Auxin activates the plasma membrane H+-ATPase by phosphorylation during hypocotyl elongation in Arabidopsis

Abstract

The phytohormone auxin is a major regulator of diverse aspects of plant growth and development. The ubiquitin-ligase complex SCF(TIR1/AFB) (for Skp1-Cul1-F-box protein), which includes the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) auxin receptor family, has recently been demonstrated to be critical for auxin-mediated transcriptional regulation. Early-phase auxin-induced hypocotyl elongation, on the other hand, has long been explained by the acid-growth theory, for which proton extrusion by the plasma membrane H(+)-ATPase is a functional prerequisite. However, the mechanism by which auxin mediates H(+)-ATPase activation has yet to be elucidated. Here, we present direct evidence for H(+)-ATPase activation in etiolated hypocotyls of Arabidopsis (Arabidopsis thaliana) by auxin through phosphorylation of the penultimate threonine during early-phase hypocotyl elongation. Application of the natural auxin indole-3-acetic acid (IAA) to endogenous auxin-depleted hypocotyl sections induced phosphorylation of the penultimate threonine of the H(+)-ATPase and increased H(+)-ATPase activity without altering the amount of the enzyme. Changes in both the phosphorylation level of H(+)-ATPase and IAA-induced elongation were similarly concentration dependent. Furthermore, IAA-induced H(+)-ATPase phosphorylation occurred in a tir1-1 afb2-3 double mutant, which is severely defective in auxin-mediated transcriptional regulation. In addition, α-(phenylethyl-2-one)-IAA, the auxin antagonist specific for the nuclear auxin receptor TIR1/AFBs, had no effect on IAA-induced H(+)-ATPase phosphorylation. These results suggest that the TIR1/AFB auxin receptor family is not involved in auxin-induced H(+)-ATPase phosphorylation. Our results define the activation mechanism of H(+)-ATPase by auxin during early-phase hypocotyl elongation; this is the long-sought-after mechanism that is central to the acid-growth theory.

Figure 1.

Auxin-induced hypocotyl elongation and activation of the plasma membrane H⁺-ATPase via phosphorylation. A, Effect of auxin on hypocotyl elongation. Hypocotyl sections from 3-d-old etiolated Arabidopsis seedlings were treated with 10 µm IAA (IAA; red circles), 0.01% dimethyl sulfoxide (DMSO) as a vehicle (Mock; blue circles), and 10 µm IAA and 1 mm sodium orthovanadate (IAA+Vana; white circles) after depletion of endogenous auxin. The elongation of hypocotyl sections was measured after these treatments. Values are means ± se; nf 20. Similar results were obtained in two additional independent assessments. B, Effect of auxin on H⁺-ATPase phosphorylation in hypocotyl sections. Endogenous auxin-depleted hypocotyl sections were incubated with 10 µm IAA for the times indicated (min). The amounts of H⁺-ATPase and the phosphorylation status of the penultimate Thr in the C terminus were determined by immunoblot analysis with anti-H⁺-ATPase (H⁺-ATPase) and anti-pThr-947 (pThr₉₄₇) antibodies, respectively. Arrowheads indicate the positions of the H⁺-ATPase. C, Kinetics of IAA-induced elongation and IAA-induced H⁺-ATPase phosphorylation. The plot shows the relative signal intensity of the immunoblot bands cross-reacted to anti-pThr-947 (red circles) and anti-H⁺-ATPase (blue circles) antibodies. Values are means ± sd; n= 3. The elongation rate (green line) was calculated from the IAA-induced hypocotyl elongation depicted in A. The signal intensity is expressed relative to the intensity of the band signal at time zero. D, H⁺-ATPase phosphorylation and binding of the 14-3-3 protein. Binding of the 14-3-3 protein was determined by protein-blot analysis using glutathione S-transferase-14-3-3 protein as a probe (14-3-3 bound). The rest of the procedure was as described for B. E, H⁺-ATPase activity in hypocotyl sections. Vanadate-sensitive ATP hydrolysis was measured by determining the inorganic phosphate released from ATP. Values are means ± sd; n= 3. * P < 0.05, results of paired Student’s t tests. For D and E, endogenous auxin-depleted hypocotyl sections were treated with 10 µm IAA (IAA) or 0.01% DMSO (Mock) for the times indicated.

Figure 2.

Dose responses of H⁺-ATPase phosphorylation and hypocotyl elongation to exogenous auxin application. A, Dose response of H⁺-ATPase phosphorylation to IAA. Endogenous auxin-depleted hypocotyl sections were incubated for 30 min in the presence of IAA at the concentrations indicated. The phosphorylation level of the H⁺-ATPase was quantified as the ratio of the signal intensity from the phosphorylated H⁺-ATPase to that from H⁺-ATPase and is expressed relative to the phosphorylation level of the hypocotyls that were not treated with auxin (graph at bottom). Values are means ± sd; n= 3 independent experiments. The rest of the procedure was as described in the legend to Figure 1B. B, Dose response of hypocotyl elongation to IAA. Hypocotyl elongation for 30-min periods was measured in the presence of IAA at the concentrations indicated. Values are means ± se; n= 20. Similar results were obtained in two additional independent measurements. C, Correlation between the H⁺-ATPase phosphorylation level and IAA-induced hypocotyl elongation using the data in A and B (r= 0.986).

Figure 3.

Effect of IAA on gene expression. qRT-PCR analysis of the H⁺-ATPase genes AHA1 and AHA2 and the known auxin-inducible genes KAT1 and IAA1 is shown. Total RNA was obtained from the hypocotyl sections 20 min after the application of 10 µm IAA (IAA) and 0.01% DMSO (Mock). Values are means ± sd; n= 3. * P < 0.01; ns, not significant at P > 0.05.

Figure 4.

Auxin-induced H⁺-ATPase phosphorylation and hypocotyl elongation in the tir1-1 afb2-3 and axr1-3 mutants. Hypocotyl sections depleted of endogenous auxin were incubated for 30 min in the absence (−) or the presence (+) of 10 µm IAA. A, IAA-induced H⁺-ATPase phosphorylation. H⁺-ATPase phosphorylation (pThr₉₄₇) levels and the amounts of H⁺-ATPase (H⁺-ATPase) were determined by immunoblot analysis using specific antibodies; the bottom plot depicts the phosphorylation level of the H⁺-ATPase. Values are means ± sd; n= 3 independent experiments. The rest of the procedure was performed as described in the legend to Figure 2A. B, Auxin-induced hypocotyl elongation. Hypocotyl elongation during periods of 30 min was measured. Values are means ± se; n= 15. Similar results were obtained in two additional independent measurements. * P < 0.01; ns, not significant at P > 0.05.

Figure 5.

Effect of the auxin antagonist PEO-IAA on auxin responses. Hypocotyl sections depleted of endogenous auxin were treated with 100 µm PEO-IAA (PEO) or 0.1% DMSO (Control) for 60 min and then incubated for 30 min in the absence (Mock) or presence (IAA) of 10 µm IAA. A, Effect of PEO-IAA on H⁺-ATPase phosphorylation. Values are means ± sd; n= 3 independent experiments. Other details are provided in the legend to Figure 4A. B, Effect of PEO-IAA on auxin-induced hypocotyl elongation. Hypocotyl elongation was measured for 30-min periods. Values are means ± se; n= 15. Similar results were obtained in two additional independent measurements. C, Effect of PEO-IAA on the expression of the auxin-inducible genes KAT1 and IAA1. Relative expression levels of the genes were determined by qRT-PCR analysis. Values are means ± sd; n= 3. * P < 0.01; ** P < 0.05; ns, not significant at P > 0.05.

Figure 6.

Effect of the proteasome inhibitor MG132 on auxin responses. Hypocotyl sections depleted of endogenous auxin were treated with 50 µm MG132 (MG132) or 0.1% DMSO (Control) for 60 min and then incubated for 30 min in the absence (Mock) or presence (IAA) of 10 µm IAA. A, Effect of MG132 on H⁺-ATPase phosphorylation. Details are provided in the legend to Figure 4A. Values are means ± sd; n= 3 independent experiments. B, Effect of MG132 on auxin-induced hypocotyl elongation. Hypocotyl elongation in 30-min periods was measured. Values are means ± se; n= 15. Similar results were obtained in two additional independent measurements. C, Effect of MG132 on the expression of the auxin-inducible genes KAT1 and IAA1. Relative expression levels of the genes were determined by qRT-PCR analysis. Values are means ± sd; n= 3. * P < 0.01; ** P < 0.05; ns, not significant at P > 0.05.

Figure 7.

Inhibition of auxin-induced H⁺-ATPase phosphorylation and hypocotyl elongation by protein phosphatase inhibitors. Hypocotyl sections depleted of endogenous auxin were preincubated with 1 µm CA (CA), 1 µm OA (OA), or 1% DMSO (Mock) for 60 min and then incubated for 30 min in the absence (−) or presence (+) of 10 µm IAA. A, Effect of protein phosphatase inhibitors on IAA-induced H⁺-ATPase phosphorylation. Values are means ± sd; n= 3 independent experiments. * P < 0.01. The rest of the procedure was as described in the legend to Figure 4A. B, Effects of protein phosphatase inhibitors on IAA-induced hypocotyl elongation. Hypocotyl elongation was measured during 30-min periods. Values are means ± se; n= 15. Similar results were obtained in two additional independent measurements. * P < 0.01.