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. 2016 Oct 13;7:13181. doi: 10.1038/ncomms13181

Figure 4. TCP20/TCP22 bind to the TCP-binding site (TBS) for CCA1 activation.

Figure 4

(a) TCP20 associates with the TBS-containing region of CCA1 promoter in vivo. ChIP assays involved use of the anti-Flag antisera with tcp20-2 pTCP20::TCP20-Flag pCCA1::LUC2 complementation line (CL #20) or 35S::HA-GFP-Flag transgenic line. Data are mean±s.d. (n=3 technical replicates). Asterisk indicates that TCP20 preferentially binds with the amplicon ‘b' (Student's t test; *P<0.005). Similar results were observed in two independent experiments. (b) EMSA confirmed the specific binding of TCP20 and TCP22 with the TBS. DNA–protein complex was competed with excess (20 × or 40 ×) unlabelled wild-type (TBS) or mutated (mTBS) probes. (c) Transient assay in Arabidopsis protoplasts of TCP20 and TCP22 possessing activator activity toward promoters containing TBS but not mTBS. Effector and reporter constructs are illustrated. LUC2 reporter activity was normalized to GUS activity from the transfection control 35S::GUS. Data are mean±s.d. (n=3). Asterisks indicate that TCP20 or TCP22 preferentially activates the reporter gene driven by promoters containing TBS (mini35S was used as a negative control) (Student's t test; *<0.005). Similar results were observed in three independent experiments.