analyyysis of photosynthesis via chlorophyll fluorescence ...€¦ · fluorescence kinetic...
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Biophysical & physicochemical methodsBiophysical & physicochemical methods for analyzing plants in vivo and in situ (I):
Analysis of Photosynthesis via y yChlorophyll Fluorescence Kinetics
Necessary for energy transfer: stable S1-state
S2S2
intersystem crossing
S1T1intersystem crossing EET
h·ν
h νabsorption
inters stem crossingphosphoresscence
photochemistry
h·νabsorption
fluorescence
intersystem crossingintersystem crossing
S0
Chlorophyll
Necessary for energy transfer: Overlap of emission/absorption bandsp p
Mg-Chl a Absorption in Aceton Mg-Chl a Fluoreszenz in Aceton
Abs
orpt
ion
550 600 650 700 750Wellenlänge / nm
Adjustment of absorption bands by chemical modification
From: Lawlor DWLawlor DW
(1990) Thieme, Stuttgart,
377S377S
From: Barber J (1978) Rep
Prog Phys 41 1158-9941, 1158-99
Energy transfer – funnel principle (II): Scheme in cyanobacteria (Trichodesmium)
Transmission of filters for selective excitation
(Trichodesmium)
CarCar
PUB = Phycourobilin
PC = Phyco-cyanin
PE = Phyco-erythrin
ChlRC
(Chl)
APC =Allo-
Phyco-cyanin
Energy transfer – funnel principle (II): Scheme in higher plants
Aus: Horton P, Ruban AV, Walters RG (1996) Annu Rev Plant Physiol Plant Mol Biol 47: 655-84
Energy transfer – funnel principle (III): Transfer times between Chls towards & in PSIIRCTransfer times between Chls towards & in PSIIRC
From: vanGrondelle R, Novoderezhkin VI, 2006, PCCP8, 793-807
Mechanisms of energy transfer between chlorophylls
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)each other, e.g. special pair) : direct transfer of S1 excited state (Dexter Mechanism)
Larger distance, requires overlap of absoprtion/emission spectra: Transfer by induktive
D* A
Resonance („Förster-Mechanism“)
D* A
Fast adaptation to irradance changes: combination of LHCII-aggregation with xanthophyll cyclegg g p y y
ViolaxanthinViolaxanthinZeaxanthinLHCII
From: http://photosynthesis.peterhorton.eu/research/lightharvesti
ng.aspx (Horton lab web page)Horton P, Johnson MP, Perez-Bueno ML, Kiss AZ, Ruban
AV (2008) FEBS Journal 275, 1069-79
Fast adaptation to irradance changes: combination of LHCII-aggregation with xanthophyll cyclegg g p y y
Violaxanthin Zeaxanthin
From: http://photosynthesis.peterhorton.eu/research/lightharvesting.aspx (Horton lab web page)Horton P, Johnson MP, Perez-Bueno ML, Kiss AZ, Ruban AV (2008) FEBS Journal 275, 1069-79
Biophysical aspects of photosynthetic electron transport A) Photosystem II reaction centre:) y
special pair chlorophyll and pheophytins
From: Barber J, 2003, QuartRevBiophys36, 71-89
Mechanism of charge separation 1. Special pair chlorophylls (=P680) accept excitons from antenna 2. P680 transfers an electron to ChlD1 (“initial charge separation”)D1 ( g p ) 3. Within a few ps, the electron is further transferred to Phe ( P680+ / Phe-) “primary
charge separation”
Biophysical aspects of photosynthetic electron transportA) Photosystem II reaction centre:) y
speeds of electron transfer
Biophysical aspects of photosynthetic electron transportB) Cytochrome b6f complex:
Functional characteristics t f f PQ t
) y 6 pmechanism
transfers 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 g pheme x group Electrons are transferred
from the heme b groups tofrom the heme b groups to PC via a “Rieske” [2Fe2S]-cluster and a heme f group Cyclic electron transport Cyclic electron transport
occurs via coupling of ferredoxin to heme x
From: Cramer WA Zhang H Yan J Kurisu GFrom: Cramer WA, Zhang H, Yan J, Kurisu G, Smith JL, 2006, AnnRevBiochem75_769-90
Biophysical aspects of photosynthetic electron transportC) Plastocyanin
Functional characteristics
C) Plastocyanin
Functional characteristics Oxidised (Cu2+) plastocyanin accepts
electron from Cytb6f complex, Reduced ( Cu+) plastocyanin diffuses to
the PSIRC Plastocyanin releases the electron y
(Cu+ Cu2+) rigid protein structure facilitates fast red/ox-
changes but recent data show that copperchanges, but recent data show that copper binding still causes changes in structure (“induced rack” rather than “entatic state”)
From: Shibata N, Inoue T, Nagano C, Nishio N, Kohzuma T, Onodera K, Y hi ki F S i Y K i Y 1999 J Bi l Ch 274 4225 30Yoshizaki F, Sugimura Y, Kai Y, 1999, J Biol Chem. 274: 4225-30
Biophysical aspects of photosynthetic electron transportD) Photosystem I reaction centreD) Photosystem I reaction centre
Funtional characteristics: i h ti primary charge separation:
special pair (=P700, Chl a / Chl a’ heterodimer), releases e- to A0 via A (both Chl a)
-580 mV
e- transport via A1 (phylloquinone) and the [4Fe4S]-clusters Fx, FA and FB to the [4Fe4S]-cluster of ferredoxin -705 mV
-520 mV
P700 is re-reduced by plastocyanin-800 mV
-1000 mV
+430 mV
From: Nelson N, Yocum CF, 2006, AnnRevPlantBiol 57, 521-65
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 relaxationtransport after PSII via feedback), thermal relaxation ("nonphotochemical quenching") and fluorescence.--> 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 ecosystemtissue, plant or even ecosystem.
Examples of Applications- biophysical investigations of mechanisms of photosynthesis
t di f th ff t f bi ti d bi ti t l t- studies of the effects of abiotic and biotic stress on plants- ecophysiological studies- fruit quality assessmentfruit quality assessment
The basis for measurement of photosynthesis via fluorescence kinetics: competition for the S1 excited state
S2
p
S2
intersystem crossing
S1T1
intersystem crossing
h·ν
h νabsorption
inters stem crossingphosphoresscence
photochemistry
h·νabsorption
fluorescence
intersystem crossingintersystem crossing
S0
Chlorophyll
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)
b/w measuring CCD camera
detection module
double TV filter (neutral, var. PC-controlled, long pass, short pass)
iris aperture
light source (LED)
camera
Fiberoptics-adapter for spectrometer
double TV-tube mot w. 2
t t
double TV-adapter
mirrors (flat, concave), switching mirror
beam unifying prismEyepiece
spectrometer outputs
shutter (manual, computer controlled)
mot filter &
C-mount adapter for colour photo CCD camera Mot. Filter wheel
Mot. Filter wheelobjective
mot. filter & reflector cube
microscope stand
object
standtransmitted light sourcecondenser
excitation module
Küpper H, Aravind P, Leitenmaier B, Trtilek M, Šetlík I (2007) New Phytol175, 655-74
Küpper H, Šetlík I, Trtilek M, Nedbal L (2000) Photosynthetica 38(4), 553-570
Fluorescence kinetic microscopyMethods of data processing
Method 1: images of fluorescence parameters
To obtain images of fluorescence parametersfluorescence parameters, frames within the relevant time periods are selected and the necessaryand the necessary mathematical operations are performed on every pixel.
False colour image of Fm Chl fluorescence calculated from fluorescence kinetic film
False colour map of Fv/Fm, showing the differences in this parameter over the entire image.
Method 2: kinetics of selected areas (objects)To obtain kinetic traces, the relevant regions are gselected on a captured frame or parameter image. The kinetics of all pixels within the selected areas are averaged.
Manual selection of objects for kinetic analysis.
Fluorescence induction of selected objects, showing all differences in kinetics for representative cells.
Cd-stressed Thlaspi caerulescensImages of PS II activity parametersg y p
C
Cu
Cd
Fluorescence yield Efficiency of PS II Light saturation Electron flow trhough yduring Fm
yFv/Fm
gFm/Fp
gPS II during actinic irradiance (Fm'-Ft)/Fm'
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 Fm of a leaf stressed with 50µM Cd2, showing unusually bright cells
Image of Fm of the same sample as on the left, but with lower magnification
Image of F /F of the same sample Image of F /F of the same sample Image of F /F of the sameImage of Fv/Fm of the same sample as above, showing the homogeneously high photosynthtic activity of a healthy leaf of this plant
Image of Fv/Fm of the same sample as above, showing the low photosynthtic activity of the bright cells
Image of Fv/Fm of the same sample as on the left, but with lower magnification
Cd-stress and acclimation in T. caerulescens & T. fendleri:histograms of Fv/Fm
30
T. caerulescens
C 20
T. fendleri
A
10
20C
Con
trol
10
20
Con
trol
A
0
C
20 D
ed
0
C
20 B
ed
0
10
Stre
sse
E0
10
Stre
sse
10
20 E
clim
atin
g
0Acc
20 F
ted
0
10
Acc
limat
0.0 0.2 0.4 0.6 0.8 1.0 Fv / Fm
Spectrally resolved fluorescence kinetic parameters (II)
Spectrally resolved fluorescence kinetic parameters (I)
bright II
FluorescenceAbsorption
cenc
e
bright IIbright InormallowFo activelowFo inactivept
ion
bright Inormallow Fo
Fluo
resc lowFo inactive
Abs
orp
450 500 550 600 650 700 750 800450 500 550 600 650 700 750
Wavelength / nmCarWavelength / nm
PUB = Phycourobilin
PC = Phyco-cyanin
PE = Phyco-erythrin Chl
RC (Chl)
APC =Allo-
Phyco-cyanin
Purification of Trichodesmium phycobiliproteinsfor deconvoluting spectrally resolved in vivo fluorescence
kinetics and absorption spectraPhycourobilin
isoforms Diazotrophic cell
basicantu
m y
ield
Phycobiliprotein purification + characterisation: Küpper H
Phycourobilin isoforms
(II)
m)ax
imum
)
basic fluorescence yield F0
ores
cenc
e qu
characterisation: Küpper H, Andresen E, Wiegert S, Šimek
M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787 155-167
Phycoerythrinisoforms
red
max
imum
ised
to re
d m
Rel
ativ
e flu
o
Method of deconvolution:Küpper H, Seibert S, Aravind P
(Bioenergetics) 1787, 155-167
Ph i orm
alis
ed to
ence
(nor
mal
i
PSII activity Fvm y
ield
(2007) Analytical Chemistry 79, 7611-7627
Phycocyaninisoforms
ores
cenc
e (n
o
Fluo
resc
e y v
ence
qua
ntum
Allophycocyanin
Fluo
ativ
e flu
ores
cR
ela
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
Sequence of uncoupling events in a bright II cell“events in a „bright II cell
Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167
Light limitation: acclimation by reversible phycobiliprotein coupling: basic fluorescence yield F0
relative frequency
Andresen E, Adamska I, Šetlikova E, Lohscheider J, Šimek M, Küpper H, (2009) unpublished data
All slides of my lectures can be downloaded from my workgroup homepage
i k t d D t t f Bi l W k Kü l bwww.uni-konstanz.de Department of Biology Workgroups Küpper lab,
or directlyo d ect y
http://www.uni-konstanz.de/FuF/Bio/kuepper/Homepage/AG_Kuepper_Homepage.html
AND:AND:please remember the lecture of Prof. Harald Paulsen in the “Fachbereichsseminar”,
5 May 2011, 17:15, M629:Watching the light-harvesting fold: fluorescence and EPR (electron paramagnetic g g g ( p g
resonance) studies on the assembly of the LHC II (light harvesting complex II).
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