Lecture 6 Photosynthesis

I. Introduction

Interesting website

http://gened.emc.maricopa.edu/bio/bio181/BIOBK/BioBookPS.html

1. Light reaction

(1). Localization: thylakoid membranes

(2). End products: ATP and NADPH

2. Dark reaction

(1). Localization: stroma

(2). End products: sugar

II. Historical Background

1. Light

(1). Light wave and energy of a photon (Planck's law)

(2). Action spectra measure the biological effect as a function of wavelength.

(3). Measuring light

(4). Photosynthetically active radiation

2. Research history

(1). Stephen Hales, 1727

(2). Joseph Priestly, 1771

(3). Ingen-Housz 1779

(4). Julius Sachs, 1864

(5). C. B. van Niel, 1920-1930, Chromatium vinosum

light, bacteria

nCO₂ + 2nH₂A (CH₂O) _n + 2nA + nH₂O

(6). Robert Hill, 1939, Hill reaction, isolated chloroplast thylakoids

e^-

4Fe³⁺ + 2H₂O 4Fe²⁺ + O₂ + 4H⁺

III. Photoreceptors

1. Chlorophylls contain a porphyrin head and a phytol tail.

2. Carotenoids: accessory pigments: C₄₀ terpenoids.

3. Pheophytin

4. Phycobilins: straight-chain tetrapyrroles.

5. Cryptochrome: hidden pigment, cryptochromes comprise a family of flavin-type blue/UV-A light receptors. Plant cryptochromes were first characterized in Arabidopsis and share similarities in both protein structure and chromophore composition with the microbial DNA photolyases. The cry1 regulates many blue light responses in Arabidopsis, including the inhibition of hypocotyl elongation and the induction of anthocyanin synthesis, and the entrainment of the circadian clock by blue light. Although cry2 is also involved in mediating blue light inhibition of hypocotyl elongation, cry2 functions primarily in the regulation of floral initiation in response to photoperiod.

6. The UV-B receptors (280-320 nm)

7. Flavonoids

8. Betacyanins

Photosynthesis: Light Reaction

I. Structure of the Photosynthetic Apparatus

1. Palisade cells

2. Proton pumping in guard cells

(1). Proton pumping generates ion gradients that regulate guard cell turgor.

(2). Ionic concentrations

(3). Mechanisms mediate the stomatal response to light

(4). Control of stomatal movements

a. Light and CO₂

b. Water status and temperature

c. Circadian rhythms

3. The chloroplast

(1). Thylakoids contain integral membrane proteins

(2). Structures of two bacterial reaction centers of the purple bacterium Rhodopseudomonas viridis (resolved by x-ray crystallography, 1984-1989, Hartmut Michel, Johann Deisenhofer, and Robert Huber).

(3). PSI and PSII are spatially separated in the thylakoid membrane

4. Efficiency of photosynthesis

(1). Quanta yield (Warburg, 1920)

yield of photochemical products

f =

total number of quanta absorbed

(2). Photosynthetic energy conversion depends on cooperation between many pigment molecules and a group of electron transfer proteins.

5. Two photosystems

II. Organization of Light-Absorbing Antenna Systems

1. The antenna funnels energy to the reaction center

2. Many antenna complexes have a common structural motif

Light-harvesting complex protein (LHCI and LHCII)

III. Mechanisms of Electron and Proton Transport

1. Four thylakoid protein complex carry out electron and proton transport

Z scheme for O₂-evolving photosynthetic organisms.

2. Energy is captured when excited chlorophyll reduces an electron acceptor molecule.

3. Reaction center chlorophyll of the two photosystems absorb at different wavelengths (P700 and P680).

4. PSII reaction center is a multisubunit pigment-protein complex

(1). Core of PSII consists of D1 (32 kDa) and D2 (34 kDa).

(2). P680, chlorophylls, pheophytins, carotenoids, and plastoquinones

(3). Antenna complexes: 43 kDa and 47 kDa

(4). Peripheral membrane O₂-evolving proteins: 33, 23 and 18 kDa

5. Water is oxidized to oxygen by PSII

(1). 2H₂O ® O₂ + 4H⁺ + 4e^-

(2). The S state mechanism

(3). Protons produced are released into lumen of the thylakoid.

(4). Cofactors: 4 Mn, 4 Cl^- and Ca²⁺.

(5). Y_Z: an electron carrier, a radical formed from a tyrosine residue in the D protein of the PSII reaction center protein.

6. Pheophytin and two quinones are early electron acceptors of PSII

7. Electron flow through the cytochrome b₆-f complex transports protons to the thylakoid lumen

(1). Components

2 b-type cytochromes, 2 c-type cytochromes, a Rieske iron-sulfur protein

(2). Mechanism of e^- and H⁺ transfer in the cytochrome b₆-f complex

Q cycle: two e^- and four H⁺ are transferred

Two plastoquinones are oxidized, and one oxidized plastoquinone is reduced to hydroquinone form.

8. Plastoquinone and plastocyanin is putative e carrier between PSII and I

(1). Cytochrome b₆-f complex is equally distributed between the grana and stroma regions of the membrane.

(2). Plastoquinone or plastocyanin is probably carrier to connect the two PSs.

(3). Plastocyanin: a small (10.5 kDa) copper-containing protein.

(4). Cyclic electron flow: e^- from the reducing side (ferredoxin) of PSI through the cytochrome b₆-f complex and back to P700.

9. PSI reaction center reduces NADP⁺

(1). Components

a. P700, chlorophylls

b. Polypeptides, 66-70 kDa, 4-25 kDa, 8 kDa (bound iron-sulfur centers)

c. Three electron carriers A_0, A₁(vitamin K), X (4Fe-4S)

d. Three membrane associated iron-sulfur proteins (bound ferredoxin)

e. The soluble flavoprotein ferredoxin-NADP reductase reduces NADP⁺ to NADPH.

10. Oxidation of water: The S-state model

(1). Oxygen-evolving machinery of PSII progresses through five successive states of increasing oxidation, S₀ through S₄, with S₄ being a strong oxidant capable of oxidizing water.

(2). Each charge separation event in P680 yields P680⁺, ultimately oxidizes the charge accumulator, advancing it to the next S-state and increasing the charge by +1. The only state from which O₂ is evolved is S₄.

(3). In the dark-adapted chloroplasts, the charge accumulator is predominantly in the S₁ state, resulting in a maximal yield of O₂ on the third flash.

(4). First flash advances the oxidation state from S₁ to S₂. Each cycle requires four flashes because four photons are required per O₂ evolved.

(5). Reactions are complicated by “misses’’, “double hits” and “relaxations”.

11. A chemiosmotic mechanism converts the energy stored in chemical and electric potentials to ATP-photophosphorylation

(1). The ATP synthetase (coupling factor, 400 kDa) and proton motive force

Dp = DEm – 59 (pHi - pHo)

(2). Similarity of photosynthetic and respiratory electron flow.

12. Regulation and repair of the photosynthetic apparatus

(1). Carotenoids serve as both accessory pigments and photoprotective agents.

(2). Thylakoid stacking permits energy partitioning between the photosystems.

a. Thylakoid membranes contain a protein kinase that can phosphorylate a specific threonine residue on the surface of LHCII.

b. LHCII phosphorylated: delivers more energy to PSI.

d. Plastoquinone accumulates in the reduced state: kinase is activated.

(3). Some xanthophylls also participate in energy dissipation.

(4). PSII is easily damaged. PSI is protected from active oxygen species.

IV. Genetics, Assembly, and Evolution of Photosynthetic systems

1. Chloroplast genes exhibit non-Mendelian patterns of inheritance.

2. Many chloroplast proteins are imported from the cytoplasm.

3. The biosynthesis and breakdown of chlorophyll are complex pathways.

4. Evolution

Lec 7 Photosynthesis II: Carbon Metabolism

I. The C3 Photosynthetic Carbon Reduction (PCR) Cycle (Calvin Cycle)

Carboxylation RuBP carboxylase (Rubisco)

RuBP + CO₂ + H₂O 3-phosphoglycerate

ATP + NADPH ADP + Pi + NADP+

Reduction

3-phosphoglycerate Triose phosphate

Sucrose, starch

ATP ADP

Regeneration

Triose phosphate RuBP

1. The PCR cycle was elucidated using radioactive carbon compounds

Melvin Calvin, James Bassham 1950s-1965, Chlorella

2. The C3 PCR regenerates its own biochemical components

6 CO₂ + 11 H₂O + 12 NADPH + 18 ATP

fructose6-P + 12 NADP⁺ + 6 H⁺ + 18 ADP + 17 Pi

4. The regulation of the C3 PCR cycle

(1). Light-dependent enzyme activation

a. Activation of RuBP carboxylase involves formation of a carbamate-Mg²⁺ complex on the e-amino group of a lysine within the active site of the enzyme. Activation is promoted by increasing pH and [Mg²⁺].

b. Rubisco activase

c. Light stimulates enzyme activity via a covalent thiol-based oxidation-reduction system (the reduced sulfhydryl is the active form).

Ferredoxin:thioredoxin reductase, Thioredoxin, Target enzyme

(2). Ionic changes in the stroma

During light illumination, protons are pumped from the stroma into the lumen of the thylakoids in exchange for Mg²⁺

In the stroma, [Mg²⁺] and pH (7 to 8) increased.

(3). Transport processes in the chloroplast envelope

II. The C2 Photorespiratory Carbon Oxidation (PCO) Cycle

1. Photosynthetic CO₂ fixation and photorespiratory oxygenation of RuBP are competing reactions at the same active site of RuBP carboxylase.

(1). In the chloroplast

RuBP + O₂ 2-phosphoglycolate + 3-phosphoglycerate + 2H⁺

phosphoglycolate + H₂O glycolate (CH₂OHCOO^-) + HOPO₃^2-

(2). In the peroxisome

Glycolate oxidase

glycolate + O₂ glyoxylate (CHOCOO^-) + H₂O₂

Catalase

H₂O₂ H₂O + O₂

Glycolate:glutamate aminotransferase

glyoxylate + glutamate glycine + a-ketoglutarate

(3). In the mitochondrion

Glycine decarboxylase

glycine + NAD⁺ + H₄-folate NADH + H⁺ + CO₂ + NH₃ + methylene H₄-folate

Serine hydroxymethyltransferase

methylene H₄-folate + H₂O + glycine serine + H₄-folate

(4). Back to the peroxisome

Serine aminotransferase

serine + a-ketoglutarate hydroxypyruvate + glutamate

Hydroxypyruvate reductase

hydroxypyruvate + NADH ⁺ H⁺ glycerate + NAD⁺

(5). Back to the chloroplast

Glycerate kinase

glycerate + ATP 3-phosphoglycerate + ADP + H⁺

2. Competition between the carboxylation and oxygenation reactions

(1). Carboxylation/oxygenation = 2.5-3

(2). Thermodynamic efficiency for C3 PCR is reduced from 90% to 54%.

3. Factors affect carboxylation and oxygenation in leaf

(1). Kinetic properties of RuBP carboxylase

(2). Prevailing temperature

(3). Concentrations of CO₂ and O₂

III. The C₄ Photosynthetic Carbon Assimilation (PCA) Cycle

1. Characteristics in leaf anatomy

(1). Mesophyll cells and the bundle sheath (kranz) cells

(2). The C₄ PCA cycle increases CO₂ concentration in the bundle sheath cells since a unique enzyme exists in the mesophyll cells

Phosphoenolpyruvate carboxylase

Phosphoenolpyruvate + CO₂ Oxaloacetate + Pi

2. The C₄ PCA cycle

(1). Assimilation of CO₂ to C₄ acids in the mesophyll cells.

(2). Transport of C₄ acids to the bundle sheath cells.

(3). Decarboxylation of the C₄ acids (CO₂ C₃ cycle) in the bundle sheath cells.

(4). Transport of the C₃ acids back to the mesophyll cells and regenerate PEP.

3. The variation of the C4 PCA cycle

(1). Variants differ principally in

a. The nature of the C3 (pyruvate or alanine) returned to the mesophyll.

b. The enzyme catalyzing the decarboxylation step in the bundle sheath cell.

(2). Varians

a. The NADP-malic enzyme type (maize, sugarcane)

NADP malate dehydrogenase (in the mesophyll chloroplast)

OAA + NADPH + H⁺ malate + NADP⁺

NADP malic enzyme (in the bundle sheath chloroplast)

malate + NADP⁺ pyruvate + CO₂ + NADPH + H⁺

b. The NAD-malic enzyme type

Aspartate aminotransferase (in the mesophyll chloroplast)

OAA + glutamate aspartate + a-ketoglutarate

Aspartate aminotransferase (in bundle sheath mitochondrion)

aspartate + a-ketoglutarate OAA + glutamate

NAD malate dehydrogenase (in bundle sheath mitochondrion)

OAA + NADPH + H⁺ malate + NADP⁺

NAD malic enzyme (in the bundle sheath mitochondrion)

malate + NADP⁺ pyruvate + CO₂ + NADPH + H⁺

Alanine aminotransferase

pyruvate + glutamate alanine + a-ketoglutarate

c. The phosphoenolpyruvate carboxykinase type

Aspartate aminotransferase (in mesophyll chloroplast)

OAA + glutamate aspartate + a-ketoglutarate

Aspartate aminotransferase (in bundle sheath cytosol)

aspartate + a-ketoglutarate OAA + glutamate

PEP carboxykinase (in the bundle sheath cytosol)

OAA + ATP PEP + CO₂ + ADP

Alanine aminotransferase

pyruvate + glutamate alanine + a-ketoglutarate

3. The final step of the C₄ PCA cycle is common to all the three variants.

Pyruvate, orthophosphate dikinase (in the mesophyll chloroplast)

pyruvate + HOPO₃^2- + ATP PEP + AMP + H₂P₂O₇^2-

5. Energy cost

CO₂ (mesophyll) + 2 ATP + H₂O CO₂ (bundle sheath) + 2 ADP + 2 Pi

IV. Crassulacean Acid Metabolism (CAM)

1. Transpiration ratio

CAM 50-100 g H₂O/g CO₂

C₄ 250-300 g H₂O/g CO₂

C₃ 400-500 g H₂O/g CO₂

CAM plants have high water-use efficiency.

2. In CAM plants, the formation of the C₄ acids is temporally, but not spatially, separated from decarboxylation and refixation.

3. CAM plants open their stomates at night and keep them closed during the day to achieve their high water-use efficiency.

(1). At night, stomates open, CO₂ assimilates, and malate stores in the vacuole.

(2). Upon illumination, stomates close, the malic acid is recovered from the vacuole undergoes decarboxylation, CO₂ released is fixed via the C₃ cycle.

4. CAM PEP carboxylase exists in two forms

(1). The night form (Enz-ser-OP) is insensitive to malate, the day form (Enz-ser-OH) is inhibited by malate.

(2). Expression of CAM gene is susceptible to environmental control.

V. Synthesis of Sucrose and Starch

1. Starch is synthesized and stored in the chloroplast while sucrose synthesis takes place in the cytosol.

(1). Sucrose synthesis: cytosol

Sucrose-phosphate synthetase

UDPglucose + fructose-6-phosphate ↔ UDP + sucrose-6-phosphate

Sucrose-phosphate phosphatase

Sucrose-6-phosphate + H₂O ↔ sucrose + Pi

(2). Starch synthesis: chloroplast

ADPglucose phosphorylase

ADP + glucose-1-phosphate ↔ ADP-glucose + PPi

Starch synthase

ADP-glucose + a-(1®4)-glucan ↔ ADP + a-(1®4)-glucosyl-glucan

Q enzyme

(3). Chloroplast fructose 1,6-bisphosphate phosphatase is regulated by the thioredoxin system and is insensitive to fructose 2,6-bisphosphate and AMP. However, cytosolic fructose 1,6-bisphosphate phosphatase is regulated by fructose 2,6-bisphosphate.

2. The key metabolites that regulate the synthesis of sucrose and starch

[orthophosphate] and [triose-phosphate] in cytosol and chloroplast

[fructose 2,6-bisphosphate] in cytosol

Phosphate/triose phosphate translocator in chloroplast envelope

(1). Chloroplast enzyme ADPglucose pyrophosphate is stimulated by triose phosphate, 3-phosphoglycerate and is inhibited by orthophosphate.

(2). Abundance of orthophosphate in cytosol ® inhibits starch synthesis in chloroplast and promotes the export of triose phosphate into cytosol.

(4). Fructose 2,6-bisphosphate stimulates the activity of PPi-dependent fructose-6-phosphate kinase and inhibits the activity of fructose-1,6-bisphosphate phosphatase.

Physiological and Ecological Effects

I. Rate Limiting Reactions of Photosynthesis

1. Effects of light intensity and temperature on rates of photosynthesis in continuous light (p234-236)

(1). Light compensation point

(2). At low light intensity, the limiting of PS is the photochemical reaction. At high light intensity, the limiting is the thermochemical reaction.

2. CO₂ and photosynthesis in leaves

(1). CO₂ compensation point

(2). CO₂ enrichment in the greenhouse

(3). Define the photosynthetic CO₂ assimilation (A)

(Ca - Ci) (Ca - Ci) g

A = =

1.6 Pr 1.6 P

1.6 AP

Ci = Ca − (von Caemmerer and Farquhar, 1981)

g

(4). Water use efficiency

A Ca - Ci

=

E (e_i - e_a) 1.6

Lec 8 Translocation and Distribution of Photoassimilates

I. Translocation of Photoassimilates

1. Pathways of translocation

(1). The structure of phloem

Sieve (tube) elements

Sieve-tube members

Sieve plate, sieve plate pores

P-protein (PP1, the phloem filament protein

PP2, the phloem lectin

Callose

Sieve cells

Companion cells

Companion cells and transfer cells

Albuminous cells

(2). The composition of phloem exudate

a. Sugar is translocated in phloem sieve elements-with radioactive labels.

d. Sugars are translocated in nonreducing form: sucrose, raffinose, stachyose, and verbascose

e. Nitrogenous compounds

(3). P-protein and callose (a b-(1®3) glucan): function in sealing off damaged sieve elements by plugging up the sieve-plate pores.

2. Sources and sinks

(1). Source: a net exporter or producer of photoassimilates.

(2). Sink: a net importer or consumer of photoassimilates.

(3). Plant development affects the importance of sinks.

II. Mechanism of Translocation in the Phloem

1. The pressure-flow hypothesis (Ernst Münch, 1926)

(1). The flow of solution in the sieve elements is driven by an osmotically generated pressure gradient between source and sink.

(2). Energy driven phloem loading: a high osmotic pressure in the sieve elements of the source tissue leads to a steep drop in the water potential.

(3). Water enters the sieve elements in the source and increases turgor pressure.

(4). Phloem unloading at the receiving ends: a lower osmotic pressure in sink.

(5). Water and its dissolved solutes move by bulk flow from the area of high pressure (source) to the area of low pressure (sink).

(6). Yw of the phloem rises above that of the xylem, water leaves the phloem ® decreases turgor pressure of the phloem sieve elements of the sink.

2. Prediction of the pressure-flow model

(1). Sieve-plate pores are open. Transport of mass flow is bidirectional.

(2). Rate of translocation is relatively insensitive to energy supply of tissues.

(3). Pressure gradients in sieve elements are sufficient to drive a mass flow.

3. Phloem loading

(1). Transport steps involved:

a. Triose phosphate ® from the chloroplast to the cytosol ® sucrose

b. Sucrose ® from mesophyll to sieve elements-companion cell complex.

c. Symplastic and apoplastic pathways.

(2). Phloem loading of sugar requires metabolic energy: sugar-H⁺ cotransport

a. Treating source tissues with respiratory inhibitors inhibits phloem loading.

b. PCMBS: interfere with carrier proteins involved in the transport of sucrose across the plasma membrane.

4. Phloem unloading and sink-to-source transition

(1). Phloem unloading into receiver cells can be symplastic or apoplastic.

(2). Transport into sink tissues depends on metabolic energy.

Two possible routes for apoplastic sugar unloading

a. In the storage parenchyma cells of sugarcane: insensitive to PCMBS

acid invertase

sucrose + H₂O glucose + fructose

b. In legume seeds, the embryo unloading is sensitive to anoxia, low temperature, metabolic poisons and PCMBS.

5. Assimilate allocation and partitioning

(1). Harvest index: the ratio of usable plant material to total biomass.

(2). Allocation: the regulation of the diversion of fixed carbon into the various metabolic pathways.

(3). Key enzymes regulate allocation in source leaves: Starch synthesis in chloroplast coordinated with sucrose synthesis in cytosol.

(4). Partitioning: differential distribution of photoassimilates within plant.

a. Factors

(a). The nature of vascular connections between source and sinks.

(b). The proximity of the sink to the source.

(c). Sink strength

b. Sink strength = sink size x sink activity

sink size: total weight of the sink tissue (dry weight)

sink activity: the rate of uptake of assimilates per unit weight of sink tissue

(5). Changes in the source-to-sink ratio, turgor pressure, chemical messengers.

a. Changes in sieve element turgor pressure: an important message in long-distance communication between sinks and sources.

b. Plant hormones: ABA and IAA

c. Xenobiotic chemicals