Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron

Abstract

Iron (Fe) is necessary for all living cells, but its bioavailability is often limited. Fe deficiency limits agriculture in many areas and affects over a billion human beings worldwide. In mammals, NRAMP2/DMT1/DCT1 was identified as a major pathway for Fe acquisition and recycling. In plants, AtNRAMP3 and AtNRAMP4 are induced under Fe deficiency. The similitude of AtNRAMP3 and AtNRAMP4 expression patterns and their common targeting to the vacuole, together with the lack of obvious phenotype in nramp3-1 and nramp4-1 single knockout mutants, suggested a functional redundancy. Indeed, the germination of nramp3 nramp4 double mutants is arrested under low Fe nutrition and fully rescued by high Fe supply. Mutant seeds have wild type Fe content, but fail to retrieve Fe from the vacuolar globoids. Our work thus identifies for the first time the vacuole as an essential compartment for Fe storage in seeds. Our data indicate that mobilization of vacuolar Fe stores by AtNRAMP3 and AtNRAMP4 is crucial to support Arabidopsis early development until efficient systems for Fe acquisition from the soil take over.

Figure 1

Tissue-specific expression of AtNRAMP4 is induced under Fe starvation. (A–D) Histochemical staining of GUS reporter activity in proAtNRAMP4-GUS seedlings grown for 7 days either on Fe replete medium (ABIS+100 μM FeHBED, A) or under Fe starvation (ABIS without Fe+100 μM ferrozine, B). Scale bar is 1 cm. (C) Detail of the junction between root and hypocotyl in a seedling grown under Fe starvation showing expression in vascular tissues. Scale bar is 0.6 mm. (D) Cross section through the root of a seedling grown under Fe starvation showing preferential expression in phloem tissues. Scale bar is 20 μm. (E) Quantitative analysis of GUS activity in protein extracts from roots or shoots of proAtNRAMP4-GUS seedlings. Seedlings were grown for a week on Fe-replete medium and then transferred either to +Fe (black bar) or −Fe (white bar) medium. Mean activities±s.e. (n=5 independent lines). (F) AtNRAMP4 protein abundance is upregulated under Fe starvation. Western blot experiment showing relative abundance of AtNRAMP4 protein in roots and shoots of Fe-starved (−Fe) or Fe-replete (+Fe) A. thaliana ecotype WS seedlings. 20 μg of proteins was loaded in each lane. AtNRAMP4 migrated at 48 kDa. Growth conditions as in (E).

Figure 2

Identification of a knockout mutant carrying a T-DNA insertion in AtNRAMP4 gene. (A) Localization of the T-DNA insertions in nramp3-1 and nramp4-1 mutant alleles. (B) AtNRAMP4 protein is not detectable in nramp4-1 homozygous mutant line. Western blot showing abundance of AtNRAMP4 protein in roots and shoots of Fe-starved or -replete A. thaliana wild type or nramp4-1 homozygous seedlings. 20 μg of proteins was loaded in each lane. Growth conditions as in Figure 1E and F. Note that anti-AtNRAMP4 antibodies recognize two additional proteins that are not affected in nramp4-1 mutants. (C) Seedling development is not affected differentially in wild type, nramp3-1 and nramp4-1 grown under a range of Fe supply. Mean root length±s.e. (n=15–25) of wild type (black bars), nramp3-1 (light gray bars) or nramp4-1 mutant (dark gray bars) measured after 16 days of growth on ABIS media containing 0.1–100 μM FeHBED (as indicated) as the sole source of Fe.

Figure 3

nramp3 nramp4 double knockout mutant exhibits a transient phenotype during germination on synthetic medium. (A) Four days after sowing on ABIS medium containing 50 μM FeEDTA as sole source of Fe, nramp3 nramp4 mutant exhibits pale cotyledon and reduced root elongation in comparison to wild type. (B) Ten days after sowing, the mutant phenotype is no longer obvious: the cotyledons have greened and the root growth almost caught up with that of the wild type seedlings. Scale bar is 20 mm.

Figure 4

nramp3 nramp4 double mutant seedlings are hypersensitive to low Fe nutrition conditions. (A) Seedlings of wild type, nramp3 nramp4 and nramp3 nramp4 complemented by AtNRAMP3 gene (left panels) or AtNRAMP4 gene (right panels) grown for 10 days either with low Fe supply (upper panels, 0.15 μM FeHBED) or with high Fe supply (lower panels, 50 μM FeHBED). Under low Fe supply, nramp3 nramp4 seedling development is arrested. Fe supplementation or expression of either AtNRAMP3 or AtNRAMP4 gene rescues nramp3 nramp4 mutant phenotype. Scale bar: 2 cm. (B) Dose-dependent effect of Fe supply on nramp3 nramp4 mutant phenotype. Mean root length±s.e. (n=15–25) of wild type (black bars) or nramp3 nramp4 mutant (white bars) measured after 14 days of growth on ABIS media containing 0.1–100 μM FeHBED (as indicated) as the sole source of Fe.

Figure 5

AtNRAMP3 and AtNRAMP4 are critical for plant survival on Fe-limiting soils. Wild type (front) or nramp3 nramp4 mutant (back) seedlings 6 days after sowing either on humic soil (A) or on calcareous soil (B). Note the bleached appearance of nramp3 nramp4 seedlings on calcareous soil (B, back). Wild type (front) or nramp3 nramp4 mutant (back) plants 12 days after sowing either on humic soil (C) or on calcareous soil (D). nramp3 nramp4 seedlings did not develop into adult plants on calcareous soil (D, back).

Figure 6

AtNRAMP3, AtNRAMP4 and IRT1 are expressed sequentially during germination. Histochemical staining of proAtNRAMP3-GUS (A, D, G), proAtNRAMP4-GUS (B, E, H) and proIRT1-GUS (C, F, I) seedlings germinated under Fe starvation (ABIS without Fe+100 μM ferrozine) one (A–C), two (D–F) or three (G–I) days after sawing. Scale bars are 0.5 mm (A–C), 1 mm (D–F) and 3 mm (G–I). At days 1 and 2, the embryos have been extracted from the seed teguments. Similar results were obtained with independent lines of proAtNRAMP3-GUS and proAtNRAMP4-GUS.

Figure 7

AtNRAMP4 is localized on vacuolar and punctuate structures in cotyledon cells. (A–G) Immunolocalization in PEG sections of 2-day-old Arabidopsis seedling cotyledons. (A–D) Fluorescence detection with anti-rabbit antibody coupled to FITC (green images). Upper panels: overviews of the cotyledons; lower panels: staining at the cellular level. Scale bars are 10 μm. (A, B) AtNRAMP4 staining of internal membranes of wild type seeds. (C) AtNRAMP4 staining of nramp4-1 mutants: nramp4-1 seeds are devoid of staining. (D) γ-TIP staining of wild type seeds. (E–G) Colocalization of AtNRAMP4 and γTIP. AtNRAMP4 was detected with FITC (green fluorescence). γ-TIP was detected using Cy3 (red fluorescence), colocalization (yellow) is shown in the merged picture. (E) Vacuolar and (F) punctuate patterns of AtNRAMP4/γ-TIP staining. (G) AtNRAMP4 staining of cells in the absence of γ-TIP antibody. No Cy3 staining was detectable. Differential interference contrast pictures are shown at the right side of each panel.

Figure 8

nramp3 nramp4 double mutants fail to retrieve Fe from seed vacuole globoids. (A–D) Transmission electron microscopy images of cotyledon parenchyma cells from wild type (A, C) or nramp3 nramp4 (B, D) either in dry seeds (A, B) or 2 days after sowing (C, D). Left panels: transmission image; right panels: Fe localization (red) obtained through imaging of inelastically scattered electrons (ESI) superimposed on the transmission image. Visible structures are labelled according to the Arabidopsis Atlas (Mansfield and Bowman, 1994): cw, cell wall; lb, lipid body; g, globoid; psv, protein storage vacuole. Scale bar is 1 μm. Empty globoid cavities correspond to globoid compartments from which the globoid crystal has been ejected during sectioning. (E–H) Representative EDX spectra of globoids from wild type (E, G) or nramp3 nramp4 (F, H) either in dry seeds (E, F) or 2 days after sowing (G, H). ^*Titanium and copper originate mostly from the grids holding the sections.