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