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From protein dynamics to physiology: New Insights into Phytochrome B mediated photomorphogenesis Christian Fleck Center for Biological Systems Analysis University of Freiburg, Germany Plant, Light, Action! All mechanisms throughout plant life cycle are regulated by light far-red red blue UV-A Plant photoreceptors photoreceptor genes evolutionary precursor photoresponses UV-B receptor — — hypocotyl growth flavonoid synthesis cryptochormes CRY1 CRY2 photolyases hypocotyl growth flavonoid synthesis flower induction phototropins PHOT1 PHOT2 bacterial light, oxygen, voltage receptors phototropism stomata opening chloroplast movement bacterial two-component histidine kinases hypocotyl growth flower induction flavonoid synthesis root growth shade avoidance greening etc. phytochromes PHYB PHYC PHYD PHYE PHYA Phytochrome characteristics • Dimeric protein of about 125kDa • Two reversibly photointerconverting forms: • Phytochrome B: – – – – Abundant in red light (660nm) Pfr is light stable Low Fluence Response in red light Early, transient, nuclear speckles late, stable, nuclear speckles – Mediated actions: • • • • • Growth of hypocotyl length Magnitude of cotyledon area Regulation of chlorophyll synthesis Induction of flowering Shade avoidance 5 weeks old A.thaliana (wt) Phytochrome characteristics • Overlapping absorption spectra k1 Pr Pfr k2 ⇒ wavelength dependent photoequilibrium • Adjustable parameters: – spectral composition of incident light – light intensity (photon flux) – duration of irradiation protein dynamics can be changed by switching on/off the light Developmental programs Alternative developmental programs during early plant growth: light-dependent de-etiolation Skotomorphogenesis Photomorphogenesis darkness white light How do the phytochromes influence hypocotyl growth? • How is the phytochrome dynamics changed by light? • How do hypocotyls grow? • How can we connect the mesoscopic protein dynamics with the macroscopic hypocotyl growth? Time resolved hypocotyl growth Darkness Continuous red light phyB-9 Col WT phyB-GFP No active phytochromes present Active phytochromes present The logistic growth function • Population or organ growth (Verhulst, 1837) – Growth rate is proportional to existing population and available resources • Small population: exponential growth; growth rate α>0 • Large population: saturated/inhibited growth due to environmental factors; inhibition coefficient βL>0 – Growth is given by Experimental investigations of growth patterns • Sachs (1874): ”large period of growth”: – growth velocity increases, reaches a maximum, growth velocity decreases • Backman (1931): S-shaped growth curve is called “growth cycle”, integration of the “large period” • BUT: symmetry is not given – the period of increasing velocity is of greater amplitude than the period of decreasing velocity • Growth is characterized by: – asymmetric S-curve – asymmetric bell-shape of velocity function describes the “large period” – decrease of velocity takes longer than increase -> growth rate is not constant over time The biological growth function Biological Growth Environmental time rate limitation Variation of γ γ determines the asymmetry of L and dL/dt Variation of α/γ α/γ determines initial growth profile Fit dark grown data The underlying protein pool dynamics Speckle formation phyB-GFP dark phyB-GFP 24h red Time resolved experiments for the protein dynamics How does active phytochrome come into play? A. Hussong Modified growth rate Multi-experiment fit FRAP Dark reversion phyB-GFP phyB-YFP Hypocotyl growth Fluence rate response Col WT A. Hussong, S.Kircher Pfr degradation Col WT Prediction: fluence rate response of a phyB overexpressing hypocotyl phyB-GFP Sensitivities: Effect of parameter variation on hypocotyl length k3 k1 kdr k2 k4 kdfr kr k5 kS The importance of the expression level Wagner et al. Plant Cell (1991) WT OX-R OX-A Khanna et al. Plant Cell (2007) Leivar et al. Plant Cell (2008) Al-Sady et al. PNAS (2008) WT OX-R OX-A phyB-OX leads to hypersensitivity PIFs regulate hypocotyl growth by modulating phyB levels • Expression strength (phyB level) is determined on protein level • Hypocotyl growth is determined on organ level What is functional relation between hypocotyl length and phyB level? Hypocotyl growth and phyB expression level • Growth function for light grown seedlings: • Pool dynamics is quite fast, i.e., steady states are reached quickly in comparison to hypocotyl growth ⇒ • Analytical solution for hypocotyl L can be derived: determines expression level for t>>tc, i.e., if hypocotyl growth has reached steady state for t<tc Functional and quantitative relation between expression level and hypocotyl length Khanna et al., Plant Cell (2007) Leivar et al., Plant Cell (2008) Al-Sady et al., PNAS (2008) A. Hussong (unpublished data) Conclusions • Quantitative understanding of phytochrome B dynamics • Phenomenological model captures many features of phyB mediated photomorphogenesis • Physiology is most sensitive to changes in photoreceptor expression level • Excellent quantitative agreement between mesoscopic protein dynamics and macroscopic physiology Outlook • Wavelength dependence of the phytochrome dynamics • Phytochromes form dimers: how does this change the overall dynamics and when is this important? • PIF - PHYB interaction: phyB degrades PIF3, but there is also a PIF3 mediated phyB degradation. How does this double negative feedback work? • PHYB abundance is circadian clock regulated. How is this achieved and how does light feed into the clock? Acknowledgements Institute of Physics Center for Systems Biology Faculty of Biology Andrea Hussong Julia Rausenberger Stefan Kircher Eberhard Schäfer Jens Timmer