Fig. 5. ERF55 and ERF58 regulate the expression of genes encoding ABA metabolic enzymes and increase ABA levels.
a Completion of germination of wild-type (Col-0), erf55-1 erf58-2 double mutant, and ERF58ox (two independent lines) seeds on ddH2O and increasing concentrations of ABA. Seeds were exposed to W light for 6 h, and then incubated in the dark at 22 °C for 7 days before scoring for completion of germination. Data points show mean cumulative germination percentages of three replicates ±SD. b RT-qPCR analysis of ABA2 expression levels in Col-0 and erf55-1 erf58-2 seeds germinating under phyA-ON/OFF or phyB-ON/OFF conditions; data for other genes encoding ABA metabolic enzymes are shown in Supplementary Fig. 12. Light conditions are described in Supplementary Fig. 6a, b. ACT7 was used as an internal control. Values are means of three replicates ±SD. c Quantification of endogenous ABA levels in Col-0, erf55-1 erf58-2, and ERF58ox seeds after 24 h germination in the dark. Values are means of three replicates ±SD. Letters indicate levels of significance as determined by t-test and Holm–Bonferroni method; p < 0.05. d, e Schematic representation of the ABA2 promoter. The promoter fragment used for EMSA is shown in e. f EMSA. Biotin-labelled ABA2 −52…−222 promoter fragments containing a wild-type or mutated DRE motif were incubated with MBP-ERF58 or MBP alone (negative control). Samples were analysed by native PAGE. The gel was blotted onto a nylon membrane and signals were detected by streptavidin-coupled horseradish peroxidase and ECL. FP, free probe. The experiment was repeated six times with similar results. Data for ERF55 are shown in Supplementary Fig. 13a. g ChIP-qPCR. Seeds of Col-0 p35S:HA-YFP-ERF58 (line 21C) and Col-0 p35S:YFP-HA (negative control) were germinated and subject to light treatments as described in Fig. 4a. Chromatin was isolated and HA-YFP-ERF58 bound DNA fragments were purified using α-GFP coupled magnetic beads. qPCR and specific primers were used to detect the ABA2 −52…−222 promoter fragment in the eluate fraction. ACT7 was used for normalisation. Values show enrichment of the ABA2 promoter fragment in the eluate fraction compared to the input fraction and are means of three replicates ±SD. a, b, g Different letters indicate significant differences as determined by two-way ANOVA followed by post-hoc Tukey’s HSD test. a, b p < 0.05. g p < 0.01.