Light-Dependent Reactions of Photosynthesis

Home , P680, P700, Photoinhibition, Plastocyanin

Light-dependent reactions of photosynthesis

Tom Donald

• Photosynthesis overview • Evolution and diversity of photosynthesis • Light and pigments • The light response curve and quantum efficiency • Plastids and chloroplasts • Structure and function of photosynthetic complexes • Pathways of electron transport • Damage avoidance and repair: Acclimations to light • Monitoring light reactions • Optimizing and improving photosynthesis • Artificial photosynthesis • Photosynthetic fungi and animals

“High energy”, Light-dependent reactions reduced The first step is the capture of carbon light energy as ATP and Energy input reducing power, NADPH from sunlight ATP NADPH

Oxygen is Light-independent reactions released as The second step is the transfer of energy and reducing power from “Low energy”, a byproduct ATP and NADPH to CO , to produce oxidized carbon 2 high-energy, reduced sugars in carbon dioxide

6 CO2 + 6 H2O  C6H12O6 + 6 O2

2 NADPH e− 2 H+ 2 NADP+

2 H O O + 2 H+ + 2 e− ADP ATP Chloroplast 2 2 + H

The LIGHT reactions take place in the thylakoid membranes

The CARBON-FIXING reactions take place in the chloroplast stroma

Adapted from Kramer, D.M., and Evans, J. R. (2010). The importance of energy balance in improving photosynthetic productivity. Plant Physiol.155: 70–78.

Prokaryotes Eukaryotes

Gloeobacter violaceus, Chlamydomonas the only cyanobacterium PLANT without thylakoid reinhardtii, a model green algae membranes Light-induced differentiation

Proplastid Chloroplast Synechocystis spp. PCC6803, a model cyanobacterium

Reproduced with permission © Annual Reviews of Plant Biology Nickelsen, J. and Rengstl, B. (2013).Photosystem II assembly: From cyanobacteria to plants. Annu. Rev. Plant Biol. 64: 609-635.

2 NADPH Cytochrome e− 2 H+ The reactions b6f complex 2 NADP+ require several ADP ATP large multi-protein Photosystem complexes: two I (PSI) light harvesting photosystems (PSI 2 H O O + 2 H+ + 2 e− 2 2 + and PSII), the H cytochrome b6f complex, and ATP Photosystem II (PSII) ATP synthase synthase

Adapted from Kramer, D.M., and Evans, J. R. (2010). The importance of energy balance in improving photosynthetic productivity. Plant Physiol.155: 70–78.

First step of photochemistry

Chl* e−

Chlorin ring Photon captures photons

H+

H2O + Chlorophyll is Chl Chl e− held in pigment- O2 Photon capture by protein + complexes in a chlorophyll excites the Chl is reduced by highly organized chlorophyll (Chl*). Chl* stripping an electron manner can lose an electron to from water, releasing become oxidized oxygen and protons chlorolphyll (Chl+)

Buchanan, B.B., Gruissem, W. and Jones, R.L. (2015) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists.

Photochemistry (first step in photosynthesis) Chl* Fluorescence Photon Non-photochemical quenching (e.g., heat)

3Chl* Alternatively, it can convert to a damaging triplet state

Chl

The fate of captured light energy and photosynthetic efficiency depend on many factors including temperature, water availability, nutrient availability, stress etc.

Light reactions The light reactions and carbon-fixing reactions are linked ADP + ATP NADP NADPH through pools of ATP/ADP and Carbon-fixing reactions NADPH/NADP+

For example…. • High temperature affects protein complex stability and thylakoid membrane fluidity • Low temperatures slow enzyme-catalyzed reactions

• Drought causes stomata to close, lowering CO2 uptake and carbon-fixing reactions • Nutrient deficiency or toxicity can affect electron transfer machinery

© 2015 American Society of Plant Biologists Oxygenic photosynthesis requires TWO photosystems Strong −1.5 PSII PSI P700* is a very strong reductant reductant – strong Reductants donate P700* electrons to other enough to donate species −1.0 electrons to NADP+

P680* e−

−0.5 NADP+ NADPH

Energy 0.0

Redox Redox potentialeV + 0.5 P700 4 e− P700 2 H2O Strong P680+ is a very strong oxidant 1.0 4 H+ O oxidant – strong enough to Oxidizers remove 2 P680+ electrons from other pull electrons from H2O species P680

Reductants donate P700* electrons to other species −1.0

P680* e−

−0.5 PQ NADP+ NADPH

Cyt b6f Energy 0.0

Redox Redox potentialeV PC + 0.5 P700 4 e− P700 2 H2O Strong oxidant 1.0 The electron transport chain 4 H+ O generates proton-motive force that Oxidizers remove 2 P680+ electrons from other drives ATP production species P680

Strong −1.5 PSII PSI reductant

Reductants donate P700* electrons to other species −1.0

P680* e−

−0.5 PQ NADP+ NADPH

Cyt b6f Energy 0.0

Redox Redox potentialeV PC 0.5 P700+ 4 e− P700 2 H2O Strong oxidant 1.0 The electron transport chain 4 H+ O generates proton-motive force that Oxidizers remove 2 P680+ electrons from other drives ATP production species P680

PSI Cyclic electron transport: P700* • Involves PSI • Does not involve PSII • Involves the electron transport chain • Results in ATP production PQ • Does not liberate O2 • Does not produce NADPH Cyt b6f

PC P700+ P700

The electron transport chain generates proton-motive force that drives ATP production

© 2015 American Society of Plant Biologists The photosystems are embedded in thylakoid membranes Plastid STROMA 2 NADPH LUMEN Cytochrome b6f (Cyt b6f) is a multiprotein e− 2 H+ membrane-embedded + complex 2 NADP STROMA

Thylakoid PQ Cyt b6f Membrane − 2 H O 4 e PSI 2 PSII PC Plastoquinone (PQ) is 4 H+ O 2 a small molecule and LUMEN mobile electron carrier Plastocyanin (PC) is a PQ small protein and mobile electron carrier

2 NADPH + − ATP 2 H e ADP + Pi ATP Synthase 2 NADP+ H+

Thylakoid Cyt b6f Membrane − 2 H O 4 e PSI 2 PSII

4 H+ O2 [H+] Proton gradient from high (in) to low (out)

Light-dependent reactions Carbon-fixing reactions Each CO2 fixed requires 3 ATP CO2 and 2 NADPH Rubisco ADP ATP Carboxylation Ribulose-1,5- bisphosphate NADPH

+ H+ H Calvin- NADP+ Benson Regeneration Cycle ATP Energy Glyceraldehyde 3- phosphate (GAP) input ADP + Pi

For every 3 CO fixed, one NADPH Reducing 2 1 x GAP power input GAP is produced for NADP+ + H+ biosynthesis and energy

Adapted from: Buchanan, B.B., Gruissem, W. and Jones, R.L. (2000) Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists.

Most There are two types of reaction centers, Type I & Type II photosynthetic Each type is found in various photosynthetic bacteria prokaryotes use Both types are found in cyanobacteria and chloroplasts only a single Type II are pheophytin- Type I are iron-sulfur type of reaction quinone reaction centers reaction centers center and do not release oxygen

Note that oxygenic photosynthesis PSI requires Type I & Type II PSII reaction centers working in series O2

Reprinted from Allen, J.P. and Williams, J.C. (1998). Photosynthetic reaction centers. FEBS Letters. 438: 5-9 with permission from Elsevier.

© 2015 American Society of Plant Biologists Type I & Type II reaction centers are broadly distributed in prokaryotes Several bacterial lineages Cyanobacteria, Type I have some photosynthetic chloroplasts members, indicating that lateral gene transfer has Type I + Type II played an important role in the Type II evolution of photosynthesis.

Type I

Type II

Reprinted from Macalady, J.L., Hamilton, T.L., Grettenberger, C.L., Jones, D.S., Tsao, L.E. and Burgos, W.D. (2013). Energy, ecology and the distribution of microbial life. Phil. Trans. Roy. Soc. B: 368: 20120383. by permission of the Royal Society. See also Blankenship, R.E. (2010). Early evolution of photosynthesis. Plant Physiol. 154: 434–438;

Phylum Discovery Reaction Pigments Colloquial name center Cyanobacteria 1800s Type I + Type II Chl a,b,c,d

Proteobacteria 1800s Type II BChl a,b Purple sulfur / nonsulfur bacteria

Chlorobi 1906 Type I BChl a,c, d, e; Chl a Green sulfur bacteria

Chloroflexi 1974 Type II BChl a,c Filamentous anoxygenic phototrophs

Firmicutes 1983 Type I BChl g; Chl a Heliobacteria Acidobacteria 2007 Type I BChl a,c

Adapted from Raymond, J. (2008). Coloring in the tree of life. Trends Microbiology. 16: 41-43.

Chlorobi Cyanobacteria Cyanobacteria Rhodopseudomonas

present

time

Gene duplication: PsA → PsaB

Ancestral

reaction past center Fe-S cluster as electron acceptor

With kind permission from Springer Science+Business Media Nelson, N. (2013). Evolution of photosystem I and the control of global enthalpy in an oxidizing world. Photosynth. Res. 116: 145-151.

Mammals Plants Oxygenic Algae photosynthesis Eukaryotes Cyanobacteria Origin of Phototropic bacteria? Earth Life 4 3 2 1 0 Billion years before present

Stromatolites are nearly 3 billion years old and may have been formed by oxygen- producing cyanobacteria

Adapted from Des Marais, D.J. (2000). When did photosynthesis emerge on Earth? Science. 289: 1703-1705 and Xiong, J. and Bauer, C.E. (2002). Complex evolution of photosynthesis. Annu. Rev. Plant Biol. 53: 503-521. NASA; Ruth Ellison

A single primary Ancestral endosymbiotic event* about cyanobacterium 1.5 billion years ago gave rise to chloroplasts in chlorophytes (green algae and plants), red algae, and glaucophytes *A second more recent event that gave rise to Paulinella Glaucophytes Rhodophytes Chlorophytes chromatophora is described later Green plants

This lesson focuses on Brown algae, photosynthesis as it diatoms, Secondary, tertiary dinoflagellates, endosymbiosis occurs in plants, green euglenoids…. algae and cyanobacteria

Reprinted with permission from Rumpho, M.E., Pelletreau, K.N., Moustafa, A. and Bhattacharya, D. (2011). The making of a photosynthetic animal. J. Exp. Biol. 214: 303-311.

A huge variety of photosynthetic euglenoids, diatoms, dinoflagellates and other algae are products of secondary (or tertiary) endosymbiosis; the engulfment of a primary (or secondary) endosymbiont

Reproduced with permission © Annual Reviews from Keeling, P.J. (2013). The number, speed, and impact of plastid endosymbioses in eukaryotic evolution. Annu. Rev. Plant Biol. 64: 583-607.

Eukaryotic Primary eukaryotic host amoeba host Plastid Nucleus

Rhodophytes chromatophores (Red algae) Mitochondrion

Free-living β-Cyanobacterium 5 μm Chlorophytes (Green algae) Photosynthetic amoeba Plants Paulinella chromatophora

Reprinted by permission from Macmillan Publishers Ltd: from Gould, S.B. (2012). Evolutionary genomics: Algae's complex origins. Nature. 492: 46-48. Reproduced with permission © Annual Reviews Gould, S.B., Waller, R. F., and McFadden, G. (2008). Plastid evolution. Annu. Rev. Plant Biol. 59: 491 – 517; See also Mackiewicz, P., Bodył, A. and Gagat, P. (2012). Possible import routes of proteins into the cyanobacterial endosymbionts/plastids of Paulinella chromatophora. Theory in Biosciences. 131: 1-18.

(Gould, Nature 2012) (Gould, Nature 2012) (Gould, Nature 2012) Summary: Evolution and diversity of photosynthesis

Photosynthetic Photosynthetic bacteria eukaryotes

Anoxygenic Oxygenic (H2O as electron donor)

Cyanobacteria

Purple Green Purple Green sulfur sulfur nonsulfur nonsulfur bacteria bacteria bacteria bacteria Primary Rhodophytes, endosymbiosis glaucophytes, Viridiplantae (plants, chlorophytes)

Brown algae, diatoms, Secondary, tertiary dinoflagellates, endosymbiosis euglenoids….

Light travels at a fixed speed, c (3 x 108 m/sec). Frequency (ν) is inversely proportional to wavelength (λ): Energy is proportional to frequency: E = hν and ν = c / λ inversely proportional to wavelength. Therefore, shorter wavelength light (e.g., UV) has higher energy than longer wavelength light

Wavelength 1 nm 1 μm 1 mm 1 m

Gamma rays UV IR Radio waves

X rays Visible Microwaves 400 500 600 Wavelength nm nm nm

High energy Low energy High frequency Low frequency Short wavelength Long wavelength

visible spectrum (400 – 700 nm)

sec)

- 2

Ultraviolet The sun emits light at a range of different wavelengths, but much of the very short wavelength

light is absorbed by Earth’s

mol photos / m μ

( atmosphere Spectra of hittinglightleaf 300 400 500 600 700 800 Wavelength (nm) The earth’s atmosphere blocks much of the short wavelength light

NASA

All chlorophyll-based photosynthesis systems use chlorophyll a Chlorophyll a Different antenna systems use

different subsets of accessory Chlorophyll b pigments which expand the range of β-carotene light absorbed Phycoerythrin

Phycocyanin pigments • Chlorophyll b is found in land Accessory plants, green algae and

cyanobacteria (normalized)

• Carotenoids are found in all Absorption spectra of spectra Absorption

chlorophyll-based photosynthesis pigments photosynthetic systems • Phycoerythrin are found in cyanobacteria and non-green 300 400 500 600 700 800 algae, and phycocyanin in Wavelength (nm) cyanobacteria

In cyanobacteria, accessory pigments are arranged in phycobilisomes Antenna pigments transfer light energy to the reaction center

Antenna complex Photosystem Antenna complex In green algae and plants accessory pigments are embedded in the thylakoid membranes

Govindjee and Shevela, D. (2011). Adventures with cyanobacteria: a personal perspective. Frontiers in Plant Science. 2: 28; Reprinted by permission from Macmillan Publishers Ltd: Scholes, G.D., Fleming, G.R., Olaya-Castro, A. and van Grondelle, R. (2011). Lessons from nature about solar light harvesting. Nat. Chem. 3: 763-774.

Absorbance The photosynthetic action spectrum of a spectrum shows the rate of green plant photosynthesis that occurs when a single wavelength of light shines on a plant Absorbance spectra Chlorophyll a A different action spectrum can be Chlorophyll b measured in other organisms as a β -carotene function of their accessory pigments 300 400 500 600 700 800 Wavelength (nm)

Reaction center chlorophylls absorb maximally at 680 and 700 nm. Longer wavelength light does not have sufficient energy to drive photosynthesis in plants

Tetrapyrrole ring with Mg in center

Chlorophyll a

Chlorophyll b β-carotene Chlorophyll a Phycoerythrin

Phycocyanin pigments

Accessory Accessory

Absorption spectra of spectra Absorption photosynthetic pigments photosynthetic

300 400 500 600 700 800 Wavelength (nm)

Phycocyanobilin β-carotene (linear tetrapyrrole)

different absorption spectra

Bacteriochlorophyll a

Bacteriochlorophyll b

sec)

- 2

mole photons / m

( Spectra of hittinglightleaf 300 400 500 600 700 800 Wavelength (nm) Small changes in side chains are sufficient to change the absorption spectra of (bacterio)chlorophylls

Protoporphyrin IX is a precursor of Mg-containing chlorophyll and Fe-containing heme

Protochlorophyllide Chlorophyllide Attaches phytyl tail Chlorophyll synthase

Rissler, H.M., Collakova, E., DellaPenna, D., Whelan, J. and Pogson, B.J. (2002). Chlorophyll biosynthesis. Expression of a second Chl I gene of magnesium chelatase in Arabidopsis supports only limited chlorophyll synthesis. Plant Physiol. 128: 770-779; Yamazaki, S., Nomata, J. and Fujita, Y. (2006). Differential operation of dual protochlorophyllide reductases for chlorophyll biosynthesis in response to environmental oxygen levels in the cyanobacterium Leptolyngbya boryana. Plant Physiol. 142: 911-922.

Why does the rate of At low light CO2 consumption level intensities the off at higher light relationship intensities? between CO2 consumption and light intensity is linear. Why?

At low light intensities, why is there is a production (negative consumption) of CO2?

Skillman, J.B. (2008). Quantum yield variation across the three pathways of photosynthesis: not yet out of the dark. J. Exp. Bot. 59: 1647-1661 by permission of Oxford University Press .

© 2015 American Society of Plant Biologists Quantifying photosynthesis: The light response curve At low light intensities, As light intensity increases photosynthesis is light above the light saturation limited, so as more point, photosynthetic reaction photons are absorbed rate is determined by light- more CO2 is fixed independent reactions

Plants have mitochondria

and respire, consuming O2 and producing CO2. In the light they are net CO2 consumers, but in the dark production is greater than This is the light compensation point: consumption The amount of light needed to balance photosynthetic CO2 consumption to respiratory CO2 production

Skillman, J.B. (2008). Quantum yield variation across the three pathways of photosynthesis: not yet out of the dark. J. Exp. Bot. 59: 1647-1661 by permission of Oxford University Press .

In this study, the quantum What factors yield is 0.05 mol CO2 fixed influence per mol absorbed photons quantum yield? (the slope of the line)

Note that the quantum yield can only be measured in light- limiting conditions where the relationship between

absorbed light and CO2 fixation is linear

Skillman, J.B. (2008). Quantum yield variation across the three pathways of photosynthesis: not yet out of the dark. J. Exp. Bot. 59: 1647-1661 by permission of Oxford University Press .

In this study, the quantum What factors yield is 0.05 mol CO2 fixed influence per mol absorbed photons quantum yield? (the slope of the line) • Light absorbance by photosynthetic vs non-photosynthetic pigments Note that the quantum • Balance in excitation yield can only be measured in light- energy between PSI limiting conditions where and PSII the relationship between • (For CO yield, absorbed light and CO2 2 fixation is linear activities of downstream processes) • Temperature

Skillman, J.B. (2008). Quantum yield variation across the three pathways of photosynthesis: not yet out of the dark. J. Exp. Bot. 59: 1647-1661 by permission of Oxford University Press .

© 2015 American Society of Plant Biologists Plastids and chloroplasts: Essential organelles for most plant cells Plant cell (outlined) with Envelope (double many green chloroplasts A single chloroplast membrane, resembles prokaryotic membranes) with specialized transporters Stroma Thylakoid membranes

Besides photosynthesis, several metabolic pathways occur in plastids including N and S assimilation, and the synthesis of secondary metabolites, pigments, and hormones

Kristian Peters; Louisa Howard, Dartmouth microscopy facility; and3k and caper437

Seeds, embryonic, Dark grown meristems and photosynthetic reproductive tissue tissues Leaf Flower, fruit

Storage of starch, oils and proteins

Immage credit LadyofHats; see also Sakamoto W., Miyagishima S., and Jarvis P. (2008). Chloroplast Biogenesis: Control of Plastid Development, Protein Import, Division and Inheritance. The Arabidopsis Book 6:e0110. doi:10.1199/tab.0110

flagella These images show the thylakoids (green), chloroplast starch grains (brown) and a special region called the pyrenoid (py) in which carbon- fixing reactions take place nucleus

Engel, B.D., Schaffer, M., Kuhn Cuellar, L., Villa, E., Plitzko, J.M. and Baumeister, W. (2015). Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography. eLife. 4: e04889.

DARK

Prolamellar body Transition to in etioplast lamellar layers

Primary lamellar layers

Grana layers Pribil, M., Labs, M. and Leister, D. (2014). Structure and dynamics of thylakoids in land plants. J. Exp. Bot. 65: 1955-1972 by permission of Oxford University Press . Von Wettstein, D., Gough, S. and Kannangara, C.G. (1995). Chlorophyll biosynthesis. Plant Cell. 7: 1039-1057..

Grana

Grana stacks

Membranes at the margins are non- appressed (green) and those within the grana stacks (red) are appressed. Different complexes are found in appressed vs non- appressed regions of the thylakoids

Louisa Howard, Austin, J.R., et al and Staehelin, L.A. (2006). Plastoglobules Are lipoprotein subcompartments of the chloroplast that are permanently coupled to thylakoid membranes and contain biosynthetic enzymes. Plant Cell. 18: 1693-1703; see also Staehelin, L.A. (1976). Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro. J. Cell Biol. 71: 136-158.

lumen State 1 PSII

Cyt b f PSII packing into appressed membranes 6 LHCII

PSII is mainly found in appressed regions, Grana ATP synthase and PSI in ATP synthase b f unappressed regions, and Cyt 6 PSI-LHCI supercomplex Cyt b6f is distributed throughout

Daum, B., et al., (2010). Arrangement of Photosystem II and ATP synthase in chloroplast membranes of spinach and pea. Plant Cell. 22: 1299-1312; Nagy, G., et al. and Minagawa, J. (2014). Chloroplast remodeling during state transitions in Chlamydomonas reinhardtii as revealed by noninvasive techniques in vivo. Proc. Natl. Acad. Sci. USA. 111: 5042-5047.

The first step of photosynthesis is light capture by pigments in thylakoid membranes of chloroplasts.

β-carotene

Stroma (electro- negative side)

Lumen (electro- positive side)

Cytochrome Photosystem II Photosystem I b6f complex

Reprinted by permission from Baniulis, D., Yamashita, E., Zhang, H., Hasan, S.S. and Cramer, W.A. (2008). Structure–Function of the Cytochrome b6f Complex†. Photochemistry and Photobiology. 84: 1349-1358.

complexes, PSII, Cyt b6f & PSI

e− e−

e−

e− e− H2O O2 H+

Cytochrome Photosystem II Photosystem I b6f complex

PSII is a multi-protein Dimer structure of PSII The conserved complex that functions as a reaction center dimer. This diagram core is made of representsConserved a monomer reaction up proteins D1 center core and D2, and inner-antenna H O CP43 CP47 proteins CP43 2 and CP47 + 1/2 O2 + 2 H D2 D1 The oxygen-evolving

Mn4CaO5 cluster is on the luminal side and shielded by the more divergent extrinsic proteins Divergent extrinsic proteins

Reprinted by permission from Calderone, V., Trabucco, M., Vujičić, A., Battistutta, R., Giacometti, G.M., Andreucci, F., Barbato, R. and Zanotti, G. (2003). Crystal structure of the PsbQ protein of Photosystem II from higher plants. EMBO reports. 4: 900-905; Reprinted with permission © Annual Reviews Nickelsen, J. and Rengstl, B. (2013).Photosystem II assembly: From cyanobacteria to plants. Annu. Rev. Plant Biol. 64: 609 – 634.

Cyanobacteria & red algae harvest light through peripheral antenna systems

The peripheral antenna and light- harvesting complex (LHC) are different between Same core Plants and green algae cyanobacteria and harvest light through chloroplasts membrane-embedded light harvesting complexes

LHCII

Reprinted with permission © Annual Reviews Nickelsen, J. and Rengstl, B. (2013).Photosystem II assembly: From cyanobacteria to plants. Annu. Rev. Plant Biol. 64: 609 – 634.

© 2015 American Society of Plant Biologists Proteins in PSII can be characterized by SGC, SDS-PAGE and EM Protein complexes can The subunit composition of each be separated by sucrose band is determined by SDS-PAGE

gradient centrifugation

Lighter

EM can also reveal complex composition

Heavier

Reprinted with permission from Caffarri, S., Kouřil, R., Kereïche, S., Boekema, E.J. and Croce, R. (2009). Functional architecture of higher plant Photosystem II supercomplexes. EMBO J. 28: 3052-3063.

© 2015 American Society of Plant Biologists Proteins’ roles are to orient and position pigment molecules Numerous chlorophylls, β carotenes and other small molecules are held in position by PSII proteins

Neveu,Curtis

(1) Light converts reaction center chlorophyll (P680) STROMA to excited form P680* PSII (2) Electron leaves P680*, forming P680+ (photo- oxidation, charge separation) (3) The electron is transferred to Pheophytin (Pheo), forming Pheo− − − (4) Pheo passes the electron to QA to produce QA (5) Q − passes the electron to Q to produce Q − (5) A B B (4) (5) − QB → QB e-− (4) − QA → QA (3) e-− (3) Pheo → Pheo− e-− P680 → P680* → P680+ (1) (2) LUMEN

2 H+ Diffusion through lipid − 2 e bilayer to Cyt b6f

PQH diffuses Plastoquinone (PQ) Plastoquinonol (PQH ) 2 2 through the lipid at Q site of PSII B bilayer, carrying protons and Plastoquinone (PQ) at the QB site electrons. is reduced to PQ2 − which picks up to protons from the stroma to

form PQH2 (plastoquinol)

See Cramer, W. A., Hasan, S.S., and Yamashita, E. (2011). The Q cycle of cytochrome bc complexes: A structure perspective. Biochim. Biophys. Acta - Bioenerg. 1807: 788–802.

The OEC’s catalytic center core is an inorganic

Mn4CaO5 cluster which performs the mechanistically-challenging reaction of removing four tightly-bound electrons and four protons from water to form dioxygen

LUMEN

Reprinted from Vogt, L., Vinyard, D.J., Khan, S. and Brudvig, G.W. (2015). Oxygen-evolving complex of Photosystem II: an analysis of second-shell residues and hydrogen-bonding networks. Curr. Opin. Chem. Biol. 25: 152-158 with permission from Elsevier. Neveu,Curtis

PSII To Cyt c6f

− 2− PQB / PQB / PQB e−

− PQA / PQA e− Pheo / Pheo−

e−

Luminal side Chl / Chl+

e− H2O O2 H+

Reprinted from Senge, M. O., Ryan, A. A., Letchford, K. A., MacGowan, S. A., & Mielke, T. (2014). Chlorophylls, symmetry, chirality, and photosynthesis. Symmetry. 6: 781-843

LHCII trimers Each monomer of LHCII (green) from spinach includes one surrounding polypeptide, 14 chlorophylls PSII dimers and four carotenoids (grey)

The rate of energy transfer from LHCII to the reaction center is regulated in response to light environment, metabolic state etc.

Reprinted by permission from Macmillan Publishers Ltd Liu, Z., Yan, H., Wang, K., Kuang, T., Zhang, J., Gui, L., An, X. and Chang, W. (2004). Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution. Nature. 428: 287-292; see also Drop, B., Webber-Birungi, M., Yadav, S.K.N., Filipowicz-Szymanska, A., Fusetti, F., Boekema, E.J. and Croce, R. (2014). Light-harvesting complex II (LHCII) and its supramolecular organization in Chlamydomonas reinhardtii. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1837: 63-72..

PSII Reaction Center PSI Reaction Center

Cytochrome b6f complex Stroma (electro- negative side)

Lumen (electro- positive side)

Mobile plastoquinone

(PQH2) carries electrons from PSII to Cyt b6f

Reprinted by permission from Baniulis, D., Yamashita, E., Zhang, H., Hasan, S.S. and Cramer, W.A. (2008). Structure–Function of the Cytochrome b6f Complex†. Photochem. Photobiol 84: 1349-1358.

© 2015 American Society of Plant Biologists Stroma Cyt b6f structure Cyt b6f is made from eight (from cyanobacteria) polypeptides Electrons are transferred Lumen including Cyt from PQH through Cyt b f b6 and Cyt f 2 6 and ultimately passed to plastocyanin (PC). This is accompanied by the transport Redox centers include four of H+ from stroma to lumen hemes and an Fe2S2 cluster Stroma

PQH2 H+ From − PSII e Cyt b6f Lumen

e− PC To PSI

Reprinted from Hasan, S.S., Yamashita, E., Baniulis, D. and Cramer, W.A. (2013). Quinone-dependent proton transfer pathways in the photosynthetic cytochrome b6f complex. Proc. Natl. Acad. Sci. USA 110: 4297- 4302; See also Tikhonov, A. (2013). pH-Dependent regulation of electron transport and ATP synthesis in chloroplasts. Photosynth. Res. 116: 511-534.. Stroebel, D., Choquet, Y., Popot, J.-L. and Picot, D. (2003). An atypical haem in the cytochrome b6f complex. Nature. 426: 413-418.

Plastoquinone (PQ) is reduced to

plastoquinol (PQH2), then oxidized to plastoquinone (it cycles). This involves oxidation–reduction and deprotonation–protonation, which couples proton translocation and electron transfer

PQ PQH2

First half Q cycle Second half Q cycle Cycle completion

PQH2 delivers two protons to Another PQH2 delivers two protons to lumen, one lumen, one electron to PC. electron to PC. PQ, two electrons and two

PQ returns to PSII protons regenerate PQH2 which cycles again

+ Cyt b6f H

e− e− PQ − PQ PQ

e e− e−

PSII e− PSII e− PQH PQH PQH2 2 PC 2 PC

2 H+ 2 H+

PSII Reaction Center PSI Reaction Center

Cytochrome b6f complex Stroma (electro- negative side)

Lumen (electro- positive side)

Electrons are passed from Cyt b6f to PSI by plastocyanin (PC) or

sometimes in algae Cyt c6 Reprinted by permission from Baniulis, D., Yamashita, E., Zhang, H., Hasan, S.S. and Cramer, W.A. (2008). Structure–Function of the Cytochrome b6f Complex†. Photochemistry and Photobiology. 84: 1349-1358.

PSI is a complex of 17 protein subunits, dominated by PsaA and PsaB, and 178 prosthetic groups,

mainly chlorophyll

Reprinted from Amunts, A. and Nelson, N. (2009). Plant photosystem I design in the light of evolution. Structure. 17: 637-650 with permission from Elsevier.

Stroma To NADPH Electron transport chain Ferredoxin e−

Fe4S4 (x3) e−

Phylloquinone e− Lumen Chlorophyll PSI complex showing Plastocyanin e− proteins and electron transport chain

Reprinted by permission from Macmillan Publishers Ltd Jordan, P., Fromme, P., Witt, H.T., Klukas, O., Saenger, W. and Krausz, N. (2001). Three-dimensional structure of cyanobacterial photosystem I at 2.5 Å resolution. Nature. 411: 909-917, © 2015 David Goodsell & RCSB Protein Data Bank See also . Amunts, A., Drory, O. and Nelson, N. (2007). The structure of a plant photosystem I supercomplex at 3.4 Å resolution. Nature. 447: 58-63

Looking at the complex from the lumen; the membrane is in the plane of the slide

Proteins Pigments PSI core

Chlamydomonas Two rows of LHCI

Pea complex Single row of LHCI LHCI

Reprinted from Mazor, Y., Borovikova, A. and Nelson, N. (2015). The structure of plant photosystem I super-complex at 2.8 Å resolution. eLife. 4: e07433. Minagawa, J. (2013). Dynamic reorganization of photosynthetic supercomplexes during environmental acclimation. Front. Plant Sci. 4: 513.

From PSI Ferredoxin (Fd) Ferredoxin: e- NADP+ reductase

H+ + 2 e−

Ferredoxin is a small iron- Fe S containing 2 2 NADP+ NADPH Fe2S2 protein

Reduced Fd can also pass electrons to other enzymes

Reprinted from Mulo, P. (2011). Chloroplast-targeted ferredoxin-NADP+ oxidoreductase (FNR): Structure, function and location. Biochim. Biophy. Acta (BBA) - Bioenergetics. 1807: 927-934 with permission from Elsevier.

ATP couples the dissipation of the proton gradient to ATP synthesis

ATP ADP + Pi ATP Synthase H+

+ H+ + H+ H H + + + + H H H H+ H H+ Thylakoid lumen

Daum, B., Nicastro, D., Austin, J., McIntosh, J.R. and Kühlbrandt, W. (2010). Arrangement of Photosystem II and ATP synthase in chloroplast membranes of spinach and pea. Plant Cell. 22: 1299-1312.

The F1 subunit has three α and Rotary mechanism of ATP three β subunits, synthase Video link and one each of γ, δ and ε

Stalk 2 nm

F0 subunit: 10 – 15 copies of subunit c assembled as a ring Electron flow through F0 turns the within the thylakoid central stalk subunit generating membrane torque to energize ATP synthesis

Reprinted by permission from Macmillan Publishers Ltd: from Abrahams, J.P., Leslie, A.G.W., Lutter, R. and Walker, J.E. (1994). Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria. Nature. 370: 621-628.; Seelert, H., Poetsch, A., Dencher, N.A., Engel, A., Stahlberg, H. and Muller, D.J. (2000). Structural biology: Proton-powered turbine of a plant motor. Nature. 405: 418-419. See also Groth, G. and Pohl, E. (2001).

The structure of the chloroplast F1-ATPase at 3.2 Å resolution. J. Biol. Chem. 276: 1345-1352.

ATP Synthase

Video link

The efforts of hundreds of scientists across several decades have revealed detailed structures and mechanisms for each of the unusual, important, and beautiful photosynthetic complexes, comprising dozens of proteins and hundreds of pigments and other cofactors

Reprinted by permission from Baniulis, D., Yamashita, E., Zhang, H., Hasan, S.S. and Cramer, W.A. (2008). Structure–Function of the Cytochrome b6f Complex. Photochemistry and Photobiology. 84: 1349-1358.

Linear electron transport Cyclic electron transport Water-water cycle

• Electrons transferred • Electrons cycle with no • Electrons transferred from H O to H O from H2O to NADPH net production of NADPH 2 2 • Involves PSII, Cyc b f, • Involves PSII, Cyc b6f, • Involves Cyc b6f and PSI 6 and PSI • Generates pmf for ATP and PSI • Generates NADPH and synthesis but not NADPH • Generates pmf for ATP pmf for ATP synthesis synthesis but not NADPH H+ H+ H+ NADPH H2O

H+ H+ H+

H2O H2O

LET (diagrammed by the Z-scheme) is only one possible electron transport pathway. It is the only scheme that results in NADPH, but other schemes can produce ATP and protect against photodamage

The Mehler reaction or water-water cycle is thought to contribute to Cyclic electron transport photoprotection (CET) balances production of ATP and NADPH and helps alleviate photodamage Reprinted by permission from Baniulis, D., Yamashita, E., Zhang, H., Hasan, S.S. and Cramer, W.A. (2008). Structure–Function of the Cytochrome b6f Complex†. Photochem. Photobiol. 84: 1349-1358.

+ H2O  PSII  PQ  Cyt b6f  PC PSI  Fd  NADP  NADPH

Ferredoxin e− e−

e−

e- Plastoquinone e− 2 H2O O2 4 H+ Plastocyanin Cytochrome Photosystem II Photosystem I b6f complex Reprinted by permission from Baniulis, D., Yamashita, E., Zhang, H., Hasan, S.S. and Cramer, W.A. (2008). Structure–Function of the Cytochrome b6f Complex†. Photochem. Photobiol. 84: 1349-1358.

© 2015 American Society of Plant Biologists Stoichiometry of electron transport yields 1. Assuming a quantum 5. The Calvin-Benson efficiency of 0.8, for 2… 4 e− are cycle requires 3 ATP each 5 photons required to for each 2 NADPH captured by PSII, 4 e− reduce + enter LEF from PSII… 2 NADP 6. ATP synthase + + 2 H to requires 14 H+ to NADPH… produce 3 ATP

3… and 4 H+ are released by the + oxidation of H O 7. Therefore, at least 2 H 2 must be contributed by 4. The 4 e− from PSII lead CEF to meet the to 8 H+ released into the requirements of the lumen during the Q cycle Calvin-Benson cycle

Minagawa, J. (2013). Dynamic reorganization of photosynthetic supercomplexes during environmental acclimation. Front. Plant Sci. 4: 513.

pass from PSI to Cyt b6f

As a result, protons are moved into the lumen (powering ATP synthesis) but electrons are not passed to NADP+. Thus, cyclic electron transport alters the ratio of ATP to NADPH produced as compared to linear electron transport. It also alleviates photoinhibition.

Reprinted from Shikanai, T. (2014). Central role of cyclic electron transport around photosystem I in the regulation of photosynthesis. Curr. Opin. Biotechnol. 26: 25-30 by permission from Elsevier..

(1) PSI  Fd PQ  Cyt b6f PC PSI

+ (2) PSI  Fd NADP  PQ  Cyt b6f PC PSI

Scheme 1 requires PGR5 (PROTON GRADIENT REGULATED5) and PGRL1 (PGR-LIKE1) Scheme 2 involves NAD(P)H dehydrogenase (NDH)

Reprinted from Shikanai, T. (2014). Central role of cyclic electron transport around photosystem I in the regulation of photosynthesis. Curr. Opin. Biotechnol. 26: 25-30 by permission from Elsevier..

H2O  PSII  PQ  Cyt b6f PC  PSI  O2 H2O2  H2O SOD APX − H O O2 O2· 2 2 H2O e− e− SOD = superoxide dismutase

e− APX = ascorbate peroxidase

Like CET, the water-water cycle - produces ATP but not NADPH. It e − e usually is restricted to the early period 2 H2O after the transition from dark to light O2 4 H+ when reductant cannot be used because the intermediates of the Calvin Benson cycle have not built up. Cytochrome Photosystem II Photosystem I b6f complex Reprinted by permission from Baniulis, D., Yamashita, E., Zhang, H., Hasan, S.S. and Cramer, W.A. (2008). Structure–Function of the Cytochrome b6f Complex†. Photochem. Photobiol. 84: 1349-1358.

reduced PQH2 by reducing O2

Chlororespiration Water- alleviates excitation water pressure on electron Chloro- cycle transport respiration

Reprinted with permission from Rumeau, D., Peltier, G. and Cournac, L. (2007). Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response. Plant Cell Environ. 30: 1041-1051. See also Nawrocki, W.J., Tourasse, N.J., Taly, A., Rappaport, F. and Wollman, F.-A. (2015). The plastid terminal oxidase: Its elusive function points to multiple contributions to plastid physiology. Annu. Rev. Plant Biol. 66: 49 -74.

FLV1/FLV3 carry O2 H2O FLV2/FLV4 function out a Mehler-like in alternative reaction that FLV1/FLV3 electron transfer regenerates NADP+ and alleviate PSII excitation pressure NADP+ NADPH

FNR H+

FLV2/FLV4

H+

H2O

Reprinted with permission from Allahverdiyeva, Y., Suorsa, M., Tikkanen, M. and Aro, E.-M. (2015). Photoprotection of photosystems in fluctuating light intensities. J. Exp. Bot. 66: 2427-2436. Zhang, P., Eisenhut, M., Brandt, A.-M., Carmel, D., Silén, H.M., Vass, I., Allahverdiyeva, Y., Salminen, T.A. and Aro, E.-M. (2012). Operon flv4-flv2 provides cyanobacterial photosystem II with flexibility of electron transfer. Plant Cell. 24: 1952-1971.

In linear electron transport,

water (H2O) is the electron RUBISCO donor and NADP+ the electron acceptor. 3 GAP In cyclic electron transport, electrons transferred from PSI are returned to PSI.

The water-water cycle is a variation in which electrons transferred from water are

passed to O2, reducing it to H2O.

The relative contributions of each are determined by metabolic supply and demand

At high light intensities, excess light can damage photosynthetic machinery

Optimal conditions

Rate Rate of photosynthesis Stressed conditions At low light intensities, light intensity is the The metabolic and dominant factor physiological state of limiting photosynthesis the plant or cell determines how much light is “too much”

Light intensity

Adapted from Li, Z., Wakao, S., Fischer, B.B. and Niyogi, K.K. (2009). Sensing and responding to excess light. Annu. Rev.Plant Biol. 60: 239-260.

Photon-excited chlorophyll forms excited singlet chlorophyll 1Chl* NPQ 1Chl* can return to its ground state through: P Photochemistry F Fluorescence D Dissipation (e.g., heat, NPQ)

Alternatively, it can convert to the excited triplet state 3Chl* which can transfer energy to oxygen to produce singlet oxygen with subsequent damage due to reactive oxygen species

Reprinted by permission from Macmillan Publishers Ltd Demmig-Adams, B. and Adams, W.W. (2000). Photosynthesis: Harvesting sunlight safely. Nature. 403: 371-374.

Decrease light incidence through dynamic changes to antenna complex A plant defective in energy dissipation is susceptible to high-light induced bleaching and death

Antenna complex Release excess energy as heat or Reaction fluorescence center

lutein Detoxify reactive oxygen side- zeaxanthin products of excess excitation energy (e.g., antioxidant production)

Li, Z., Ahn, T.K., Avenson, T.J., Ballottari, M., Cruz, J.A., Kramer, D.M., Bassi, R., Fleming, G.R., Keasling, J.D. and Niyogi, K.K. (2009). Lutein accumulation in the absence of zeaxanthin restores nonphotochemical quenching in the Arabidopsis thaliana npq1 mutant. Plant Cell. 21: 1798-1812. Havaux, M. and Niyogi, K.K. (1999). The violaxanthin cycle protects plants from photooxidative damage by more than one mechanism. Proc. Natl. Acad. Sci. USA. 96: 8762-8767; Adapted from Li, Z., Wakao, S., Fischer, B.B. and Niyogi, K.K. (2009). Sensing and responding to excess light. Annu. Rev.Plant Biol. 60: 239-260

Top view Side view Leaf reorientation or curling are

adaptations to minimize light damage. Leaf reorientation can also decrease Lowlight heat damage

Arctostaphylos patula

Chloroplasts move to sides of cells to decrease light High lightHigh interception Selaginella Eucalyptus rossii lepidophylla

Oikawa, K., Kasahara, M., Kiyosue, T., Kagawa, T., Suetsugu, N., Takahashi, F., Kanegae, T., Niwa, Y., Kadota, A. and Wada, M. (2003). CHLOROPLAST UNUSUAL POSITIONING1 is essential for proper chloroplast positioning. Plant Cell. 15: 2805-2815. Donald Hoburn; Walter Siegmund; Kristian Peters

Low light: More light harvesting complexes (LHCII) High light: Greater photosynthetic capacity (PSII etc) Also more photoprotection

Arabidopsis grown in low (black bars) or high (white bars). Chloroplast components relative to total leaf chlorophyll

Reprinted by permission from Walters, R.G. (2005). Towards an understanding of photosynthetic acclimation. J. Exp. Bot. 56: 435-447.

1Chl*

Photon Non-photochemical quenching (NPQ)

“Non-photochemical quenching” encompasses several components: Chl qE = Energy-dependent quenching: The xanthophyll cycle qT = State transition: Conformational changes in LHCII qI = Photoinhhibition: Light-induced reduction in quantum yield as a consequence of damage

LHC PSII LHC PSII [H+] Unquenched: Energy-dependent VDE 2. VDE converts High efficiency violaxanthin to zeaxanthin, quenching: + [H ] + transfer of light 1. Lumen acidification [H ] leading to light energy energy to PSII activates Violaxanthin dissipation in LHCII reaction center [H+] De-epoxidase [H+]

Zeaxanthin Violaxanthin epoxidase de-epoxidase (ZE) (VDE)

High light / low luminal pH induces VDE, which catalyzes the conversion of violaxanthin to zeaxanthin In low light / high luminal pH, the reaction is reversed by ZE

Hieber, A.D., Kawabata, O. and Yamamoto, H.Y. (2004). Significance of the lipid phase in the dynamics and functions of the xanthophyll cycle as revealed by PsbS overexpression in tobacco and in- vitro de-epoxidation in monogalactosyldiacylglycerol micelles. Plant Cell Physiol 45: 92-102 by permission of Oxford University Press.

Zeaxanthin accumulation (due to VDE activation) causes a rearrangement of LHCII and RCII, which decreases light transfer to RCII

The structural changes cause more light energy to be dissipated as heat

Reprinted with permission from Ruban, A.V. (2015). Evolution under the sun: optimizing light harvesting in Photosynthesis. J. Exp. Bot. 66: 7 – 23; Müller-Moulé, P., Conklin, P.L. and Niyogi, K.K. (2002). Ascorbate Deficiency Can Limit Violaxanthin De-Epoxidase Activity in Vivo. Plant Physiol. 128: 970-977.

© 2015 American Society of Plant Biologists Zeaxanthin and lutein also have roles as antioxidants and in photoprotection Chlamydomonas mutants deficient in zeaxanthin and lutein production are more susceptible to photo-oxidation

Interestingly, these two xanthophyll pigments (obtained from dietary sources) also protect human eyes from phototoxic damage by accumulating in the macula (orange color)

Niyogi, K.K., Björkman, O. and Grossman, A.R. (1997). The roles of specific xanthophylls in photoprotection. Proc. Natl. Acad. Sci. USA 94: 14162-14167.

When PSII, PSI and downstream metabolism are balanced, the PQ pools is distributed between PQ

(oxidized) and PQH2 (reduced)

High light (or light that favors PSII) or conditions that decrease downstream metabolism lead to an over-reduction

of the PQ pool

Rosso, D., Bode, R., Li, W., Krol, M., Saccon, D., Wang, S., Schillaci, L.A., Rodermel, S.R., Maxwell, D.P. and Hüner, N.P.A. (2009). Photosynthetic redox imbalance governs leaf sectoring in the Arabidopsis thaliana variegation mutants immutans, spotty, var1, and var2. Plant Cell. 21: 3473-3492.

LHCII kinase phosphorylates LHCII. Some LHCII relocates to PSI

Accumulation of PQH2 activates LHCII kinase

Minagawa, J. (2013). Dynamic reorganization of photosynthetic supercomplexes during environmental acclimation. Front. Plant Sci. 4: 513.

ATP synthase LHCII Cyt b f trimer 6 PSI-LHCI (quenched) supercomplex State 2

A current model indicates that state transitions balance PSII and PSI mainly by quenching LHCII energy transfer to PSII

Nagy, G., Ünnep, R., et al.. and Minagawa, J. (2014). Chloroplast remodeling during state transitions in Chlamydomonas reinhardtii as revealed by noninvasive techniques in vivo. Proc. Natl. Acad. Sci. USA 111: 5042-5047. See also Ünlü, C., Drop, B., Croce, R. and van Amerongen, H. (2014). State transitions in Chlamydomonas reinhardtii strongly modulate the functional size of Photosystem II but not of photosystem I. Proc. Natl. Acad. Sci. USA 111: 3460-3465.

The D1 protein of PSII is susceptible to photodamage, and when its rate of damage exceeds the rate of repair, photosynthesis is inhibited.

Reprinted by permission from Takahashi, S. and Murata, N. (2008). How do environmental stresses accelerate photoinhibition? Trends Plant Sci 178-182.

Reprinted by permission from Nath, K., Jajoo, A., Poudyal, R.S., Timilsina, R., Park, Y.S., Aro, E.-M., Nam, H.G. and Lee, C.H. (2013). Towards a critical understanding of the Photosystem II repair mechanism and its regulation during stress conditions. FEBS Lett. 587: 3372-3381.

Photosynthetic When supply > demand, control of elevated NADPH & ATP levels NPQ plastoquinol feed back and induce reoxidation photoprotection (red arrows)

pH-induced promotes conversion of violaxanthin (V) Metabolic imbalances, drought, into zeaxanthin (Z) cold, pathogen infection and other factors can decrease flux through the Calvin-Benson cycle

Reprinted with permission from Schöttler, M.A., Tóth, S.Z., Boulouis, A. and Kahlau, S. (2015). Photosynthetic complex stoichiometry dynamics in higher plants: biogenesis, function, and turnover of ATP synthase and the cytochrome b6f complex. J. Exp. Bot. 66: 2373-2400.

Photochemistry Metabolism Growth Translatome Transcriptome

Electron transport affected Protein degradation and PSII repair Δ pH Changes in antenna size Reduced PQ pool Increased xanthophyll pool Changes in PSI/ PSII stoichiometry Seconds Minutes Hours Days

NPQ kinetics 10 – 200 s 10 – >30 min Dark qE qI 10 – 60 s 10 – 60 min qT

Adapted from Jahns, P. and Holzwarth, A.R. (2012). The role of the xanthophyll cycle and of lutein in photoprotection of Photosystem II. Biochim. Biophys. Acta Bioenergetics. 1817: 182-193. Dietz, K.-J. (2015). Efficient high light acclimation involves rapid processes at multiple mechanistic levels. J. Exp. Bot. 66: 2401-2414. Erickson, E., Wakao, S. and Niyogi, K.K. (2015). Light stress and photoprotection in Chlamydomonas reinhardtii. Plant J. 82: 449-465.

High temperatures Light reactions Nutrient deficiency increase membrane or toxicity affects permeability and ADP ATP NADP+ electron transport decrease proton- NADPH motive force Carbon-fixing reactions Cold temperature slows enzyme- Drought-induced catalyzed reactions stomatal closure

prevents CO2 uptake

Retrograde signals Possible signals include compounds are biogenic (during derived from heme biosynthesis, development) and ROS, nucleotide derivatives and operational (during many more function)

Reprinted with permission from Macmillan Publishing Ltd from Jarvis, P. and López-Juez, E. (2013). Biogenesis and homeostasis of chloroplasts and other plastids. Nat Rev Mol Cell Biol. 14: 787-802; see also Dietz, K.-J. (2015). Efficient high light acclimation involves rapid processes at multiple mechanistic levels. J. Exp. Bot. 66: 2401-2414l Chi, W., Sun, X. and Zhang, L. (2013). Intracellular signaling from plastid to nucleus. Annu. Rev. Plant Biol. 64: 559-582; Estavillo, G.M., Chan, K.X., Phua, S.Y. and Pogson, B.J. (2013). Reconsidering the nature and mode of action of metabolite retrograde signals from the chloroplast. Frontiers Plant Sci. 3: 300.

Although the photosynthetic machinery is susceptible to photoinhibitory damage, it also has several strategies for photoprotection

Reprinted from Takahashi, S. and Badger, M.R. (2011). Photoprotection in plants: A new light on Photosystem II damage. Trends Plant Sci. 16: 53–60. with permission from Elsevier

Photosynthesis can be monitored by O2 production and CO2 consumption

Alternatively, photosynthesis can be monitored spectroscopically

• PSII activity can be measured by chlorophyll fluorescence

• PSI activity can be measured by the absorbance change at 810~830 nm

• Transthylakoid proton motive force (pmf) and proton flux can be measured by electrochromic shift (ECS)

NASA; Steveadcuk

Light Light Gas exchange Closed system in a leaf can be Open system measured in a Air in Air out closed or open (flow through) system

Light on Light off Light on Light off [CO2] is

measured by O2 infrared gas O2 analysis (IRGA) Ambient [O2] and [O ] by an

2 Ambient [CO2] in chamber in

CO oxygen air exiting in CO2 Concentration Concentration Concentration Concentration 2 electrode

When a chlorophyll a molecule absorbs light, it is excited qP from its ground state to its singlet excited state (Chl*), which can return to the ground state via one of three pathways: Chl* fluorescence (1) The excitation energy can be transferred to reaction Photon NPQ centers to drive photosynthesis (photochemistry, qP) (2) Energy can be re-emitted as chlorophyll fluorescence (3) It can be released as heat (non-photochemical quenching, NPQ) Chl

Photochemistry The practical application is Fluorescence that fluorescence can be PSII (longer quantified under various chlorophyll wavelength) conditions to measure Incident photosynthetic activities light NPQ (heat)

Q → Q − A pulse of saturating light B B leads to a transient pulse of e-− fluorescence as reaction − QA → QA centers close (become e-− reduced) and light is emitted Pheo → Pheo− as fluorescence -− e Reaction centers open with P680 → P680* → P680+ measuring beam on, closed after saturating pulse Reaction center transiently closed

Murchie, E.H. and Lawson, T. (2013). Chlorophyll fluorescence analysis: a guide to good practice and understanding some new applications. J. Exp. Bot. 64: 3983-3998 by permission of Oxford University Press.

A saturating flash transiently closes reaction centers, resulting in maximum Chl fluorescence. With measuring light only, the reaction centers are fully opened, resulting in minimum Chl fluorescence.

Photochemistry A very bright light Fm

pulse is mainly PSIIX

chlorophyll o F

emitted as

- fluorescence;

reaction centers m

= F =

close until they Saturating v F

can be re-oxidized pulse Fm Fluorescence

F In dim light Photochemistry o

(measuring light), PSII 0 Saturating pulse the energy is chlorophyll Very low nearly all used for Measuring light on level light Fo photochemistry

Fo = baseline fluorescence, reaction centers open

Fm = maximal fluorescence when reaction centers closed

(Fm− Fo) = Fv = variable fluorescence

Fv / Fm = maximum quantum efficiency of PSII chemistry (values less than ~0.8 indicate stress or photoinhibition)

NPQ and qP can be distinguished by the difference in fluorescence when reaction centers are open or closed (Fm′)

Due to NPQ

Due to photochemistry (photochemical quenching, pQ)

F ' is steady state ′ ′ ′ fluorescence in the Fm – F = Fq light Fq′ / Fm′ = ΦPSII ΦPSII = quantum efficiency of PSII

• Fm increases as NPQ reverses • Different components of NPQ have different relaxation kinetics (e.g., qE reverses more rapidly than qI.)

P700+ Fully oxidized

P700+ e- P700 Fully reduced

e- P700 An increase in P700 oxidation ratio = A / B reduction rate of P700+ under actinic light Reduced P700 does not P700+ can indicate absorb 810 nm light, but an induction of cyclic Max P700+ induced by far red and flash + oxidized P700 does electron transport

The proton-motive Electrochromic shift (ECS) is force (electrical and the change in light absorbing

Without With pH gradient) across electric electric properties undergone by the thylakoid can be field field pigments when subjected to a changing electric field

measured by its Absorbance effect on pigment Wavelength absorbance spectra

Witt, H.T. (1979). Energy conversion in the functional membrane of photosynthesis. Analysis by light pulse and electric pulse methods: The central role of the electric field. Biochim. Biophy. Acta Bioenergetics. 505: 355-427.

ΔECS: light-induced transthylakoid

pmf (proton motive force)  : relaxation ECS 0.015 0.003 time constant 0.010 0.002  ECS

 0.005 ECS  Light induces pmf, 0.001 0.000 therefore ECS ECS

pH Optical density, relative Optical density, relative 0.000 -0.005 light off light off light on 1/EC: proton conductance, proportional to ATP synthase -400 -200 0 200 400 -10 0 10 20 30 40 50 activity. In other words, the faster Time, ms Time, s the ECS decays, the more active ATP synthase

With kind permission from Springer Science+Business Media from Zhang, R. and Sharkey, T. (2009). Photosynthetic electron transport and proton flux under moderate heat stress. Photosynth. Res. 100: 29-43; see also Baker, N.R., Harbinson, J., and Kramer, D.M. (2007). Determining the limitations and regulation of photosynthetic energy transduction in leaves. Plant Cell Environ. 30: 1107–1125..

VERY fast = charge separation through PSI & PSII

Fast = Cyt b6f electron flow Slow decay = H+ flow

through ATP synthase

ECS

Light pulse

Inhibitors can PSII inhibitors distinguish PSI from PSII

With kind permission from Springer Science+Business Media from Bailleul, B., Cardol, P., Breyton, C., and Finazzi, G. (2010). Electrochromism: a useful probe to study algal photosynthesis. Photosynth. Res. 106: 179–189.

Fluorescence measurements can be coupled to imaging systems to measure photosystem efficiency across many size scales – within a leaf or within a plant.

F’

Photosynthetic efficiency of Technologies are PSII photochemistry (F q′/F m′) being developed to monitor solar-induced fluorescence from space

Murchie, E.H. and Lawson, T. (2013). Chlorophyll fluorescence analysis: a guide to good practice and understanding some new applications. J. Exp. Bot. 64: 3983-3998; NASA; NASA/Caltech; Guanter, L., et al. (2014). Global and time-resolved monitoring of crop photosynthesis with chlorophyll fluorescence. Proc. Natl. Acad. Sci. USA 111: E1327-E1333; Joiner, J., et al., (2011). First observations of global and seasonal terrestrial chlorophyll fluorescence from space. Biogeosciences. 8: 637-651..

The light-absorbing and emitting properties of the photosynthetic complexes allow their activities to be monitored with great precision. These methods have been developed using intact leaves and cells, but to some extent can be applied to field- and global-level monitoring

Reprinted by permission from Baniulis, D., Yamashita, E., Zhang, H., Hasan, S.S. and Cramer, W.A. (2008). Structure–Function of the Cytochrome b6f Complex†. Photochem. Photobiol. 84: 1349-1358.

Crop plants grow in very unnatural conditions, different than those in which natural selection has acted. Can photosynthetic light- dependent reactions be turbo-charged for higher yields?

© 2015 American Society of Plant Biologists Harvest other light wavelengths? Another suggestion is to increase green-light absorption by introducing phycoerythrin from red algae (who wants black leaves?)

Current oxygenic photosynthesis

A hypothetical system in which PSI is replaced by a bacterial reaction center that uses longer wavelength light. (Note that the system would have to be extensively engineered to bridge the gap between water oxidation and NADPH reduction in the absence of PSI.) Hypothetical long-wavelength oxygenic photosynthesis

Ort, D.R., et al. (2015). Redesigning photosynthesis to sustainably meet global food and bioenergy demand. Proc. Natl. Acad. Sci. USA 112: 8529–8536 See also Croce, R. and van Amerongen, H. (2014). Natural strategies for photosynthetic light harvesting. Nat Chem Biol. 10: 492-501 Blankenship, R.E., et al. and Sayre, R.T. (2011). Comparing photosynthetic and photovoltaic efficiencies and recognizing the potential for improvement. Science. 332: 805-809. .

Light penetration into culture vessels is a problem for algal biofuels production. By engineering cyanobacteria with truncated light antennas (phycocyanin deletion mutants), productivity increased (due to improved light penetrance and less excess light dissipated from surface cells).

Reprinted from Kirst, H., Formighieri, C. and Melis, A. (2014). Maximizing photosynthetic efficiency and culture productivity in cyanobacteria upon minimizing the phycobilisome light-harvesting antenna size. Biochim. Biophys. Acta - Bioenergetics. 1837: 1653-1664; IGV; Biotech

In a “smart In a typical canopy, canopy”, upper upper leaves leaves would have intercept too much smaller antenna light and must complexes and a dissipate some as more vertical heat, meanwhile orientation, shading lower permitting more leaves light to reach lower leaves for greater overall efficiency

Ort, D.R., et al. (2015). Redesigning photosynthesis to sustainably meet global food and bioenergy demand. Proc. Natl. Acad. Sci. USA 112: 8529–8536.

Manipulating photoprotective pathways can enhance stress Increase flow of resistance and photosynthetic electrons through productivity of crop plants. Enhance PSII repair CEF or water- water cycle

What are the metabolic Increase NPQ costs of enhanced responsiveness photoprotection?

Murchie, E.H. and Niyogi, K.K. (2011). Manipulation of Photoprotection to Improve Plant Photosynthesis. Plant Physiol. 155: 86-92.

The energy that reaches the earth from the sun in Solar panels make electricity that is hard to an hour is equivalent to “all the energy humankind store currently uses in a year”

The goal of artificial photosynthesis is to develop cheap, durable ways to make fuels from sunlight (like a leaf)

Barber, J. and Tran, P.D. (2013). From natural to artificial photosynthesis. J. Roy. Soc. Interface. 10: 20120984; Photos by Tom Donald

The four fundamental steps that comprise photosynthesis can be 1. Light harvesting engineered using catalysts

2. Charge separation

3. Water 4. Proton oxidation reduction

Cogdell, R.J., Gardiner, A.T., Molina, P.I. and Cronin, L. (2013). The use and misuse of photosynthesis in the quest for novel methods to harness solar energy to make fuel. Phil. Trans. Royal Soc. A: Mathematical Physical Engineering Sci 371: 20110603 by permission of the Royal Society.

Semiconducting They can be materials can augmented with carry out charge electrocatalysts separation

They can be wired together as a Z- scheme

Barber, J. and Tran, P.D. (2013). From natural to artificial photosynthesis. J. Roy. Soc. Interface. 10: 20120984 by permission of the Royal Society.

A bioelectrochemical cell. Water- splitting and proton-reducing electrodes are immersed into a culture of bacteria that convert H 2 A photoelectrical cell that mimics the to isopropanol electron transfer reactions of PSII

Torella, J.P., Gagliardi, C.J., Chen, J.S., Bediako, D.K., Colon, B., Wray, J.C., Silver, P.A., and Nocera, D.G. (2015). Efficient solar-to-fuels production from a hybrid microbial–water-splitting catalyst system. Proc. Natl. Acad. Sci. USA. 112: 2337- 2342. Reproduced from Duan, L., Tong, L., Xu, Y. and Sun, L. (2011). Visible light-driven water oxidation-from molecular catalysts to photoelectrochemical cells. Energy Environ. Sci. 4: 3296-3313 with permission of The Royal Society of Chemistry. .

As an alternative to chlorophyll based systems, bacteriorhodopsins are being explored for solar-driven chemistry.

Retinal is the chromophore

Bacteriorhodopsin uses light energy to pump protons (or other ions) which can be used for ATP synthesis Structure of bacteriorhodopsin

See for example Claassens, N.J., Volpers, M., Martins dos Santos, V.A.P., van der Oost, J., and de Vos, W.M. (2013). Potential of proton-pumping rhodopsins: Engineering photosystems into microorganisms. Trends Biotechnol. 31: 633 – 642.

This lesson focuses on photosynthesis in cyanobacteria and the products of primary endosymbiosis Other, unrelated eukaryotic organisms can serve as hosts to photosynthetic bacteria and algae: Fungi • Lichen Animals Secondary, tertiary • Corals endosymbiosis Brown algae, • Flatworms diatoms, • Sea slugs dinoflagellates, • Salamanders euglenoids….

Reprinted with permission from Rumpho, M.E., Pelletreau, K.N., Moustafa, A. and Bhattacharya, D. (2011). The making of a photosynthetic animal. J. Exp. Biol. 214: 303-311.

Lichen have many forms

Lichen are mutualistic associations Algal cell Fungus of fungi (blue) and green algae or cyanobacteria (green).

The photosynthetic partner (phycobiont) resides outside the fungal cells (mycobiont). The phycobiont provides the mycobiont with reduced carbon in return for shelter, nutrients and water.

Photos by Vernon Ahmadjian courtesy of BSA; jdurant

© 2015 American Society of Plant Biologists Photosynthetic animals: Reef-building corals Coral bleaching is the loss of the symbiont. Bleaching is caused by stress, particularly heat, and often leads to coral death

Bleached

Symbiodinium dinoflagellates live within the bodies of Various species of corals and provide coral and their them with fixed dinoflagellates carbon

Credit: Todd LaJeunesse, Penn State University. Fournier, A. (2014). The story of symbiosis with zooxanthellae, or how they enable their host to thrive in a nutrient poor environment. BioSciences Master Reviews. Weis, V.M. (2008). Cellular mechanisms of Cnidarian bleaching: stress causes the collapse of symbiosis. J. Exp. Biol. 211: 3059-3066. Allisonmlewis

Symsagittifera roscoffensis Symsagittifera

Life cycle of the photosynthetic photosynthetic the cycle of Life worm

Bailly, X., Laguerre, L., Correc, G., Dupont, S., Kurth, T., Pfannkuchen, A., Entzeroth, R., Probert, I., Vinogradov, S., Lechauve, C., Garet-Delmas, M.-J., Reichert, H. and Hartenstein, V. (2014). The chimerical and multifaceted marine acoel Symsagittifera roscoffensis: from photosymbiosis to brain regeneration. Front. Microbiol. 5: 498.

The algae provides the animal with oxygen and photosynthate, and benefits from the animal’s nitrogenous waste

Recently, algae (red fluorescent spots) were found within the animal’s cells, the first evidence Spotted salamander for endosymbiosis in a vertebrate Ambystoma maculatum

Kerney, R., Kim, E., Hangarter, R.P., Heiss, A.A., Bishop, C.D. and Hall, B.K. (2011). Intracellular invasion of green algae in a salamander host. Proc. Natl. Acad. Sci. USA 108: 6497-6502. Camazine

Vaucheria litorea (algae) The plastids ingested from the algae stay viable for several months within the sea slug – this “plastid stealing” is known as Chloroplasts in sea slug kleptoplasty

Ingested chloroplasts are stored in animal’s cells Elysia chlorotica (sea slug)

Pelletreau, K.N., Bhattacharya, D., Price, D.C., Worful, J.M., Moustafa, A. and Rumpho, M.E. (2011). Sea slug kleptoplasty and plastid maintenance in a metazoan. Plant Physiol. 155: 1561-1565. Rumpho, M.E., Pelletreau, K.N., Moustafa, A. and Bhattacharya, D. (2011). The making of a photosynthetic animal. J. Exp. Biol. 214: 303-311.

The Z-scheme is a familiar way to depict the light- harvesting reactions of photosynthesis

Core reactions are conserved, but variation in tolerances and The core chemical reactions capacitieis of each of the have remained essentially reactions allows for dynamic the same for > 2.5 billion responses and adaptations to years, since the days of the variable light, temperature etc. first cyanobacteria

Ruth Ellison

Variable Responses Protecting plants from photo-oxoidative damage, Light harvesting particularly when they are Chloroplast and leaf stressed, may be a fruitful movements, accumulation of approach to improving pigments and proteins photosynthesis

Light intensity, Energy dissipation and photon balancing qE, quenching in LHC qT, state transition Variable wavelength, Chloroplast chemistry angle, duration Transthylakoid pH/ pmf

PQ / PQH2

Variable Whole-plant physiology * * environmental stress

Photoprotection Variable Plastid terminal oxidase Light and stress- Antioxidant production induced reactive oxygen species

Ort, D.R., et al. (2015). Redesigning photosynthesis to sustainably meet global food and bioenergy demand. Proc. Natl. Acad. Sci. USA 112: 8529–8536.; IGV Biotech; Caltech; Joint Center for Artificial Photosynthesis

STRESS PHOTOSYNTHESIS

DEVELOPMENT

We need to understand photosynthesis within the context of a whole plant, considering the effects of development, stress, age, demand for photosynthate and all other factors that affect it.

Tom Donald; Tom Donald; See Allahverdiyeva, Y., Battchikova, N., Brosché, M., Fujii, H., Kangasjärvi, S., Mulo, P., Mähönen, A.P., Nieminen, K., Overmyer, K., Salojärvi, J. and Wrzaczek, M. (2015). Integration of photosynthesis, development and stress as an opportunity for plant biology. New Phytol. 208: 647–655; FEMA