Auxin Activates the Plasma Membrane H⁺-ATPase by Phosphorylation during Hypocotyl Elongation in Arabidopsis

The phytohormone auxin is a major regulator of diverse aspects of plant growth and development. The ubiquitin-ligase complex SCF TIR1/AFB (for Skp1-Cull-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.