ZmLG2 directly regulates the expression of ZmBEH1. A, ChIP-Seq shows that ZmLG2 binds to the ZmBEH1 promoter. B, EMSA of ZmLG2 binding to the promoter of ZmBEH1. Top, diagram of the promoter fragments of ZmBEH1. The locations of four Biotin-labeled probes named P1-P4 are indicated by black lines. Red arrows indicate the probes that can be bound by ZmLG2. Bottom, ZmLG2 directly binds to P2-P4 probes with different affinities in EMSA. Black arrows indicate the positions of the bound complex and free probe. GST is the control of ZmLG2-GST. C, Top, diagram of the P4 native and mutated (m1 to m7) fragments acting as probes in EMSA. The ZmLG2 binding motif is labeled in red, and the nucleotide substitutions in the mutated fragments (m1 to m7) are labeled in blue. Bottom, ZmLG2-GST, but not GST by itself, specifically binds to the Biotin-labeled ZmBEH1 P4 probe. Ten- and 100-fold excess amounts of the unlabeled probe were used to compete for the binding of the ZmLG2 protein to the labeled probe. m1 to m7 unlabeled mutated probes were used as competitors (m-competitor) at 100-fold in EMSA. “−” indicates the absence of this component. “+” indicates the presence. Black arrows indicate the positions of the bound complex and free probe. D, Dual-luciferase assay shows relative transactivation of ZmLG2 to the ZmBEH1 promoter. The coding sequence of ZmLG2 driven by the 35S promoter was used as an effector, and the empty vector (EV) was used as an effector control in the transient luciferase assay performed in maize protoplasts. The vector with the REN gene driven by a 35S promoter and firefly luciferase (LUC) gene driven by the promoter sequence from ZmBEH1 was used as the reporter. The LUC/REN ratio represents the relative activity of the promoters. n = 5. E, RT-qPCR analyses show the relative expression of ZmBEH1 in the ligular region of WT and lg2 mutants. Total RNA was isolated from the ligular region of V3 leaves when the seedlings were grown for 18 d. n = 7. Error bars indicate SD. Statistical significance was determined by a two-sided t test: *P < 0.05, **P < 0.01.
