Arabidopsis florigen FT binds to diurnally oscillating phospholipids that accelerate flowering

Abstract

Arabidopsis FT protein is a component of florigen, which transmits photoperiodic flowering signals from leaf companion cells to the shoot apex. Here, we show that FT specifically binds phosphatidylcholine (PC) in vitro. A transgenic approach to increase PC levels in vivo in the shoot meristem accelerates flowering whereas reduced PC levels delay flowering, demonstrating that PC levels are correlated with flowering time. The early flowering is related to FT activity, because expression of FT-effector genes is increased in these plants. Simultaneous increase of FT and PC in the shoot apical meristem further stimulates flowering, whereas a loss of FT function leads to an attenuation of the effect of increased PC. Specific molecular species of PC oscillate diurnally, and night-dominant species are not the preferred ligands of FT. Elevating night-dominant species during the day delays flowering. We suggest that FT binds to diurnally changing molecular species of PC to promote flowering.

Figure 1. FT protein binding to phosphatidylcholine (PC).

(a) Membrane-lipid overlay assay with different glycerolipids. A specific binding to PC was observed. Purified, soluble His-tagged FT protein was incubated with a membrane containing different glycerolipid spots as indicated. Lipid-protein binding was visualized with a peroxidase-coupled anti-His antibody and chemiluminescence. A representative membrane of three biological replicates is shown. (b) Comparison of FT binding to PC and PE. Different amounts of di18:1-PC or di18:1-PE were spotted onto the membrane and probed with His-tagged FT protein. (c) FT binding to PC and PE purified from Arabidopsis. Total lipid was extracted from rosette leaves grown in LD, and PC and PE were purified and quantified for spotting on the membrane. (d) Liposome association assay to confirm specific binding of FT to PC. Purified soluble His-tagged FT protein was incubated with liposomes containing different PC:PE ratios. After collecting the liposomes by centrifugation, the portion of FT bound to the liposomes was detected by western blotting using anti-His antibodies. A representative blot of three biological replicates is shown. The left lane shows the position of the protein size marker (25 kDa).

Figure 2. Alterations in PC levels affect flowering time.

(a–c) Transgenic plant lines that harbour pOP::amiPECT1, 35S::GRLhG4 showed constitutively reduced PECT1 expression (a), increased PC levels (b) and early flowering (c). (d) pFD::amiPECT1 transgenic plants showed a significantly early flowering phenotype. (e) pFD::PECT1 transgenic plants showed late flowering, opposite to the effect observed in (d). Data were averaged by three biological replicates (a,b) and 16 plants (c–e) with standard deviation as error bars. Asterisks show statistical significance (P-value <0.01, Student’s t-test).

Figure 3. Impact of alterations in PC level on the expression of known flowering time control genes.

(a,b) Increased expression of SOC1 (a) and AP1 (b) in pFD::amiPECT1 plants of 7, 9 and 14 days after germination under LD condition. In each time point, seven independent transgenic lines and wild type as a control (indicated as 1–7 and W) were examined with three biological replicates with standard deviation as error bars. (c) Stable expression of pFD::amiPECT1 in ft-10 tsf-1 plants attenuated the early flowering effect of pFD::amiPECT1 observed in the wild-type background. (d) Stable expression of pFD::amiPECT1 in an FT-overexpressing line (pGAS1::FT, ft-10 tsf-1) further accelerated the flowering time. Data from 16 transgenic plants were averaged with standard deviation as error bars. Y axes in (c,d) are the number of leaves at flowering time relative to the control plants that do not harbor the pFD::amiPECT1 transgene.

Figure 4. Diurnal oscillation of PC molecular species and selective binding of FT to PC species dominant at daytime.

(a–d) Lipidomic profiling of PC and PE species under SD (a and c, respectively) or LD (b and d, respectively). Seedlings were germinated and grown for 2 weeks under SD, shifted to either LD or SD and time-course dependent harvesting started 24 h after shifting the plants. Data show averages and standard deviation of four biological replicates. Grey boxes indicate the dark period. (e) Membrane-lipid overlay assay of different PC molecular species with His-tagged FT protein. (f) Weaker binding of FT to di18:3-PC at different concentration of lipids. The photos of the membranes after binding in (e) and (f) are representative of three replicates. (g) Leaf number at flowering time of CS8035 plants under LD. Data were averaged by 16 plants with standard deviation as error bars.