Rapid and reversible root growth inhibition by TIR1 auxin signalling

Abstract

The phytohormone auxin is the information carrier in a plethora of developmental and physiological processes in plants¹. It has been firmly established that canonical, nuclear auxin signalling acts through regulation of gene transcription². Here, we combined microfluidics, live imaging, genetic engineering and computational modelling to reanalyse the classical case of root growth inhibition³ 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.

Fig. 1.

Nanomolar concentrations of auxin reversibly inhibit root growth. (A). Schematics of auxin fluxes in the root tip during gravitropism. (B). The vRootChip device with flow channels and pressure valves are colored in green and red, respectively (left). The side view of the device positioned in the vertical microscope (middle) and a root growing in the channel (right). (C). A timelapse of a DII-Venus root treated with 10nM IAA; IAA medium contains a fluorescent tracer shown in magenta. (D). Quantification of root growth rate (GR) during addition of 10nM IAA, shown in magenta. Normalized to GR precedent to IAA addition, mean of 4 roots, +SD. (E). The dependence of the response growth rate on [IAA]_ext. See D for “response GR”. Data points are means of N =3,8,9,3,16,7,16 roots for [IAA]_ext =1,2,5,10,50,100,1000nM ±SD. Best fit (red): IC50=1.44nM, GRmin=0.13 (F). Quantification of a repetitive treatment of roots with 10nM IAA (magenta). Mean of 7 roots +SD.

Fig. 2.

Roots rapidly adapt growth rate to auxin application and removal. (A,B).The reaction of two individual roots to the application (A) and removal (B) of 10nM IAA. The presence of IAA shown as the intensity of the fluorescent tracer (magenta), temporal resolution is 30s. (C,D). Roots rapidly resume the growth rate after 40 minutes (C) and 80 minutes (D) of 10nM IAA treatment. Means of 4 and 6 roots for (C,D), respectively, +SD. (E,F). TIR1-dependent protein synthesis in the root tips was determined by the luminescence intensity of DR5::luciferase. The first detectable increase in the signal is marked by arrowhead (E). At 40 and 80 minutes (dotted lines) the transcriptional response is fully activated (F). 100nM IAA with 1µM NPA was added at timepoint 0. (E) is a zoomed region from (F); means of 5 root tips, ±SD.

Fig.3.

The rapid root growth inhibition depends on auxin levels inside the cell. (A). A schematics of auxin fluxes in a root epidermal cell. (B). Auxin influx carrier AUX1-YFP is expressed in the root epidermal cells. Scale bar = 50µm. (C). The aux1 mutant is resistant to 5nM IAA but reacts to 50nM IAA. Mean normalized values of 3 (5nM), 2 (50nM control) and 5 (50nM aux1) roots, ±SD. (D). The dose response of aux1 is shifted towards higher IAA concentrations by ~10 fold (control is the same as in Fig.1E). Fit: IC₅₀^aux=17.2nM = 11.8*IC₅₀^col . Data points are means of N = 10,4,2,9,7 roots for [IAA]_ext =5,10,20,50,1000nM respectively +-SD; the 1000nM was done on agar plate. (E). The aux1 mutant reacts little to 5nM IAA (magenta) but its reaction to membrane permeable NAA (100nM, green) is comparable to control roots. Mean normalized values of 5 (aux1) and 2 (control) roots, ±SD. (F). The auxin efflux inhibitor NPA (5µM, gray) triggers root growth inhibition. Mean normalized values of 5 roots, +SD. (G). Auxin accumulation (magenta, simulated) is faster than growth inhibition during application of 5nM IAA (gray, mean experimental values of 1-GR). Note time delay between [IAA]_cell = α^col · IC₅₀ and (1 – GR) = 0.5. Accumulation ratio α^col ≅ 30 (Supp.text). Shaded regions correspond to 1 SD (gray, n=6 roots) or variation due to SD of parameter values (magenta, simulation results). (H). The modelled theoretical inhibition for aux1 and control roots.

Fig.4.

The root growth inhibition requires the TIR1/AFB-Aux/IAA auxin co-receptor. (A). tir triple mutant shows a defect in the response to 10nM IAA; compare to Fig.1 D, F. Mean normalized growth rate, 5 and 2 roots for tir triple and control, +SD. (B). The dose response of the tir triple mutant is shifted towards higher IAA concentrations: IC₅₀=17.8nM (control is the same as in Fig.1E). Data points are means of N= 4,5,4,8,9 roots for [IAA]_ext =2,5,10,20,50nM respectively ±SD. (C). Addition of PEO-IAA results in a rapid reversion of the growth inhibition and an increase in the DII-Venus marker intensity. IAA 1nM is shaded in magenta, addition of PEO-IAA (10µM) is marked by gray stripes. DII-Venus intensity (green) was measured in the lateral root cap cells. Growth rates and DII-Venus intensities of two independent roots are shown. (D). cvxIAA causes root growth inhibition only in the synthetic ccvTIR1 line. Addition of cvxIAA (50nM) is marked by the tracer fluorescence in blue. (E). Quantification of the ccvTIR1 (red) and controlTIR1 (black) roots to 50nM cvxIAA (blue). Means of 5 roots for each genotype, ±SD. (F). The formation of the TIR1/AFB-auxin-Aux/IAA complex negatively regulates root growth, likely by a signaling branch that is distinct form the transcriptional regulation function. The synthetic ccvTIR1 receptor and the cvxIAA ligand are shown in blue.