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

Plant photoreceptors photoreceptor phytochromes phototropins hypocotyl growth flower induction flavonoid synthesis root growth shade avoidance greening etc. photoreceptor phytochromes phototropins cryptochormes UV-B receptor evolutionary precursor — bacterial two-component histidine kinases bacterial light, oxygen, voltage receptors photolyases genes CRY1 CRY2 PHOT1 PHOT2 PHYA PHYB PHYC PHYD PHYE blue UV-A red far-red photo- responses hypocotyl growth flavonoid synthesis phototropism stomata opening chloroplast movement flavonoid synthesis flower induction

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 Pr Pfr k1 k2 Overlapping absorption spectra ⇒ 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 Active phytochromes present No 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 time Growth rate Environmental 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 dark phyB-GFP 24h red Speckle formation

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 Pfr degradation phyB-GFP Col WT phyB-YFP Hypocotyl growth Fluence rate response Col WT A. Hussong, S.Kircher

Prediction: fluence rate response of a phyB over-expressing hypocotyl phyB-GFP

Sensitivities: Effect of parameter variation on hypocotyl length k3 k4 k1 kdr kdfr k2 kr kin kS k5

The importance of the expression level WT OX-R OX-A Wagner et al. Plant Cell (1991) ⇒ phyB-OX leads to hypersensitivity Khanna et al. Plant Cell (2007) Leivar et al. Plant Cell (2008) ⇒ PIFs regulate hypocotyl growth by modulating phyB levels Al-Sady et al. PNAS (2008) 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) Al-Sady et al., PNAS (2008) Leivar et al., Plant Cell (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