Phosphate availability alters lateral root development in Arabidopsis by modulating auxin sensitivity via a mechanism involving the TIR1 auxin receptor

Abstract

The survival of plants, as sessile organisms, depends on a series of postembryonic developmental events that determine the final architecture of plants and allow them to contend with a continuously changing environment. Modulation of cell differentiation and organ formation by environmental signals has not been studied in detail. Here, we report that alterations in the pattern of lateral root (LR) formation and emergence in response to phosphate (Pi) availability is mediated by changes in auxin sensitivity in Arabidopsis thaliana roots. These changes alter the expression of auxin-responsive genes and stimulate pericycle cells to proliferate. Modulation of auxin sensitivity by Pi was found to depend on the auxin receptor TRANSPORT INHIBITOR RESPONSE1 (TIR1) and the transcription factor AUXIN RESPONSE FACTOR19 (ARF19). We determined that Pi deprivation increases the expression of TIR1 in Arabidopsis seedlings and causes AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) auxin response repressors to be degraded. Based on our results, we propose a model in which auxin sensitivity is enhanced in Pi-deprived plants by an increased expression of TIR1, which accelerates the degradation of AUX/IAA proteins, thereby unshackling ARF transcription factors that activate/repress genes involved in LR formation and emergence.

Figure 1.

Effect of Pi Availability on LR Formation. (A) Wild-type (Col-0) seedlings were grown on the surface of agar plates containing 0.1× MS medium under P+ (1 mM) or P− (10 μM) for the indicated days after germination. Data are presented for LRP formation and emerged LRs (LRs) at different time points after germination. Values shown represent the mean of three groups of 15 seedlings ± se. (B) Micrographs showing the LRPs in seedlings grown in P+ or P− at 1 and 2 dpg. Bar = 50 μm. (C) Kinetic assay of primary root growth. Mean values were plotted at the indicated days after seed germination (n = 15). Different letters in (A) are used to indicate means that differ significantly (P < 0.05). [See online article for color version of this figure.]

Figure 2.

Effect of Pi Availability on LR Formation in the Phosphate-Insensitive lpi3 Arabidopsis Mutant. Wild-type and lpi3 seedlings were grown for 13 d on vertically oriented agar plates. (A) Photograph of seedlings growing on P+ (left) or P− (right). Bar = 1 cm. (B) Nomarski images of the meristems of wild-type (Col-0) and lpi3 plants growing in P+ (left) or P− (right). Bar = 50 μm. (C) Mean values (±se) of primary root length and LR number are presented (n = 15 seedlings). Different letters are used to indicate means that are significantly different (P < 0.05). [See online article for color version of this figure.]

Figure 3.

Effect of Auxin Transport Inhibition on LR Development in Arabidopsis Seedlings. (A) Arabidopsis (Col-0) seedlings were grown for 4 d on P+ and P− with 1 μM of NPA and transferred to medium with or without 1 μM NPA or to medium containing 1 μM NPA with or without 10⁻⁸ M NAA for 48 h. The seedlings were cleared and the number and stage of LRP recorded according to Zhang et al. (1999). The data represent the mean ± se of the number of LR primordia per plant (n = 10). Different letters indicate that the means differ significantly (P < 0.05). (B) Arabidopsis (Col-0) seedlings were grown for 4 d on P+ or P− with 1 μM of NPA and transferred to medium with (+) or without (−) NPA and 10⁻⁶ M NAA for 48 h. The seedlings were cleared, and the division and cellular proliferation in the pericycle were analyzed. Nomarski micrographs are representative of at least 10 plants analyzed. In all cases, the data represent three independent experiments. Bar = 50 μm. [See online article for color version of this figure.]

Figure 4.

Effect of Pi Availability on the Temporal and Spatial Expression Patterns of the Auxin-Responsive Gene Marker DR5:uidA in Arabidopsis Roots. (A) DR5:uidA seedlings grown for 5 d in P+ (right) or P− (left), immediately transferred to liquid media with various concentrations of auxin (IAA) for 6 h, and then subjected to GUS staining or quantification. Panels for 0, 10⁻⁸, and 10⁻⁶ M IAA correspond to the root region where LR have emerged, and that for 10⁻⁷ M illustrates changes in DR5:uidA expression in the root region where LRP are being formed. Photographs are representative of at least 15 stained plants. Bar = 50 μm. (B) Close-up Normarski image of 5-d-old seedlings showing the expression pattern of DR5:uidA in pericycle cells. Micrograph is representative of at least 15 stained plants. Bar = 50 μm. (C) Fluorometric analysis of the expression of the GUS reporter gene, DR5:uidA. Bars represent the average ± se of three independent experiments (n = 15). Different letters indicate that the means differ significantly (P < 0.05). GUS activity is presented as pmol methylumbelliferone per microgram of protein per minute and was measured 2, 4, and 6 dpg, as indicated.

Figure 5.

Effects of the tir1-1 Mutation and TIR1 Overexpression on LR Formation in Response to Pi Availability. Seedlings of Arabidopsis Col-0, the tir1-1 and tir1-1 afb2 afb3 mutants, and P35S:TIR1-14 were grown in P+ or P−. Seedlings were photographed 13 dpg. (A) to (H) Changes in root system morphology and LR number of seedlings grown in P+ ([A] to [D]) and P− ([E] to [H]). Photographs are representative of at least 15 plants analyzed. Bar = 1 cm. (I) LR number in the wild type, tir1, and the TIR1 overexpressor at 13 dpg. Values shown represent the mean of 15 seedlings ± se. Letters represent statistically different means (P < 0.05). [See online article for color version of this figure.]

Figure 6.

Effect of Pi Availability on the TIR1 Expression Pattern. (A) to (F) Expression patterns of TIR1:uidA in 6-d-old seedlings grown in P+ ([A] to [C]) or in P− ([D] to [F]). Photographs are representative of at least 15 stained plants. Bars = 50 μm. (G) Nomarski optics close-up of the central cylinder of seedlings grown in P− in the same root region presented in (F). (H) GUS activity of TIR1:uidA seedlings grown in P+ or P− for the indicated times. Letters represent statistically different means (P < 0.05). (I) Real time-PCR analysis of TIR1 in total RNA of roots isolated from 6-d-old seedlings. White bars represent growth in P+, and black bars represent growth in P−.

Figure 7.

Effect of Pi Availability on AXR3 Degradation. Four-day-old HS:AXR3NT-GUS seedlings grown in P+ or P− media were heat shocked for 2 h at 37°C and then transferred for 20 min to liquid media containing various concentrations of NAA. (A) HS:AXR3NT-GUS seedlings were stained for histochemical GUS activity for the indicated periods of time. Bars = 200 μm. (B) Fluorometric quantification of GUS activity. Relative activity is expressed as a percentage of the 20-min level. Each data point shows the mean of three independent experiments.

Figure 8.

Effect of Pi Availability on LR Formation in Auxin Response Mutants. LR number for 13-d-old wild-type (Col-0), arf7-1, arf19-1, arf7 arf19, and slr Arabidopsis seedlings was determined. The data represent the means ± se of seedlings (n = 15) grown in P+ (white bars) and P− (black bars). Different letters are used to indicate means that differ significantly (P < 0.05).