Fig. 4: PIF4a binds to a negative cis-element in the FT2 genomic region.

a Genomic organisation of FT2 paralogs in Populus tremula. Black boxes indicate genomic regions of the two paralogs from the start to the stop codons. The red box shows the E-box elements (NACGTG, CACNTG). b Yeast one-hybrid assays of PIF4 binding to the E-box region. The indicated plasmid combinations were co-transformed into a yeast reporter strain, and interactions were assessed by growth on selective media and colourimetric assay for β-Galactosidase activity. 50 mM 3-amino-1,2,4-triazole (3-AT) was used to suppress autoactivation⁸⁴. The combinations of proFT1_301bp and PtrSVL served as a positive control^22,36. Negative controls are represented by the vector combinations of proFT1_301bp + PIF4a and proFT2_E-box+ PtrSVL. c Relative enrichment of intergenic fragments after CUT&Tag quantified by qPCR. Values are normalised against the input DNA. The unrelated elongation factor 1 alpha (EF1a, Potra2n6c14161) served as a negative control. d Diurnal expression of FT2b in leaves of the proFT2_E-box-8 CRISPR plants grown for 40 days in 21 °C LD. Height growth (e) and bud set scores (f) of WT and the proFT2_E-box-8 mutant. Plants were grown in LD at 21 °C for 77 days, then shifted to SD at 21 °C. Representative pictures of whole trees in LD and shoot apices in SD are shown in the right panel. Bars indicate a length of 10 cm. The dotted line marked bud set score 2 indicates the stage of growth cessation²⁶. Data are presented as mean ± SEM (n = 2 for b, d; n = 3 for c; n = 10 for e, f). Fisher’s LSD post hoc tests (two-sided, uncorrected for multiple comparisons) were performed following ANOVA to compare different plasmid combinations (b), various chromatin positions (c), and distinct time points (d, e, f). The statistically significant differences are indicated by non-matching letters in (b) (p < 0.05). Asterisks in (c, d, e and f) indicate the levels of statistical significance, * p < 0.05, ** p < 0.01. See also Supplementary Fig. 4 and Supplementary Table 1, 2. Source data are provided as a Source Data file.