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. 2017 Aug 3;8:1347. doi: 10.3389/fpls.2017.01347

FIGURE 2.

FIGURE 2

IFR1 accumulation is triggered by the SOR1-dependent pathway. (A) Samples were taken before (0 h, +S) and during the course of hydrogen production induced by sulfur deprivation of a wild type cell line (16–48 h; –S). IFR1 accumulation was analyzed with an IFR1-specific antiserum (αIFR1) and equal protein loading confirmed by colloidal Coomassie staining (CBB). (B) Comparison of IFR1 mRNA levels in the sor1 mutant (Fischer et al., 2012) and its parental strain (4A+) with samples taken in the late exponential phase. mRNA levels were determined by RTqPCR and the IFR1 transcript level in 4A+ was set to 1. Median and interquartile range shown in the box-and-whisker diagram are derived from two biological replicates, each including nine technical replicates (n = 18). (C) Representative immunoblot (αIFR1) showing IFR1 accumulation during growth of mutant sor1 and its parental strain (4A+) in nutrient-replete TAP medium for 3 days. Relative band intensities (Dens.) determined by densitometric scanning of immunblot signals are given relative to the band intensity of the 4A+ sample at t48 h (set to 1). (D) Position of the octanucleotide motif CAACGTTG described to represent an electrophile response element (ERE; Fischer et al., 2012) implicated in the genetic response to reactive electrophile species (RES) and SOR1-dependent signaling relative to the start codon (ATG) of the 4.87 kbp IFR1 gene, comprising exons, introns and untranslated regions (UTRs). (E) IFR1 mRNA levels determined by RTqPCR following dark treatment of WT cell cultures with DBMIB (5 μM) and 2-(E)-hexenal (500 μM) for 24 h. The mRNA level of the solvent control sample was set to 1. Median and interquartile range shown in the box-and-whisker diagram are derived from two biological replicates, each including six technical replicates (n = 12). (F) Immunoblot (αIFR1) showing IFR1 accumulation distinct time points (6–24 h) after the addition of DBMIB (5 μM) or only solvent (Control) to a liquid TAP culture of the C. reinhardtii wild type CC124 and subsequent dark incubation for 24 h.