EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana

Abstract

The EIR1 gene of Arabidopsis is a member of a family of plant genes with similarities to bacterial membrane transporters. This gene is expressed only in the root, which is consistent with the phenotypes of the eir1 mutants-the roots are agravitropic and have a reduced sensitivity to ethylene. The roots of eir1 mutants are also insensitive to the excess auxin produced by alf1-1 and fail to induce an auxin-inducible gene in the expansion zone. Although they fail to respond to internally generated auxin, they respond normally to externally applied auxin. Expression of the EIR1 gene in Saccharomyces cerevisiae confers resistance to fluorinated indolic compounds. Taken together, these data suggest that the EIR1 protein has a root-specific role in the transport of auxin.

Figure 1

Tropic responses are blocked in roots of eir1-3. (A) Seedlings were grown on plates placed at 90° for 5 days, and then manually reoriented so that the roots were parallel to the surface of the earth. The direction of new root growth was monitored after 48 hr. Wild type (bottom) reorients root growth in response to gravity. eir1-3 (top) no longer responds to such changes. (B) Growth pattern of wild type (left) and eir1-3 (right) primary roots on 2% agar plates. Wavy growth on the surface is no longer visible in eir1-3. On reaching the bottom of the plate, wild-type roots (C) grow in a spiral pattern; eir1-3 (D) grows irregularly with random turns in direction.

Figure 2

Dose response curves of root growth from wild-type plants and eir1 mutants [(•) Col-O; (▪) Ws; (○) eir1-1; () eir1-3]. Root elongation determined at 12 DAG was normalized to root growth on unsupplemented medium (100%). Standard deviations are shown as bars; molarities used are indicated.

Figure 3

The EIR1. (A) Schematic representation of an EcoRI fragment isolated from phage λ5-3 (see text for details). The bars indicate the nine exons of EIR1. Those segments presumed to be translated are black. Two mutations are indicated beneath the line: Insertion of Ac in eir1-3 after amino acid 133 and base substitution of the intron/exon junction in eir1-1. The shaded bar above the line indicates the genomic fragment amplified by inverse PCR (see text). (RI) EcoRI; (H) HinDIII; (Ba) BamHI; (X) XbaI; (B) BclI. (B) Southern blot made with genomic DNA from ecotype Ws hybridized with a 600-bp HinDIII fragment (hatched bar above the line in A). (Left) High stringency wash; (right) low stringency wash. (C) The gravitropism defect of eir1-1 is suppressed by transformation with the EcoRI fragment shown in A. The plant at the top is the recipient, eir1-1/BRL3-2 in the middle is the transformant, and Col-O at the bottom is wild type.

Figure 4

(A) Alignment of deduced amino acid sequences of EIR1, the rice homolog REH1, and the two putative Arabidopsis homologs AEH1 and AEH2. For EIR1 and REH1, ORFs of the cDNAs were deduced. The protein sequences of AEH1 and AEH2 were deduced from the genomic sequences by identifying canonical splice donor and acceptor sites. Identical residues are boxed and dashes indicate gaps in the sequence. Black lines correspond to the 10 potential transmembrane domains shared by all four proteins. Potential, conserved N-glycosylation sites are in bold letters. An arrow indicates the cleavage site of a potential amino-terminal signal peptide found for EIR1, REH1, and AEH1. (B) Hydrophobicity plots of EIR1, REH1, AEH1, and AEH2 according to Kyte and Doolittle (1982). Positive values indicate hydrophobic areas. (C) Alignment of the conserved amino-terminal (top) and carboxy-terminal (bottom) transmembrane domains of EIR1 and REH1 with a number of selected bacterial transporters (for more details, see text). Identical residues are boxed. The bold letters represent positions where exchanges are conservative (L,I,V,M; A,S,T; F,W,Y; N,Q; D,E; and K,R) and shared by EIR1 and at least two other sequences. Dashes indicate gaps in the alignment.

Figure 5

The phenotype of eir1 is restricted to the root. (A) From left to right: Wild type (Col-O), eir1-3, eto3-1, and eir1-3 eto3-1 were germinated under constant illumination or in the dark. Pictures were taken at 5 DAG (light) and at 2–3 DAG (dark), respectively. The image of the eir1 eto3 mutant grown in the dark reveals the similarity of its hypocotyl and that of eto3. With time the root of eir1 eto3 (arrow) becomes much longer than that of eto3. Average primary root lengths of light-grown plants are as follows: Col-O, 37 ± 3 mm; eir1-3, 44 ± 4 mm; eto3-1, 8 ± 1 mm; eir1-3 eto3-1, 22 ± 4 mm. (B) From left to right: Wild type, ctr1-1, eir1-3, and eir1-3 ctr1-1 at ∼10 DAG. Note the intermediate root elongation phenotype of the double mutant. The aerial part of eir1-3 ctr1-1 still resembles ctr1-1. Average primary root lengths were as follows: ctr1-1, 6 ± 1 mm; eir1-3 ctr1-1, 16 ± 2 mm.

Figure 6

RS–PCR performed with tissue-specific cDNA of Col-O plants. (Top) Expression of ACT2 is detectable in all tissues tested after 30 cycles. (Middle) An EIR1-specific amplification product derived from root cDNA after 60 cycles. No signal was detectable in any other tissues tested. (Bottom) Southern blot of EIR1-specific amplification products after 30 cycles using a transcript-specific oligonucleotide as a probe.

Figure 7

AtIAA2::GUS expression in primary roots: Wild-type roots exhibit asymmetric GUS-staining in the elongation zone on root bending. In eir1-3, expression of the auxin reporter no longer responds to gravistimuli.

Figure 8

(A) AtIAA2::GUS expression upon hormone and TIBA treatment. From left to right: wild type (top) and eir1-3 (bottom) plants were grown on standard medium (PNA), or for 24 hr on PNA supplemented with 1 μm NAA, 1 μm ACC, or 5 μm TIBA. In untreated plants expression of the auxin reporter is restricted to the tip and the stele of the roots. eir1-3 no longer responds to ACC and TIBA treatment. (B) eir1-3 suppresses the inhibition of root elongation in alf1-1. (Top) The plants are ∼6 DAG. From left to right: wild type, alf1-1, eir1-1 alf1-1, and eir1-1. (Bottom) Comparison of the primary root–hypocotyl junction of older plants. Excessive lateral and adventitious root formation is not suppressed in the eir1-1 alf1-1 double mutant.

Figure 9

EIR1 expressed in S. cerevisiae. (A) Growth of both wild-type and gef1 strains on 5-fluoro-indole (30 μl of a 100 mm stock solution) is rescued by constitutive expression of EIR1. Similar experiments expressing either ClC-0 or a mutated version of EIR1 failed to restore yeast growth in the presence of fluorinated indolic compounds. (B) Immunolocalization of HA-tagged EIR1 protein (i, iii). Most of the signal is localized to the plasma membrane. Weaker signals proximal to the nucleus (indicated by the DAPI staining in ii and iv) could be ER localization (bar, 1 μm). Controls were performed with strains expressing EIR1 without HA tag and by omitting the primary antibody during immunostaining. No specific signals could be detected in these experiments.