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. 2014 Aug 7;10(8):e1004519. doi: 10.1371/journal.pgen.1004519

Figure 5. SCL27 activates MIR171 gene expression in a feedback manner.

Figure 5

(A) qPCR analysis of MIR171a, MIR171b and MIR171c expression in Col, scl6 scl22 scl27 and LUC-rSCL27-OX plants. Relative expression levels of MIR171 genes were normalized to that of ACTIN2, and the relative expression in WT plants was set as 1. Error bars represent the s.d. (n = 3). Two biological replicates were performed with similar results. (B) 35S::6xMYC-rSCL27-GR/scl6 scl22 scl27 transgenic plants were treated with DEX (10 µM) or untreated (Mock) for 20 days. Bars = 1 cm. (C) Chlorophyll content of the plants shown in (B). ** indicates p value (Student's t-test) <0.01; Error bars indicate the s.d. (n = 4). (D) Relative expression of PORC, MIR171a, MIR171b and MIR171c in the plants shown in (B). Expression levels were normalized to that of ACTIN2. Expression levels in plants without DEX were set as 1 for each gene. Error bars represent the s.d. (n = 3). Two biological replicates were performed with similar results. (E) Relative activity of the MIR171a promoter. pMIR171a::LUC was transformed into N. benthamiana leaves with or without co-transformation of 6xMYC-rSCL27. Relative LUC activities were normalized to the 35S::REN internal control. Error bars indicate the s.d. (n = 4). Three biological replicates showed similar results. (F) Schematic diagram of MIR171a promoter regions V (−726 bp to −495 bp), VΙ (−260 bp to −71 bp) and VΙΙ (the precursor of MIR171a), which were used for ChIP experiments. (G) Relative enrichment of MIR171a promoter fragments in the immuno-precipitates. Leaves of 3-week-old Col and 6xMYC-rSCL27-OX plants were used for ChIP experiments. The enriched DNA fragments were quantified using qPCR. The β-TUBULIN-2 promoter was used as a reference. Error bars indicate the s.d. (n = 3). Similar results were obtained from three independent immuno-precipitation experiments.