A role for auxin redistribution in the responses of the root system architecture to phosphate starvation in Arabidopsis

Abstract

The changes in root system architecture (RSA) triggered by phosphate (P) deprivation were studied in Arabidopsis (Arabidopsis thaliana) plants grown for 14 d on 1 mM or 3 microM P. Two different temporal phases were observed in the response of RSA to low P. First, lateral root (LR) development was promoted between days 7 and 11 after germination, but, after day 11, all root growth parameters were negatively affected, leading to a general reduction of primary root (PR) and LR lengths and of LR density. Low P availability had contrasting effects on various stages of LR development, with a marked inhibition of primordia initiation but a strong stimulation of activation of the initiated primordia. The involvement of auxin signaling in these morphological changes was investigated in wild-type plants treated with indole-3-acetic acid or 2,3,5-triiodobenzoic acid and in axr4-1, aux1-7, and eir1-1 mutants. Most effects of low P on RSA were dramatically modified in the mutants or hormone-treated wild-type plants. This shows that auxin plays a major role in the P starvation-induced changes of root development. From these data, we hypothesize that several aspects of the RSA response to low P are triggered by local modifications of auxin concentration. A model is proposed that postulates that P starvation results in (1) an overaccumulation of auxin in the apex of the PR and in young LRs, (2) an overaccumulation of auxin or a change in sensitivity to auxin in the lateral primordia, and (3) a decrease in auxin concentration in the lateral primordia initiation zone of the PR and in old laterals. Measurements of local changes in auxin concentrations induced by low P, either by direct quantification or by biosensor expression pattern (DR5::beta-glucuronidase reporter gene), are in line with these hypotheses. Furthermore, the observation that low P availability mimicked the action of auxin in promoting LR development in the alf3 mutant confirmed that P starvation stimulates primordia emergence through increased accumulation of auxin or change in sensitivity to auxin in the primordia. Both the strong effect of 2,3,5-triiodobenzoic acid and the phenotype of the auxin-transport mutants (aux1, eir1) suggest that low P availability modifies local auxin concentrations within the root system through changes in auxin transport rather than auxin synthesis.

Figure 1.

Effect of P availability on root architecture parameters. Wild-type Columbia seedlings were grown for 14 d in the presence of low (3 μm; ○) or high (1 mm; •) P concentration on vertically oriented agar plates. Average values (±sd) of eight seedlings, calculated daily from d 7 to d 14 after sowing, are given for the length of the entire root system (A), the PR (B), the cumulated length of the LRs (C), the LR number (D), the PR elongation rate (E), and the LR elongation rate (F). Significant differences (t test) are indicated by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

Effect of P availability on both primordia number and meristematic activity. A, Histochemical GUS staining of transgenic (CycB1::GUS) Arabidopsis plants that were cultivated on high (1 mm) or low (3 μm) P medium on vertically oriented agar plates. Meristematic activity was estimated according to staining intensity; plain boxes correspond to emerged LRs, black dots represent intensely stained primordia, and white dots correspond to weakly stained primordia. Bar = 1 cm. B and C, Schematic representation of individual entire PRs (B) of plants grown on high or low P medium for 9 d or apical part of the PR (C) of a 14-d-old plant that had developed after d 9. Bars = 1 cm.

Figure 3.

Effect of P availability on the number of second-order primordia initiated on LRs. Transgenic (CycB1::GUS) Arabidopsis plants were cultured for 14 d on vertically oriented agar plates containing high (1 mm) or low (3 μm) P medium and histochemically GUS stained to easily and unambiguously score all initiated primordia. Data correspond to the average number of primordia (±sd) scored on LRs of different length of eight plants. Seedlings were cultured on low (white bars) or high (black bars) P medium. Probabilities are indicated by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, Not significant.

Figure 4.

Morphology of hormone-treated wild-type seedlings and auxin mutants. Wild-type Columbia or mutant seedlings were grown for 14 d on high (1 mm) P medium on vertically oriented agar plates. IAA and TIBA correspond to wild-type seedlings, Col-0 ecotype, grown on 0.1 μm IAA or 0.1 μm TIBA, respectively. axr4, aux1, and eir1 indicate axr4-1, aux1-7, and eir1-1 mutant seedlings. Bars = 1 cm.

Figure 5.

Effect of P availability and hormone treatments on LR density and LR elongation rate of Arabidopsis. Seedlings were grown for 9 d on low (white bars) or high (black bars) P medium on vertically oriented agar plates. IAA corresponds to wild-type seedlings, Col-0 ecotype, grown on 0.1 μm IAA. axr4, aux1, and eir1 indicate axr4-1, aux1-7, and eir1-1 mutant seedlings. Data correspond to the average value (±sd) of eight seedlings. Significant differences (t test) are indicated by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, Not significant.

Figure 6.

Effect of P availability and hormone treatments on PR elongation rate, LR density, and LR elongation rate of Arabidopsis. Seedlings were grown for 14 d on low (white bars) or high (black bars) P medium on vertically oriented agar plates. IAA and TIBA correspond to wild-type seedlings, Col-0 ecotype, grown in presence of 0.1 μm IAA or 0.1 μm TIBA, respectively. axr4, aux1, and eir1 correspond to the axr4-1, aux1-7, and eir1-1 mutant seedlings. Data correspond to the average value (±sd) of eight seedlings. Significant differences (t test) are indicated by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, Not significant.

Figure 7.

Effect of P availability and hormone treatments on Arabidopsis leaf area. Wild-type Columbia or mutant seedlings were grown for 14 d on low (white bars) or high (black bars) P medium on vertically oriented agar plates. Aerial parts were excised and scanned, and projected leaf area was quantified. IAA and TIBA correspond to wild-type seedlings, Col-0 ecotype, grown in presence of 0.1 μm IAA or 0.1 μm TIBA, respectively. axr4, aux1, and eir1 correspond to the axr4-1, aux1-7, and eir1-1 mutant seedlings. Data correspond to the average value (±sd) of eight seedlings. Significant differences (t test) are indicated by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, Not significant.

Figure 8.

Effect of P availability and IAA treatment on PR elongation rate and LR density of Arabidopsis. Auxin-untreated (circles) and IAA-treated (triangles) Col-0 seedlings were grown for 14 d on low (white marks) or high (black marks) P medium on vertically oriented agar plates. Average values (±sd) of eight seedlings, calculated daily from d 7 to d 14 after sowing, are given for the PR elongation rate (A) and the LR density (B). Significant differences (t test) are indicated by asterisks: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 9.

Free IAA levels measured in seedlings grown for 14 d on high or low P medium. Free IAA levels were measured from leaves, PR central part (from the last basal emerged LR downward but excluding the last centimeter), PR apex (1 cm), long LRs (>1.5 cm), and short LRs (<1.5 cm) of seedlings grown for 14 d. Data correspond to the average value (±se) of three independent cultures of Columbia seedlings cultured on high (black bars) or low (white bars) P medium or in the presence of 0.1 μm TIBA on high (hatched bars) or low (dotted bars) P medium.

Figure 10.

Effect of P availability on auxin-responsive reporter DR5::GUS expression in PR apex and LRs. Transgenic (DR5::GUS) Arabidopsis plants were cultured for 14 d on high (A, C, E, G, I, K, M, and O) or low (B, D, F, H, J, L, N, and P) P medium followed by histochemical GUS staining. The expression pattern of the DR5::GUS was observed in the PR apical meristem (A–D) and from primordia initiation to postemergence of LRs (E–P). Primordia stages were named according to Malamy and Benfey (1997): stage I (E and F), stage II (G and H), stage V (I and J), stage VI (K and L), emergence (M and N), and LR elongation (O and P). Bars = 50 μm.

Figure 11.

Effect of P availability on root architecture of alf3 mutant. The alf3 mutant seedlings were grown for 14 d on high (+P) or low (−P) P medium on vertically oriented petri dishes. Bar = 1 cm.

Figure 12.

Schematic model of auxin redistribution within the root system in response to P starvation.