Figure 4.

DET1 inhibit the transcriptional activation of ABI5 by FHY3. A and B, Co-IP analyses showing the protein interaction between DET1 and FHY3 dependent on light (A) and enhanced by ABA (B). In A, 4-d-old etiolated MYC-DET1 seedlings (DD) being irradiated with light (DD-L) for 1 h were used to perform Co-IP assays. In B, 4-d-old etiolated MYC-DET1 seedlings treated without (Mock) or with ABA (30 μm) for the indicated times under DD or DD-L conditions were used to perform Co-IP assays. In A and B, MYC-DET1 fhy3 was used as a negative control to indicate the positions (asterisk) of FHY3 protein. C and D, ChIP-qPCR assays showing the association of DET1 with the ABI5 promoter is dependent on FHY3 (C) and enhanced by ABA under light (D). In C, 4-d-old etiolated seedlings (DD) after being irradiated with light (DD-L) for 3 h were used to perform ChIP assays. In D, 4-d-old etiolated seedlings were treated without (Mock) or with ABA (30 μm) for 3 h under DD or DD-L conditions, and then used to perform a ChIP assay. E and F, Transient expression assays showing DET1 represses the transcriptional activation of ABI5 by FHY3. In E, the transformed Arabidopsis protoplasts were first incubated without (Mock) or with 0.3 μm of ABA under darkness for 16 h (DD), then irradiated with light (DD-L) or not for 3 h and used to measure the expression of LUC reporter. In F, the abundances of FHY3-3FLAG and DET1-3FLAG were detected by anti-FLAG antibodies. Data are means of five biological replicates, and error bars represent sd. In C, D, and F, asterisks indicate the statistical significance by Student's t test (*P <0.05, **P <0.01, and ***P <0.001).