FRD3, a member of the multidrug and toxin efflux family, controls iron deficiency responses in Arabidopsis

Abstract

We present the cloning and characterization of an Arabidopsis gene, FRD3, involved in iron homeostasis. Plants carrying any of the three alleles of frd3 constitutively express three strategy I iron deficiency responses and misexpress a number of iron deficiency-regulated genes. Mutant plants also accumulate approximately twofold excess iron, fourfold excess manganese, and twofold excess zinc in their shoots. frd3-3 was first identified as man1. The FRD3 gene is expressed at detectable levels in roots but not in shoots and is predicted to encode a membrane protein belonging to the multidrug and toxin efflux family. Other members of this family have been implicated in a variety of processes and are likely to transport small organic molecules. The phenotypes of frd3 mutant plants, which are consistent with a defect in either iron deficiency signaling or iron distribution, indicate that FRD3 is an important component of iron homeostasis in Arabidopsis.

Figure 1.

Fe(III) Chelate Reductase Activity. (A) frd3 and man1 exhibit constitutive Fe(III) chelate reductase activity. Plants grown with (+) or without (−) Fe(III) EDTA for 3 days were assayed for Fe(III) chelate reductase activity. (B) frd3 and man1 are recessive and allelic. All plants were grown with Fe(III) EDTA for 3 days. Data shown are means and se values of nine plants. Experiments were performed at least twice, and representative data sets are shown. FW, fresh weight.

Figure 2.

Expression of Iron Deficiency Responses. (A) frd3 constitutively expresses IRT1 and FRO2. An RNA gel blot with 5 μg of total root RNA per lane that was probed with IRT1 and FRO2 is shown. RNA was extracted from plants grown with (+) or without (−) Fe(III) EDTA for 3 days. UBQ5 is shown as a loading control. (B) frd3 accumulates IRT1 protein. An immunoblot of 10 μg of total root protein per lane that was probed with anti-IRT1 antibody is shown. Protein was extracted from roots of plants grown with (+) or without (−) Fe(III) EDTA for 3 days. A similar gel was stained with Coomassie blue to check for equal loading (data not shown). (C) frd3 does not accumulate ferritin (FER) protein. An immunoblot of 30 μg of total shoot protein per lane that was probed with anti-ferritin antibody is shown. Protein was extracted from shoots of plants grown with (+) or without (−) Fe(III) EDTA for 3 days. A similar gel was stained with Coomassie blue to check for equal loading (data not shown). These experiments were repeated twice, and similar results were obtained. Col, Columbia wild type.

Figure 3.

frd3 Alleles Accumulate More Fe, Mn, and Zn in Their Shoots. Pooled samples of 2-week-old shoots from plants grown on B5 plates were subjected to elemental analysis. This experiment was repeated, and similar results were obtained. Col, Columbia wild type.

Figure 4.

frd3-1 Exhibits Constitutive Acidification. After being grown with (+Fe) or without (−Fe) Fe(III) EDTA for 3 days, plants were transferred to plates containing bromocresol purple for 18 h. (A) Wild type (ecotype Columbia). (B) frd3-1. Iron-deficient wild type and iron-sufficient and iron-deficient frd3-1 reduced the pH of the medium to <5.2, as indicated by the yellow color. Iron-sufficient wild type caused the pH to increase to >7.0, as indicated by the purple color.

Figure 5.

Positional Cloning and Structure of the FRD3 Gene. (A) The region of chromosome 3 containing FRD3. The chromosome is depicted by the uppermost horizontal line with the flanking markers C6 and g4119. Below that are three BACs from the Arabidopsis Genome Initiative (2000) minimal tiling path: MLP3, F17A17, and T8G24. Markers (see Methods), the number of recombinant chromosomes from the 1640 examined, and the final 55-kb interval containing FRD3 are shown below. The striped bar indicates the segment of genomic DNA used to complement frd3-1. (B) Complementation of frd3-1. An 11-kb segment of genomic DNA, when expressed in frd3-1, restores the repression of Fe(III) chelate reductase activity in plants grown for 3 days in the presence of Fe(III) EDTA. Values shown are means of nine individual plants, and error bars depict se. Col, Columbia wild type. (C) Predicted topology of the FRD3 protein. The 12 transmembrane domains, as predicted by HMMTOP, and the location and nature of the mutations carried by the three mutant alleles are shown. (D) Intron/exon structure of FRD3. The narrow lines depict intron sequences, and the boxes depict exon sequences. The closed boxes correspond to the open reading frame, and the open boxes correspond to the 5′ and 3′ untranslated regions. Line lengths are approximately to scale.

Figure 6.

FRD3 Expression Levels Depend on Iron Status and Genotype. The RNA gel blot used in Figure 3 was reprobed with FRD3. The expression level of FRD3 was normalized to UBQ5 and is shown in arbitrary units. Results are presented as a graph to emphasize the very large differences in expression levels. Values shown are means of three replicate experiments, and error bars indicate se. Col, Columbia wild type.

Figure 7.

Dendrogram Showing Amino Acid Sequence Similarity Relationships among Selected MATE Family Members. Multiple sequence alignments were determined using the BCM Search Launcher, and the dendrogram was produced using MEGA 2.1. Bootstrap values are shown next to each junction.

Figure 8.

Alignment of the Amino Acid Sequences of Selected MATE Family Members. Identical residues are shown on a black background, and conservative substitutions are shown on a gray background. Lines depict FRD3 transmembrane domains (TM I to TM XIII) as predicted by HMMTOP. Multiple sequence alignments were determined using the BCM Search Launcher, and residues were shaded using BoxShade 3.21.