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. 2013 Jun 10;110(26):10854–10859. doi: 10.1073/pnas.1302592110

Fig. 4.

Fig. 4.

THB8 is required for NO-dependent hypoxic growth and induction of anaerobic genes. (A) Aerobically and illuminated grown cells (0 h) were incubated for the indicated time points in open (+) or sealed (−) flasks in the dark. The bars at the bottom indicate the following: white, light/aerobiosis (+O2); gray, dark/aerobiosis (+O2); black, dark/anaerobiosis (−O2). (B) Cultures were incubated for 2.5 h anaerobically in the dark (2.5−) and then transferred to open beakers in the dark (+, indicated by a gray arrow) or kept in anaerobiosis (−, indicated by a black bar) in the dark for the indicated time points in hours. (C) Wild-type CC-124 and transformants amiTHB8-1 and -2 were grown aerated in the light and then incubated anaerobically in the dark for 7 h. RNA was extracted from the precultures and after dark–anoxic incubation. Below the individual graphs, fold-changes of the individual transcripts in the wild type, calculated using the ΔΔC(t) method related to values obtained from preculture RNA, are depicted. (D) Strain CC-124 and amiTHB8-1 and -2 grown without or with 100 µM PTIO. Each plate with or without PTIO was incubated under aerobic (+O2) or hypoxic (−O2) conditions in the light. (E) Cells were grown on TAP agar in flasks. Anaerobic conditions were achieved by N2 purging of gas tightly sealed vessels. NO was added as NO/N2 mixture. The crr1 mutant, which does not grow under hypoxic conditions (41), was used as control for maintained anaerobiosis. (D and E) The result of one experiment is depicted. The same results were obtained by two repetitions. (F) Light-grown C. reinhardtii cultures (0 h) were transferred to two open beakers in the dark, and DEA-NONOate was added to one culture to a final concentration of 10 µM (+NO). RNA was isolated after 45 min. (A, B, C, and F) Transcript abundances relative to RCK1, whose amounts stay constant during anaerobiosis, were determined by qPCR and multiplied by 1,000. (A and B) Averages ± SD of three independent experiments are shown. (C and E) The average of two independent analyses is depicted; error bars indicate the SD.