Volume 7, Issue 2, Pages 377-387 (February 2014) The Arabidopsis Floral Repressor BFT Delays Flowering by Competing with FT for FD Binding under High Salinity  Jae Yong Ryu, Hyo-Jun Lee, Pil Joon Seo, Jae-Hoon Jung, Ji Hoon Ahn, Chung-Mo Park  Molecular Plant  Volume 7, Issue 2, Pages 377-387 (February 2014) DOI: 10.1093/mp/sst114 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 fd-2 Flowering Is Insensitive to High Salinity. (A) Flowering phenotype of fd-2 mutant under high salinity. Six-week-old plants grown in soil containing 150mM NaCl were photographed (left panel). The rosette leaf numbers of approximately 30 plants were counted and averaged for each plant genotype (right panel). Statistical significance of the countings was determined by the Student’s t-test (* P < 0.01). Bars indicate standard error of the mean. (B) ft-10 flowering under high salinity. Plants were grown in soil containing 150mM NaCl until flowering. Countings of approximately 30 plants were averaged and statistically treated for each plant genotype (t-test, * P < 0.01). (C) Percent changes of flowering time under high salinity. Five calculations of the percent changes, each consisting of 20 plants, were averaged and statistically treated (t-test, * P < 0.01). Molecular Plant 2014 7, 377-387DOI: (10.1093/mp/sst114) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 BFT Is Linked with FD in Flowering Time Control. Plants were grown in soil containing 150mM NaCl under LD until flowering. The rosette leaf numbers of approximately 30 plants were averaged and statistically treated for each plant genotype (t-test, * P < 0.01). Bars indicate standard error of the mean. (A) Flowering time of plants that overexpress FT or BFT gene driven by the CaMV 35S promoter. The 35S:FT x 35S:BFT plants were also included in the countings. (B) Flowering time of ft-10 mutant and 35S:BFT transgenic plants. The 35S:BFT x ft-10 plants were also included in the countings. (C) Flowering time of fd-2 mutant and 35S:BFT transgenic plants. The 35S:BFT x fd-2 plants were also included in the countings. (D) Flowering time of bft-2 and fd-2 mutants. The bft-2 x fd-2 double mutant was also included in the countings. Molecular Plant 2014 7, 377-387DOI: (10.1093/mp/sst114) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 BFT Interacts with FD in the Nucleus. (A) Interaction of BFT with FD in yeast cells. Cell growth on selective media without Leu, Trp, His, and Ade (-QD) represents positive interactions (upper panel). Three measurements of β-Gal activity were averaged and statistically treated (bottom panel). Different letters represent significant difference at P < 0.05 (one-way ANOVA with Fisher’s post hoc test). (B) BiFC assays. The nYFP–FD and cYFP–BFT constructs were transiently co-expressed in Arabidopsis protoplasts and visualized by differential interference contrast microscopy (DIC) and fluorescence microscopy. Bars = 20 µm. Molecular Plant 2014 7, 377-387DOI: (10.1093/mp/sst114) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Interaction of BFT with FD Is Mediated by the C-Terminal Domain of FD. The BFT–FD interactions were analyzed in yeast cells. Yeast two-hybrid assays were performed as described in Figure 3A. T282A represents a mutant FD form, in which T-282 was mutated to A. The T282A mutation has been shown to disrupt FT–FD interaction (Abe et al., 2005). Molecular Plant 2014 7, 377-387DOI: (10.1093/mp/sst114) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 BFT Inhibits FD Function by Competing with FT. (A) Expression constructs used in yeast three-hybrid assays. (B) Effects of BFT on FT–FD interaction. In the yeast three-hybrid assays, BFT gene was expressed under the control of the Met-repressible promoter (pMET25–BFT). Therefore, the BFT gene is expressed on selective media without Leu, Trp, His, and Met (–LWHM) but is not expressed on selective media without Leu, Trp, and His (–LWH). AD, activation domain; BD, DNA-binding domain. Five measurements were averaged and statistically treated (t-test, * P < 0.01). Bars indicate standard error of the mean. (C) In vitro pull-down assays. Recombinant MBP–BFT and HIS–FT fusion proteins were prepared in Escherichia coli cells. FD polypeptides were prepared by in vitro translation in the presence of 35S-methionine. Input represents 5% of the FD polypeptides used in each assay. MBP protein was also included as control in the assays. Part of Coomassie blue-stained gel is displayed at the bottom. kDa, kilodalton. (D) Effects of FT and BFT on the transcriptional activation activity of FD. GAL4 transient expression assays were performed as previously described (Miura et al., 2007; Yang et al., 2011). ARF5M is transcriptional activator control. Biological triplicates were averaged and statistically treated. Different letters represent significant difference at P < 0.05 (one-way ANOVA with Fisher’s post hoc test). (E) Competition of BFT with FT for binding to FD. Under high-salt conditions, BFT competitively interacts with FD. Molecular Plant 2014 7, 377-387DOI: (10.1093/mp/sst114) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 Salt Induction of BFT Gene Is Independent of FT. (A) Diurnal expression patterns of BFT gene under high salinity. Plants grown on MS-agar plates for 2 weeks were further incubated for 24h in MS-liquid cultures containing 150mM NaCl. Salt treatments were initiated at ZT0. Whole plants were harvested at various ZT points for the extraction of total RNA. Transcript levels were determined by RT–qPCR. Biological triplicates were averaged and statistically treated (t-test, * P < 0.01). Bars indicate standard error of the mean. (B) Absolute quantitation of FT and BFT transcripts under high salinity. Col-0 plants grown on MS-agar plates for 7 d were exposed to high salt at ZT0. Whole plants were harvested at ZT8. The set of standards contains 10-fold serial dilutions of BFT or FT transcripts. The regression line from the dilution curve was used to determine the concentrations of BFT and FT transcripts. Molecular Plant 2014 7, 377-387DOI: (10.1093/mp/sst114) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 Working Model of BFT Function. Under salt stress, BFT gene is induced, which would correlate with the high-level accumulation of the BFT protein. BFT interrupts the FT–FD interaction, resulting in delayed flowering. Salt-mediated FT transcription is independent of FD. It seems that the salt -mediated suppression of the FT gene also contributes to the delayed flowering under high salinity. Molecular Plant 2014 7, 377-387DOI: (10.1093/mp/sst114) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions