Fig. 5

Mutations in the first microtubule-binding region of CC1 impair salt tolerance of plants because of mis-regulated microtubule organization upon salt stress. a Cellulose levels in seedlings grown as in b. Values are means ± S.D. expressed as % cellulose of wild-type seedlings grown on MS control media. Dots represent individual data points of the corresponding bars. N ≥ 3 biological replicates with two technical replicates each from three-independent experiments. Welch’s unpaired t-test; **p-value ≤ 0.01. b Seedlings germinated and grown for 2 days on MS plates and then transferred to either MS control plates or MS plates supplemented with 150 mM NaCl and grown for additional 5 days. Scale bar = 2 mm. Please be aware that the images were stitched with Leica LAS X Life Science software. c Quantification of hypocotyl elongation of seedlings grown on MS plates for 3 days and then transferred to either MS control plates or MS plates supplemented with 100 mM NaCl and grown for additional 4 days. Dots represent individual data points of the corresponding bars. Values are mean ± S.D., n = 30 seedlings, 10 seedlings each per three-independent experiments. Welch’s unpaired t-test; ***p-value ≤ 0.001. d GFP-CC1YYAA and mCh-TUA5 in dual-labeled 3-day-old cc1cc2 etiolated hypocotyls (left panels; single frame, right panels; time average projections). Scale bars = 5 μm. e Fluorescence intensity plot of GFP-CC1YYAA and tdT-CESA6 from transect in d along the depicted yellow line. Note that the GFP signal does not substantially correlate with the mCherry signal. f Quantification of GFP-CC1 and GFP-CC1YYAA fluorescent foci on cortical microtubules in a 50 × 50 pixel area of five individual time-lapse images, n = 5 cells from 5 seedling and three independent experiments (box plots: Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum). Welch’s unpaired t-test; ***p-value ≤ 0.001. g Quantification of microtubule and GFP-CC (GFP-CC1 or GFP-CC1YYAA) coverage at the cell cortex and plasma membrane, respectively, after exposure of cc1cc2 seedlings to 200 mM NaCl as in an experiment shown in Supplementary Fig. 7f. Time indicates time after salt exposure. Values are mean ± S.E.M., n = 27 cells from three seedlings per time point and three-independent experiments. Two-way ANOVA analysis of microtubule coverage; p ≤ 0.001 (genotype), p ≤ 0.001 (time), p ≤ 0.001 (genotype × time). Two-way ANOVA analysis of GFP-CC protein density; p ≤ 0.01 (genotype), p ≤ 0.001 (time), p ≤ 0.001 (genotype × time). h Quantification of microtubule bundling after exposure of cc1cc2 GFP-CC1 /GFP-CC1YYAA seedlings to 200 mM NaCl as in an experiment shown in Supplementary Fig. 7f. The salt-adjusted microtubule array in GFPCC1 seedlings shows increased bundling after exposure to salt while the array GFPCC1YYAA seedlings does not. Dots represent individual data points of the corresponding bars. Values are mean ± S.E.M., n = 27 cells from three seedlings per time point and three-independent experiments. Two-way ANOVA analysis of microtubule bundling (excluding T2 and T28); p ≤ 0.001 (genotype), p ≤ 0.001 (time), p ≤ 0.001 (genotype × time)