| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online March 2, 2007; 10.1104/pp.106.094474 Plant Physiology 143:1761-1773 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Iron Acquisition by Phytosiderophores Contributes to Cadmium Tolerance1,[OA]Molecular Plant Nutrition, Institute of Plant Nutrition, University of Hohenheim, 70593 Stuttgart, Germany (A.R.M., E.B.S., U.E.P., G.S., N.v.W.); Soil Science and Plant Nutrition, Faculty of Agriculture, Cukurova University, 01330 Adana, Turkey (B.E.); and Institute for Analytical Sciences, 44139 Dortmund, Germany (H.H., G.W.)
Based on the ability of phytosiderophores to chelate other heavy metals besides iron (Fe), phytosiderophores were suggested to prevent graminaceous plants from cadmium (Cd) toxicity. To assess interactions between Cd and phytosiderophore-mediated Fe acquisition, maize (Zea mays) plants were grown hydroponically under limiting Fe supply. Exposure to Cd decreased uptake rates of 59Fe(III)-phytosiderophores and enhanced the expression of the Fe-phytosiderophore transporter gene ZmYS1 in roots as well as the release of the phytosiderophore 2'-deoxymugineic acid (DMA) from roots under Fe deficiency. However, DMA hardly mobilized Cd from soil or from a Cd-loaded resin in comparison to the synthetic chelators diaminetriaminepentaacetic acid and HEDTA. While nano-electrospray-high resolution mass spectrometry revealed the formation of an intact Cd(II)-DMA complex in aqueous solutions, competition studies with Fe(III) and zinc(II) showed that the formed Cd(II)-DMA complex was weak. Unlike HEDTA, DMA did not protect yeast (Saccharomyces cerevisiae) cells from Cd toxicity but improved yeast growth in the presence of Cd when yeast cells expressed ZmYS1. When supplied with Fe-DMA as a Fe source, transgenic Arabidopsis (Arabidopsis thaliana) plants expressing a cauliflower mosaic virus 35S-ZmYS1 gene construct showed less growth depression than wild-type plants in response to Cd. These results indicate that inhibition of ZmYS1-mediated Fe-DMA transport by Cd is not related to Cd-DMA complex formation and that Cd-induced phytosiderophore release cannot protect maize plants from Cd toxicity. Instead, phytosiderophore-mediated Fe acquisition can improve Fe uptake in the presence of Cd and thereby provides an advantage under Cd stress relative to Fe acquisition via ferrous Fe.
Under certain challenging environmental conditions, plant roots may enhance the release of root exudates that can either increase the mobilization of essential mineral elements in the rhizosphere or inhibit uptake of toxic mineral elements from the soil solution (Marschner, 1995  ). These contrasting functions might apply even for one molecule or class of molecules depending on the physiological and environmental context. For example, citrate and other dicarboxylates are released by phosphorus-deficient or iron (Fe)-deficient plant roots to mobilize phosphate from sparingly soluble calcium (Ca), Fe, or aluminum (Al) precipitates. While Fe chelation by citrate promotes Fe solubility and subsequent Fe uptake, Al chelation by citrate decreases Al uptake (Neumann and Römheld, 2000). Thus, several plant species release citrate or other carboxylates in response to elevated Al concentrations in the soil solution to protect their roots from Al toxicity (Kochian et al., 2004).
Under Fe deficiency, graminaceous plant species release phytosiderophores, which are hexadentate metal chelators with high affinity for complex formation with Fe(III). The whole Fe(III)-phytosiderophore complex is subsequently taken up by Fe deficiency-inducible transporters of the YS1/YSL protein family (Römheld and Marschner, 1986
Plants represent the major route for the entry of Cd into the food chain. In humans and mammals, Cd might then cause genotoxic and cytotoxic effects, leading finally to the inhibition of cell proliferation and apoptosis. In plants, Cd can cause the inhibition of photosynthesis, respiration, and nitrogen metabolism as well as a decrease in water and mineral nutrient uptake (Deckert, 2005
Inhibitory effects of Cd on the uptake of metal micronutrients have been described for different transport systems and growth conditions. With regard to transport processes across plant membranes, Cd efficiently competed with Fe, Mn, or Zn transport by AtIRT1 (Eide et al., 1996 It was therefore the aim of this study to evaluate a role of the phytosiderophore 2'-deoxymugineic acid (DMA) in preventing Cd toxicity in maize plants and to analyze possible interactions of Cd with Fe acquisition. To characterize the Cd-chelating potential of DMA, we conducted Cd mobilization tests from different substrates by DMA, carried out competition studies with Fe and Zn, and verified complex formation by high-resolution mass spectrometry (MS). We then investigated whether the presence of DMA protects yeast (Saccharomyces cerevisiae) or plant cells from Cd toxicity and how this relates to Fe uptake systems. Finally, we tested the hypothesis whether phytosiderophore-mediated Fe uptake in transgenic Arabidopsis (Arabidopsis thaliana) plants may decrease Cd sensitivity.
Uptake and Translocation of Fe Are Decreased in the Presence of Cd
As a prerequisite to verify a role of phytosiderophores in the alleviation of Cd toxicity in strategy II plants, we first investigated the effect of Cd on growth and metal accumulation in maize plants precultured under adequate or limiting Fe supply. Despite 12 or 24 h of growth in the absence or presence of increasing Cd concentrations, biomass formation of roots and shoots was still unaffected by Cd irrespective of the Fe nutritional status (Fig. 1A
). As expected, Cd accumulation in roots and shoots increased with the duration of Cd exposure and amount of Cd supply (Fig. 1B). Roots and shoots of plants that were grown under Fe deficiency, however, accumulated on average 2-fold more Cd than Fe-sufficient plants. At the same time, Fe concentrations in the shoot tissue of Cd-treated plants were unaffected by Cd exposure (Fig. 1C) and remained above a critical level of 66 µg g–1 (Marschner, 1995
We then investigated a direct effect of Cd on Fe-DMA uptake. Uptake rates of 59Fe-labeled Fe-DMA were determined in the presence of Cd or after pretreating roots for 24 h with Cd. In the presence of 25 µM Cd only during the uptake assay, 59Fe-DMA uptake rates decreased by approximately 20% in Fe-sufficient roots relative to nontreated roots (Fig. 2 ). However, when roots were preincubated with Cd for 12 h, Fe-DMA uptake rates decreased by approximately 50% irrespective of whether Cd was still present or not during the uptake period. In Fe-deficient roots, uptake rates of Fe(III)-DMA were at an approximately 2.5 times higher level than in Fe-sufficient roots, but Cd showed similar quantitative effects, with Cd preincubation of roots leading to the strongest suppression of Fe(III)-DMA uptake rates. This experiment suggested that inhibition of Fe-DMA uptake is an early event in Cd-mediated toxicity.
Translocation rates of 59Fe showed exactly the same relative differences among Cd treatments as root uptake rates but at an approximately 20-fold lower level (data not shown). To better quantify this observation, we compared the Fe concentration in the xylem sap of Fe-deficient maize plants after Fe resupply in the presence or absence of Cd. While the bleeding rate of the xylem sap remained unaffected by Cd (data not shown), Fe concentrations in the xylem sap of decapitated maize plants decreased by approximately 50% in response to Cd supply (Fig. 3 ). Cd-treated plants translocated an almost equimolar amount of Cd and Fe to the shoots. These lower Fe translocation rates in the presence of Cd most likely reflected the inhibition of Fe-DMA uptake by Cd (Fig. 2) and suggested that 48 h of Cd exposure before determining total Fe concentrations in shoots (Fig. 1C) was obviously too short to significantly decrease Fe concentrations in the shoot tissue.
Cd Up-Regulates the Fe-Deficiency Stress Response in Maize Roots
To characterize the Fe nutritional status of Cd-treated maize plants, expression of the major Fe-phytosiderophore transporter in Fe-deficient maize roots, ZmYS1 (Curie et al., 2001
Phytosiderophores Poorly Mobilize Cd To investigate a role of phytosiderophores in Cd chelation, an aliquot of a soil being naturally rich in heavy metals was extracted by phytosiderophores or by the synthetic chelator diaminetriaminepentaacetic acid (DTPA). In agreement with ammonium nitrate-extractable amounts of metals (see "Materials and Methods"), DTPA efficiently mobilized Zn, Cu, and to a lesser extent also Cd (Table I ), confirming that Cd mobilization is increased in the presence of this ligand. In contrast, DMA efficiently mobilized Cu and Zn but almost no Cd, suggesting a particularly low affinity of phytosiderophores to Cd in comparison to synthetic chelators.
Because metal-binding forms and equilibrium reactions in soils are complex and difficult to predict, we reevaluated the Cd mobilization capacity by phytosiderophores using a synthetic resin loaded with a single metal in incubation assays with DMA for comparison to the synthetic chelator HEDTA that is a weaker ligand for Cd, Zn, and Cu than DTPA (Norvell, 1991 ). As expected, both chelators mobilized Fe(III) from the resin and even more efficiently Cu(II) (Fig. 5
), which is in agreement with a slightly higher complex formation constant of DMA with Cu(II) than with Fe(III) (Murakami et al., 1989). Notably, Cd mobilization was also high for HEDTA but not detectable in the case of DMA (Fig. 5). This was unexpected with regard to the metal adsorption properties reported for the resin, which has been reported to bind Cu even more firmly than Cd (manufacturer's protocol, Bio-Rad). Taken together, two independent mobilization assays showed that the phytosiderophore DMA is apparently a weak or inefficient ligand for the chelation of Cd relative to Zn, Cu, or Fe(III).
Low Competitivity of Cd in Metal-Phytosiderophore Complex Formation
Nano-electrospray-Fourier transform ion cyclotron resonance-MS (nano-ESI-FTICR-MS) was then employed to verify the formation of Cd-DMA complexes in aqueous solutions. This type of high-resolution MS has recently been shown to accurately discriminate among metal chelate species that differ in even less than 0.02 mass-to-charge ratio (m/z; Weber et al., 2006
As an alternative approach to characterize the complex stability of Cd-DMA relative to other metals, Fe(III) and Zn were added to a 10-µM Cd-DMA solution before the Cd-DMA complex was redetermined by nano-ESI-FTICR. Addition of Fe(III) in an equimolar ratio to Cd led to an almost complete disappearance of the Cd-DMA complex (Fig. 6B). Employing Zn as a competing metal, which has an approximately 5 orders of magnitude lower stability constant with DMA relative to Fe(III) (Murakami et al., 1989 ), also led to an almost complete dissociation of Cd from the ligand. Even though these competition experiments were conducted in simplified solutions that did not consider the presence of other chelating and competing substances or redox reactions that might occur simultaneously in complex soil solutions, they indicated that Cd was rapidly outcompeted by Fe(III) and Zn from the DMA complex. This was in strong agreement with the observation that Cd was not chelated by DMA in the presence of other heavy metals (Table I). When equimolar Cd concentrations were added to compete with Fe(III) or Zn(II) in a preformed DMA complex, Cd did not significantly displace any Fe(III) or Zn from the DMA complex (Fig. 6C), confirming that DMA plays a minor role in Cd complexation.
Because the presence of other metals leads to a destabilization of the Cd-DMA complex (Fig. 6C), a short-term uptake experiment was conducted in which root uptake of DMA-chelated 109Cd was compared to that of free 109Cd2+ in the absence of competing metals. For that purpose, wild-type maize plants were starved for Fe, which led to an increase in the uptake capacity for 59Fe-labeled Fe(III)-DMA (Fig. 7A
). Fe-deficient ys1 mutant plants lacking functional ZmYS1 expression (Schaaf et al., 2004a
Phytosiderophores Cannot Protect Yeast from Cd Toxicity
Because Hill et al. (2002)
Overexpression of the ZmYS1 Gene Alleviates Strategy I Plants from Cd Toxicity in the Presence of Fe-DMA Following the observations that ZmYS1-expressing yeast cells grew slightly better in the presence of Cd (Fig. 8B) and that overall Cd uptake rates were independent of phytosiderophores (Fig. 7), we compared Cd sensitivity of Fe acquisition via Fe2+ or Fe-DMA in planta. For this purpose, the ZmYS1 cDNA was overexpressed under control of a cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis. Homozygote T2 plants were obtained based on segregation analysis on kanamycin. Three independent lines were selected in the T3 generation and confirmed for expression of the ZmYS1 gene in a northern-blot analysis (Fig. 9A ). Transcript levels of ZmYS1 were high in roots of the lines 3 and 9, but low in line 11. Plants of these lines were then pregerminated in 0.5 Murashige and Skoog medium for 8 d before being transferred to agar plates containing either Fe-phytosiderophores or Fe-EDTA as a Fe source and continued to grow for 10 d. In the absence of Cd, wild-type and transgenic lines grew similarly well and free of any visual symptoms under supply of either Fe form (Fig. 9B). In the presence of Cd, however, wild-type plants and transgenic lines grown on Fe-EDTA showed an approximately 30% decrease in shoot biomass accompanied by severe chlorosis (Fig. 9, C and D). These Cd-induced symptoms were similar in all lines. When Fe was supplied as Fe-DMA, however, the two CaMV35S-ZmYS1-transformed lines with highest expression levels exhibited no significant decrease in shoot fresh weight in response to 15 µM Cd and only an approximately 15% decrease in response to 30 µM Cd relative to control plants not exposed to Cd (Fig. 9C). With regard to plant development, shoot biomass of the two transgenic lines 3 and 9 and leaf chlorophyll levels of line 3 were superior to those of wild-type plants grown on Fe-DMA. Taken together, these results indicated that Cd-induced growth repression in wild-type plants is similar under supply of Fe-EDTA or Fe-DMA as Fe source, but that Cd stress is alleviated in transgenic plants expressing ZmYS1 provided that Fe-DMA is made available.
Cd Chelation by Phytosiderophores Is Too Weak to Protect Plant Roots from Cd Uptake
In nongraminaceous plant species, the Fe2+ transporter IRT1 has been identified as a major transport pathway for Cd uptake in Fe-deficient roots (Cohen et al., 1998
First, the phytosiderophore DMA hardly mobilized any Cd from a soil substrate that was naturally rich in Cd (Table I). While phytosiderophores may solubilize Cd from precipitated Cd3(PO4)2 even in the presence of Fe(OH)3 (Shenker et al., 2001
A weak complex formation was then identified as a major reason for the inefficiency of phytosiderophores to mobilize Cd. Chemical analysis by nano-ESI-FTICR MS of an aqueous solution to which Cd(II) and DMA were added separately proved formation of an intact complex (Fig. 6A). This Cd(II)-DMA complex was formed in a 1:1 stoichiometry, comparable to that of Fe(III)-DMA (Weber et al., 2006
Most likely as a consequence of weak complex formation, DMA also failed to protect yeast cells from Cd toxicity. In contrast to the addition of the synthetic chelator HEDTA, DMA supplementation did not alleviate yeast wild-type cells from Cd stress (Fig. 8). The inability of DMA to alleviate yeast cells from Cd toxicity was recently reported by Nakanishi et al. (2006)
With regard to transport processes across plant membranes, Cd has been reported to efficiently compete with Fe, Mn, or Zn transport by AtIRT1 (Eide et al., 1996
Based on the findings that Cd induces Fe-deficiency stress responses and IRT1-mediated Cd accumulation in strategy I plants (Cohen et al., 1998
As ZmYS1 did not contribute to Cd uptake (Fig. 7), Fe-phytosiderophore transport in general might improve Fe nutrition under Cd stress in grasses relative to nongraminaceous plants. To confer transgenic Arabidopsis plants the possibility of taking up Fe via phytosiderophores, we expressed ZmYS1 constitutively and grew these plants on Fe(III)-EDTA or Fe(III)-DMA. Under supply of Fe(III)-EDTA and in the presence of Cd, wild-type and transgenic Arabidopsis lines exhibited severe chlorosis, whereas transgenic lines expressing ZmYS1 at higher levels remained greener and showed less growth suppression when Fe(III)-DMA was the Fe source (Fig. 9, B and C). In comparison, Connolly et al. (2002)
Plant Culture and Growth Conditions Maize (Zea mays) seeds (UH002 inbred line or ys1) were rinsed for 3 min in 70% ethanol for 10 min in 15% hydrogen peroxide solution and finally in distilled water. Seeds were germinated in the dark between filter papers soaked with CaSO4-saturated solution for 4 to 5 d and transferred to a half-strength nutrient solution without Fe. After 2 d, seedlings were transferred to a full nutrient solution containing 2.0 mM Ca(NO3)2, 0.7 mM K2SO4, 0.5 mM MgSO4, 0.1 mM KCl, 0.1 mM KH2PO4, 1.0 µM H3BO3, 0.5 µM MnSO4, 0.5 µM ZnSO4, 0.2 µM CuSO4, and 0.01 µM (NH4)6Mo7O24. In Fe-sufficient treatments, 100 µM Fe(III)-EDTA was supplied. The nutrient solution was renewed every 2 or 3 d. Plants were grown hydroponically under aerated and nonsterile conditions in a climate chamber under the following conditions: 16/8 h light/dark; light intensity 280 µmol photons m–2 s–1; temperature 25°C/20°C; and 60% humidity. In all experiments, plants were harvested at the same time of day (usually 7 h after onset of light).
For short-term uptake experiments of radiolabeled substrates, plants were transferred 1 d before the experiment to 0.5-L pots containing nutrient solution without micronutrients. Plants were then transferred to fresh nutrient solution supplemented with 10 µM 109CdSO4, 109Cd-DMA, or 10 µM 59Fe(III)-DMA (the latter two with 10% excess of DMA), which had been prepared 1 d before the experiment and shaken overnight. The specific activity was 6.2 GBq/mol for both radioisotopes. To remove the extraplasmatic Cd, plants were rinsed with 1 mM CaSO4 solution for 10 min, whereas the Fe-DMA-treated plants were washed with 1 mM CaSO4 solution and bipyridyl solution under N2 flushing to create reductive conditions and remove apoplasmic Fe (Bienfait et al., 1985
Xylem sap was collected from maize plants precultured under Fe deficiency for 23 d. Plants were resupplied with 100 µM Fe(III)-EDTA in the presence or absence of 25 µM CdCl2 48 h before being detopped. Liquid appearing on the excised stem surface during the first few minutes was taken off before a flexible tube was fixed onto the cut end of the stem. Xylem sap exuded for 3 h was collected with a Pasteur pipette and stored at –20°C until further analysis.
The phytosiderophore DMA used in all experiments was obtained from root exudates of wheat (Triticum aestivum) cv Ares plants, because wheat roots release large amounts of DMA. Seeds were surface rinsed as above and germinated for 6 d in the dark in quartz sand moistened with saturated CaSO4 solution. Seedlings were transferred to continuously aerated nutrient solution. Plants were grown for 2 weeks and nutrient solution was exchanged every 2 to 3 d. After 1 week of preculture under Fe deficiency, root exudates were collected daily. The root system of approximately 50 plants per pot was first rinsed in deionized water for 30 min and then transferred to 500 mL deionized water for 4 h under continuous aeration, starting 2 h after onset of the light period (Cakmak et al., 1998
Maize seeds (cv UH002) were surface sterilized with 96% ethanol for 3 min followed by incubation in 3% sodium hypochloride for 30 min. Seeds were then soaked with autoclaved CaSO4-saturated solution at room temperature during 2 h before being placed on solid half-strength modified Murashige and Skoog medium (without Fe) containing 1% Suc and 0.7% agar (Difco, Becton Diekison). Seeds were incubated in the dark at 28°C for 3 d. Individual seedlings were then transferred to sterile glass tubes containing 12 mL Fe-free nutrient solution in which the plantlets were held by inserted 5-mL pipette tips. Plantlets continued to grow during 4 d before transfer to Fe-free nutrient solution in the absence or presence of 25 µM CdCl2 for 24 h. For collection of root exudates, plants were transferred to sterile ultrapure water (Elga) for a period of 6 h. A drop test on solid Luria-Bertani medium from collected exudates of each sample was performed to verify that the solution was free of microbial contamination. DMA concentrations were determined by HPLC according to Neumann et al. (1999)
To obtain Cd or Cu-loaded Chelex 100, 4.4 g of Na-loaded resin (Bio-Rad) were washed with 100 mL 1-M HCl in a glass bead column and rinsed with an excess of ultrapure water (Elga) until water drops reached a pH of 5.5 to 6.0. Resins were stirred in 200 mL 50-µM CdCl2 or CuSO4 for 15 min. The suspensions obtained were poured back onto a glass bead column and washed with ultrapure water until washing solutions were free of Cd or Cu, as verified by atomic absorption spectroscopy. In addition, the commercially available Fe-loaded resin Chelex 100 was used (Bio-Rad). The metal-loaded resins were then kept in a 10-mM MES-Tris buffered solution, pH 5.5. For the mobilization test, 2 mL of resin suspension were added to 8 mL water or chelate solution (5 µM HEDTA or DMA at pH 5.5) and shaken at 100 rpm at room temperature for 1 h. Filtrates (Blue ribbon no. 5893, Schleich und Schüll) were analyzed for Cd and Cu concentrations by inductively coupled plasma optical emission spectrometry and Fe by atomic absorption spectroscopy. A Rendzina soil with a pH (in CaCl2) of 6.4 and 1.4% total organic matter was collected from an agricultural area in Bonndorf, Germany and used for metal mobilization tests. The total concentrations of Cd, Cu, and Zn were 17.0, 53.0, and 443.0 mg kg–1, respectively, while NH4NO3-extractable concentrations of Cd, Cu, and Zn were 96.0, 137.5, and 193.3 µg kg–1, respectively. Five grams of homogenized and dried soil was shaken in 20 mL water (control) or 20 mL 50-µM chelate (DTPA or DMA) at 175 rpm for 1 h and filtered (Blue ribbon no. 5893, Schleich und Schüll). Filtrates were analyzed for Cd, Cu, and Zn concentrations by inductively coupled plasma optical emission spectrometry.
The Cd(II) complex was prepared from an aqueous DMA solution (20 µM in 50 mM ammonium bicarbonate, pH 7.3) by adding an appropriate amount of a 1-mM aqueous solution of Cd(II) chloride (Merck). The pH was readjusted to 7.3 with HCl or aqueous ammonia solution, if necessary. The competition experiments with Fe(III) and Zn(II) were carried out by adding the respective amount of a 1-mM solution of Fe(III) chloride hexahydrate (Roth) or Zn(II) acetate dihydrate (Roth). To improve the comparability of the different measurements, L-Trpe (Roth) was added as an internal standard yielding a final concentration of 20 µM. All signal intensities were analyzed relative to the L-Trpe signal intensity (m/z 203.0826). Before measurement, the samples were diluted with methanol (liquid chromatography-MS grade, Riedel-de Haën) to 70% to avoid problems induced by high surface tension of pure water and to facilitate a stable spray at reduced needle voltage (without electrical discharge). The theoretical masses and isotope distributions for the complexes were calculated using the Xcalibur software LTQ FT version 1.4.2 (Thermo Electron). All nano-ESI-FTICR-MS experiments were carried out using a LCQ FT FTICR hybrid mass spectrometer (Thermo Electron) equipped with a 7.0 Tesla actively shielded superconducting magnet and nano-ESI source. Gold-plated nano-ESI pipettes (tip i.d. approximately 1 µm) were from MasCom. The instrument was operated in negative ionization mode. Ion transmission into the linear trap and signal intensity was automatically optimized for maximum ion signal of the Fe(III)-DMA complex. The parameters were: source voltage, 0.8 to 1.0 kV; capillary voltage, 20 V; capillary temperature, 100°C; and tube lens voltage, 80 V. The targets for the full scan linear trap and FTICR cell were 3 x 104 and 2 x 105, respectively. The resolving power of the FTICR mass analyzer was set to 200,000 (full width at half maximum at m/z = 400). Full scan FTICR mass spectra in the mass range m/z 150 to 800 were acquired using a single microscan, and displayed mass spectra were averaged from 50 single spectra. The instrument was calibrated externally using 0.01% solution of 85% phosphoric acid in water/methanol (1:1, v/v).
For growth complementation, the yeast (Saccharomyces cerevisiae) strains fet3fet4 (DEY1453, MATa/MAT
Using a BamHI restriction site, the open reading frame (ORF) containing a 32-bp 5'-untranslated region of ZmYS1 in pDR196 (Schaaf et al., 2004b
Total RNA was isolated by Trizol extraction (Invitrogen). RNA (20 µg per lane) was separated by electrophoresis on MOPS-formaldehyde agarose gels, blotted onto Hybond-N+ nylon membranes (Amersham Biosciences), and cross-linked to the membrane by incubation at 80°C for 2 h. The ORF of ZmYS1 was used as a probe for hybridization to total RNA. Membrane hybridization, probe labeling, and membrane washing were conducted essentially as described (Loqué et al., 2005
For growth test of Arabidopsis plants on agar plates, seeds of wild-type and transgenic plants were germinated and grown for 8 d on modified 0.5 Murashige and Skoog medium (Duchefa), 0.5% Suc, solidified with 1% Difco agar (Becton Diekison), and transferred to the same medium supplemented with metals at indicated concentrations. Plants were grown in a growth chamber with the following conditions: 10/14 h light/dark; light intensity, 120 µmol photons m–2 s–1; and temperature 22°C/19°C at day/night. Chlorophyll concentrations were measured according to Moran and Porath (1980) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers U27590 and AF186234.
We thank Susanne Reiner, Jennifer Haeberle, Silvia Kirchner, and Maria Ruckwied, University of Hohenheim, for excellent technical assistance, and David Eide, University of Wisconsin-Madison, for providing the yeast strains. Received December 8, 2006; accepted February 15, 2007; published March 2, 2007.
1 This work was supported by the Deutsche Forschungsgemeinschaft, Bonn (grant nos. WI1728/6–1 to N.v.W. and WE 2422/5–1 to G.W.), and by the German Academic Exchange Agency, Bonn (fellowship to A.R.M.). 
2 These authors contributed equally to the paper. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Nicolaus von Wirén (vonwiren{at}uni-hohenheim.de).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.094474 * Corresponding author; e-mail vonwiren{at}uni-hohenheim.de; fax 49–711–45923295.
Awad F, Römheld V (2000) Mobilization of heavy metals from contaminated calcareous soils by plant born, microbial and synthetic chelators and their uptake by wheat plants. J Plant Nutr 23: 1847–1855[Web of Science] Bienfait HF, Briel W, Mesland-Mul NT (1985) Free space iron pools in roots: generation and mobilization. Plant Physiol 78: 596–600 Cakmak I, Erenoglu B, Gülüt KY, Derici R, Römheld V (1998) Light-mediated release of phytosiderophores in wheat and barley under iron or zinc deficiency. Plant Soil 202: 309–315[CrossRef][Web of Science] Clemens S (2006) Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants. Biochimie 88: 1707–1719[Medline] Clemens S, Antosiewicz DM, Ward JM, Schachtman DP, Schroeder JI (1998) The plant cDNA LCT1 mediates the uptake of calcium and cadmium in yeast. Proc Natl Acad Sci USA 95: 12043–12048 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743[CrossRef][Web of Science][Medline] Cohen CK, Fox TC, Garvin DF, Kochian LV (1998) The role of iron-deficiency stress responses in stimulating heavy-metal transport in plants. Plant Physiol 116: 1063–1072 Cohen CK, Garvin DF, Kochian LV (2004) Kinetic properties of a micronutrient transporter from Pisum sativum indicate a primary function in Fe uptake from the soil. Planta 218: 784–792[CrossRef][Web of Science][Medline] Collins RN, Merrington G, Mclaughlin MJ, Morel JL (2003) Organic ligand and pH effects on isotopically exchangeable cadmium in polluted soils. Soil Sci Soc Am J 67: 112–121 Connolly EL, Fett JP, Guerinot ML (2002) Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation. Plant Cell 14: 1347–1357 Curie C, Panaviene Z, Loulergue C, Dellaporta SL, Briat JF, Walker EL (2001) Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature 409: 346–349[CrossRef][Medline] Deckert J (2005) Cadmium toxicity in plants: is there any analogy to its carcinogenic effect in mammalian cells? Biometals 18: 475–481[CrossRef][Web of Science][Medline] DiDonato RJ, Roberts LA, Sanderson T, Eisley RB, Walker EL (2004) Arabidopsis yellow stripe-like2 (YSL2): a metal-regulated gene encoding a plasma membrane transporter of nicotianamine-metal complexes. Plant J 39: 403–414[CrossRef][Web of Science][Medline] Eide D, Broderius M, Fett J, Guerinot ML (1996) A novel iron-regulated metal transporter from plants identified by functional expression in yeast. Proc Natl Acad Sci USA 93: 5624–5628 Garnier L, Simon-Plas F, Thuleau P, Agnel JP, Blein JP, Ranjeva R, Montillet JL (2006) Cadmium affects tobacco cells by a series of three waves of reactive oxygen species that contribute to cytotoxicity. Plant Cell Environ 29: 1956–1969[CrossRef][Medline] Gietz D, St Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20: 1425 Hart JJ, Welch RM, Norvell WA, Kochian LV (2002) Transport interactions between cadmium and zinc in roots of bread and durum wheat seedlings. Physiol Plant 116: 73–78[CrossRef][Medline] Hill KA, Lion LW, Ahner BA (2002) Reduced Cd accumulation in Zea mays: a protective role for phytosiderophores? Environ Sci Technol 36: 5363–5368[Medline] Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, Wada Y, Watanabe S, Matsuhashi S, Takahashi M, et al (2006) Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+. Plant J 45: 335–346[CrossRef][Web of Science][Medline] Jalil A, Selles F, Clarke JM (1994) Effect of cadmium on growth and the uptake of cadmium and other elements by durum wheat. J Plant Nutr 17: 1839–1858[Web of Science] Jensen LT, Culotta VC (2002) Regulation of Saccharomyces cerevisiae FET4 by oxygen and iron. J Mol Biol 318: 251–260[CrossRef][Web of Science][Medline] Kochian LV, Hoekenga OA, Pineros MA (2004) How do crop plants tolerate acid soils? Mechanisms of aluminum tolerance and phosphorous efficiency. Annu Rev Plant Biol 55: 459–493[CrossRef][Medline] Koike S, Inoue H, Mizuno D, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2004) OsYSL2 is a rice metal-nicotianamine transporter that is regulated by iron and expressed in the phloem. Plant J 39: 415–424[CrossRef][Web of Science][Medline] Koleli N, Eker S, Cakmak I (2004) Effect of zinc fertilization on cadmium toxicity in durum and bread wheat grown in zinc-deficient soil. Environ Pollut 131: 453–459[CrossRef][Medline] Korshunova YO, Eide D, Clark WG, Guerinot ML, Pakrasi HB (1999) The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range. Plant Mol Biol 40: 37–44[CrossRef][Web of Science][Medline] Larbi A, Morales F, Abadia A, Gogorcena Y, Lucena JJ, Abadia J (2002) Effects of Cd and Pb in sugar beet plants grown in nutrient solution: induced Fe deficiency and growth inhibition. Funct Plant Biol 29: 1453–1464[CrossRef] Li L, Kaplan J (1998) Defects in the yeast high affinity iron transport system result in increased metal sensitivity because of the increased expression of transporters with a broad transition metal specificity. J Biol Chem 273: 22181–22187 Loqué D, Ludewig U, Yuan L, von Wirén N (2005) Tonoplast intrinsic proteins AtTIP2;1 and AtTIP2;3 facilitate NH3 transport into the vacuole. Plant Physiol 137: 671–680 Marschner H (1995) Mineral Nutrition of Higher Plants. Academic Press, New York Mench M, Martin E (1991) Mobilization of cadmium and other metals from two soils by root exudates of Zea mays L., Nicotiana tabacum L. and Nicotiana rustica L. Plant Soil 132: 187–196[Web of Science] Mills RF, Francini A, Ferreira da Rocha PSC, Baccarini PJ, Aylett M, Krijger GC, Williams LE (2005) The plant P1B-type ATPase AtHMA4 transports Zn and Cd and plays a role in detoxification of transition metals supplied at elevated levels. FEBS Lett 579: 783–791[CrossRef][Web of Science][Medline] Moran R, Porath D (1980) Chlorophyll determination in intact tissues using N,N-dimethylformamide. Plant Physiol 65: 478–479 Murakami T, Ise K, Hayakawa M, Kamei S, Takagi S (1989) Stabilities of metal complexes of mugineic acid and their specific affinities for iron(III). Chem Lett (Jpn) 18: 2137–2140 Nakanishi H, Ogawa I, Ishimaru Y, Mori S, Nishizawa NK (2006) Iron deficiency enhances cadmium uptake and translocation mediated by the Fe2+ transporters OsIRT1 and OsIRT2 in rice. Soil Sci Plant Nutr 52: 464–469[CrossRef] Neumann G, Haake C, Römheld V (1999) Improved HPLC-method for determination of phytosiderophores in root washings and tissue extracts. J Plant Nutr 22: 1389–1402[Web of Science] Neumann G, Römheld V (2000) The release of root exudates as affected by the plant's physiological status. In R Pinton, Z Varanini, Z Nannipieri, Z Varanni, eds, The Rhizosphere: Biochemistry and Organic Substances at the Soil-Plant Interface. Marcel Dekker, New York, pp 41–94 Norvell W (1991) Reactions of metal chelates in soils and nutrient solutions. In JJ Mortvedt, ed, Micronutrients in Agriculture, Ed 2. Soil Science Society of America, Madison, WI, pp 187–228 Pence NS, Larsen PB, Ebbs SD, Letham DLD, Lasat MM, Garvin DF, Eide D, Kochian LV (2000) The molecular physiology of heavy metal transport in the Zn/Cd hyperaccumulator Thlaspi caerulescens. Proc Natl Acad Sci USA 97: 4956–4960 Perfus-Barbeoch L, Leonhardt N, Vavasseur A, Forestier C (2002) Heavy metal toxicity: cadmium permeates through calcium channels and disturbs the plant water status. Plant J 32: 539–548[CrossRef][Web of Science][Medline] Roberts LA, Pierson AJ, Panaviene Z, Walker EL (2004) Yellow stripe1: expanded roles for the maize iron-phytosiderophore transporter. Plant Physiol 135: 112–120 Römheld V, Marschner H (1986) Evidence for a specific uptake system for iron phytosiderophores in roots of grasses. Plant Physiol 80: 175–180 Schaaf G, Erenoglu BE, von Wirén N (2004a) Physiological and biochemical characterization of metal-phytosiderophore transport in graminaceous species. Soil Sci Plant Nutr 50: 989–995 Schaaf G, Honsbein A, Meda AR, Kirchner S, Wipf D, von Wirén N (2006) AtIREG2 encodes a tonoplast transport protein involved in iron-dependent nickel detoxification in Arabidopsis thaliana roots. J Biol Chem 281: 25532–25540 Schaaf G, Ludewig U, Erenoglu BE, Mori S, Kitahara T, von Wirén N (2004b) ZmYS1 functions as a proton-coupled symporter for phytosiderophore- and nicotianamine-chelated metals. J Biol Chem 279: 9091–9096 Schmidt W (2003) Iron solutions: acquisition strategies and signaling pathways in plants. Trends Plant Sci 8: 188–193[CrossRef][Web of Science][Medline] Shenker M, Fan TWM, Crowley DE (2001) Phytosiderophores influence on cadmium mobilization and uptake by wheat and barley plants. J Environ Qual 30: 2091–2098 Suzuki M, Takahashi M, Tsukamoto T, Watanabe S, Matsuhashi S, Yazaki J, Kishimoto N, Kikuchi S, Nakanishi H, Mori S, et al (2006) Biosynthesis and secretion of mugineic acid family phytosiderophores in zinc-deficient barley. Plant J 48: 85–97[CrossRef][Web of Science][Medline] Thomine S, Lelievre F, Debarbieux E, Schroeder JI, Barbier-Brygoo H (2003) AtNRAMP3, a multispecific vacuolar metal transporter involved in plant responses to iron deficiency. Plant J 34: 685–695[CrossRef][Web of Science][Medline] Tolay I, Erenoglu B, Römheld V, Braun HJ, Cakmak I (2001) Phytosiderophore release in Aegilops tauschii and Triticum species under zinc and iron deficiencies. J Exp Bot 52: 1093–1099 Treeby M, Marschner H, Römheld V (1989) Mobilization of iron and other micronutrient cations from a calcareous soil by plant-borne, microbial, and synthetic metal chelators. Plant Soil 114: 217–226[CrossRef][Web of Science] Vert G, Grotz N, Dedaldechamp 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: 1223–1233 von Wirén N, Römheld V, Shioiri T, Marschner H (1995) Competition between micro-organisms and roots of barley and sorghum for iron accumulated in the root apoplasm. New Phytol 130: 511–521[CrossRef][Web of Science] Walter A, Romheld V, Marschner H, Mori S (1994) Is the release of phytosiderophores in zinc-deficient wheat plants a response to impaired iron utilization? Physiol Plant 92: 493–500[CrossRef] Weber G, von Wirén N, Hayen H (2006) Analysis of iron(II)/iron(III) phytosiderophore complexes by nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Rapid Commun Mass Spectrom 20: 973–980[CrossRef][Web of Science][Medline] Yoshihara T, Hodoshima H, Miyano Y, Shoji K, Shimada H, Goto F (2006) Cadmium inducible Fe deficiency responses observed from macro and molecular views in tobacco plants. Plant Cell Rep 25: 365–373[CrossRef][Web of Science][Medline] Zhang FS, Römheld V, Marschner H (1989) Effect of zinc deficiency in wheat on the release of zinc and iron mobilizing root exudates. J Plant Nutr Soil Sci 152: 205–210[CrossRef] This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
ade2/+can1/can1 his3/his3 leu2/leu2 trp1/trp1 ura3/ura3 fet3-2:HIS3/fet3-2:HIS3/fet4-1:LEU2/fet4-1:LEU2) and wild type (DY1457, MAT
