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).