FIGURE 3.

IFR1 knock-down causes diminished tolerance toward RES in C. reinhardtii. (A) Immunodetection of IFR1 protein (αIFR1) in the parental strain (PCS; wild type CC124) and IFR1 knock-down strains (IFR1_1 and IFR1_6) detected after 48 h of cultivation in sulfur deplete medium. A colloidal Coomassie stained gel (CBB) served as loading control. Different amounts of proteins were used and band intensities (lower bar diagram) determined by densitometric analysis (1x PCS set to 100%). (B,C) IFR1 accumulation in PCS and IFR1 knock-down strains grown for 24 h in TAP supplemented with DBMIB (5 μM) or 2-(E)-hexenal (500 μM). (D) Growth inhibition by reactive oxygen species determined for the PCS and the two IFR1 knock-down strains during 24 h of growth in TAP supplemented with 4 μM rose Bengal (RB), 15 μM neutral red (NR), or 0.5 μM methyl viologen (MV). Optical densities (determined at 680 and 750 nm) and cell counts are given relative to the untreated/solvent-control sample (set to 1). Error bars indicate standard errors derived from three biological replicates including technical replicates (n = 3). Asterisks indicate significant differences between PCS and knock-down strains according to a two-tailed Student’s t-test (p < 0.05). (E,F) Growth inhibition following treatment of PCS and IFR1 knock-down strains with 5 μM DBMIB and 500 μM 2-(E)-hexenal for 9 or 24 h in TAP medium. Standard errors are derived from three biological replicates, including technical replicates (n = 3). Except for the data indicated by asterisks (p > 0.05) differences between PCS and knock-down strains were significant according to a two-tailed Student’s t-test (p < 0.05).