Phytoplankton Pigments THE REACTIONS OF PHOTOSYNTHESIS Overall Rxn: 12 H2O + 6 CO2 WATER LIGHT CARBON DIOXIDE Net Rxn: 6 CO2+ 6 H2O + lightà C6H12O6 + 6 O2 Simplest form: CO2 + H2O + lightà CH2O + O2 6 O2 + C6H12O6 + 6 H2O OXYGEN GLUCOSE WATER PLANT ANATOMY Chloroplasts are highly structured, membrane-rich organelles. Outer membrane Inner membrane Thylakoids Granum Stroma • Stroma (Not To Be Confused With Stomata!!!) – Dense fluid within surrounding thylakiod PLANT ANATOMY •  Chloroplasts: –  Light energy is captured and converted in chloroplasts –  Light energy is passed through an ETC which converts it into a usable, chemical form Cyanobacteria: Also have phycobilosomes PHOTOSYNTHETIC PIGMENTS •  Chlorophylls: –  Reflect mainly green light »  Chlorophyll a, b, c, d »  Divinyl chlorophyll •  Carotenoids: –  Reflect mainly orange and yellow »  Fucoxanthin »  Beta-carotene •  Phycobilins (proteins): –  Reflect mainly blue-green or reddish »  Phycocyanin (blue-green algae) »  Phycoerythrin PHOTOSYNTHETIC PIGMENTS H 2C Ring Structure In Head Absorbs Light CHO in chlorophyll b CH3 in chlorophyll a CH H 3C N N CH2CH3 Mg N N H 3C CH2 CH2 C CH3 COCH3 O OO O CHLOROPHYLL a & b CH2 Tail PHOTOSYNTHETIC PIGMENTS CH3 CH3 H 3C CH β-carotene H 3C CH CH3 CH3 HO http://www.dougbushphoto.com/#!/portfolio/G0000iA5Vj5t_zO4/I0000z_64QFk3bKE DIFFERENT PIGMENTS ABSORB DIFFERENT WAVELENGTHS OF LIGHT Amount of light absorbed Chlorophyll a Chlorophyll b Carotenoids 400 500 600 Wavelength of light (nm) 700 Phycobiliproteins, bilin variation, and group III CA regulation. David M. Kehoe PNAS 2010;107:9029-9030 ©2010 by National Academy of Sciences ABSORBPTION SPECTRUM •  Every pigment has a characteristic Absorption Spectrum •  Optimal λ •  Optimal emission (fluorescence) •  This provides a “fingerprint” chlorophyll b % ABS chlorophyll a λ (nanometers) Example: 9 months of data from San Francisco Bay Ciliate' 0%' Eus=gmatophyte' 3%' Euglenophyte' 3%' Ciliate' Euglenophyte' 0%' 0%' Cyanobacteria' 0%' Cryptophyte' 2%' Raphidophyte' 0%' Prasinophyte' 5%' Unknown'coccoid' 0%' Chrysophyte' 0%' Chlrophyte' 0%' Cyanobacteria' 3%' Cryptophyte' 10%' Eus<gmatophyte' Prasinophyte' 0%' 0%' Raphidophyte' 0%' Unknown'coccoid' 3%' Dinoﬂagellate' 26%' Chrysophyte' 0%' Chlrophyte' 0%' Dinoﬂagellate' 11%' Chemtax Diatom' 65%' Diatom' 69%' Microscopy Central Bay Chlorophyll Diatoms Dinoflagellates Cryptophytes Cyanobacteria Eustigmat. Chlorophytes Euglenophytes Raphidophytes Prasinophytes Chrysophytes > 5 μg L-1 South Bay 0 μg L-1 Flow Cytometry http://www.whoi.edu/science/B/Olsonlab/insitu2001.htm Chlorophyll a emits red light when excited with blue or red light ! Fluorescence HEAT Log (Irradiance, W m-2)! Each fluorometer has unique properties, even though they all work the same way… Flow Cytometer! Sea Tech! Sunlight! PAM! -8! -6! P&P! -4! Turner Designs! -2! 0! Log (Time, s)! 2! 4! Courtesy of JJ Cullen Variable Fluorescence • Start with a pulse of weak light--this will cause weak (background) fluorescence and is called the probe flash 1 F 0 Time Variable Fluorescence • Turn the lights all the way up (actinic light) and you get maximum fluorescence, directly proportional to the # of functional chl molecules 1 F 0 Time Variable Fluorescence 1 F 0 Fm Fv Fo Time • Turn the lights all the way up (actinic light) and you get maximum fluorescence, directly proportional to the # of functional chl molecules Variable Fluorescence • Leave the light on long enough, and the dark reactions (photochemical quenching) take over…. 1 F 0 ….leave it on even longer, and non-photochemical quenching starts Time Variable Fluorescence • Leave the light on long enough, and the dark reactions (photochemical quenching) take over…. 1 F 0 ….leave it on even longer, and non-photochemical quenching starts Time Variable Fluorescence •  Fv/Fm (Fm-Fo/Fm) provides an indication of relative “health”, or whether there is damage to the photosystem •  Short-term changes (seconds) provide an indication of photosynthetic efficiency (quantum yield) •  Long-term changes (seconds-minutes) provide an indication of adaptability •  Do the same thing in ambient light, get an indication of photosynthetic rates “Exposure to higher irradiances and elevated ultraviolet dosage may have depressed the Fv/Fm values of cells in surface waters relative to those at depth (Fig. 4). Consequently, the magnitude of Fv/Fm from underway mapping may not be representative of cells at depth after day 5” Chlorophyll Fluorescence •  Good qualitative indicator of biomass, but it’s NOT quantitative! •  Affected by temperature, irradiance, previous light history, species composition, nutrient status, etc. • We use it because it’s specific to autotrophs, even though it is very flawed… Next-generation Imaging Flow Cytometry New instruments, such as the Amnis system, combine flow cytometry with imaging—you get the advantage of having a microscope-like image combined with lasers, counting every particle, etc… Phytoplankton Functional Types Size Trophic Status Functional Type Genus/ Species/ Strain Picoplankton (0.2-2.0 µm) Heterotroph Cyanophyte >500 species, Chlorophyte Unknown # of strains… Nanoplankton (2-20 µm) Mixotroph Cryptophyte Pyrrophyte Microplankton (20-100 µm) Autotroph Bacillariophyte Cell Counts 1E6 cells/L H. Akashiwo Pseudo-nitzschia Karenia Dinophysis Karlodinium Akashiwo Alexandrium Anabaena Aphanizomenon Oscillatoria Planktothrix Synechococcus* 1, 10, 100 E3 µm^3/mL H. Akashiwo Pseudo-nitzschia Karenia Dinophysis Karlodinium Akashiwo Alexandrium Anabaena Aphanizomenon Oscillatoria Planktothrix Synechococcus Year Phytoplankton Functional Types Most common classification is based on size: • Picoplankton • Nanoplankton • Net plankton Or Group: • Cyanobacteria • PicoEukaryotes • Nanoflagellates • Coccolithophores • Dinoflagellates • Diatoms Or Function: • “good” or “bad” • Nitrogen fixers • Coccolithophores Zooplankton" Phytoplankton" Nutrients" Returning to our box model—we’ve been describing who and how much biomass is in our “P” box. Now we want to talk about how fast that box is changing…. Light and Nutrients Regulate Growth—knowing which one is more limiting allows you to predict the response to eutrophication