Fig. 4: CRISPR mutants of CaLB1 show salt sensitivity.

a Expression of CaLB1 and ALIX after 30- or 90 min treatment with 150 mM NaCl normalized against ACTIN8. Both plots show the results of three experiments each of which is the average of technical quadruplicates. For CaLB1: NaCl 0 min/30 min p = 0.0270 (*0.01 < p < 0.05), NaCl 0 min/90 min p = 0.816 (ns: not significant, p > 0.05), for ALIX: NaCl 0 min/30 min p = 0.692 (ns not significant), NaCl 0 min/90 min p = 0.303 (ns). b Seedlings containing CaLB1pro:CaLB1-GFP, ALIXpro:ALIX-GFP, or UBQ10pro:GFP-ATG8a were treated for 2 h with 150 mM NaCl. Immunoblotting was performed using an anti-GFP antibody. An anti-UGPase antibody was used for loading control. The experiment was conducted three times, and one representative result is shown. c Photographs of 7-day-old wild-type, calb1-1, and calb1-1 expressing CaLB1pro: CaLB1-GFP seedlings were transferred after three days to media with or without 100 mM NaCl and were grown for further 4 days. Scale bars: 5 mm. d Ratio of primary root length with and without NaCl treatment as in (c). Wild type (n = 53 and 49 for +/− NaCl), calb1-1 (n = 53 and 65 for +/− NaCl), calb1-1/CaLB1-GFP (n = 100 for both +/− NaCl). The root length after NaCl treatment was divided by the average root length of untreated seedlings. Wild type/calb1-1 p = 1.83 × 10⁻⁶ (***p < 0.001), wild type/calb1 with CaLB1-GFP p = 0.734 (ns). The experiment was conducted three times, and one representative result is shown. e Total extracts were prepared from 7-day-old seedlings as in (c) and subjected to immunoblotting with an anti-GFP antibody. An anti-UGPase antibody was used for loading control. The experiment was conducted three times, and one representative result is shown. a, b, d, e Source data are provided as a Source Data file. a, d Box plot: center line, median; box limits, first and third quartiles; whiskers, 1.5x interquartile range Two-tailed t-test, no equal variance.