Fig. S1. Protoplast detachment from cell walls during apparent (A) and incipient (B) plasmolysis. (A) An epidermal strip was treated with 0.38M NaCl. Protoplasts of all the cells are completely detached from the walls at the tips of cells. (B) The extent of protoplast detachment in cells treated with 0.2M NaCl is lower: compare cell tips of rectangular (white asterisks) or acute (black asterisks) shapes in the two micrographs. Images were obtained using light microscopy equipped with a Nomarski differential interference contrast system. Note the appearance of wrinkles that form due to osmotic treatment, on the outer periclinal wall surface facing the protoplast. Scale bars = 50 µm.

Fig. S2. Micrographs taken from a fragment of the epidermis strip shown in Fig. 4A. (A) Cell outlines observed under light microscopy. (B) Images of different portions of the outer periclinal wall of an exemplary cell (this cell is marked by asterisk here and in Fig. 4A) showing an arrangement of wrinkles on the inner wall surface. The wrinkle patterns at cell center (B2) and differently shaped cell tips (B1 – rectangular, B3 – acute) were observed at higher magnification and at various focal planes, under light microscopy equipped with a Nomarski differential interference contrast system. The epidermis strip was treated with 0.2M NaCl, causing incipient plasmolysis. Scale bars = 50 µm (A); 25 µm (B).

Fig. S3. Exemplary vertical deflection AFM images in which the cellulose microfibril arrangement was analyzed. Images are the patches of the inner wall surface of cells #3 (A) and #5 (B) shown in Fig. 5C. The original AFM images (A1, B1) were divided into 36 square-shaped portions (white lines in A2 and B2), and microfibril arrangement was analyzed in every portion of each image (A2, B2). Red line segments show individual microfibrils that were digitized. Black arrows point to the direction of the cell long axis.

Fig. S4. Variation in the areal strain of cell outlines. Measurements were performed using in vivo replicas. (A) Histogram of strain in area assessed for outlines of all the examined cells (n = 70). (B) Variation in outline areal strain of individual samples. Difference in the strain of samples treated with the different osmoticum is not statistically significant (t-test, p = 0.189). Samples 3 and 5 are the only two samples that differ significantly in median areal strain (p < 0.05; Kruskal-Wallis non-parametric test). In (B) the thick line within each box represents median, box delimits the first and third quantiles, whiskers extend from each end of the box to the adjacent values in the data as long as the most extreme values are within 1.5 times the interquartile range from the box limits; crosses represent outliers.

Fig. S5. Reappearance of wrinkles on the protoplast-facing surface of the outer periclinal wall after the second round of osmotic treatment. Micrographs were taken from the same portion of epidermal strip treated with 0.38M NaCl causing apparent plasmolysis (A), with deionized water (B), and again with the same osmoticum. Note that wrinkles that are apparent after the first osmotic treatment (A) disappear when the osmoticum is replaced by water (B), but reappear after the second osmotic treatment (C). Micrographs were obtained using light microscopy equipped with a Nomarski differential interference contrast system. Scale bar = 50 µm.

Fig. S6. Model validation. Histograms of areal strain of outer periclinal walls (A) and cell volume strain (B) upon turgor pressure loss, obtained from experiment (in vivo confocal microscopy, MorphoGraphX; n = 110 for areal strain; n = 70 for strain in volume) and model (n = 66; marginal cells were excluded). Because the areal strain is small its measurement is more affected by segmentation errors than the strain in volume. (C) The map of cell volume strain upon the pressure loss, computed using the mechanical model.