Gravitropism of Arabidopsis thaliana roots requires the polarization of PIN2 toward the root tip in meristematic cortical cells

Abstract

In the root, the transport of auxin from the tip to the elongation zone, referred to here as shootward, governs gravitropic bending. Shootward polar auxin transport, and hence gravitropism, depends on the polar deployment of the PIN-FORMED auxin efflux carrier PIN2. In Arabidopsis thaliana, PIN2 has the expected shootward localization in epidermis and lateral root cap; however, this carrier is localized toward the root tip (rootward) in cortical cells of the meristem, a deployment whose function is enigmatic. We use pharmacological and genetic tools to cause a shootward relocation of PIN2 in meristematic cortical cells without detectably altering PIN2 polarization in other cell types or PIN1 polarization. This relocation of cortical PIN2 was negatively regulated by the membrane trafficking factor GNOM and by the regulatory A1 subunit of type 2-A protein phosphatase (PP2AA1) but did not require the PINOID protein kinase. When GNOM was inhibited, PINOID abundance increased and PP2AA1 was partially immobilized, indicating both proteins are subject to GNOM-dependent regulation. Shootward PIN2 specifically in the cortex was accompanied by enhanced shootward polar auxin transport and by diminished gravitropism. These results demonstrate that auxin flow in the root cortex is important for optimal gravitropic response.

Figure 1.

Effect of Brefeldin on Arabidopsis Root Elongation and Gravitropism. For assaying elongation (A), 5-d-old seedlings were transferred to plates containing brefeldin and incubated for 48 h. For assaying gravitropism ([B] and [C]), the seedlings were incubated on inhibitor plates for 18 h and then gravity stimulation applied by rotating the plate by 90°. Bars and symbols plot mean ± se from three independent experiments, with 10 to 12 seedlings per experiment. Asterisks represent the statistical significance between an experimental and control mean as judged by Student's t test: ** P < 0.01 and *** P < 0.001. In (B), brefeldin treatments were significant (P < 0.001) at all times past zero except for 1 μM, which was significant at P < 0.01 at 9 h and otherwise not significant. (A) Root elongation. (B) Curvature of root tips plotted against time after reorientation. (C) Root elongation during gravitropism measured for the experiment in (B). [See online article for color version of this figure.]

Figure 2.

Effect of Brefeldin on the Localization of PIN2 and PIN1. Five-day-old Arabidopsis seedlings were treated for 48 h before being fixed and processed for immunofluorescence with anti-PIN2 and anti-PIN1 antibodies. The images are single confocal sections representative of three to four separate experiments, with four to six roots imaged per treatment for each experiment. Arrowheads illustrate polarity. For PIN2, the left-most column shows glancing views of the epidermis, and the central column shows roughly median planes, of which the right-most column shows magnified views. For PIN1, the left column represents the median views, and the right column shows magnified views of the left column. e, epidermis; c, cortex. Bars = 20 μm for columns 1, 2, and 4 and 10 μm for columns 3 and 5.

Figure 3.

Brefeldin Alters Auxin Responsiveness. (A) Five-day-old IAA2-GUS transgenic seedlings were treated for 48 h, stained in a buffer containing 1 mM X-gluc for 1 h at 37°C, and cleared for photography. For IAA treatment, seedlings were transferred to plates containing IAA and incubated for 4 h prior to staining. Images are representative of 20 to 30 seedlings, stained in two to three separate experiments. Bar = 100 μm. (B) Five-day-old DR5-mGFP transgenic seedlings were treated for 18 h in the vertical position and then rotated by 90° for 3 h prior to imaging. Two representative images are shown out of 20 seedlings imaged in two separate experiments. Arrowheads indicate DR5-GFP expression in the peripheral layers, which was generally asymmetric in controls, weak or absent on 3 μM, and symmetric on 10 μM brefeldin. Bar = 100 μm.

Figure 4.

Brefeldin Enhances Shootward Auxin Transport in the Root. Five-day-old seedlings were treated for 48 h, and transport of tritiated IAA over 1 h was measured as described in Methods. Bars plot mean ± se of three replicate experiments, each done in triplicate. Asterisks represent the statistical significance between control and brefeldin-treated means, as judged by Student's t test: * P < 0.02, ** P < 0.01, and *** P < 0.001. [See online article for color version of this figure.]

Figure 5.

Roots of pp2aa1 Respond to Brefeldin More Strongly Than Those of the Wild Type. Five-day-old seedlings were transferred to treatment plates and either incubated for 18 h and then rotated by 90° ([A] and [B]) or incubated for 48 h (C). Bars plot mean ± se from three to five independent experiments, with 10 to 12 seedlings per experiment. Asterisks represent the statistical significance between the means for each genotype, as judged by Student's t test: * P < 0.02, ** P < 0.01, and *** P < 0.001. (A) Root tip orientation after 6 h of stimulation. (B) Root elongation of the same plants shown in (A). (C) Root elongation of vertical plants.

Figure 6.

Brefeldin Alters the Morphology of pp2aa1 Roots. Five-day-old seedlings were transferred to 10 μM brefeldin plates for 48 h and then stained in 2 μM FM 4-64 dye for 15 min. Left columns show the whole root, center columns the meristem, and right columns the transition zone. Images represent single confocal sections of surface views, obtained from three separate experiments, with five roots imaged per treatment for each experiment. Bars = 100 μm for left columns and 50 μm for middle and right columns. [See online article for color version of this figure.]

Figure 7.

Cortical PIN2 in pp2aa1 Is Hypersensitive to Brefeldin-Induced Relocation. Five-day-old seedlings were treated for 48 h before being fixed and processed for immunostaining with an anti-PIN2 antibody. Images represent single confocal sections of median views, obtained from two separate experiments, with 8 to 10 roots imaged per treatment for each experiment. Arrowheads illustrate PIN2 polarity. c, cortex; e, epidermis. Bar = 10 μm.

Figure 8.

PIN1 in pp2aa1 Is Not Translocated on Low Concentrations of Brefeldin. Five-day-old seedlings treated for 48 h before being fixed and processed for immunostaining with an anti-PIN1 antibody. Images are single confocal sections, representative of two separate experiments, with eight to ten roots imaged per treatment in each experiment. Arrowheads illustrate PIN1 polarity. Bar = 10 μm.

Figure 9.

Brefeldin Alters the Localization of PP2AA1-GFP. Five-day-old PP2AA1-GFP transgenic seedlings were treated for 48 h and imaged with confocal fluorescence microscopy. Images were captured using the same confocal settings and are representative of 40 roots obtained from at least five independent experiments. Arrowheads indicate nuclei. Top panel shows the transition zone; bottom panel shows the elongation zone. Bars = 50 μm. [See online article for color version of this figure.]

Figure 10.

Brefeldin Immobilizes a Fraction of PP2AA1-GFP. Five-day-old PP2AA1-GFP transgenic seedlings were treated for 48 h before being imaged with confocal fluorescence microscopy and subjected to photobleaching. Symbols plot mean ± se of three separate experiments, with three or four roots imaged per treatment for each experiment. In each root, three to five cells were photobleached. In (A) and (B), white brackets show the approximate region used for bleaching and for measurement of fluorescence intensity. (A) Control. (B) Brefeldin (10 μM). (C) Quantification of recovery. [See online article for color version of this figure.]

Figure 11.

Brefeldin Increases the Abundance of PINOID Kinase. Five-day-old PID-YFP transgenic seedlings were treated for 48 h and imaged with confocal fluorescence microscopy. Images are single confocal sections of surface views, representative of 30 seedlings obtained from four independent experiments, and were captured using the same confocal settings. For immunoblotting, 5-d-old seedlings were treated for 4 h with 50 μM brefeldin. Total proteins were extracted, resolved by SDS-PAGE, and probed with a monoclonal antibody against YFP. The blot is representative of three independent experiments. Bar = 50 μm. (A) PID-YFP localization in the meristem. (B) Immunoblot. Lanes 1 and 3 are control; lanes 2 and 4 are brefeldin treated. Lanes 1 and 2 were loaded with 10 μg total protein, and lanes 3 and 4 were loaded with 20 μg. [See online article for color version of this figure.]

Figure 12.

Gravitropism, Root Elongation, and Cortical PIN2 Translocation in pid-9 Show a Wild-Type Sensitivity to Brefeldin. For gravitropism, seedlings were treated vertically for 18 h and then rotated by 90°. Symbols plot mean ± se from three independent experiments with 10 to 12 seedlings per experiment. For root elongation, 5-d-old seedlings were treated vertically for 48 h. For PIN2 localization, 5-d-old seedlings were treated for 48 h before being fixed and processed for immunostaining with an anti-PIN2 antibody. Images represent single confocal sections of median views, obtained from two separate experiments, with 8 to 10 roots imaged per treatment for each experiment. Arrows illustrate PIN2 polarity. In (B), no significant difference between means for the two genotypes was found. c, cortex; e, epidermis. Bar = 20 μm. (A) Gravitropism. (B) Root elongation. (C) PIN2 localization. [See online article for color version of this figure.]

Figure 13.

The Quadruple Mutant (pid pid2 wag1 wag2) Is Resistant to Brefeldin-Induced Inhibition of Root Elongation and Gravitropism. For gravitropism, 5-d-old wild-type and 7-d-old quadruple mutant seedlings were treated vertically for 24 h and then rotated by 90°. The gravity response was measured after 24 h of gravity stimulation. For root elongation, elongation rate of the same seedlings was measured. Symbols plot mean ± sd from two independent experiments with seven seedlings per experiment for pid pid2 wag1wag2 and 10 seedlings for the wild type. Asterisks represent the statistical significance between brefeldin and DMSO-treated seedlings, as judged by Student's t test: ** P < 0.01. For the quadruple mutant, the effect of brefeldin was not significant in either assay. (A) Gravitropism after 24 h of gravity stimulation. (B) Root elongation after 48 h of brefeldin treatment.