Fig. 5

Transcriptional condensates formed by heterotypic interaction between paralogous TFAM proteins repress AN expression to synchronize flowering. A Representative images (upper) showing the interactions between ALOG proteins in BiFC assays. The fluorescence intensity of indicated yellow lines (bottom) showing the heterogenous condensates formed from interactions between TFAM proteins. Scale bars, 2 μm. B Quantitative data showing the percentage of cells with condensates formed by interactions between TFAM proteins in nuclei. Data are presented as three biological replicates ± SD (n = 72, 67, 69, 60, 69, 71). C Cross-mixing phase separation reactions using recombinantly expressed mCherry-TMF fusion proteins and GFP fusion proteins of TFAMs. Protein concentration, 15 μM; NaCl concentration, 25 mM. Scale bar, 20 μm. D Quantification data showing the intersection between TMF droplets and TFAM1/2/3/11 droplets, respectively. Pearson’s coefficient was calculated by ImageJ. Data are presented as three biological replicates ±SD (n = 3). E Stereoscope images (upper) of the micro-dissected transitional meristem for real-time PCR (bottom). White dashed line indicates the dissection position. The relative expression of AN was normalized to WT using UBIQUITIN (UBI) as an internal control. Data are presented as three replicates ± SD (n = 3, ***P < 0.001, Student t-test). Three independent experiments with similar results were carried out. Scale bars, 100 μm. F Schematics of constructs used to analyze transcriptional activity. G Transcriptional repression of AN by transcriptional condensates formed by TMF, TFAM and variant TFAM proteins as indicated. The ratio of GUS to LUC indicates relative transcriptional activity. LUC served as an internal control. Data are presented as six biological replicates from two independent experiments. Data are means ±SD (n = 6, ***P < 0.001, Student t-test). H BiFC assays visualizing the condensates formed by TMF and TFAMs in tobacco leaves. Scale bar, 50 μm. I Representative images for primary inflorescences of WT, an and higher-order mutants of an and tfams. Scale bars, 1 cm