Plant materials and growth conditions
The Arabidopsis thaliana Columbia (Col-0) ecotype was used as the main experimental organism. Seeds of Col-0 (N70000), fd-3 (SALK_054421), ft-10 (GK_290E08) and other transgenic plants were surface-sterilized with 70% ethanol for 10 min, rinsed with 99% ethanol for 5 min, air-dried and stratified at 4 °C for 3 days before sowing. Plants were grown on soil under long-day conditions (16 h light:8 h dark cycles) or were grown vertically on plates containing 1% agar supplemented with half-strength Murashige and Skoog (MS) medium (pH 5.7) at 22 °C with a light intensity of 160–180 μmol m−2 s−1 provided by LED bulbs (Philips F17T8/TL841 17 W).
Plasmid construction
To generate different epitope-tagged fusions of FD, the genomic fragment carrying FD promoter (2,930 bp), the full-length coding region and the 3′ untranslated region (1,982 bp) were amplified from Col-0 genomic DNA. DNA encoding 3HA-3Flag and 2HA-mVenus57 tags were synthesized and amplified with PrimeSTAR GXL DNA Polymerase (Takara Bio) for subcloning. Overlapping PCR was then performed to obtain genomic fusions with FD and the epitope tags. Corresponding PCR fragments were then cloned into a modified binary vector PER8 using a HiFi DNA Assembly Kit (NEB). The constructs were transformed into fd-3 mutants using the floral dip method. The same strategy (with genomic fusions) was used to construct gGRFs::2HAmScarlet-I58-GRFs in Col-0 (GRF2 and GRF6), grf7 (SALK_084141)or grf8 (SALK_148929) mutants, and three genomic constructs carrying the 9,149-bp promoter, the FT coding region fused with different tags (gFT::FT-mVenus, gFT::FT-Venus-Halo-Venus and gFT::FT-ALFA59), and a 3,159-bp downstream sequence were transformed separately into ft-10 (GK-290E08) plants. SUC2::pp2A.1-mCherry was transformed in to Col-0 background. Primers used to amplify these sequences are listed in Supplementary Table 4.
Generation of Arabidopsis mutants and transgenic plants
Corresponding Col-0, fd-3 or ft-10 mutant plants were grown in the greenhouse under LD conditions and were transformed by the floral dip method using Agrobacterium tumefaciens strain GV3101. The resulting transgenic T1 seeds were screened on half-strength MS medium with supplemented hygromycin for 7 LD and then they were transferred to soil for the measurement of flowering time.
RNA extraction and RT–qPCR analysis
Total RNA was extracted from 13-day-old seedlings grown in long-day conditions using the RNeasy plant Mini Kit (QIAGEN) with an on-column DNase (QIAGEN) treatment. cDNA was synthesized from 1 μg RNA using a QuantiTect Reverse Transcription Kit (QIAGEN). Real-time PCR was performed with iQ SYBR Green Supermix (Bio-Rad) in a CFX384 Touch Real-Time PCR Detection System (Bio-Rad). The reference gene ACTIN2 was used for normalization. Three technical replicates for each of three independent biological replicates were performed for each experiment and representative results are presented. The primers used for qRT–PCR are listed in Supplementary Table 4.
Immunoblot assays
For western blots, approximately 30 mg of tissue from 13-day-old seedlings grown in long-day conditions was ground into fine powder with liquid nitrogen with a TissueLyser system (QIAGEN). Total protein was extracted using denaturing buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 30 mM EDTA pH 8.0, 4% (w/v) SDS, 20% (v/v) glycerol, 20 mM β-mercaptoethanol (Sigma-Aldrich), 20 mM DTT, 2 mM PMSF (Sigma-Aldrich), 1× Protease Inhibitor Cocktail (PIC, Sigma-Aldrich, P9599), 1× Phosphatase Inhibitor Cocktail 2 (PIC2, Sigma-Aldrich, P5726), 1× Phosphatase Inhibitor Cocktail 3 (PIC3, Sigma-Aldrich, P0044), 80 μM MG132 (Sigma-Aldrich), and 0.01% bromophenol blue) in a 1:5 (w/v) ratio and was boiled at 95 °C for 10 min. Protein samples were centrifuged at 16,000g for 5 min at room temperature and the supernatants were transferred to a new low-protein-binding tube and separated by SDS–PAGE.
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