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From protein dynamics to physiology: New Insights into Phytochrome B mediated photomorphogenesis
Christian FleckCenter for Biological Systems
AnalysisUniversity of Freiburg, Germany
Plant, Light, Action!
All mechanisms throughout plant life cycle are regulated by light
Plant photoreceptors
hypocotyl growthflower induction
flavonoid synthesisroot growth
shade avoidancegreening
etc.
photoreceptor
phytochromes
phototropins
cryptochormes
UV-B receptor
evolutionaryprecursor
—
bacterial two-component
histidinekinases
bacterial light,oxygen, voltage
receptors
photolyases
genes
CRY1CRY2
—
PHOT1PHOT2
PHYA
PHYBPHYCPHYDPHYE
blu
e
UV
-Are
dfa
r-re
d
photo-responses
hypocotyl growthflavonoid synthesis
phototropismstomata opening
chloroplast movement
hypocotyl growthflavonoid 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
• 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
• Overlapping absorption spectra Pr Pfr
k1
k2
⇒ wavelength dependent photoequilibrium
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
No active phytochromes present
Darkness
phyB-9Col WTphyB-GFP
Continuous red light
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
Growthrate
Environmentallimitation
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
phyB-GFP
Time resolved experiments for the protein dynamics
How does active phytochrome come into play?
A. Hussong
Modified growth rate
Multi-experiment fit
A. Hussong, S.Kircher
phyB-GFP
phyB-YFP
Col WT
Col WT
FRAP Dark reversion Pfr degradation
Hypocotyl growth Fluence rate response
Prediction: fluence rate response of a phyB over-expressing hypocotyl
phyB-GFP
Sensitivities: Effect of parameter variation on hypocotyl length
k5
k2
k4
kS
k3
kr
k1 kdfrkdr
kin
The importance of the expression level
WT OX-R OX-A 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?
• 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:
Hypocotyl growth and phyB expression level
for t<tc
for t>>tc, i.e., if hypocotyl growth has reached steady state
determines expression level
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
Faculty of BiologyInstitute of PhysicsCenter for Systems Biology
Andrea Hussong
Eberhard Schäfer
Stefan Kircher
Julia Rausenberger
Jens Timmer