(A) Immunolocalization of PIN1 (red) and α-tubulin (green) in a thick section through the surface of the meristem. Scale bar: 10 µm. (B) Close-up of the double PIN1–microtubule immunosignal in the boundary domain: PIN1 (red) and microtubule (green) patterns are correlated. Scale bar: 5 µm. (C) Examples of different degrees of correlation between microtubule bundle orientation and PIN1 localization, as quantified in (D). (D) Quantifications of the different classes of behavior in the center zone (CZ) and peripheral zone (PZ) of the meristem (n = 614 cells). (E–J) Correlations between PIN1 polarities and microtubule orientations similar to those seen in (B) are observed in living plants expressing PIN1-GFP (red) and TagRFP-MAP4 (green) in the boundary domain (E–H) and in incipient primordia (asterisk) (I and J). Scale bars for (E), (G), and (I): 5 µm.

(A) PIN1 behavior at the meristem surface after oryzalin treatment. The absence of cell division and the enlargement of the cells are consistent with the impact of microtubule depolymerization on growth. Nevertheless, differential expression of PIN1 is maintained, and new peaks of PIN1 expression arise 20 h (middle panel) and 47 h (right panel) after microtubule depolymerization. The red dot marks the same cell at the three time points. Scale bar: 50 µm. (B) Close-up of the surface of a meristem expressing PIN1-GFP 67 h after microtubule depolymerization. The PIN1 signal is weaker in the boundary and is polarized in a divergent pattern, as observed in untreated meristems. Scale bar: 20 µm. (C) Transverse sections through a PIN1-GFP meristem treated with oryzalin. As primordia arise, the GFP signal is also detected at sites where the provasculature is initiated (arrowhead), as observed in unteated meristems. Scale bar: 50 µm. (D) Kinetics of PIN1-GFP reorientation at the surface of an oryzalin-treated meristem. The GFP signal (grayscale) is color coded (upper panels) and represented as histograms (lower panels) to better visualize the differences in GFP signals from one time point to another (from left to right 21 h, 27 h, and 42 h after oryzalin treatment). The GFP signal switches from one side of the cell to another, showing that PIN1 retains the ability to reorient in the absence of microtubules. (E) Close-ups of the surface of the same meristem expressing PIN1-GFP before and 47 h after oryzalin treatment. The PIN1-GFP signal becomes broader within the cell after long-term oryzalin treatment. The red dot marks the same cell at the two time points.

(A) Surface of a clv3-2 GFP-MBD meristem before isoxaben treatment, with magnified insert (later time points after treatment shown in [C] and [D]). Note the presence of strong GFP signal on all sides for many of the cells, indicating a random alignment of microtubules in those cells. Scale bar: 20 µm. (B) Theoretical impact of isoxaben on patterns of stress (red) and strain (green) in a cylindrical pressure vessel. The main direction of stress in a cylindrical pressure vessel is circumferential. As plant cells reinforce their walls parallel to the main stress, the residual axial stress drives a strain perpendicular to the main stress. If cellulose deposition is inhibited, the strain follows the stress pattern, i.e., the circumferential strain becomes higher than the axial strain. (C) Impact of isoxaben on microtubule behavior in the clv3-2 GFP-MBD line at different time points after application (microtubule arrangements before application are shown in [A]). Cell growth continues, and microtubules gradually form thick bundles. There is no apparent coordination in the orientations. The red dot marks the same cell at the three time points. Scale bar: 40 µm. (D) Close-ups from (C) showing microtubule orientations in relation to the cell shapes after isoxaben treatment. (E) Surface of a GFP-MBD meristem 70 h after the isoxaben treatment. Note the presence of circumferential bundles of microtubules. Scale bar: 20 µm. (F) Close-up from (E) showing that the microtubules become circumferential even at the tip of the meristem, and this can be correlated to the dome shape of the meristem in the wild-type background. The red dotted lines represent the position of the anticlinal walls (reconstructed from sections through the stack). Scale bar: 10 µm. (G) Surface of a meristem expressing PIN1-GFP before and 20 h after isoxaben treatment. The GFP signal becomes localized to a subdomain of the plasma membrane. Scale bar: 30 µm. (H) Close-ups from (G): the GFP signal is concentrated on the circumferential membrane near a vertex. (I) Surface of a meristem expressing PIN1-GFP 16 h after isoxaben treatment, showing a preferential localization of PIN1 on the circumferential membranes.

(A) Schematic representation of the interactions leading to a pattern-forming behavior in the model. Auxin (a) is transported out of cell j to cell i by the PIN1 (P) proteins localized to the membrane, P_ji. Auxin concentration in each cell affects the elasticity (E_i and E_j) of the adjacent wall, which influences the mechanical stress (S_ij and S_ji) perceived in both parts of the wall between the cells as a result of the force F. For example a_i>a_j leads to E_i<E_j, which in turn causes S_ij<S_ji. The cycling of PIN1 between cytosol, P_j, and the membrane, P_ji, depends on these stresses, and larger S_ij causes stronger allocation of P_j to P_ji. (B) Example of the auxin pattern created spontaneously in the model by applying uniform tension to a two-dimensional template. (C) Spacing of emerging auxin peaks can be controlled by adjustment of model parameters. Patterns of auxin distribution obtained in two different one-dimensional simulations of the model show different arrangements of auxin peaks (compare red versus green peak profiles). (D–G) Comparison of the behavior of the new model with a previously proposed auxin transport model [1] in the case of the response to ablation of a cell. In the auxin-concentration-based model, removal of the cell in the region of uniform auxin distribution (D) causes only minor response of the PIN1 polarization in the cells neighboring the removed cell (E). In the stress-based model, we observed much stronger, outward PIN1 polarization in the cells closest to the ablated region (G), as compared to the situation prior to ablation (F). The ablation results for the new model provide a better fit to the experimental data (see Figures 2 and 4).

Time series showing changing localization of PIN1-GFP in response to laser ablation. (A) A vertical file of cells (marked “X”) was targeted with a pulsed laser. PIN1 localization was first detected to change by 2 h after laser treatment. Scale bar: 10 µm. (B) Visualization of membrane marker 29-1 fused to YFP together with PIN1-CFP after laser ablation shows that the relocalization response is specific to PIN1. Scale bar: 10 µm. (C) Co-alignment of PIN1-GFP (red) and microtubules (green) after ablation. Ablated cells are stained with propidium iodide (blue). Scale bar: 5 µm.

(A) Confocal projection showing the orientation of microtubule arrays at the pin1 mutant meristem summit after laser ablation. (B) Close-up of cells in (A), showing circumferential microtubule orientation in cells surrounding ablation site. Cells at least one cell distant from the wound exhibit circumferential orientation. Both microtubules (C) and PIN1 (D) reorient circumferentially around wounds 24 h after NPA treatment. Note that in (D) PIN1 is oriented away from the wound in cells several cells distant from the wound. Similar behavior is observed for microtubules (E) and PIN1 (F) after 2,4-D treatment. Dead cells are marked by propidium iodide staining (red). Note that two cells separated by an intervening cell are ablated in E. Scale bar for (A): 10 µm. Scale bars for (B–F): 5 µm.

Visualization of PIN1-GFP (red) and TagRFP-MAP4 (green) before (A and B) and after (C and D) laser-induced cell ablation (asterisk) in a pid mutant background. After ablation microtubules reorient around the wound similarly to wild-type. In contrast, PIN1 repolarization in the pid mutant is significantly reduced and remains uncorrelated with microtubule orientations (arrows). Scale bar: 5 µm.