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

2 WHAT DOES PHOTOBIOLOGY MEAN? Photosynthesis Photomorphogenesis Cirvadian RhythmUltraviolet Radiation

3 PHOTOBIOLOGY Part 1: Photosynthesis and Fluorescence Part 2: Photoacclimation/-adaptation Part 3: Photoprotection

4 Part 1: Photosynthesis and Fluorescence

5 PHOTOSYNTHESIS

6 LIGHT ABSORPTION

7 THE PHOTOSYNTHETIC APPARATUS

8 PSII LHCII Cyt bfPSILHCI ATPase

9 LIGHT ABSORPTION Photochemistry Antenna pigments PS II Photochemistry Antenna pigments PS II The absorbed light energy is funneled by excitation transfer into the RC’s, where energy conversion by charge separation takes place.

10 excited state ground state molecule absorbs photon photon Increasing energy LIGHT ABSORPTION

11 EXCITATION ENERGY TRANSFER Light Reaction Center Antenna Excitation transfer Electron transfer Donor Acceptor e-e- e-e-

12 PSII LHCII Cyt bfPSILHCI 2H 2 OO 2 + 4H + 2H + PQ PQH 2 2H + PC Fd NADP + H + NADPH H+H+ ATPase ADP + Pi ATP ELECTRON TRANSFER

13 LIGHT ABSORPTION AND ENERGY TRANSFER

14 PHOTOSYNTHESIS AND FLUORESCENCE

15 excited state ground state molecule absorbs photon photon excited state ground state Photochemistry Fluorescence Heat PHOTOSYNTHESIS AND FLUORESCENCE

16 Heat Fluorescence Photochemistry Antenna pigments PS II PHOTOSYNTHESIS AND FLUORESCENCE

17 Heat Fluorescence Photochemistry Antenna pigments PS II Non-light -tress conditions PHOTOSYNTHESIS AND FLUORESCENCE

18 Whitmarsh & Govindjee (2002) Photochemistry = 1 Fluorescence = 0 Photochemistry = 0 Fluorescence = 1 PHOTOSYNTHESIS AND FLUORESCENCE

19 PS = 0 NPQ = 0 PS = 1 NPQ = 0 CHLOROPHYLL FLUORESCENCE MEASUREMENT Fv/Fm = (Fm-Fo)/Fm Fm = maximum fluorescence (RC’s closed) Fo = minimum fluorescence (RC’s open) (higher plants – 0.85, macroalgae usually lower)

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

21 Chondrus crispus Hanelt et al. (1992) Fv/Fm – Diurnal and spatial variation Depth (m) Macrocystis pyrifera Colombo-Pallotta (2007)

22 Littoral Sublittoral van de Poll et al. (2001) Fv/Fm – Comparison of stress responses between species

23 1  PS  0 1  NPQ  0 CHLOROPHYLL FLUORESCENCE MEASUREMENT Fv/Fm  F/Fm’ PS = 0 1  NPQ  0

24  F/Fm’ – EFFECTIVE QUANTUM YIELD Used to describe the variation in the photochemical efficiency of PSII under illuminated conditions. Measurement of this parameter at certain irradiance value.  Indicator of the ability of an organism to move electrons beyond PSII (ETR)  F/Fm’ = (Fm’-F)/Fm’

25 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 same as absorbance (quantifies how much of the incident light is absorbed by an object). ELECTRON-TRANSPORT RATE (ETR)– CURVES

26 ELECTRON-TRANSPORT RATE (ETR)– CURVES ETR = Irradiance   F/Fm’  0,5  Absorptance Relative ETR = Irradiance   F/Fm’  0,5 (Ralph et al. 2002) -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

27 Macrocystis pyrifera Colombo-Pallotta et al. (2006) ETR– CURVES AS AN ANALOGUE TO P-E- CURVES

28 CHLOROPHYLL FLUORESCENCE EXTENSIVELY USE DUE TO: NON-DESTRUCTIVE NON-INVASIVE RAPID SENSITIVE IN REAL-TIME Since 1995 the number of articles published applying chlorophyll fluorescence on the analysis of the photosynthetic performance in macroalgae and seagrasses has increased more than five times.

29 The Chl fluorometer should be capable of measuring the fluorescence yield in a non-intrusive way:  very low measuring light (i.e. exciting light) intensity for assessment of the fluorescence yield of a dark-adapted sample  the detection system has to be very selective to distinguish between fluorescence excited by the measuring light and the much stronger signals caused by ambient and actinic light (full sun light, saturating light pulses for assessment of maximum fluorescence)  fast time response to resolve the rapid changes in fluorescence yield upon dark-light and light-dark transitions PAM fluorometers: Pulse-Amplitude-Modulated fluorometers FLUOROMETERS

30  Allows measurement of fluorescence in the presence of actinic light (light absorbed by the photosynthetic apparatus to drive photosynthesis) How? – Measuring light is modulated and the fluorescence amplifier is highly selective for the modulated signal (yield of chlorophyll fluorescence) - 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) Pulse-Amplitude-Modulated Fluorometers Distinguish between fluorescence and ambient light

31 Pulse-Amplitude-Modulated Fluorometers MINI-PAM DUAL-PAM IMAGE-PAM DIVING-PAM XE-PAM