Conversion of endogenous indole-3-butyric acid to indole-3-acetic acid drives cell expansion in Arabidopsis seedlings

Abstract

Genetic evidence in Arabidopsis (Arabidopsis thaliana) suggests that the auxin precursor indole-3-butyric acid (IBA) is converted into active indole-3-acetic acid (IAA) by peroxisomal beta-oxidation; however, direct evidence that Arabidopsis converts IBA to IAA is lacking, and the role of IBA-derived IAA is not well understood. In this work, we directly demonstrated that Arabidopsis seedlings convert IBA to IAA. Moreover, we found that several IBA-resistant, IAA-sensitive mutants were deficient in IBA-to-IAA conversion, including the indole-3-butyric acid response1 (ibr1) ibr3 ibr10 triple mutant, which is defective in three enzymes likely to be directly involved in peroxisomal IBA beta-oxidation. In addition to IBA-to-IAA conversion defects, the ibr1 ibr3 ibr10 triple mutant displayed shorter root hairs and smaller cotyledons than wild type; these cell expansion defects are suggestive of low IAA levels in certain tissues. Consistent with this possibility, we could rescue the ibr1 ibr3 ibr10 short-root-hair phenotype with exogenous auxin. A triple mutant defective in hydrolysis of IAA-amino acid conjugates, a second class of IAA precursor, displayed reduced hypocotyl elongation but normal cotyledon size and only slightly reduced root hair lengths. Our data suggest that IBA beta-oxidation and IAA-amino acid conjugate hydrolysis provide auxin for partially distinct developmental processes and that IBA-derived IAA plays a major role in driving root hair and cotyledon cell expansion during seedling development.

Figure 1.

Peroxisomal mutants are defective in IBA-to-IAA conversion. A, Auxin response of Col-0, tir1-1, axr2-1, pex6-1, pxa1-1, acx1-2 acx2-1, chy1-10, and ibr1-2 ibr3-1 ibr10-1. Mean primary root lengths (+se; n ≥ 12) of 8-d-old seedlings grown under yellow-filtered light at 22°C on medium supplemented with ethanol (mock), 100 nm IAA, or 10 μm IBA are shown. B, Suc dependence of Col-0, tir1-1, axr2-1, pex6-1, pxa1-1, acx1-2 acx2-1, chy1-10, and ibr1-2 ibr3-1 ibr10-1. Mean hypocotyl lengths (+se; n = 12) of seedlings grown in the absence or presence of 0.5% Suc were measured 4 d after transfer of 1-d-old seedlings to the dark. C, In planta conversion of IBA to IAA. Eight-day-old Col-0, tir1-1, axr2-1, pex6-1, pxa1-1, acx1-2 acx2-1, chy1-10, and ibr1-2 ibr3-1 ibr10-1 light-grown seedlings were incubated for 1 h in the presence of 10 μm [¹³C₈-¹⁵N₁]IBA and the resultant [¹³C₈-¹⁵N₁]IAA levels were measured. Wt, Col-0.

Figure 2.

ibr mutants display root hair elongation defects. A to F, Histograms of root hair lengths of 5-d-old vertically grown Col-0 (Wt; A), pen3-4 (B), ibr1-2 (C), ibr3-1 (D), ibr10-1 (E), and ibr1-2 ibr3-1 ibr10-1 (F) seedlings. Root hairs were measured using NIH Image software (n = 500 total root hairs from at least 10 seedlings). Bars depicting median root hair lengths are shaded. [See online article for color version of this figure.]

Figure 3.

Auxin application restores root hair elongation to ibr mutants. A, Photographs of 5-d-old Col-0 and ibr1-2 ibr3-1 ibr10-1 seedling roots vertically grown under yellow-filtered light at 22°C on medium supplemented with ethanol (mock) or 100 nm IAA. Scale bar = 1 mm. B, Mean root hair lengths (+se) of 5-d-old Col-0, ibr1-2 ibr3-1 ibr10-1, and pen3-4 seedlings vertically grown under yellow-filtered light at 22°C on medium supplemented with ethanol (mock), 100 nm IAA, 100 nm NAA, or 10 μm NPA. Root hair lengths were measured using NIH Image software (n = 500 total root hairs from at least 12 seedlings). C to K, Histograms of root hair lengths from section B of Col-0 (C–E), ibr1-2 ibr3-1 ibr10-1 (F–H), and pen3-4 (I–K) grown on IAA (C, F, and I), NAA (D, G, and J), or NPA (E, H, and K). Histograms for mock-treated seedlings are shown in every section for comparison. Wt, Col-0. [See online article for color version of this figure.]

Figure 4.

ibr1 ibr3 ibr10 suppresses pen3 root hair hyperelongation. A, Mean primary root lengths (+se) of 8-d-old Col-0, pen3-4, ibr1-2 ibr3-1 ibr1-10, and pen3-4 ibr1-2 ibr3-1 ibr10-1 seedlings grown under yellow-filtered light at 22°C on medium supplemented with ethanol (mock), 8 μm IBA, 1 μm 2,4-DB, or 100 nm 2,4-D (n ≥ 15). B to E, Histograms of root hair lengths of 5-d-old vertically grown Col-0 (B), pen3-4 (C), ibr1-2 ibr3-1 ibr10-1 (D), and pen3-4 ibr1-2 ibr3-1 ibr10-1 (E) seedlings. Root hair lengths were measured using NIH Image software (n = 500 total root hairs from at least 15 seedlings). Bars depicting median root hair lengths are shaded. Wt, Col-0. [See online article for color version of this figure.]

Figure 5.

The ilr1 iar3 ill2 triple auxin-conjugate hydrolase mutant displays defects in root hair elongation. Histograms of root hair lengths of 5-d-old vertically grown Ws-2 (Wt; A) and ilr1-1 iar3-2 ill2-1 (B) seedlings. Root hair lengths were measured using NIH Image software (n = 500 total root hairs from at least 15 seedlings). Bars depicting median root hair lengths are shaded. [See online article for color version of this figure.]

Figure 6.

The ibr1 ibr3 ibr10 mutant has small cotyledons but normal hypocotyl elongation. A, Mean hypocotyl lengths (+se) of 8-d-old Col-0 (Wt), ibr1-2 ibr3-1 ibr10-1, Ws-2 (Wt), and ilr1-1 iar3-2 ill2-1 seedlings grown under yellow-filtered light at 22°C or 28°C. B, Photograph of 7-d-old Col-0 (Wt), ibr1-2 ibr3-1 ibr10-1, Ws-2 (Wt), and ilr1-1 iar3-2 ill2-1 light-grown seedlings. C, Mean cotyledon areas (+se) of 7-d-old Col-0 (Wt), ibr1-2 ibr3-1 ibr10-1, Ws-2 (Wt), and ilr1-1 iar3-2 ill2-1 light-grown seedlings (n ≥ 33). D, ibr1 ibr3 ibr10 suppresses pen3 enlarged cotyledons. Col-0 (Wt), pen3-4, ibr1-2 ibr3-1 ibr10-1, and pen3-4 ibr1-2 ibr3-1 ibr10-1 seeds were stratified for 3 d at 4°C prior to growth at 22°C under white light. Mean cotyledon areas (±se) of light-grown seedlings during growth following germination (n ≥ 51). Measurements were made using NIH Image software.