Skip to main content
. 2015 Jul 7;169(1):391–402. doi: 10.1104/pp.15.00943

Figure 3.

Figure 3.

TTG1 is directly suppressed by FUS3 during seed development. A, Induced FUS3 activity transcriptionally represses TTG1 expression during seed development. fus3-3 35S:FUS3-GR siliques at 12 d after pollination were mock treated (Mock) or treated with 10 µm dexamethasone (Dex), 5 µm cycloheximide (Cyc), or 5 µm cycloheximide plus 10 µm dexamethasone (Cyc+Dex). TTG1 expression was examined after 2 or 4 h of treatment by quantitative real-time PCR analyses of three independently collected replicates. Results were normalized against the expression of EF1aA4. Asterisks indicate significant differences in TTG1 expression in dexamethasone-treated samples compared with their respective controls (two-tailed paired Student’s t test, P < 0.05). Error bars denote sd of triplicate assays. B, Western analysis of FUS3-6HA in nuclear extracts or immunoprecipitated fractions of wild-type or fus3-3 35S:FUS3-6HA siliques at 12 d after pollination. Nuclear extracts served as the input (I), while immunoprecipitated fractions by anti-HA antibody were used as the eluate (E). Immunoblot analysis was performed using anti-HA antibody. C, Schematic diagram of the TTG1 genomic region. The coding region and other genomic regions are represented by black and white boxes, respectively. Arrowheads indicate the putative binding sites (CATG [CA/TG]) of FUS3. Four DNA fragments (P1–P4) spanning these sites were examined by ChIP enrichment test as shown in D. D, ChIP analysis of FUS3-6HA binding to the TTG1 genomic region. To detect FUS3-6HA binding during seed development, ChIP analysis was performed using the input and eluate described in B. Similar ChIP analysis was also performed on rosette leaves. A TUB2 fragment was amplified as a negative control. Asterisks indicate significant differences in the enrichment fold at P3 compared with that of other DNA fragments tested (two-tailed paired Student’s t test, P < 0.05). Error bars denote sd of triplicate assays. E, List of the putative FUS3-binding element (native probe) in the TTG1 promoter and its mutated version (mutated probe). These probes are part of the 60-bp TTG1 sequences used for EMSA assays shown in F. F, EMSA assay shows the binding of GST-FUS3 to the conserved CATGTG motif in the TTG1 promoter. GST-FUS3 protein was incubated with biotinylated native or mutated probe. Nonlabeled native or mutated probes in 100-fold molar excess relative to the biotinylated native probe were used as cold competitors. A supershift band was observed when anti-GST antibody was added. Col-0, Ecotype Columbia.