Iron acquisition by phytosiderophores contributes to cadmium tolerance

Abstract

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.

Figure 1.

Fe deficiency increases Cd accumulation in maize plants. Fifteen-day-old Fe-sufficient and Fe-deficient plants were harvested after 12, 24, or 48 h following addition of 0, 1, 5, or 25 μm CdCl₂ to the nutrient solution. A, Dry matter of shoot and roots. B, Cd concentrations. C, Fe concentrations. Significant differences among Fe-deficient (−Fe) and Fe-sufficient (+Fe) plants as determined by ANOVA followed by the Tukey test are indicated by an asterisk (P < 0.05; n = 4). Bars indicate means ± sd.

Figure 2.

Cd inhibits Fe-phytosiderophore uptake in maize roots. Uptake rates of ⁵⁹Fe-labeled Fe-DMA in roots of maize plants precultured in the presence or absence of Cd. Fe-sufficient and Fe-deficient plants were either pretreated with 25 μm Cd for 24 h prior to the Fe-DMA uptake experiment or Cd was added during the uptake period, or both treatments were combined. ⁵⁹Fe-labeled Fe-DMA was supplied at 10 μm for 60 min. Bars indicate means ± sd, and significant differences among different Cd treatments within the Fe-deficient (−Fe) and Fe-sufficient (+Fe) plants as determined by ANOVA followed by the Tukey test are indicated by different letters (P < 0.05, n = 4).

Figure 3.

Cd stress decreases the Fe concentration in the xylem sap of Fe-deficient plants. Fe and Cd concentrations in the xylem bleeding sap of Fe-resupplied plants grown in the absence (control) or presence of 25 μm Cd. Plants were precultured for 23 d under Fe deficiency before Fe and Cd were added to the nutrient solution for a period of 48 h. Significant differences between control and Cd treatments as determined by ANOVA and Tukey test are indicated by an asterisk (P < 0.05, n = 4). n.d., Not detected.

Figure 4.

Cd induces Fe-deficiency stress responses in maize. A, RNA gel-blot analysis of ZmYS1 expression in roots from hydroponically grown plants that were precultured for 20 d in absence of Fe or in the presence of 100 μm Fe(III)-EDTA before addition of 0, 1, 5, or 25 μm CdCl₂ for 12, 24, or 48 h to the nutrient solution. Plants were harvested at the same time, and total RNA from roots was used for hybridization to the complete ORF of ZmYS1. Ethidium bromide-stained gel blots are shown as a control. B, Rate of DMA release within 6 h from roots of 10-d-old maize plants grown under axenic conditions. The plants were incubated in the absence or presence of 25 μm Cd for 24 h before being transferred to ultrapure water for collection of the exudates. Bars indicate means ± sd (n = 5).

Figure 5.

The phytosiderophore DMA does not mobilize Cd from a Cd-loaded resin. Mobilization of Cd, Fe, and Cu was determined from a Cd-, Fe-, or Cu-loaded Chelex resin using water, a HEDTA, or DMA solution at pH 5.5 as eluent for metal extraction. sds in all treatments were too low to become visualized (n = 4).

Figure 6.

Phytosiderophores form a weak complex with Cd as revealed by nano-ESI-FTICR-MS. A, ESI ionization mass spectra of Cd-DMA complexes in the negative ionization mode. Top graph, Calculated mass spectrum of the Cd(II)-DMA complex ([DMA − 3H + Cd(II)]⁻). Bottom graph, Section of the mass spectrum (m/z 150–800) of a 20-μm DMA solution supplemented with 10 μm Cd(II); resolution (full width at half maximum) 200,000. B and C, Competition studies between Cd and Fe or Zn for phytosiderophore complex formation. Addition of 10 μm Fe(III) or Zn(II) to a 10-μm Cd(II)-DMA solution (left) or of 10 μm Cd(II) to 10 μm Fe(III)-DMA or Zn(II)-DMA solution (right). Mass spectra were recorded by nano-ESI-FTICR after 30 min of incubation. Experiments were repeated several times with similar results, and representative values of one experiment are shown.

Figure 7.

Cd uptake by maize roots is not prevented by phytosiderophore chelation. A, Short-term uptake rates of ⁵⁹Fe-labeled Fe-DMA in roots of 17-d-old plants of wild-type maize (UH002) or of the ys1 mutant precultured under Fe-sufficient (+Fe) or Fe-deficient (−Fe) conditions for the whole growth period. B, Short-term uptake rates of ¹⁰⁹Cd-labeled CdSO₄ or Cd-DMA in the same plant lines as used in A. An inbred line (UH002) and the ys1 mutant were compared. Bars indicate means ± sd and significant differences are indicated by different letters as determined by ANOVA and Tukey test (P < 0.05; n = 4).

Figure 8.

Phytosiderophores do not protect yeast from Cd toxicity but allow growth of ZmYS1-transformed yeast cells on Cd. A, Growth complementation assay of wild-type yeast and the Fe-uptake defective mutant fet3fet4 transformed with the empty pDR195 vector, pDR196-ZmYS1, or pDR195-AtIRT1. B, Growth complementation assay of the same strains as in A but in the presence of Cd. Except for the 45-μm Fe-EDTA treatment, all media (YNB-ura) were buffered with 50 mm MES-Tris at pH 7.4 or 5.5 and supplemented with 10 μm Fe. For drop tests, yeast precultures in YNB-ura + 45 μm Fe-EDTA were pelleted, washed in Na-EDTA solution, and resuspended in water to achieve an optical density of 1.0 at 600 nm. Cells were diluted by 10-fold, and 10 μL of each dilution were spotted on YNB-ura plates supplemented or not with Cd and/or the chelators HEDTA or DMA. Plates were photographed after 5 d of incubation at 28°C.

Figure 9.

Functional expression of ZmYS1 improves growth of Arabidopsis plants under Cd stress in the presence of Fe phytosiderophore. A, RNA gel-blot analysis of Arabidopsis root RNA extracted from wild-type or homozygous T3 plants transformed with a CaMV35S:ZmYS1 construct. ZmYS1 expression was detected in hydroponically grown, Fe-sufficient plants. Ethidium bromide-stained gel blots are shown as a loading control. B, Growth phenotype of plants grown on modified 0.5 Murashige and Skoog agar plates supplemented with 20 μm Fe(III)-EDTA or Fe(III)-DMA. Plants in bottom section were grown in the presence of 15 μm CdCl₂. Plates of representative plants were photographed 10 d after transfer of seedlings to the treatments. Bar = 3 cm. C, Quantitative analysis of shoot growth after plant cultivation as described in B with different Fe-binding forms and Cd concentrations. D, Chlorophyll concentrations in shoots of plants as grown in B. Significant differences at P < 0.05 as determined by ANOVA followed by Tukey test are indicated by different letters (n = 7). Presented data are representative for three independent experiments.