Fig 1: Modification of ICE1 and general SUMOylation upon cold exposure are inhibited in fugu5.(A) Cold acclimation induces ICE1 SUMOylation which then activates CBFs and leads to the expression of downstream targets for the establishment of freezing tolerance. (B) Determination of the amount of ICE1 in Col-0, fugu5-1 and ice1-2 seedlings. Amount of TUBULIN was detected as loading control. Plants grown in long day conditions for 3 weeks. Black arrow indicates modified ICE1 dimer whereas red arrow indicates unmodified ICE1 dimer. (C) Comparison of the amount of the ICE1 under normal conditions (22°C) and after cold treatment (4°C, 3 hr) in Col-0 and fugu5-1 seedlings. 10-days-old liquid grown seedlings were used for total protein extraction. Anti-ICE1 was used as primary antibody. (D) Western blots comparing SUMOylation levels of Col-0 and fugu5-1 under normal conditions (22°C) and after cold treatment (4°C, 3 hr). (E) Western blots demonstrating the total SUMOylation in Col-0, fugu5-1, fugu5-3, UBQ:AVP1 and UBQ:PPa5-GFP under normal conditions (22°C) and after cold treatment (4°C, 3 hr). (D) and (E) 10 days old liquid grown seedlings were used for total protein extraction. Anti-SUMO1/2 was used as primary antibody. Whole lanes were measured for the calculation of protein amounts using ImageJ. cFBPase detection was used for normalization. Error bars represent SD of n = 2 biological replicates. Asterisk indicates significant difference compared to Col-0 (Student’s t test; *p<0.05, ***p<0.001).10.7554/eLife.44213.013Figure 3—source data 1.(D-E) Comparison of the amount of the total SUMOylation with and without cold treatment.