Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

The Influence of Cadmium Stress on the Content of Mineral Nutrients and Metal-Binding Proteins in Arabidopsis halleri

The Influence of Cadmium Stress on the Content of Mineral Nutrients and Metal-Binding Proteins in... Water Air Soil Pollut (2012) 223:5445–5458 DOI 10.1007/s11270-012-1292-4 The Influence of Cadmium Stress on the Content of Mineral Nutrients and Metal-Binding Proteins in Arabidopsis halleri Ewa Przedpełska-Wąsowicz & Aleksandra Polatajko & Małgorzata Wierzbicka Received: 10 March 2012 /Accepted: 25 July 2012 /Published online: 23 August 2012 The Author(s) 2012. This article is published with open access at Springerlink.com Abstract We investigated the influence of cadmium content and mineral nutrients were evidenced by our stress on zinc hyperaccumulation, mineral nutrient study. We identified more than ten low-molecular- uptake, and the content of metal-binding proteins in weight (<100 kDa) Cd-binding proteins in Cd-treated Arabidopsis halleri. The experiments were carried out plants. These proteins are unlikely to be phytochelatins using plants subjected to long-term cadmium exposure or metallothioneins. We hypothesize that low- (40 days) in the concentrations of 45 and 225 μM molecular-weight Cd-binding proteins can be involved 2+ Cd . Inductively coupled plasma-mass spectrometry, in cadmium resistance in A. halleri. size exclusion chromatography coupled with plasma- . . mass spectrometry, and laser ablation inductively cou- Keywords Arabidopsis halleri Cadmium . . pled plasma-mass spectrometry used for ablation of Cadmium-binding proteins Hyperaccumulator polyacylamide gels were employed to assess the con- Metal accumulation Zinc tent of investigated elements in plants as well as to identify metal-binding proteins. We found that A. hal- Abbreviations leri is able to translocate cadmium to the aerial parts in ICP-MS Inductively coupled plasma-mass high amounts (translocation index >1). We showed spectrometry that Zn content in plants decreased significantly with LA-ICP-MS Laser ablation inductively coupled the increase of cadmium content in the growth medium. plasma-mass spectrometry Different positive and negative correlations between Cd LMW Low molecular weight PAGE Polyacrylamide gel electrophoresis PVP Polyvinylpyrrolidone Electronic supplementary material The online version of this SEC-ICP-MS Size exclusion chromatography article (doi:10.1007/s11270-012-1292-4) contains coupled with plasma-mass supplementary material, which is available to authorized users. spectrometry E. Przedpełska-Wąsowicz (*) M. Wierzbicka Department of Molecular Plant Physiology, Institute of Botany, Faculty of Biology, University of Warsaw, 1 Introduction Miecznikowa 1, 02-096 Warsaw, Poland e-mail: [email protected] Arabidopsis halleri (Brassicaceae) is a perennial spe- cies occurring in Europe and East Asia (Al-Shehbaz A. Polatajko and O’Kane 2002). Apart from natural mountainous ISAS-Institute for Analytical Sciences, P.O. Box 101352, 44013 Dortmund, Germany habitats, it occurs also in areas polluted with heavy 5446 Water Air Soil Pollut (2012) 223:5445–5458 metals (Ernst 1990; Pauwels et al. 2006). The species histidine, nicotiamine), organic acids such as: malate is well known for its’ tolerance to zinc and cadmium and and citric acid, phytochelatins, and metallothioneins the ability to hyperaccumulate these metals. A. halleri,a (Szpunar 2005; Fenik et al. 2007; Polatajko et al. close wild relative of Arabidopsis thaliana,isamodel 2007; Maestri et al. 2010). Braude et al. (1980) men- species in studies focused on the problem of metal toler- tioned that cadmium shows a tendency to accumulate ance and hyperaccumulation in plants (Pauwels et al. in a protein fraction within the plant cell. This is 2006; Verbruggen et al. 2009;Maestrietal. 2010; particularly interesting, as most of the proteins regu- Meyer et al. 2010, 2011; Gode et al. 2012). lating metal homeostasis of the plant cell, are also Many studies have investigated zinc hyperaccumula- involved in detoxification, regulation of the cell cycle, tion in plants (Lasat and Kochian 1998, 2000;Pauwels proliferation, and apoptosis (Garcia et al. 2006). To et al. 2006; Broadley et al. 2007; Verbruggen et al. 2009; understand thoroughly plant response to environmen- Maestri et al. 2010; Meyer et al. 2010). In case of tal stress caused by metal ions, it is necessary to cadmium hyperaccumulation, however, the problem localize, identify, and quantify metal-containing mac- seems to be less studied. Hitherto, only four species romolecules. This task is particularly challenging from are known to hyperaccumulate cadmium. Since these the analytical point of view. Hyphenated techniques plants are also zinc hyperaccumulators, it suggests a for biological systems such as size exclusion chroma- common genetic basis of both phenomena tography coupled with plasma-mass spectrometry (Verbruggen et al. 2009). Nonetheless, little is known (SEC-ICP-MS) or laser ablation inductively coupled regarding correlation between hyperaccumulation of plasma-mass spectrometry (LA-ICP-MS) can be zinc and cadmium. regarded as a solution of this problem. Recently, this Zinc and cadmium belong to the most intensively technique was used for direct ablation of polyacryl- studied metals in terms of their impact on plants. Zinc amide gels (PAGE) (Szpunar 2005; Fenik et al. 2007; is a micronutrient essential to plant growth; however, Polatajko et al. 2007). its excess can cause toxic effects. Cadmium is one the We employed these techniques in order to investi- most frequent and the most dangerous inorganic pol- gate the influence of the Cd stress on mineral nutrient lutants. Although both metals share similar chemical uptake and metal-binding proteins content in A. properties (they have similar atomic radius, similar halleri. oxidation state in chemical compounds, and share We tested the following hypotheses: similar geochemical properties), cadmium shows higher tendency to bond with sulfur and higher mo- 1. A. halleri is a Cd hyperaccumulator. 2. There is a correlation between Cd uptake and the bility in soils and in whole ecosystems (Emsley 1991; Kabata-Pendias 2010). It has been hypothesized that content of mineral nutrients. both metals can share similar pathway while entering 3. Cd ions are bound by low-molecular-weight the plant organism (Zhao et al. 2006). In contrast to (<100 kDa) metal-binding proteins. zinc, which is essential for plant growth, cadmium is not found in any natural chemical compound in living organisms. Cadmium is widely studied in the context 2 Materials and Methods of environmental pollution and its’ impact on human health (di Toppi and Gabbrielli 1999). Although the 2.1 Plant Material metal is toxic to plants, it is known for its easy uptake (Kabata-Pendias 2010). Strong affinity to sulfhydryl Experiments were carried out using A. halleri plants. groups is one of the most important biochemical char- Seeds were obtained from the Pb/Zn mining area in acteristics of cadmium. The metal, however, can also Boleslaw near Olkusz (S Poland), a region highly pol- easily bind to functional groups containing nitrogen or luted with heavy metals (Przedpelska and Wierzbicka oxygen (Polatajko et al. 2007). 2007;Abratowskaet al. 2012). Plants were cultivated Research on metal-chelating compounds in organ- for 40 days in growth chamber under controlled tem- isms responsible for the metal homeostasis of the cell perature conditions (24±4 °C), relative humidity of 65± showed that different chemical compounds can be 4 %, light intensity of ∼120 μmol/m /s, and photoperiod involved in this process including amino acids (e.g., of 8:16 h. Water Air Soil Pollut (2012) 223:5445–5458 5447 −1 2.2 Plant Cultivation and Metal Exposure (10 ng mL ) as an internal standard. The double focus- ing sector field ICP-MS (ELEMENT 2, ThermoFisher Seeds collected from the field were germinated in Petri Scientific, Bremen, Germany) coupled to the Cetac dishes on filter paper moistened with diluted Knop autosampler (ASX-500, Omaha, Nebraska, USA) was nutrient solution (1:8 dilution) supplemented with A- used for multi-element analysis (Cd, Zn, Mg, S, Mn, Fe, Z mixture of trace elements (Strebeyko 1967), in the Cu, Mo). Translocation index was calculated as de- light at the temperature of 24 °C±1 °C. About scribed by Branquinho et al. (2007). Statistical signifi- 7 days old, seedlings were transferred into pot boxes cance of the observed differences between the control containing perlite as a substrate. The perlite was and experimental groups was tested by Kruskal–Wallis washed daily with Knop’s nutrient solution of the test using Statistica 9.1. Shape of the distribution within following chemical composition—200 g/l Ca analyzed groups and the variance were checked prior to (NO ) ×4H O, 71.5 g/l KNO , 35.5 g/l KCl, 71.5 g/ the analysis in order to ensure that the assumption of the 3 2 2 3 l MgSO , 71.5 g/l KH PO , 28 g/l EDTA-Fe, and trace homoscedasticity of the data is not broken. To test for 4 2 4 2+ elements (including Zn at the concentration of correlations between cadmium and other analyzed ele- 0.4 μM), pH06. ments, we used coefficient of determination (R ). The plants were divided into control group and two Regression lines and 95 % confidence intervals were experimental groups (30 plants each). Experimental calculated. Significance tests were carried out at 5 % groups were treated every second day with cadmium significance level. All the statistical analyses were per- added to the nutrient solution as Cd(NO ) ×4 H Oto formed using Statistica 9.1 (Statsoft Inc.) 3 2 2 2+ achieve the concentrations of 45 and 225 μMCd . Cd concentrations employed were chosen on the basis 2.4 Protein Extraction of pilot experiments (data not shown). We intended to use Cd doses that would enable plants to accumulate Proteins were extracted from shoots and roots of the maximal amount of Cd in their tissues without visible control and Cd-treated plants following the sample prep- effects of toxicity. Forty-day-old plants were har- aration protocol—5gofleavesand3gofroots were vested, divided into shoot and root portions, and then homogenized with 6 mL of 50 mM HEPES-NaOH washed several times in distilled water. By the shoot (pH 7.6) containing 2 % PVP and protease inhibitor portions, all the aerial portions of plant including stem cocktail (Complete, EDTA-free) using a ultrasonic probe and leaves are meant. To ensure complete removal of homogenization technique (Branson, SONIFIER, all the components of the growth medium (including Schwäbisch Gmünd, Germany). The homogenization 2+ Cd ), roots were washed for 5 min in 20 mM EDTA. method involved 5×30-s treatments until a well- Subsequently, samples were immediately frozen in homogenized extract was obtained. In order to obtain liquid nitrogen and ground using a pestle and mortar. cytosol free from organelle samples were centrifuged at To avoid protein unfolding and a loss of metals from 105,000g for1 hat4°C usinga SorvallDiscovery 90SE their native structures, samples were placed on dry ice ultracentrifuge (ThermoFisher, Dreieich, Germany). The after grounding. A part of the sampled material was supernatant was then centrifuged for 30 min at 4,000 rpm used in protein extraction, and the remaining portion using a Heraeus SEPATECH centrifuge (ThermoFisher, was lyophilized. Dreieich, Germany). The aliquots were stored at −80 °C for further investigations. The method employed has been 2.3 Quantification of Cd, Zn, and Mineral Nutrients optimized by Polatajko et al. (2011). For quantification of the total metal content, 0.2 g of lyophilized leaf and 0.1 g of root samples were digested 2.5 SEC-ICP-MS Analysis of Leaves and Roots Extracts with 6 mL of a HNO –H O mixture (5:1) using a 3 2 microwave-assisted digestion with closed vessels Cadmium–protein complexes from shoots and roots (Anton Paar, Graz, Austria). Triplicate extracts from all extracts were probed by size exclusion chromatography the samples were taken. Digested samples were diluted (SEC, Dionex, Germering, Germany) coupled to ICP- with Milli-Q water to 50 mL and analyzed by ICP-MS MS (ELEMENT 2, Thermo Fisher Scientific, Bremen, using an external calibration curve with Rh Germany). SEC-purified fraction was also used for 5448 Water Air Soil Pollut (2012) 223:5445–5458 identification of Zn- and Cu-proteins. The method in leaves as well as in roots (Fig. 1a). In plants treated 2+ employed has been optimized by Polatajko et al. (2011). with lower dose of cadmium (45 μMCd ), concentra- −1 tion of the metal in roots was 265 μgg d.w., whereas in 2+ 2.6 PAGE-LA-ICP-MS Analysis of Leaves and Roots plants treated with the higher dose (225 μMCd ), it −1 Extracts was 835 μgg d.w. We found that in treated plants −1 cadmium concentration in shoots was 940 μgg d.w. −1 The separation of cadmium-containing proteins was and 2,525 μgg d.w, respectively, for lower and higher done by using anodal native 10 % polyacrylamide gel dose. Total concentration of the metal in treated plants electrophoresis (1D AN-PAGE) with a discontinuous was always higher in shoots than in roots. Tris/Tricine system according to the manufacturer’s The translocation index, calculated by dividing the instructions. A Nd:YAG laser (Minilight II, concentration of the metal in shoots by the concentra- Continuum, Santa Clara, USA) coupled to the sector tion of the metal in roots, varied narrowly between 3 filed ICP-MS was used for laser ablation (LA) of the and 3.5 for both tested cadmium doses. Cd-binding proteins on the membrane. For a detailed Experiments showed that that zinc content in cadmium- description of the background of the chemical analy- treated plants was significantly lower (Fig. 1b). While in −1 ses, the reader is referred to Polatajko et al. (2007). the control group zinc content was 175 μgg d.w., in treated plants, it decreased by 37 % and 70 % in case of 2+ 2+ lower (45 μMCd ) and higher (225 μMCd )cadmi- 3 Results um dose, respectively. In roots, zinc content followed the same pattern being the highest in control plants −1 3.1 The Influence of Cadmium on Biomass Production (125 μgg d.w.) and decreasing by 58 % and 71 % in both cadmium-treated groups. The Zn root–shoot trans- Cd-treated plants showed no signs that could be asso- location index calculated for each experimental group ciated with the toxic effect of Cd ions. Leaves showed was 1.2 and 2.1, respectively. no signs of chlorosis or necrosis; tissues were well The values of the translocation indices showed hydrated (no signs of decreased turgidity). No signs of hyperaccumulation of both tested metals (Cd and Zn) growth inhibition or differences in development were by A. halleri. There was no significant difference in recorded between control and treated plants. Mean dry accumulation between investigated isotopes: Cd vs. 114 66 68 weight of shoots was 2.2, 1.8, and 1.65 g for the Cd and Zn vs. Zn. control and two experimental groups, respectively. Mean dry weight of roots was 0.3, 0.3, and 0.27 g, 3.3 The Relationships Between Cd and Mineral respectively. Nevertheless, there was no significant Nutrient Concentrations in Shoots and Roots difference in biomass production between control and treated plants, regardless of the concentration of Statistical analysis showed the presence of significant Cd in the growth medium (Kruskal–Wallis test, p0 correlations between the concentration of cadmium 0.1955 for shoot samples, p00.3594 for root samples). and other macro- (Mg, S) and microelements (Mn, The above-mentioned observations show that A. hal- Fe, Cu, Zn, Mo). Correlation analysis uncovered that leri is tolerant to tested cadmium doses. The fact that no significant and strong negative correlation exists be- signs of toxicity were observed while A. halleri accu- tween concentrations of zinc and cadmium (R 0 mulated Cd in its tissues in high amounts (particularly in −0.8487, p<0.05inroots; R 0−0.9922, p<0.05 in shoots, see below) additionally confirms this notion. shoots). Magnesium showed significant correlation with cadmium concentration in both roots and shoots, 3.2 Cd and Zn Accumulation however, in roots, the coefficient of determination showed negative (R 0−0.9125, p<0.05), whereas in Results obtained for quantitative multi-element analysis shoots positive (R 00.8765, p<0.05) value. No other relative to the dry mass are presented in Fig. 1a, b. investigated element showed significant correlation Results showed that cadmium concentration in experi- with cadmium concentration in roots at the signifi- mental plants (40 days of treatment) increased with cance level of 5 %. In shoots, significant correlations increasing concentration of the metal in growth medium were also found between cadmium and manganese Water Air Soil Pollut (2012) 223:5445–5458 5449 Fig. 1 Total concentration Cd 112 of Cd (a) and Zn (b)in A. 2500 Cd 114 halleri extracts from shoots and roots, respectively. Metal concentration was quantified using ICP-MS Control 45 µM 225 µM Control 45 µM 225 µM group Cd-treated Cd-treated group Cd-treated Cd-treated group group group group Shoots Roots b Zn 66 Zn 68 Control 45 µM 225 µM Control 45 µM 225 µM group Cd-treated Cd-treated group Cd-treated Cd-treated group group group group Shoots Roots 2 2 (R 00.9593, p<0.05), cadmium and iron (R 0 3.4 Cd-Binding Proteins −0.8046, p<0.05), as well as between cadmium and copper (R 0−0.7307, p<0.05). There was no Using ICP-MS, we measured the content of Cd- significant correlation between cadmium and sulfur binding proteins in shoots and roots. We found that, 2 2 (R 00.2593, p>0.05inroots, R 00.1064, p>0.05 in plants treated with 45 μM Cd, the content of Cd- in shoots). Regression lines along with coefficients binding proteins was about 8.6 and 9.6 μg/g f.w. in of determination (R ) and results of significance shoots and roots, respectively. In plants treated with testing for each analyzedelement canbefound 225 μM Cd, these values were about 52.5 and in electronic supplementary material (Fig. ESM 1, 33.5 μg/g f.w., respectively. We did not record the Fig. ESM 2). presence of Cd-binding proteins in the control group. Cd concentration µg/g d.w. Zn concentration µg/g d.w. 5450 Water Air Soil Pollut (2012) 223:5445–5458 2+ The control electropherograms (Fig. 2a, d) showed Cd . The same methodology enabled us to detect two the lack of Cd-binding proteins in both shoots and signals and one broad peak showing the presence of roots. Application of SEC-ICP-MS enabled us to de- Cd-binding proteins in roots and shoots obtained from tect one signal for Cd-binding protein in roots plants treated with 225 μM solution of cadmium (Fig. 2b) as well as in shoots (Fig. 2e), during the (Fig. 2c, f). All the identified Cd-proteins had small examination of the plant material treated with 45 μM molecular weight of <13 kDa. Apart from Cd-proteins, High molecular Low molecular High molecular Low molecular weight fractions weight fractions weight fractions weight fractions Control Control Cu 63 Cu 63 group group a Zn 66 Zn 66 30000 Cd 112 30000 Cd 112 20000 20000 10000 10000 0 0 45 µM 45 µM Cu 63 Cu 63 Cd-treated Cd-treated e Zn 66 Zn 66 group group 30000 Cd 112 30000 Cd 112 20000 20000 10000 10000 225 µM 225 µM Cu 63 Cu 63 Cd-treated Cd-treated Zn 66 f Zn 66 group group 30000 Cd 112 30000 Cd 112 20000 20000 0 0 0 10203040 0 10203040 Time, min Time, min 112 + 66 + Fig. 2 a-f SEC-ICP-MS electropherogram of Cd , Zn , with 45 μMCd(e), plants treated with 225 μMCd(f)]. The 64 + 112 + and Cu intensity in A. halleri extracts from roots [control signal from Cd is designated by the solid line; the signal 66 + plants (a), plants treated with 45 μMCd (b), plants treated with from Zn is designated by the dashed line, and the signal from 64 + 225 μMCd (c)], and shoots [control plants (d), plants treated Cu is designated by the dotted line Intensity, cps Intensity, cps Intensity, cps void void void 67 kDa 67 kDa 67 kDa 43 kDa 43 kDa 43 kDa 13 kDa 13 kDa 13 kDa Intensity, cps Intensity, cps Intensity, cps void void void 67 kDa 67 kDa 67 kDa 43 kDa 43 kDa 43 kDa 13 kDa 13 kDa 13 kDa Water Air Soil Pollut (2012) 223:5445–5458 5451 two clear signals for Zn-binding proteins were also strongly. We noticed that intensity of the signal from recorded in shoot extracts from of Cd-treated plants Zn-binding protein increased slightly in samples treated (Fig. 2a–f). We also recorded two signals of Cu- with the highest dose of cadmium. binding proteins in all the analyzed extracts (Fig. 2a–f). Application of PAGE-LA-ICP-MS (laser ablation in- One of the Cu-binding proteins was eluted together with ductively coupled plasma-mass spectrometry used for one of the Cd-binding protein (Fig. 2c, f). Both Cu- ablation of polyacylamide gels) enabled us to record binding proteins identified by us showed low molecular Cd-binding proteins with higher resolution than the one weight of <13 kDa. It should be noted that intensity of offered by SEC-ICP-MS (Fig. 3a, b). Figure 3a presents the signal from the bigger protein increased with the electropherogram of Cd-binding proteins in roots. In 2+ increase of cadmium content in growth medium in sam- samples treated with 45 μMCd , signal below 75 kDa ples from roots and shoots. The intensity of the signal is smeared and looks like a distribution of the metal from the smaller Cu-binding protein was smaller in Cd- through the gel. In roots of plants treated with 225 μM 2+ treated than in control plants in roots. In shoots, signal Cd , four bands at 35, 25, 20, and ∼19 kDa were intensity from the smaller molecule did not differ so recorded. In leaves from Cd-treated plants (regardless Fig. 3 SDS-PAGE gel stained with Coomassie blue (right panels) and 112 + Cd intensity for PAGE- LA-ICP-MS (left panels)of A. halleri extracts from roots (a) and from shoots (b) 5452 Water Air Soil Pollut (2012) 223:5445–5458 thedoseapplied), one band at ∼75 kDa was recorded uncertain until recently. It has been shown that A. halleri (Fig. 3b). Additionally, two bands at 50 and 20 kDa were has a potential to hyperaccumulate cadmium (Dahmani- recorded in leaf extracts from plants treated with 225 μM Muller et al. 2000;Bertetal. 2002). Recently, a growing 2+ Cd (Fig. 3b). number of experimental studies confirms the ability of We showed for the first time that Cd-binding pro- the plant to hyperaccumulate cadmium (Bert et al. 2003; teins with low molecular weight (<100 kDa) are pres- Cosio et al. 2004;Zhao et al. 2006; Verbruggen et al. ent in A. halleri and that the amount of Cd bound to 2009;Maestrietal. 2010). The majority of above- these proteins correlated with the amount of the metal mentioned studies, however, were carried out in hydro- in the growth medium. The presence of two Zn- and ponics and employed short-term cadmium exposure in two Cu-binding proteins were recorded in control and high doses. metal-exposed plants. Our results have shown that A. halleri is able to −1 accumulate cadmium up to 1,000 μgg d.w. in roots −1 andupto2,500 μgg d.w. in shoots without any visible 4 Discussion symptoms of phytotoxicity. Similar Cd content in shoots −1 (2,700 mg kg d.w.) was obtained by Küpper et al. 4.1 Experimental Conditions (2000) under laboratory conditions. Interestingly, how- ever, Küpper et al. (2000)and Bert et al.(2003) showed Our experiments were carried out employing perlite and that Cd content in shoots was lower than in roots. Similar relatively long metal treatment using cadmium doses pattern of Cd translocation has been observed in Thlaspi mimicking concentrations of this metal found in polluted caerulescens by Lombi et al. (2000) and Zhao et al. soils (Nriagu and Pacyna 1988;Nriagu 1996; Yanai et al. (2006). We suppose that difference in the pattern of Cd 2006). During pilot experiments (data not shown), we translocation might have been caused by different grow- tried to adjust cadmium dose in such a way as to avoid ing conditions. Küpper et al. (2000), Lombi et al. (2000), the emergence of visible toxic effects like chlorosis, Bert et al. (2003), and Zhao et al. (2006) carried out their necrosis, or decreased biomass production. Di Toppi experiments employing hydroponic approach, while we and Gabbrielli (1999) indicate that growth conditions have grown plants using perlite as a semi-hydroponic play crucial role in experiments focused on cadmium culture system and long-term cadmium exposure. tolerance in plants. According to them, Cd tolerance in Moreno-Jimenez et al. (2007) have summarized higher plants is a natural or artificially given capacity, results concerning different patterns of mercury trans- regulated by interactions between genetic and environ- location in plants depending on the type of substrate mental factors, to bear high levels of Cd exposure for a used during cultivation. They showed that root to long time, without appreciable detrimental effects on shoot translocation of the metal was significantly metabolism. It seems that employing short-term Cd treat- higher in plants grown in perlite when compared with ment leads to activation of more or less efficient detox- those grown under hydroponic conditions. Our data ification mechanism that is present in all higher plants (di seem to support these observations. Toppi and Gabbrielli 1999). Application of long-term Cd Also, Vazquez and Carpena-Ruiz (2005), Moreno- treatment (i.e., chronic stress) gives an opportunity to get Jimenez et al. (2007), and Sobrino-Plata et al. (2009) insights into plant response in which all the homeostatic have noticed that the type of substrate used during mechanisms of the cell are involved. In that way, we are cultivation significantly affects the uptake of heavy able to assess the real level of Cd tolerance in a given metals. The abovementioned authors paid particular species. From all the above-mentioned reasons, we de- attention to the differences between classic hydropon- cided to employ long-term (40 days) treatment with ics and perlite-based cultivation system. It seems that cadmium spanning almost the entire life cycle of inves- growing plants in almost completely inert substrate tigated plants (from a seedling to the mature organism). such as perlite mimics well the conditions for root growth found in natural soils (Olympios 1999; 4.2 Cd Accumulation Robbins and Evans 2005) including adequate humid- ity, stimulation of root growth, etc. (Moreno-Jimenez Although A. halleri is a well-known zinc hyperaccumu- et al. 2007; Sobrino-Plata et al. 2009). At the same lator, its status as a cadmium hyperaccumulator was time, access to nutrients and heavy metals is more Water Air Soil Pollut (2012) 223:5445–5458 5453 limited under perlite cultivation than in pure hydroponic 4.4 The Correlation Between Cd and Mineral Nutrient conditions (Sobrino-Plata et al. 2009). Küpper et al. Accumulation (2000), Lombi et al. (2000), Bert et al. (2003), and Zhao et al. (2006)showed that A. halleri accumulated It has been shown that the influence of cadmium on Cd in the amounts higher than 0.01 % d.w., what has the content of mineral nutrients in plants can depend been suggested as a criterion for Cd hyperaccumulation on the concentration of cadmium, growth conditions, by Brooks (1998). Our results agree with these findings. species, or ecotype being investigated or even on plant We have also shown, however, that the plant is able to organ under study (Zhang et al. 2002; Cai et al. 2010). translocate the metal from roots to shoots in such a way Interactions between cadmium and essential mineral that the ratio of the metal content in roots and in shoots is elements have been studied in potatoes (Gonçalves et higher than 1. This criterion of metal hyperaccumulation al. 2009), different ecotypes of wheat (Zhang et al. has been proposed by McGrath and Zhao (2003). 2002), several Cd-tolerant, and non-tolerant ecotypes of rice (Liu et al. 2003; Cai et al. 2010), in Betula 4.3 The Correlation Between Cd and Zn pendula—a pioneer species colonizing post-industrial Hyperaccumulation polluted areas (Gussarsson et al. 1996) as well as in cadmium hyperaccumulators: Brasica juncea (Jiang et Cd hyperaccumulation has been shown in all known al. 2004), T. caerulescens (Roosens et al. 2003), and Zn hyperaccumulating plants. This suggests that ge- Sedum alfredii (Yang et al. 2004). This wide literature netic basis of both phenomena might be, at least par- reveals, however, a very complicated and often con- tially, the same (Verbruggen et al. 2009). Our results tradictory picture of the problem. indicate negative correlation between Cd and Zn con- In the present work, we show for the first time that tent in both shoots and roots in A. halleri. The higher cadmium influences the uptake of mineral nutrients in content of cadmium was recorded; the lower was the A. halleri. Our data show that the content of cadmium 2+ 2+ 2+ 2+ content of zinc. and mineral nutrients (Mg ,Mn ,Fe ,Cu , and 2+ The same phenomenon, suggesting a competition Zn ) was significantly correlated. At the same time, between zinc and cadmium, has been also observed by the content of mineral nutrients recorded by us in Zhao et al. (2006). On the basis of an experiment investigated plants ranged within normal limits for carried out during 3 weeks under hydroponic condi- wild plants (Kabata-Pendias 2010). It seems from the tions, they demonstrated that increase in Zn content in above-mentioned reasons that correlations between the growth medium caused decrease in Cd content in the content of cadmium and mineral nutrients might plants. Investigating the effect of Zn and Cd treatment not be an effect of a simple specific competition be- on the content of these metals in plant tissues, Küpper tween these elements but rather an outcome of the mechanism involved in maintaining ion homeostasis. et al. (2000) showed that increase in Zn concentration caused the decrease of cadmium content in shoots by It can be hypothesized that the ability to tolerate and 50 %. The existence of a competition between Zn and hyperaccumulate heavy metals by A. halleri may re- Cd has been also evidenced by Cosio et al. (2004), sult from the ability of the species to maintain ion who demonstrated that increased amount of Cd in the homeostasis under cadmium stress. growth medium reduced Zn accumulation. An important difference between the authors cited 4.5 Analysis of Metal-Binding Proteins above and our experiments is that, in our study, concen- tration of Zn in the growth medium remained on constant It is estimated that about 40 % of all proteins contain level of 0.4 μM and was similar to the concentration heavy metal ion in their structure (Garcia et al. 2006). found in natural, unpolluted soils (Grimme 1968). Metal-containing proteins may be divided into two Our results seem to support the hypothesis put groups: metalloproteins and metal-binding proteins. forward by Zhao et al. (2006) that A. halleri is able The first group is characterized by strong affinity to hyperaccumulate Cd through the Zn pathway and between protein and metal ion. The second group that, in case of increased Cd content in the growth shows significantly weaker affinity between the two medium, Zn is accumulated in lower amounts due to types of molecules. This fact makes the analysis of the competition between both metals. metal binding proteins an extremely difficult task as 5454 Water Air Soil Pollut (2012) 223:5445–5458 metals can be easily lost during the procedure of macromolecules and to conduct their preliminary sample preparation. Hence, it is necessary here to identification. Our research is an important contribu- employ chemical techniques of high resolution for tion to the problem. protein separation and identification. In our research, we used SEC-ICP-MS and PAGE-LA-ICP-MS. Both 4.6 Metal-Binding Proteins methods are widely accepted as useful techniques for bioinorganic speciation analysis (Szpunar 2005; We showed for the first time that control and Cd- Garcia et al. 2006; Polatajko et al. 2007, 2011). treated plants of A. halleri differ significantly at mo- SEC-ICP-MS enabled us to identify dominant coordi- lecular level. Studies carried out hitherto were focused nation complexes of cadmium of molecular weight mainly on the isolation and identification of Cd- lower than 13 kDa in treated plants. New-generation binding proteins (e.g., Bartolf et al. 1980;Rauser columns applied in SEC-ICP-MS enable the research- 1984; Kumar and Prasad 2004; Kastenholz 2006; er to identify intact coordination complexes (Persson Fenik et al. 2007; Polatajko et al. 2007, 2011). Our et al. 2006). The multi-elemental capacity of the meth- study enabled us not only to identify these proteins but od is one of its major advantages (Persson et al. 2006; also to show that the pattern of their occurrence differ Polatajko et al. 2011). We employed this tool to elu- between control and Cd-treated plants, as well as cidate the problem whether, apart from Cd, other im- between groups treated with different doses of cadmi- portant elements (such as Zn and Cu) can also interact um. These differences were shown in plants without with proteins. In our case, the use of SEC-ICP-MS any signs of Cd toxicity. We think that this finding enabled us to identify proteins forming coordination gives a new perspective on the phenomenon of metal 112 66 complexes with the following isotopes: Cd, Zn, hyperaccumulation. Cu. SEC-ICP-MS has, however, some limitations in In Cd-treated plants, we identified Cd-binding pro- resolution when compared with other techniques teins of low molecular weight (LMW): several pro- (Persson et al. 2006). The application of size exclusion teins of <13 kDa, proteins of 20, 25, and 50 kDa, and chromatography together with inductively coupled of about 19, 35, 45 and 75 kDa in size. In control plasma mass spectrometry enabled us, however, to plants, we did not record the presence of these mole- detect and identify metal binding proteins in A. halleri. cules, which can suggest that Cd-binding proteins are The application of PAGE-LA-ICP-MS enabled us to involved in plant response to cadmium. Fenik et al. identify more cadmium-binding proteins with higher (2007) showed the presence of numerous Cd-binding molecular weight, alongside with relatively small cad- proteins in Cd-resistant lines of Nicotiana plumbagi- mium loss during experimental procedures. Cd-binding nifolia. They showed the presence of LMW Cd- proteins identified using this method ranged between 19 binding proteins of 19, 34, and 40 kDa and five and 75 kDa. It is now widely accepted that PAGE-LA- proteins of <13 kDa in size in Cd-treated plants. In ICP-MS is a powerful tool in research on metal binding cadmium-sensitive A. thaliana, the presence of high- complexes in plant material rich in proteins (Polatajko et molecular-weight Cd-binding protein of approximate- al. 2011). One important limitation of the technique is, ly 200 kDa in size has been recorded by Kastenholz however, that multi-elemental analysis is not possible on (2006). The presence of LMW Cd-binding proteins in a single-blot membrane. The method, however, allowed Cd-tolerant ecotypes treated with cadmium can be the detection of some rare Cd-proteins with resolution considered as an indirect proof of the significance of significantly better than the one offered by SEC-ICP- these molecules in plant response to cadmium stress. MS. These investigations allowed us to show that long- Our experiments showed that the amount of Cd- term cadmium exposure alters the protein composition proteins correlated with the amount of Cd present in of a plant. the growth medium. Study carried out by Kumar and Prasad (2004) showed also that there is a positive Investigation of metal-binding proteins requires a lot of effort and the use of advanced methods. Studies correlation between Cd bioaccumulation and the con- in this subject should be focused on the structure and centration of Cd-binding proteins. Hence, application biological role of these proteins. It seems to us that the of long-term Cd exposure instead of short-term, first and the most important step on this way is to “acute” treatment seems to be more appropriate to localize and quantify the content of metal-binding the authors, as it should promote Cd-protein synthesis. Water Air Soil Pollut (2012) 223:5445–5458 5455 Our results evidenced also the presence of LMW Molecular weight of Cd–metallothionein complexes (<13 kDa) Cu- and Zn-binding proteins in A. halleri.It range between 6 and 7 kDa (Sabolic et al. 2002). suggests the presence of specific proteins with very Mejare and Bulow (2001), however, put forward the low molecular weight and high affinity to divalent hypothesis that the expression of cysteine-free metal metal ions in investigated plants. It seems that two chelating proteins is less toxic for cells than the ex- 2+ 2+ metabolically important metal ions (Cu and Zn ) pression of cysteine-rich molecules. According to can form complexes with proteins similar to Cd- them, this scenario is particularly relevant for plants binding proteins, as they were characterized by almost in which synthesis of such molecules is controlled by a identical elution time in size exclusion chromatogra- constitutive promoter and it is not adjusted by the phy. On the basis of our studies, however, it is difficult metal concentration inside the cell (Mejare and to answer whether Cd, Cu, and Zn bind to the same or Bulow 2001). Moreover, Gussarsson (1994) showed different proteins. that increase in sulfur content under cadmium stress Identification of Cd-binding proteins in A. halleri was correlated with the increased content of some Cd- can be of importance in our understanding of the binding peptides containing sulfur. We showed that mechanisms of cadmium tolerance and hyperaccumu- sulfur content in treated plants was not correlated with lation in plants. It has been shown that Cd-binding Cd. Therefore we think, that molecules of the molec- proteins can contain cysteine or histidine (Mejare and ular weight < 13 kDa cannot be associated with metal- Bulow 2001). Both amino acids form complexes with lothioneins. Hence, there are premises that, in small metal ions relatively easily by binding to the thiol proteins identified by us, Cd is not bound by sulfhy- group in case of the former or to nitrogen from imid- dryl groups that are common in phytochelatins and azole functional group (that can easily form coordina- metallothioneins. It is widely known that histidine can act as a very tion bonds with metal cations) in case of the latter. 2+ 2+ 2+ It is known that cysteine-rich proteins such as metal- efficient ligand for metal ions such as Cd ,Zn ,Cu , 2+ 2+ lothioneins and phytochelatins form stable complexes Ni ,and Co (Wierzbicka et al. 2007; Verbruggen et al. with cadmium (Mejare and Bulow 2001; Fenik et al. 2009). This amino acid is also thought to be one of the 2007; Meyer et al. 2011). Hitherto, numerous studies most important compounds responsible for the chelation stressed the importance of phytochelatins (oligomers of of metal ions in hyperaccumulators (Haydon and glutathione) in plant response to heavy metals (e.g., di Cobbett 2007; Verbruggen et al. 2009). Additionally, Toppi and Gabbrielli 1999; Szpunar 2005; Memon and our previous research on the speciation of Cd in Schroder 2009; Meyer et al. 2011). It is usually accepted Allium cepa epidermal cells showed the presence of that molecular weight of Cd–phytochelatin complexes specific cadmium ligand—the histidine (Wierzbicka et range between 2.5 and 10 kDa, depending on the plant al. 2007). This can suggest that LMW proteins identified species and physiological status of investigated speci- by us and able to bind Cd could contain histidine—the mens (Persson et al. 2006). It is also widely accepted amino acid playing a key role in heavy metal detoxifi- that this mechanism of Cd detoxification plays a key cation in hyperaccumulators. This hypothesis requires, role in Cd-sensitive ecotypes (Ebbs et al. 2002). In case however, further studies and evaluation. of tolerant plants, or Cd hyperaccumulators such as A. It is worth noting that all the investigated plants halleri, the role of phytochelatins is almost negligible showed high level of Cd-tolerance and differed signif- (Ebbs et al. 2002; Fenik et al. 2007; Meyer et al. 2011). icantly in terms of the pattern of Cd-binding proteins. It seems, therefore, that amino acids in the form of This can suggest that these proteins can be involved in glutathione could be involved in the protection against cadmium detoxification as it was suggested previously oxidative stress caused by heavy metal ions. In this case, by several authors (Fenik et al. 1995; Woolhouse other molecules have to act as detoxification agents. For 1983; Meyer et al. 2011). all the above-mentioned reasons, it seems to us that Cd- binding proteins of molecular weight <13 kDa cannot be interpreted as phytochelatins. 5 Conclusions Metallothioneins are another group of chemical compounds that are thought to play role in plant resis- We showed that A. halleri is able to translocate Cd to tance to cadmium (e.g., Mejare and Bulow 2001). aerial parts in high amounts (translocation index >1) 5456 Water Air Soil Pollut (2012) 223:5445–5458 Cai, Y., Lin, L., Cheng, W., Zhang, G., & Wu, F. (2010). that confirmed its status as Cd hyperaccumulator. Our Genotypic dependent effect of exogenous glutathione on experiments showed that Cd influenced the uptake of Cd-induced changes in cadmium and mineral uptake and different mineral nutrients. Our results suggest that Cd accumulation in rice seedlings (Oryza sativa). Plant, Soil and Zn can be hyperaccumulated by A. halleri through and Environment, 56, 516–525. Cosio, C., Martinoia, E., & Keller, C. (2004). Hyperaccumula- a common pathway. The data obtained by us point out tion of cadmium and zinc in Thlaspi caerulescens and that Cd is bound by low-molecular-weight metal- Arabidopsis halleri at the leaf cellular level. Plant Physi- binding proteins of the molecular mass <100 kDa. ology, 134, 716–725. These proteins are unlikely to be phytochelatins or Dahmani-Muller, H., van Oort, F., Gelie, B., & Balabane, M. (2000). Strategies of heavy metal uptake by three plant metallothioneins. We hypothesize that these proteins species growing near a metal smelter. Environmental Pol- can play a role in broadly understood Cd detoxifica- lution, 109, 231–238. tion process in A. halleri. di Toppi, L. S., & Gabbrielli, R. (1999). Response to cadmium in higher plants. Environmental and Experimental Botany, Acknowledgments The authors would like to thank two anon- 41, 105–130. ymous reviewers for their comments that improved our manuscript Ebbs, S., Lau, I., Ahner, B., & Kochian, L. (2002). Phytochela- and to Dr. Pawel Wasowicz, for critically reading the paper. tin synthesis is not responsible for Cd tolerance in the Zn/ Cd hyperaccumulator Thlaspi caerulescenes (J. and C. Open Access This article is distributed under the terms of the Presl). Planta, 214, 635–640. Creative Commons Attribution License which permits any use, Emsley, J. (1991). Oxford chemistry guides the elements. Ox- distribution, and reproduction in any medium, provided the ford: Clarendon. original author(s) and the source are credited. Ernst, W. (1990). Mine vegetation in Europe. In J. Shaw (Ed.), Heavy metal tolerance in plants: Evolutionary aspects (pp. 21–37). Boca Raton: CRC Press. Fenik, S. I., Trofimyak, T. B., & Blyum, Y. B. (1995). Mecha- References nisms of development of plant tolerance to heavy metals. Uspekhisovremennoĭbiologii, 115, 259–275. Fenik, S. I., Solodushko, V. G., Kalinyak, T. B., & Blume, A. B. Abratowska, A., Wąsowicz, P., Bednarek, P., Telka, J., & Wierzbicka, (2007). The role of Cd-binding proteins and phytochelatins M. (2012). Morphological and genetic distinctiveness of the in the formation of cadmium resistance in Nicotiana plum- metallicolous and non-metallicolous populations of Armeria baginifolia cell lines. Cytology and Genetics, 41,9–15. maritima s. l. (Plumbaginaceae) in Poland. Plant Biology, 14, Garcia, J. S., de Magalhaes, C. S., & Arruda, M. A. Z. (2006). Trends 586–595. in metal-binding and metalloprotein analysis. Talanta, 69,1–15. Al-Shehbaz, I. A., & O’Kane, S. L., Jr. (2002). Taxonomy and Gode, C., Decombeix, I., Kostecka, A., Wąsowicz, P., Pauwles, phylogeny of Arabidopsis (Brassicaceae). The Arabidopsis M., Courseaux, A., & Saumitou-Laprade, P. (2012). Nuclear Book. doi:10.1199/tab.0009. microsatellite loci for Arabidopsis halleri (Brassicaceae), a Bartolf, M., Brenna, E., & Price, C. A. (1980). Partial charac- model species to study plant adaptation to heavy metals. terization of a cadmium-binding protein from the roots of American JournalofBotany, 99(2), e49–e52. cadmium-treated tomato. Plant Physiology, 66, 438–441. Gonçalves, J. F., Antes, F. G., Maldaner, J., Pereira, L. B., Tabaldi, Bert, V., Bonnin, I., Saumitou-Laprade, P., de Laguerie, P., & Petit, D. (2002). Do Arabidopsis halleri from nonmetallic- L. A., Rauber, R., et al. (2009). Cadmium and mineral nutrient accumulation in potato plantlets grown under cad- olous populations accumulate zinc and cadmium more mium stress in two different experimental culture conditions. effectively than those from metallicolous populations? Plant Physiology and Biochemistry, 47,814–821. New Phytologist, 155,47–57. Grimme, H. (1968). Adsorption of Mn, Co, Cu and Zn by Bert, V., Meerts, P., Saumitou-Laprade, P., Salis, P., Gruber, W., goethite from dilute solutions. Zeitschrift für Pflanzener- & Verbruggen, N. (2003). Genetic basis of Cd tolerance nährung und Bodenkunde, 121,58–65. and hyperaccumulation in Arabidopsis halleri. Plant and Soil, 249,9–18. Gussarsson, M. (1994). Cadmium-induced alterations in nutri- Branquinho, C., Serrano, H. C., Pinto, M. J., & Martins-Loucao, ent composition and growth of Betula pendula seedlings: M. A. (2007). Revisiting the plant hyperaccumulation cri- The significance of fine root as a primary target for cadmi- teria to rare plants and earth abundant elements. Environ- um toxicity. Journal of Plant Nutrition, 17, 2151–2163. mental Pollution, 146, 437–443. Gussarsson, M., Asp, H., Adalsteinsson, S., & Jensén, P. (1996). Braude, G. L., Nash, A. M., Wolf, W. J., Carr, R. L., & Chaney, Enhancement of cadmium effects on growth and nutrient R. L. (1980). Cadmium and lead content of soybean prod- composition of birch (Betula pendula) by buthioninesulphox- ucts. Journal of Food Science, 45, 1187–1199. imine (BSO). Journal of Experimental Botany, 47,211–215. Broadley, M. R., White, P. J., Hammond, J. P., Zelko, I., & Lux, Haydon, M. J., & Cobbett, C. S. (2007). Transporters of ligands for A. (2007). Zinc in plants. New Phytologist, 173, 677–702. essential metal ions in plants. New Phytologist, 174, 499–506. Brooks, R. R. (1998). Plants that hyperaccumulate heavy met- Jiang, X. J., Luo, Y. M., Liu, Q., Liu, S. L., & Zhao, Q. G. als: their role in phytoremediation, microbiology, archae- (2004). Effects of cadmium on nutrient uptake and trans- ology, mineral exploration, and phytomining. Oxford- New location by Indian mustard. Environmental Geochemistry York: CAB International. and Health, 26, 319–324. Water Air Soil Pollut (2012) 223:5445–5458 5457 Kabata-Pendias, A. (2010). Trace elements in soils and plants. Nriagu, J. O. (1996). A history of global metal pollution. Sci- Boca Raton: CRC Press. ence, 272, 223–224. Kastenholz, B. (2006). Comparison of the electrochemical be- Nriagu, J. O., & Pacyna, J. M. (1988). Quantitative assessment havior of the high molecular weight cadmium proteins in of worldwide contamination of air, water and soils by Arabidopsis thaliana and in vegetable plants on using trace-metals. Nature, 333, 134–139. preparative native continuous polyacrylamide gel electro- Olympios, C. M. (1999). Overview of soilless culture: Advantages, phoresis (PNC-PAGE). Electroanalysis, 18, 103–106. constraints and perspectives for its use in Mediterranean Kumar, G. P., & Prasad, M. N. V. (2004). Cadmium-inducible countries. Cahiers Options Méditerranéennes, 31, 307–324. proteins in Ceratophyllum demersum L. (a fresh water Pauwels, M., Frerot, H., Bonnin, I., & Saumitou-Laprade, P. macrophyte): Toxicity bioassays and relevance to cadmium (2006). A broad-scale analysis of population differentiation detoxification. Bulletin of Environmental Contamination for Zn tolerance in an emerging model species for tolerance and Toxicology, 73, 174–181. study: Arabidopsis halleri (Brassicaceae). Journal of Evo- Küpper, H., Lombi, E., Zhao, F. J., & McGrath, S. P. (2000). lutionary Biology, 19, 1838–1850. Cellular compartmentation of cadmium and zinc in relation Persson, D., Hansen, T. H., Holm, P. E., Schjørring, J. K., to other elements in the hyperaccumulator Arabidopsis Cakmak, I., & Husted, S. (2006). Multi-elemental specia- halleri. Planta, 212,75–84. tion analysis of barley genotypes differing in tolerance to Lasat, M. M., & Kochian, L. V. (1998). Physiological basis for Cd toxicity using LC-ICP-MS and ESI-TOF-MS. Journal Zn hyperaccumulation in Thlaspicaerulescens.InH.E. of Analytical Atomic Spectrometry, 21, 996–1005. Flores, J. P. Lynch, & D. Eissenstat (Eds.), Radical biolo- Polatajko, A., Azzolini, M., Feldmann, I., Stuezel, T., & Jakubowski, gy: Advances and perspectives on the function of plant N. (2007). Laser ablation-ICP-MS assay development for roots (pp. 139–149). Rockville: American Society of Plant detecting Cd- and Zn-binding proteins in Cd-exposed Spina- Physiologists. cia oleracea L. Journal of Analytical Atomic Spectrometry, Lasat, M. M., & Kochian, L. V. (2000). Physiology of Zn hyper- 22,878–887. accumulation in Thlaspi caerulescens. In N. Terry & G. Polatajko, A., Feldmann, I., Hayen, H., & Jakubowski, N. Bañuelos (Eds.), Phytoremediation of contaminated soil (2011). Combined application of a laser ablation-ICP-MS and water (pp. 159–169). Boca Raton: Lewis Publishers. assay for screening and ESI-FTICR-MS for identification Liu, J. G., Liang, J. S., Li, K. Q., Zhang, Z. J., Yu, B. Y., Lu, X. of a Cd-binding protein in Spinacia oleracea L. after L., et al. (2003). Correlations between cadmium and min- exposure to Cd. Metallomics, 3, 1001–1008. eral nutrients in absorption and accumulation in various Przedpelska, E., & Wierzbicka, M. (2007). Arabidopsis arenosa genotypes of rice under cadmium stress. Chemosphere, 52, (Brassicaceae) from a lead-zinc waste heap in southern 1467–1473. Poland—A plant with high tolerance to heavy metals. Lombi, E., Zhao, F. J., Dunham, S. J., & McGrath, S. P. (2000). Plant and Soil, 299,43–53. Cadmium accumulation in populations of Thlaspi caerules- Rauser, W. E. (1984). Isolation and partial purification of cens and Thlaspi goesingense. New Phytologist, 145,11–20. cadmium-binding protein from roots of the grass Agrostis Maestri, E., Marmiroli, M., Visioli, G., & Marmiroli, N. (2010). gigantea. Plant Physiology, 74, 1025–1029. Metal tolerance and hyperaccumulation: Costs and trade- Robbins, J. A., & Evans, M. R. (2005). Growing media for con- offs between traits and environment. Environmental and tainer production in a greenhouse or nursery: Part I (compo- Experimental Botany, 68,1–13. nents and mixes). Fayetteville: University of Arkansas. McGrath, S. P., & Zhao, F. J. (2003). Phytoextraction of metals Roosens, N., Verbruggen, N., Meerts, P., Ximénez-Embun, P., & and metalloids from contaminated soils. Current Opinion Smith, J. A. C. (2003). Natural variation in cadmium in Biotechnology, 14, 277–282. tolerance and its relationship to metal hyperaccumulation Mejare, M., & Bulow, L. (2001). Metal-binding proteins and for seven populations of Thlaspi caerulescens from west- peptides in bioremediation and phytoremediation of heavy ern Europe. Plant, Cell & Environment, 26, 1657–1672. metals. Trends in Biotechnology, 19,67–73. Sabolic, I., Ljubojevic, M., Herak, K. C. M., & Brow, D. (2002). Memon, A. R., & Schroder, P. (2009). Implications of metal Cd-MT cause endocytosis of brush-border transporters in accumulation mechanisms to phytoremediation. Environ- rat proximal tubules. American Journal of Physiology. mental Science and Pollution Research, 16, 162–175. Renal Physiology, 283, 1389–1402. Meyer, C. L., Kostecka, A. A., Saumitou-Laprade, P., Creach, A., Sobrino-Plata, J., Ortega-Villasante, C., Flores-Caceres, M. L., Castric, V., Pauwels, M., & Frerot, H. (2010). Variability of Escobar, C., Del Campo, F. F., & Hernandez, L. E. (2009). zinc tolerance among and within populations of the pseudo- Differential alterations of antioxidant defenses as bioindi- metallophyte species Arabidopsis halleri and possible role of cators of mercury and cadmium toxicity in alfalfa. Chemo- directional selection. New Phytologist, 185,130–142. sphere, 77, 946–954. Meyer, C. L., Peisker, D., Courbot, M., Craciun, A. R., Cazale, Strebeyko, P. (1967). Introduction to plant physiology. Warsaw: A. C., Desgain, D., et al. (2011). Isolation and character- PWRiL. ization of Arabidopsis halleri and Thlaspi caerulescens Szpunar, J. (2005). Advances in analytical methodology for phytochelatin synthases. Planta, 234(1), 83–95. bioinorganic speciation analysis: Metallomics, metallopro- Moreno-Jimenez, E., Penalosa, J. M., Esteban, E., & Carpena- teomics and heteroatom-tagged proteomics and metabolo- Ruiz, R. O. (2007). Mercury accumulation and resistance mics. Analyst, 130, 442–465. to mercury stress in Rumex induratus and Marrubium Vazquez, S., & Carpena-Ruiz, R. O. (2005). Use of perlite in vulgare grown in perlite. Journal of Plant Nutrition and cadmium plant studies: An approach to polluted soil condi- Soil Science, 170, 485–494. tions. Journal of Environmental Monitoring, 7,1355–1358. 5458 Water Air Soil Pollut (2012) 223:5445–5458 Verbruggen, N., Hermans, C., & Schat, H. (2009). Molecular Thlaspi caerulescens. Environmental Pollution, 139,167– mechanisms of metal hyperaccumulation in plants. New 175. Phytologist, 182, 781–781. Yang, X. E., Long, X. X., Ye, H. B., He, Z. L., Stoffella, P. J., & Wierzbicka, M. H., Przedpełska, E., Ruzik, R., Ouerdane, L., Calvert, D. V. (2004). Cadmium tolerance and hyperaccu- Połeć-Pawlak, K., Jarosz, M., Szpunar, J., & Szakiel, A. mulation in a new Zn-hyperaccumulating plant species (2007). Comparison of the toxicity and distribution of cad- (Sedum alfredii Hance). Plant and Soil, 259, 181–189. mium and lead in plant cells. Protoplasma, 231,99–111. Zhang, G. P., Fukami, M., & Sekimoto, H. (2002). Influence of Woolhouse, H. W. (1983). Toxicity and tolerance in the cadmium on mineral concentrations and yield components responses of plants to metals. In O. L. Lange, P. S. Nobel, in wheat genotypes differing in Cd tolerance at seedling C. B. Osmond, & H. Ziegler (Eds.), Encyclopedia of plant stage. Field Crops Research, 77,93–99. physiology: Physiological plant ecology (pp. 245–310). Zhao, F. J., Jiang, R. F., Dunham, S. J., & McGrath, S. P. (2006). Berlin: Springer. Cadmium uptake, translocation and tolerance in the hyper- Yanai, J., Zhao, F. J., McGrath, S. P., & Kosaki, T. (2006). Effect accumulator Arabidopsis halleri. New Phytologist, 172, of soil characteristics on Cd uptake by the hyperaccumulator 646–654. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Water, Air, and Soil Pollution Pubmed Central

The Influence of Cadmium Stress on the Content of Mineral Nutrients and Metal-Binding Proteins in Arabidopsis halleri

Przedpełska-Wąsowicz, Ewa; Polatajko, Aleksandra; Wierzbicka, Małgorzata
Water, Air, and Soil Pollution , Volume 223 (8) – Aug 23, 2012
Download PDF
14 pages

Loading next page...
 
/lp/pubmed-central/the-influence-of-cadmium-stress-on-the-content-of-mineral-nutrients-OU9wAj1HrH

References (74)

Publisher
Pubmed Central
Copyright
© The Author(s) 2012
ISSN
0049-6979
eISSN
1573-2932
DOI
10.1007/s11270-012-1292-4
Publisher site
See Article on Publisher Site

Abstract

Water Air Soil Pollut (2012) 223:5445–5458 DOI 10.1007/s11270-012-1292-4 The Influence of Cadmium Stress on the Content of Mineral Nutrients and Metal-Binding Proteins in Arabidopsis halleri Ewa Przedpełska-Wąsowicz & Aleksandra Polatajko & Małgorzata Wierzbicka Received: 10 March 2012 /Accepted: 25 July 2012 /Published online: 23 August 2012 The Author(s) 2012. This article is published with open access at Springerlink.com Abstract We investigated the influence of cadmium content and mineral nutrients were evidenced by our stress on zinc hyperaccumulation, mineral nutrient study. We identified more than ten low-molecular- uptake, and the content of metal-binding proteins in weight (<100 kDa) Cd-binding proteins in Cd-treated Arabidopsis halleri. The experiments were carried out plants. These proteins are unlikely to be phytochelatins using plants subjected to long-term cadmium exposure or metallothioneins. We hypothesize that low- (40 days) in the concentrations of 45 and 225 μM molecular-weight Cd-binding proteins can be involved 2+ Cd . Inductively coupled plasma-mass spectrometry, in cadmium resistance in A. halleri. size exclusion chromatography coupled with plasma- . . mass spectrometry, and laser ablation inductively cou- Keywords Arabidopsis halleri Cadmium . . pled plasma-mass spectrometry used for ablation of Cadmium-binding proteins Hyperaccumulator polyacylamide gels were employed to assess the con- Metal accumulation Zinc tent of investigated elements in plants as well as to identify metal-binding proteins. We found that A. hal- Abbreviations leri is able to translocate cadmium to the aerial parts in ICP-MS Inductively coupled plasma-mass high amounts (translocation index >1). We showed spectrometry that Zn content in plants decreased significantly with LA-ICP-MS Laser ablation inductively coupled the increase of cadmium content in the growth medium. plasma-mass spectrometry Different positive and negative correlations between Cd LMW Low molecular weight PAGE Polyacrylamide gel electrophoresis PVP Polyvinylpyrrolidone Electronic supplementary material The online version of this SEC-ICP-MS Size exclusion chromatography article (doi:10.1007/s11270-012-1292-4) contains coupled with plasma-mass supplementary material, which is available to authorized users. spectrometry E. Przedpełska-Wąsowicz (*) M. Wierzbicka Department of Molecular Plant Physiology, Institute of Botany, Faculty of Biology, University of Warsaw, 1 Introduction Miecznikowa 1, 02-096 Warsaw, Poland e-mail: [email protected] Arabidopsis halleri (Brassicaceae) is a perennial spe- cies occurring in Europe and East Asia (Al-Shehbaz A. Polatajko and O’Kane 2002). Apart from natural mountainous ISAS-Institute for Analytical Sciences, P.O. Box 101352, 44013 Dortmund, Germany habitats, it occurs also in areas polluted with heavy 5446 Water Air Soil Pollut (2012) 223:5445–5458 metals (Ernst 1990; Pauwels et al. 2006). The species histidine, nicotiamine), organic acids such as: malate is well known for its’ tolerance to zinc and cadmium and and citric acid, phytochelatins, and metallothioneins the ability to hyperaccumulate these metals. A. halleri,a (Szpunar 2005; Fenik et al. 2007; Polatajko et al. close wild relative of Arabidopsis thaliana,isamodel 2007; Maestri et al. 2010). Braude et al. (1980) men- species in studies focused on the problem of metal toler- tioned that cadmium shows a tendency to accumulate ance and hyperaccumulation in plants (Pauwels et al. in a protein fraction within the plant cell. This is 2006; Verbruggen et al. 2009;Maestrietal. 2010; particularly interesting, as most of the proteins regu- Meyer et al. 2010, 2011; Gode et al. 2012). lating metal homeostasis of the plant cell, are also Many studies have investigated zinc hyperaccumula- involved in detoxification, regulation of the cell cycle, tion in plants (Lasat and Kochian 1998, 2000;Pauwels proliferation, and apoptosis (Garcia et al. 2006). To et al. 2006; Broadley et al. 2007; Verbruggen et al. 2009; understand thoroughly plant response to environmen- Maestri et al. 2010; Meyer et al. 2010). In case of tal stress caused by metal ions, it is necessary to cadmium hyperaccumulation, however, the problem localize, identify, and quantify metal-containing mac- seems to be less studied. Hitherto, only four species romolecules. This task is particularly challenging from are known to hyperaccumulate cadmium. Since these the analytical point of view. Hyphenated techniques plants are also zinc hyperaccumulators, it suggests a for biological systems such as size exclusion chroma- common genetic basis of both phenomena tography coupled with plasma-mass spectrometry (Verbruggen et al. 2009). Nonetheless, little is known (SEC-ICP-MS) or laser ablation inductively coupled regarding correlation between hyperaccumulation of plasma-mass spectrometry (LA-ICP-MS) can be zinc and cadmium. regarded as a solution of this problem. Recently, this Zinc and cadmium belong to the most intensively technique was used for direct ablation of polyacryl- studied metals in terms of their impact on plants. Zinc amide gels (PAGE) (Szpunar 2005; Fenik et al. 2007; is a micronutrient essential to plant growth; however, Polatajko et al. 2007). its excess can cause toxic effects. Cadmium is one the We employed these techniques in order to investi- most frequent and the most dangerous inorganic pol- gate the influence of the Cd stress on mineral nutrient lutants. Although both metals share similar chemical uptake and metal-binding proteins content in A. properties (they have similar atomic radius, similar halleri. oxidation state in chemical compounds, and share We tested the following hypotheses: similar geochemical properties), cadmium shows higher tendency to bond with sulfur and higher mo- 1. A. halleri is a Cd hyperaccumulator. 2. There is a correlation between Cd uptake and the bility in soils and in whole ecosystems (Emsley 1991; Kabata-Pendias 2010). It has been hypothesized that content of mineral nutrients. both metals can share similar pathway while entering 3. Cd ions are bound by low-molecular-weight the plant organism (Zhao et al. 2006). In contrast to (<100 kDa) metal-binding proteins. zinc, which is essential for plant growth, cadmium is not found in any natural chemical compound in living organisms. Cadmium is widely studied in the context 2 Materials and Methods of environmental pollution and its’ impact on human health (di Toppi and Gabbrielli 1999). Although the 2.1 Plant Material metal is toxic to plants, it is known for its easy uptake (Kabata-Pendias 2010). Strong affinity to sulfhydryl Experiments were carried out using A. halleri plants. groups is one of the most important biochemical char- Seeds were obtained from the Pb/Zn mining area in acteristics of cadmium. The metal, however, can also Boleslaw near Olkusz (S Poland), a region highly pol- easily bind to functional groups containing nitrogen or luted with heavy metals (Przedpelska and Wierzbicka oxygen (Polatajko et al. 2007). 2007;Abratowskaet al. 2012). Plants were cultivated Research on metal-chelating compounds in organ- for 40 days in growth chamber under controlled tem- isms responsible for the metal homeostasis of the cell perature conditions (24±4 °C), relative humidity of 65± showed that different chemical compounds can be 4 %, light intensity of ∼120 μmol/m /s, and photoperiod involved in this process including amino acids (e.g., of 8:16 h. Water Air Soil Pollut (2012) 223:5445–5458 5447 −1 2.2 Plant Cultivation and Metal Exposure (10 ng mL ) as an internal standard. The double focus- ing sector field ICP-MS (ELEMENT 2, ThermoFisher Seeds collected from the field were germinated in Petri Scientific, Bremen, Germany) coupled to the Cetac dishes on filter paper moistened with diluted Knop autosampler (ASX-500, Omaha, Nebraska, USA) was nutrient solution (1:8 dilution) supplemented with A- used for multi-element analysis (Cd, Zn, Mg, S, Mn, Fe, Z mixture of trace elements (Strebeyko 1967), in the Cu, Mo). Translocation index was calculated as de- light at the temperature of 24 °C±1 °C. About scribed by Branquinho et al. (2007). Statistical signifi- 7 days old, seedlings were transferred into pot boxes cance of the observed differences between the control containing perlite as a substrate. The perlite was and experimental groups was tested by Kruskal–Wallis washed daily with Knop’s nutrient solution of the test using Statistica 9.1. Shape of the distribution within following chemical composition—200 g/l Ca analyzed groups and the variance were checked prior to (NO ) ×4H O, 71.5 g/l KNO , 35.5 g/l KCl, 71.5 g/ the analysis in order to ensure that the assumption of the 3 2 2 3 l MgSO , 71.5 g/l KH PO , 28 g/l EDTA-Fe, and trace homoscedasticity of the data is not broken. To test for 4 2 4 2+ elements (including Zn at the concentration of correlations between cadmium and other analyzed ele- 0.4 μM), pH06. ments, we used coefficient of determination (R ). The plants were divided into control group and two Regression lines and 95 % confidence intervals were experimental groups (30 plants each). Experimental calculated. Significance tests were carried out at 5 % groups were treated every second day with cadmium significance level. All the statistical analyses were per- added to the nutrient solution as Cd(NO ) ×4 H Oto formed using Statistica 9.1 (Statsoft Inc.) 3 2 2 2+ achieve the concentrations of 45 and 225 μMCd . Cd concentrations employed were chosen on the basis 2.4 Protein Extraction of pilot experiments (data not shown). We intended to use Cd doses that would enable plants to accumulate Proteins were extracted from shoots and roots of the maximal amount of Cd in their tissues without visible control and Cd-treated plants following the sample prep- effects of toxicity. Forty-day-old plants were har- aration protocol—5gofleavesand3gofroots were vested, divided into shoot and root portions, and then homogenized with 6 mL of 50 mM HEPES-NaOH washed several times in distilled water. By the shoot (pH 7.6) containing 2 % PVP and protease inhibitor portions, all the aerial portions of plant including stem cocktail (Complete, EDTA-free) using a ultrasonic probe and leaves are meant. To ensure complete removal of homogenization technique (Branson, SONIFIER, all the components of the growth medium (including Schwäbisch Gmünd, Germany). The homogenization 2+ Cd ), roots were washed for 5 min in 20 mM EDTA. method involved 5×30-s treatments until a well- Subsequently, samples were immediately frozen in homogenized extract was obtained. In order to obtain liquid nitrogen and ground using a pestle and mortar. cytosol free from organelle samples were centrifuged at To avoid protein unfolding and a loss of metals from 105,000g for1 hat4°C usinga SorvallDiscovery 90SE their native structures, samples were placed on dry ice ultracentrifuge (ThermoFisher, Dreieich, Germany). The after grounding. A part of the sampled material was supernatant was then centrifuged for 30 min at 4,000 rpm used in protein extraction, and the remaining portion using a Heraeus SEPATECH centrifuge (ThermoFisher, was lyophilized. Dreieich, Germany). The aliquots were stored at −80 °C for further investigations. The method employed has been 2.3 Quantification of Cd, Zn, and Mineral Nutrients optimized by Polatajko et al. (2011). For quantification of the total metal content, 0.2 g of lyophilized leaf and 0.1 g of root samples were digested 2.5 SEC-ICP-MS Analysis of Leaves and Roots Extracts with 6 mL of a HNO –H O mixture (5:1) using a 3 2 microwave-assisted digestion with closed vessels Cadmium–protein complexes from shoots and roots (Anton Paar, Graz, Austria). Triplicate extracts from all extracts were probed by size exclusion chromatography the samples were taken. Digested samples were diluted (SEC, Dionex, Germering, Germany) coupled to ICP- with Milli-Q water to 50 mL and analyzed by ICP-MS MS (ELEMENT 2, Thermo Fisher Scientific, Bremen, using an external calibration curve with Rh Germany). SEC-purified fraction was also used for 5448 Water Air Soil Pollut (2012) 223:5445–5458 identification of Zn- and Cu-proteins. The method in leaves as well as in roots (Fig. 1a). In plants treated 2+ employed has been optimized by Polatajko et al. (2011). with lower dose of cadmium (45 μMCd ), concentra- −1 tion of the metal in roots was 265 μgg d.w., whereas in 2+ 2.6 PAGE-LA-ICP-MS Analysis of Leaves and Roots plants treated with the higher dose (225 μMCd ), it −1 Extracts was 835 μgg d.w. We found that in treated plants −1 cadmium concentration in shoots was 940 μgg d.w. −1 The separation of cadmium-containing proteins was and 2,525 μgg d.w, respectively, for lower and higher done by using anodal native 10 % polyacrylamide gel dose. Total concentration of the metal in treated plants electrophoresis (1D AN-PAGE) with a discontinuous was always higher in shoots than in roots. Tris/Tricine system according to the manufacturer’s The translocation index, calculated by dividing the instructions. A Nd:YAG laser (Minilight II, concentration of the metal in shoots by the concentra- Continuum, Santa Clara, USA) coupled to the sector tion of the metal in roots, varied narrowly between 3 filed ICP-MS was used for laser ablation (LA) of the and 3.5 for both tested cadmium doses. Cd-binding proteins on the membrane. For a detailed Experiments showed that that zinc content in cadmium- description of the background of the chemical analy- treated plants was significantly lower (Fig. 1b). While in −1 ses, the reader is referred to Polatajko et al. (2007). the control group zinc content was 175 μgg d.w., in treated plants, it decreased by 37 % and 70 % in case of 2+ 2+ lower (45 μMCd ) and higher (225 μMCd )cadmi- 3 Results um dose, respectively. In roots, zinc content followed the same pattern being the highest in control plants −1 3.1 The Influence of Cadmium on Biomass Production (125 μgg d.w.) and decreasing by 58 % and 71 % in both cadmium-treated groups. The Zn root–shoot trans- Cd-treated plants showed no signs that could be asso- location index calculated for each experimental group ciated with the toxic effect of Cd ions. Leaves showed was 1.2 and 2.1, respectively. no signs of chlorosis or necrosis; tissues were well The values of the translocation indices showed hydrated (no signs of decreased turgidity). No signs of hyperaccumulation of both tested metals (Cd and Zn) growth inhibition or differences in development were by A. halleri. There was no significant difference in recorded between control and treated plants. Mean dry accumulation between investigated isotopes: Cd vs. 114 66 68 weight of shoots was 2.2, 1.8, and 1.65 g for the Cd and Zn vs. Zn. control and two experimental groups, respectively. Mean dry weight of roots was 0.3, 0.3, and 0.27 g, 3.3 The Relationships Between Cd and Mineral respectively. Nevertheless, there was no significant Nutrient Concentrations in Shoots and Roots difference in biomass production between control and treated plants, regardless of the concentration of Statistical analysis showed the presence of significant Cd in the growth medium (Kruskal–Wallis test, p0 correlations between the concentration of cadmium 0.1955 for shoot samples, p00.3594 for root samples). and other macro- (Mg, S) and microelements (Mn, The above-mentioned observations show that A. hal- Fe, Cu, Zn, Mo). Correlation analysis uncovered that leri is tolerant to tested cadmium doses. The fact that no significant and strong negative correlation exists be- signs of toxicity were observed while A. halleri accu- tween concentrations of zinc and cadmium (R 0 mulated Cd in its tissues in high amounts (particularly in −0.8487, p<0.05inroots; R 0−0.9922, p<0.05 in shoots, see below) additionally confirms this notion. shoots). Magnesium showed significant correlation with cadmium concentration in both roots and shoots, 3.2 Cd and Zn Accumulation however, in roots, the coefficient of determination showed negative (R 0−0.9125, p<0.05), whereas in Results obtained for quantitative multi-element analysis shoots positive (R 00.8765, p<0.05) value. No other relative to the dry mass are presented in Fig. 1a, b. investigated element showed significant correlation Results showed that cadmium concentration in experi- with cadmium concentration in roots at the signifi- mental plants (40 days of treatment) increased with cance level of 5 %. In shoots, significant correlations increasing concentration of the metal in growth medium were also found between cadmium and manganese Water Air Soil Pollut (2012) 223:5445–5458 5449 Fig. 1 Total concentration Cd 112 of Cd (a) and Zn (b)in A. 2500 Cd 114 halleri extracts from shoots and roots, respectively. Metal concentration was quantified using ICP-MS Control 45 µM 225 µM Control 45 µM 225 µM group Cd-treated Cd-treated group Cd-treated Cd-treated group group group group Shoots Roots b Zn 66 Zn 68 Control 45 µM 225 µM Control 45 µM 225 µM group Cd-treated Cd-treated group Cd-treated Cd-treated group group group group Shoots Roots 2 2 (R 00.9593, p<0.05), cadmium and iron (R 0 3.4 Cd-Binding Proteins −0.8046, p<0.05), as well as between cadmium and copper (R 0−0.7307, p<0.05). There was no Using ICP-MS, we measured the content of Cd- significant correlation between cadmium and sulfur binding proteins in shoots and roots. We found that, 2 2 (R 00.2593, p>0.05inroots, R 00.1064, p>0.05 in plants treated with 45 μM Cd, the content of Cd- in shoots). Regression lines along with coefficients binding proteins was about 8.6 and 9.6 μg/g f.w. in of determination (R ) and results of significance shoots and roots, respectively. In plants treated with testing for each analyzedelement canbefound 225 μM Cd, these values were about 52.5 and in electronic supplementary material (Fig. ESM 1, 33.5 μg/g f.w., respectively. We did not record the Fig. ESM 2). presence of Cd-binding proteins in the control group. Cd concentration µg/g d.w. Zn concentration µg/g d.w. 5450 Water Air Soil Pollut (2012) 223:5445–5458 2+ The control electropherograms (Fig. 2a, d) showed Cd . The same methodology enabled us to detect two the lack of Cd-binding proteins in both shoots and signals and one broad peak showing the presence of roots. Application of SEC-ICP-MS enabled us to de- Cd-binding proteins in roots and shoots obtained from tect one signal for Cd-binding protein in roots plants treated with 225 μM solution of cadmium (Fig. 2b) as well as in shoots (Fig. 2e), during the (Fig. 2c, f). All the identified Cd-proteins had small examination of the plant material treated with 45 μM molecular weight of <13 kDa. Apart from Cd-proteins, High molecular Low molecular High molecular Low molecular weight fractions weight fractions weight fractions weight fractions Control Control Cu 63 Cu 63 group group a Zn 66 Zn 66 30000 Cd 112 30000 Cd 112 20000 20000 10000 10000 0 0 45 µM 45 µM Cu 63 Cu 63 Cd-treated Cd-treated e Zn 66 Zn 66 group group 30000 Cd 112 30000 Cd 112 20000 20000 10000 10000 225 µM 225 µM Cu 63 Cu 63 Cd-treated Cd-treated Zn 66 f Zn 66 group group 30000 Cd 112 30000 Cd 112 20000 20000 0 0 0 10203040 0 10203040 Time, min Time, min 112 + 66 + Fig. 2 a-f SEC-ICP-MS electropherogram of Cd , Zn , with 45 μMCd(e), plants treated with 225 μMCd(f)]. The 64 + 112 + and Cu intensity in A. halleri extracts from roots [control signal from Cd is designated by the solid line; the signal 66 + plants (a), plants treated with 45 μMCd (b), plants treated with from Zn is designated by the dashed line, and the signal from 64 + 225 μMCd (c)], and shoots [control plants (d), plants treated Cu is designated by the dotted line Intensity, cps Intensity, cps Intensity, cps void void void 67 kDa 67 kDa 67 kDa 43 kDa 43 kDa 43 kDa 13 kDa 13 kDa 13 kDa Intensity, cps Intensity, cps Intensity, cps void void void 67 kDa 67 kDa 67 kDa 43 kDa 43 kDa 43 kDa 13 kDa 13 kDa 13 kDa Water Air Soil Pollut (2012) 223:5445–5458 5451 two clear signals for Zn-binding proteins were also strongly. We noticed that intensity of the signal from recorded in shoot extracts from of Cd-treated plants Zn-binding protein increased slightly in samples treated (Fig. 2a–f). We also recorded two signals of Cu- with the highest dose of cadmium. binding proteins in all the analyzed extracts (Fig. 2a–f). Application of PAGE-LA-ICP-MS (laser ablation in- One of the Cu-binding proteins was eluted together with ductively coupled plasma-mass spectrometry used for one of the Cd-binding protein (Fig. 2c, f). Both Cu- ablation of polyacylamide gels) enabled us to record binding proteins identified by us showed low molecular Cd-binding proteins with higher resolution than the one weight of <13 kDa. It should be noted that intensity of offered by SEC-ICP-MS (Fig. 3a, b). Figure 3a presents the signal from the bigger protein increased with the electropherogram of Cd-binding proteins in roots. In 2+ increase of cadmium content in growth medium in sam- samples treated with 45 μMCd , signal below 75 kDa ples from roots and shoots. The intensity of the signal is smeared and looks like a distribution of the metal from the smaller Cu-binding protein was smaller in Cd- through the gel. In roots of plants treated with 225 μM 2+ treated than in control plants in roots. In shoots, signal Cd , four bands at 35, 25, 20, and ∼19 kDa were intensity from the smaller molecule did not differ so recorded. In leaves from Cd-treated plants (regardless Fig. 3 SDS-PAGE gel stained with Coomassie blue (right panels) and 112 + Cd intensity for PAGE- LA-ICP-MS (left panels)of A. halleri extracts from roots (a) and from shoots (b) 5452 Water Air Soil Pollut (2012) 223:5445–5458 thedoseapplied), one band at ∼75 kDa was recorded uncertain until recently. It has been shown that A. halleri (Fig. 3b). Additionally, two bands at 50 and 20 kDa were has a potential to hyperaccumulate cadmium (Dahmani- recorded in leaf extracts from plants treated with 225 μM Muller et al. 2000;Bertetal. 2002). Recently, a growing 2+ Cd (Fig. 3b). number of experimental studies confirms the ability of We showed for the first time that Cd-binding pro- the plant to hyperaccumulate cadmium (Bert et al. 2003; teins with low molecular weight (<100 kDa) are pres- Cosio et al. 2004;Zhao et al. 2006; Verbruggen et al. ent in A. halleri and that the amount of Cd bound to 2009;Maestrietal. 2010). The majority of above- these proteins correlated with the amount of the metal mentioned studies, however, were carried out in hydro- in the growth medium. The presence of two Zn- and ponics and employed short-term cadmium exposure in two Cu-binding proteins were recorded in control and high doses. metal-exposed plants. Our results have shown that A. halleri is able to −1 accumulate cadmium up to 1,000 μgg d.w. in roots −1 andupto2,500 μgg d.w. in shoots without any visible 4 Discussion symptoms of phytotoxicity. Similar Cd content in shoots −1 (2,700 mg kg d.w.) was obtained by Küpper et al. 4.1 Experimental Conditions (2000) under laboratory conditions. Interestingly, how- ever, Küpper et al. (2000)and Bert et al.(2003) showed Our experiments were carried out employing perlite and that Cd content in shoots was lower than in roots. Similar relatively long metal treatment using cadmium doses pattern of Cd translocation has been observed in Thlaspi mimicking concentrations of this metal found in polluted caerulescens by Lombi et al. (2000) and Zhao et al. soils (Nriagu and Pacyna 1988;Nriagu 1996; Yanai et al. (2006). We suppose that difference in the pattern of Cd 2006). During pilot experiments (data not shown), we translocation might have been caused by different grow- tried to adjust cadmium dose in such a way as to avoid ing conditions. Küpper et al. (2000), Lombi et al. (2000), the emergence of visible toxic effects like chlorosis, Bert et al. (2003), and Zhao et al. (2006) carried out their necrosis, or decreased biomass production. Di Toppi experiments employing hydroponic approach, while we and Gabbrielli (1999) indicate that growth conditions have grown plants using perlite as a semi-hydroponic play crucial role in experiments focused on cadmium culture system and long-term cadmium exposure. tolerance in plants. According to them, Cd tolerance in Moreno-Jimenez et al. (2007) have summarized higher plants is a natural or artificially given capacity, results concerning different patterns of mercury trans- regulated by interactions between genetic and environ- location in plants depending on the type of substrate mental factors, to bear high levels of Cd exposure for a used during cultivation. They showed that root to long time, without appreciable detrimental effects on shoot translocation of the metal was significantly metabolism. It seems that employing short-term Cd treat- higher in plants grown in perlite when compared with ment leads to activation of more or less efficient detox- those grown under hydroponic conditions. Our data ification mechanism that is present in all higher plants (di seem to support these observations. Toppi and Gabbrielli 1999). Application of long-term Cd Also, Vazquez and Carpena-Ruiz (2005), Moreno- treatment (i.e., chronic stress) gives an opportunity to get Jimenez et al. (2007), and Sobrino-Plata et al. (2009) insights into plant response in which all the homeostatic have noticed that the type of substrate used during mechanisms of the cell are involved. In that way, we are cultivation significantly affects the uptake of heavy able to assess the real level of Cd tolerance in a given metals. The abovementioned authors paid particular species. From all the above-mentioned reasons, we de- attention to the differences between classic hydropon- cided to employ long-term (40 days) treatment with ics and perlite-based cultivation system. It seems that cadmium spanning almost the entire life cycle of inves- growing plants in almost completely inert substrate tigated plants (from a seedling to the mature organism). such as perlite mimics well the conditions for root growth found in natural soils (Olympios 1999; 4.2 Cd Accumulation Robbins and Evans 2005) including adequate humid- ity, stimulation of root growth, etc. (Moreno-Jimenez Although A. halleri is a well-known zinc hyperaccumu- et al. 2007; Sobrino-Plata et al. 2009). At the same lator, its status as a cadmium hyperaccumulator was time, access to nutrients and heavy metals is more Water Air Soil Pollut (2012) 223:5445–5458 5453 limited under perlite cultivation than in pure hydroponic 4.4 The Correlation Between Cd and Mineral Nutrient conditions (Sobrino-Plata et al. 2009). Küpper et al. Accumulation (2000), Lombi et al. (2000), Bert et al. (2003), and Zhao et al. (2006)showed that A. halleri accumulated It has been shown that the influence of cadmium on Cd in the amounts higher than 0.01 % d.w., what has the content of mineral nutrients in plants can depend been suggested as a criterion for Cd hyperaccumulation on the concentration of cadmium, growth conditions, by Brooks (1998). Our results agree with these findings. species, or ecotype being investigated or even on plant We have also shown, however, that the plant is able to organ under study (Zhang et al. 2002; Cai et al. 2010). translocate the metal from roots to shoots in such a way Interactions between cadmium and essential mineral that the ratio of the metal content in roots and in shoots is elements have been studied in potatoes (Gonçalves et higher than 1. This criterion of metal hyperaccumulation al. 2009), different ecotypes of wheat (Zhang et al. has been proposed by McGrath and Zhao (2003). 2002), several Cd-tolerant, and non-tolerant ecotypes of rice (Liu et al. 2003; Cai et al. 2010), in Betula 4.3 The Correlation Between Cd and Zn pendula—a pioneer species colonizing post-industrial Hyperaccumulation polluted areas (Gussarsson et al. 1996) as well as in cadmium hyperaccumulators: Brasica juncea (Jiang et Cd hyperaccumulation has been shown in all known al. 2004), T. caerulescens (Roosens et al. 2003), and Zn hyperaccumulating plants. This suggests that ge- Sedum alfredii (Yang et al. 2004). This wide literature netic basis of both phenomena might be, at least par- reveals, however, a very complicated and often con- tially, the same (Verbruggen et al. 2009). Our results tradictory picture of the problem. indicate negative correlation between Cd and Zn con- In the present work, we show for the first time that tent in both shoots and roots in A. halleri. The higher cadmium influences the uptake of mineral nutrients in content of cadmium was recorded; the lower was the A. halleri. Our data show that the content of cadmium 2+ 2+ 2+ 2+ content of zinc. and mineral nutrients (Mg ,Mn ,Fe ,Cu , and 2+ The same phenomenon, suggesting a competition Zn ) was significantly correlated. At the same time, between zinc and cadmium, has been also observed by the content of mineral nutrients recorded by us in Zhao et al. (2006). On the basis of an experiment investigated plants ranged within normal limits for carried out during 3 weeks under hydroponic condi- wild plants (Kabata-Pendias 2010). It seems from the tions, they demonstrated that increase in Zn content in above-mentioned reasons that correlations between the growth medium caused decrease in Cd content in the content of cadmium and mineral nutrients might plants. Investigating the effect of Zn and Cd treatment not be an effect of a simple specific competition be- on the content of these metals in plant tissues, Küpper tween these elements but rather an outcome of the mechanism involved in maintaining ion homeostasis. et al. (2000) showed that increase in Zn concentration caused the decrease of cadmium content in shoots by It can be hypothesized that the ability to tolerate and 50 %. The existence of a competition between Zn and hyperaccumulate heavy metals by A. halleri may re- Cd has been also evidenced by Cosio et al. (2004), sult from the ability of the species to maintain ion who demonstrated that increased amount of Cd in the homeostasis under cadmium stress. growth medium reduced Zn accumulation. An important difference between the authors cited 4.5 Analysis of Metal-Binding Proteins above and our experiments is that, in our study, concen- tration of Zn in the growth medium remained on constant It is estimated that about 40 % of all proteins contain level of 0.4 μM and was similar to the concentration heavy metal ion in their structure (Garcia et al. 2006). found in natural, unpolluted soils (Grimme 1968). Metal-containing proteins may be divided into two Our results seem to support the hypothesis put groups: metalloproteins and metal-binding proteins. forward by Zhao et al. (2006) that A. halleri is able The first group is characterized by strong affinity to hyperaccumulate Cd through the Zn pathway and between protein and metal ion. The second group that, in case of increased Cd content in the growth shows significantly weaker affinity between the two medium, Zn is accumulated in lower amounts due to types of molecules. This fact makes the analysis of the competition between both metals. metal binding proteins an extremely difficult task as 5454 Water Air Soil Pollut (2012) 223:5445–5458 metals can be easily lost during the procedure of macromolecules and to conduct their preliminary sample preparation. Hence, it is necessary here to identification. Our research is an important contribu- employ chemical techniques of high resolution for tion to the problem. protein separation and identification. In our research, we used SEC-ICP-MS and PAGE-LA-ICP-MS. Both 4.6 Metal-Binding Proteins methods are widely accepted as useful techniques for bioinorganic speciation analysis (Szpunar 2005; We showed for the first time that control and Cd- Garcia et al. 2006; Polatajko et al. 2007, 2011). treated plants of A. halleri differ significantly at mo- SEC-ICP-MS enabled us to identify dominant coordi- lecular level. Studies carried out hitherto were focused nation complexes of cadmium of molecular weight mainly on the isolation and identification of Cd- lower than 13 kDa in treated plants. New-generation binding proteins (e.g., Bartolf et al. 1980;Rauser columns applied in SEC-ICP-MS enable the research- 1984; Kumar and Prasad 2004; Kastenholz 2006; er to identify intact coordination complexes (Persson Fenik et al. 2007; Polatajko et al. 2007, 2011). Our et al. 2006). The multi-elemental capacity of the meth- study enabled us not only to identify these proteins but od is one of its major advantages (Persson et al. 2006; also to show that the pattern of their occurrence differ Polatajko et al. 2011). We employed this tool to elu- between control and Cd-treated plants, as well as cidate the problem whether, apart from Cd, other im- between groups treated with different doses of cadmi- portant elements (such as Zn and Cu) can also interact um. These differences were shown in plants without with proteins. In our case, the use of SEC-ICP-MS any signs of Cd toxicity. We think that this finding enabled us to identify proteins forming coordination gives a new perspective on the phenomenon of metal 112 66 complexes with the following isotopes: Cd, Zn, hyperaccumulation. Cu. SEC-ICP-MS has, however, some limitations in In Cd-treated plants, we identified Cd-binding pro- resolution when compared with other techniques teins of low molecular weight (LMW): several pro- (Persson et al. 2006). The application of size exclusion teins of <13 kDa, proteins of 20, 25, and 50 kDa, and chromatography together with inductively coupled of about 19, 35, 45 and 75 kDa in size. In control plasma mass spectrometry enabled us, however, to plants, we did not record the presence of these mole- detect and identify metal binding proteins in A. halleri. cules, which can suggest that Cd-binding proteins are The application of PAGE-LA-ICP-MS enabled us to involved in plant response to cadmium. Fenik et al. identify more cadmium-binding proteins with higher (2007) showed the presence of numerous Cd-binding molecular weight, alongside with relatively small cad- proteins in Cd-resistant lines of Nicotiana plumbagi- mium loss during experimental procedures. Cd-binding nifolia. They showed the presence of LMW Cd- proteins identified using this method ranged between 19 binding proteins of 19, 34, and 40 kDa and five and 75 kDa. It is now widely accepted that PAGE-LA- proteins of <13 kDa in size in Cd-treated plants. In ICP-MS is a powerful tool in research on metal binding cadmium-sensitive A. thaliana, the presence of high- complexes in plant material rich in proteins (Polatajko et molecular-weight Cd-binding protein of approximate- al. 2011). One important limitation of the technique is, ly 200 kDa in size has been recorded by Kastenholz however, that multi-elemental analysis is not possible on (2006). The presence of LMW Cd-binding proteins in a single-blot membrane. The method, however, allowed Cd-tolerant ecotypes treated with cadmium can be the detection of some rare Cd-proteins with resolution considered as an indirect proof of the significance of significantly better than the one offered by SEC-ICP- these molecules in plant response to cadmium stress. MS. These investigations allowed us to show that long- Our experiments showed that the amount of Cd- term cadmium exposure alters the protein composition proteins correlated with the amount of Cd present in of a plant. the growth medium. Study carried out by Kumar and Prasad (2004) showed also that there is a positive Investigation of metal-binding proteins requires a lot of effort and the use of advanced methods. Studies correlation between Cd bioaccumulation and the con- in this subject should be focused on the structure and centration of Cd-binding proteins. Hence, application biological role of these proteins. It seems to us that the of long-term Cd exposure instead of short-term, first and the most important step on this way is to “acute” treatment seems to be more appropriate to localize and quantify the content of metal-binding the authors, as it should promote Cd-protein synthesis. Water Air Soil Pollut (2012) 223:5445–5458 5455 Our results evidenced also the presence of LMW Molecular weight of Cd–metallothionein complexes (<13 kDa) Cu- and Zn-binding proteins in A. halleri.It range between 6 and 7 kDa (Sabolic et al. 2002). suggests the presence of specific proteins with very Mejare and Bulow (2001), however, put forward the low molecular weight and high affinity to divalent hypothesis that the expression of cysteine-free metal metal ions in investigated plants. It seems that two chelating proteins is less toxic for cells than the ex- 2+ 2+ metabolically important metal ions (Cu and Zn ) pression of cysteine-rich molecules. According to can form complexes with proteins similar to Cd- them, this scenario is particularly relevant for plants binding proteins, as they were characterized by almost in which synthesis of such molecules is controlled by a identical elution time in size exclusion chromatogra- constitutive promoter and it is not adjusted by the phy. On the basis of our studies, however, it is difficult metal concentration inside the cell (Mejare and to answer whether Cd, Cu, and Zn bind to the same or Bulow 2001). Moreover, Gussarsson (1994) showed different proteins. that increase in sulfur content under cadmium stress Identification of Cd-binding proteins in A. halleri was correlated with the increased content of some Cd- can be of importance in our understanding of the binding peptides containing sulfur. We showed that mechanisms of cadmium tolerance and hyperaccumu- sulfur content in treated plants was not correlated with lation in plants. It has been shown that Cd-binding Cd. Therefore we think, that molecules of the molec- proteins can contain cysteine or histidine (Mejare and ular weight < 13 kDa cannot be associated with metal- Bulow 2001). Both amino acids form complexes with lothioneins. Hence, there are premises that, in small metal ions relatively easily by binding to the thiol proteins identified by us, Cd is not bound by sulfhy- group in case of the former or to nitrogen from imid- dryl groups that are common in phytochelatins and azole functional group (that can easily form coordina- metallothioneins. It is widely known that histidine can act as a very tion bonds with metal cations) in case of the latter. 2+ 2+ 2+ It is known that cysteine-rich proteins such as metal- efficient ligand for metal ions such as Cd ,Zn ,Cu , 2+ 2+ lothioneins and phytochelatins form stable complexes Ni ,and Co (Wierzbicka et al. 2007; Verbruggen et al. with cadmium (Mejare and Bulow 2001; Fenik et al. 2009). This amino acid is also thought to be one of the 2007; Meyer et al. 2011). Hitherto, numerous studies most important compounds responsible for the chelation stressed the importance of phytochelatins (oligomers of of metal ions in hyperaccumulators (Haydon and glutathione) in plant response to heavy metals (e.g., di Cobbett 2007; Verbruggen et al. 2009). Additionally, Toppi and Gabbrielli 1999; Szpunar 2005; Memon and our previous research on the speciation of Cd in Schroder 2009; Meyer et al. 2011). It is usually accepted Allium cepa epidermal cells showed the presence of that molecular weight of Cd–phytochelatin complexes specific cadmium ligand—the histidine (Wierzbicka et range between 2.5 and 10 kDa, depending on the plant al. 2007). This can suggest that LMW proteins identified species and physiological status of investigated speci- by us and able to bind Cd could contain histidine—the mens (Persson et al. 2006). It is also widely accepted amino acid playing a key role in heavy metal detoxifi- that this mechanism of Cd detoxification plays a key cation in hyperaccumulators. This hypothesis requires, role in Cd-sensitive ecotypes (Ebbs et al. 2002). In case however, further studies and evaluation. of tolerant plants, or Cd hyperaccumulators such as A. It is worth noting that all the investigated plants halleri, the role of phytochelatins is almost negligible showed high level of Cd-tolerance and differed signif- (Ebbs et al. 2002; Fenik et al. 2007; Meyer et al. 2011). icantly in terms of the pattern of Cd-binding proteins. It seems, therefore, that amino acids in the form of This can suggest that these proteins can be involved in glutathione could be involved in the protection against cadmium detoxification as it was suggested previously oxidative stress caused by heavy metal ions. In this case, by several authors (Fenik et al. 1995; Woolhouse other molecules have to act as detoxification agents. For 1983; Meyer et al. 2011). all the above-mentioned reasons, it seems to us that Cd- binding proteins of molecular weight <13 kDa cannot be interpreted as phytochelatins. 5 Conclusions Metallothioneins are another group of chemical compounds that are thought to play role in plant resis- We showed that A. halleri is able to translocate Cd to tance to cadmium (e.g., Mejare and Bulow 2001). aerial parts in high amounts (translocation index >1) 5456 Water Air Soil Pollut (2012) 223:5445–5458 Cai, Y., Lin, L., Cheng, W., Zhang, G., & Wu, F. (2010). that confirmed its status as Cd hyperaccumulator. Our Genotypic dependent effect of exogenous glutathione on experiments showed that Cd influenced the uptake of Cd-induced changes in cadmium and mineral uptake and different mineral nutrients. Our results suggest that Cd accumulation in rice seedlings (Oryza sativa). Plant, Soil and Zn can be hyperaccumulated by A. halleri through and Environment, 56, 516–525. Cosio, C., Martinoia, E., & Keller, C. (2004). Hyperaccumula- a common pathway. The data obtained by us point out tion of cadmium and zinc in Thlaspi caerulescens and that Cd is bound by low-molecular-weight metal- Arabidopsis halleri at the leaf cellular level. Plant Physi- binding proteins of the molecular mass <100 kDa. ology, 134, 716–725. These proteins are unlikely to be phytochelatins or Dahmani-Muller, H., van Oort, F., Gelie, B., & Balabane, M. (2000). Strategies of heavy metal uptake by three plant metallothioneins. We hypothesize that these proteins species growing near a metal smelter. Environmental Pol- can play a role in broadly understood Cd detoxifica- lution, 109, 231–238. tion process in A. halleri. di Toppi, L. S., & Gabbrielli, R. (1999). Response to cadmium in higher plants. Environmental and Experimental Botany, Acknowledgments The authors would like to thank two anon- 41, 105–130. ymous reviewers for their comments that improved our manuscript Ebbs, S., Lau, I., Ahner, B., & Kochian, L. (2002). Phytochela- and to Dr. Pawel Wasowicz, for critically reading the paper. tin synthesis is not responsible for Cd tolerance in the Zn/ Cd hyperaccumulator Thlaspi caerulescenes (J. and C. Open Access This article is distributed under the terms of the Presl). Planta, 214, 635–640. Creative Commons Attribution License which permits any use, Emsley, J. (1991). Oxford chemistry guides the elements. Ox- distribution, and reproduction in any medium, provided the ford: Clarendon. original author(s) and the source are credited. Ernst, W. (1990). Mine vegetation in Europe. In J. Shaw (Ed.), Heavy metal tolerance in plants: Evolutionary aspects (pp. 21–37). Boca Raton: CRC Press. Fenik, S. I., Trofimyak, T. B., & Blyum, Y. B. (1995). Mecha- References nisms of development of plant tolerance to heavy metals. Uspekhisovremennoĭbiologii, 115, 259–275. Fenik, S. I., Solodushko, V. G., Kalinyak, T. B., & Blume, A. B. Abratowska, A., Wąsowicz, P., Bednarek, P., Telka, J., & Wierzbicka, (2007). The role of Cd-binding proteins and phytochelatins M. (2012). Morphological and genetic distinctiveness of the in the formation of cadmium resistance in Nicotiana plum- metallicolous and non-metallicolous populations of Armeria baginifolia cell lines. Cytology and Genetics, 41,9–15. maritima s. l. (Plumbaginaceae) in Poland. Plant Biology, 14, Garcia, J. S., de Magalhaes, C. S., & Arruda, M. A. Z. (2006). Trends 586–595. in metal-binding and metalloprotein analysis. Talanta, 69,1–15. Al-Shehbaz, I. A., & O’Kane, S. L., Jr. (2002). Taxonomy and Gode, C., Decombeix, I., Kostecka, A., Wąsowicz, P., Pauwles, phylogeny of Arabidopsis (Brassicaceae). The Arabidopsis M., Courseaux, A., & Saumitou-Laprade, P. (2012). Nuclear Book. doi:10.1199/tab.0009. microsatellite loci for Arabidopsis halleri (Brassicaceae), a Bartolf, M., Brenna, E., & Price, C. A. (1980). Partial charac- model species to study plant adaptation to heavy metals. terization of a cadmium-binding protein from the roots of American JournalofBotany, 99(2), e49–e52. cadmium-treated tomato. Plant Physiology, 66, 438–441. Gonçalves, J. F., Antes, F. G., Maldaner, J., Pereira, L. B., Tabaldi, Bert, V., Bonnin, I., Saumitou-Laprade, P., de Laguerie, P., & Petit, D. (2002). Do Arabidopsis halleri from nonmetallic- L. A., Rauber, R., et al. (2009). Cadmium and mineral nutrient accumulation in potato plantlets grown under cad- olous populations accumulate zinc and cadmium more mium stress in two different experimental culture conditions. effectively than those from metallicolous populations? Plant Physiology and Biochemistry, 47,814–821. New Phytologist, 155,47–57. Grimme, H. (1968). Adsorption of Mn, Co, Cu and Zn by Bert, V., Meerts, P., Saumitou-Laprade, P., Salis, P., Gruber, W., goethite from dilute solutions. Zeitschrift für Pflanzener- & Verbruggen, N. (2003). Genetic basis of Cd tolerance nährung und Bodenkunde, 121,58–65. and hyperaccumulation in Arabidopsis halleri. Plant and Soil, 249,9–18. Gussarsson, M. (1994). Cadmium-induced alterations in nutri- Branquinho, C., Serrano, H. C., Pinto, M. J., & Martins-Loucao, ent composition and growth of Betula pendula seedlings: M. A. (2007). Revisiting the plant hyperaccumulation cri- The significance of fine root as a primary target for cadmi- teria to rare plants and earth abundant elements. Environ- um toxicity. Journal of Plant Nutrition, 17, 2151–2163. mental Pollution, 146, 437–443. Gussarsson, M., Asp, H., Adalsteinsson, S., & Jensén, P. (1996). Braude, G. L., Nash, A. M., Wolf, W. J., Carr, R. L., & Chaney, Enhancement of cadmium effects on growth and nutrient R. L. (1980). Cadmium and lead content of soybean prod- composition of birch (Betula pendula) by buthioninesulphox- ucts. Journal of Food Science, 45, 1187–1199. imine (BSO). Journal of Experimental Botany, 47,211–215. Broadley, M. R., White, P. J., Hammond, J. P., Zelko, I., & Lux, Haydon, M. J., & Cobbett, C. S. (2007). Transporters of ligands for A. (2007). Zinc in plants. New Phytologist, 173, 677–702. essential metal ions in plants. New Phytologist, 174, 499–506. Brooks, R. R. (1998). Plants that hyperaccumulate heavy met- Jiang, X. J., Luo, Y. M., Liu, Q., Liu, S. L., & Zhao, Q. G. als: their role in phytoremediation, microbiology, archae- (2004). Effects of cadmium on nutrient uptake and trans- ology, mineral exploration, and phytomining. Oxford- New location by Indian mustard. Environmental Geochemistry York: CAB International. and Health, 26, 319–324. Water Air Soil Pollut (2012) 223:5445–5458 5457 Kabata-Pendias, A. (2010). Trace elements in soils and plants. Nriagu, J. O. (1996). A history of global metal pollution. Sci- Boca Raton: CRC Press. ence, 272, 223–224. Kastenholz, B. (2006). Comparison of the electrochemical be- Nriagu, J. O., & Pacyna, J. M. (1988). Quantitative assessment havior of the high molecular weight cadmium proteins in of worldwide contamination of air, water and soils by Arabidopsis thaliana and in vegetable plants on using trace-metals. Nature, 333, 134–139. preparative native continuous polyacrylamide gel electro- Olympios, C. M. (1999). Overview of soilless culture: Advantages, phoresis (PNC-PAGE). Electroanalysis, 18, 103–106. constraints and perspectives for its use in Mediterranean Kumar, G. P., & Prasad, M. N. V. (2004). Cadmium-inducible countries. Cahiers Options Méditerranéennes, 31, 307–324. proteins in Ceratophyllum demersum L. (a fresh water Pauwels, M., Frerot, H., Bonnin, I., & Saumitou-Laprade, P. macrophyte): Toxicity bioassays and relevance to cadmium (2006). A broad-scale analysis of population differentiation detoxification. Bulletin of Environmental Contamination for Zn tolerance in an emerging model species for tolerance and Toxicology, 73, 174–181. study: Arabidopsis halleri (Brassicaceae). Journal of Evo- Küpper, H., Lombi, E., Zhao, F. J., & McGrath, S. P. (2000). lutionary Biology, 19, 1838–1850. Cellular compartmentation of cadmium and zinc in relation Persson, D., Hansen, T. H., Holm, P. E., Schjørring, J. K., to other elements in the hyperaccumulator Arabidopsis Cakmak, I., & Husted, S. (2006). Multi-elemental specia- halleri. Planta, 212,75–84. tion analysis of barley genotypes differing in tolerance to Lasat, M. M., & Kochian, L. V. (1998). Physiological basis for Cd toxicity using LC-ICP-MS and ESI-TOF-MS. Journal Zn hyperaccumulation in Thlaspicaerulescens.InH.E. of Analytical Atomic Spectrometry, 21, 996–1005. Flores, J. P. Lynch, & D. Eissenstat (Eds.), Radical biolo- Polatajko, A., Azzolini, M., Feldmann, I., Stuezel, T., & Jakubowski, gy: Advances and perspectives on the function of plant N. (2007). Laser ablation-ICP-MS assay development for roots (pp. 139–149). Rockville: American Society of Plant detecting Cd- and Zn-binding proteins in Cd-exposed Spina- Physiologists. cia oleracea L. Journal of Analytical Atomic Spectrometry, Lasat, M. M., & Kochian, L. V. (2000). Physiology of Zn hyper- 22,878–887. accumulation in Thlaspi caerulescens. In N. Terry & G. Polatajko, A., Feldmann, I., Hayen, H., & Jakubowski, N. Bañuelos (Eds.), Phytoremediation of contaminated soil (2011). Combined application of a laser ablation-ICP-MS and water (pp. 159–169). Boca Raton: Lewis Publishers. assay for screening and ESI-FTICR-MS for identification Liu, J. G., Liang, J. S., Li, K. Q., Zhang, Z. J., Yu, B. Y., Lu, X. of a Cd-binding protein in Spinacia oleracea L. after L., et al. (2003). Correlations between cadmium and min- exposure to Cd. Metallomics, 3, 1001–1008. eral nutrients in absorption and accumulation in various Przedpelska, E., & Wierzbicka, M. (2007). Arabidopsis arenosa genotypes of rice under cadmium stress. Chemosphere, 52, (Brassicaceae) from a lead-zinc waste heap in southern 1467–1473. Poland—A plant with high tolerance to heavy metals. Lombi, E., Zhao, F. J., Dunham, S. J., & McGrath, S. P. (2000). Plant and Soil, 299,43–53. Cadmium accumulation in populations of Thlaspi caerules- Rauser, W. E. (1984). Isolation and partial purification of cens and Thlaspi goesingense. New Phytologist, 145,11–20. cadmium-binding protein from roots of the grass Agrostis Maestri, E., Marmiroli, M., Visioli, G., & Marmiroli, N. (2010). gigantea. Plant Physiology, 74, 1025–1029. Metal tolerance and hyperaccumulation: Costs and trade- Robbins, J. A., & Evans, M. R. (2005). Growing media for con- offs between traits and environment. Environmental and tainer production in a greenhouse or nursery: Part I (compo- Experimental Botany, 68,1–13. nents and mixes). Fayetteville: University of Arkansas. McGrath, S. P., & Zhao, F. J. (2003). Phytoextraction of metals Roosens, N., Verbruggen, N., Meerts, P., Ximénez-Embun, P., & and metalloids from contaminated soils. Current Opinion Smith, J. A. C. (2003). Natural variation in cadmium in Biotechnology, 14, 277–282. tolerance and its relationship to metal hyperaccumulation Mejare, M., & Bulow, L. (2001). Metal-binding proteins and for seven populations of Thlaspi caerulescens from west- peptides in bioremediation and phytoremediation of heavy ern Europe. Plant, Cell & Environment, 26, 1657–1672. metals. Trends in Biotechnology, 19,67–73. Sabolic, I., Ljubojevic, M., Herak, K. C. M., & Brow, D. (2002). Memon, A. R., & Schroder, P. (2009). Implications of metal Cd-MT cause endocytosis of brush-border transporters in accumulation mechanisms to phytoremediation. Environ- rat proximal tubules. American Journal of Physiology. mental Science and Pollution Research, 16, 162–175. Renal Physiology, 283, 1389–1402. Meyer, C. L., Kostecka, A. A., Saumitou-Laprade, P., Creach, A., Sobrino-Plata, J., Ortega-Villasante, C., Flores-Caceres, M. L., Castric, V., Pauwels, M., & Frerot, H. (2010). Variability of Escobar, C., Del Campo, F. F., & Hernandez, L. E. (2009). zinc tolerance among and within populations of the pseudo- Differential alterations of antioxidant defenses as bioindi- metallophyte species Arabidopsis halleri and possible role of cators of mercury and cadmium toxicity in alfalfa. Chemo- directional selection. New Phytologist, 185,130–142. sphere, 77, 946–954. Meyer, C. L., Peisker, D., Courbot, M., Craciun, A. R., Cazale, Strebeyko, P. (1967). Introduction to plant physiology. Warsaw: A. C., Desgain, D., et al. (2011). Isolation and character- PWRiL. ization of Arabidopsis halleri and Thlaspi caerulescens Szpunar, J. (2005). Advances in analytical methodology for phytochelatin synthases. Planta, 234(1), 83–95. bioinorganic speciation analysis: Metallomics, metallopro- Moreno-Jimenez, E., Penalosa, J. M., Esteban, E., & Carpena- teomics and heteroatom-tagged proteomics and metabolo- Ruiz, R. O. (2007). Mercury accumulation and resistance mics. Analyst, 130, 442–465. to mercury stress in Rumex induratus and Marrubium Vazquez, S., & Carpena-Ruiz, R. O. (2005). Use of perlite in vulgare grown in perlite. Journal of Plant Nutrition and cadmium plant studies: An approach to polluted soil condi- Soil Science, 170, 485–494. tions. Journal of Environmental Monitoring, 7,1355–1358. 5458 Water Air Soil Pollut (2012) 223:5445–5458 Verbruggen, N., Hermans, C., & Schat, H. (2009). Molecular Thlaspi caerulescens. Environmental Pollution, 139,167– mechanisms of metal hyperaccumulation in plants. New 175. Phytologist, 182, 781–781. Yang, X. E., Long, X. X., Ye, H. B., He, Z. L., Stoffella, P. J., & Wierzbicka, M. H., Przedpełska, E., Ruzik, R., Ouerdane, L., Calvert, D. V. (2004). Cadmium tolerance and hyperaccu- Połeć-Pawlak, K., Jarosz, M., Szpunar, J., & Szakiel, A. mulation in a new Zn-hyperaccumulating plant species (2007). Comparison of the toxicity and distribution of cad- (Sedum alfredii Hance). Plant and Soil, 259, 181–189. mium and lead in plant cells. Protoplasma, 231,99–111. Zhang, G. P., Fukami, M., & Sekimoto, H. (2002). Influence of Woolhouse, H. W. (1983). Toxicity and tolerance in the cadmium on mineral concentrations and yield components responses of plants to metals. In O. L. Lange, P. S. Nobel, in wheat genotypes differing in Cd tolerance at seedling C. B. Osmond, & H. Ziegler (Eds.), Encyclopedia of plant stage. Field Crops Research, 77,93–99. physiology: Physiological plant ecology (pp. 245–310). Zhao, F. J., Jiang, R. F., Dunham, S. J., & McGrath, S. P. (2006). Berlin: Springer. Cadmium uptake, translocation and tolerance in the hyper- Yanai, J., Zhao, F. J., McGrath, S. P., & Kosaki, T. (2006). Effect accumulator Arabidopsis halleri. New Phytologist, 172, of soil characteristics on Cd uptake by the hyperaccumulator 646–654.

Journal

Water, Air, and Soil Pollution – Pubmed Central

Published: Aug 23, 2012

There are no references for this article.