photobiology 1-ps and fluorescence

PHOTOBIOLOGY PHOTOBIOLOGY 

1Nadine Schubert

Instituto de Ciencias del Mar y Limnología de la UNAM  1Unidad de Sistemas Arrecifales, Puerto Morelos, México

WHAT DOES PHOTOBIOLOGY MEAN?

Photosynthesis Photomorphogenesis Cirvadian Rhythm Ultraviolet Radiation

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PHOTOBIOLOGY PHOTOBIOLOGY 

Part 1:  Photosynthesis and FluorescencePart 1:  Photosynthesis and Fluorescence

Part 2: Photoacclimation/Part 2: Photoacclimation/‐‐adaptationadaptation

Part 3: PhotoprotectionPart 3: Photoprotection

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pp

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Part 1:Part 1:h h d lh h d lPhotosynthesis and FluorescencePhotosynthesis and Fluorescence

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PHOTOSYNTHESIS

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LIGHT ABSORPTIONLIGHT ABSORPTION

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THE PHOTOSYNTHETIC APPARATUSTHE PHOTOSYNTHETIC APPARATUS

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THE PHOTOSYNTHETIC APPARATUSTHE PHOTOSYNTHETIC APPARATUS

PSIILHCII Cyt bf PSI LHCIATPase

y

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LIGHT ABSORPTION

The absorbed light energy is

Antenna pigmentsAntenna pigments

The absorbed light energy is

funneled by excitation transfer

into the RC’s, where energy , gy

conversion by charge

separation takes place.

9Photochemistry

PS II

Photochemistry

PS II

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LIGHT ABSORPTIONLIGHT ABSORPTION

photonexcited state

p

molecule b b g

ener

gy

absorbs photon

crea

sing

10ground state

In

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EXCITATION ENERGY TRANSFER

Excitation transfer Electron transfer

Light Acceptore-

Reaction C tCenter

11AntennaDonor

e-

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ELECTRON TRANSFER

ADP + Pi ATP

2H+ Fd

NADP + H+ NADPH

PSIILHCII Cyt bf PSI LHCIPQH2 ATPase

yPQ

122H2O O2+ 4H+

2H+

PC H+

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LIGHT ABSORPTION AND ENERGY TRANSFER

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PHOTOSYNTHESIS AND FLUORESCENCE

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PHOTOSYNTHESIS AND FLUORESCENCE

photonexcited state

pexcited state

molecule b b

Photochemistry

absorbs photon Fluorescence

15ground state

ground state

Heat

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PHOTOSYNTHESIS AND FLUORESCENCE

Antenna pigments

Heat Fluorescence

PS II

16Photochemistry

PS II

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PHOTOSYNTHESIS AND FLUORESCENCE

Antenna pigments

Non-light -tress conditions

Heat Fluorescence

PS II

17Photochemistry

PS II

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PHOTOSYNTHESIS AND FLUORESCENCE

Photochemistry = 1Fluorescence = 0

Photochemistry = 0Fluorescence = 1Fluorescence 0 Fluorescence 1

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Whitmarsh & Govindjee (2002)

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CHLOROPHYLL FLUORESCENCE MEASUREMENT

PS = 0PS   0NPQ = 0

Fv/Fm = (Fm‐Fo)/Fm 

Fm = maximum fluorescence (RC’s closed)Fo = minimum fluorescence (RC’s open) 

PS = 1NPQ = 0

(higher plants – 0.85, macroalgae usually lower)

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Fv/Fm – MAXIMUM QUANTUM YIELD

Quantum yield: Probability that the energy of a photon absorbed will be used for photosynthesis (i.e. enters in the 

e‐ ‐ transport chain)

⇓⇓Indicator of photosynthetic efficiency

M i i ld

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Maximum quantum yield: requires complete relaxation of the competing mechanisms with the photochemical energy conversion

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Fv/Fm – Diurnal and spatial variationChondrus crispus

Dep

th (m

)

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Macrocystis pyrifera

Colombo-Pallotta (2007)21

Hanelt et al. (1992)

Fv/Fm – Comparison of stress responses between species

Littoral Sublittoral Sublittoral

Littoral SublittoralSublittoral

22van de Poll et al. (2001)

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CHLOROPHYLL FLUORESCENCE MEASUREMENT

Fv/Fm ∆F/Fm’PS 0Fv/Fm ∆F/FmPS = 01 > NPQ > 0

1 > PS > 01 > NPQ > 0

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∆F/Fm’ – EFFECTIVE QUANTUM YIELD

Used to describe the variation in the photochemical ffi i f PSII d ill i t d ditiefficiency of PSII under illuminated conditions. 

Measurement of this parameter at certain irradianceMeasurement of this parameter at certain irradiance value. ⇓⇓

Indicator of the ability of an organism to move electrons beyond PSII (ETR)

24∆F/Fm’ = (Fm’‐F)/Fm’

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ELECTRON‐TRANSPORT RATE (ETR)– CURVES

ETR = Irradiance ⋅ ∆F/Fm’ ⋅ 0,5 ⋅ Absorptance (Genty et al. 1989)

∆F/Fm’ = effective quantum yield (under light)

0,5 = Assumption that 50% of these quanta are absorbed by PSII

Absorptance = fraction of incident light that is absorbed by the photosynthetic tissue. Not the p g y p y

same as absorbance (quantifies how much of the incident light is absorbed by an object).

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ELECTRON‐TRANSPORT RATE (ETR)– CURVES

ETR = Irradiance ⋅ ∆F/Fm’ ⋅ 0,5 ⋅ Absorptance

Relative ETR = Irradiance ⋅ ∆F/Fm’ ⋅ 0,5 (Ralph et al. 2002)

ETR h b ti h t i ti h b t i li ti-ETR: when absorption characteristics change between species, acclimations, seasons…

- rel. ETR: use only when it is sure that there are no differences in the absorption characteristics

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ETR– CURVES AS AN ANALOGUE TO P‐E‐ CURVES

Macrocystis pyrifera

27Colombo-Pallotta et al. (2006)

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CHLOROPHYLL FLUORESCENCE

EXTENSIVELY USE DUE TO:

NON DESTRUCTIVE• NON‐DESTRUCTIVE• NON‐INVASIVE

• RAPID• RAPID• SENSITIVE

• IN REAL‐TIMEIN REAL‐TIME

Since 1995 the number of articles published applyinghl h ll fl th l i f th

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chlorophyll fluorescence on the analysis of thephotosynthetic performance in macroalgae and seagrasseshas increased more than five times.

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FLUOROMETERS

The Chl fluorometer should be capable of measuring the fluorescence yield ina non-intrusive way:

very low measuring light (i.e. exciting light) intensity for assessment of thefluorescence yield of a dark-adapted sample

the detection system has to be very selective to distinguish betweenfluorescence excited by the measuring light and the much stronger signalscaused by ambient and actinic light (full sun light saturating light pulses forcaused by ambient and actinic light (full sun light, saturating light pulses forassessment of maximum fluorescence)

fast time response to resolve the rapid changes in fluorescence yield upon

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fast time response to resolve the rapid changes in fluorescence yield upondark-light and light-dark transitions

PAM fluorometers: Pulse-Amplitude-Modulated fluorometers 29p

Pulse‐Amplitude‐Modulated Fluorometers

Distinguish between fluorescence and ambient light

→ Allows measurement of fluorescence in the presence of actinic light (light absorbed by the photosynthetic apparatus to drive g ( g y p y pp

photosynthesis)

How? – Measuring light is modulated and the fluorescenceHow? Measuring light is modulated and the fluorescence amplifier is highly selective for the modulated signal (yield of

chlorophyll fluorescence)

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- pulse-modulated measuring light can be generated either by a light-emitting diode (LED; most PAM fluorometers) or a flash

discharge lamp (i e XE-PAM) 30discharge lamp (i.e. XE PAM)

Pulse‐Amplitude‐Modulated Fluorometers

DUAL-PAMIMAGE-PAM

MINI-PAM

31DIVING PAM XE-PAM 31DIVING-PAM XE-PAM