Extended Data Fig. 7: The phosphorylation status at T282 does not affect the in vivo stability of FD protein, and 14-3-3 proteins, rather than FT protein, repress FD condensation in vivo and in vitro.

a, Flowering time [rosette leaf number (R) and cauline leaf number (C)] of T3 homozygous populations of transgenic fd-3 mutant plants carrying gFD::3HA-3Flag-FD^WT, gFD::3HA-3Flag-FD^T282E or gFD::3HA-3Flag-FD^T282A. Different letters represent significant differences among genotypes (P < 0.05, using one-way ANOVA followed by Tukey’s pairwise multiple comparison, P = 1.11 * 10⁻¹⁶). The sample sizes were as follows: n = 16 for gFD::3HA-3Flag-FD^WT, n = 14 for gFD::3HA-3Flag-FD^T282E, n = 12 for gFD::3HA-3Flag-FD^T282A, and n = 11 and 12 for Col-0 and fd-3 seedlings, respectively, all grown under long-days (LDs). b, RT-qPCR analysis of FD mRNA levels in shoot apices of transgenic fd-3 mutant plants carrying gFD::3HA-3Flag-FD^WT, gFD::3HA-3Flag-FD^T282E, gFD::3HA-3Flag-FD^T282A, as well as Col-0 and fd-3 plants. All values are normalized to ACTIN2 levels. Data are the mean ± SEM of three biological replicates (n = 3). Statistical significance was determined by pairwise two-sided t-test [P (FD^WT vs. FD^T282E) = 0.1914; P (FD^WT vs. FD^T282A) = 0.0444; P (FD^T282E vs. FD^T282A) = 0.0210]. Asterisks indicate significant differences (*P < 0.05); n.s., not significant (P > 0.05). Shoot apices were harvested from seedlings grown for 7 LDs. c, Western blotting analysis of the abundance of 3HA-3Flag-FD proteins in 7 LD-grown seedlings of the genotypes described in (a). d,e, Confocal images of shoot apical meristem cells of fd-3 plants expressing gFD::mVenus-FD (green) WT #19 or T282A #3. Cell walls (blue) were stained with Renaissance 2200. Scale bars = 2 μm. f, RT-qPCR analysis of FD mRNA levels in shoot apices of transgenic fd-3 mutant plants carrying gFD::2HA-mVenus-FD^WT and gFD::2HA-mVenus-FD^T282A. All values are normalized to ACTIN2 levels. Data are the mean ± SEM of three biological replicates (n = 3). Statistical significance was determined by pairwise two-sided t-test [P (FD^{WT #19} vs. FD^{WT #22}) = 0.8709; P (FD^{WT #19} vs. FD^{T282A #9}) = 0.1293; P (FD^{WT #19} vs. FD^{T282A #3}) = 0.8384; P (FD^{WT #22} vs. FD^{T282A #9}) = 0.0445; P (FD^{WT #22} vs. FD^{T282A #3}) = 0.9998; P (FD^{T282A #9} vs. FD^{T282A #3}) = 0.0401]. Asterisks indicate significant differences (*P < 0.05); n.s., not significant (P > 0.05). Shoot apices were harvested from seedlings grown for 7 LDs. g, Western blotting analysis of the abundance of 2HA-mVenus-FD proteins in 7 LD-grown seedlings of the genotypes described in (d) and (e). Western blots represent one of two independent biological replicates in (c) and (g). ACTIN was used as the loading control. h, i, Confocal images of shoot apical meristem cells of fd-3 or fd-3 ft-10 plants expressing gFD::mCherry-FD wild type (WT). Scale bars = 20 μm. h.1,i.1, Close-up images of cells from (N) and (O), respectively. Scale bars = 2 μm. j, Volcano plots showing differential protein abundance for FD in ft-10 mutants based on label-free quantification. The log₂-transformed fold change between genotypes is plotted against the -log₁₀ of the adjusted p-value. Statistical significance was assessed using a two-sided t-test with permutation-based false discovery rate (FDR) correction (AdaPT method). Proteins with log₂ (fold change) > 1 and FDR-adjusted p < 0.05 are highlighted in distinct colors as significant outliers. k.1, Schematic of protein fusion used for in vitro phase separation assay and phase diagram of mScarlet1-FD2-285^T282E droplets. k, In vitro phase separation of mScarlet1-FD2-285^T282E droplets in the present or absent of GRF7 or FT. Scale bars = 10 μm. l, RNA-seq data for FD target genes in shoot apices of Col-0 wild type and fd-3 mutant across various developmental stages²⁰. Data are the mean ± SEM of three biological replicates (n = 3). m, Schematic of FD protein fusion used for in vitro phase separation assay. n, o, Phase diagram of FD^T282E and FD^T282A droplets, respectively. Formation of FD^T282E protein droplets was captured by optical microscopy. Scale bars = 10 μm. p, Gel-shift analysis of the interactions between single MBP-FD or combination of GRF7 or protease to remove the MBP tag from MBP-FD fusion proteins with wild-type (WT) or mutant SEP3 DNA probes. The confocal images in d,e,h,i are representative of three independent meristems. The results in k,n,o are representative of three independent experiments.