Transcript PowerPoint-Präsentation - Biological Background I

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
far-red
red
blue UV-A
Plant photoreceptors
photoreceptor
genes
evolutionary
precursor
photoresponses
UV-B receptor
—
—
hypocotyl growth
flavonoid synthesis
cryptochormes
CRY1
CRY2
photolyases
hypocotyl growth
flavonoid synthesis
flower induction
phototropins
PHOT1
PHOT2
bacterial light,
oxygen, voltage
receptors
phototropism
stomata opening
chloroplast movement
bacterial
two-component
histidine
kinases
hypocotyl growth
flower induction
flavonoid synthesis
root growth
shade avoidance
greening
etc.
phytochromes
PHYB
PHYC
PHYD
PHYE
PHYA
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
• Overlapping absorption spectra
k1
Pr
Pfr
k2
⇒ 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
No active phytochromes present
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 Growth Environmental
time
rate
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
Speckle formation
phyB-GFP
dark
phyB-GFP
24h red
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
phyB-GFP
phyB-YFP
Hypocotyl growth
Fluence rate response
Col WT
A. Hussong, S.Kircher
Pfr degradation
Col WT
Prediction: fluence rate response of a phyB overexpressing hypocotyl
phyB-GFP
Sensitivities: Effect of parameter variation on hypocotyl
length
k3
k1
kdr
k2
k4
kdfr
kr
k5
kS
The importance of the expression level
Wagner et al.
Plant Cell (1991)
WT OX-R OX-A
Khanna et al.
Plant Cell (2007)
Leivar et al.
Plant Cell (2008)
Al-Sady et al.
PNAS (2008)
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?
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)
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
Institute of Physics
Center for Systems Biology
Faculty of Biology
Andrea Hussong
Julia Rausenberger
Stefan Kircher
Eberhard Schäfer
Jens Timmer