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, a-(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.

The phytohormone auxin is the information carrier in a plethora of developmental and physiological processes in plants. It has been firmly established that canonical, nuclear auxin signalling acts through regulation of gene transcription. Here, we combined microfluidics, live imaging, genetic engineering and computational modelling to reanalyse the classical case of root growth inhibition by auxin. We show that Arabidopsis roots react to addition and removal of auxin by extremely rapid adaptation of growth rate. This process requires intracellular auxin perception but not transcriptional reprogramming. The formation of the canonical TIR1/AFB-Aux/IAA co-receptor complex is required for the growth regulation, hinting to a novel, non-transcriptional branch of this signalling pathway. Our results challenge the current understanding of root growth regulation by auxin and suggest another, presumably non-transcriptional, signalling output of the canonical auxin pathway.

Growth regulation tailors plant development to its environment. A showcase is response to gravity, where shoots bend up and roots down 1 . This paradox is based on opposite effects of the phytohormone auxin, which promotes cell expansion in shoots, while inhibiting it in roots via a yet unknown cellular mechanism 2 . Here, by combining micro uidics, live imaging, genetic engineering and phosphoproteomics in Arabidopsis thaliana, we advance our understanding how auxin inhibits root growth. We show that auxin activates two distinct, antagonistically acting signalling pathways that converge on the rapid regulation of the apoplastic pH, a causative growth determinant. Cell surface-based TRANSMEMBRANE KINASE1 (TMK1) interacts with and mediates phosphorylation and activation of plasma membrane H + -ATPases for apoplast acidi cation, while intracellular canonical auxin signalling promotes net cellular H + -in ux, causing apoplast alkalinisation. The simultaneous activation of these two counteracting mechanisms poises the root for a rapid, ne-tuned growth modulation while navigating complex soil environment. MainAuxin, a major growth regulator in plants, acts oppositely in shoots and roots. In shoots, canonical/intracellular auxin TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) receptors by downstream transcriptional regulation activate H + -pumps to acidify the apoplast a promote cell elongation 3,4 , in accordance with the Acid Growth Theory, which postulates that low apoplastic pH promotes growth 5 . In roots of many species including Arabidopsis, auxin inhibits growth. These contrasting responses are the basis for positive versus negative bending of roots and shoots in response to gravity and light 1 . The inhibitory auxin effect in roots also involves TIR1/AFB receptors but its rapid timing points towards an unknown non-transcriptional signalling branch 6 . Besides, a cell surface-based pathway involving TMK1 regulates development 7 , including differential growth in the apical hook 8 , while its role in auxin-regulated root growth remains unclear. Hence, the auxin signalling mechanism and the downstream processes for regulating root growth remain elusive.In this study, we revealed antagonistic action of intracellular TIR1/AFB and cell surface TMK1 auxin signalling converging on regulation of apoplastic pH, which we con rm as the key cellular mechanism allowing immediate and sensitive root growth regulation. Growth inhibition correlates with H + -in uxAuxin rapidly inhibits root growth through a non-transcriptional branch of TIR1/AFB signalling 6 . Although several cellular processes, including cortical microtubule (CMT) reorientation 9,10 , vacuolar fragmentation 11 and apoplastic pH changes [12][13][14] have been implicated, the causal mechanism remains unidenti ed.

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Blue light (BL) receptor phototropins activate the plasma membrane H(+)-ATPase in guard cells through phosphorylation of a penultimate threonine and subsequent binding of the 14-3-3 protein to the phosphorylated C-terminus of H⁺-ATPase, mediating stomatal opening. To date, detection of the phosphorylation level of the guard cell H⁺-ATPase has been performed biochemically using guard cell protoplasts (GCPs). However, preparation of GCPs from Arabidopsis for this purpose requires >5,000 rosette leaves and takes >8 h. Here, we show that BL-induced phosphorylation of guard cell H⁺-ATPase is detected in the epidermis from a single Arabidopsis rosette leaf via an immunohistochemical method using a specific antibody against the phosphorylated penultimate threonine of H⁺-ATPase. BL-induced phosphorylation of the H⁺-ATPase was detected immunohistochemically in the wild type, but not in a phot1-5 phot2-1 double mutant. Moreover, we found that physiological concentrations of the phytohormone ABA completely inhibited BL-induced phosphorylation of guard cell H⁺-ATPase in the epidermis, and that inhibition by ABA in the epidermis is more sensitive than in GCPs. These results indicate that this immunohistochemical method is very useful for detecting the phosphorylation status of guard cell H⁺-ATPase. Thus, we applied this technique to ABA-insensitive mutants (abi1-1, abi2-1 and ost1-2) and found that ABA had no effect on BL-induced phosphorylation in these mutants. These results indicate that inhibition of BL-induced phosphorylation of guard cell H⁺-ATPase by ABA is regulated by ABI1, ABI2 and OST1, which are known to be early ABA signaling components for a wide range of ABA responses in plants.