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