Localized iron supply triggers lateral root elongation in Arabidopsis by altering the AUX1-mediated auxin distribution

Abstract

Root system architecture depends on nutrient availability, which shapes primary and lateral root development in a nutrient-specific manner. To better understand how nutrient signals are integrated into root developmental programs, we investigated the morphological response of Arabidopsis thaliana roots to iron (Fe). Relative to a homogeneous supply, localized Fe supply in horizontally separated agar plates doubled lateral root length without having a differential effect on lateral root number. In the Fe uptake-defective mutant iron-regulated transporter1 (irt1), lateral root development was severely repressed, but a requirement for IRT1 could be circumvented by Fe application to shoots, indicating that symplastic Fe triggered the local elongation of lateral roots. The Fe-stimulated emergence of lateral root primordia and root cell elongation depended on the rootward auxin stream and was accompanied by a higher activity of the auxin reporter DR5-β-glucuronidase in lateral root apices. A crucial role of the auxin transporter AUXIN RESISTANT1 (AUX1) in Fe-triggered lateral root elongation was indicated by Fe-responsive AUX1 promoter activities in lateral root apices and by the failure of the aux1-T mutant to elongate lateral roots into Fe-enriched agar patches. We conclude that a local symplastic Fe gradient in lateral roots upregulates AUX1 to accumulate auxin in lateral root apices as a prerequisite for lateral root elongation.

Figure 1.

Effect of Homogeneous and Localized Fe Supplies on Lateral Root Development of Arabidopsis Plants. (A) and (B) Root architecture of wild-type plants (accession Col-0) in response to Fe supply. Seedlings were grown on half-strength MS medium without Fe for 7 d before being transferred to SAPs containing half-strength MS and 75 μM ferrozine. Fe(III)-EDTA (μM) was added at the indicated concentrations to all three segments ([A]; homogeneous supply) or only to the middle segment ([B]; localized supply). Plants were scanned after 15 d of growth on Fe treatments, and representative plants are shown. Horizontal lines represent the borders between the three segments. (C) to (H) Lateral root density ([C], [E], and [G]) and average lateral root length ([D], [F], and [H]) in the top ([C] and [D]), middle ([E] and [F]), and bottom ([G] and [H]) segment. Bars indicate means ± se; n = 7 plates containing three plants. Different letters indicate significant differences among means (P < 0.05 by Tukey’s test).

Figure 2.

Defective Lateral Root Development in the arf7 arf19 Double Mutant Causes Leaf Chlorosis under Localized Fe Supply. (A) and (B) Wild-type (Col-0) and arf7 arf19 seeds were germinated on Fe-free, half-strength MS medium for 7 d. Seedlings were then transferred to segmented agar plates supplied with 75 μM ferrozine and with 50 μM Fe-EDTA in all three segments (homogeneous) or only in the middle segment (localized). (A) Photographs were taken after 15 d of treatments, and representative plants are shown. (B) Chlorophyll concentrations of whole shoots of wild-type and arf7 arf19 plants grown under homogeneous or localized supply of 50 μM Fe. Bars represent means ± se; n = 6 plates containing three plants. Asterisk denotes a significant difference according to Student’s t test (P < 0.05). FW, fresh weight; ns, not significant.

Figure 3.

Lateral Root Development in Response to Localized Fe Supply in Transgenic Plants with Deregulated Expression of FIT. Lateral root density (A) and average lateral root length (B) in wild-type (Col-0), Pro35S:FIT, and fit mutant plants as affected by the Fe concentration supplied to the middle segment. Seeds were germinated on Fe-free, half-strength MS medium for 7 d. Then, seedlings were transferred to segmented agar plates supplied with Fe in the middle segment at the indicated concentrations. After 15 d, the number of visible lateral roots (>0.5 mm) and mean lateral root length in the middle segment were determined by image analysis. Bars indicate means ± se, n = 7 to 12 plates with three plants per plate. Different letters indicate significant differences among means (P < 0.05 by Tukey’s test).

Figure 4.

Lateral Root Development in Wild-Type and irt1 Plants in Response to Localized Fe Supply. Density (A) and average length (B) of lateral roots in wild-type (Col-0) and irt1 mutant plants as affected by the Fe concentration supplied to the middle segment. Wild-type and irt1 seeds were germinated on Fe-free, half-strength MS medium for 7 d. Seedlings were then transferred to segmented agar plates supplied with Fe in the middle segment at the indicated concentrations. After 15 d, the number and average length of visible lateral roots (>0.5 mm) in the middle segment were determined by image analysis. Shoot chlorophyll concentrations were determined after 15 d of growth (C). Bars represent means ± se; n = 7 replicates consisting of three plants. Different letters indicate significant differences among means (P < 0.05 by Tukey’s test).

Figure 5.

Effect of Shoot Fe Supply on the Development of Lateral Roots in Wild-Type (Col-0) and irt1 on a Tetrapartite Agar Plate. (A) Experimental setup for supplying Fe to shoots. Segment a was supplemented with 100 μM citrate or 100 μM Fe(III)-citrate, segment c was supplied with 10 to 600 μM Fe-EDTA, and segments b and d were Fe deficient. (B) to (E) Chlorophyll concentrations (B), shoot biomass (C), density (D), and average length (E) of lateral roots of wild-type (Col-0) and irt1 plants after growth for 14 d under increasing concentrations of Fe-EDTA in agar segment c. Bars indicate means ± se; n = 6 to 7 plates with three plants each. Different letters indicate significant differences among means (P < 0.05 by Tukey’s test). FW, fresh weight. [See online article for color version of this figure.]

Figure 6.

Lateral Root Development in Wild-Type and frd3-1 Plants in Response to Localized Fe Supply. Lateral root density (A), lateral root length (B), and chlorophyll concentration (C) in the shoots of wild-type (Col-gl) and frd3-1 mutant plants. Seeds were germinated on Fe-deficient, half-strength MS medium for 7 d before transfer to segmented agar plates locally supplied with Fe(III)-EDTA only to the middle segment. Plant roots were scanned and the chlorophyll concentration determined after 15 d on Fe treatments. Bars represent means ± se; n = 7 to 12 plates with three seedlings per plate. Different letters indicate significant differences among means (P < 0.05 by Tukey’s test). FW, fresh weight.

Figure 7.

Effect of Homogeneous or Localized Fe Supply on Cell Division, Differentiation, and Elongation. (A) Seedlings of a ProCYCB1;1:GUS line were germinated on Fe-deficient medium and transferred to segmented agar plates supplemented with 75 μM ferrozine and with the indicated Fe(III)-EDTA concentrations only in the middle segment (localized supply) or in all three segments (homogeneous supply). After 7 d, lateral roots from the middle segment were assayed for GUS activity ([A], left panel) and length of rhizodermal cells ([A], right panel). Arrowheads indicate the boundaries of two consecutive epidermal cells. Bars = 25 μm. (B) and (C) Length of lateral root meristems (B) and length of individual rhizodermal cells (C). Bars represent means ± se; n = 3 to 4 lateral roots from >14 seedlings. Asterisks denote a significant difference according to Student’s t test (P < 0.01).

Figure 8.

Role of Auxin in Fe-Dependent Lateral Root Elongation. (A) and (B) Expression of the auxin-responsive reporter DR5-GUS in emerged (A) and elongating (B) lateral roots. Fe-EDTA (10, 50, or 200 μM) was supplied either homogenously to all three agar segments or locally to the middle agar segment. Lateral roots were analyzed by light microscopy after 7 d of treatments (n > 10 seedlings). The experiments were repeated twice and representative lateral roots are shown. Bars = 100 μm. (C) Experimental setup for supplying NPA either to leaves or to the primary root tip. (D) Elongation rate of lateral roots of wild-type (Col-0) plants supplied locally with 50 μM Fe and treated with the auxin transport inhibitor NPA on leaves or on the primary root tip. Seeds were germinated on Fe-free, half-strength MS medium for 7 d. Seedlings were then transferred to segmented agar plates supplied with 50 μM Fe in the middle segment and on which the shoot or the primary root tip was placed over a fourth segment. After 7 d, 5 μM NPA was supplied either to the leaves or to the primary root tip. Lateral root length was measured every day from days 6 to 9 to determine lateral root growth rate before and after NPA treatments. Bars indicate means ± se; n = 6 plates with three plants each. Different letters indicate significant differences among means (P < 0.05 by Tukey’s test).

Figure 9.

Auxin Accumulation Responds to Internal Fe Levels of the Root. Expression of the auxin-sensitive reporter DR5-GFP in lateral roots of wild-type (Col-0) and irt1 plants in response to localized supply of 50 μM Fe and the concomitant shoot supplementation of 100 μM citrate or Fe-citrate. Seven-day-old seedlings germinated on Fe-deficient medium were transferred to segmented agar plates containing 50 μM Fe-EDTA in the middle segment. Lateral roots were analyzed by confocal laser scanning microscopy after 7 d of treatments (n > 10 seedlings). The experiments were repeated twice, and representative lateral roots are shown. Bars = 100 μm. [See online article for color version of this figure.]

Figure 10.

The Effect of Localized Fe on Lateral Root Elongation Is Dependent on AUX1. (A) and (B) Density (A) and length (B) of lateral roots in wild-type (Col-0) and aux1-T plants after 15 d on treatments. Bars represent means ± se; n = 7 plates with three seedlings per plate. Different letters indicate significant differences among means (P < 0.05 by Tukey’s test). (C) ProAUX1:AUX1:YFP expression in lateral roots from the upper and middle segments in wild-type plants grown on a homogeneous or localized supply of 50 μM Fe. Bars = 50 μm. (D) Relative YFP fluorescence in lateral root tips of plants grown under homogeneous or localized supply of 10, 50, or 200 μM Fe. (E) Relative YFP fluorescence in lateral root apices from the upper and middle segment of plants grown under homogeneous or localized supply of 50 μM Fe. Bars represent means ± se; n > 10 seedlings. One asterisk and two asterisks denote a significant difference according to Student’s t test at P < 0.05 or P < 0.01, respectively.

Figure 11.

Model for the Regulation of Lateral Root Development under Localized Availability of Fe. Uptake of Fe via the high-affinity Fe²⁺ transporter IRT1 increases the symplastic root Fe pool, which can be replenished by Fe derived from the shoot or other parts of the root. The local enrichment in symplastic root Fe leads to an upregulation of the auxin importer AUX1, which channels auxin from the rootward auxin stream toward the lateral root tip. This response is reinforced by the integration of systemic Fe deficiency signals from other parts of the root. Increased rootward auxin movement in lateral roots triggers the longitudinal elongation of mature cells, subsequently resulting in enhanced lateral root elongation. [See online article for color version of this figure.]