Analyse der Photosynthese über Chlorophyll- Fluoreszenzkinetik

Transkript

1 Ausgewählte biophysikalische Methoden zur Analyse von Pflanzen in vivo und in situ (I): Analyse der Photosynthese über Chlorophyll- Fluoreszenzkinetik

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3 Voraussetzung der Energieübertragung: stabiler S1-Zustand S2 intersystem crossing S1 h ν h ν intersystem T1 crossing EET photochemistry absorption phosphoresscence intersystem crossing absorption intersystem crossing fluorescence S0 Chlorophyll

4 Voraussetzung der Energieübertragung: Überlappende Emissions-/Absorptionsbanden

5 Energieübertragung: Trichter-Prinzip

6 Energieübertragung Kurze Entfernung (nur evtl. eng benachbarte Pigmentmoleküle im selben Protein): direkter Transfer von Anregungszuständen Größere Entfernung: Transfer per induktiver Resonanz ( Förster-Mechanismus)

7 Regulation der Energieübertragung (I) Höhere Pflanzen, div. Algen: State transitions durch Bewegung des LHC II. Diese wird durch eine vom Redox-Status des Elektronentransports abhängige Phosphorylierung kontrolliert.

8 Regulation der Energieübertragung (I) Cyanobakterien und Rotalgen: State transitions durch Bewegung der Phycobilisomen. Klassisches Modell: Einige Cyanobakterien machen jedoch sehr viel komplexere State transitions, dazu mehr am Ende des Kurses im Zusammenhang mit der Stickstoff-Fixierung

9 Regulation der Energieübertragung (II) Minuten bis Stunden: - Änderung der Epoxidation der Xantophylle (Xantophyll-Zyklus) --> Anpassung an Lichtintensität

10 Regulation der Energieübertragung (II) Mechanismen der Energieregulierung durch Carotinoide

11 Voraussetzung der Energieübertragung: stabiler S1-Zustand S2 intersystem crossing S1 h ν h ν intersystem T1 crossing EET photochemistry absorption phosphoresscence intersystem crossing absorption intersystem crossing fluorescence S0 Chlorophyll

12 Chlorophyll-Fluoreszenz

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18 Wichtigste Symbole in der Chlorophyll-Fluoreszenzmessung

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24 Cd-stressed Thlaspi caerulescens Images of PS II activity parameters C Cu Cd Fluorescence yield during F m Efficiency of PS II F v /F m Light saturation F m /F p Electron flow trhough PS II during actinic irradiance (F m '-F t )/F m '

25 The Fluorescence Kinetic Microscope dichroic mirror b/w measuring CCD camera image intensifier b/w measuring CCD camera colour photo CCD camera projective projective Fibre optic coupling projective single lens, lens system filter (neutral, long pass, short pass) iris aperture light sources (short arc, flashlamp, tungsten) mirrors (flat, concave), switching mirror beam unifying prism shutter (manual, computer controlled) objective object condenser Küpper H, Šetlík I, Trtilek M, Nedbal L (2000) Photosynthetica 38(4),

26 The Konstanz version of the Fluorescence Kinetic Microscope detection module Fiberopticsadapter for spectrometer double TVadapter for TV-tube mot b/w measuring CCD camera double TVtube mot w. 2 outputs 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 Eyepiece beam unifying prism shutter (manual, computer controlled) C-mount adapter for colour photo CCD camera mot. filter & reflector cube objective object Filter wheel for switching excitation while maintaining emission filters excitation module condenser microscope stand transmitted light source

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28 Fluorescence kinetic microscopy Methods of data processing Method 1: images of fluorescence parameters To obtain images of fluorescence parameters, frames within the relevant time periods are selected and the necessary mathematical operations are performed on every pixel. False colour image of F m Chl fluorescence calculated from fluorescence kinetic film False colour map of F v /F m, 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 selected 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.

29 Low Mn 2+ -treatment of Thlaspi caerulescens: Photosynthetic oscillations in isolated protoplasts...

30 Cd-stress in the Zn-/Cd-hyperaccumulator T. caerulescens images of PSII activity parameters Image of F m of an unstressed mature leaf Image of F m of a leaf stressed with 50µM Cd 2, showing unusually bright cells Image of F m of the same sample as on the left, but with lower magnification Image of F v /F m of the same sample as above, showing the homogeneously high photosynthtic activity of a healthy leaf of this plant Image of F v /F m of the same sample as above, showing the low photosynthtic activity of the bright cells Image of F v /F m of the same sample as on the left, but with lower magnification