Figure 6.

BOA Binds to the Promoter of CCA1 and Regulates Its Expression in Vivo and in Vitro.

(A) BOA binds to CCA1 promoter in vivo. Leaf samples from Col-0 plants maintained under 12-h-light/12-h-dark photoperiods were collected at Zeitgeber time 7 (ZT7) and 15 (ZT15) for chromatin preparation. Anti-BOA antibody was used to enrich BOA-bound chromatins (ChIP). Serum collected from rabbits prior to immunization (Prebleed) was used in control reactions. A mock ChIP without serum was included as negative control. DNA samples from ChIP assays and 5% of the amount of input chromatin were amplified using CCA1 promoter-specific primers. PCR products were resolved in 1.2% agarose gels.

(B) BOA activates transcription from the CCA1 promoter in vitro. In vitro transcription assays were conducted using rice whole-cell extract. Plasmid pCCA1t:GUS was used as reporter gene. RNA polymerase II inhibitor α-amanitin (5 μg/mL) was added to the control reaction. In vitro transcription assays with 500 ng (+) or 1 μg (++) of BOA protein or without BOA were included.

(C) Sequences of PBS4 probes used in EMSAs.

(D) BOA binds to PBS4 of CCA1. EMSAs were conducted using BOA purified from E. coli with or without nonlabeled competitor with molar ratios as labeled. Arrow indicates bands of the BOA-PBS4 complex.

(E) BOA activates CCA1 expression in vivo. Transgenic Arabidopsis lines 35S:BOA-8 and 35S:BOA-19 carrying pC-35S:BOA and wild-type plants (Col-0) were maintained under 12-h-light/12-h-dark photoperiods before they were released to continuous light (LL) for sample collection starting from 21 d after sowing. RNAs from those samples were subjected to qRT-PCR analysis to quantify CCA1 expression. Each data point reflects the mean of three repeats ± sd.