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. 2019 Aug 29;15(8):e1008377. doi: 10.1371/journal.pgen.1008377

Fig 7. BZU2/ZmMUTE binds to the E-box motifs located in the PAN1 and PAN2 promoters.

Fig 7

(A) RT-qPCR analysis of the expression of the genes associated with stomatal development indicates repression of transcription in bzu2-1 mutant plants. RNA was taken from samples excised up to ~1.5 cm from the base of second and third leaves. RT-qPCR values are expressed as the mean ± SD compared to that of the internal control (ZmUbiquitin 2). Error bars indicate SD, n = 3, Student’s t test, **P < 0.01. Assays were done in triplicate. (B) ChIP-qPCR results indicating that the promoter fragments of PAN1and PAN2, can be amplified from the immunoprecipitates pulled down by the anti-GFP antibody. The sequences used for ChIP-qPCR contain the E-box cis-elements located within the PAN1 and PAN2 promoters. Second and third leaf base sections (~1.5 cm) of 8-day-old seedlings were harvested for the chromatin immunoprecipitation (ChIP) experiments. Sequence from regions of the PAN1 and PAN2 promoters lacking the E-box cis-elements were used as controls. Error bars indicate SD, n = 3, Student’s t test, **P<0.01. (C) The E-box motifs in the promoters of PAN1 and PAN2. The bHLH transcription factor binding the cis-element consensus sequence (E-box) and their locations in the promoters of PAN1 and PAN2 (-750 - -1). The E-box (6 bp) motif logo was produced by MEME based on previously studied motifs. The relative position of E-box cis-elements in the promoters of PAN1 and PAN2 as detected by ChIP-qPCR. (D) The DNA fragments used for yeast one-hybrid and EMSA assays. The E-box is underlined, and the mutated sequences are highlighted in red. (E) Yeast one-hybrid assays showing BZU2/ZmMUTE binding to the E-box motifs present in the PAN1 and PAN2 promoters. (F) EMSA assays showing specific binding of BZU2/ZmMUTE to the E-box motifs in the PAN1 and PAN2 promoters. EMSA was performed with probes that were biotin-labeled (biotin probe) or unlabeled (cold probe). E-box-containing DNA fragments (top panel) and recombinant BZU2/ZmMUTE protein; specific combinations are shown above the autoradiograph. Unlabeled fragments were added gradually in 2-, 5- or 10- fold excess, as indicated.