The essential basic helix-loop-helix protein FIT1 is required for the iron deficiency response

Abstract

Regulation of iron uptake is critical for plant survival. Although the activities responsible for reduction and transport of iron at the plant root surface have been described, the genes controlling these activities are largely unknown. We report the identification of the essential gene Fe-deficiency Induced Transcription Factor 1 (FIT1), which encodes a putative transcription factor that regulates iron uptake responses in Arabidopsis thaliana. Like the Fe(III) chelate reductase FRO2 and high affinity Fe(II) transporter IRT1, FIT1 mRNA is detected in the outer cell layers of the root and accumulates in response to iron deficiency. fit1 mutant plants are chlorotic and die as seedlings but can be rescued by the addition of supplemental iron, pointing to a defect in iron uptake. fit1 mutant plants accumulate less iron than wild-type plants in root and shoot tissues. Microarray analysis shows that expression of many (72 of 179) iron-regulated genes is dependent on FIT1. We demonstrate that FIT1 regulates FRO2 at the level of mRNA accumulation and IRT1 at the level of protein accumulation. We propose a new model for iron uptake in Arabidopsis where FRO2 and IRT1 are differentially regulated by FIT1.

Figure 1.

Steady State Levels of mRNA and Protein of Iron Uptake Genes in fit1-1 Plants. Wild-type and fit1-1 plants were grown on B5 plates for 12 d, then transferred to iron sufficient (+) or iron deficient (−) media for 3 d. RNA and total protein samples were prepared from root (R) and shoot (S) tissues. (A) FIT1, FRO2, or IRT1 cDNAs were used to probe individual RNA gel blots, and the corresponding ethidium bromide–stained rRNA is shown as a loading control. (B) An IRT1 affinity-purified peptide antibody was used to detect IRT1 protein at ∼35 kD.

Figure 2.

Localization of FIT1 to Iron-Deficient Roots. (A) to (C) GUS staining of a representative T3 transgenic line expressing a FIT1-GUS translational fusion protein. Plants were germinated directly on iron-deficient plates containing hygromycin (25 mg/mL). (A) Staining of a day 2 seedling. (B) The main root of a day 9 seedling. (C) Main and lateral roots of a day 9 seedling. (D) and (E) In situ hybridization was performed on 10-μm longitudinal root sections of day 7 plants grown on iron-deficient plates using a FIT1 antisense probe (D) or a FIT1 sense probe (E).

Figure 3.

Growth Phenotype, Rescue, and Complementation of fit1-1 Plants. Wild-type and fit1-1 plants were germinated on B5 plates. T2 fit1-1 transgenic plants complemented with a genomic fragment containing FIT1 (fit1-1:FIT1) were selected on B5 medium containing hygromycin (25 mg/mL). After 11 d, wild-type, fit1-1, and fit1-1:FIT1 plants were transferred to soil for 3 weeks. (A) No added iron. (B) Plants were watered two times per week with 0.5 g/L of Sequestrene as an additional iron source.

Figure 4.

fit1-1 Plants Lack Inducible Fe(III) Chelate Reductase Activity. Wild-type and fit1-1 plants were grown on B5 plates for 12 d, then transferred to iron-sufficient (Fe+) or iron-deficient (Fe−) plates for 3 d. Fe(III) chelate reductase activity of a pool of five plant roots was measured, in triplicate, using the ferrozine assay (Yi and Guerinot, 1996). Error bars indicate standard deviation.

Figure 5.

Iron Content of Wild-Type and fit1-1 Plants. Wild-type and fit1-1 plants were grown on B5 plates for 12 d, then either harvested or transferred to iron-sufficient (+Fe) or iron-deficient (−Fe) media for 3 d. Plants were pooled and harvested into root and shoot samples, and two biological sets of tissue were subjected to elemental analysis. Standard deviations were calculated, and statistically significant differences are indicated (*).

Figure 6.

Expression of Iron Uptake Genes in 35S:FIT1 Plants. Wild-type plants and three independent homozygous 35S:FIT1 transgenic lines were grown on B5 plates for 12 d, then transferred to iron-sufficient (+) or iron-deficient (−) media for 3 d. (A) RNA was prepared from root (R) and shoot (S) tissues. FIT1, FRO2, or IRT1 cDNAs were used to probe individual RNA gel blots, and the corresponding ethidium bromide–stained rRNA is shown as a loading control. (B) Total protein was prepared, and an IRT1 affinity-purified peptide antibody was used to detect IRT1 protein in wild-type plants and a single 35S-FIT1 line.

Figure 7.

Promoter Analysis of Potential FIT1 Binding Sites. The promoter regions of 37 iron-regulated genes that are also deregulated in fit1-1 were analyzed for the occurrence of the E-box motif 5′-CANNTG-3′, which is a potential FIT1 binding site. The number of sites found in each upstream region is indicated to the left of the schematic, which represents 1000 bp of sequence upstream of the translational start. Approximate positions of E-box sites are indicated by filled squares, which are not drawn to scale.

Figure 8.

Model of IRT1 Protein Regulation by FIT1. FIT1 may prevent IRT1 protein turnover through inhibition of an unknown factor (product of Gene X), allowing IRT1 protein to accumulate under iron-deficient conditions. Failure to activate Gene X in fit1-1 plants would prevent IRT1 protein accumulation.