Auxin response in Arabidopsis under cold stress: underlying molecular mechanisms

Abstract

To understand the mechanistic basis of cold temperature stress and the role of the auxin response, we characterized root growth and gravity response of Arabidopsis thaliana after cold stress, finding that 8 to 12 h at 4 degrees C inhibited root growth and gravity response by approximately 50%. The auxin-signaling mutants axr1 and tir1, which show a reduced gravity response, responded to cold treatment like the wild type, suggesting that cold stress affects auxin transport rather than auxin signaling. Consistently, expression analyses of an auxin-responsive marker, IAA2-GUS, and a direct transport assay confirmed that cold inhibits root basipetal (shootward) auxin transport. Microscopy of living cells revealed that trafficking of the auxin efflux carrier PIN2, which acts in basipetal auxin transport, was dramatically reduced by cold. The lateral relocalization of PIN3, which has been suggested to mediate the early phase of root gravity response, was also inhibited by cold stress. Additionally, cold differentially affected various protein trafficking pathways. Furthermore, the inhibition of protein trafficking by cold is independent of cellular actin organization and membrane fluidity. Taken together, these results suggest that the effect of cold stress on auxin is linked to the inhibition of intracellular trafficking of auxin efflux carriers.

Figure 1.

Effect of Cold Stress on Arabidopsis Root Gravity Response and Elongation. Five-day-old seedlings were transferred to new agar plates and subjected to cold treatment (4°C) for various time lengths. After the cold treatment, gravity stimulation was provided to the roots by rotating the plates 90° at 23°C. Data for root tip orientation and elongation were collected for 3, 6, and 9 h. Vertical bars represent mean ± se. Data are from three independent experiments with 10 to 12 seedlings per experiment. (A) Curvature of root tips plotted against time after reorientation. (B) Root elongation.

Figure 2.

Effect of Cold Stress on Auxin Signaling Mutants. Root gravity response and elongation of Columbia-0, axr1-3, and tir1-1 were compared after 12 h of 4°C treatment. Gravity response and root elongation were measured as described in Figure 1. Vertical bars represent mean ± se. Data are from three independent experiments with 10 to 12 seedlings per experiment. (A) Curvature of root tips plotted against time after reorientation. (B) Root elongation.

Figure 3.

Cold Stress Alters the Auxin Response in Arabidopsis Roots. Five-day-old IAA2-GUS seedlings were treated at 4°C for 12 h. Seedlings were stained in a buffer containing 1 mM X-gluc for 1 h at 37°C and cleared for photography. These are representative images of 30 seedlings for (A) and (B) and 25 seedlings for (C) stained in at least three separate experiments. Bars = 50 μm. (A) Control roots (top panel) and cold-treated roots (bottom panel). (B) Seedlings treated with 1 μM IAA for 30 min, control roots (top panel), and cold-treated roots (bottom panel). (C) Monitoring the formation of asymmetric auxin gradient in gravity stimulated roots. Left panels represent control roots, and right panels represent cold stressed roots.

Figure 4.

Effect of Cold Stress on Root Basipetal Auxin Transport. Five-day-old Columbia seedlings were subjected to cold stress at 4°C for 12 h, and root basipetal transport of auxin was measured for 1 h at room temperature immediately after the cold treatment. The same transport assay was performed for the 12 h cold-treated roots followed by incubation at 23°C for 9 h. The experiments were done in triplicate and repeated at least three times (control n = 96; 12 h cold treatment n = 150; 12 h cold treatment followed by incubation at 23°C for 9 h = 50). Vertical bars represent mean ± se.

Figure 5.

Cold Stress Affects the Intracellular Dynamic Cycling of PIN2 but Not Its Polar Localization. Five-day-old PIN2:PIN2–GFP transgenic seedlings were treated at 4°C for 12 h. Seedlings grown at 23°C ([A], [B], [E], and [F]) and cold-treated seedlings ([C], [D], [G], and [H]). Roots treated with 20 μM BFA for 30 min ([E] to [H]). The images were captured using the same confocal setting and are representative of 40 roots obtained from at least five independent experiments. Middle panels ([A'] to [G']) represent zoomed images of the left panels. Bars = 50 μm in (A) to (H) and 10 μm in zoomed images. [See online article for color version of this figure.]

Figure 6.

Cold Stress Inhibits the Lateral Relocalization and Intracellular Cycling of PIN3. Five-day-old PIN3:PIN3–GFP transgenic seedlings were treated at 4°C for 12 h. Seedlings grown at 23°C ([A], [B], and [E]) and cold-treated seedlings ([C], [D], and [F]). After the cold treatment, gravity stimulation was provided to the roots by rotating the plates 90° at 23°C. The large arrows in the middle panel show the direction of gravity. PIN3 relocalization was imaged after 3 h of gravistimulation ([B] and [D]). Arrowheads indicate symmetric ([A], [C], and [D]) and lateral localizations (B) of PIN3. To observe the intracellular cycling of PIN3, control or cold-treated roots were treated with 50 μM BFA for 30 min ([E] and [F]). The images were captured using the same confocal setting and are representative of 20 roots obtained from three independent experiments. Bars = 10 μm. [See online article for color version of this figure.]

Figure 7.

Effect of Cold Stress on Endosomal Movement. Five-day-old GFP-ARA7 transgenic plants were cold stressed at 4°C for 12 h and subjected to real-time imaging. (A) to (C) show the trafficking in control plants at 23°C; (D) to (F) show the trafficking under cold stress. Effect of BFA on endosomal movement ([G] to [I]) grown at 23°C and ([J] to [L]) under cold stress. The roots were treated with 20 μM BFA for 30 min. Movies were captured for 30 s with 3-s time lapse between frames. Merged image is composed by superimposing frame 1 and frame 10. Color codes are green for frame 1 and red for frame 10. Images are representative of 20 roots obtained from three independent experiments. Bar = 10 μm.

Figure 8.

Effect of Cold Stress on the Intracellular Trafficking of PM Protein LTI6b. Five-day-old EGFP-LTI6b seedlings were treated at 4°C for 12 h. (A), (B), (E), and (F) show seedlings grown at 23°C, and (C), (D), (G), and (H) show cold-treated seedlings. (E) to (H) show roots treated with 20 μM BFA for 30 min. Middle panels ([A'], [C'], [E'], and [G']) represent zoomed images of left panels. The images were captured using same confocal setting and are representative of 30 roots obtained from five independent experiments. Bars = 10 μm. [See online article for color version of this figure.]

Figure 9.

Effect of Cold Stress on Actin Cytoskeleton Structure. Five-day-old GFP-ABD2-GFP transgenic seedlings were subjected to cold treatment at 4°C for 12 h. Seedlings grown at 23°C ([A] to [C]), cold-treated seedlings ([D] to [F]), and seedlings treated with 5 μM TIBA for 30 min at 23°C ([G] to [I]). The images were captured using same confocal setting and are representative of 30 roots obtained from at least four independent experiments. Images are projections of 10 to 12 optical sections. Bar = 50 μm. [See online article for color version of this figure.]

Figure 10.

Effect of Cold Stress on Microtubule Cytoskeleton Structure. Five-day-old TUB6-GFP transgenic seedlings were incubated at 23°C or cold stressed at 4°C for 12 h and imaged using the same confocal setting. The images are representative of 30 seedlings obtained from three independent experiments. Seedlings grown at 23°C ([A] and [B]) and cold-treated seedlings ([C] and [D]). Images are projections of 10 to 12 optical sections. Bar = 10 μm. [See online article for color version of this figure.]

Figure 11.

Effect of Membrane Rigidification on Root Gravity Response and PIN2 Cycling. Five-day-old Columbia-0 seedlings were transferred to new agar plates containing 3% DMSO for 12 h at 23°C. After the DMSO treatment, seedlings were transferred to new agar plates without DMSO, and gravity response and root elongation were measured as described in Figure 1 [A] and [B]. Five-day-old PIN2:PIN2-GFP seedlings were treated with or without 3% DMSO for 12 h ([a] and [b]). Control and 12-h 3% DMSO–treated seedlings were treated with 20 μM BFA for 30 min ([c] and [d]). The images were captured using same confocal setting and are representative of 40 roots obtained from at least five independent experiments. Right panel ([a'] to [d']) represents zoomed images of left panel. Bars = 10 μm. (A) Curvature of root tips plotted against time after reorientation. (B) Root elongation. Vertical bars represent mean ± se. Data are from three independent experiments with 20 seedlings per experiment. (C) The intracellular trafficking of PIN2 protein in DMSO treatment was investigated using PIN2:PIN2–GFP transgenic plants. [See online article for color version of this figure.]