from protein dynamics to physiology: new insights into phytochrome b mediated photomorphogenesis...
TRANSCRIPT
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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
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Plant, Light, Action!
All mechanisms throughout plant life cycle are regulated by light
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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
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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)
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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
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Developmental programs
Alternative developmental programs during early plant growth: light-dependent de-etiolation
Skotomorphogenesis
Photomorphogenesis
darkness white light
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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?
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Time resolved hypocotyl growth
No active phytochromes present
Darkness
phyB-9Col WTphyB-GFP
Continuous red light
Active phytochromes present
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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
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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
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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
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The underlying protein pool dynamics
dark
phyB-GFP
24h red
Speckle formation
phyB-GFP
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Time resolved experiments for the protein dynamics
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How does active phytochrome come into play?
A. Hussong
Modified growth rate
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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
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Prediction: fluence rate response of a phyB over-expressing hypocotyl
phyB-GFP
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Sensitivities: Effect of parameter variation on hypocotyl length
k5
k2
k4
kS
k3
kr
k1 kdfrkdr
kin
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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?
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• 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
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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)
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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
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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?
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Acknowledgements
Faculty of BiologyInstitute of PhysicsCenter for Systems Biology
Andrea Hussong
Eberhard Schäfer
Stefan Kircher
Julia Rausenberger
Jens Timmer