The Biphasic Root Growth Response to Abscisic Acid in Arabidopsis Involves Interaction with Ethylene and Auxin Signalling Pathways

Abstract

Exogenous abscisic acid (ABA) is known to either stimulate or inhibit root growth, depending on its concentration. In this study, the roles of ethylene and auxin in this biphasic effect of ABA on root elongation were investigated using chemical inhibitors and mutants. Inhibitors of ethylene perception and biosynthesis and an auxin influx inhibitor were all found to block the inhibitory effect of high ABA concentrations, but not the stimulatory effect of low ABA concentrations. In addition, three ethylene-insensitive mutants (etr1-1, ein2-1, and ein3-1), two auxin influx mutants (aux1-7, aux1-T) and an auxin-insensitive mutant (iaa7/axr2-1) were all insensitive to the inhibitory effect of high ABA concentrations. In the case of the stimulatory effect of low ABA concentrations, it was blocked by two different auxin efflux inhibitors and was less pronounced in an auxin efflux mutant (pin2/eir1-1) and in the iaa7/axr2-1 auxin-insensitive mutant. Thus it appears that the stimulatory effect seen at low ABA concentrations is via an ethylene-independent pathway requiring auxin signalling and auxin efflux through PIN2/EIR1, while the inhibitory effect at high ABA concentrations is via an ethylene-dependent pathway requiring auxin signalling and auxin influx through AUX1.

Keywords: Arabidopsis; abscisic acid (ABA); auxin signalling; auxin transport; ethylene biosynthesis; ethylene signalling; hormone; root elongation.

FIGURE 1

Biphasic effect of applied exogenous ABA on the growth of primary root over the 6-day treatments. (A) Total primary root length. (B) Primary root elongation rate. Four-day old Arabidopsis wild-type Col-8 seedlings with similar root length were chosen and transferred to newly made 0.02 × B5 medium (1 mM KNO₃, 0.5% sucrose) with various ABA concentrations (black circle, control; white circle, 0.1 μM ABA; black triangle, 1 μM ABA; white triangle, 10 μM ABA). Primary root length was marked after transplanting and the increase of primary root were measured every day. The root elongation rate was calculated for each day. The values are means, and the vertical bars represent standard errors. Data analysed using one-way ANOVA with Tukey’s post hoc test and different letters indicate significant differences among ABA treatments in the same day at P < 0.05. Eta² of one-way ANOVA: (A) 0.776 (day 6); (B) 0.694 (day 3); 0.737 (day 6). Seedling numbers: control, n = 14; 0.1 μM ABA, n = 9–14; 1 μM ABA, n = 10–14; 10 μM ABA, n = 11–14. At least three independent experiments were performed and similar results obtained and reported.

FIGURE 2

Ethylene biosynthesis and signalling inhibitors altered root responses to ABA treatments. AVG: ethylene biosynthesis inhibitor. STS: ethylene signalling inhibitor. (A) The effects of AVG after 1 day. (B) The effects of AVG over a 4-day period. (C) The effects of STS during the first 2-day period. (D) The effects of STS during 4 days. Col-8 seedlings were germinated, chosen and transferred to medium as described in Figure 1. The medium was treated with various ABA and AVG/STS concentrations (μM). Primary root length was marked after transplanting and the increase of primary root were measured every day. The root elongation rate was calculated for the 1 or 2 days and 4 days after treatments on average. The values are means, and the vertical bars represent standard errors of the means. Data analysed using one-way ANOVA with Tukey’s post hoc test and different letters indicate significant differences cross all treatments at P < 0.05. Eta²: (A) 0.650; (B) 0.622; (C) 0.729; (D) 0.817. Seedling numbers: (A) n = 14; (B) n = 9–14; (C) n = 9–12; (D) n = 7–12. At least three independent experiments were performed and similar results obtained and reported.

FIGURE 3

Various responses of root growth to seven ABA treatments in three ethylene insensitive mutants and wild-type. Primary root elongation rates 1 day after treatment: (A) Col-8 wild-type; (C) etr1-1; (E) ein2-1; (G) ein3-1, and over a 4-day treatment: (B) Col-8 wild-type; (D) etr1-1; (F) ein2-1; (H) ein3-1. Seedlings of each line were germinated, chosen and transferred to medium with various ABA concentrations (μM) as described in Figure 1. Primary root length was marked after transplanting and the increase of primary root were measured every day. The root elongation rate was calculated for 1 and 4 days after treatments on average. Only one genotype was used in each experiment (n = 14), and results for each genotype came from combining two sets of independent experiments. All eight experiments were done consecutively from 17/07/2013 (day/month/year) to 26/08/2013. The values are means, and the vertical bars represent standard errors of the means. Data analysed using one-way ANOVA with Tukey’s post hoc test and different letters indicate significant differences among ABA treatments at P < 0.05. Eta²: (A) 0.773; (B) 0.842; (C) 0.333; (D) 0.635; (E) 0.237; (F) 0.660; (G) 0.810; (H) 0.827. Seedling numbers: (A) n = 28; (B) n = 21–28; (C) n = 28; (D) n = 22–28; (E) n = 28; (F) n = 21–28; (G) n = 28; (H) n = 27–28. At least three independent experiments were performed and similar results obtained and reported.

FIGURE 4

Auxin influx and efflux inhibitors altered root responses to ABA. NPA, N-1-naphthylphthalamidic acid, auxin efflux inhibitor; TIBA, 2,3,5-triiodobenzoic acid, auxin efflux inhibitor; CHPAA, 3-chloro-4-hydroxyphenylacetic acid, auxin influx inhibitor. Primary root elongation rates (A) 1 day after treatment and (B) over a 4-day treatment period. Col-8 seedlings were germinated, chosen and transferred to medium as described in Figure 1. The medium was treated with various ABA concentrations and 0.1% DMSO or 10 μM NPA/TIBA/CHPAA. Primary root length was marked after transplanting and the increase of primary root were measured every day. The root elongation rate was calculated for 1 day and over a 4-day treatment on average. The values are means, and the vertical bars represent standard errors of the means. Data analysed using one-way ANOVA with Tukey’s post hoc test and different letters indicate significant differences cross all treatments at P < 0.05. Eta²: (A) 0.834; (B) 0.951. Seedling numbers: (A) n = 10–12; (B) n = 3–12. At least three independent experiments were performed and similar results obtained and reported.

FIGURE 5

Auxin relevant mutants showed both auxin signalling and auxin transport are required for root growth response to ABA treatments. Primary root elongation rate after the first 24 h treatment of: (A) wild-type Col-8, pin2/eir1-1, aux1-T, iaa7/axr2-1 (eta² of one-way ANOVA: 0.835, 0.517, 0.118, 0.087); (C) Col-8, pin4-3, pin7-2, tir1-1 (eta² of one-way ANOVA: 0.798, 0.840, 0.808, 0.798); (E) Col-8, aux1-7, pin3-4, pin3-5 (eta² of one-way ANOVA: 0.773, 0.419, 0.854, 0.881). Average primary root elongation rate over the 4-day treatment of: (B) Col-8, pin2/eir1-1, aux1-T, iaa7/axr2-1 (eta² of one-way ANOVA: 0.740, 0.724, 0.324, 0.616); (D) Col-8, pin4-3, pin7-2, tir1-1 (eta² of one-way ANOVA: 0.790, 0.884, 0.883, 0.828); (F) Col-8, aux1-7, pin3-4, pin3-5 (eta² of one-way ANOVA: 0.908, 0.705, 0.906, 0.826). A–F were results from three experiments separately. In each experiment, seedlings of each line were germinated, chosen and transferred to medium with various ABA concentrations (μM) as described in Figure 1. Primary root length was marked after transplanting and the increase of primary root were measured every day. The root elongation rate was calculated for 1 and 4 days after treatments on average. The values are means, and the vertical bars represent standard errors of the means. Data analysed using one-way ANOVA with Tukey’s post hoc test and different letters indicate significant differences among ABA treatments in each genotype at P < 0.05. Seedling numbers: (A) n = 12; (B) n = 6–12; (C) n = 12; (D) n = 3–12; (E) n = 8; (F) n = 4–8. Similar experiments were done for at least three times with different mutant combinations and similar results were obtrained.

FIGURE 6

A model for the involvement of ethylene and auxin in root growth responses to different ABA treatments. In the model, ABA regulates root growth through two distinct pathways: (1) an ethylene-independent stimulatory pathway that operates at low [ABA] and requires auxin signalling and auxin efflux through PIN2/EIR1; and (2) an ethylene-dependent inhibitory pathway that operates at high [ABA] and that also requires auxin signalling and auxin influx through AUX1. Ethylene regulates root growth through downstream auxin is based on the report that aux1-T mutant exhibited ACC-resistant root growth (Růžička et al., 2007).