Extended Data Fig. 8: The A283W substitution mutation disrupts the interaction between the phosphomimic T282E FD and 14-3-3 proteins, resulting in FD condensation both in vivo and in vitro.

a, Multiple sequence alignment of the C4 (SAP) domain segment in group A bZIP proteins of Arabidopsis. Mutations in Mu9 are also shown. Amino-acid residues conserved among all proteins are highlighted in red. b, Modeled structure of the FD^SAP–GRF7 complex with mutations on residue T282E (left) and T282E/A283W (right). c, Size-exclusion chromatography and gel analysis of GRF7 protein. d, Size-exclusion chromatography and gel analysis of MBP-FD^{2–285(T282E)} and GRF7 proteins. e, Size-exclusion chromatography and gel analysis of MBP-FD^{2–285(T282E/A283W)} and GRF7 proteins. Data in d,e are representative of two independent experiments with similar results. f, Schematic of FD protein fusion used for in vitro phase separation assay. g, Phase diagram of FD^T282E (top) and FD^T282E/A283W (bottom) droplets with or without GRF7 protein. Formation of FD^T282E and FD^T282E/A283W protein droplets was captured by optical microscopy. Scale bars = 10 μm. Data are representative of three independent experiments. h,i, Confocal images of shoot apical meristem cells of fd-3 plants expressing gFD::mVenus-FD^T282E or gFD::mVenus-FD^T282E/A283W, respectively. Cell walls (blue) and nuclei (magenta) were stained with Direct Red 23 and DAPI, respectively. Scale bars = 20 μm. The confocal images are representative of three independent meristems. j, Flowering time (total leaf number, TLN) of transgenic fd-3 mutant plants carrying gFD::mVenus-FD^T282E/A283W. 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); n = 21 for gFD::mVenus-FD^T282E/A283W, n = 16 for fd-3, and n = 13 for Col-0 seedlings respectively, all grown under long-days (LDs).