TCP4 physically interacts with FBH1 but not CDF1, and acts additively with FBH1 to activate <i>CO</i> transcription.

<p>(A) Yeast two-hybrid (Y2H) assay showing the interaction between TCP4 and FBH1. (B) LUC complementation imaging (LCI) assay detecting the interaction among TCP4, FBH1 and CDF1. (C) Co-immunoprecipitation (Co-IP) assay showing the interaction between TCP4 and FBH1 in <i>Arabidopsis</i>. The asterisk indicates unspecific signal. (D-F) FLIM-FRET measurement of the interaction between TCP4 and FBH1 in <i>Arabidopsis</i> protoplasts. In (D), the CFP and YFP fluorescence signals were observed with the confocal microscope at 24 h after transformation. BF, bright filed; bars = 10 μm. In (E), the white arrowheads indicate the CFP fluorescence lifetime images of the nuclei of representative cells expressing the indicated protein combinations, and the average fluorescence lifetimes are encoded by color as indicated by the scale at right side. Bars = 20 μm. (F) shows the CFP fluorescence intensity decay curves in indicated combinations. The average fluorescence lifetimes represented by τ are separately marked above the curves of TCP4-CFP/YFP (mean ± SD, n = 7 nuclei), TCP4-CFP/FBH1-YFP (mean ± SD, n = 17 nuclei) and TCP4-CFP/CDF1-YFP (mean ± SD, n = 15 nuclei) combinations. ROI, region of interest. (G-I) Transcriptional activity assays in <i>N</i>. <i>benthamiana</i> illustrating the additive effects of TCP4 and FBH1 in activating <i>CO</i> transcription. A representative leaf image is shown in (G), and (H) shows the quantification of the relative luminescence intensities (mean ± SD, n = 18). In (I) the expression levels of <i>TCP4</i> and <i>FBH1</i> in the infiltrated <i>N</i>. <i>benthamiana</i> leaf areas were determined by qRT-PCR (mean ± SD, n = 5). Results were normalized to <i>NbACTIN1</i> (<i>NbACT1</i>). Asterisks in (H) and (I) denote significant differences against the combination 1 at <i>P</i> < 0.01 (**, Student’s <i>t</i> test).