analysis of photosynthesis via chlorophyll fluorescence
TRANSCRIPT
Biophysical & physicochemical methodsfor analyzing plants in vivo and in situ (I):
Analysis of Photosynthesis viaChlorophyll Fluorescence Kinetics
Chlorophyll
S0
S2
S1T1
h·ν
h·ν
intersystem crossing
absorption
absorptionfluorescence
intersystem crossingintersystem crossing
phosphoresscence
intersystem crossing EET
photochemistry
Necessary for energy transfer:stable S1-state
5 5 0 6 0 0 6 5 0 7 0 0 7 5 0
Abs
orpt
ion
W e ll e n l ä n g e / n m
M g - C h l a A b so rp t io n i n A c et o n M g - C h l a F lu o re sz en z in A c et o n
Necessary for energy transfer:Overlap of emission/absorption bands
Energy transfer – funnel principle (I): Scheme in green plants
PUB = Phycourobilin
PC = Phyco-cyanin
PE = Phyco-erythrin
ChlRC
(Chl)
APC =Allo-
Phyco-cyanin
Transmission of filters forselective excitation
Car
Energy transfer – funnel principle (II): Scheme in cyanobacteria (Trichodesmium)
Energy transfer between chlorophylls
D* A
Larger distance, requires overlap of absoprtion/emission spectra: Transfer by induktiveResonance („Förster-Mechanism“)
D* A
Short distance, requires overlap of molecular orbitals ( only Chls in extremely short distance to each other, e.g. special pair) : direct transfer of S1 excited state (Dexter-Mechanism)
Transfer times between Chls towards / in PSIIRC
From: vanGrondelle R, Novoderezhkin VI, 2006, PCCP8, 793-807
Biophysical aspects of photosynthetic electron transport A) Photosystem II reaction centre:
special pair chlorophyll and pheophytins
Mechanism of charge separation1. Special pair chlorophylls (=P680) accept excitons from antenna2. P680 transfers an electron to ChlD1 (“initial charge separation”)3. Within a few ps, the electron is further transferred to Phe ( P680+ / Phe-) “primary charge separation”
From: Barber J, 2003, QuartRevBiophys36, 71-89
Biophysical aspects of photosynthetic electron transportA) Photosystem II reaction centre:
speeds of electron transfer
From: Cramer WA, Zhang H, Yan J, Kurisu G, Smith JL, 2006, AnnRevBiochem75_769-90
Functional characteristicstransfers e- from PQ to
plastocyanin (PC), It uses the difference in
potential betwen QB and PC for translocating a proton via 2x2 heme b groups and 2x1 heme x groupElectrons are transferred
from the heme b groups to PC via a “Rieske” [2Fe2S]-cluster and a heme f groupCyclic electron transport
occurs via coupling of ferredoxin to heme x
Biophysical aspects of photosynthetic electron transportB) Cytochrome b6f complex:
mechanism
From: Shibata N, Inoue T, Nagano C, Nishio N, Kohzuma T, Onodera K, Yoshizaki F, Sugimura Y, Kai Y, 1999, J Biol Chem. 274: 4225-30
Functional characteristicsOxidised (Cu2+) plastocyanin accepts
electron from Cytb6f complex, Reduced ( Cu+) plastocyanin diffuses to
the PSIRCPlastocyanin releases the electron
(Cu2+ Cu+)rigid protein structure facilitates fast red/ox-
changes, but recetn data show that copper binding still causes changes in structure (“induced rack” rather than “entatic state”)
Biophysical aspects of photosynthetic electron transportC) Plastocyanin
Photosynthesis related Proteins with metal centresD) Photosystem I reaction centre
From: Nelson N, Yocum CF, 2006, AnnRevPlantBiol 57, 521-65
Funtional characteristics: primary charge separation:
special pair (=P700, Chl a / Chl a’ heterodimer), releases e- to A0 via A (both Chl a) e- transport via A1 (phylloquinone) and the
[4Fe4S]-clusters Fx, FA and FB to the [4Fe4S]-cluster of ferredoxinP700 is re-reduced by plastocyanin
+430 mV
-1000 mV
-800 mV
-705 mV
-520 mV
-580 mV
Measurement of in vivo chlorophyll fluorescence kinetics
Why?The quantum yield of in vivo chlorophyll fluorescence depends on a competition for excitons between photochemistry (including electron transport after PSII via feedback), thermal relaxation ("nonphotochemical quenching") and fluorescence.
Examples of Applications- biophysical investigations of mechanisms of photosynthesis- studies of the effects of abiotic and biotic stress on plants- ecophysiological studies- fruit quality assessment
--> The fluorescence quantum yield and especially its change in response to changes in actinic irradiance allows a detailed assessment of photosystem II function und thus the vitality of a cell, tissue, plant or even ecosystem.
Chlorophyll
S0
S2
S1T1
h·ν
h·ν
intersystem crossing
absorption
absorptionfluorescence
intersystem crossingintersystem crossing
phosphoresscence
intersystem crossing
photochemistry
The basis for measurement of photosynthesis via fluorescence kinetics: competition for the S1 excited state
Chlorophyll-Fluoreszenz
Wichtigste Symbole in der Chlorophyll-Fluoreszenzmessung
Biophysical measurements in vivo with temporal, spatial and spectral resolution: the Fluorescence Kinetic Microscope
dichroic mirror
single lens, lens system
filter (neutral, var. PC-controlled, long pass, short pass)
iris aperture
light source (LED)
mirrors (flat, concave), switching mirror
beam unifying prism
shutter (manual, computer controlled)
microscope stand
Mot. Filter wheel
excitation module
transmitted light sourcecondenser
objective
object
mot. filter & reflector cube
b/w measuring CCD camera
Eyepiece
Fiberoptics-adapter for spectrometer
C-mount adapter for colour photo CCD camera
detection module
double TV-tube mot w. 2 outputs
double TV-adapter
Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175, 655-74
Mot. Filter wheel
Küpper H, Šetlík I, Trtilek M, Nedbal L (2000) Photosynthetica 38(4), 553-570
Fluorescence kinetic microscopy
False colour image of Fm Chl fluorescence calculated from fluorescence kinetic film
Manual selection of objects for kinetic analysis.
Fluorescence induction of selected objects, showing all differences in kinetics for representative cells.
Methods of data processingMethod 1: images of fluorescence parameters
False colour map of Fv/Fm, showing the differences in this parameter over the entire image.
To obtain images of fluorescence parameters, frames within the relevant time periods are selected and the necessary mathematical operations are performed on every pixel.
Method 2: kinetics of selected areas (objects)To obtain kinetic traces, the relevant regions are selected on a captured frame or parameter image. The kinetics of all pixels within the selected areas are averaged.
Cd-stressed Thlaspi caerulescensImages of PS II activity parameters
Fluorescence yield during Fm
Efficiency of PS II Fv/Fm
Light saturationFm/Fp
Electron flow trhoughPS II during actinic irradiance (Fm'-Ft)/Fm'
C
Cu
Cd
Spatial heterogeneity of photosynthetic oscillationsover the leaf surface
Insets: fluorescence emission images (Fp); the white bar represents 100 µm
Cd-stress in the Zn-/Cd-hyperaccumulator T. caerulescens:images of PSII activity parameters
Image of Fm of an unstressed mature leaf
Image of Fv/Fm of the same sample as above, showing the homogeneously high photosynthtic activity of a healthy leaf of this plant
Image of Fm of a leaf stressed with 50µM Cd2, showing unusually bright cells
Image of Fv/Fm of the same sample as above, showing the low photosynthtic activity of the bright cells
Image of Fm of the same sample as on the left, but with lower magnification
Image of Fv/Fm of the same sample as on the left, but with lower magnification
0
10
20
30
T. caerulescens
C
Con
trol
0
10
20 D
Stre
ssed
0
10
20 E
Accl
imat
ing
0.0 0.2 0.4 0.6 0.8 1.00
10
20 F
Fv / Fm
Acc
limat
ed
0
10
20
T. fendleri
Con
trol
A
0
10
20 B
Str
esse
d
Cd-stress and acclimation in T. caerulescens & T. fendleri:histograms of Fv/Fm
Cd-Akklimation im Zn-/Cd-hyperaccumulator T. caerulescens
Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol., 10.1111/j.1469-8137.2007.02139.x.
0.00.10.20.30.40.50.60.70.8
dark acclimated light acclimated at the end of the recovery period
control palisade mesophyllF v/ F
m
; Φ
PSII =
(Fm'-F
t')/ F
m' spongy mesophyll
0.00.10.20.30.40.50.60.70.8
NPQ
= (F
m-F
m')/
Fm
Mrz Mai Jul Sep Nov0.00.10.20.30.40.50.60.70.8
Sat
urat
ion
= (F
p-F0)/(
F m-F
0)
DateMrz Mai Jul Sep Nov
10 µM Cd2+
palisade mesophyll spongy mesophyll
Mrz Mai Jul Sep NovDate
Mrz Mai Jul Sep Nov
Spectrally resolved fluorescence kinetic parameters (II)
450 500 550 600 650 700 750 800Wavelength / nm
Fluo
resc
ence
bright IIbright InormallowFo activelowFo inactive
Fluorescence
PUB = Phycourobilin
PC = Phyco-cyanin
PE = Phyco-erythrin Chl
RC (Chl)
APC =Allo-
Phyco-cyanin
Car
Absorption
450 500 550 600 650 700 750Wavelength / nm
Abs
orpt
ion
bright Inormallow Fo
Spectrally resolved fluorescence kinetic parameters (I)
morning noon evening night
HL
LL
PUB PCPE Chl RCAPC
Spectrally resolved fluorescence kinetic parameters (II)
Purification of Trichodesmium phycobiliproteinsfor deconvoluting spectrally resolved in vivo fluorescence
kinetics and absorption spectra
Method of deconvolution:Küpper H, Seibert S, Aravind P (2007) Analytical Chemistry
79, 7611-7627
Phycobiliprotein purification + characterisation: Küpper H,
Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I
(2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-
167
Phycourobilin isoforms
Phycourobilin isoforms
(II)
Phycoerythrinisoforms
Phycocyaninisoforms
Allophycocyanin
Fluo
resc
ence
(nor
mal
ised
to re
d m
axim
um)
Fluo
resc
ence
(nor
mal
ised
to re
d m
axim
um)
Diazotrophic cell
basic fluorescence yield F0
PSII activity Fv
Rel
ativ
e flu
ores
cenc
e qu
antu
m y
ield
Rel
ativ
e flu
ores
cenc
e qu
antu
m y
ield
Deconvolution of spectrally resolved in vivo fluorescence kinetics shows reversible coupling of individual
phycobiliproteins
Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167
Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167
Sequence of uncoupling events in a „bright II cell“
Andresen E, Adamska I, Šetlikova E, Lohscheider J, Šimek M, Küpper H, (2009) unpublished data
Light limitation: acclimation by reversible phycobiliprotein coupling: basic fluorescence yield F0
relative frequency