Role of auxin-induced reactive oxygen species in root gravitropism

Abstract

We report our studies on root gravitropism indicating that reactive oxygen species (ROS) may function as a downstream component in auxin-mediated signal transduction. A transient increase in the intracellular concentration of ROS in the convex endodermis resulted from either gravistimulation or unilateral application of auxin to vertical roots. Root bending was also brought about by unilateral application of ROS to vertical roots pretreated with the auxin transport inhibitor N-1-naphthylphthalamic acid. Furthermore, the scavenging of ROS by antioxidants (N-acetylcysteine, ascorbic acid, and Trolox) inhibited root gravitropism. These results indicate that the generation of ROS plays a role in root gravitropism.

Figure 1

Generation of ROS during gravitropism in maize roots. A, Time course of gravistimulation-induced ROS generation. The roots were oriented horizontally for the indicated time and then cut into two parts. Zone 1 (black bars) contains the apical end of root to 0.4 cm, and zone 2 (hatched bars) contains 0.4 to 0.8 cm from root tip. SA, Salicylic acid-treated maize roots. The segments were subjected to ROS measurement assay (OXIS, Portland, OR). Values are the means ± se for five independent experiments. B, Asymmetric generation of ROS by gravistimulation. Gravistimulated or control roots were dissected and stained with a 0.003% (w/v) dihydrorhodamine-123 solution for 10 min. Fluorescence intensity of oxidized rhodamine was observed with a fluorescence microscope (Zeiss, Jena, Germany; excitation = 485 nm, emission = 535 nm). Experiments were repeated at least five times with similar results.

Figure 2

Effect of asymmetric application of H₂O₂ on root curvature. A, Induction of curvature in horizontal roots by asymmetric application of H₂O₂. Agar (1.5%, w/v) blocks were immersed into 1 mm H₂O₂ in 5 mm MES buffer [2-(N-morpholino)ethanesulfonic acid, pH 6.8] and then put on the lower side (●) or the upper side (▪) of roots held horizontally. ▵, Control root showing normal gravitropism. Values are the means ± se for five independent experiments. B, Induction of curvature in vertical roots by asymmetric application of H₂O₂.

Figure 3

Effect of NAC on gravitropic root curvature. A and B, Suppression of gravitropic curvature by pretreatment with NAC. Roots were immersed in 1 mm NAC for 2 h and then oriented horizontally to induce gravitropism. ○, Control maize roots; ●, NAC-treated roots. C, Induction of curvature by asymmetric application of NAC. Agar (1.5%, w/v) block was soaking into 10 mm NAC solution in 5 mm MES buffer (pH 6.8) and then put on the lower side (▪) or the upper side (●) of roots held horizontally. ▵, Control roots, which were subjected to gravistimulation. Values are the means ± se for five independent experiments.

Figure 4

Auxin-induced ROS generation. A, Agar blocks (1.5%, w/v) were incubated in 5 μm IAA in 5 mm MES (pH 6.8) and then placed on the indicated region. ROS generation was detected by fluorescence microscopy. B, Transient generation of ROS by auxin. Intracellular ROS generation in protoplast was measured by 5 μm 2′,7′-dichlorofluorescein diacetate (DCF-DA; Molecular Probes, Eugene, OR) and flow cytometry (FACScan, Becton-Dickinson, Bedford, NJ). Shaded area means control fluorescence intensity. Protoplasts were incubated with 5 μm IAA for 5 min (thin line), 10 min (thick line), 20 min (dashed line), or 30 min (dot line). C, Effect of N-(1-naphthyl)phthalamic acid (NPA) on ROS-induced gravitropism. Roots were immersed in 5 μm NPA in 5 mm MES buffer (pH 6.8) and then gravistimulated. H₂O₂-containing (●) or control agar (○) was then placed on the lower side of roots held horizontally. Values are the means ± se for five independent experiments.