Fig. 4.

Redox-mediated regulation of REVOLUTA-DNA-binding capability and influence of the PAS domain. (A,B) Redox-DPI-ELISAs. The DNA-protein interaction assays were performed by using 5′ biotinylated complementary annealed oligonucleotides coupled to a streptavidin-coated ELISA plate. Crude E. coli extracts (25 µg) expressing recombinant REV or REVΔPAS were pre-incubated with different concentrations of DTT and H₂O₂ to examine a redox state-dependent binding of REV. In order to test the reversibility of the redox effect, high concentrations of H₂O₂ were added first and then oxidizing conditions were reversed by addition of DTT. After binding, biotinylated DNA-protein complexes were detected using anti His-HRP conjugated antibodies. Results for REV binding site 1 of the WRKY53 promoter are shown. E. coli BL21 cells transformed with the empty vector were used as background control. (C) Non-radioactive electrophoretic mobility shift assays. Purified GST-REV protein was incubated with a biotinylated oligonucleotide containing the HB9-binding motif (Wenkel et al., 2007) in the presence of different redox conditions. After gel electrophoresis and subsequent blotting, the biotinylated DNA probe was detected with a HRP-strepatividin substrate. (D,E) Chromatin-immunoprecipitation qPCR assays of 35S::FLAG-GR-REVd plants. Twelve-day-old seedlings (D) and 7-week-old transgenic plants (E) were treated with mock substrate (0.1% ethanol), DEX or 0.1% H₂O₂ and DEX. H₂O₂ was given 15 min prior to 45 min of DEX induction. Fold enrichment for the same primer sets as in Fig. 1 is shown.