Global Compression Reorients Cortical Microtubules in Arabidopsis Hypocotyl Epidermis and Promotes Growth  Sarah Robinson, Cris Kuhlemeier  Current Biology  Volume 28, Issue 11, Pages 1794-1802.e2 (June 2018) DOI: 10.1016/j.cub.2018.04.028 Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 1 Determining the Relevant Conditions for Using ACME to Monitor Microtubule Dynamics (A) Ablations were made in the center of 3- and 4-DAS hypocotyls (35S::GFP-MBD) using 405-nm ablation laser. The cells were imaged immediately after. (B) The same cells were observed 18–24 hr later. The maximum microtubule orientation is indicated. The length of the line represents the average orientation of the microtubules, and its length is proportional to the anisotropy of the microtubules. Five out of six hypocotyls showed clear microtubule reorientation around the wound. Scale bars: 50 μm. (C) A diagram of the ACME setup showing a seedling attached between two gray plates. The direction of movement/force application is indicated (arrow). The plates are attached to a force sensor (f.s) in a feedback loop via software (cpu). (D) A representative trace of the force exerted by a 3-DAS 35S::GFP-MBD seedling when held in distilled water and after NaCl solution was added (dashed line). (E) In water, the samples were fully turgid. (F) Plasmolysis of the sample was observed when the force was stable after NaCl treatment. Cell wall (propidium iodide, PI), red; protoplast (35S::GFP-MBD), green. (G) An example trace of the force generated by a 3-DAS hypocotyl held in a fixed position in ACME for 25 hr. (H and I) The sample was initially straight (H), but during the experiment, it buckled (I). (J and K) Average microtubule angle was calculated using FibrilTool. The line is proportional to the anisotropy of the microtubule array. The microtubules are initially perpendicular to the sample (J) but become perpendicular (K). (L) In order to prevent force from building up, the plates move apart. An example of the distance that the plates move while maintaining a 5-DAS sample at zero force for 33 hr is shown. Scale bars: 50 μm. See also Video S1. Current Biology 2018 28, 1794-1802.e2DOI: (10.1016/j.cub.2018.04.028) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 2 Analyzing Microtubule Responses to Tension and Compression. 3- to 5-DAS hypocotyls (35S::GFP-MBD) were held under 1 mN compression (n = 7), 1 mN tension (n = 7), or zero force (n = 5) for 16–24 hr. An example hypocotyl held at zero force (A and B), 1 mN tension (C and D), and 1 mN compression (E and F) is shown. Average microtubule orientation before treatment (A, C, and E) and after (B, D, and F) was found per cell using FibrilTool. The average orientation is shown, and the length is proportional to the anisotropy. (G–I) The average microtubule angle was transformed to be between 0° and 90°, and the average was taken per sample in the first and last image for the different treatments: zero (G), tension (H), and compression (I). 0° is parallel to the axis of the sample, which is also the orientation of the stress. Statistically significant differences are indicated with an asterisk (p < 0.05). Samples held under compression had a significantly different orientation at the end of the experiment compared to the start (start = 28° ± 6 SEM, end = 47° ± 4 SEM, p = 0.029); samples held under tension (start = 30° ± 2 SEM, end = 39° ± 4 SEM, p = 0.1) or at zero force (start = 34° ± 5 SEM, end = 36° ± 7 SEM, p = 0.8) did not change significantly. (J) The cells were classified as having an average angle closer to 0°, 45°, or 90° with respect to the axis of the sample. The percentage of cells that are in each of these categories was calculated. The maximum percentage of cells that are at 90° for each sample is shown. The samples under compression and tension differ significantly from each other in terms of the maximum percentage of cells with microtubules at 90° (compression = 49% ± 5 SEM, tension = 17% ± 5 SEM, p = 0.0013). Neither treatment differs significantly from the zero treatment (34% ± 5 7.6 SEM, n = 5). (K–M) Four 3- to 5-DAS seedlings were held under 1 mN compression for 6–10 hr, then 1 mN tension for 10 hr. The average microtubule angle was found per cell every 4 hr. For the same sample, we computed the microtubule angles before the start of the experiment (K), during compression (L), and during tension (M). (N) The mean microtubule angle was computed per sample at the start of the experiment and when it was held under compression and tension. The microtubule angle differs significantly when the sample is under tension (31° ± 1.9 SEM, n = 13 measurements from four independent samples) versus compression (51° ± 3.1 SEM, measurement from four independent samples). Boxplots show the median and interquartile range (IQR), with the whiskers showing the data that extends beyond the IQR by less than 1.5 × IQR. Boxes with the same letter do not differ significantly (p > 0.05). Scale bars, 50 μm. (O) The orientation of the microtubules in the different layers prior to the treatments. (P–R) Microtubule orientation was quantified from the same samples as Figures 2A–2J in the outer epidermal wall, the inner epidermal wall, and the cortex layer in one image 4–8 hr after force application. (P) The microtubules in the outer wall of the epidermis. (Q) The microtubules in the inner wall of the epidermis. (R) The microtubules in the cortex. Bars show the mean percentage of cells with that orientation and the error bars show SD. Letters are assigned to bars based on them having the same mean percentage as the early zero treatment (p < 0.05). An additional comparison was made between the percentage of cells at 90° in the compressed and tensile state; this was significant in the epidermal cells (∗). See also Figures S1 and S2. Current Biology 2018 28, 1794-1802.e2DOI: (10.1016/j.cub.2018.04.028) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 3 A Simulation of Tissue Stress in a Model Hypocotyl during Application of External Stress (A) A finite element model was constructed in Abaqus to simulate the tissue stresses in the hypocotyl. A simplified axis-symmetric geometry was used. The model was constrained at the base and pressurized. At the top, there is a variable boundary (orange arrows) that is used to stretch or compress the sample. The inner walls represent the cortex and the outer walls the epidermis; the different layers can be assigned different material properties and different pressures. (B) The model can be visualized as a cylinder. To avoid the influence of the boundary and top, we only consider the microtubule orientation in the center (square box). (C–F) The results of the simulation showing the direction of maximum (red) and minimum (yellow) tensile stress, including a zoomed-in-view square box. The outer layer is colored green, and the inner is blue. (C) The material was initially defined to be isotropic. The maximum tensile stress direction (red) is in the circumferential orientation. (D–F) The inner layer was defined to be anisotropic with an elastic modulus that is ten times lower in the longitudinal direction while the epidermis remained isotropic. The pressure in the inner layer was set to be four times higher than the pressure in the outer layer. (D) The model was allowed to expand. The maximum stress direction is circumferential in the inner layers and longitudinal in the outer layer. (E) The variable boundary was specified to displace in the longitudinal direction 1.5 times more than when unconstrained. The orientation of stresses was the same as for the unconstrained model. (F) The simulation was restricted to only 0.6 times as much as the unconstrained model in the longitudinal direction. The maximum direction of stress in both layers was circumferential. See also Figure S3 and Video S2. Current Biology 2018 28, 1794-1802.e2DOI: (10.1016/j.cub.2018.04.028) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 4 Sample Strain at Different Forces (A–C) The strain was calculated for each hypocotyl in each image. Strain of each sample when held at zero force (A; n = 5), 1 mN of tension (B; n = 7), and 1 mN of compression (C; n = 7). (D) A comparison of the strain shown after 16 hr for the different treatments. Boxplots show the median and interquartile range (IQR), with the whiskers showing the data that extends beyond the IQR by less than 1.5 × IQR. Boxes with the same letter do not differ significantly (p > 0.05). Samples held under relative compression showed more strain after 16 hr (mean = 17.7% ± 2.4 SEM) compared to samples held at zero force (mean = 5.2% ± 1.6 SEM, p = 0.002) and seedlings under relative tension (mean = 10.0% ± 3.0 SEM, p = 0.07). (E) The percentage of cells with microtubules at 90° to the axis of the sample and direction of applied force. Color coded by the total strain shown by the patch of cells in the sample. The compressed samples with a higher percentage of cells at 90° showed more strain (blue-green) than the samples where microtubule reorientation was not observed (red-orange). See also Figure S4. Current Biology 2018 28, 1794-1802.e2DOI: (10.1016/j.cub.2018.04.028) Copyright © 2018 Elsevier Ltd Terms and Conditions