Fig. 5: Identifying cis-regulatory elements and TFs that regulate C4 genes in Ftri. | Nature Communications

Fig. 5: Identifying cis-regulatory elements and TFs that regulate C4 genes in Ftri.

From: A dominant role of transcriptional regulation during the evolution of C4 photosynthesis in Flaveria species

Fig. 5

a Integrated Genome Viewer (IGV) of RNA-seq reads and two-biological replicates of ATAC-seq reads of three PEPC1s in Ftri (C4). b Word cloud shows the log2-transformed frequency of enriched cis-regulatory elements (CREs) identified through Monte Carlo permutation testing (FDR < 0.05) in accessible chromatin regions (ACR)-CREs associated with C4 genes based on ATAC-seq in Ftri. c, d Bar plots show the top five most abundant TF families of all annotated TFs and TFs that are from the C4 gene-TF regulatory network, respectively, with the latter were termed as C4GRN TFs. e C4 gene-TF regulatory network of the C4 species Ftri. Lines represent predicted regulatory interactions between TFs and C4 genes. ERFs are highlighted in pink. f Electrophoretic mobility shift assay (EMSA) was performed with ERF TFs and cy5-labled partial DNA sequence (probe) of their regulated C4 genes’ promoter from Ftri. Labeled probes were incubated with GST-TF-10xHis protein. GST represents GST−10xHis protein without TF. For competition analysis, the binding reaction was performed with addition of 200-fold of corresponding unlabeled probe. Bands corresponding to DNA-protein complexes (shift) or free probes are indicated by arrows. Predicted ERF motif was shown on the bottom of each panel. The EMSA experiment was conducted in three biological replicates and one representative result (f) was shown. g Structure of reporter and effector plasmids for transient transcription assay. For the reporter constructs, 35S promoter, C4 gene (CA1.1 or PEPC1.1) promoter, firefly luciferase (LUC) and Renilla luciferase (REN) are indicated. For the effector construct, ERF TF (ERF12 or ERF61) was driven by the 35S promoter. h, i ERF12 and ERF61 activates the promoters of CA1.1 and PEPC1.1 (Significance was calculated with two tailed T-test, n = 3 biological replicates). Data are presented as mean values ± SD. Leaf epidermal cells of Nicotiana benthamiana were transfected with infiltration buffer (Mock), reporter DNA (pCA1.1 or pPEPC1.1) with Flag, reporter DNA with ERF-Flag (ERF12 or ERF61). Source data are provided as a Source Data file.

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