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. 2023 Jan 5;14:67. doi: 10.1038/s41467-022-35542-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Relative stiffness as a function of time using the drop functions displayed in Fig. 2e as inputs for pressure. All simulations were performed over the 100-parameter value sets best-fitting iku2 experimental data using iku2 drop function as an input for pressure. Plain curves display the mean dynamics and confidence intervals show standard deviations. b pELA1::3X-VENUS-N7 expression in Col-0 and iku2 seeds at the heart embryo stage. Scale bars: 50 µm. c Mean signal of pELA1::3X-VENUS-N7 reporter in nuclei of Col-0 and iku2 seeds (intensity unit/pixel). Seeds were classified according to embryo developmental stage (one experiment, Col-0, Globular: n = 15, Triangular/Heart: n = 68, Torpedo: n = 46; iku2, Globular: n = 25, Triangular/Heart: n = 58, Torpedo: n = 30). Fluorescent signals were compared using two-sided Student tests without adjustments for multiple comparisons, **p < 0.01, ***p < 0.001. d, f, h Signal from LM19 (d), 2F4 (f) and JIM5 (h) immunolocalizations on Col-0 and iku2 testas at 4-5 DPA. Scale bars: 20 µm. e, g, i Signal intensity from LM19 (e), 2F4 (g) and JIM5 (i) immunolocalizations in wall 3 of Col-0 and iku2 seeds as a function of time (LM19: two independent experiments, Col-0: 3 DPA: n = 9, 4 DPA: n = 9, 5 DPA: n = 10, 6 DPA: n = 8, 7–9 DPA: n = 9; iku2: 3 DPA: n = 9, 4 DPA: n = 9, 5 DPA: n = 9, 6 DPA: n = 9, 7–9 DPA: n = 9; 2F4: three independent experiments, Col-0: 3 DPA: n = 9, 4 DPA: n = 9, 5 DPA: n = 9, 6 DPA: n = 9, 7–9 DPA: n = 9; iku2: 3 DPA: n = 8, 4 DPA: n = 9, 5 DPA: n = 9, 6 DPA: n = 9, 7–9 DPA: n = 9, JIM5: three independent experiments, Col-0: 3 DPA: n = 9, 4 DPA: n = 9, 5 DPA: n = 10, 6 DPA: n = 8, 7–9 DPA: n = 9; iku2: 3 DPA: n = 9, 4 DPA: n = 9, 5 DPA: n = 9, 6 DPA: n = 9, 7–9 DPA: n = 9). Signal intensities were compared using two-sided Student tests without adjustments for multiple comparisons, *p < 0.05, **p < 0.01, *p < 0.001. j Maximum projection and z-section of a z-stack of WT seeds at 5 DPA expressing the membrane marker LTi6b-GFP (green) and stained with Propidium Iodide (magenta) without indentation or after a 40 µm depth indentation with a conical tip to rupture testa walls. A wall was considered to be ruptured when some intracellular PI staining was found in the underlying cell layer. Scale bars, 20 µm. k Quantification of testa wall rupture in Col-0 and iku2 following a 40 µm indentation with a conical tip. Two independent experiments (Col-0: n = 35, iku2: 36). Wall rupture frequencies were compared using a two-sided χ² test, ***p < 0.00001. In all boxplots, the centerline shows the median; the box limits show the upper and lower quartiles; the whiskers correspond to 1.5× interquartile range. Individual points are superimposed in black.