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Chlorophyll fluorescence lecture

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Berkley Walker

on 13 November 2015

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Transcript of Chlorophyll fluorescence lecture

To infer photosynthetic biochemistry through curve fitting
To estimate leaf energetics

Why does chlorophyll fluoresce?
Absorption
Emission
Absorption
Emission
Heat (Fast)
E
0
E
1
E
2
This explains why blue and red are absorbed, but chlorophyll fluorescence is only red
The observation
Spectra of emission
Cerovic et al, Plant, Cell & Environment (25) 1663-1676, 2002.

Absorbed in two peaks, fluoresces at one
Fluorescent peak shifted from absorption
Fluorescence (=emission) much smaller than absorption (~2%)
The color of fluorescence
But why is it shifted?
Stoke's Shift
Longer wavelength, less energy
Why does this shift occur?
Slight shift to longer wavelength with emission
Not intuitive, have to know equations, analogies help
Molecular mechanisms of PS - Robert Blankenship (278-305)
Equations that explain observations at the atomic scale
Word on Quantum mechanics
http://usersguidetotheuniverse.com/
This is an simplification showing only electronic energy states
Light is absorbed when the frequency matches the energy gap
Really an energy landscape (nD) that depends on bond length
Vibrational states within electronic states define energy gaps
http://www.life.illinois.edu/govindjee/biochem494/Abs.html
Electrons start in the ground vibrational state
Can be excited from 0 to 0
... or from 0 to 1...
...or to 2 or 3.
Absorbed photons can have a spread of frequencies
(this is why peaks are broad)
Like a ball rolling down the energy curve, electrons rapidly relax to the ground vibrational state
Emissions (fluorescence) can then come from the decay to any of the ground electronic vibrational states
usually the decay is to an excited vibrational state of the ground electronic state
These emissions have less energy, therefore, are a longer wavelength
Stoke's Shift explained
Why do I fluoresce?
How can my fluorescence be used to probe PSII?
What can this knowledge teach me about photosynthesis?
Fluorescence and PSII
Heat
Kautsky and Hirsch
Decline inversely related to CO fixation
Fluorescence to energy fates
Neil Baker, Ann. Rev. Plant Biology (59) 89-113, 2008.


2
CO fixation
2
Absorption
Heat (Fast)
E
2
E
1
E
0
Fluorescence
What's wrong with this picture for chlorophyll?
Fluorescence is one of three major quenchers of excitation energy
Heat
Photochemistry
PSII quenches fluorescence (takes energy from chlorophyll) when the Q site is oxidized
Fluorescence and PSII
When it is oxidized it is ready to receive an electron and is "open"
When it is reduced it can't receive an electron and it is "closed"
A
PSII quenching
PSII
Photosystem II can be closed due to downstream backup or damage
Photosystem I is an excellent quencher, so all the fluorescence observed comes from PSII
Decoding quenching
Fluorescence
Heat
Photochemistry
How can we use fluorescence to understand heat dissipation and photochemistry?
DCMU
Heat (nonphotochemical, NPQ) quenching changes
photochemical
non-photochemical (heat)
DCMU to resolve quenching
Only works in algae
Hard to probe a living system when you kill it
Saturation pulses to decode quenching
Heat (NPQ)
Photochemistry
Use rapid, saturating flashes of light to fully reduce Q and resolve photochemical quenching and NPQ
Fluorescence
A
One way to maximally reduce Q ...
A
Requires "pulse-amplitude modulated fluorometry" (PAM) to separate background fluorescence from chlorophyll fluorescence
Fluorescence quenching analysis using modulated fluorescence
DOB
In the dark Q maximally oxidized and PSII is maximally open
Fluorescence is at a minimum because there is little NPQ and PSII quenches efficiently
Weak non-actinic measuring light
A
Fluorescence yield when PSII is fully closed
Actinic light turned on
PSII close
NPQ increases
Far-red opens PSII
Fluorescence in action
Wasteful (but protective) heat dissipation
Light use efficiencies
Maximum quantum efficiency
Measured on dark-adapted leaves
Healthy leaves ~0.82
Decreases with stress and damage
Maximum quantum efficiency
Operating quantum efficiency
Photon to electron efficiency
What can this be used for?
Linear electron flux
Linear electron flux
Most energy in the light goes to photosynthesis
Two electrons reduce one NADPH
Two NADPH are needed to reduce one CO
Can we estimate gas exchange from fluorescence?
2
Yes, for C4 at least...
What about C3?
Have to account for NADPH demand of photorespiration
Can be done with leaf models of photosynthesis
Requires some assumptions, but is possible
Can be used to estimate mesophyll conductance
Poll Time!
Getting Good measurements
Who will be/has been using a LiCor 6400 to do fluorescence?
Read the manual
Refer to Licor Fluorometer manual for more information.
Berkley's Checklist
Optimal measuring intensity
The square flash calibration
Who wants to know how to properly use the 6400 for measurements?
Knowing is half the battle!
Zero fluorescence signal
Optimal measuring intensity
Square flash calibration
Optimal saturation intensity
Saturation with multi-phase flash
Saturating flash
What is saturating?
A
Saturation flashes need to fully reduce the Q site
Optimal measuring intensity
Measuring pulses have to be bright enough to create a good fluorescence signal, but not bright enough to drive photosynthesis
Too strong of measuring light in the dark will decrease maximum quantum efficiency measurements
Zero fluorescence signal
Bad zero will offset all your measurements
Me
Loriaux, S. D., T. J. Avenson, J. M. Welles, D. K. McDermitt, R. D. Eckles, B. Riensche and B. Genty. 2013. Closing in
on maximum yield of chlorophyll fluorescence using a single multiphase flash of sub-saturating intensity. Plant, Cell &
Environment. doi: 10.1111/pce.12115
Saturating flash
Fluorescence increases hyperbolically with flash intensity
This allows maximum fluoresence to be extrapolated from sub-saturating pulses
This extrapolation should be less impacted by multiple PSII turnovers
Requires a series of sub-saturating pulses
Evidence for multiple PSII turnovers which decrease F
m
The multiphase flash
Extrapolates to maximum fluorescence from a single, multi-phase flash
Saturating flash
Optimum flash intensity routine
Highest intensity without photo-damage
Why is fluorescence just red?
Why is fluorescence shifted from absorbed spectra?
How can fluorescence be used as an indicator of plant stress?
How can fluorescence be used to estimate electron transfer rates
What are main considerations to take good fluorescence measurements?
Emission spectra is also mirrored (Stoke's shift)
All fluorescence is relative
Rubisco
CO
2
O
2
Photorespiration
CO
2
Sugars/biomass
Photosynthesis
Kinetics determine reaction rate
CO release per V
2
o
Respiration
Rubisco kinetics determine rates of net CO assimilation!
2
Calvin Cycle Limitation
(J )
Rubisco Limited
(V )
CO compensation point
2
Theory and use of chlorophyll fluorescence
Using the leaf photosynthesis model
cmax
max
CO compensation point
2
Uncertainties in many parts of the model
Be consistent with technique
Relative comparisons strongest
Cost per carboxylation:
3 ATP and 2 NAD(P)H
Cost per oxygenation:
3.5 ATP and 2 NAD(P)H
Compare modeled electron demand of photosynthesis and photorespiration with measured LEF
Why might they be different?
Full transcript