ABI4 is a direct target gene of EIN3 in the regulation of AsA biosynthesis. A, Expression of ABI4 in Col-0 and ein3 eil1 in response to treatment with ethylene or ABA. Seven-day-old seedlings were treated with ethylene or ABA for 2 h. B and C, Transient expression assays testing the capability of EIN3 to regulate the expression of ABI4 in tobacco leaves (B) and Arabidopsis protoplasts (C). D, Top, sequence analysis of the ABI4 promoter. EBSs (TACAT and TTCAAA) are shown. The numbers indicate the distance away from the start codon. DNA fragments (P1, P2, and P3) were used for the ChIP assay. Bottom, ChIP-qPCR assays of EIN3 binding to the ABI4 promoter. The AtTUB4 promoter (TUB4-P) was used as an internal control. E, Transient expression assays testing the capability of EIN3 to regulate the expression of ABI4 in tobacco leaves when the ABI4 promoter contained mutations in the binding motif (M1, M2, and M3 indicate the ABI4 promoter motifs that contain a mutation). F, EMSA testing the binding of EIN3N to the promoter of ABI4 in vitro. As indicated, unlabeled probe was used as Competitor and those with a mutated binding motif as mCompetitor. Values in A and C to E are means ± sd (n = 3). Statistically significant differences are indicated by different letters in A (P < 0.05, ANOVA with Tukey’s test).