AUX1-mediated root hair auxin influx governs SCFTIR1/AFB-type Ca2+ signaling

Abstract

Auxin is a key regulator of plant growth and development, but the causal relationship between hormone transport and root responses remains unresolved. Here we describe auxin uptake, together with early steps in signaling, in Arabidopsis root hairs. Using intracellular microelectrodes we show membrane depolarization, in response to IAA in a concentration- and pH-dependent manner. This depolarization is strongly impaired in aux1 mutants, indicating that AUX1 is the major transporter for auxin uptake in root hairs. Local intracellular auxin application triggers Ca²⁺ signals that propagate as long-distance waves between root cells and modulate their auxin responses. AUX1-mediated IAA transport, as well as IAA^- triggered calcium signals, are blocked by treatment with the SCF^TIR1/AFB - inhibitor auxinole. Further, they are strongly reduced in the tir1afb2afb3 and the cngc14 mutant. Our study reveals that the AUX1 transporter, the SCF^TIR1/AFB receptor and the CNGC14 Ca²⁺ channel, mediate fast auxin signaling in roots.

Fig. 1

Auxin-induced plasma membrane depolarization and associated H⁺-fluxes in root-hair cells. a Experimental set-up: Intracellular microelectrodes measured the membrane potential of bulging root-hair cells and auxin was applied with an application pipette by back pressure. b Representative changes in membrane potential evoked by 1 s IAA pulses (10⁻⁸ to 10^–5 M, arrow indicates time point of stimulation). c Dose–response curves of plasma membrane depolarization at a range of IAA concentrations, given as the percentage of the maximal depolarization (red symbols) and maximum rate of voltage change (black symbols). Apparent K_m values according to Michaelis–Menten fitting were 53 ± 6 nM (max depol) and 300 ± 133 nM (max velocity) IAA (n = 6 ± s.e.m.). Note that K_m values were calculated from undiluted auxin concentrations inside application pipettes. d H⁺-flux kinetics determined with ion-selective electrodes, scanning in close proximity of root-hair cells. After 3 min, IAA was applied to a final concentration of 10 µM (black bar, curves are interrupted at time point of IAA stimulation), n = 11 ± s.e.m. e Impact of extracellular pH on the IAA-induced depolarization (arrow, 1 s stimulation with 10 µM IAA). f IAA-induced depolarization as a function of extracellular H⁺-concentration, given as percentage of the depolarization rate at pH 4.5. Similar values were obtained for an IAA conc. of 10 µM (closed symbols) and 0.3 µM (open symbols). Apparent K_m value according to Michaelis–Menten fitting was 910 ± 500 nM (i.e., pH 6.0; n = 6 for 10 µM IAA and n = 10 for 0.3 µM IAA). Error bars represent s.e.m.

Fig. 2

IAA-induced membrane depolarization and H⁺-influx are AUX1-dependent. a Maximal depolarization rate and initial change of H⁺-flux, evoked by 10 µM IAA in wild-type and aux1-2 (Ler), aux1-7 (Col-0), aux1-22 (Col-0), aux1-T (Ws), and wav5-33 (Ler). Values for mutants are given as the percentage of the response of the respective accessions (for mutants n = 10 ± SE for depolarization and n = 12 ± s.e.m. for H⁺-flux, wild-type responses were measured with n = 11 to 13 ± s.e.m.). Asterisks indicate significant reductions (Students t-test, p < 0.05). b IAA-induced max. depolarization rates, for Ler (closed symbols) and wav5-33 (open symbols). Michaelis–Menten fits revealed half-maximal depolarization rates at 67 ± 54 nM for Ler and 1.7 ± 1.6 µM for wav5-33. Note that K_m values were calculated from undiluted auxin concentrations inside application pipettes. Differential values of both black curves (red curve) point to a dominant AUX1-dependent uptake at [IAA] below 10⁻⁶ M, whereas unspecific transporters contribute at higher IAA-levels (Ler n = 6 to 8 ± s.e.m.; wav5-33 n = 5 ± s.e.m.). c IAA-dependent changes in the membrane potential of wild-type and AUX1-mutant, grown at standard and phosphate starved conditions. Representative traces of A. thaliana Ler and the wav5-33 mutant following stimulation with 0.3 µM IAA during a period of 1 s (as indicated by arrow). d Dose–response curve of the phosphate concentration in the growth medium, plotted against the maximum rate of depolarization. Error bars show s.e.m. (n = 14 (Ler) and 9 (wav5-33)). Asterisks mark significant differences (Students t-test, p < 0.05)

Fig. 3

Auxin induces Ca²⁺-influx and increase of the cytosolic-free Ca²⁺-concentration of root-hair cells. a Average ion flux kinetics determined with ion-selective electrodes (H⁺ black symbols, left axis; Ca²⁺ red symbols, right axis, n = 12 ± s.e.m.). After 3 min, IAA was applied to a final concentration of 10 µM (black bar, curves are interrupted at time point of application). b IAA-evoked changes of the cytosolic-free Ca²⁺-concentration in root-hair cells, determined with R-GECO1. Images of a single root-hair cell, before, during, and after the application of 10 µM IAA. IAA was applied at t = 0.5 min. False colored images indicate R-GECO1 fluorescence intensity, relative to the average value in ROI (red line) just before IAA application (color code above the panels). The scale bar represents 20 µm. c Simultaneous measurement of IAA-induced depolarization (black trace, left axes) and change in R-GECO1 fluorescence intensity (red trace, right axes) of a ROI that includes the nucleus, in a single root-hair cell (arrow: time point of 1 s application of 10 µM IAA). Representative measurement from 26 experiments. d IAA-dependent changes in R-GECO1 fluorescence intensity determined across the root and representative of 7 experiments for each IAA concentration (indicated by colored lines). e Max. depolarization rate (closed bars) and slope of R-GECO1 intensity change (open bars) of single root-hair cells to 1 s pulses of 10 µM IAA and auxin analogs, as indicated. Data are given as percentage of the response to 3-IAA (n = 8 ± s.e.m. for BA and n = 16 ± s.e.m. for auxins), asterisks indicate significant reductions (Students t-test, p < 0.05). f Max. depolarization rate (closed bars) and slope of R-GECO1 intensity change (open bars) of single root-hair cells to a 1 s pulses of 10 µM IAA. Root-hair cells were measured under control conditions, or after 20 min pretreatment with 20 µM auxinole. Bars represent percentage of control (n = 10 ± s.e.m. for control and 11 ± s.e.m. for auxinole), asterisks indicate significant reductions (Students t-test, p < 0.05)

Fig. 4

The auxin-induced plasma membrane depolarization as well as H⁺- and Ca²⁺-influx are dependent on intracellular auxin perception. a IAA-dependent changes in the membrane potential of wild-type and receptor mutant root hairs. Representative traces of A. thaliana Col-0 in the presence (green) and absence (black) of 20 µM auxinole as well as of the tir1-1 (blue) and tir1-1afb2-3afb3-4 (red) mutants following a 1 s stimulation with 10 µM IAA (arrow). b Maximal depolarization rate (black bars) and initial change of H⁺- (gray bars) and Ca²⁺-flux (open bars), evoked by 10 µM IAA in roots of Col-0 (n = 14 ± s.e.m. for depolarization; n = 10 ± s.e.m. for ion fluxes), Col-0 pretreated with 20 µM auxinole (n = 6 ± s.e.m. for depolarization; n = 4 ± s.e.m. for ion fluxes), as well as in tir1-1 (n = 8 ± s.e.m. for depolarization; n = 16 ± s.e.m. for ion fluxes) and in tir1-1afb2-3afb3-4 (n = 6 ± s.e.m. for depolarization; n = 9 ± s.e.m. for ion fluxes). Values are given as the percentage of the wild-type response. Asterisks indicate significant reductions (Students t-test, p < 0.05)

Fig. 5

Cytosolic application of IAA triggers a lateral Ca²⁺ wave in roots. a IAA was applied iontophoretically together with Lucifer yellow (LY) via an intracellular double-barreled microelectrode. Panel upper left, transmitted light signal, note microelectrode on right. Panel lower left, LY fluorescence signal at indicated time value. Panels middle and right, false color images of R-GECO1 intensity, normalized to the average value of ROI1 just before stimulation with IAA, as indicated by the scale on left. Time after experiment onset is given. IAA was injected with a current of -1 nA to the cell in ROI1, from t = 0.5 to 1.5 min. Representative measurement from 20 experiments. The scale bar represents 20 µm. b Representative time-dependent changes in the fluorescence signal of LY (green line, right axis) and the R-GECO1 signals of ROI1 (black line, left axis) and ROI2 (red line, left axis), relative to their average fluorescence intensity at the start of injection. The light gray bar indicates the period of iontophoretic injection of IAA. Representative measurement from 20 experiments. c, d Responses of A. thaliana Col-0 root-hair cells to iontophoretical intracellular injection of IAA, 2-NAA, and IAA in roots pretreated with 20 µM auxinole. c Average value of max. depolarization. d Average changes in R-GECO1 signals of ROI1 (closed bars) and ROI2 (open bars, n = 20 ± s.e.m. for IAA; n = 11 ± s.e.m. for 2-NAA and n = 6 ± s.e.m. for IAA w/auxinole), asterisks indicate significant differences (Students t-test, p < 0.05). e Average net Ca²⁺- (red) and H⁺- (black) flux kinetics of wild-type (closed symbols) and cngc14-2 (open symbols) roots. After 3 min, IAA was applied to a final concentration of 10 µM (black bar), curves are interrupted at time point of IAA stimulation, n = 17 ± s.e.m. for Col-0 Ca²⁺-fluxes, n = 18 ± s.e.m. for cngc14-2 Ca²⁺-fluxes, n = 7 ± s.e.m. for H⁺-fluxes. f IAA-dependent changes in the membrane potential of wild-type and cngc14-2 mutant root hairs. Average traces of A. thaliana Col-0 (black) and of the cngc14-2 (red) mutant following a 1 s stimulation with 10 µM IAA (arrow) (n = 6 ± s.e.m. for Col-0 and n = 7 ± s.e.m. for cngc14-2)

Fig. 6

Long-distance Ca²⁺- and IAA-signaling. a Fluorescence image of a single root-hair cell stimulated by intracellular application of IAA and LY, over a period of 5 min. All other panels, false color images indicating changes in R-GECO1 intensity, normalized to the average value at ROI2 at the start of IAA-injection (indicated by the color bar on the left). The time after the start of the experiment is given. Representative for 8 measurements. The scale bar represents 100 µm. b Changes in R-GECO1 signal intensity, plotted against time, in ROI1 (green line) and ROI2 (red line) of root stimulated with 3-IAA, or in ROI1 of root stimulated with 2-NAA (black line). Data are presented relative to the average fluorescence intensity in ROI of interest, at the start of injection. Black bar indicates the period of IAA injection into a root epidermal cell in close proximity to ROI1. Average values are shown (n = 8 ± s.e.m. for IAA and n = 8 ± s.e.m. for 2-NAA). c Fluorescence image of a single root-hair cell stimulated by 5 min intracellular application of IAA and LY. All other panels, changes in fluorescence intensity of the auxin-signaling sensor DII-Venus triggered by cytosolic application of IAA, bar on the right indicates fluorescence intensities relative to the average intensity of the ROI at the start of the experiment. Representative for 14 experiments. The scale bar represents 100 µm. d Changes in DII-Venus signal intensity, relative to the start of the experiment. Single root epidermal cells were stimulated with 2-NAA (black curves), or 3-IAA (red curves), in the presence (open circles) or absence (closed circles) of 128 µM La³⁺ in the bath. Data were obtained from ROI at ~400 µm distance, towards the root tip, from the stimulated root epidermal cell (red circles in a and c). Average values are shown (n = 14 ± s.e.m. w/o La³⁺ and n = 6 ± s.e.m. w/La³⁺)

Fig. 7

Model for fast auxin signaling in root cells. Following its uptake via the high-affinity IAA/H⁺-symporter AUX1 (a) auxin is perceived by a nuclear (b) and a cytosolic fraction of the SCF^TIR1/AFB receptor complex (c). Although nuclear perception results in changes at the transcriptional level via proteasomal degradation of Aux/IAA-class transcriptional repressors (d), a functional complex between the auxinole-sensitive cytosolic SCF^TIR1/AFB receptor and CNGC14 activates La³⁺-sensitive Ca²⁺-influx upon auxin perception (e) resulting in elevated cytosolic Ca²⁺ levels (f). Increased [Ca²⁺]_cyt feeds back on AUX1-mediated auxin transport, possibly through phosphorylation of AUX1 via Ca²⁺-dependent kinases (g) and on CNGC14 activity via the Ca²⁺-dependent interaction of CNGC14 with calmodulin (h)