Plasticity of Arabidopsis root gravitropism throughout a multidimensional condition space quantified by automated image analysis

Abstract

Plant development is genetically determined but it is also plastic, a fundamental duality that can be investigated provided large number of measurements can be made in various conditions. Plasticity of gravitropism in wild-type Arabidopsis (Arabidopsis thaliana) seedling roots was investigated using automated image acquisition and analysis. A bank of computer-controlled charge-coupled device cameras acquired images with high spatiotemporal resolution. Custom image analysis algorithms extracted time course measurements of tip angle and growth rate. Twenty-two discrete conditions defined by seedling age (2, 3, or 4 d), seed size (extra small, small, medium, or large), and growth medium composition (simple or rich) formed the condition space sampled with 1,216 trials. Computational analyses including dimension reduction by principal components analysis, classification by k-means clustering, and differentiation by wavelet convolution showed distinct response patterns within the condition space, i.e. response plasticity. For example, 2-d-old roots (regardless of seed size) displayed a response time course similar to those of roots from large seeds (regardless of age). Enriching the growth medium with nutrients suppressed response plasticity along the seed size and age axes, possibly by ameliorating a mineral deficiency, although analysis of seeds did not identify any elements with low levels on a per weight basis. Characterizing relationships between growth rate and tip swing rate as a function of condition cast gravitropism in a multidimensional response space that provides new mechanistic insights as well as conceptually setting the stage for mutational analysis of plasticity in general and root gravitropism in particular.

Figure 1.

A schema of the automated image acquisition, analysis, and data-mining workflow. A, Seven CCD cameras acquired electronic images of seedlings growing along the surface of agar-filled petri plates maintained vertically, then rotated 90° to induce gravitropism. B, Images acquired at 2-min intervals streamed from the cameras to storage discs and database. C, Custom image-processing algorithms extracted the root midline. D, Analysis of the midlines returned tip angle and growth rate as a function of time. A relational database stored the extracted results with the metadata. E, Post acquisition, the database was queried with analysis algorithms designed to identify patterns and quantify relationships between key parameters of the gravitropic response. F, The analyses involved standard statistics, classification, and signal processing.

Figure 2.

The three axes of the condition space. A, Seedling age formed one axis of the condition space. The root of a seedling between 2, 3, and 4 d (left) doubled in length (right) on average. B, The size of the seed formed another axis. Mechanical sieving separated seeds (left) into subpopulations with different mean sizes and distributions (right) quantified by image analysis. C, Composition of the agar growth medium (either a simple mix of KCl and CaCl₂ or a rich mixture of all essential elements). D, The complete set of tip angle and growth rate versus time data acquired in 22 different condition combinations.

Figure 3.

Tip angle development throughout the conditional space. A, Average tip angle development for each seed size class. B, Principal components coefficients of the entire root data set colored according to seed size class. C, Average tip angle development across developmental time. D, Principal components coefficients colored according to developmental age. E, Average tip angle development of seedlings grown on either rich or simple medium. F, Principal components coefficients of all individuals colored according to media condition. The contours in each principle components plot outline where 75% of the data for each population should lie.

Figure 4.

k-means clustering of tip angle responses into three distinct classes. A, Average tip angle development of each k-means defined class. B, Average growth rates of the k-means defined tip angle responses. C, Representative roots at 10 h after gravistimulation from each of the classes. In addition to showing differences in final root length and tip angle between the classes, there is an apparent difference in root width. D, Average growth rate plotted against root width at 10 h of gravistimulation. The data are colored according to k-means class.

Figure 5.

Distribution of class A, B, and C roots throughout the condition space. Percentage of roots falling into each class was determined by k-means clustering for plants on simple medium only. Class A roots increased with seed size and decreased with age.

Figure 6.

Relationship between tip angle and average growth rate throughout the condition space. PCA was performed on the entire population of tip angle responses. Plotted is PC1 of the tip angle versus the average growth rate of the same individual for each individual in the data set. Growth rates below 0.10 mm h⁻¹ reasonably predict tip angle development of an individual. Growth rate is not a good predictor of tip angle when the root grows faster than 0.10 mm h⁻¹. The line is a Boltzman equation fit to the data.

Figure 7.

The relationship between tip angle and growth rate varies throughout the conditional space. Shown in gray in each figure are the PC1 coefficients and average growth rate for all 1,216 individuals in the data set, as was shown in Figure 6. In vermillion are the PC1 coefficients and average growth rates for the individuals at each developmental age and seed size on simple media.

Figure 8.

Relationship between swing rate and tip angle. A, The tip angle at the time of maximum swing rate versus the time when swing rate was maximal. The dashed lines show that the average maximum swing rate of the data set occurred when the tip angle was 30° on average, and 2.5 h after gravistimulation, on average. B, The average swing rate (gray) and the average tip angle (black) response of the entire data set.

Figure 9.

Patterns in swing rate and time to reach maximum swing rate throughout the condition space. A, Average swing rates of roots coming from each seed size class. B, Tip angle and time to maximum swing rate by seed size class for each individual root. C, Average swing rates of roots over developmental time. D, Tip angle and time at the maximum swing rate grouped by developmental age. E, Average swing rates of roots grown on simple or rich medium. F, Tip angle and time at the maximum swing rate grouped by growth medium. The enlarged points within these plots indicate the population means for each condition.

Figure 10.

Distribution of curvature along the root axis during gravitropism throughout the condition space. Color-coded curvature (K in units of mm⁻¹) is shown at each point along the midline of the root apex (y axis) beginning 60 μm from the tip (top edge of each section) and extending back 945 μm at each time point (x axis) over the 10-h experiment. The plots, which are the averages of at least 40 roots per condition, show where along the root midline, when, and to what extent curvature developed after reorientation, at each position in the condition space.