Root gravitropism is regulated by a transient lateral auxin gradient controlled by a tipping-point mechanism

Abstract

Gravity profoundly influences plant growth and development. Plants respond to changes in orientation by using gravitropic responses to modify their growth. Cholodny and Went hypothesized over 80 years ago that plants bend in response to a gravity stimulus by generating a lateral gradient of a growth regulator at an organ's apex, later found to be auxin. Auxin regulates root growth by targeting Aux/IAA repressor proteins for degradation. We used an Aux/IAA-based reporter, domain II (DII)-VENUS, in conjunction with a mathematical model to quantify auxin redistribution following a gravity stimulus. Our multidisciplinary approach revealed that auxin is rapidly redistributed to the lower side of the root within minutes of a 90° gravity stimulus. Unexpectedly, auxin asymmetry was rapidly lost as bending root tips reached an angle of 40° to the horizontal. We hypothesize roots use a "tipping point" mechanism that operates to reverse the asymmetric auxin flow at the midpoint of root bending. These mechanistic insights illustrate the scientific value of developing quantitative reporters such as DII-VENUS in conjunction with parameterized mathematical models to provide high-resolution kinetics of hormone redistribution.

Fig. 1.

(A) Schematic diagram of auxin redistribution predicted by the Cholodny and Went hypothesis following a gravity stimulus (19, 20). (B) Schematic representation of signaling parameters monitored by DII-VENUS compared with DR5::VENUS. (C) Confocal images of DII-VENUS and mDII-VENUS root tips at denoted times (in minutes) following a 90° gravity stimulus. Images were obtained by confocal microscopy and representative of 20 samples. Yellow channel: VENUS. Red channel: cell walls stained with propidium iodide to reveal their cellular organization. (Scale bar: 50 μm.)

Fig. 2.

(A) Quantification of the dose- and time-dependent degradation of the DII-VENUS signal by exogenous auxin concentrations (IAA, 1–1,000 nM); points are means ± 1 SE of at least eight independent replicates (∼100 nuclei quantified in each replicate); solid lines show the fitted model results. (B) Schematic model of the network of interactions that relate the DII-VENUS reporter to auxin. (C) The equations that describe the model dynamics: a system of four ODEs coupled to an equation representing the conservation of TIR1/AFB receptors. The total concentration of TIR1/AFB receptors is denoted by [TIR1]_T and the remaining parameters are labeled in the schematic in B. (D) The reduced model equations: a single ODE that depends on four groupings of the model parameters and the auxin influx rate. Parameter grouping represents the ratio between the auxin influx during auxin treatment and the basal (steady-state) auxin influx, α₀.

Fig. 3.

(A) DII-VENUS reporter roots show an asymmetric distribution following a gravity stimulus, peaking in the middle of the response. The angle of the root tip is shown on each image following a 90° reorientation. The ratios of fluorescence intensity between the upper and lower sides of the roots are also shown. (Scale bar: 50 μm.) (B) The fold change in DII-VENUS ratio (upper/lower tissue signals) plotted against time following a 90° gravitropic stimulus. Black crosses show experimental data points (n = 96), and lines show the fitted simulation results: Red, blue, and green lines are, respectively, the signal from the lower side, the signal from the upper side, and the ratio. (C) Dynamic changes in auxin distribution between upper and lower root tissues following a gravity stimulus (t = 0). (D) Transcript profiling of several auxin-inducible genes in root apical tissues minutes after a 90° gravitropic stimulus. Data are mean ± 1 SE of four technical and four biological replicates, representing ∼180 roots. (E) The auxin redistribution in terms of the angle of reorientation following a gravity stimulus.

Fig. 4.

DII-VENUS signal 2 h after a gravity stimulus in root tissues of (A) wild type and (B) starchless mutant pgm-1. (Scale bar: 50 μm.) (C–E) Statoliths visualized within columella cells of root tips at angles of (C) 0°, (D) 40°, and (E) 90° to the horizontal, with schematics of “tipping point” model where statoliths within columella cells redirect auxin distribution at the root tip (denoted by arrows).