Gene expression, phenotypic, and immunoblot analysis of Arabidopsis protease loss-of-function mutants rbl11, rbl10, and ftsh11 grown under standard and cold (4 °C) conditions. Plants were grown under standard conditions (21 °C day and night temperature, 10 h day length and 120 µE light intensity) for 3 wks and then treated with cold (4 °C day and night temperature, 10 h day length, and 120 µE light intensity). A) Gene expression levels of RBL11, FTSH11, and RBL10 in the Wt by qRT-PCR under standard growth conditions (0 d at 4 °C) and several days during cold treatment (7, 14, 28, 42, and 70 d at 4 °C). Data represent relative mean expression levels of 3 biological replicates and are normalized to standard conditions (0 d at 4 °C) using UBQ as an internal control. B) Phenotypic analysis of rbl11, rbl10, and ftsh11 Arabidopsis plants. Images were taken from plants grown under standard conditions and after chilling for 7 and 56 d. C) Wt and rbl11 rosettes were cut after 70 d of cold treatment and rotated to highlight the wilted leaves. D) Number of wilted leaves per plant in Wt and rbl11 after 70 d of cold treatment. E) Immunoblot analysis via FAX1 antibody in isolated chloroplast envelopes of Wt, rbl11, rbl10, and ftsh11 from plants grown under standard conditions (21 °C) and plants grown for 7 d at 4 °C. Immunoblot detection via Tic110 antibody is used as a control and shows an equal loading of the protein samples at 3 µg per lane. Error bars in A) represent ± Sem. Error bars in D) are ± Sd. Asterisks indicate significant differences between Wt and mutant using a t test: ***P-value ≤ 0.001 (Supplementary Data Set 3).