Phosphorylation of phytochrome B inhibits light-induced signaling via accelerated dark reversion in Arabidopsis

Abstract

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (λmax = 730 nm) and inactive Pr (λmax = 660 nm) forms in a red/far-red-dependent fashion and regulates, as molecular switch, many aspects of light-dependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein-protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB-yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyB(Ser86Asp) Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light-induced nuclear import and interaction of phyB(Ser86Asp)-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB-green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling.

Figure 1.

In Planta Phosphorylation and Expression Analysis of phyB-GFP. (A) Phytochrome B is phosphorylated in vivo. Wild-type (Col-0) or transgenic plants expressing the phyB-GFP fusion protein were grown in darkness (cD) or in cR light at 40 µmol m⁻² s⁻¹ fluence rate for 4 d on wet filter papers. Total protein extracts were prepared and treated (+) or not (−) with λ phosphatase (λPPase), and the different forms of phyB-GFP protein were separated by Zn-Phos-tag PAGE and visualized using antibody-specific for GFP. The lanes contain equal amounts of total protein as shown by the comparable levels of ACTIN. (B) Levels of the phyB-GFP, phyB^Ser86Ala-YFP, and phyB^Ser86Asp-YFP fusion proteins are identical in the selected transgenic phyB-9 lines. Transgenic phyB-9 seedlings were grown in darkness for 4 d on wet filter paper, and the expression levels of the various fusion proteins were determined by immunoblot analysis using anti-GFP antibody. The lanes contain equal amounts of total protein as shown by the comparable levels of tubulin.

Figure 2.

Phenotypic Characterization of Seedlings Expressing phyB^Ser86Ala-YFP and phyB^Ser86Asp-YFP. (A) Hypocotyl growth inhibition of phyB^Ser86Ala-YFP seedlings exhibits extreme hypersensitivity to R light. Nontransgenic wild-type (Col-0) (closed square), and phyB-9 (cross) as well as transgenic phyB-9 seedlings expressing the phyB-GFP (closed triangle), phyB^Ser86Asp-YFP (open circle), and phyB^Ser86Ala-YFP (open triangle) fusion proteins were grown for 4 d at 22°C in darkness or at the indicated fluence rates of cR light on wet filter paper. Hypocotyl length was determined using the MetaMorph image analysis software, and the fluence rate response is shown as relative hypocotyl length to dark-grown samples (n = 50). Error bars indicate se. (B) Cotyledon expansion is impaired in transgenic seedlings expressing the phospho-mimic phyB^Ser86Asp-YFP grown under low-intensity R light. Nontransgenic wild-type (Col-0; 1) and phyB-9 (2) as well as transgenic phyB-9 seedlings expressing the phyB-GFP (3), phyB^Ser86Ala-YFP (4), and phyB^Ser86Asp-YFP (5) fusion proteins were grown for 4 d on Murashige and Skoog medium without sugar at 1 µmol m⁻² s⁻¹ R light. The cotyledon surface area was measured using MetaMorph image analysis software (n = 40, error bars represent se). Asterisks indicate significant difference from the wild type as determined by Student’s t test (P < 0.001). (C) Transgenic seedlings expressing the phospho-mimic phyB^Ser86Asp-YFP display hyposensitivity to shade. Nontransgenic wild-type (1) and phyB-9 (2) as well as transgenic P35S:PHYB-GFP (3), P35S:PHYB^Ser86Ala-YFP (4), and P35S:PHYB^Ser86Asp-YFP (5) expressing phyB-9 seedlings were grown for 7 d in high R/FR or for 3 d in high R/FR followed by 4 d in low R/FR at 20°C. The experiment was performed under constant light conditions (PAR = 130 µmol m⁻² s⁻¹). Hypocotyl lengths shown were measured using ImageJ software (n = 31 to 40). Error bars indicate se.

Figure 3.

Phosphorylation of phyB Ser-86 Affects the Nuclear Translocation of the Photoreceptor. (A) Initial rate of light-induced accumulation of the phospho-mimic phyB^Ser86Asp-YFP fusion protein in the nucleus is reduced. phyB-9 seedlings expressing the phyB-GFP, phyB^Ser86Asp-YFP, or phyB^Ser86Ala-YFP fusion proteins were grown for 5 d in darkness and subsequently exposed for 2 h to the indicated fluences of R light. Quantification of the nuclear accumulation of the various fusion proteins was performed as described (Pfeiffer et al., 2012). Nuclear fluorescence normalized to dark levels is shown (n = 50). Error bars indicate se. (B) PhyB^Ser86Asp-YFP photobodies are detected only after irradiation with high intensity R light. phyB-9 seedlings expressing the phyB-GFP, phyB^Ser86Asp-YFP, and phyB^Ser86Ala-YFP fusion proteins were grown for 5 d in darkness and subsequently exposed for 6 h to the indicated fluences of R light. Representative pictures are shown (n = 50). Bars = 10 µm.

Figure 4.

Phosphorylation of Ser-86 Affects Photobody Dissociation but Not the Abundance of phyB. (A) phyB^Ser86Ala-YFP photobodies exhibit increased stability under FR light irradiation. Transgenic phyB-9 seedlings expressing the phyB-GFP (S86S), phyB^Ser86Ala-YFP (S86A), and phyB^Ser86Asp-YFP (S86D) fusion proteins were grown for 5 d in darkness and then exposed to 24 h of R light (22 µmol m⁻² s⁻¹) followed by irradiation with FR light for 5 min (20 µmol m⁻² s⁻¹). phyB-associated photobodies were monitored as described by Pfeiffer et al. (2012). The experiments were repeated three times (n = 50), and representative pictures are shown. Bars = 10 µm. (B) Substitutions of Ser-86 do not alter cR-induced degradation of the various phyB fusion proteins. Transgenic phyB-9 seedlings were grown in darkness for 4 d on wet filter paper and exposed to cR (0.1 µmol m⁻² s⁻¹) as indicated. The levels of the various fusion proteins were determined by immunoblot analysis using anti-GFP antibody. The lanes contain equal amounts of total protein as shown by the comparable levels of actin.

Figure 5.

Phosphorylation of Ser-86 Regulates the Dark Reversion of phyB. (A) Under nonsaturating light conditions, R/FR reversible binding of PIF3 to the phospho-mimic phyB^Ser86Asp-YFP is reduced when compared with phyB-GFP in vitro. Liquid cultures of yeast cells expressing the AD-PIF3 and PHYB(N651)-BD (black bars) or PHYB(N651)^Ser86Ala-BD (gray bars) or PHYB(N651)^Ser86Asp-BD (white bars) fusion proteins were grown overnight in darkness in the presence of exogenously supplied chromophore. The overnight cultures were halved and irradiated with the indicated fluence rate of R light for 10 min and then returned again to darkness. The β-galactosidase enzyme activity was measured 4 h after the light pulse. Error bars indicate se of three independent experiments. (B) Substitution of Ser-86 to Ala or Asp does not alter photoconversion of recombinant phyB photoreceptors in vitro. Liquid yeast cultures supplemented with exogenously added chromophore and expressing the phyB-YFP, phyB^Ser86Ala-YFP, and phyB^Ser86Asp-YFP photoreceptors were grown overnight in darkness. Photoconversion of the Pfr conformer of the various fusion proteins to Pr was measured as described by Kunkel et al. (1993). The fluence rate of the reverting FR light (20 µmol m⁻² s⁻¹) and the amount of Pfr, as the relative amount of Pfr (%) is shown. Pfr (%) at photoequilibrium = 100%. Error bars indicate se of three independent experiments. (C) Dark reversion of the Pfr of the phospho-mimic phyB^Ser86Asp-YFP fusion protein is accelerated compared with phyB-GFP or phyB^Ser86Ala-YFP in vitro. Liquid yeast cultures expressing the recombinant fusion proteins were grown overnight and reconstituted with phycocyanobilin. Samples of identical density were prepared, irradiated for 5 min with saturating R light, and transferred into darkness. Dark reversion of phyB-YFP, phyB^Ser86Asp-YFP, and phyB^Ser86Ala-YFP Pfr was measured as described (Kunkel et al., 1995). The relative amount of Pfr (%) to the total amount of phytochrome (Ptot) is shown. Error bars indicate se of three independent experiments. (D) The phospho-mimic phyB^Ser86Asp-YFP Pfr also rapidly dark reverts in planta. Eighty milligrams of 4-d-old etiolated Arabidopsis seedlings were irradiated for 5 min with saturating R light and incubated afterwards in darkness. The total amounts of phyB-GFP (closed triangle), phyB^Ser86Asp-YFP (closed diamond), and phyB^Ser86Ala-YFP (closed circle) and dark reversion of phyB-GFP (open triangle), phyB^Ser86Asp-YFP (open diamond), and phyB^Ser86Ala-YFP (open circle) Pfr were measured as described (Eichenberg et al., 1999). The relative amount of Pfr (%) to the total amount of phytochrome (Ptot) is shown. Error bars indicate se of three independent experiments.