Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation

Abstract

Iron, an essential nutrient, is not readily available to plants because of its low solubility. In addition, iron is toxic in excess, catalyzing the formation of hydroxyl radicals that can damage cellular constituents. Consequently, plants must carefully regulate iron uptake so that iron homeostasis is maintained. The Arabidopsis IRT1 gene is the major transporter responsible for high-affinity iron uptake from the soil. Here, we show that the steady state level of IRT1 mRNA was induced within 24 h after transfer of plants to iron-deficient conditions, with protein levels peaking 72 h after transfer. IRT1 mRNA and protein were undetectable 12 h after plants were shifted back to iron-sufficient conditions. Overexpression of IRT1 did not confer dominant gain-of-function enhancement of metal uptake. Analysis of 35S-IRT1 transgenic plants revealed that although IRT1 mRNA was expressed constitutively in these plants, IRT1 protein was present only in the roots when iron is limiting. Under these conditions, plants that overexpressed IRT1 accumulated higher levels of cadmium and zinc than wild-type plants, indicating that IRT1 is responsible for the uptake of these metals and that IRT1 protein levels are indeed increased in these plants. Our results suggest that the expression of IRT1 is controlled by two distinct mechanisms that provide an effective means of regulating metal transport in response to changing environmental conditions.

Figure 1.

Characterization of IRT1 Antiserum. Wild-type plants were grown for 2 weeks on B5 medium and transferred to either iron-deficient or iron-sufficient medium, and roots and shoots were harvested at 3 days after the transfer. Protein samples were prepared from each tissue sample and used to prepare protein gel blots. IRT1 protein was detected using affinity-purified antiserum raised against a synthetic peptide. The reaction of proteins prepared from iron-deficient roots with preimmune serum is shown as a control. Numbers at left indicate molecular mass (kD).

Figure 2.

Time Course of IRT1 mRNA and Protein Abundance Patterns in Response to Iron-Deficient and Iron-Sufficient Growth Conditions. (A) Wild-type plants were grown for 2 weeks on B5 medium and transferred to iron-deficient medium, and roots were harvested at 0, 12, 24, 36, 48, and 72 h and 6 days after the transfer. RNA and protein samples were prepared from each tissue sample and used to prepare RNA and protein gel blots. The IRT1 cDNA was used to probe the RNA gel blot. Ethidium bromide–stained rRNA is shown as a control for loading. IRT1 protein was detected using an IRT1 affinity-purified peptide antibody. (B) Wild-type plants were grown for 2 weeks on B5 medium, transferred to iron-deficient medium for 3 days, and transferred a second time to iron-sufficient medium. Roots were harvested at various times as indicated, and RNA gel blot and immunoblot analyses were performed as described above.

Figure 3.

RNA Gel Blot Analysis of Wild-Type and Transgenic 35S-IRT1 Plants. The IRT1 cDNA was used to probe a RNA gel blot containing RNA prepared from the shoots (A) and roots (B) of plants grown for 3 days on either iron-sufficient (+) or iron-deficient (−) medium. RNA from wild-type plants (WT) was electrophoresed next to RNA from the transgenic lines (lines 1 to 4). Ethidium bromide–stained rRNA is shown as a control for loading.

Figure 4.

Protein Gel Blot Analysis of Wild-Type and Transgenic 35S-IRT1 Plants. IRT1 protein was detected using the affinity-purified IRT1 peptide antibody. Each lane contained 10 μg of protein extracted from roots (R) or shoots (S) of plants grown for 3 days on either iron-sufficient (+) or iron-deficient (−) medium. Protein extracted from wild-type (WT) plants was electrophoresed next to protein extracted from transgenic lines 1 and 2 (A) and transgenic lines 3 and 4 (B).

Figure 5.

Time Course of IRT1 mRNA and Protein Abundance Patterns in 35S-IRT1 Plants. 35S-IRT1 plants (line 4) were grown on B5 plates for 2 weeks, transferred to iron-deficient plates for 3 days, and transferred again to iron-sufficient plates. Roots were harvested at 0, 6, 12, 18, 24, 36, and 48 h after the second transfer. RNA and protein samples were prepared from each tissue sample and used to prepare RNA and protein gel blots. The IRT1 cDNA was used to probe the RNA gel blot. Ethidium bromide–stained rRNA is shown as a control for loading. IRT1 protein was detected using the IRT1 affinity-purified peptide antibody.

Figure 6.

Zinc Affects the Abundance of IRT1 mRNA and Protein. Wild-type (WT [A]) and 35S-IRT1 (line 4) transgenic (B) plants were grown for 2 weeks on B5 plates. Seedlings were transferred subsequently and grown for 3 days on iron-sufficient plates (lane 1), iron-deficient plates (lane 2), iron-sufficient plates supplemented with 500 μM zinc (lane 3), iron-deficient plates supplemented with 100 μM zinc (lane 4), or iron-deficient plates supplemented with 500 μM zinc (lane 5). RNA and protein samples were prepared from each root sample and used to prepare RNA and protein gel blots. The IRT1 cDNA was used to probe the RNA gel blots. Ethidium bromide–stained rRNA is shown as a control for loading. IRT1 protein was detected using the IRT1 affinity-purified peptide antibody.

Figure 7.

Time Course of IRT1 mRNA and Protein Abundance Patterns in Wild-Type Plants Grown on Plates Containing Zinc. Wild-type plants were grown on B5 plates for 2 weeks, transferred to iron-deficient plates for 3 days, and transferred again to iron-deficient medium supplemented with 100 μM zinc. Roots were harvested at 0, 6, 12, 24, 36, 48, and 72 h after the second transfer. RNA and protein samples were prepared from each tissue sample and used to prepare RNA and protein gel blots. The IRT1 cDNA was used to probe the RNA gel blot. Ethidium bromide–stained rRNA is shown as a control for loading. IRT1 protein was detected using the IRT1 affinity-purified peptide antibody.

Figure 8.

Time Course of IRT1 mRNA and Protein Abundance Patterns in Wild-Type Plants Grown on Plates Containing Cadmium. Plants were grown on B5 plates for 2 weeks, transferred to iron-deficient plates for 3 days, and transferred again to iron-deficient medium supplemented with 90 μM CdSO₄. Roots were harvested at 0, 12, 24, 36, 48, and 72 h and 6 days after the second transfer. RNA and protein samples were prepared from each tissue sample and used to prepare RNA and protein gel blots. The IRT1 cDNA was used to probe the RNA gel blot. Ethidium bromide–stained rRNA is shown as a control for loading. IRT1 protein was detected using the IRT1 affinity-purified peptide antibody.

Figure 9.

Root Growth of 35S-IRT1 Transgenic Plants on Plates Containing Cadmium. Seedlings (wild type [WT] and transgenic line 4) were grown on B5 plates for 8 days before being transferred to iron-deficient plates that contained 50 μM CdSO₄. Plates were placed in the growth chamber in the vertical orientation such that root growth occurred along the surface of the agar, and root growth was measured every 24 h. Results are means of six independent measurements, and bars indicate standard error of mean. The experiment was performed twice.

Figure 10.

Sensitivity of 35S-IRT1 Transgenic Plants to Cadmium. Seedlings were grown for 2 weeks on B5 plates before being transferred to plates that were either iron deficient (−) or iron deficient plus 90 μM CdSO₄. Seedlings were allowed to grow for 6 days more before being transferred for photography. Wild-type (WT) plants are shown next to transgenic line 4 plants.

Figure 11.

Elemental Analysis of Wild-Type and 35S-IRT1 Transgenic Plants Grown on Plates Containing Cadmium. Plants were grown on B5 plates before being transferred to iron-deficient medium that contained 90 μM CdSO₄. Plants were allowed to grow for 6 days, at which time the roots and shoots were harvested separately and subjected to elemental analysis. Approximately 50 plants were pooled for each experiment, and results are means of three (WT) or six (35S-IRT1) independent experiments. DW, dry weight. Bars indicate standard error of mean.