IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth

Abstract

Plants are the principal source of iron in most diets, yet iron availability often limits plant growth. In response to iron deficiency, Arabidopsis roots induce the expression of the divalent cation transporter IRT1. Here, we present genetic evidence that IRT1 is essential for the uptake of iron from the soil. An Arabidopsis knockout mutant in IRT1 is chlorotic and has a severe growth defect in soil, leading to death. This defect is rescued by the exogenous application of iron. The mutant plants do not take up iron and fail to accumulate other divalent cations in low-iron conditions. IRT1-green fluorescent protein fusion, transiently expressed in culture cells, localized to the plasma membrane. We also show, through promoter::beta-glucuronidase analysis and in situ hybridization, that IRT1 is expressed in the external cell layers of the root, specifically in response to iron starvation. These results clearly demonstrate that IRT1 is the major transporter responsible for high-affinity metal uptake under iron deficiency.

Figure 1.

Molecular Characterization of the irt1-1 Knockout Mutant. (A) Scheme of the T-DNA integration site in irt1-1. The location of the T-DNA insertion within the sequence of the IRT1 gene is shown. Exons are boxed. (B) Detection of IRT1 mRNA. RT-PCR was performed on total RNA extracted from wild-type (WT) and irt1-1 iron-deficient roots. EF1α-specific primers were included as a control. (C) Immunoblot analysis of IRT1. Total proteins were extracted from wild-type and irt1-1 plants grown in the presence (+) or absence (−) of iron. The blot was probed with antibodies directed against IRT1. R, roots; S, shoots.

Figure 2.

Phenotype of irt1-1. Phenotype of 15-day-old (top) or 4-week-old (bottom) plants grown in soil. WT, wild-type plant; irt1-1, irt1-1 mutant; irt1-1 +/−, irt1-1 heterozygous plant; irt1-1:IRT1, transgenic irt1-1 mutant expressing functional IRT1; irt1-1 + Fe, irt1-1 watered with 0.5 g/L Sequestrene.

Figure 3.

Reduced Leaf Iron Content in irt1-1. Leaf iron content was assessed on 4-week-old plants grown in soil. DW, dry weight; WT, wild-type plant; irt1-1, homozygous mutant; irt1-1 +/−, heterozygous plant; irt1-1:IRT1, irt1-1 mutant transformed with a functional IRT1; irt1-1 + Fe, irt1-1 watered with 0.5 g/L Sequestrene.

Figure 4.

Defect of ⁵⁵Fe Accumulation in irt1-1. Accumulation of ⁵⁵Fe was performed on wild-type (WT) and irt1-1 plants grown in iron-sufficient (+) or iron-deficient (−) conditions for 4 days and incubated for 48 h in the presence of 10 μM ⁵⁵Fe-EDTA. DW, dry weight.

Figure 5.

Deregulation of the Iron-Deficiency Responses in irt1-1. (A) Root ferric chelate reductase activity in wild-type (WT) and irt1-1 plants grown in iron-sufficient (+) or iron-deficient (−) conditions for 4 days. FW, fresh weight. (B) Expression of iron deficiency–induced genes. Twenty micrograms of total RNA extracted from wild-type or irt1-1 roots of plants grown in iron-sufficient or iron-deficient conditions for 3 days was blotted and hybridized with FRO2 and IRT2 cDNA probes. (C) Quantification relative to the 25S rRNA of the RNA gel blots presented in (B).

Figure 6.

IRT1 Is a Multispecific Metal Ion Transporter in Planta. (A) Elemental analysis of wild-type (WT) and irt1-1 roots. Plants were grown in the presence (+Fe) or absence (−Fe) of iron for 5 days, and roots were processed for inductively coupled plasma mass spectrometry analysis (see Methods). DW, dry weight. (B) Phenotype of 15-day-old wild-type and irt1-1 plants grown in soil and irrigated either with water (wild type and irt1-1) or with a solution of 600 μM Sequestrene (irt1-1 + Fe), 500 μM ZnCl₂ (irt1-1 + Zn), 500 μM MnCl₂ (irt1-1 + Mn), or 50 μM CoCl₂ (irt1-1 + Co) as indicated.

Figure 7.

Reduced Cadmium Sensitivity of irt1-1. (A) Phenotype of wild-type (WT) and irt1-1 plants grown in the presence of cadmium. Plants were grown in the presence or absence of iron (+Fe and −Fe) and in the presence or absence of 20 μM cadmium (+Cd and −Cd) for 8 days. (B) Cadmium content in roots of the iron-deficient plants described in (A) and processed for inductively coupled plasma mass spectrometry analysis. DW, dry weight.

Figure 8.

Subcellular Localization of the IRT1 Protein. Subcellular localization of IRT1-GFP (A) and GFP alone (C) in transfected Arabidopsis protoplasts. Chlorophyll autofluorescence, shown in red (indicated by arrows in [A]), is superimposed on GFP fluorescence in (A) and (C). (B) and (D) show the transmission view of the same fields shown in (A) and (C), respectively.

Figure 9.

Root Periphery Localization of IRT1 Expression. (A) Enzymatic GUS assay was performed on six independent transgenic lines expressing an IRT1 promoter–GUS fusion protein and grown in the presence of 50 μM Fe-EDTA (+Fe) or in the absence of added iron (−Fe). No significant GUS activity was detected in wild-type plants (data not shown). MU, methylumbelliferone. (B) to (F) GUS histochemical staining of the transgenic lines. (B) Twelve-day-old plantlets grown as in (A) in the presence of iron. (C) Twelve-day-old plantlets grown as in (A) in the absence of iron. (D) and (E) Enlargement of the iron-deficient roots shown in (C) to highlight the weak GUS staining in the meristematic zone (D) and the strong staining in the root hairs and in the root lateral branching zone (E). (F) Three-micrometer cross-section of an iron-deficient root shown in (C). Cross sections were counterstained with Schiff dye. c, cortex; ep, epidermis; vc, vascular cylinder. Bar = 100 μm. (G) In situ hybridization experiments were performed on longitudinal sections (10 μm) of iron-deficient roots using an IRT1 antisense probe. (H) In situ hybridization experiments were performed on longitudinal sections (10 μm) of iron-deficient roots using a sense probe.

Figure 10.

IRT1 Expression in Flower. (A) Spatial distribution of IRT1 expression using 10 μg of total RNA isolated from roots (R), rosette leaves (rL), cauline leaves (cL), floral stalk (fS), flowers (F), and siliques (S) of 6-week-old soil-grown plants. (B) Developmental analysis of IRT1 expression in flowers using 10 μg of total RNA extracted from flowers of 6-week-old plants before pollination (F1) and after pollination (F2). (C) and (D) Histochemical localization of GUS activity in flowers of 6-week-old GUS transgenic plants grown in soil. (C) Flower. (D) Enlarged view of a stained anther filament. (E) Analysis of IRT1 expression in response to iron status. Gel blot analysis was performed using 15 μg of total RNA isolated from roots (R) and flowers (F) of 6-week-old plants irrigated with either water (H₂O) or 0.5 g/L Sequestrene (Fe).