From protein dynamics to physiology: New Insights into Phytochrome B mediated photomorphogenesis

1. From protein dynamics to physiology: New Insights into Phytochrome B mediated photomorphogenesis Christian Fleck Center for Biological Systems Analysis University of Freiburg, Germany

2. Plant, Light, Action! All mechanisms throughout plant life cycle are regulated by light

3. Plant photoreceptors evolutionary precursor photo-responses photoreceptor genes — UV-B receptor — hypocotyl growthflavonoid synthesis CRY1 CRY2 photolyases cryptochormes hypocotyl growth flavonoid synthesisflower induction blue UV-A bacterial light, oxygen, voltage receptors phototropism stomata openingchloroplast movement PHOT1 PHOT2 phototropins hypocotyl growth flower induction flavonoid synthesisroot growthshade avoidancegreening etc. bacterial two-component histidine kinases PHYB PHYC PHYD PHYE red phytochromes far-red PHYA

4. 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 Phytochrome characteristics 5 weeks old A.thaliana (wt) • Dimeric protein of about 125kDa • Two reversibly photointerconverting forms:

5. k1 Phytochrome characteristics Pr Pfr 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

6. Developmental programs darkness white light Alternative developmental programs during early plant growth: light-dependent de-etiolation Skotomorphogenesis Photomorphogenesis

7. 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?

8. phyB-9 Col WT phyB-GFP Time resolved hypocotyl growth Darkness Continuous red light Active phytochromes present No active phytochromes present

9. 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

10. 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

11. Variation of γ Fit dark grown data Variation of α/γ The biological growth function Biological time Growth rate Environmentallimitation ⇒ γ determines the asymmetry of L and dL/dt ⇒ α/γ determines initial growth profile

12. Speckle formation The underlying protein pool dynamics phyB-GFP dark phyB-GFP 24h red

13. Time resolved experiments for the protein dynamics

14. How does active phytochrome come into play? A. Hussong Modified growth rate

15. 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

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

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

18. Wagner et al.Plant Cell (1991) Khanna et al.Plant Cell (2007) Leivar et al.Plant Cell (2008) Al-Sady et al.PNAS (2008) The importance of the expression level WT OX-R OX-A 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?

19. Hypocotyl growth and phyB expression level determines 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: for t>>tc, i.e., if hypocotyl growth has reached steady state for t<tc

20. Functional and quantitative relation between expression level and hypocotyl length Khanna et al., Plant Cell (2007) Al-Sady et al., PNAS (2008) A. Hussong (unpublished data) Leivar et al., Plant Cell (2008)

21. 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

22. 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?

23. Eberhard Schäfer Acknowledgements Andrea Hussong Stefan Kircher Institute of Physics Center for Systems Biology Faculty of Biology Julia Rausenberger Jens Timmer