Figure 10. C53 is crucial for ER stress tolerance.

(A) Atc53 mutant is insensitive to carbon starvation. Phenotypes before (0 d) and after 9 days carbon starvation (9 d) of 7-day-old seedlings, n ≥ 20 seedlings per genotype. (B) Atc53 mutant is insensitive to nitrogen starvation. Phenotypes before (0 d) and after 14 days nitrogen starvation (14 d) of 7-day-old seedlings, n ≥ 20 seedlings per genotype. (C) Atc53 mutant is sensitive to phosphate starvation. Root-length quantification of seven-day-old seedlings which were transferred to media with or without Pi supplement (+Pi, -Pi), and imaged after 2 days. (D) Atc53 mutants are sensitive to ER stress induced by tunicamycin. Root-length quantification of 7-day-old seedlings grown on half strength MS media without sucrose treated with 100 ng/mL tunicamycin (Tm). Bottom Panel, Root-length quantification of 7-day-old seedlings. n≈125 seedlings per genotype and treatment. Left Panel, Example of 7-day-old seedlings grown in described conditions. Scale bars = 5 mm. Left, non-treated seedlings. Right, seedlings grown at 100 ng/mL Tm. Right Panel, Root length of each genotype was compared pairwise with the wild type (Col-0) for each specific treatment condition. (E) Main molecular players in the ufmylation pathway. UFSP2: UFM1-specific protease two that matures UFM1, exposing the terminal glycine residue. UBA5: the E1 activating enzyme, UFC1: E2 conjugating enzyme, UFL1: E3 ligase (F) Ufmylation pathway mutants are sensitive to ER stress triggered by tunicamycin. Root length quantification of 7-day-old seedlings grown on half strength MS media without sucrose treated with 100 ng/mL tunicamycin (Tm). Left panel, Root length quantification of 7-day-old seedlings. n≈100 seedlings per genotype and treatment. Right Panel, Representative images of 7-day-old seedlings grown in described conditions. Scale bars, 5 mm. To the left are non-treated seedlings, to the right are seedlings grown at 100 ng/mL Tm. (G) AtC53^sAIM mutant does not complement tunicamycin sensitivity phenotype. Root length quantification of indicated 7-day-old seedlings grown on half strength MS media without sucrose in control conditions (Ctrl) or treated with 100 ng/mL tunicamycin (Tm). T1 transgenic lines were used. n = 12 seedlings per genotype and treatment. Data represent the median with its interquartile range. Root length of each genotype was compared pairwise with the wild type (Col-0) for each treatment condition. Significant differences compared to control treatment (Ctrl) are indicated with * when p value ≤ 0.05, ** when p value ≤ 0.01, and *** when p value ≤ 0.001.

Figure 10—figure supplement 1. C53 and the UFMylation machinery are essential for ER stress tolerance.

(A) Atc53 mutant is sensitive to phosphate starvation. Seedlings were germinated for 5 days on +Pi media prior to transfer for 2 days to medium supplemented with or without Pi (-Pi, +Pi) and imaged after 2 days (Naumann et al., 2019). See Figure 10c for quantification. Scale bars, 1 cm. (B, C) Atc53 and UFMylation pathway mutants display lower survival on tunicamycin-containing plates. Seedlings were grown on control or 150 ng/mL tunicamycin containing half-strength MS media for two weeks and were evaluated for survival. Left Panel shows an example of the plates. Right Panel, Quantification of living and dead seedlings. Data represents six plates with 4 × 36 seedlings (n = 216). Survival percentage was compared pairwise to the survival of the wildtype (Col-0). Data represent the median with its interquartile range. (D, E) Ufmylation mutants are insensitive to carbon and nitrogen starvation. (D) Phenotypes before (0 d) and after 9 days carbon starvation (9 d). n ≥ 25. Seven-day-old seedlings per genotype were used. (E) Phenotypes before (0 d) and after 14 days nitrogen starvation (14 d). n ≥ 25. Seven-day-old seedlings per genotype were used. (F) Marchantia polymorpha c53 mutants are sensitive to tunicamycin. 14-day-old plants were transferred to control or 2 µg/ml tunicamycin containing agar plates. Right Panel, Quantification of the plant area 21 days after transfer. Data shown as mean ± SD. (G) The AtC53^sAIM mutant does not complement tunicamycin sensitivity phenotype. Left Panel, Representative images of 7-day-old seedlings grown on half strength MS media without sucrose in control conditions (Ctrl) or treated with 100 ng/mL tunicamycin (Tm). At least 10 independent T1 transgenic lines were analyzed to measure the root lengths. See Figure 10g for quantification. Significant differences compared to control treatment (Ctrl) are indicated with * when p value ≤ 0.05, ** when p value ≤ 0.01, and *** when p value ≤ 0.001. Right Panel, western blot analyses of AtC53 complementation lines showing expression of the tested constructs used in Figure 10G. (H) Knockdown of C53 leads to increased BiP3 chaperone levels. Knock down and complementation assays showing C53 function is important for ER stress tolerance in HeLa cells.