables the comparison in terms of two out of three critical factors. In our results, AF followed the order Zn > Cu > Pb = Cd, while TF followed the order Zn > Cd > Cu > Pb (Fig. 4), which agrees with available data [17]. In all combinations, bioaccumulation of one metal was found to be affected by the presence of a second metal, inhibited or increased, ex. presence of other metals in binary combinations increased TF for Pb (Fig. 4). When essential metals (Cu or Zn) were available, more of Pb was transported to the leaves. This agrees with our observation of Pb distribution toward vascular tissue in cross-sections of roots and stems of Cu + Pb and Zn + Pb combinations. In treatment with non-essential Pb and Cd + Pb, more of Pb was

distributed toward stems and no high signal intensity at the center of stem cross-sections was observed (Fig. 3A). Even though other metals increased translocation of Pb, the AF for Pb was higher only in combination Zn + Pb, compared to the treatment with Pb. This agrees with previous reports that the presence of Zn increased Pb accumulation [18–20]. Although this is not a new observation, the molecular explanation for such a synergistic action between Zn and Pb is still elusive. Available data on metal transporters show that no metal trafficking proteins are shared for these two elements, which could explain absence of competition [17,21]. Increased Zn concentration in soil could stimulate apoplastic transport of ions or/and activate non-specific ion channels. This might explain our results that in Zn + Pb combination not only more Pb is accumulated, but also more of Pb is directed toward stem, which could be expected with increased apoplastic transport. Binary combinations with other trace metals increased AF for Cd. As for translocation factor, it was increased for Cd in presence of Pb and Zn, but the effect of each metal was different: Zn increased accumulation of Cd in leaves, while Pb – in stems. Additionally, presence of Pb had a stimulating effect on the Cd accumulation, but at the expense of lowered Pb uptake in Cd + Pb combination. We observed also an increased Zn and Cu uptake in combinations with Cd. TF*AF for Cd was highest in combinations Cd + Pb and Zn + Cd. In cross-sections of Cu + Cd and Zn + Cd treated roots we observed Cd distribution towards center (the vascular tissue), suggesting efficient translocation. In Zn + Cd combination high signal intensity was observed also in stem, 35

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Fig. 4. Effect of separate and binary combinations of metals (Cu, Zn, Cd, Pb) on metal accumulation (accumulation factor, AF) and translocation to shoots (translocation factor, TF) in B. juncea L. seedling parts (roots, stems and leafs). AF is the ratio of metal content accumulated in the seedlings (in their roots, stems and leaves) divided by a metal content available for the plant to uptake from the medium. TF is the ratio of metal content accumulated in the shoots of seedlings divided by metal content in the whole plant. TF*AF.

excess. Redox-active metal ions can participate in Haber–Weiss and Fenton reactions and thereby trigger the formation of hydroxyl radical. Non-essential metals such as Cd and Pb intrude into plant cells at the expense of essential ions. The phytotoxicity of these metals is caused by their interaction with proteins, displacement of essential ions from binding sites and stimulation of reactive oxygen species (ROS) generation. Despite the differences in metal functions and properties, the effects of their toxicity are very similar [15,35,38]. Inhibition of growth and reduction of biomass belong to the main responses of higher plants to heavy metal toxicity. The decline in biomass production could be explained, in part, due to the inhibition of both, the cell division and cell elongation, by heavy metals [26]. In the present study, in case of separate treatments Pb was least inhibiting to biomass production (14% of biomass production inhibition compared to control) and Cu was a strongest inhibitor (78% of inhibition). Binary combinations had more inhibitory effect on biomass production than separate treatments, except for Zn + Cd (with only 38% of biomass production inhibition). Cu + Cd, Cu + Zn, Pb + Cd, Cu + Pb combinations not only inhibited biomass production, but also decreased seedlings primary biomass from before the treatment, with 54%, 41%, 41% and 38% decrease in biomass, respectively. These results suggest a synergistic effect of toxicity in binary combinations on B. juncea growth. Increased metal accumulation results mainly in higher energy demand to cope with stress, thus leading to decreased biomass production [27]. Observed decrease in FW of seedlings might be caused by disturbance in water balance and high energy expenses. Similar inhibitory effects have also been observed in other plants species [20,19,13,28]. Interestingly, combination of two essential metals – Cu and Zn – had a stronger impact on seedlings than what was observed in case of separate treatments with each of these metals. Transition metals are physiologically strong prooxidants. Many of them, e.g., Cu, Co, Ni or Mn in th presence of Fe participate in Haber-Weiss and Fenton reactions, resulting in the production of the most dangerous molecule being created in biological systems: hydroxyl radical. The Haber-Weiss and Fenton reactions could be one of the reasons behind the high toxicity of Cu + Zn combination. However, our results on accumulation and translocation (Fig. 4) indicate additionally that in combination Cu + Zn translocation of both metals to the shoots is impaired. The micronutrient deficiency in photosynthetically active parts could be one of the reasons for toxicity. The effect of combination of zinc and Cd on metal accumulation and plant stress was complex – seedlings’ biomass in combination was

implicating transport to leaves, which agrees with ICP-MS results on Cd translocation in this variant (Fig. 3B, Table S4). In case of two studied micronutrients – Cu and Zn – we observed different behaviors. TF for Cu was lower in every binary combination tested (compared to treatment with only Cu), while for Zn TF was lower only in combination Cu + Zn. Additionally, TF for Cu in the Cu + Zn combination was decreased for almost a factor of 2 (without changing AF). This observation suggests an antagonism between Cu and Zn, not at the entry point in roots, but probably later during xylem loading/ unloading. Arabidopsis P-type ATPases HMA2/4 could be implicated in this process, because they serve as xylem loading transporters for Cu and Zn [22]. It would also explain why the competition is absent at the root surface – during metal uptake different transporters are involved in the Cu and Zn trafficking, probably with low overlap in specificity. Pb in Cu + Pb combination decreased Cu accumulation and stimulated distribution of Cu towards the leaves. An antagonistic effect of Pb on the accumulation of Cu may be caused by the competition between metals at the plant uptake site. Similarly, [23] reported reduced uptake of Cu in binary combinations with Pb in Brassica chinensis. This argues with the report [19] that combination of Cu and Pb can stimulate Cu accumulation in Cucumis sativus, though the interactions are dose-dependent. Lead had a synergistic effect on Zn accumulation in combination Zn + Pb. TF*AF for Cu was lower in every combination than in treatment with only Cu. Previous study [19] showed that Cu and Cd act antagonistically, which results in decreased accumulation of both metals. In most plants, metals are predominantly accumulated in the roots. As a consequence, shoot:root ratios of metal concentrations are generally substantially below unity in these plants. By comparison, in hyperaccumulators metal concentrations are substantially higher in the leaves and much lower in the roots. There are several factors which can affect the uptake mechanism of heavy metals such as: root zone, the plant species, bioavailability of the metal, environmental condition, chemical properties of the contaminant, chelating agent added, properties of medium [24]. Plants exposed to high levels of heavy metals causes reduction in photosynthesis, water uptake, and nutrient uptake. Plants grown in soil containing high levels of heavy metals show visible symptoms of injury reflected in terms of chlorosis, growth inhibition, browning of root tips, and finally death [25]. Copper and zinc are essential for plants and most other organisms due to their chemical properties such as redox-activity (Cu, Fe) or Lewis acid strength (Zn). These properties, however, are also the reason why they can be toxic in 36

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shoot metal concentrations that can be considered toxic to plants – and thus inhibiting photosynthesis – are as follows: 30–300 mg kg−1 Pb, 20–100 mg kg−1 Cu, and 100–400 mg kg−1 Zn [33]. Such an interpretation could explain our results (though with higher limits for Indian mustard is a more tolerant plant), if we considered metal concentration only in leaves, e.g. Cu + Pb had a strong inhibitory effect on biomass production but a low impact on chlorophyll b content (leaf Pb content: 46.387 mg kg−1, shoot Pb content: 272.36 mg kg−1), while Zn + Pb had lower inhibitory effect on biomass but a high impact on chlorophyll b (leaf Pb content: 139.171 mg kg−1, shoot Pb content: 624.38 mg kg−1). Similarly to our setup, experiments were performed in binary combinations (Cd + Pb), (Cu + Pb) and (Cu + Cd) at 0.1, 1.0 and 10.0 μg/ml [23]. Authors reported that Cu + Cd variant was the most toxic, a combination Cd + Pb exhibited synergistic toxic effect, while Cu + Pb was least toxic [23]. This agrees with our results on glutathione content. Trace metals generate high levels of reactive oxygen species, which damage cell constituents and alter cell oxido-reactive balance [34,26]. Plant cells protect themselves by employing defense systems, which include antioxidative enzymes (like superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase) and non-enzymatic antioxidants such as ascorbic acid, cysteine and non-protein thiols [35]. Glutathione content and the ratio of its reduced state (GSH) to oxidized (GSSG) are considered key players in maintaining oxydoreductive homeostasis in cells [36,37]. The glutathione assay chosen by us uses isolation in an acidic environment to ensure the removal of peptides and proteins and provides a suitable environment for the action of 2vinylpyridine [10]. 2-Vinylpyridine reacts with glutathione at slightly acidic pH values where spontaneous formation of glutathione disulfide is minimal. Because NADPH dependent conversion of GSSG to GSH catalysed by glutathione reductase is a rate limiting step in the ascorbate-glutathione pathway, plants under high stress struggle to synthesize new GSH, which leads to increased total glutathione content. Plants that had more time in adjusting and eventually overcame the stress, usually have higher GSH to GSSG ratio than control plants [35,38]. In our study, the glutathione levels in aerial parts showed no significant changes upon treatment with metals (data not shown), but significant changes in the levels of GSH and GSSG were observed in the roots. All treatments caused increased glutathione content compared to control, and significant differences were observed between separate and binary treatments. Cu, which is very reactive, increased GSH content and decreased GSH:GSSG ratio in B. juncea shoots, while plants subjected to Cd and Pb had increased ratio and total glutathione content. Binary combinations caused a large decrease to GSH:GSSG ratio, especially in variants that were most inhibiting in terms of biomass production – Cu + Pb, Cu + Cd, Cu + Zn and Pb + Cd. Least inhibiting combinations – Zn + Pb and Zn + Cd – also had higher GSH content, though the difference was not as high as for other binary treatments. These results indicate the potential of B. juncea to tolerate metal inducing oxidative stress and suggest that this plant could be employed for the phytoextraction of Cd and Pb, on soils polluted with Cd, Cd and Zn, or Pb and Zn, respectively. Interactions observed during our experiments are shown in Table 1.

inhibited by 38% compared to control, which is less than the effect of separate Zn and Cd treatment (51% and 56%, respectively), suggesting lowered toxicity. In the same time, accumulation and shoot translocation factors were increased for both Zn and Cd, suggesting improved metal uptake, accumulation and translocation. Therefore, we observe a case, where metal toxicity is lowered even as more of the metals is accumulated and transported to the shoots. Researchers [28] proposed that Zn may protect plants from Cd toxicity by enhancing activity of antioxidative enzymes such as superoxide dismutase (a Zn-binding enzyme), and by competing with Cd for binding to −SH groups of enzymes and membrane proteins. Micro- and macronutrients are fundamentally important for plant physiology [15,17]. Because Cu and Zn are essential metals, they have to be always present in the Hoagland solution used for the plant culture and cannot be ommited from the experiment [2,41]. Contrarily, during the experiment, plants (except for deliberately treated variants) were not grown with Cd and Pb. Therefore, the levels of accumulated metals significantly differ for essential and non-essential metals in control plants, with the latter occurring at very low levels (Table S4). The level of Pb and Cd in control plants remained in our experiment at a background level, reflecting also the capacity of the method used for the analysis of metal content (ICP-MS)[11]. In the treated variants, metal content in plants was increased for the metals used in the treatments, whereas the content of essential metals (Cu, Zn) was either decreased or increased, depending on the treatment. To illustrate it, we have examined the fold change of the essential nutrients (Cu, Zn) and their distribution in plants treated with metals, in comparison to control plants (Fig. S5). Cu and Zn content in plants treated with other metal ions (for Cu: in Cd, Pb, Zn, Zn + Cd, Cd + Pb, Zn + Pb treatments; for Zn: in Cd, Pb, Zn, Cd + Pb, Cu + Pb, Cu + Cd treatments) was affected, as shown in Fig. S5. Cu concentration was lowered in roots and leaves, increased in the stem, and in total – lowered by a factor of around 2. Zn content changed in a more diverse way, it was induced by a factor of 1.5-2 in whole plants, with stems having up to 4 or 5 times more of Zn than in control. Plants treated with Cd, Pb and Cd + Pb were generally distributing more essential metals toward their stems. The physiological state of B. juncea and its photosynthetical activity in the presence of metals were indirectly assessed by measuring changes in the chlorophyll a and b content in leaves. The changes in chlorophyll content are universal response to metal stress, which might induce interactions of metal ions with eSH groups of enzymes of chlorophyll and carotenoid biosynthesis. Additionally, the decrease in net photosynthesis can be a consequence of reduced absorption of essential mineral nutrients [29]. Many authors reported that plants treated with heavy metals like Cu, Cd, Cr or Pb show decrease of chlorophyll ratio a/b, decomposed or decreased content of PS II chlorophyll molecules, changes in sub-microstructure of chloroplasts and damaged chloroplast membranes [30–32]. Combination Zn + Pb had the strongest impact on chlorophyll b, comparable only with the influence of separate Cu treatment. It is surprising, because Zn + Pb had second lowest effect on biomass production among all combinations. This could be explained with increased metal translocation and accumulation in leaves, which may have disturbed the delicate network of macro- and micronutrients in leaves. A study [6] showed that shoots of B. juncea plants were more sensitive to Pb toxicity than the roots. According to another study [33], Table 1 Summary of interactions observed between metals. Interaction

During uptake to root

During translocation to shoot

Interaction observed also in:

Zn and Pb Cu and Pb Cu and Zn Cu and Cd Zn and Cd Cd and Pb

synergy competition weak interaction − > competition in favor of Zn weak interaction − > synergy competition competition in favor of Cd

synergy synergy with compartmentation – Pb to stem, Cu to leaves competition weak interaction − > competition in favor of Cd synergy in favor of Cd synergy

[12,18,17,19] [22] (competition)

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[18] (competition) [38–40]

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4.1. Limitations For our studies, we chose the concentration of 50 μM (separately) and 25 μM (binary combinations), because according to our earlier experiments, these are the concentrations at which metals, both essential and non-essential, cause negative changes in plants, but are not lethal. Our parameters were based on our own experience and literature data indicating the level of metals in soil and soil solution, both in Poland and in the world [41–44]. The use of such a concentration enables us to observe changes under the influence of heavy metals in the plant, for example: translocation of metals, activation of antioxidative and detoxicative systems, without causing necrosis in the plant. When we tested the binary combinations of metals administered at 50 μM of each metal, the toxicity was too high, whereas the administration of 25 μM of metals in individual treatments did not allow to observe the extent of negative changes that would be comparable. We had to limit the number of tested variants, because of the difficulty to collect the necessary amount of material (parts of seedlings) during one growth season (although seedlings can be grown in hydroponics throughout the whole year, we observe higher levels of stress in seedlings grown during winter and late autumn, which could affect our results). The choice of the concentrations and their combinations is evidently a limitation of our research. 4.2. Conclusion This study was conducted to characterise influence of metal crosstalk in binary metal combinations on metal uptake, translocation and physiological aspects of response to metals in Indian mustard seedlings. Results show that metals interact with each other during metal uptake to roots and metal transport to shoots. Binary combinations of metals resulted generally in a greater reduction of B. juncea L. biomass and glutathione content than separate treatments with metals. We observed one very strong relation: synergistic response between Zn and Pb resulting in an increased accumulation of two metals. This interaction could have a high potential impact on phytoextraction efficiency. Additionally, our results suggest that ICP-MS and LA-ICP-MS techniques should be used together in a characterisation of metal uptake and distribution: one method can indicate mean metal accumulation of a pool of samples, while the other – individual metal accumulation patterns. Moreover, we believe that metal interactions should be studied separately in roots, stems and leaves (instead of a standard division of samples to roots and shoots). This approach allows additional analysis of metal translocation throughout the plant, with respect to three main metal transport bottlenecks – during root uptake, xylem loading and uptake to leaves. Conflict of interest Authors report no conflict of interest. Acknowledgments This research was partially supported by the National Science Center in Poland [project numbers N N305 381138 and 2013/11/N/ NZ9/00070]. KA is also a beneficiary of program ‘KNOW Poznan RNA Centre, 01/KNOW2/2014'. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jtemb.2017.05.007. References [1] B. Dhir, S.A. Nasim, P. Sharmila, P.P. Saradhi, Heavy metal removal potential of

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