Figure 6.

MdNAC5 regulates the expression of MdERF3 and MdACS1 by binding to their promoters. (A) Y1H assay showing that MdNAC5^A and MdNAC5^T bind to the MdERF3 and MdACS1 promoters. The schematic diagram of truncated 350-bp fragments with NACRS elements on MdERF3 and MdACS1 promoters. The fusion vectors pGADT7-MdNAC5^A/MdNAC5^T and pABAi-MdERF3/MdACS1 were cotransformed in Y1H on the SD/−Leu (−LT) medium. Single colonies were dissolved in 6 μl of sterile 0.1% NaCl, diluted to 10⁻¹ to 10⁻³, and finally transferred to the SD/−Leu/AbA²⁰⁰ medium. (B) EMSA showing that MdNAC5^A and MdNAC5^T bind to the promoters of MdERF3 and MdACS1. The shifted probes and free DNA probes are indicated by black arrows. “+” and “–” demonstrate the presence and absence of the protein and the probe appeared, individually. (C) Dual-luciferase assay revealing that MdNAC5^A and MdNAC5^T bind to the MdERF3 and MdACS1 promoters. The white color indicates high luciferase activity, and the black color indicates low luciferase activity. The bar chart shows the relative activity of LUC/REN and set at least three repetitions. (D) CHIP-qPCR assay showing that MdNAC5^A and MdNAC5^T bind to the MdERF3 and MdACS1 promoters. F1–F4 indicate the position of qPCR detection for the MdERF3 promoter, and S1–S4 indicate the position of qPCR detection for the MdACS1 promoter. Actin serves as an internal control. Values are means of three replicates ± SD (Turkey’s test, ^***P < 0.001, ^**  P < 0.01, ^*P < 0.05).