TCP4-dependent induction of CONSTANS transcription requires GIGANTEA in photoperiodic flowering in Arabidopsis
Fig 3
TCP4 physically associates with CO promoter and activates its transcription.
(A to C) Histochemical GUS staining images from plants harboring the GUS reporter controlled by the class II TCP promoters. Images from whole seedlings, cotyledons, and the first set of leaves in TCP3:GUS (A), TCP4:GUS (B), and TCP10:GUS (C) plants grown in LD are shown. Scale bar, 0.5 mm. (D) A diagram of the CO locus and the locations of 7 amplicons used in ChIP analysis. The gray box represents 5′-UTR and the white box represents the first exon. Red boxes represent class II TCP binding sites (GGACCA). (E) Results of ChIP analysis using SUC2:mTCP4-3F6H plants against the CO promoter harvested at different times of day are shown. mTCP4 contains synonymous mutations in the miR319-binding site. 10-day-old plants grown in LD were harvested at Zeitgeber time (ZT) 4, 13, and 22. The UBQ10 locus was used as a control. Significant differences from WT harvested on the same ZT are indicated by asterisks (one-tailed Student’s t-test, p<0.005). Data represent means ± SEM (n = 4). (F) A schematic diagram of different lengths of the CO promoter with the location of the TCP-binding site used in (H) is depicted. Red boxes represent TCP binding sites and a pink box represents a mutated TCP binding site (TCPmut). (G) The representative western blot images of effectors (GFP and mTCP4) and the reference [Renilla Luc (RLuc)] in each combination analyzed in (H) and quantitative results of the effector levels (relative to RLuc levels) obtained from 3 independent biological replicates are shown. (H) The results of the luciferase reporter assay in N. benthamiana are shown. The effects of TCP4 on firefly luciferase (Luc) activities controlled by 1,000-bp of the CO promoter (gray bars), 500-bp (white bars), and 500-bp with a mutation on the TCP-binding site (dark gray bars) are tested. The activities of firefly Luc were normalized by the activities of RLuc. Asterisks denote significant differences in each combination (Bonferroni-corrected student’s t-test, p<0.05). Data represent means ± SEM (n = 3).