Membrane potential reflects the difference between cytoplasmic and apoplastic electrical potentials and is essential for cellular operation. The application of the phytohormone auxin (IAA) causes instantaneous membrane depolarization in various cell types 1 – 6 , making depolarization a hallmark of IAA-induced rapid responses. In root hairs, depolarization requires functional IAA transport and TIR1/AFB signalling 5 but its physiological significance is not understood. Specifically in roots, auxin triggers rapid growth inhibition 7 – 9 (RGI); a process required for gravitropic bending. RGI is initiated by the TIR1/AFB co-receptors, AFB1 paralogue playing a crucial role 10 , 11 . The nature of the underlying rapid signalling is unknown, as well as the molecular machinery executing it. Even though the growth and depolarization responses to auxin show remarkable similarities, the significance of membrane depolarization for root growth inhibition and gravitropism is unclear. Here, by combining the DISBAC 2 (3) voltage sensor with microfluidics and vertical-stage microscopy, we show that rapid auxin-induced membrane depolarization tightly correlates with rapid RGI. Rapid depolarization and RGI require the AFB1 auxin co-receptor. Finally, AFB1 is essential for the rapid formation of membrane depolarization gradient across the gravistimulated root. These results clarify the role of AFB1 as the central receptor for rapid auxin responses.

The existence of an electric gradient across membranes is essential for a cell operation. In plants, application of the growth regulator auxin (IAA) causes almost instantaneous membrane depolarization in various cell types, making membrane depolarization a hallmark of the rapid non-transcriptional responses to IAA. Auxin triggers rapid root growth inhibition; a process that underlies gravitropic bending. The growth and depolarization responses to auxin show remarkable similarities in dynamics, requirement of auxin influx and the involvement of the TIR1/AFB auxin coreceptors, but whether auxin-induced depolarization participates in root growth inhibition remains unanswered. Here, we established a toolbox to dynamically visualize membrane potential in vivo in Arabidopsis thaliana roots by combining the DISBAC2(3) fluorescent probe with microfluidics and vertical stage microscopy. This way we show that auxin-induced membrane depolarization tightly correlates with rapid root growth inhibition and that the cells of the transition zone/early elongation zone are the most responsive to auxin. Further, we demonstrate that auxin cycling in and out of the cells through AUX1 influx and PIN2 efflux is not essential for membrane depolarization and rapid root growth inhibition but acts as a facilitator of these responses. The rapid membrane depolarization by auxin instead strictly depends on the AFB1 auxin receptor, while the other TIR1/AFB paralogues contribute to this response. The lack of membrane depolarization in the afb1 mutant explains the lack of the immediate root growth inhibition. Finally, we show that AFB1 is required for the rapid depolarization and rapid growth inhibition of cells at the lower side of the gravistimulated root. These results are instrumental in understanding the physiological significance of membrane depolarization for the gravitropic response of the root and clarify the role of AFB1 as the receptor central for rapid auxin responses, adding another piece to the puzzle in understanding the biology of the phytohormone auxin.

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