PMC

Extent of deviation from vertical growth of control roots, roots with S3 and tip cells ablated, roots with S1 and S2 cells ablated, roots with flank columella cells ablated, and roots with central columella cells ablated. Roots were maintained vertically and the curvature was measured after 5 h. Ablating the inner columella stories (S1 and S2 cells) caused the greatest deviation from vertical growth, as shown by the largest mean and sd.

Time course of curvature of roots with intact caps and roots with cap cells ablated. Downward curvature of the roots was measured for 12 h following a 90° horizontal reorientation. Note that a single central S2 cell shown in F is actually a total of four cells, two flank and two central columella cells. Each data point represents a mean ± se of at least 10 roots.

Digitized bright-field images of S1 and S2 columella stories collected from the vertical stage microscope and used for measurement of amyloplast (arrows) sedimentation velocities in a vertical root (A) and in a root 5 min after a 135° reorientation (B). Although amyloplasts in the columella cells of a vertical root appear clumped, individual amyloplasts could be resolved from actual video sequences of sedimentation. After the root was rotated horizontally, the course of an individual amyloplast was tracked through digitized video images of sedimentation before individual frames were selected to measure displacement. ci, Columella initials. Bar = 10 μm.

A, Schematic diagram of S3 columella cells illustrating the method used for columella cell ablations. After the target cells were ablated, roots were stained with propidium iodide and imaged with a confocal microscope, and optical sections (Z sections) were taken at the plane indicated by the arrows. The first section was taken at the plane of the peripheral cap cells (root surface). Exclusion of propidium iodide from the peripheral cap cells shows that these cells were not damaged, despite being in the path of the laser, as the power density of the laser was only strong enough to ablate cells at its focal plane. The laser was focused sequentially on all four Z axis planes of columella cells and the defined cells at the laser focal plane were ablated. The diagram illustrates two cells ablated in S3 at each focal plane for a total of eight cells (gray boxes). B, Examples of ablation patterns used for gravitropism studies. Bar = 50 μm.

Induction time of control Arabidopsis roots with intact caps and roots with columella cells ablated. After cell ablations, seedlings were kept vertical for 2 h, stimulated at 90° for a single 2- to 30-min period, and then rotated axially on a 1-rpm clinostat. After 3 h on the clinostat, the curvature was measured and plotted against the logarithm of the stimulation time. Presentation times were calculated from the regression equations for y = 0°. A, Inner versus outer story ablations. Presentation time was 1.16 min for control roots, 1.28 min for S3/tip cell ablations, and 7.13 min for S1/S2 cell ablations. B, Individual story ablations. Presentation time was 2.55 min for S1 cell ablations and 3.53 min for S2 cell ablations. C, Individual stories intact. The presentation time was 2.62 min for roots with only S2 cells intact and 4.85 min for roots with S1 cells intact. D, Central columella versus flank columella cell ablations. Presentation time was 4.07 min for roots with the central columella cells ablated and 1.91 min for roots with the flank columella cells ablated. Correlation coefficients for the regression lines are 0.99 (controls, tip and S3 cells, S1 and S2 cells, and S1 cells ablated), 0.98 (central), 0.97 (S2 cells intact), 0.96 (S2 cells ablated), 0.95 (flank), and 0.87 (S1 cells intact). Each data point represents a mean ± se of 15 to 30 roots.

A, Transmission detector image of the root cap of a 3-d-old Arabidopsis seedling. Amyloplasts (arrowheads) are clearly visible in the columella cells. B, Line diagram of the root cap depicted in A. In two dimensions, the columella cells (numbered) are typically organized into three horizontal stories and four vertical files. As a guide to the ablation experiments described in the succeeding figures, horizontal tiers are classified into three stories (S1–S3): S1, cells 9–12; S2, cells 5–8; and S3, cells 1–4. Vertical files are classified as flank columella cells, cells 1, 5, and 9 and 4, 8, and 12; and central columella cells, cells 2, 6, and 10 and 3, 7, and 11. tc, Tip cells; pc, peripheral cells. C, Ablation of S3 columella cells resulted in morphological distortion of the cells (arrows), whereas adjacent cells remained intact. D, Fluorescence image of the same root stained with propidium iodide, which enters the ablated cells (arrows) and is excluded by live cells. E, Rotational sequence of a three-dimensional confocal data set of an Arabidopsis root cap with the S1 columella cells and S3 peripheral cap cells ablated. Ablation was successful, as shown by the entry of propidium iodide (arrows). Note that the S1 ablations did not cause any damage to peripheral cap cells or adjacent stories and that peripheral cap cell ablations did not damage the inner cap cells. Bar = 25 μm.