ABCG36/PEN3/PDR8 Is an Exporter of the Auxin Precursor, Indole-3-Butyric Acid, and Involved in Auxin-Controlled Development

Abstract

The PDR-type ABCG transporter, ABCG36/PDR8/PEN3, is thought to be implicated in the export of a few structurally unrelated substrates, including the auxin precursor, indole-3-butyric acid (IBA), although a clear-cut proof of transport is lacking. An outward facing, lateral root (LR) location for ABCG36 fuelled speculations that it might secrete IBA into the rhizosphere. Here, we provide strong evidence that ABCG36 catalyzes the export of IBA - but not of indole-3-acetic acid - through the plasma membrane. ABCG36 seems to function redundantly with the closely related isoform ABCG37/PDR9/PIS1 in a negative control of rootward IBA transport in roots, which might be dampened by concerted, lateral IBA export. Analyses of single and double mutant phenotypes suggest that both ABCG36 and ABCG37 function cooperatively in auxin-controlled plant development. Both seem to possess a dual function in the control of auxin homeostasis in the root tip and long-range transport in the mature root correlating with non-polar and polar expression profiles in the LR cap and epidermis, respectively.

Keywords: ABC transporter; ABCG; IAA; IBA; PDR; PEN3; auxin; plant development.

FIGURE 1

Heterologous expression of ABCG36 results in enhanced plasma membrane export of the native and synthetic auxins, IBA and 2.4-D. (A) Yeast expressing ABCG36 or ABCG37 retain significantly less IBA and 2.4-D indicating higher export. (B) Enhanced IBA and 2.4-D export from Nicotiana benthamiana protoplasts prepared after Agrobacterium-mediated leaf transfection with 35S:ABCG36. Significant differences (unpaired t-test with Welch’s correction, p < 0.05) to vector control are indicated by an ‘a’ (mean ± SE; n ≥ 4 transport experiments generated from independent yeast transformations or tobacco transfections). (C) ABCG36 is expressed on the plasma membrane revealed by confocal imaging of ABCG36-GFP (upper panel) and Western analyses of sucrose gradient fractions prepared from tobacco leaves transfected with ABCG36:ABCG36-GFP. Microsomes were separated by a linear 10–50% sucrose gradient and plasma membrane (PIP2)-positive fractions 9–13 (corresponding to 37–50% sucrose) were probed with anti-GFP (lower panel).

FIGURE 2

ABCG36 transports the native and synthetic auxins, IBA and 2.4-D, in Arabidopsis. (A) Enhanced IBA and 2.4-D efflux from leaf protoplasts prepared from ABCG36 (35S:ABCG36) and ABCG37 (35S:ABCG37-GFP) gain-of-function plants. (B) Reduced IBA and 2.4-D efflux from leaf protoplasts prepared from indicated ABCG36 loss-of-function plants. Significant differences (unpaired t-test with Welch’s correction, p < 0.05) to Wt (Col Wt) are indicated by an ‘a’ (mean ± SE; n ≥ 4 transport experiments generated from independent protoplast preparations).

FIGURE 3

ABCG36 functions in rootward IBA transport in the Arabidopsis root. Rootward (A, acropteal) and shootward (B, basipetal) root transport of ³H-IBA and ¹⁴C-IAA assayed in parallel. Significant differences (unpaired t-test with Welch’s correction, p < 0.05) between WT and mutant alleles are indicated by an ‘a’ (mean ± SE; n ≥ 4 transport experiments). Both radiotracers were applied to root-shoot junctions and root tips by diffusion into plant tissues from agar beads functioning as source (Lewis and Muday, 2009); 5 mm segments from the source were used for quantification. (C) ABCG36-GFP is localized to the periphery of epidermal cells but reveals a non-polar expression in cells of the lateral root cap (LRC). Confocal image of a 5 dag root tip carrying ABCG36:ABCG36 (PEN3:PEN3-GFP) in abcg36-3/pen3-3 (Underwood and Somerville, 2017). (D) Putative model on the role of ABCG36 in polar transport of IBA. ABCG36 contributes to the rootward (acropetal) transport of IBA, which based on (A,B) seems to be shared by ABCG37 (not shown here). Lateral, outward-facing PM expression in the WT root epidermis (left panel) suggests lateral IBA excretion into the rhizosphere, taking IBA out of the polar IBA stream provided by so far uncharacterized apical-basal IBA transporters. Deletion of ABCG36 in the mutant (right panel) would enhance polar IBA transport, which is in line with our data. Additionally, ABCG36 seems to contribute to local auxin homeostasis in the very root tip correlating with a predominant non-polar expression in the LRC (dark gray). Absence of ABCG36 might thus abolish IBA export resulting in DR5-GFP activation (Figure 4) and root hypersensitivity on IBA (Strader and Bartel, 2009).

FIGURE 4

Auxin responses and quantification of free IBA and IAA in abcg36. Root auxin responses visualized by the auxin-responsive DR5 reporter in the root tip of 5 dag seedlings (A) grown on solvent (DMSO) or 5 μM IBA (B, quantification of fluorescence by image analysis of confocal sections). Significant differences (unpaired t-test with Welch’s correction, p < 0.05) to corresponding solvent controls are indicated by an ‘a,’ while differences between IBA-treated WT and mutant alleles are marked by a ‘b’ (mean ± SE; n ≥ 20 images). Bar: 20 μm. (C) Free IBA quantified by GC–MS is reduced in abcg36/37 roots. Significant differences (unpaired t-test with Welch’s correction, p < 0.05) between WT and mutant alleles are indicated by ‘a’ (mean ± SE; n = 4). Shoot quantification is shown in Supplementary Figure 2.

FIGURE 5

ABCG36 contributes to auxin-controlled plant development. (A) Hypocotyl elongation of 5 dag seedlings after transfer onto solvent control or IBA (5 μM) plates after 3 days at 28°C. 22°C control is shown in Supplementary Figure 3. (B) Hook opening angle of etiolated seedlings grown for 3 days in the dark and transferred onto solvent control or IBA (5 μM) plates in the dark after 4 days. (C) Root elongation of 5 dag seedlings after transfer onto solvent control or IBA (5 μM) plates after 24 h. (D) Quantification of emerged lateral roots of 5 dag seedlings after transfer onto solvent control or IBA (5 μM) plates after 7 days. Significant differences (unpaired t-test with Welch’s correction, p < 0.05) between corresponding WT and mutant alleles are marked with an ‘a,’ differences to corresponding WT solvent control with a ‘b’ (means ± SE; n = 4 sets of > 20 seedlings each). For root bending assays, 5 dag seedlings were transferred to solvent control (E) or NPA (10 μM) plates (F), and root bending angles were judged after 12 h in the dark.