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 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

4. 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)

5. Phytochrome characteristicsPr
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

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

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. Time resolved hypocotyl growth
Darkness
Continuous red light
phyB-9
Col WT
phyB-GFP
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. 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

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

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 lengthk3 k4 k1
kdr
kdfr k2 kr
kin kS k5

18. 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?

19. 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

20. 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)

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