Elevated nicotianamine levels in Arabidopsis halleri roots play a key role in zinc hyperaccumulation

Abstract

Zn deficiency is among the leading health risk factors in developing countries. Breeding of Zn-enriched crops is expected to be facilitated by molecular dissection of plant Zn hyperaccumulation (i.e., the ability of certain plants to accumulate Zn to levels >100-fold higher than normal plants). The model hyperaccumulators Arabidopsis halleri and Noccaea caerulescens share elevated nicotianamine synthase (NAS) expression relative to nonaccumulators among a core of alterations in metal homeostasis. Suppression of Ah-NAS2 by RNA interference (RNAi) resulted in strongly reduced root nicotianamine (NA) accumulation and a concomitant decrease in root-to-shoot translocation of Zn. Speciation analysis by size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry showed that the dominating Zn ligands in roots were NA and thiols. In NAS2-RNAi plants, a marked increase in Zn-thiol species was observed. Wild-type A. halleri plants cultivated on their native soil showed elemental profiles very similar to those found in field samples. Leaf Zn concentrations in NAS2-RNAi lines, however, did not reach the Zn hyperaccumulation threshold. Leaf Cd accumulation was also significantly reduced. These results demonstrate a role for NAS2 in Zn hyperaccumulation also under near-natural conditions. We propose that NA forms complexes with Zn(II) in root cells and facilitates symplastic passage of Zn(II) toward the xylem.

Figure 1.

Steady State NAS2 Transcript Levels and NA Concentrations in Roots of Hydroponically Grown A. halleri ssp halleri Individuals Representing Six Different European Populations. The individuals analyzed were clones of individuals collected from German and Polish populations at either metalliferous (yellow) or nonmetalliferous (green) sites in Oker (Ok), Langelsheim (Lan), Rodacherbrunn (Rod), Miasteczko Slaskie (MS), Bibiela (Bib), and Muchowiec (Much) (Table 1). A. halleri ssp gemmifera from Japan (white) and A. thaliana Col-0 (black) were included for comparison. Plants were grown in control medium for 5 weeks. (A) NAS2 transcript abundance in roots, expressed relative to EF1α, as determined by real-time RT-PCR. Values are arithmetic means ± sd of three independent experiments (three plants of each genotype were pooled for each data point). (B) Root NA concentrations. NA was extracted with water, derivatized with Fmoc-Cl, and quantified by UPLC-ESI-QTOF-MS using a ¹⁵N-labeled standard. Values are arithmetic means ± sd of n = 3 to 8 independent experiments (three plants of each genotype were pooled for each data point) per species or population. Statistical significance was determined using one-way analysis of variance followed by a Tukey test. Asterisks denote significant differences compared with the mean of A. thaliana: *P < 0.05, **P < 0.01, and ***P < 0.001. f.w., fresh weight.

Figure 2.

Steady State NAS2 Transcript Levels and NA Concentrations in Roots of Hydroponically Grown A. halleri Wild-Type (Langelsheim) and Ah-NAS2-RNAi Plants. Plants were grown in control medium for 5 weeks. (A) NAS2 transcript abundance in roots, expressed relative to EF1α, as determined by real-time RT-PCR. WT, wild type. (B) Root NA concentrations. NA was extracted with water, derivatized with Fmoc-Cl, and quantified by UPLC-ESI-QTOF-MS using a ¹⁵N-labeled standard. Values in (A) and (B) are arithmetic means ± sd of three independent experiments (three plants of each genotype were pooled for each data point). Statistical significance was determined using one-way analysis of variance followed by a Tukey test. Asterisks denote significant differences compared with the wild-type mean: *P < 0.05 and ***P < 0.001. f.w., fresh weight. (C) Root NA concentrations shown as a function of NAS2 transcript levels.

Figure 3.

Zn Accumulation in Hydroponically Grown A. halleri Wild-Type (Langelsheim) and Ah-NAS2-RNAi Plants. Plants were cultivated in a medium containing 10 μM ZnSO₄. Tissues were harvested after 5 weeks, digested, and analyzed by ICP-OES. Shown in (A) and (B) are values for roots and leaves, respectively. For (C), Zn shoot:root ratios of Zn concentrations were calculated from data shown above. All values are arithmetic means ± sd, n = 4 to 6 with three replicate clones per genotype pooled per experiment. Asterisks denote significant differences compared with the wild-type (WT) mean: *P < 0.05 and **P < 0.01. In (D), shoot:root ratios of Zn concentrations are shown as a function of NA concentrations. d.w., dry weight.

Figure 4.

Shoot:Root Ratios of Mn and Fe Concentrations in Hydroponically Grown A. halleri Wild-Type (Langelsheim) and Ah-NAS2-RNAi Plants. Root and leaf tissues were harvested after 5 weeks, digested, and analyzed by ICP-OES for Mn (A) and Fe (B). Values are arithmetic means ± sd (n = 3, with three replicate clones per genotype pooled per experiment) of shoot:root concentration ratios for plants grown either in control medium (white) or in the presence of 10 μM Zn²⁺ (black). Asterisks denote significant differences compared with the wild-type (WT) mean: *P < 0.05.

Figure 5.

Confocal Imaging of the Loosely Bound Fraction of Zn in Roots of the Wild Type (Langelsheim) and Ah-NAS2-RNAi Line 1-2. (A) Roots of plants cultivated in a hydroponic solution containing 1 μM ZnSO₄ were consecutively stained with Zinpyr and propidium iodide to visualize Zn (green) and cell walls (red), respectively. WT, wild type. Bars = 50 μm. (B) Relative quantification of Zn was performed by normalizing the Zinpyr signal to the propidium iodide signal for 112 (wild type) and 96 (RNAi 1-2) stacks obtained for roots of eight individual clones each (error bars = sd, ***P < 0.001). A second set of experiments with six individuals each yielded comparable results (2.4-fold stronger signal for RNAi line 1-2).

Figure 6.

Speciation of Zn in Root and Shoot Tissue for the Wild Type (Langelsheim) and the Ah-NAS2-RNAi Line 1-2. Tris/NaCl extracts of root (left) and shoot (right) tissue of the wild type (black lines) and the Ah-NAS2-RNAi line 1-2 (red lines) were subjected to SEC-ICP-MS analysis. Data for Zn ([A] and [B]) and S ([C] and [D]) are shown. Note that the figure shows low molecular mass Zn fractions that in shoots represent only a very small fraction of extractable Zn, most of which is present as free Zn²⁺ (cf. Table 2). Day-to-day variability in ion intensity was normalized using an external standard. Shown here are data from one experiment representative of three independent experiments (Table 2). WT, wild type.

Figure 7.

Phenotypic Analysis of A. halleri Wild-Type (Langelsheim) and Ah-NAS2-RNAi Plants on Soil Collected at Sites of Native A. halleri Populations. (A) Photographs of wild-type (WT; Langelsheim) plants and two representative RNAi lines (control transformant 0-7 and NAS2-suppressed line 11-1) taken 4 weeks after transfer of clones into field-collected soils containing different soil Zn levels. Soils were collected at sites of native A. halleri populations in the Harz Mountains in Germany with Zn concentrations ranging from background levels (noncontaminated) to heavily Zn contaminated (Table 4). Please note additional Cd contamination in these soils. (B) and (C) Leaf Zn (B) and Cd (C) concentrations in wild-type A. halleri (Langelsheim) (light yellow), the control transformant line 0-7 (dark yellow), and the three NAS2-suppressed lines 1-2 (green), 7-12 (blue), and 11-1 (red), as analyzed by ICP-OES. Values are arithmetic means ± sd of n = 3 to 9 individual plants from three independent experiments. Asterisks denote significant differences compared with the wild-type mean: *P < 0.05, **P < 0.01, and ***P < 0.001. Please note that the NAS2-suppressed lines 1-2, 7-12, and 11-1 all showed significantly different Zn accumulation on soils Harz 1 and Harz 2 when compared with the control transformant line 0-7. The same applies to Cd accumulation on soil Harz 1. d.w., dry weight.