Figure 8.
GhCTR1 is a downstream target of GhARF6-1 and GhARF23-2. A) Y1H assay showing that GhARF6-1 and GhARF23-2 directly bind to the promoter of GhCTR1. JG-45, empty pJG-45 vector without inserted protein; Laczi, empty pLaczi vector without inserted promoter. B)N. benthamiana transient expression assay showing the binding of GhARF6-1 to GhCTR1 promoter. C) Quantification of LUC activity in B). The expression of REN is used as an internal control. Mean values ± Sd (n = 3) are shown. The expression of REN was used as internal control. LUC, luciferase; REN, renilla. The P values were calculated using an unpaired 2-tailed Student’s t test. D)N. benthamiana transient expression assay showing the binding of GhARF23-2 to GhCTR1 promoter. E) Quantification of LUC activity in D). The expression of REN is used as an internal control. Mean values ± Sd (n = 3) are shown. The expression of REN was used as internal control. LUC, luciferase; REN, renilla. The P values were calculated using an unpaired 2-tailed Student’s t test. F) Y1H assay showing that GhARF6-1 and GhARF23-2 directly bind to the second ARF-binding site (S2) in the promoter of GhCTR1. JG-45, empty pJG-45 vector without inserted protein; Laczi, empty pLaczi vector without inserted promoter. S1 and S2 are predicted ARF-binding sites. m, mutant. G)N. benthamiana transient expression assay showing the binding of GhARF6-1 to the S2 binding site in the promoters of GhCTR1. H) Quantification of LUC activity in G). The expression of REN is used as an internal control. Mean values ± Sd (n = 3) are shown. The expression of REN was used as internal control. LUC, luciferase; REN, renilla. The P values were calculated using an unpaired 2-tailed Student’s t test. I)N. benthamiana transient expression assay showing the binding of GhARF23-2 to the S2 binding site in the promoters of GhCTR1. J) Quantification of LUC activity in I). The expression of REN is used as an internal control. LUC, luciferase; REN, renilla. Mean values ± Sd (n = 3) are shown. The expression of REN was used as internal control. The P values were calculated using an unpaired 2-tailed Student’s t test. K) and M) EMSA showing that GhARF6-1 (K) and GhARF23-2 (M) directly bind to the F1 fragment of the GhCTR1 promoter. CK, control. L) and N) Competitive EMSA using a biotin-labeled F1 fragment of the GhCTR1 incubated with GhARF6-1 (L) and GhARF23-2 (N), competing with different concentrations of cold probes (without biotin label) containing the intact or mutated binding site. CK, control; m, mutant.

GhCTR1 is a downstream target of GhARF6-1 and GhARF23-2. A) Y1H assay showing that GhARF6-1 and GhARF23-2 directly bind to the promoter of GhCTR1. JG-45, empty pJG-45 vector without inserted protein; Laczi, empty pLaczi vector without inserted promoter. B)N. benthamiana transient expression assay showing the binding of GhARF6-1 to GhCTR1 promoter. C) Quantification of LUC activity in B). The expression of REN is used as an internal control. Mean values ± Sd (n = 3) are shown. The expression of REN was used as internal control. LUC, luciferase; REN, renilla. The P values were calculated using an unpaired 2-tailed Student’s t test. D)N. benthamiana transient expression assay showing the binding of GhARF23-2 to GhCTR1 promoter. E) Quantification of LUC activity in D). The expression of REN is used as an internal control. Mean values ± Sd (n = 3) are shown. The expression of REN was used as internal control. LUC, luciferase; REN, renilla. The P values were calculated using an unpaired 2-tailed Student’s t test. F) Y1H assay showing that GhARF6-1 and GhARF23-2 directly bind to the second ARF-binding site (S2) in the promoter of GhCTR1. JG-45, empty pJG-45 vector without inserted protein; Laczi, empty pLaczi vector without inserted promoter. S1 and S2 are predicted ARF-binding sites. m, mutant. G)N. benthamiana transient expression assay showing the binding of GhARF6-1 to the S2 binding site in the promoters of GhCTR1. H) Quantification of LUC activity in G). The expression of REN is used as an internal control. Mean values ± Sd (n = 3) are shown. The expression of REN was used as internal control. LUC, luciferase; REN, renilla. The P values were calculated using an unpaired 2-tailed Student’s t test. I)N. benthamiana transient expression assay showing the binding of GhARF23-2 to the S2 binding site in the promoters of GhCTR1. J) Quantification of LUC activity in I). The expression of REN is used as an internal control. LUC, luciferase; REN, renilla. Mean values ± Sd (n = 3) are shown. The expression of REN was used as internal control. The P values were calculated using an unpaired 2-tailed Student’s t test. K) and M) EMSA showing that GhARF6-1 (K) and GhARF23-2 (M) directly bind to the F1 fragment of the GhCTR1 promoter. CK, control. L) and N) Competitive EMSA using a biotin-labeled F1 fragment of the GhCTR1 incubated with GhARF6-1 (L) and GhARF23-2 (N), competing with different concentrations of cold probes (without biotin label) containing the intact or mutated binding site. CK, control; m, mutant.

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