Fig.Fig. 11 22 33 44 55 66 77 88

99 1010 1111 1212 1313 1414 1515 1616 1717

1818 1919 2020

44

Energy conservation in photosynthesis: Harvesting Sunlight

The primary function of leaves is photosynthesis.

Focus of this chapter (1)

• The structure of higher plant leaves with respect to the interception of light

• Photosynthesis as the reduction of carbon dioxide to carbohydrate

• The photosynthetic electron transport chain, its organization in the thylakoid membrane, and its role in generating reducing potential and ATP

• Problems encounters by chloroplasts when they are subjected to varying amount of light

Focus of this chapter (2)

• The dynamic nature of the thylakoid membrane, showing how changes in the organization of light-harvesting apparatus influence the absorption and distribution of light energy

• The role of carotenoids as accessory pigments and in photoprotection of chlorophyll and

• The use of herbicides that specifically interact with photosynthetic electron transport

The structure of the leaf

• The architecture of a typical higher plant leaf is particularly well suited to absorb light.

• The photosynthetic tissues (mesophyll) are located between the two epidermal layers.

• Dicotyledonous leaf is structurally different from monocotyledonous leaf.

The structure of dicotyledonous leaf

• One-to-three layers of palisade mesophyll cells forms the upper photosynthetic tissue.

• Below is the spongy mesophyll cells.

The structure of dicotyledonous leaf

• Palisade mesophyll cells are elongated, cylindrical cells with the long axis perpendicular to the surface of the leaf.

• Spongy mesophyll cells are irregular with lots of air spaces between the cells.

• Monocotyledonous leaf lack the distinction between palisade and spongy mesophyll cells.

The structure of monocotyledonous leaf

Comparison between mesophyll cells

• Palisade mesophyll generally have larger numbers of chloroplasts than spongy mesophyll.

Sieve effect

• When light passes through the first layer of cells (palisade mesophyll cells) without being absorbed, we call this sieve effect.

• The sieve effect is due to the fact that chlorophyll is not uniformly distributed throughout cells but instead is confined to the chloroplasts.

Sieve effect

• To reduce sieve effect, plant develops multiple layers of photosynthetic cells.

• The reflection, refraction, and scattering of light inside leaf may also reduce sieve effect.

Photosynthesis

• Photosynthesis can be viewed as a photochemical reduction of CO2.

• In the 1920s, C.B. van Niel discovered the O2 produced from photosynthesis is from water.

• In 1939 Robert Hill found light reaction still can happen in isolated chloroplast when no CO2 is consumed and no carbohydrate was produced.

• In the early 1940s S. Ruben and M. Kamen showed O2 produced from photosynthesis is from water by using O18 labeled water.

Photosynthetic electron transport

The principle function of the light-dependent reactions of photosynthesis is therefore to generate the NADPH

and ATP required for carbon reduction.

Photosynthetic electron transport

• The effect of photosynthetic electron transport chain is to extract low-energy electrons from water and raise the energy level of those electrons to produce a strong reductant NADPH.

• The energy plant used to raise the energy level of those electrons is the light energy trapped by chlorophyll.

Photosynthetic electron-transport chain

• Two large, multimolecular complexes, photosystem I (PSI) and photosystem II (PSII), linked with a third multiprotein aggregate called the cytochrome complex, form the photosynthetic electron-transport chain.

Photosystems

• Photosystems contain several different proteins together with a collection of chlorophyll and carotenoid molecules that absorb photons.

• Most of the chlorophyll in the photosystem functions as antenna chlorophyll.

Photosystems

• The antenna chlorophyll absorb light but do not participate in photochemical reactions. It pass its energy to the next chlorophyll by either inductive resonance or radiationless energy transfer.

Reaction center of photosystem

• For PSII, each reaction center consisted of two chlorophyll a called reaction center chlorophyll.

• Reaction center chlorophyll is the lowest-energy absorbing chlorophyll in the PSII complex (energy sink).

Energy transfer efficiency of Photosystem

• The design of photosystems ensure efficient energy transfer. Only about 10% of the energy is lost during the whole transfer process (from antenna to reaction center chlorophyll).

Why photosystems?

• The principle advantage of associating a single reaction center with a large number of antenna chlorophyll molecules is to increase efficiency in the collection and utilization of light energy.

Why photosystems?

• Even in bright sunlight, an individual chlorophyll will only be struck not more than a few times per second. However, energy transfer only takes s. So it is more economical not to make every chlorophyll into reaction center.

Light-harvesting complexes (LHC) are closely associated

with photosystems

Light-harvesting complexes (LHC)

• Light harvesting complex (also consisted of chlorophyll and proteins) serves as extended antenna systems for harvesting additional light energy.

• In chloroplast, there are two LHCs. The one associated with PSI is named LHCI and the one associated with PSII is named LHCII, accordingly.

Light-harvesting complexes (LHC)

• All the chlorophyll b are contained in LHCs. Most of the chloroplast pigments (70%) are in LHCs.

• LHCI has a chlorophyll a/b ratio about 4 and it is tightly bound to PSI.

• LHCII has a chlorophyll a/b ratio about 1.2. Besides owning most of the chloroplast chlorophyll (50~60%), LHCII also contains most of the chlorophyll b and xanthophyll.

Photosynthetic electron transport chain

PSII pheophytin

• P680 is located at the lumenal side of reaction center.

• When excited, the excited P680 (P680*) is rapidly (10-12s) photooxidized as it passes an electron to pheophytin (primary electron acceptor).

pheophytin

• Pheophytin is a form of chlorophyll a with the Mg2+ replaced by two hydrogens.

• The photo-oxidation of P680 is then followed by charge separation (P680+Pheo-).

Pheophytin a R1 =-CH3; R

2 = phytyl

Pheophytin b R1 = -CHO; R2 = phytyl

phytyl

Pheophytin

P680 pheophytin

• Noted the direction of electron movement in PSII. P680 is located at the lumen side of PSII, then the electron is transferred to pheophytin, which is more towards the stromal side, so electron will not recombine with P680+.

Pheophytin QA PQ

• Reaction proteins D1 and D2 orient specific redox carriers of the PSII reaction center so the probability of charge recombination is further reduced.

Pheophytin QA PQ

• D2 contains QA (quinone electron acceptor) which will accept electrons from pheophytin within picoseconds.

• Then electron from QA will be passed to plastoquinone (PQ), a quinone bound transiently to the binding site on D1 protein (QB).

Plastoquinone (PQ)

• The reduction of plastoquinone (PQ) to plastoquinol (PQH2) lowering the affinity of this molecule for the binding site.

• After plastoquinol is released from the reaction center, another molecule of PQ will occupy the empty space.

Oxygen-evolving complex (OEC)

• P680+ got its electron directly from a cluster of four Mn2+ associated with a small complex of proteins.

• OEC is located on the lumen side of the thylakoid membrane and bound to the D1 and D2 proteins of PSII reaction center.

Oxygen-evolving complex (OEC)

• The OEC proteins functions to stabilize the Mn2+ cluster.

• Chloride ion (Cl-) is also required for the water splitting function.

Oxygen-evolving complex (OEC)

• To generate one molecule of O2, four electrons must be withdrawn from two molecule of H2O. This suggest that OEC should be able to store charges (and experiment results agree with this).

PQ cyt b6f complex

• After plastoquinol is released from PSII, it diffuses through the membrane until reaches cytochrome b6f complex.

• Because plastoquinol has to reach cyt b6f by diffusion, this is the slowest step in photosynthetic electron transport (milliseconds).

Cytb6f complex

• Electron is then transferred from plastoquinol to Rieske iron-sulfur (FeS) protein cytochrome f (all on the lumenal side).

• Then electrons are picked up by plastocyanin (PC).

Plastocyanin (PC)

• Plastocyanin is a small peripheral protein that is able to diffuse freely along the lumenal surface of the thylakoid membrane.

PC PSI

• PC is then transfer electron to PSI.

• The reaction center chlorophyll (P700) first become P700*, then photooxidized to P700+ and give its electron to a molecule of chlorophyll a.

Photosystem I

• The electron is then passed to a quinone (phylloquinone).

• Electron transfer then proceeds through a series of Fe-S centers and ultimately to the soluble iron-sulfur protein, ferredoxin (Fdx).

Ferredoxin NADP+

• Ferredoxin-NADP+ reductase (Fd-NADP+ reductase) then uses ferredoxin to reduce NADP+.

Although PSI do accept electrons from plastocyanin, PSI …

…can also be activated by light.

• When PSI is directly activated by light, the electron it lost is satisfied by withdrawing an electron from reduced PC.

Photosynthetic efficiency

• The efficiency of photosynthesis can be expressed as quantum yield ().

• Quantum yield is number of photochemical product produced per photon absorbed.

Noncyclic electron transport

• When electron transport is operating according to the figure above, electrons are continuously supplied from water and withdrawn as NADPH. This flow-through form of electron transport is known as noncyclic or linear electron transport.

Cyclic electron transport

• Cyclic electron transport is referring to a condition when electrons from PSI is transported not to Fd-NADP+-reductase but to a Fd-PQ reductase.

Photophosphorylation

The light-driven production of ATP by chloroplasts is known as

photophosphorylation.

How is ATP generated?

The light-driven accumulation of protons in the lumen by oxidation of water and PQ-cytochrome proton pump is the energy source of ATP production.

How cytb6f complex moves protons (H+) across the

membrane• The most widely

accepted model for this question is known as the Q-cycle.

Q-cycle (1)

Q-cycle (2)

ATP synthase complex

Thylakoid ATP synthase complex : 400kDa, 9 subunit.CF1 (hydrophilic stromal part): 33CF0 (transmembrane segment): I II III12IV

Binding change mechanism of ATP synthesis by the CF0-CF1

complex

O-site (open): available to bind ADP and PiL-site (loose): ADP and Pi are loosely boundT-site (tight): nucleotide-binding siteProton translocation conformation change rotation of interconversion of these sites

Lateral heterogeneity

• Lateral heterogeneity is referring to the condition that two photosystems (PSI and PSII) are distributed unevenly.

• PSI is mainly located in the stromal membranes and PSII is in the granal membranes.

• ATP synthase is found mostly in stromal membrane.

• Cytochrome b6f complex is distributed evenly.

Lateral heterogeneity

Lateral heterogeneity

• Lateral heterogeneity is referring to the uneven distribution of PSI, PSII, and ATP synthase complexes on thylakoid membranes.

Lateral heterogeneity

PSII/LHCII

PSI/LHCI and ATP synthase

Cytb6f is uniformly distributed

Consequences of lateral heterogeneity

• The ratio between PSI and PSII is adjustable.

• Output of NADPH and ATP can be adjusted to meet cellular demands because non-cyclic and cyclic photophosphorylations can happen more or less simultaneously.

Role of LHCII in photosynthesis

• LHCII contains more than half of the chlorophyll a and almost all of the chlorophyll b, however it is not directly involved in photochemical reduction.

• Functions of LHCII:(1) increase the activity of PSII under conditions of low irradiance (shade plants)(2) regulate PSII activity when light condition fluctuates for a short period of time (phosphorylation/dephosphorylation)

Shade plants

• Plants grown under shade have more thylakoids with large grana, therefore they have higher proportion of apressed thylakoids.

• Sun plants have less LHCII but with more cytochrome b6f complex, plastoquinone, plastocyanin, ferredoxin, and ATP synthease (CF0-CF1 complex).

Phosphorylation/dephosphorylation of LHCII

• LHCII can be phosphorylated by a protein kinase. The phosphorylation causes LHCII becoming more negatively charged.

• Phosphorylated LHCII can be dephosphorylated by a protein phosphatase.

Phosphorylation/dephosphorylation of LHCII

• Under high irradiance of light, PSII will be preferentially excited (state 2). The activation of PSII will result in accumulation of PQH2, which will activate (LHCII) protein kinase.

Phosphorylation/dephosphorylation of LHCII

• The protein kinase is then phosphorylate LHCII.

• The phosphorylation makes LHCII becoming more negatively charged.

Phosphorylation/dephosphorylation of LHCII

• LHCII moves to the stromal thylakoid because charge repulsion, making PSII antenna size smaller.

• Granal thylakoid also loosens due to lack of LHCII.

Phosphorylation/dephosphorylation of LHCII

• Now PSI is preferentially excited (state 1).

• [PQH2], [PQ]• (LHCII) phosphatase

is activated.

Phosphorylation/dephosphorylation of LHCII

• Phosphatase dephosphyrylates LHCII and LHCII moves back to the granal side, which increase the antenna size of PSII.

• Granal membrane is stacked again.

Figure 4.12

LHCII and photoprotection

• PSII is the component of the thylakoid membrane that is most sensitive to excess light.

• Phosphorylation/dephosphorylation of LHCII will protect PSII from thermal damage due to excess light energy.

• Photodamage happens when excess light causes the oxidation of the D1 protein of PSII, which is slowly reversible to some extent.

Carotenoid and photoprotection

• The principle carotene in most higher plants is -carotene.

• Carotenoids serve two functions in photosynthesis: light harvesting and photoprotection.

Carotenoid and photoprotection

• Carotenoid-deficient mutant and norflurazon-treated plants (Norflurazon is an inhibitor of phytoene desaturase and subsequent blocking of carotenoid biosynthesis) are bleached in spite of their ability of chlorophyll biosynthesis is still functional.

Carotenoid and photoprotection

• Carotenoids will trap and dissipate excess excitation energy before it reaches reaction center.

• If excess excitation energy (happens during periods of peak irradiance) reaches reaction center chlorophyll, the chance of 1O2 production (reactive oxygen species, ROS) will increase, and ROS will result in cell damage, even death.

Xanthophylls

Zeaxanthin and photoprotection

• Zeaxanthin can dissipate excess excitation energy as heat.

Zeaxanthin is formed by xanthophyll cycle

Xanthophyll cycle

• Under conditions of excess light, violaxanthin is enzymatically converted to zeaxanthin through de-epoxidation.

• De-epoxidation can also be induced by a low pH in the lumen, which also happens under high light conditions.

• Violaxanthin can also act as a light-harvesting carotenoid.

Xanthophyll cycle

• Violaxanthin is a diepoxide. The de-epoxidation of it is progressing one by one, first producing antheraxanthin (monoepoxide), then zeaxanthin.

• Antheraxanthin and zeaxanthin will be converted back to violaxanthin in the dark by enzymatic actions.

Xanthophyll cycle

• Both antheraxanthin and zeaxanthin can lose excess energy in the form of heat.

• However, neither of they can transfer their energy to chlorophyll because even when they are in excited states, their energy levels are still lower than antenna chlorophylls.

Xanthophyll cycle

• Although they cannot pass their energy to antenna chlorophyll, antenna chlorophyll can transfer excess energy to them and dissipate it as heat.

Xanthophyll cycle

• So xanthophyll cycle acts as a switch, generating antheraxanthin and zeaxanthin whenever dissipation of excess energy is required but removing the zeaxanthin under conditions of low irradiance.

Potential value of xanthophyll cycle

Shade leaves Sun leaves

Xanthophyll content

13% 32%

Absorbed light used in photosynthesis

91% 12%

Light allocated to dissipation as heat

6% 79%

Mehler reaction and Asada-Halliwell pathway

• Sometimes (about 5~10%) O2 can react with electrons generated by PSI, producing superoxide radical (O2

-). This is called Mehler reaction.

• Superoxide dismutase (SOD) will remove the O2-, producing

H2O2 (peroxide). H2O2 is then reduced to water by ascorbate.

Mehler reaction and Asada-Halliwell pathway

• Plant chloroplasts normally exhibit relatively high concentrations of ascorbate (0.5~1.0 mol/mg of chlorophyll).

• This pathway is to prevent H2O2 react with O2-, producing

OH·(hydroxyl radical).

Chlororespiratory pathway – reducing O2 in the dark

O2 H2O

Chlororespiratory pathway

• Chlororespiartory pathway is probably have a role in photoprotection because it is not only operating in the dark.

• This pathway also operate in the light when organisms are exposed to excess irradiance.

The D1 repair cycle

The D1 repair cycle

• PSII reaction center exhibit an inherent lifetime because D1 polypeptide of PSII will be irreversibly damaged due to photo-oxidative damage after absorption of 105 to 107 photons.

• The life span for each D1 polypeptide of PSII reaction center is about 30 minutes.

The D1 repair cycle

marked for degradation

psbA

D1 polypeptide

• In addition to prone to photooxidation damage, D1 polypeptide is also the binding site of many herbicides. Therefore it is also called herbicide binding protein).

• Herbicides belong to urea derivative and triazine groups inhibit photosynthesis by binding to QB site of D1 polypeptide, interrupting photochemical electron transport.

Urea derivatives and triazines

Triazines

• Some plants are resistant to triazines so it can be used as a selective herbicides.

• Corn roots contain an enzyme that degrade the herbicide. Cotton sequesters the herbicide in special glands.

• Some weeds also develop resistance toward this herbicides.

Bipyridylium viologen dye herbicides

• This class of herbicides act by intercepting electrons on the reducing side of PSI, thus interrupting electron transport.

• After accepting electrons from PSI, they auto-oxidize and reduce oxygen to superoxide, which cause oxidative damage to plants.

• Herbicides in this class is also toxic to animal, therefore the usage is highly regulated.