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. 2014 Feb;26(2):552-64.
doi: 10.1105/tpc.113.115220. Epub 2014 Feb 14.

Structural Features Determining Flower-Promoting Activity of Arabidopsis FLOWERING LOCUS T

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Free PMC article

Structural Features Determining Flower-Promoting Activity of Arabidopsis FLOWERING LOCUS T

William Wing Ho Ho et al. Plant Cell. .
Free PMC article

Erratum in

  • CORRECTION.
    Plant Cell. 2020 Jun;32(6):2043-2047. doi: 10.1105/tpc.20.00239. Epub 2020 Apr 7. Plant Cell. 2020. PMID: 32265263 No abstract available.

Abstract

In Arabidopsis thaliana, the genes FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) have antagonistic roles in regulating the onset of flowering: FT activates and TFL1 represses flowering. Both encode small, closely related transcription cofactors of ∼175 amino acids. Previous studies identified a potential ligand binding residue as well as a divergent external loop as critical for the differences in activity of FT and TFL1, but the mechanisms for the differential action of FT and TFL1 remain unclear. Here, we took an unbiased approach to probe the importance of residues throughout FT protein, testing the effects of hundreds of mutations in vivo. FT is surprisingly robust to a wide range of mutations, even in highly conserved residues. However, specific mutations in at least four different residues are sufficient to convert FT into a complete TFL1 mimic, even when expressed from TFL1 regulatory sequences. Modeling the effects of these mutations on the surface charge of FT protein suggests that the affected residues regulate the docking of an unknown ligand. These residues do not seem to alter the interaction with FD or 14-3-3 proteins, known FT interactors. Potential candidates for differential mediators of FT and TFL1 activities belong to the TCP (for TEOSINTE BRANCHED1, CYCLOIDEA, PCF) family of transcription factors.

Figures

Figure 1.
Figure 1.
Random Mutagenesis Assay Strategy. Around 36,000 randomly mutated FT cDNA copies were generated by PCR. These were cloned en masse into a plant transformation vector for overexpression in wild-type plants as YFP fusions. Screening of 3320 primary transformants in short days identified 52 late-flowering, YFP-positive lines with reduced FT activity, representing 33 unique mutants. The progeny of these 33 lines was reevaluated in long days to distinguish mutations that inactivated FT (14 lines) from those that introduced neomorphic activity and delayed rather than accelerated flowering when overexpressed (19 lines). See Supplemental Figure 3 for fluorescence levels.
Figure 2.
Figure 2.
Flowering Times of T1 and T2 Lines. (A) Flowering times of T1 transformants and controls in short days. Light green and pink indicate distribution of positive and negative controls. The two cohorts on the right are a subset of the 35S:mFT-YFP population shown in the middle. Note that many plants with barely detectable fluorescence also flowered very early but were not considered further (see Figure 1 and Supplemental Figure 3). (B) Flowering time ranges of selected T2 lines in long days. Ochre indicates range between negative control and ft-10. Red type indicates residues subsequently investigated in detail. Lines in dark gray are statistically significantly later than ft-10 in a two-tailed Student’s t test with Bonferroni correction. Error bars indicate sd. See also Supplemental Table 1.
Figure 3.
Figure 3.
Intramolecular Hydrogen Bonds. Effects of Tyr-85 mutations are shown; wild-type FT is at the far left, and wild-type TFL1 is at the far right. Hydrogen bonds are shown as dashed lines. Hot pink indicates FT residues, cyan TFL1 residues, and green other substitutions.
Figure 4.
Figure 4.
Predicted Effects of Point Mutations on Surface Electrostatic Potential of FT. Residues tested in each structure are highlighted in yellow and the external loop and FT sequence signature from segment 4C (Ahn et al., 2006) in purple in the ribbon representation on the far left. The ligand binding pocket is indicated by an asterisk where visible. TFL1 is shown for comparison on the right. FT variants with FT-like activity are labeled red, those with TFL1-like activity blue, and reduced- or loss-of-function variants in black.
Figure 5.
Figure 5.
Effects of Side Chain Properties at Critical Positions on FT Activity. Flowering time ranges of T1 transformants overexpressing FT variants and controls in short days. Far left, controls; negative control is empty vector with CaMV35S promoter. Middle, charge-dependent positions; right, charge-independent positions. T1 plants were grown in short days. The mutants indicated in ochre were not significantly earlier than 35S:TFL1 using two-tailed Student’s t test with Bonferroni correction. Error bars indicate ± sd.
Figure 6.
Figure 6.
FT Mutants with TFL1-Like Activity. (A) Comparison of inflorescence tips of plants overexpressing TFL1, FT, or FT mutants with TFL1-like activity. ft-10 tsf-1 is shown for comparison on the far right. Asterisks indicate leafy shoots typical for 35S:TFL1 plants (Ratcliffe et al., 1998). Arrow points to terminal flower in 35S:FT. (B) Complementation of tfl1-1 mutant with constructs using TFL1 genomic sequences. The bottom panel shows top views of inflorescences.
Figure 7.
Figure 7.
Yeast Two-Hybrid Interaction Assays. Results from six independent replicates. The darker gray line is for comparison to GRF results. Symbols (‡) indicate significant differences between vertical comparisons using two-tailed Student’s t test with Bonferroni correction at P < 0.001. Error bars indicate ±sd.
Figure 8.
Figure 8.
Bimolecular Luciferase Complementation Assays. Young but fully expanded 4-week-old leaves were strictly chosen for infiltration. Experiments were repeated twice. Complete set of assay results is shown in Supplemental Figure 7.
Figure 9.
Figure 9.
Repulsion/Lock Models. FT binds a coactivator, while TFL1 may either merely exclude a coactivator (middle) or attract a corepressor (bottom). Blue color represents positively charged, while red and pink represent strongly and more weakly negatively charged regions. Gray arrows indicate electrostatic interactions. π-π represents stacking between aromatic groups. AR, aromatic side chain.

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