Fig. 5: Gene regulatory network of TU1.

a RNA sequencing (RNA-seq) was performed to compare transgenic edited (Tu1-CR1) and nontransgenic plants, and the results were visualized via a volcano plot. The horizontal dashed line on the plot represents the threshold of significance (q = 0.05) for the differentially expressed genes. The blue and red points represent genes whose expression was significantly downregulated and upregulated, respectively, in the transgenic Tu1 edited plants compared with the nontransgenic plants. DNA affinity purification sequencing (DAP-seq) analysis identified the motif of the TU1 protein (b) and detected a binding peak in the promoter of the ZmMADS1 gene (c). d Schematic representation of effectors and reporter constructs for dual-luciferase transient expression assays. The reporter was designed to place LUC under the control of the ZmMADS1 promoter with a mini 35S promoter. e LUC activity was significantly repressed by the overexpression of TU1 (effector) in the construct. These results suggest that TU1 directly represses the transcription of ZmMADS1. Two-tailed Student’s t test; the data are shown as the mean ± SD (n = 3). f Electrophoretic mobility shift assays (EMSAs) were performed using fragments of the ZmMADS1 promoter containing the motif. The EMSAs were conducted with at least one of the following reagents: HaloTag, Halo-TU1 protein, a biotin-labeled probe, a competitor without a biotin label, and a competitor with the mutated motif and without a biotin label. The specificity of binding was tested with competitors. The wild-type competitors dramatically decreased binding to the probes, whereas the mutated competitors had no effect on binding. +, present; –, absent. The motif is marked in blue in the sequence. g TU1 functions as a balancer in the gene regulatory network for the number of leaves above the ear; downregulates plastochron repressors WEE1 and ZmMADS1; simultaneously upregulates plastochron repressors such as Bige1, Kn1, ZmMADS3 and ub2; and activators include PIN1a, TD1 and yabby1. The plastochron in the shoot apical meristem is accelerated, and the leaf number is ultimately increased in maize. The green and blue triangles represent plastochron activators and repressors, respectively. SAM shoot apical meristem; arrow bar, upregulation, T bar downregulation, P1-5, plastochrons. The solid and dashed lines represent direct and indirect regulation, respectively. The green and blue lines represent the final positive and negative effects on leaf number, respectively. The position of the ear bud is shown. Source data are provided as a Source Data file.