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Keywords:

  • Alyssum;
  • copper;
  • hyperaccumulation;
  • iron;
  • metal homeostasis;
  • nickel;
  • oxidative stress

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The divalent cations of several transition metal elements have similar chemical properties and, when present in excess, one metal can interfere with the homeostasis of another. To better understand the role of interactions between transition metals in the development of metal toxicity symptoms in plants, the effects of exposure to excess nickel (Ni) on copper (Cu) and iron (Fe) homeostasis in the Ni hyperaccumulator plant Alyssum inflatum were examined.
  • • 
    Alyssum inflatum was hypertolerant to Ni, but not to Cu. Exposure to elevated subtoxic Ni concentrations increased Cu sensitivity, associated with enhanced Cu accumulation and enhanced root surface Cu(II)-specific reductase activity.
  • • 
    Exposure to elevated Ni concentrations resulted in an inhibition of root-to-shoot translocation of Fe and concentration-dependent progressive Fe accumulation in root pericycle, endodermis and cortex cells of the differentiation zone. Shoot Fe concentrations, chlorophyll concentrations and Fe-dependent antioxidant enzyme activities were decreased in Ni-exposed plants when compared with unexposed controls. Foliar Fe spraying or increased Fe supply to roots ameliorated the chlorosis observed under exposure to high Ni concentrations.
  • • 
    These results suggest that Ni interferes with Cu regulation and that the disruption of root-to-shoot Fe translocation is a major cause of nickel toxicity symptoms in A. inflatum.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

To survive, plants must supply thousands of transition metal-requiring apo-proteins in different locations with adequate amounts of the respective required transition metals and store excesses or nonessential transition metals in innocuous forms. Because of the high similarity between the ligand-binding preferences of divalent cations of different transition metal elements, this is a demanding task in biology, the operation of which is still poorly understood. The maintenance of metal homeostasis is particularly challenging for organisms in metal-rich environments, where plants are likely to experience episodes of exposure to large excesses of specific metal ions in the soil solution (Krämer et al., 2007). The aim of this study was to investigate the interaction of an excess of nickel (Ni) with the homeostasis of copper and iron in plants. This work was conducted using the nickel hyperaccumulator plant Alyssum inflatum originating from Ni-rich serpentine soils of western Iran (Ghaderian et al., 2007).

Metal hyperaccumulation is a characteristic of a small proportion of metallophyte and pseudometallophyte taxa, which can accumulate metals in their aboveground tissues in concentrations that are highly toxic for other plants (Baker et al., 2000). Nickel hyperaccumulator species grown in their natural habitats contain more than 1000 µg Ni g−1 shoot dry biomass and up to 47 500 µg Ni g−1 shoot dry biomass (Kelly et al., 1975; Baker & Brooks, 1989). In general, Ni hyperaccumulators are mostly found on serpentine soils which are naturally rich in the transition metals Ni, iron (Fe), cobalt (Co) and chromium (Cr) (Baker & Brooks, 1989). Identifying and developing plants suited for environmental technologies such as phytoremediation and phytomining requires a better understanding of metal homeostasis especially in metal hyperaccumulator plants, which are candidate species for use in these technologies or may serve as models for their development. In addition, the identification and physiological characterization of novel metallophyte species are crucial to exploring and preserving their biodiversity (Whiting et al., 2004).

At the physiological and molecular levels, the mechanisms that govern Ni hyperaccumulation and associated hypertolerance are not well understood. One of the characteristics of Ni hyperaccumulator plants in the genera Alyssum and Noccaea is their high chelator content, in particular free histidine, which has an important role in the high root-to-shoot Ni flux associated with Ni hyperaccumulation, as well as in Ni hypertolerance (Krämer et al., 1996; Kerkeb & Krämer, 2003; Wycisk et al., 2004; Ingle et al., 2005a; Richau et al., 2009). The high levels of histidine in Alyssum hyperaccumulators are a consequence of constitutively high expression of genes encoding rate-limiting enzymatic steps in the biosynthesis of histidine (Ingle et al., 2005a). In addition, increased glutathione biosynthesis has been observed in Thlaspi Ni hyperaccumulator species (Freeman et al., 2004), which appears to be a consequence of enhanced salicylic acid signals (Freeman et al., 2005). In the shoots of Alyssum Ni hyperaccumulators, the highest Ni concentrations have been observed in trichomes and epidermal cells (Krämer et al., 1997a; Broadhurst et al., 2004), and, quantitatively, leaf vacuoles have a prominent role in Ni storage (Ingle et al., 2008). In order to develop additional hypotheses about the physiological and molecular mechanisms of Ni hypertolerance it is necessary to better understand how Ni toxicity arises.

As an essential element, Ni is required in plants in small amounts (Brown et al., 1987; Dalton et al., 1988; Marschner, 1995), whereas much larger amounts are required of Fe, Zn and copper (Cu). One of the well-known symptoms of heavy metal toxicity in plants is oxidative stress (Dietz et al., 1999; Clemens et al., 2002; Clemens, 2006; Krämer & Clemens, 2006; Seregin & Kozhevnikova, 2006). Exposure to high concentrations of Ni was reported to cause an increase in the accumulation of proteins in sulphur metabolism and protection against reactive oxygen species (ROS) and heat-shock responses (Ingle et al., 2005b). Nickel toxicity symptoms described in plants include interveinal chlorosis, inhibition of root growth and induction of potassium (K) leakage (Woolhouse, 1983; Gabbrielli et al., 1989).

In addition, reports of the amelioration of Ni toxicity symptoms by calcium (Ca) or Fe fertilization suggest that the toxicity of Ni might result from its interference with Ca or Fe nutrition (Woolhouse, 1983; Gabbrielli & Pandolfini, 1984). Generally, the Irving–Williams series (Zn2+ < Cu+ > Cu2+ > Ni2+ > Co2+ > Fe2+ > Mn2+ > Mg2+ > Ca2+) predicts that in a given metal–ligand complex, a divalent metal cation can substitute all metal cations positioned downwards (da Silva & Williams, 1991). Accordingly, excess Ni can be predicted to interfere with the homeostasis of several other metals, most prominently that of Fe, which is required in the greatest amounts of all transition metals because of its critical roles in abundant proteins of photosynthesis and respiration, as well as in antioxidant activities and in numerous metabolic pathways (Marschner, 1995). Furthermore, excess Cu is predicted to interfere with Ni homeostasis.

The results presented here suggest that growth in high, subtoxic Ni concentrations specifically enhances Cu accumulation and Cu sensitivity of A. inflatum. Nickel toxicity in A. inflatum is at least partly a consequence of a deterioration of Fe-dependent protein functions in the shoots resulting from Ni-mediated disruption of Fe partitioning from roots into shoots.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant growth

Seeds of A. inflatum NYÁRÁDY were harvested on serpentine soils of western Iran (35°14′ N, 46°28′ E) in July 2007. Approximately 60 000 seeds were collected as a bulk sample from c. 50 individual plants, mixed thoroughly, air dried and stored at 4°C. For the growth of seedlings in sterile conditions on square Petri dishes, seeds were surface-sterilized in 70% (v : v) ethanol for 1 min, followed by incubation in a solution of 3.5% (w : v) NaOCl and 0.05% (w : v) Tween-20 for 15 min. Seeds were sowed directly on treatment media, followed by stratification at 4°C for 2 d. The culture medium was a modified quarter-strength Hoagland solution (Hoagland & Arnon, 1950) containing 1.5 mm Ca(NO3)2, 0.28 mm KH2PO4, 0.75 mm MgSO4, 1.25 mm KNO3, 0.5 µm CuSO4, 1 µm ZnSO4, 5 µm MnSO4, 25 µm H3BO3, 0.1 µm Na2MoO4, 50 µm KCl, 5 µm FeHBED (FeIII(N,N′-di-(2-hydroxybenzoyl)-ethylenediamine-N,N′-diacetic acid)), 3 mm MES and 0.8% (w : v) agarose (Seakem LE; Cambrex Bio Science Rockland, Inc., Rockland, ME, USA), with the final pH adjusted to 5.7 with KOH. Nickel and Cu treatments were prepared by including the appropriate concentrations of NiSO4 or CuSO4, respectively. Different concentrations of FeHBED were included in the media to test the effect of Fe supply on Ni toxicity. Petri dishes were positioned vertically in a growth cabinet, with a 16 h : 8 h, 21°C : 18°C light–dark cycle, with a light intensity of 60 µmol photons m−2 s−1 provided by fluorescent tubes. Root length was measured after 6 d, and seedlings were harvested 23 d after placing the Petri dishes into the growth cabinet, respectively.

For plant growth in the glasshouse, four seeds per pot were sowed in 300 ml pots filled with Perlite and watered with quarter-strength Hoagland solution (pH 5.7), composed as described earlier. Pots were put in three replicate trays (four pots per tray) and Hoagland solution poured in the trays (1 l per tray). Evaporation was compensated by daily adding of distilled water to the solutions to a defined volume. The solutions under the pots were exchanged every 5 d. Plants were grown in a 16 h : 8 h light–dark regime with additional lighting at 150 µmol photons m−2 s−1 provided by metal halide lamps (HPI-T plus 400W; Philips). Temperatures were 37°C : 17°C (maximum : minimum) in a day–night cycle with an average of 24°C. Forty-five-day-old plants were treated with 350 µm of NiSO4, and testing for amelioration of Ni toxicity by foliar spraying was begun 3 wk later. Plants in two pots per tray were sprayed daily with 100 µm FeSO4 + 100 µm citrate, and in two pots per tray with 100 µm CaSO4 + 100 µm citrate (control), until the surface of all leaves was moistened.

Element analysis

After harvest, seedlings were divided into roots and shoots. If necessary, agarose was completely removed from roots. Roots were desorbed first in an ice-cooled solution containing 1 mm MES, pH 5.7, and 5 mm CaSO4 for 10 min and then with a solution containing 1 mm MES, pH 5.7, 5 mm CaSO4 and 5 mm Na2EDTA, pH 5.7 for 5 min. Finally, roots were rinsed in ultrapure water twice, each time for 1 min. All desorption steps were performed on ice. To prepare samples for element measurements, root and shoot tissues were pooled from each Petri plate (10–12 seedlings), dried at 60°C for 3 d and equilibrated in ambient air for 1 d. Dried tissues were then weighed and digested for the measurement of elemental concentrations by inductively-coupled plasma atomic emission spectrometry (ICP-AES, IRIS Advantage HX Duo; ThermoFisher, Dreieich, Germany). Samples were digested in disposable plastic tubes in 2 ml 60% (w : v) nitric acid at room temperature overnight and then in a water bath at 90°C for 2 h. After cooling, 1 ml 30% (v : v) H2O2 was added, and tubes were again incubated at 90°C for 20 min or until clear. Samples were made up to 10 ml final volume with ultrapure water.

Measurement of root Cu(II) reductase and Fe(III) chelate reductase activities

Root surface reductase activity was measured in seedlings grown in sterile medium based on Yi & Guerinot (1996). Agarose was separated from roots very gently to prevent root injury. Each sample contained a pool of roots from five seedlings. Entire roots were submerged in the dark in solutions containing 0.1 mm Fe(III)NaEDTA and 0.3 mm FerroZine (3-(2-pyridyl)-5,6,diphenyl 1,2,4-triazine-4′,4″-disulfonic acid) in ultrapure water for measurement of Fe(III) chelate reductase activity or 0.2 mm CuSO4, 0.6 mm Na3citrate and 0.4 mm BCDS (Bathocuproine disulfonic acid) in distilled water for measurement of Cu(II) reductase activity, respectively. After 45 min for Fe(III) chelate reductase and 30 min for Cu(II) reductase, the absorbance of assay solutions was measured at 562 nm for the Fe(II)FerroZine complex and at 483 nm for the Cu(I)BCDS complex, respectively. Extinction coefficients were 28.6 mm−1 cm−1 for the Fe(II)FerroZine complex (Gibbs, 1976), and 12.25 mm−1 cm−1 for the Cu(I)BCDS complex (Welch et al., 1993).

Chlorophyll quantification and localization of Fe, hydrogen peroxide and superoxide radicals

Concentrations of chlorophyll a, b and total chlorophyll were measured based on Arnon (1949) in the shoots of seedlings grown in Petri dishes. Roots of 2-wk-old seedlings grown in sterile culture on Petri dishes were used to determine Fe localization, hydrogen peroxide and superoxide radical localization. The Fe localization was determined using Perl's stain (Green & Rogers, 2004). This reagent specifically detects Fe(III) through the formation of a blue-coloured precipitate. Perl's stain solution was prepared by mixing equal volumes of 4% (v : v) HCl and 4% (w : v) potassium ferrocyanide immediately before use. After vacuum infiltration for 15 min, roots were left in the staining solution overnight. Hand-cut cross-sections of roots were prepared from the active zone of Fe uptake where the root hairs began emerging, between 1.5 and 2.5 cm from the root tips. When cross-sections were prepared 30 min after infiltration, we observed a lower colour intensity, but identical localization patterns of Fe(III) accumulation were observed when compared with the overnight staining.

3,3′-Diaminobenzidine (DAB) forms a deep-brown product upon reaction with hydrogen peroxide in the presence of peroxidase (Thordal-Christensen et al., 1997). To image hydrogen peroxide, roots were vacuum-infiltrated with 5 mm DAB at pH 3.8 for 15 min and then kept at room temperature 10 h. Cross-sections were prepared as described earlier for the localization of Fe.

Detection of superoxide in the roots was achieved by infiltration with 6 mm nitroblue tetrazolium (NBT). Superoxide-dependent reduction of pale yellow NBT to a dark blue insoluble formazan compound (Maly et al., 1989), which appears brownish under most light sources, is a specific reaction for the visualization of superoxide. After vacuum infiltration, roots were kept at room temperature for 10 h, and then cross-sections were prepared as described for Fe localization.

Determination of the activities of superoxide dismutase, catalase and ascorbate peroxidase

Shoots were harvested from seedlings grown on Petri dishes and immediately frozen in liquid nitrogen. Frozen samples were homogenized in liquid nitrogen in precooled 2-ml Eppendorf tubes using stainless steel beads and a homogenizer at a setting of 1800 rpm for 60 s (MM 200; Retsch GmbH, Haan, Germany). Soluble proteins were extracted by adding ice-cold 50 mm Tris-Cl, pH 6.8, and vortexing for 30 s. Samples were centrifuged at 14 000 g at 4°C for 15 min, and the supernatant was used for measurement of enzyme activities. Protein concentrations were determined using the Bradford protein assay (Bradford, 1976).

Superoxide dismutase (SOD) activity was determined semiquantitatively using an in-gel staining method (Beauchamp & Fridovich, 1971). For each sample, 15 µg of total protein was loaded on a nondenaturing 10% (v : v) polyacrylamide gel. After running the gel at 200 V and 4°C in the cold room, it was incubated in 50 mm Tris-Cl, pH 8.0, for 20 min and then in 50 mm Tris-Cl, pH 8, containing 100 mg l−1 NBT and 40 µm riboflavin in the dark for 20 min. Then the gel was incubated in 0.1% (v : v) N,N,N′,N′-tetramethylethylenediamine (TEMED) in 50 mm Tris-Cl, pH 8, in the dark for 20 min. Finally, the gel was placed in 50 mm Tris-Cl, pH 8, and incubated under fluorescent light for 5 min. For the identification of different isoforms of SOD, specific inhibitors were used during the activity staining period in replicate gels (data not shown). Hydrogen peroxide, 5 mm, was used to inhibit both FeSOD and CuZnSOD, and 2 mm NaCN was used to inhibit CuZnSOD.

Catalase activity was determined using a UV spectrophotometric method (Aebi, 1983). The reaction solution contained 5 mm H2O2 in 50 mm potassium phosphate buffer, pH 7. The reaction was started by adding 100 µl protein extract to 900 µl reaction solution. The decrease in absorbance at 240 nm was monitored, and catalase activity was calculated using an absorbance coefficient for H2O2 of 0.039 mm−1 cm−1. One unit of catalase is defined as the amount of enzyme that is needed for the decomposition of 1 µmol of H2O2 in 1 min at 25°C. Decrease in absorption at 240 nm was negligible in the absence of H2O2 or in the absence of protein extract in the reaction solution.

The method for the determination of ascorbate peroxidase activity was based on Nakano & Asada (1981) and Boominathan & Doran (2002). A 50-µl sample of the protein extract was added to 900 µl of reaction solution (a mixture of 725 µl 50 mm potassium phosphate buffer, pH 7, containing 0.2 mm EDTA and 175 µl of 0.5 mm ascorbic acid). The reaction was started by adding 50 µl of 200 mm H2O2. The decrease in absorbance at 290 nm was monitored, and enzyme activity was calculated using an absorbance coefficient for ascorbic acid of 2.6 mm−1 cm−1. One unit of ascorbate peroxidase is defined as the oxidation of 1 µmol min−1 ascorbic acid at 25°C.

Statistical analysis

Single comparisons of means were performed using Student's t-test, and multiple comparisons were performed by one-way ANOVA (Tukey's HSD) using the SPSS software (version 13, for Windows; SPSS Inc., Chicago, IL, USA). Data are shown from one repetition which is representative of three independent repetitions of each experiment except for the data shown in Fig. 9.

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Figure 9. Amelioration of nickel (Ni) toxicity by iron (Fe). (a) Alyssum inflatum plants growing in the presence of 350 µm NiSO4 were sprayed daily with an iron-containing solution (left) or with a calcium-containing solution (control, right). (b–d) Effects of different concentrations of Fe and Ni in the medium on (b) root length, (c) shoot dry biomass (open bars, 0.5 µm Fe; light tinted bars, 5 µm Fe; dark tinted bars, 20 µm Fe) and (d) concentrations of total chlorophyll (open bars), chla (light tinted bars) and chlb (dark tinted bars) of seedlings grown in Petri plates on agarose media. Shown in (a) are shoots of 80-d-old plants grown on Perlite and watered with quarter-strength Hoagland solution. The nutrient solution was supplemented with 350 µm NiSO4 during the final 5 wk of the experiment, and daily spraying was carried out during the final 2 wk of the experiment. Photographs are representative of eight pots from two independent experiments. Bars, 1 cm. Different letters in (b) and (c) denote statistically significant differences (Tukey's HSD, P < 0.05; lowercase letters for 0.5 µm Fe, uppercase letters for 5 µm Fe and italicized uppercase letters for 20 µm Fe). Different letters in (d) indicate statistically significant differences between total chlorophyll concentrations in shoots treated with different concentrations of Ni (lowercase letters for 0.5 µm Fe, uppercase letters for 5 µm Fe and italicized uppercase letters for 20 µm Fe). Data presented in (b–d) are arithmetic means ± SD of n = 3 replicate samples, each representing one Petri plate and values from 10–13 seedlings per plate (b) and from one pool of 5–6 seedlings (c,d) per plate, respectively, from one experiment.

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Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Determination of Ni tolerance in A. inflatum

The effect of different concentrations of Ni on root elongation and biomass production of seedlings was analysed to determine Ni tolerance in A. inflatum. In comparison with control seedlings grown in the absence of added Ni, apparent decreases in root length (Fig. 1a) and biomass production (Fig. 1b) were observed in seedlings grown in the presence of 300 µm Ni, but this was not statistically significant. A Ni concentration of 350 µm caused significant decreases in both root length and biomass production. The concentration of 100 µm Ni was chosen as an elevated, but nontoxic Ni concentration to investigate the effects of Ni on Cu and Fe homeostasis in A. inflatum.

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Figure 1. Effect of nickel (Ni) on root elongation and biomass production of Alyssum inflatum. (a) Root lengths of 6-d-old seedlings germinated and grown in Petri plates on agarose media containing a modified quarter-strength Hoagland solution supplemented with different concentrations of Ni. (b) Root (open bars) and shoot (closed bars) biomass production. Dry biomass was determined in 23-d-old seedlings grown as in (a). Bars represent arithmetic means ± SD of n = 3 plates, each containing 10–12 replicate seedlings. Asterisks denote a statistically significant difference from the means in the control treatment with no added Ni (P < 0.05), based on ANOVA (Tukey's HSD).

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Effect of Ni on Cu tolerance

Because the affinity of Cu for binding to low-molecular-weight chelators, such as nicotianamine, histidine and other ligands, is higher than the affinity of Ni, we investigated whether a nontoxic supply of 100 µm Ni affects Cu tolerance in A. inflatum (Fig. 2). As expected (see Fig. 1), there was no statistically significant difference between A. inflatum seedlings grown in normal quarter-strength Hoagland medium containing 0.5 µm Cu and seedlings grown in the presence of an additional 100 µm Ni with respect to dry biomass production of roots (Fig. 2a) or shoots (Fig. 2b). In the absence of Ni added to the medium, exposure to Cu of up to 3 µm did not cause a reduction in either shoot or root biomass when compared with control conditions (0.5 µm Cu). By contrast, in the presence of 100 µm Ni, supplementation of the medium with 3 µm Cu resulted in a reduction in root and shoot biomass production of 79% and 61%, respectively (P < 0.05; Tukey's HSD). This suggested that the presence of an elevated, nontoxic Ni concentration in the root medium increases the sensitivity of A. inflatum to excess Cu. At 4 µm Cu a strongly reduced biomass production indicated that seedlings suffered from Cu toxicity irrespective of whether or not Ni was present in the root medium (P < 0.05; Tukey's HSD).

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Figure 2. Copper (Cu) tolerance in Alyssum inflatum in the absence (open bars) and presence of 100 µm nickel (Ni; closed bars) in the medium. (a) Root and (b) shoot biomass production. Dry biomass was determined in 23-d-old seedlings grown in Petri plates on agarose media containing a modified quarter-strength Hoagland solution supplemented with different concentrations of Ni and Cu. Bars represent arithmetic means ± SD of n = 3 replicate samples, each containing pooled material from 10–12 seedlings. Asterisks denote a statistically significant difference between mean biomass produced in the presence and absence of nickel of the respective Cu treatment (**, P < 0.01; ***, P < 0.001), based on Student's t-test. Different letters in each series (lowercase letters for –Ni and uppercase letters for +Ni) show statistically significant differences (P < 0.05) between different concentrations of Cu based on ANOVA (Tukey's HSD).

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Effects of Ni on seedling Cu accumulation and root surface Cu(II) reductase activity

Reduced growth in the presence of a combination of elevated concentrations of both Cu (3 µm) and Ni (100 µm) in the root medium, when compared with elevated Cu (3 µm) or elevated Ni (100 µm Ni) alone, could be a result of competition between Ni and Cu for metal-binding chelators or for sequestration inside the plant or, alternatively, could be a result of increased metal accumulation in the combined treatment. Thus, elemental concentrations were analysed in seedlings grown at different Cu and Ni concentrations. We did not detect any effect of the addition of different Cu concentrations to the medium on Ni accumulation in the seedlings (100 µm Ni: roots 641 ± 48, shoots 2970 ± 82 µg Ni g−1 dry biomass; 0 µm Ni: roots 46 ± 3.1, shoots 220 ± 15 µg Ni g−1 dry biomass; arithmetic means ± standard error of n = 4 Cu treatments; data not shown).

When the medium was supplemented with 100 µm Ni, a significant increase in the concentrations of Cu was observed in both roots and shoots with increasing medium Cu concentrations. In the absence of added Ni, increasing the concentrations of Cu in the medium up to 2 µm did not cause a statistically significant increase in root Cu concentrations and only a slight increase in shoot Cu concentrations (Fig. 3a,b, and legend). At all external Cu concentrations, root Cu concentrations were between 1.6-fold and 3.7-fold higher in Ni-supplemented seedlings than in seedlings grown without added Ni, and shoot Cu concentrations were between 1.5-fold and 2.0-fold higher in Ni-supplemented seedlings (Fig. 3a,b). This suggested that under Ni exposure, root Cu uptake is stimulated and root-to-shoot translocation is similarly efficient when compared with seedlings unexposed to Ni.

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Figure 3. Effect of copper (Cu) concentrations on root and shoot copper accumulation in Alyssum inflatum in the absence (open bars) and presence of 100 µm nickel (Ni; closed bars). (a) Root and (b) shoot copper concentrations in 23-d-old seedlings grown on Petri plates. Bars represent arithmetic means ± SD of n = 3 replicate samples, each consisting of material pooled from 10 to 12 seedlings. For each copper treatment, asterisks denote statistically significant differences (*, P < 0.05; **, P < 0.01) between means of plants grown in the absence and presence of Ni, based on Student's t-test. Different letters in each series (lowercase letters for –Ni and uppercase letters for +Ni) show statistically significant differences (P < 0.05) between different concentrations of Cu based on ANOVA (Tukey's HSD). A linear regression analysis indicated that seedling Cu concentrations (c(Cu)Root/Shoot) increased with medium Cu concentrations (c(Cu)Medium) in Ni-exposed seedlings (c(Cu)Root = 15.7 × c(Cu)Medium + 15.7, R2 = 0.74) and shoots (c(Cu)Shoot = 7.4 × c(Cu)Medium + 10.1, R2 = 0.62), but not in roots of seedlings grown without added Ni (c(Cu)Root = 1.7 × c(Cu)Medium + 11.6, R2 = 0.25) and only slightly in their shoots c(Cu)Shoot = 2.7 × c(Cu)Medium + 7.7, R2 = 0.54).

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Roots can take up Cu either specifically as Cu(I) after reduction of Cu(II), or as Cu(II), which can also be taken up through transporters of other divalent metal cations (Sancenon et al., 2003; Puig et al., 2007). In order to investigate whether a Cu-specific uptake pathway contributes to the observed Ni-induced increase in Cu accumulation, we tested whether root surface Cu(II) chelate reductase activity responds to Ni exposure. In seedlings cultivated in the presence of 100 µm Ni in the medium, root surface Cu(II) chelate reductase activity was 150.9% ± 17 (n = 3 pools of five seedlings) of the activity in control seedlings cultivated with no added Ni (Fig. 4). It is thus possible that at least partly, the Ni-induced increase in Cu accumulation proceeds through the specific Cu uptake pathway involving initial reduction of Cu(II) to Cu(I), which would not be subject to competition by an excess of Ni(II).

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Figure 4. Root surface copper(II) chelate reductase activity of Alyssum inflatum in the absence (−Ni) and presence of 100 µm nickel (+Ni) in the medium. Assays were conducted with whole roots of 23-d-old seedlings grown on Petri plates. Bars represent arithmetic means ± SD of n = 3 replicate samples, each consisting of roots pooled from five seedlings. The asterisk indicates a statistically significant difference (P < 0.05) between Ni treatments based on a Student's t-test.

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Effects of Ni on root surface Fe(III) chelate reductase activity and on Fe concentrations in seedlings

To determine whether the effect of Ni on root surface Cu(II) chelate reductase activity is Cu-specific or a secondary activity of a root surface Fe(III) chelate reductase (Robinson et al., 1999), the latter activity was determined in seedlings grown at different Ni concentrations. Root surface reductase activities were lower for Fe(III) than for Cu(II) irrespective of Ni supplementation (Fig. 5). In comparison with control seedlings grown without added Ni, there was no increase in root surface Fe(III) chelate reductase activity in seedlings grown in the presence of 100 or 300 µm Ni. This suggested that Ni exposure leads to an increase in the activity of a root surface reductase that can use Cu(II), but not Fe(III), as a substrate.

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Figure 5. Root surface iron(III) chelate reductase activity of Alyssum inflatum in the presence of different concentrations of nickel (Ni) in the medium. Assays were conducted with whole roots of 23-d-old seedlings grown on Petri plates. Bars represent arithmetic means ± SD of n = 3 replicate samples, each consisting of roots pooled from 10–12 seedlings. No statistically significant differences were observed between treatments (ANOVA, Tukey's HSD).

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We also determined the effect of Ni exposure on Fe accumulation in seedlings. The supplementation of the medium with either 100 or 300 µm Ni caused an approx. 5.2-fold increase in root Fe concentrations (Fig. 6a). This indicated that, under Ni exposure, the increase in root Fe accumulation is more pronounced than that of Cu. However, unlike Cu-treated seedlings, shoot Fe accumulation at 100 µm Ni and 300 µm Ni was decreased to c. 17.4% and 12.4%, respectively, of that of controls unexposed to Ni (Fig. 6b). Together, these data suggest that in A. inflatum, exposure to high, subtoxic and toxic Ni concentrations both result in an inhibition of root-to-shoot translocation of Fe, which leads to an accumulation of Fe in roots. As expected, there was a progressive increase in Ni concentrations in roots and shoots of seedlings exposed to 100 and 300 µm Ni, when compared with seedlings grown in control media without added Ni (Fig. 6c,d). Alyssum inflatum exhibited Ni partitioning typical of a Ni hyperaccumulator, with shoot : root ratios of Ni concentrations of 3.7, 5.0 and 3.8 at 0 Ni, 100 µm Ni and 300 µm Ni, respectively (calculated from data shown in Fig. 6c,d).

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Figure 6. Effect of different concentrations of nickel (Ni) in the medium on iron (Fe) and nickel accumulation in Alyssum inflatum. Iron concentrations in (a) roots and (b) shoots, and Ni concentrations in (c) roots and (d) shoots of 23-d-old seedlings grown in Petri plates on agarose media. Bars represent arithmetic means ± SD of n = 3 replicate samples, each containing pooled material from 10–12 seedlings. Different letters indicate statistically significant differences (P < 0.001) between treatments, based on ANOVA (Tukey's HSD).

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Localization of Fe in the root tissues

To investigate where Fe accumulated in the roots, we used Perl's stain to detect Fe(III) in roots of 14-d-old seedlings exposed to different concentrations of Ni in the medium. This indicated that the concentration of Ni in the medium has an effect on the localization of the accumulation of Fe in the roots (Fig. 7). No accumulation of Fe was detected by this method in transverse sections of the differentiation zone of roots of seedlings grown in the absence of added Ni (Fig. 7a). Treatment of seedlings with a nontoxic concentration of 50 µm Ni resulted in the accumulation of Fe in the vasculature (Fig. 7b). When plants were grown at 100 µm Ni, Fe accumulation was also detected in the pericycle and endodermis (Fig. 7c,d). At this Ni concentration, accumulation of Fe in the endodermis and pericycle was observed in the differentiation and the beginning of the root hair zone. At a greater distance from the root tip, accumulation of Fe was also observed in cortex cells in the root hair zone (Fig. 7e). Treatment with high concentrations of Ni (300 µm) caused an accumulation of Fe primarily in the cortex cells in the differentiation zone and above (Fig. 7f). At a toxic Ni concentration of 350 µm, no further change in Fe localization was observed (not shown). These data suggested that exposure to Ni progressively causes Fe accumulation in cell layers external to the vasculature in a concentration-dependent manner.

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Figure 7. Localization of iron(III) in roots of Alyssum inflatum grown in the presence of different concentrations of nickel (Ni) in the medium. Iron(III) was localized using Perl's stain in roots of 14-d-old seedlings exposed to (a) no Ni, (b) 50 µm Ni, (c), (d) and (e) 100 µm Ni or (f) 300 µm Ni in the medium for 14 d. c, Cortex; en, endodermis; ep, epidermis; p, pericycle; v, vasculature; blue colour, iron(III) detected by Perl's stain. Bars, 50 µm.

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Effect of Ni on chlorophyll content and on SOD, catalase and ascorbate peroxidase activities

One of the known symptoms of Ni toxicity is interveinal and whole-leaf chlorosis (Fig. 9a, right panel), which has also been reported as a symptom of Fe deficiency. Leaf chlorosis in seedlings exposed to 350 µm Ni was accompanied by a decrease in total chlorophyll, and chlorophyll a (chla) and b (chlb) concentrations (Fig. 8a). Enzyme activities were determined in leaf protein extracts for some antioxidant enzymes which require Fe-containing cofactors. In-gel staining of activities of different superoxide dismutase (SOD) isozymes showed that FeSOD activity was present in control plants and plants exposed to 100 µm Ni, but undetectable in plants exposed to a toxic concentration of 350 µm Ni (Fig. 8b). The activities of CuZnSOD and MnSOD were unaffected by the Ni concentration in the medium. Compared with controls, the activity of catalase, a haem-containing enzyme, appeared to be reduced by on average 43% at 100 µm Ni, but a statistically significant 68% decrease was only observed in plants exposed to 350 µm of Ni (Fig. 8c). The activity of ascorbate peroxidase, another haem-containing enzyme, was not reduced at 100 µm Ni and decreased by 40% in plants exposed to 350 µm Ni (Fig. 8d), when compared with controls not exposed to Ni. This suggested that the Ni-mediated decrease in root-to-shoot Fe partitioning results in decreased activities of Fe-dependent antioxidant enzymes in the shoots of A. inflatum.

image

Figure 8. Effects of nickel (Ni) on chlorophyll concentrations and the activities of superoxide dismutase, catalase and ascorbate peroxidase. Assays were conducted using total soluble protein extracts from shoots of 23-d-old seedlings of Alyssum inflatum grown in Petri plates on agarose media. All bars represent arithmetic means ± SD of n= 3 replicate samples, each containing pooled material from 10–12 seedlings. (a) Concentrations of total chlorophyll (white bars), chla (light tinted bars) and chlb (dark tinted bars); (b) activity-stained gel image for in-gel detection of superoxide dismutase isozymes; (c) catalase activities; (d) ascorbate peroxidase activities. Asterisks denote statistically significant differences compared with the control treatments without Ni added to the medium (*, P < 0.05; ***, P < 0.001) based on ANOVA (Tukey's HSD).

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Amelioration of Ni toxicity symptoms through increased iron supply

To test whether Fe supply to the shoot can ameliorate Ni toxicity, adult plants exposed to 350 µm Ni in glasshouse conditions were sprayed with iron. Daily spraying of shoots with iron as 100 µm FeSO4 + 100 µm citrate ameliorated leaf chlorosis within 2 wk (Fig. 9a, left panel), whereas leaves remained chlorotic upon spraying with 100 µm CaSO4+ 100 µm citrate (Fig. 9a, right panel).

For a more quantitative account of the effects of increased Fe supply on Ni toxicity symptoms, seedlings were grown in the presence of different Ni concentrations and Fe supplies in the media. Compared with control seedlings unexposed to Ni, root length was unchanged at 100 µm Ni and decreased by c. 50% at 350 µm Ni irrespective of the iron concentration supplied in the medium (Fig. 9b). In the presence of the normal concentration of 5 µm Fe, a toxic concentration of 350 µm Ni caused reductions in shoot dry biomass and chlorophyll concentrations to below the levels determined in the absence of added Ni (Fig. 9c,d; compare Fig. 1). These Ni toxicity symptoms were alleviated when an increased concentration of 20 µm Fe was supplied in the medium (Fig. 9c,d). When a reduced Fe concentration of 0.5 µm Ni was supplied, toxicity symptoms in shoots were observed at a Ni concentration of 100 µm, which was nontoxic in the presence of normal Fe concentrations in the medium (Fig. 9c,d; compare Fig. 1).

Localization of hydrogen peroxide and superoxide radicals in the roots

High concentrations of Fe accumulated in roots of Ni-exposed plants may cause oxidative stress through Fenton chemistry, thus contributing indirectly to Ni toxicity. The localization of hydrogen peroxide and superoxide radicals was determined histochemically in roots of seedlings exposed to 0, 100 and 300 µm Ni in the root medium (Fig. 10). Accordingly, hydrogen peroxide was accumulated mostly in the vasculature, pericycle and endodermis. The intensity and distribution of the stain increased with increasing Ni concentrations, suggesting a Ni-induced and concentration-dependent increase in H2O2 accumulation. However, there was no consistent co-localization between the regions of highest hydrogen peroxide production and highest accumulation of Fe in roots across the different Ni treatments. Superoxide radicals were localized predominantly in the vasculature, without major differences between Ni treatments (Fig. 10d–f).

image

Figure 10. Localization of hydrogen peroxide (H2O2) and superoxide radicals in transverse sections of roots of Alyssum inflatum plants exposed to different concentrations of nickel (Ni). (a–c) 3,3′-Diaminobenzidine (DAB) staining for H2O2, and (d–f) nitroblue tetrazolium (NBT) staining for superoxide radicals of roots of 14-d-old seedlings grown from seeds on medium without added Ni (a,d), supplemented with 100 µm Ni (b,e), or with 300 µm Ni (c,f). c, Cortex; en, endodermis; ep, epidermis; p, pericycle; v, vasculature; brownish colour, H2O2 detected by DAB (a–c), superoxide detected by NBT (d–f). Bars, 50 µm.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Nickel tolerance in A. inflatum

Based on root elongation (i.e. root elongation as a common measure for tolerance to heavy metals; Baker, 1987), this species exhibits Ni hypertolerance, with detectable Ni toxicity only at a concentration of 350 µm Ni in the root medium (Fig. 1). The maximum no-effect Ni concentration of A. inflatum is thus c. 10 times higher than that of Arabidopsis thaliana (data not shown). This is in agreement with the magnitude of differences in Ni tolerance observed previously between taxonomically related hyperaccumulator and nonaccumulator species (Homer et al., 1991a; Krämer et al., 1996, 1997b; Chaney et al., 1997).

Effect of Ni on Cu tolerance

Alyssum inflatum is not tolerant to Cu concentrations above 3 µm in the medium (Fig. 2). This is comparable to the level of Cu tolerance in A. thaliana (M. Hanikenne & U. Krämer, unpublished). Therefore, the mechanisms of Ni hypertolerance in A. inflatum are not effective in Cu tolerance. Multiple metal tolerances for Cu and Ni were observed in Mimulus guttatus, but these have separate genetic bases (Tilstone & Macnair, 1997).

Treatment of plants with different concentrations of Cu in the presence and absence of Ni showed that A. inflatum is more sensitive to Cu in the presence of a nontoxic concentration of 100 µm Ni than in the absence of added Ni in the medium (Fig. 2). Accumulation of Cu in both roots and shoots was higher in the presence of Ni than in the absence of added Ni in the medium (Fig. 3). This suggested that Ni stimulates Cu uptake in A. inflatum, which explains the Cu hypersensitivity observed in Ni-exposed plants. In the leaves of crop plants, critical toxicity thresholds are c. 20–30 µg Cu g−1 dry biomass (Marschner, 1995). This range of shoot Cu concentrations was approached in A. inflatum in the 2 µm Cu treatments in the presence of Ni in the medium.

Nickel exposure stimulated root surface Cu(II) chelate reductase activity (Fig. 4). Reduction of Cu(II) to Cu(I) and Fe(III) to Fe(II) are known as the first steps in Cu and Fe uptake, respectively, in plants with strategy I iron acquisition (Chaney et al., 1972; Sancenon et al., 2003; Puig et al., 2007). Copper(I) is taken up into plant cells through members of the copper transporter (COPT) family of membrane transporters (Kampfenkel et al., 1995; Sancenon et al., 2003), whereas IRT1 (iron-regulated transporter 1) is the main transport system for the uptake of Fe(II) in Arabidopsis (Vert et al., 2002). In Arabidopsis, Cu(II) can also be taken up into cells through less specific Cu uptake systems of the zinc-regulated transporter (ZRT), IRT-related protein (ZIP) family (Wintz et al., 2003). It was suggested that the reduction of both Cu(II) and Fe(III) depend on the same reductase (Welch et al., 1993; Yi & Guerinot, 1996; Cohen et al., 1997). This appears not to be the case for the Ni-stimulated Cu(II) reductase activity in A. inflatum because there was no concurrent stimulation of root surface Fe(III) chelate reductase activity (Fig. 5). Thus, Cu(II)-specific reductases appear to operate at the root surface of plants under certain growth conditions.

We do not know whether the stimulation of Cu(II) reductase activity in response to Ni exposure is a consequence of an effect of Ni on the regulation of Cu uptake or whether it is a consequence of an indirect effect of Ni on the nutritional Cu status of the plant, but the former appears more likely. In the green algae Chlamydomonas reinhardtii, excess Ni, hypoxia and Cu deficiency all cause the activation of Copper Response Regulator 1 (CRR1), which in turn activates the transcription of Cu-deficiency response genes (Kropat et al., 2005). Recently, the homologous Arabidopsis SQUAMOSA Promoter Binding Protein-Like7 (SPL7) protein was shown to activate the transcription of Cu deficiency response genes in Arabidopsis (Yamasaki et al., 2009). Although in Cu-deficient Arabidopsis plants, amounts of miR398 are upregulated and its target AtCSD2 transcript levels encoding a CuZnSOD are downregulated via SPL7, this SPL7-dependent Cu deficiency response is no longer observed in the presence of excess Ni (Yamasaki et al., 2009). Experiments in the present study on A. inflatum were conducted under normal and excess Cu conditions, under which excess Ni had no effect on CuZnSOD protein levels in A. thaliana (Yamasaki et al., 2009). Similarly, excess Ni had no effect on CuZnSOD activity in A. inflatum (see Fig. 8). Further work will be required to investigate whether or not excess Ni increases Cu accumulation by activating a yet-unidentified subset of Cu deficiency responses in Arabidopsis roots and to obtain information on the transcriptional regulation of Cu deficiency response genes in A. inflatum, for which there is currently no sequence information.

Effect of Ni on root–shoot Fe partitioning

When compared with plants grown in the absence of added Ni, a reduced concentration of Fe in the shoots and an increased accumulation of Fe in roots of plants exposed to Ni suggested that Ni interferes with Fe translocation from the root to the shoot (Fig. 6). Various effects of Ni on root and shoot Fe accumulation have been reported before in other plants, for example in Lolium perenne (Khalid & Tinsley, 1980) and Phaseolus vulgaris (Piccini & Malavolta, 1992). In Ni-exposed A. inflatum, low shoot Fe levels did not apparently result in an activation of Fe deficiency responses because the most prominent component, root surface Fe(III) chelate reductase, was not activated in Ni-exposed plants (see Fig. 5). By contrast, in Arabidopsis excess Zn stimulates root surface Fe(III) chelate reductase activity (Becher et al., 2004). The localization of root Fe accumulation using Perl's stain suggested that exposure to subtoxic concentrations of Ni may interfere with Fe release into the xylem, whereas higher concentrations of Ni progressively interfere with earlier stages of radial cell-to-cell movement of Fe in the root towards the vasculature (Fig. 7). Consequently, there appears to be competition between Ni and Fe for translocation at different sites in the root depending on the quantities of Ni present. After uptake into the root symplasm, Fe(II) is thought to form complexes with nicotianamine (Hell & Stephan, 2003), which also has a high affinity for Ni(II). In Alyssum Ni hyperaccumulators, free histidine has an important role in symplastic Ni chelation, mobility of Ni inside the roots and xylem loading of Ni as well as Ni tolerance (Krämer et al., 1996; Richau et al., 2009). So far, root-to-shoot movement of Fe in A. thaliana is known to depend on FRD3 localized in the plasma membrane of pericycle cells, where it was suggested to transport citrate into the xylem (Durrett et al., 2007). Xylem Fe(III) is thought to occur primarily as a citrate complex (Tiffin, 1966; Tiffin, 1971; Cataldo et al., 1988). Organic acids such as citrate, malate and malonate are abundant in Ni hyperaccumulators and known to chelate Ni in leaf vacuoles (Lee et al., 1978; Brooks et al., 1981; Homer et al., 1991b; Krämer et al., 2000) and in the stem (Montargès-Pelletier et al., 2008).

Detailed knowledge of the binding partners and membrane transport proteins in the pathway of Fe and Ni movement through the root and into the xylem could help to explain the concentration-dependence of the localization of Ni-induced Fe accumulation in A. inflatum in the future.

Nickel toxicity associated with impairment of Fe-dependent protein functions in the leaves

Toxic concentrations of Ni caused a decrease in leaf chlorophyll concentrations (Fig. 8). Leaf chlorosis is one of the well-known symptoms of heavy metal toxicity and of Fe deficiency in plants. Iron is a cofactor of a di-iron enzyme performing the oxidative cyclase reaction in the biosynthesis of chlorophyll (Tottey et al., 2003). Heavy metals have been proposed to inhibit chlorophyll biosynthesis directly, displace chlorophyll magnesium (Main, 1974; Goodwin-Bailey et al., 1992; Küpper et al., 1998, 2000, 2001; Asemaneh et al., 2007) or to act indirectly by causing secondary Fe deficiency (Woolhouse, 1983; Sheoran et al., 1990; Piccini & Malavolta, 1992).

In this study, the lowered Fe-dependent antioxidant enzyme activities in A. inflatum and the markedly reduced shoot Fe concentrations under exposure to toxic Ni concentrations suggested that Ni-induced Fe deficiency is at least partly responsible for Ni toxicity symptoms (Figs 6, 8). In seedlings grown in the presence of toxic concentrations of Ni in the medium, shoot FeSOD activity was much lower than in control seedlings whereas MnSOD and CuZnSOD activities remained unchanged (Fig. 8b). In A. thaliana expression of both FeSOD and CuZnSOD is under control of the Cu status (Puig et al., 2007), whereby CuZnSOD is formed in Cu-sufficient plants and FeSOD in Cu-deficient plants. As shown here (see Fig. 3), Cu concentrations in A. inflatum increased in the presence of excess Ni in the medium. However, higher Cu accumulation and reduced FeSOD activities in shoots of plants exposed to 350 µm Ni, when compared with unexposed control plants, were not accompanied by increased CuZnSOD activities. This suggests that plants were Cu-sufficient irrespective of Ni exposure. Therefore the strong decrease in FeSOD activity in shoots of plants exposed to toxic concentrations of Ni is likely to be a consequence of Ni-induced Fe deficiency in the shoot. It is interesting to note that FeSOD activity in plants exposed to a high, nontoxic concentration of 100 µm Ni is indistinguishable from that in unexposed plants, while Fe concentration in the shoot is already strongly reduced at this Ni concentration (see Fig. 6b). The reason for this remains to be established, but one possible explanation is that toxic concentrations of Ni not only interfere with Fe translocation into the shoot, but also promote the displacement of Fe in FeSOD in the shoot.

In the presence of toxic concentrations of Ni in medium, the activities of two other Fe-dependent enzymes, catalase and ascorbate peroxidase, were also decreased in the shoot (Fig. 8). As haemoproteins these antioxidant enzymes require Fe for their function. The activities of these enzymes are known to decrease under Fe deficiency, and both catalase activity and ascorbate peroxidase activity have been suggested as markers of nutritional Fe status of plants (Marschner, 1995; Fourcroy et al., 2004). In agreement with the results presented here on A. inflatum, reduced SOD and catalase activities were also observed in the shoots of wheat grown in the presence of toxic concentrations of Ni (Gajewska et al., 2006). Leaf chlorosis in A. inflatum grown in the presence of toxic Ni concentrations was ameliorated by foliar Fe spraying (Fig. 9a). Nickel toxicity symptoms in shoots, but not in roots, were also alleviated under high Fe supply and exacerbated under reduced Fe supply in the medium (Fig. 9b–d). These results further support that secondary Fe deficiency in the shoot is a major consequence of exposure to toxic Ni concentrations. Amelioration of Ni toxicity by foliar Fe spraying has been reported in several other plants (Woolhouse, 1983), suggesting that Ni-induced disruption of Fe translocation to the shoot is not restricted to A. inflatum.

Does accumulation of Fe in roots cause oxidative stress?

An imbalance between the production and detoxification of free radicals and reactive oxygen species (ROS) is known to cause oxidative stress. Under most biological conditions Ni is not considered a redox-active heavy metal. Despite this, high concentrations of Ni in the medium are known to cause oxidative stress in plants (Boominathan & Doran, 2002). Unlike Ni, excess Fe is known to participate in direct electron transfer reactions and can contribute to the generation of superoxide radicals. In the Fenton reaction Fe can produce the extremely reactive hydroxyl radical. It is thus possible that, as a consequence of exposure to high Ni concentrations, Fe accumulating in the roots of A. inflatum directly contributes to Ni-induced oxidative stress.

At 300 µm Ni, the highest amounts of Fe accumulation in roots did not colocalize with either hydrogen peroxide or superoxide radicals (Fig. 10). These data suggest that Fenton chemistry resulting from Fe accumulated in roots of Ni-exposed plants did not make a major contribution to ROS formation. It is possible that the concentration, chemical form or subcellular localization of Ni-induced Fe accumulation in the roots of A. inflatum is not conducive to a direct participation of Fe in electron transfer reactions.

Conclusions and outlook

In A. inflatum, two major effects of Ni exposure are enhanced Cu sensitivity associated with Ni-stimulated Cu accumulation and the disruption of radial transport of Fe towards the vasculature. The resulting Fe depletion in the shoot leads to a reduction in Fe-dependent antioxidant enzyme activities and may thus contribute to Ni-induced oxidative stress. These results provide a basis for comparative studies in tolerant and nontolerant plants, also using excesses of other heavy metals.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We gratefully acknowledge a scholarship to R.G. from the Ministry of Science, Research and Technology of Iran (MSRT) and Graduate School of Isfahan University. Special thanks to Naser Karimi for his help with seed collections. U.K. gratefully acknowledges funding from a DFG Heisenberg fellowship and DFG grant Kr1967/5-1.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Aebi HE. 1983. Catalase. In: BergmeyerHU, ed. Methods of enzymatic analysis, Vol. III, 3rd edn. Weinheim, Germany: Verlage Chemie, 273286.
  • Arnon DI. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology 24: 115.
  • Asemaneh T, Ghaderian SM, Baker AJM. 2007. Responses to Mg/Ca balance in an Iranian serpentine endemic plant, Cleome heratensis (Capparaceae) and a related nonserpentine species, C. foliolosa. Plant Soil 293: 4959.
  • Baker AJM. 1987. Metal tolerance. New Phytologist 106: 93111.
  • Baker AJM, Brooks RR. 1989. Terrestrial higher plants which hyperaccumulate metallic elements: a review of their distribution, ecology and phytochemistry. Biorecovery 1: 81126.
  • Baker AJM, McGrath SP, Reeves RD, Smith JAC. 2000. A review of the biological resource for possible exploitation in the phytoremediation of metal-polluted soils. In: TerryN, BanuelosGS, eds. Phytoremediation of contaminated soil and water. Boca Raton, FL, USA: CRC Press LLC, 85107.
  • Beauchamp C, Fridovich I. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry 44: 276287.
  • Becher M, Talke IN, Krall L, Krämer U. 2004. Cross-species microarray transcript profiling reveals high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator Arabidopsis halleri. Plant Journal 37: 215268.
  • Boominathan R, Doran PM. 2002. Ni-induced oxidative stress in roots of the Ni hyperaccumulator, Alyssum bertolonii. New Phytologist 156: 205215.
  • Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248254.
  • Broadhurst CL, Chaney RL, Angle JS, Maugel TK, Erbe EF, Murphy CA. 2004. Simultaneous hyperaccumulation of nickel, manganese, and calcium in Alyssum leaf trichomes. Environmental Science and Technology 38: 57975802.
  • Brooks RR, Shaw S, Marfil AA. 1981. The chemical form and physiological function of nickel in some Iberian Alyssum species. Physiologia Plantarum 51: 167170.
  • Brown PH, Welch RM, Cary E. 1987. Nickel: a micronutrient essential for higher plants. Plant Physiology 85: 801803.
  • Cataldo A, Mcfadden KM, Garland TR, Wildung RE. 1988. Organic constituents and complexation of nickel(II), iron(III), cadmium(II), and plutonium(IV) in soybean xylem exudates. Plant Physiology 86: 734739.
  • Chaney RL, Brown JC, Tiffin LO. 1972. Obligatory reduction of ferric chelates in iron uptake by soybeans. Plant Physiology 50: 208213.
  • Chaney RL, Malik M, Li YM, Brown SL, Brewer EP, Angle JS, Baker AJM. 1997. Phytoremediation of soil metals. Current Opinions in Biotechnology 8: 279284.
  • Clemens S. 2006. Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants. Biochimie 88: 17071719.
  • Clemens S, Palmgren MG, Krämer U. 2002. A long way ahead: understanding and engineering plant metal accumulation. Trends in Plant Science 7: 309315.
  • Cohen CK, Norvell WA, Kochian LV. 1997. Induction of the root cell plasma membrane ferric reductase (an exclusive role for Fe and Cu). Plant Physiology 114: 10611069.
  • Dalton AD, Russell SA, Evans HA. 1988. Nickel as a micronutrient element for plants. BioFactors 1: 1116.
  • Dietz K-J, Baier M, Krämer U. 1999. Free radicals and reactive oxygen species as mediators of heavy metal toxicity in plants. In: PrasadMNV, HagemeyerJ, eds. Heavy metal stress in plants. Berlin, Germany: Springer, 7397.
  • Durrett TP, Gassmann W, Rogers EE. 2007. The FRD3-mediated efflux of citrate into the root vasculature is necessary for efficient iron translocation. Plant Physiology 144: 197205.
  • Fourcroy P, Vansuyt G, Kushnir S, Inzé D, Briat JF. 2004. Iron-regulated expression of a cytosolic ascorbate peroxidase encoded by the APX1 gene in Arabidopsis seedlings. Plant Physiology 134: 605613.
  • Freeman JL, Garcia D, Kim D, Hopf A. Salt DE. 2005. Constitutively elevated salicylic acid signals glutathione-mediated nickel tolerance in Thlaspi nickel hyperaccumulators. Plant Physiology 137: 10821091.
  • Freeman JL, Persans MW, Nieman K, Albrecht C, Peer W, Pickering IJ, Salt DE. 2004. Increased glutathione biosynthesis plays a role in nickel tolerance in Thlaspi nickel hyperaccumulators. Plant Cell 16: 21762191.
  • Gabbrielli R, Grossi L, Vergnano O. 1989. The effects of nickel, cadmium and magnesium on the acid phosphatase activity of two Alyssum species. New Phytologist 111: 631636.
  • Gabbrielli R, Pandolfini T. 1984. Effect of Mg2+ and Ca2+ on the response to nickel toxicity in a serpentine endemic and nickel-accumulating species. Physiologia Plantarum 62: 540544.
  • Gajewska E, Sklodowska M, Slaba M, Mazur J. 2006. Effect of nickel on oxidative enzyme activities, proline and chlorophyll content in wheat shoots. Biologia Plantarum 50: 653659.
  • Ghaderian SM, Mohtadi A, Rahiminejad R, Reeves RD, Baker AJM. 2007. Hyperaccumulation of nickel by two Alyssum species from the serpentine soils of Iran. Plant Soil 293: 9197.
  • Gibbs CR. 1976. Characterization and application of FerroZine iron reagent as a ferrous iron indicator. Analytical Chemistry 48: 11971201.
  • Goodwin-Bailey CI, Woodell SRG, Loughman BC. 1992. The responses of serpentine, mine spoil and salt marsh races of Armeria maritima (Mill.) Willd. to each others’ soils. In: BakerAJM, ProctorJ, ReevesRD, eds. The vegetation of ultramafic (Serpentine) soils. Andover, UK: Intercept Limited, 375390.
  • Green LS, Rogers EE. 2004. FRD3 controls iron localization in Arabidopsis. Plant Physiology 136: 25232531.
  • Hell R, Stephan UW. 2003. Iron uptake, trafficking and homeostasis in plants. Planta 216: 541551.
  • Hoagland DR, Arnon DI. 1950. The water-culture method for growing plants without soil. California Agricultural Experimental Station Circular 347: 132.
  • Homer FA, Morrison RS, Brooks RR, Clemens J, Reeves RD. 1991a. Comparative studies of nickel, cobalt and copper uptake by some nickel hyperaccumulators of the genus Alyssum. Plant Soil 138: 195205.
  • Homer FA, Reeves RD, Brooks RR, Baker AJM. 1991b. Characterization of the nickel extract from the nickel hyperaccumulator Dichapetalum gelonioides. Phytochemistry 30: 21412145.
  • Ingle RA, Fricker MD, Smith JAC. 2008. Evidence for nickel/proton antiport activity at the tonoplast of the hyperaccumulator plant Alyssum lesbiacum. Plant Biology (Stuttg) 10: 746753.
  • Ingle RA, Mugford ST, Rees JD, Campbell MM, Smith JAC. 2005a. Constitutively high expression of the histidine biosynthetic pathway contributes to nickel tolerance in hyperaccumulator plants. Plant Cell 17: 20892106.
  • Ingle RA, Smith JAC, Sweetlove LJ. 2005b. Responses to nickel in the proteome of the hyperaccumulator plant Alyssum lesbiacum. BioMetals 18: 627641.
  • Kampfenkel K, Kushnir S, Babiychuk E, Inzé D, Van Montagu M. 1995. Molecular characterization of a putative Arabidopsis thaliana copper transporter and its yeast homologue. Journal of Biological Chemistry 270: 2847928486.
  • Kelly PC, Brooks RR, Dilli S, Jaffré T. 1975. Preliminary observations on the ecology and plant chemistry of some nickel-accumulating plants from New Caledonia. Proceedings of the Royal Society of London Series B 189: 6980.
  • Kerkeb L, Krämer U. 2003. The role of free histidine in xylem loading of nickel in Alyssum lesbiacum and Brassica juncea. Plant Physiology 131: 716724.
  • Khalid BY, Tinsley J. 1980. Some effects of nickel toxicity on rye grass. Plant Soil 55: 139144.
  • Krämer U, Baker, AJM, Hawes CR, Smith JAC, Grime, GW. 1997a. Micro-PIXE as a technique for studying nickel localisation in leaves of the hyperaccumulator plant Alyssum lesbiacum. Nuclear Instruments and Methods in Physics Research B 130: 346350.
  • Krämer U, Clemens S. 2006. Functions and homeostasis of zinc, copper, and nickel in plants. Topics in Current Genetics 14: 216271.
  • Krämer U, Cotter-Howells JD, Charnock JM, Baker AJM, Smith JAC. 1996. Free histidine as a metal chelator in plants that accumulate nickel. Nature 379: 635638.
  • Krämer U, Pickering IJ, Prince RC, Raskin I, Salt DE. 2000. Subcellular localization and speciation of nickel in hyperaccumulator and nonaccumulator Thlaspi species. Plant Physiology 122: 13431353.
  • Krämer U, Smith RD, Wenzel WW, Raskin I, Salt DE. 1997b. The role of metal transport and tolerance in nickel hyperaccumulation by Thlaspi goesingense Halacsy. Plant Physiology 5: 16411650.
  • Krämer U, Talke IN, Hanikenne M. 2007. Transition metal transport. FEBS Letters 581: 22632272.
  • Kropat J, Tottey S, Birkenbihl RP, Depège N, Huijser P, Merchant S. 2005. A regulator of nutritional copper signaling in Chlamydomonas is an SBP domain protein that recognizes the GTAC core of copper response element. Proceedings of the National Academy of Sciences, USA 102: 1873018735.
  • Küpper H, Küpper F, Spiller M. 1998. In situ detection of heavy metal substituted chlorophylls in water plants. Photosynthesis Research 58: 123133.
  • Küpper H, Lombi E, Zhao FJ, McGrath SP. 2000. Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri. Planta 212: 7584.
  • Küpper H, Lombi E, Zhao FJ, Wieshammer G, McGrath SP. 2001. Cellular compartmentation of nickel in the hyperaccumulators Alyssum lesbiacum, Alyssum bertolonii and Thlaspi goesingense. Journal of Experimental Botany 52: 22912300.
  • Lee J, Reeves RD, Brooks RR, Jaffré T. 1978. The relation between nickel and citric acid in some nickel-accumulating plants. Phytochemistry 17: 10331035.
  • Main JL. 1974. Differential responses to magnesium and calcium by native populations of Agropyron spicatum. American Journal of Botany 61: 931937.
  • Maly FE, Nakamura M, Gauchat JF, Urwyler A, Walker G, Dahinden CA, Cross AR, Jones OTG, Weck AL. 1989. Superoxide-dependent nitroblue tetrazolium reduction and expression of cytochrome b245 components by human tonsillar lymphocytes and B cell lines. Journal of Immunology 142: 12601267.
  • Marschner H. 1995. Mineral nutrition of higher plants, 2nd edn. New York, NY, USA: Academic Press.
  • Montargès-Pelletier E, Chardot V, Echevarria G, Michot LJ, Bauer A, Morel J-L. 2008. Identification of nickel chelators in three hyperaccumulating plants: an X-ray spectroscopic study. Phytochemistry 69: 16951709.
  • Nakano Y, Asada K. 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant and Cell Physiology 22: 867880.
  • Piccini DF, Malavolta E. 1992. Effect of nickel on two common bean cultivars. Journal of Plant Nutrition 15: 23432350.
  • Puig S, Andres-Colas N, Garcia-Molina A, Peñarrubia L. 2007. Copper and iron homeostasis in Arabidopsis: responses to metal deficiencies, interactions and biotechnological applications. Plant, Cell & Environment 30: 271290.
  • Richau KH, Kozhevnikova AD, Seregin IV, Voojis R, Koevoets PLM, Smith JAC, Ivanov V, Schat H. 2009. Chelation by histidine inhibits the vacuolar sequestration of nickel in roots of the hyperaccumulator Thlaspi caerulescens. New Phytologist 183: 106116.
  • Robinson NJ, Procter CM, Connolly EL, Guerinot ML. 1999. A ferric-chelate reductase for iron uptake from soils. Nature 397: 694697.
  • Sancenon V, Puig S, Mira H, Thiele DJ, Peñarrubia L. 2003. Identification of a copper transporter family in Arabidopsis thaliana. Plant Molecular Biology 51: 577587.
  • Seregin IV, Kozhevnikova AD. 2006. Physiological role of nickel and its effects on higher plants. Russian Journal of Plant Physiology 53: 257277.
  • Sheoran IS, Singal HR, Singh R. 1990. Effect of cadmium and nickel on photosynthesis and the enzymes of the photosynthetic carbon reduction cycle in pigeon pea (Cajanus cajan L.). Photosynthesis Research 23: 345351.
  • Da Silva JJRF, Williams RJP. 1991. The biological chemistry of the elements. Oxford, UK: Oxford University Press.
  • Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB. 1997. Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley–powdery mildew interaction. Plant Journal 11: 11871194.
  • Tiffin LO. 1966. Iron translocation: plant culture, exudates sampling, iron citrate analysis. Plant Physiology 45: 280283.
  • Tiffin LO. 1971. Translocation of nickel in xylem exudate of plants. Plant Physiology 48: 273277.
  • Tilstone GH, Macnair MR. 1997. Nickel tolerance and copper-nickel co-tolerance in Mimulus guttatus from copper mine and serpentine habitats. Plant Soil 191: 173180.
  • Tottey S, Block MA, Allen M, Westergren T, Albrieux C, Scheller HV, Merchant S, Jensen PE. 2003. Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide. Proceedings of the National Academy of Sciences, USA 100: 1611916124.
  • Vert G, Grotz N, Dédaldéchamp F, Gaymard F, Guerinot ML, Briat JF, Curie C. 2002. IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth. Plant Cell 14: 12231233.
  • Welch RM, Norvell WA, Schaefer SC, Shaff JE, Kochian LV. 1993. Induction of iron(III) and copper(II) reduction in pea (Pisum sativum L.) roots by Fe and Cu status: does the root-cell plasmalemma Fe(III)-chelate reductase perform a general role in regulating cation uptake? Planta 190: 555561.
  • Whiting SN, Reeves RD, Richards D, Johnson MS, Cooke JA, Malaisse F, Paton A, Smith JAC, Angle JS, Chaney RL et al . 2004. Research priorities for conservation of metallophyte biodiversity and their potential for restoration and site remediation. Restoration Ecology 12: 106116.
  • Wintz H, Fox T, Wu YY, Feng V, Chen W, Chang HS, Zhu T, Vulpe C. 2003. Expression profiles of Arabidopsis thaliana in mineral deficiencies reveal novel transporters involved in metal homeostasis. Journal of Biological Chemistry 278: 4764447653.
  • Woolhouse HW. 1983. Toxicity and tolerance in the responses of plants to metals. In: LangeOL, NobelPS, OsmondCB, ZieglerH, eds. Encyclopedia of plant physiology, new series, Vol. 12C, Berlin, Germany: Springer, 245300.
  • Wycisk K, Kim EJ, Schroeder JI, Krämer U. 2004. Enhancing the first enzymatic step in the histidine biosynthesis pathway increases the free histidine pool and nickel tolerance in Arabidopsis thaliana. FEBS Letters 578: 128134.
  • Yamasaki H, Hayashi M, Fukazawa M, Kobayashi Y, Shikanai T. 2009. SQUAMOSA promoter binding protein-Like7 is a central regulator for copper homeostasis in Arabidopsis. Plant Cell 21: 347361.
  • Yi Y, Guerinot ML. 1996. Genetic evidence that induction of root Fe(III) chelate reductase activity is necessary for iron uptake under iron deficiency. Plant Journal 10: 835844.