Overexpression of the FRO2 ferric chelate reductase confers tolerance to growth on low iron and uncovers posttranscriptional control

Abstract

The Arabidopsis FRO2 gene encodes the low-iron-inducible ferric chelate reductase responsible for reduction of iron at the root surface. Here, we report that FRO2 and IRT1, the major transporter responsible for high-affinity iron uptake from the soil, are coordinately regulated at both the transcriptional and posttranscriptional levels. FRO2 and IRT1 are induced together following the imposition of iron starvation and are coordinately repressed following iron resupply. Steady-state mRNA levels of FRO2 and IRT1 are also coordinately regulated by zinc and cadmium. Like IRT1, FRO2 mRNA is detected in the epidermal cells of roots, consistent with its proposed role in iron uptake from the soil. FRO2 mRNA is detected at high levels in the roots and shoots of 35S-FRO2 transgenic plants. However, ferric chelate reductase activity is only elevated in the 35S-FRO2 plants under conditions of iron deficiency, indicating that FRO2 is subject to posttranscriptional regulation, as shown previously for IRT1. Finally, the 35S-FRO2 plants grow better on low iron as compared with wild-type plants, supporting the idea that reduction of ferric iron to ferrous iron is the rate-limiting step in iron uptake.

Figure 1.

Time course of FRO2 and IRT1 mRNA abundance patterns in roots exposed to iron deficiency. Wild-type plants were grown for 14 d on standard medium and transferred to iron-deficient medium. Roots were harvested 0, 12, 24, 36, 48, 72, and 144 h after the transfer to iron-deficient medium. RNA was isolated from the samples and used to prepare RNA gel blots. The blots were hybridized with radiolabeled FRO2 and IRT1 cDNA probes. Ethidium bromide-stained rRNA is shown as a control for loading.

Figure 2.

Time course of FRO2 and IRT1 mRNA abundance patterns in roots after iron resupply. Wild-type plants were grown for 14 d on standard medium, transferred to iron-deficient medium for 3 d, and then transferred a second time to iron-sufficient medium. Roots were harvested 0, 12, 24, 36, 48, 72, and 144 h after the second transfer. RNA was isolated from the samples and used to prepare RNA gel blots. The blots were hybridized with radiolabeled FRO2 and IRT1 cDNA probes. Ethidium bromide-stained rRNA is shown as a control for loading.

Figure 3.

Coordinate control of FRO2 and IRT1 expression by zinc. Wild-type plants were grown for 14 d on standard medium. Plants were subsequently transferred and grown for 3 d on iron-sufficient medium (lane 1), iron-deficient medium (lane 2), iron-sufficient medium supplemented with 500 μm zinc (lane 3), iron-deficient medium supplemented with 100 μm zinc (lane 4), or iron-deficient medium supplemented with 500 μm zinc (lane 5). RNA was isolated from roots and used to prepare RNA gel blots. The blots were hybridized with radiolabeled FRO2 and IRT1 cDNA probes. Ethidium bromide-stained rRNA is shown as a control for loading.

Figure 4.

Localization of the FRO2 transcript in longitudinal sections of iron-deficient roots. A, In situ hybridization experiments were performed on longitudinal sections (10 μm) of iron-deficient roots using a FRO2 antisense probe. B, In situ hybridization experiments were performed on longitudinal sections (10 μm) of iron-deficient roots using a FRO2 sense probe.

Figure 5.

FRO2 is expressed in flowers. A, Histochemical staining of flowers from FRO2-GUS transgenic plants grown in soil. A representative individual is shown. No significant GUS activity was detected in wild-type plants (data not shown). B, Real time RT-PCR analysis of FRO2 expression in various tissues. Plants were grown for 14 d on standard medium and then transferred to either iron-deficient or -sufficient medium as indicated or were grown in soil (flowers, siliques, and stems). FRO2 values were normalized and expressed as -fold induction relative to +Fe shoots. Due to the ratio calculation, sds are not shown in this figure.

Figure 6.

Northern-blot analysis of wild-type and transgenic 35S-FRO2 plants. RNA was prepared from the roots and shoots of 17-d-old plants grown for 3 d on either iron-sufficient or -deficient medium. The radiolabeled FRO2 cDNA was used as a probe. Ethidium bromide-stained rRNA is shown as a control for loading.

Figure 7.

Assays of ferric chelate reductase activity in wild-type and 35S-FRO2 transgenic lines. Plants were grown on standard medium for 14 d and then transferred to either iron-deficient (open bars) or -sufficient (filled bars) medium for 3 d before the assay. Values are the mean of five assays. The means of wild-type and 35S-FRO2 (lines 5A and 15G) plants grown on iron-deficient medium are statistically different at P < 0.05.

Figure 8.

Assays of copper chelate reductase activity in wild-type and 35S-FRO2 transgenic plants. Plants were grown on standard medium for 14 d and then transferred to either iron-deficient (open bars) or -sufficient (filled bars) medium for 4 d before the assay. Values are the mean of five assays. The means of wild-type and 35S-FRO2 (lines 5A and 15G) plants grown on iron-deficient medium are statistically different at P < 0.05.

Figure 9.

35S-FRO2 plants (line 15G) tolerate growth on low-iron medium. Seedlings were germinated on B5 medium and then were transferred and grown for 2 weeks on low-iron medium before being photographed.