Figure 7.

GhACE1 binds to E-box elements in the promoters of GhPIP2;7 and GhEXP8, and its transcriptional activity is suppressed by GhFP2. A, The potential E-box (CANNTG) elements in the promoters of GhPIP2;7 and GhEXP8. The upright lines indicate position of E-boxes. The Chip lines indicate fragments detected in ChIP assay. The probe1/2/3/4 lines indicate fragments used in EMSA. The red bold bases in the probe1/2/3/4 sequences are E-boxes. B, ChIP-qPCR assay of GhACE1 proteins binding to the promoters of GhPIP2;7 and GhEXP8 in vivo. GhACE1-bound chromatin DNA fragments were isolated from nine DPA fibers of WT cotton, and qPCR analysis was performed with the primer sets listed in Supplemental Table S3 (see “Materials and methods”). Error bars represent sd of three biological replicates. Data were analyzed with prism7.0. CK, the control sample without anti-GhACE1 antibody. C–F, EMSA showing that GhACE1 protein binds to the E-box elements (probe1/2/3/4) of GhPIP2;7 and GhEXP8 promoters in vitro. Biotin-labeled DNA fragments (probes) were incubated with His-GhACE1 protein. An excess of the unlabeled probes or mutated probes was used to compete with the labeled probes. mCold-probe represents 200× mutated probe. a, 20× probe; b, 200× probe. G, Dual-LUC assay of transcriptional activation of GhACE1 to the target genes and inhibitive effects of GhFP2 on GhACE1 activating GhPIP2;7 and GhEXP8 promoters (pGhPIP2;7 and pGhEXP8). GhPIP2;7 and GhEXP8 promoters were fused to the LUC reporter, respectively, and the promoter activities were determined in leaves of N. benthamiana by transient dual-LUC transcriptional activation assay. The relative LUC activities were normalized to the reference REN luciferase. The corresponding effector (+) and empty vector (−) were co-filtrated. Error bars represent sd of three biological replicates. Data were analyzed with Microsoft Excel. Independent t tests demonstrated significant (P < 0.05) or very significant (P < 0.01) difference between two groups.