An integrative model for phytochrome B mediated photomorphogenesis: from protein dynamics to physiology

Abstract

Background: Plants have evolved various sophisticated mechanisms to respond and adapt to changes of abiotic factors in their natural environment. Light is one of the most important abiotic environmental factors and it regulates plant growth and development throughout their entire life cycle. To monitor the intensity and spectral composition of the ambient light environment, plants have evolved multiple photoreceptors, including the red/far-red light-sensing phytochromes.

Methodology/principal findings: We have developed an integrative mathematical model that describes how phytochrome B (phyB), an essential receptor in Arabidopsis thaliana, controls growth. Our model is based on a multiscale approach and connects the mesoscopic intracellular phyB protein dynamics to the macroscopic growth phenotype. To establish reliable and relevant parameters for the model phyB regulated growth we measured: accumulation and degradation, dark reversion kinetics and the dynamic behavior of different nuclear phyB pools using in vivo spectroscopy, western blotting and Fluorescence Recovery After Photobleaching (FRAP) technique, respectively.

Conclusions/significance: The newly developed model predicts that the phyB-containing nuclear bodies (NBs) (i) serve as storage sites for phyB and (ii) control prolonged dark reversion kinetics as well as partial reversibility of phyB Pfr in extended darkness. The predictive power of this mathematical model is further validated by the fact that we are able to formalize a basic photobiological observation, namely that in light-grown seedlings hypocotyl length depends on the total amount of phyB. In addition, we demonstrate that our theoretical predictions are in excellent agreement with quantitative data concerning phyB levels and the corresponding hypocotyl lengths. Hence, we conclude that the integrative model suggested in this study captures the main features of phyB-mediated photomorphogenesis in Arabidopsis.

Figure 1. Reaction scheme of phyB dynamics.

The superscripts c, n and ns denote the pools of phyB in the cytosol, nucleus and the nuclear bodies, respectively. The inactive and active state of the phyB conformer is denoted by Pr and Pfr, respectively. All transition parameters are described in the text.

Figure 2. Dynamics of phyB nuclear body formation in Arabidopsis seedlings via FRAP analysis.

Selected imaging data of a representative FRAP experiment on nuclear bodies of phyB-YFP are shown. Etiolated seedlings were irradiated for 24h with red light prior to analysis in a confocal laser scanning microscope. Nuclear body distribution of the photoreceptor is shown directly before (A), immediately after (B), and 3 min after (C) photobleaching the NB marked by an arrow (scale bar = 10 µm). (D) Representative raw data of FRAP experiments showing the fluorescence intensity of the bleached NB (black), of a non-bleached reference NB (red), and of the background (green) over time.

Figure 3. Dynamics of phyB NB formation in Arabidopsis seedlings.

(A–E) Representative fluorescence microscopy pictures and corresponding DIC images of phyB-GFP expressing transgenic lines show photoreceptor localization of (A) dark grown and (B–E) red light irradiated seedlings (24 h, 30 µmol m⁻²s⁻¹). After red light irradiation, seedlings were either transferred to darkness for 6 h (C) or 24 h (D), respectively. Localization after red light irradiation, followed first by 5h darkness, then by a far-red light pulse (5 min, 20 µmol m⁻²s⁻¹) and one subsequent hour of darkness is shown in (E). Irradiation with far-red light served to convert Pfr to Pr (nu = nucleus, nuS = nuclear body; scale bar = 10 µm). A schematic representation of the light treatment is shown in (F).

Figure 4. Multi-experiment fit.

Experimental data (crosses with error-bars) and the corresponding multi-experiment fit (solid lines) of the phytochrome B dynamics of Col WT (black) and over-expressing lines (red). Error-bars indicate standard errors (SE), based on biological replicates. (A) Time course of FRAP experiments, see Figure 2. (B) Time course of dark reversion kinetics after a saturating red light pulse (5 min, 22 µmol m⁻²s⁻¹). Relative Pfr amount in ABO/A⁻ line was measured in a dual-wavelength ratio spectrophotometer . (C) Red light induced phyB degradation. Semi-quantitative quantification of phyB specific immunoblot signals of Col WT seedlings irradiated with continuous red light (3 µmol m⁻²s⁻¹) and normalized to dark value is shown. One exemplary blot is shown in Figure S1A. (D) Dark growth curve of seedlings. Single seedlings (Col WT, phyB-9 mutant, phyB-GFP-1) were imaged over six days in continuous darkness and the mean hypocotyl length is presented. (E) Fluence rate response curve. Relative hypocotyl length, normalized to dark-grown seedlings, of Col WT upon growth in different fluence rates of continuous red light for four days is shown. For all experiments, we established appropriate models, presented in File S1, and performed a simultaneous multi-experiment fit to the experimental data.

Figure 5. Predicted effect of relative parameter variation on relative hypocotyl length.

The parameters were changed from 10% to 1000% of their estimated value. The relative hypocotyl length was determined according to Eqn. (4) and is plotted on the linear scale in (A) and on the logarithmic scale in (B) and (C). (A) Dynamical protein parameters k₁ (black circle), k₂ (red square), k₅ (violet diamond), k_r (orange plus), and k_in (cyan star), whose relative variation have little effect on the physiological read out. (B) Variation of k₃ (black circle) or k₄ (red square), over-expression strength z of the photoreceptor abundance relative to P₀ ^Col (violet diamond), and Pfr-degradation rate k_dfr (orange plus) had stronger effect on the relative hypocotyl length. (C) Variations of the direct growth parameter α₀β⁻¹ (black circle), K (green diamond), and the over-expression strength z (red square).

Figure 6. Fluence rate dependency.

Submodels of phyB dynamics, describing only the photochemistry (A) or including the additional process of dark reversion (B). (C) The fluence rate response curves for Col WT (black solid lines) were estimated in a multi-experiment fit, whereas the fluence rate response curves of phyB-GFP-1 (red dashed lines) were predicted on the basis of the estimated parameters for different models of phyB dynamics, describing only photochemistry (A, square), including photochemistry and dark reversion kinetics (B, circle), or including the full protein dynamics of Figure 1 (no symbols). Error bars indicate standard error of biological replicates.

Figure 7. Dark reversion experiments in vitro and in planta.

Reaction scheme (A) assuming a single first order reaction or (B) assuming the entire biochemical pool dynamics of the integrative model depicted in Figure 1. (C) Dark reversion curves calculated with the estimated dark reversion rate k_r = 0.03 [min⁻¹] (see Table 1), for assuming a single first order reaction (blue dashed line) or for the entire model (red solid line). The inset shows the fraction of the single Pfr pools relative to Ptot for (B): Pfr^c (dashed line), Pfrⁿ (red diamonds), Pfr^ns (red crosses), sum of all (solid line).

Figure 8. Role of photoreceptor abundance on hypocotyl growth.

The experimental data, i.e., phyB levels and hypocotyl lengths, are taken from Khanna et al. (red), Leivar et al. (black), Al-Sady et al. (orange) , , , and were generated in the present work (transgenic over-expressing phyB-GFP-1 to 4 lines, green). The data were normalized to the phyB level and hypocotyl length of Col WT. Using the estimated parameters from Table 1, the simulated prediction (blue solid line) was generated according to Eqn. (6). The mean values and the standard errors (error-bars) are summarized in Table S1.