Dr. Riddhi Datta

ATP and NADPH, the final product of thylakoid reaction, flow from thylakoid membranes to the stroma and drive the enzyme-catalyzed reduction of atmospheric CO₂ to carbohydrates.

These reactions in the stroma were long thought to be independent of light and were referred to as the dark reactions.

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However, products of the light reactions not only provide substrates for enzymes but also control the catalytic rate in these reactions.

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So, these reactions are now referred to as the carbon reactions of photosynthesis.

Carbon reactions

Dr. Riddhi Datta

The Calvin–Benson Cycle

• The predominant pathway of autotrophic CO₂ fixation is the Calvin–Benson cycle.

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• This pathway decreases the oxidation state of carbon from +4 in CO₂, to +2 in keto groups and 0 in secondary alcohols found in sugars.

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• Therefore, the Calvin–Benson cycle is also known as reductive pentose phosphate cycle or photosynthetic carbon reduction cycle.

Dr. Riddhi Datta

The Calvin–Benson cycle proceeds in three phases:

• Carboxylation, which covalently links atmospheric carbon (CO₂) to a carbon skeleton
• Reduction, which forms a carbohydrate (triose phosphate) at the expense of photochemically generated ATP and reducing equivalents in the form of NADPH
• Regeneration, which restores the CO₂ acceptor ribulose 1,5-bisphosphate.

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At steady state, the input of CO₂ equals the output of triose phosphates.

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The triose phosphates can have two fates:

• To serve as precursors of starch biosynthesis in the chloroplast
• To flow to the cytosol for sucrose biosynthesis

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Sucrose is loaded into the phloem sap and used for growth or polysaccharide biosynthesis in other parts of the plant.

Dr. Riddhi Datta

Carboxylation:

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One molecule of CO₂ and one molecule of H₂O react with one molecule of ribulose 1,5-bisphosphate to yield two molecules of 3-phosphoglycerate.

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Enzyme: ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco)

ribulose 1,5-bisphosphate

3-phosphoglycerate

Dr. Riddhi Datta

Reduction phase:

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Two successive reactions reduce carbon of the 3-phosphoglycerate produced by carboxylation phase:

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1. ATP formed by the light reactions phosphorylates 3-phosphoglycerate at the carboxyl group, yielding 1,3-bisphosphoglycerate.

Enzyme: 3-phosphoglycerate kinase

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2. NADPH, also generated by the light reactions, reduces 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate.

Enzyme: NADP–glyceraldehyde-3-phosphate dehydrogenase

3-phosphoglycerate

1,3-bisphosphoglycerate

glyceraldehyde 3-phosphate

3-phosphoglycerate kinase

NADP–glyceraldehyde-3-phosphate dehydrogenase

Dr. Riddhi Datta

The operation of three carboxylation and reduction phases yields six molecules of glyceraldehyde 3-phosphate.

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Three molecules of ribulose 1,5-bisphosphate (3 molecules × 5 carbons/molecule = 15 carbons total) react with three molecules of CO₂ (3 carbons total) to form six molecules of 3-phosphoglycerate which are then are reduced.

Rubisco

3-phosphoglycerate kinase

NADP–glyceraldehyde-3-phosphate dehydrogenase

3-phosphoglycerate

1,3-bisphosphoglycerate

glyceraldehyde 3-phosphate

ribulose 1,5-bisphosphate

3x 5C

3x 1C

6x 3C

6x 3C

6x 3C

Dr. Riddhi Datta

Regeneration phase:

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This phase facilitates continuous uptake of atmospheric CO₂ by restoring the CO₂ acceptor ribulose 1,5-bisphosphate.

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Six molecules of glyceraldehyde 3-phosphate are formed after three the carboxylation and reduction phases

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Five molecules of glyceraldehyde 3-phosphate (5x3C) are used to regenerate three molecules of ribulose 1,5-bisphosphate (3x5C).

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Remaining one molecule of glyceraldehyde 3-phosphate represents the net assimilation of three molecules of CO₂ and becomes available for the carbon metabolism of the plant.

Dr. Riddhi Datta

1. Two molecules of glyceraldehyde 3-phosphate are converted to two molecules of dihydroxyacetone phosphate.

Enzyme: triose phosphate isomerase

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Regeneration phase:

2

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Glyceraldehyde 3-phosphate and dihydroxy acetone phosphate are collectively designated triose phosphates.

Dr. Riddhi Datta

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2. One molecule of dihydroxyacetone phosphate undergoes aldol condensation with a third molecule of glyceraldehyde 3-phosphate to give fructose 1,6-bisphosphate.

Enzyme: aldolase

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3. Fructose 1,6-bisphosphate is hydrolyzed to fructose 6-phosphate.

Enzyme: chloroplastic fructose 1,6-bisphosphatase

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Regeneration phase:

fructose 1,6-bisphosphatase

Dr. Riddhi Datta

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4. A two-carbon unit of the fructose 6-phosphate molecule (carbons 1 and 2) is transferred to a fourth molecule of glyceraldehyde 3-phosphate to form xylulose 5-phosphate. The other four carbons of the fructose 6-phosphate molecule (carbons 3, 4, 5, and 6) form erythrose 4-phosphate.

Enzyme: transketolase

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Regeneration phase:

Dr. Riddhi Datta

5. The erythrose 4-phosphate then combines with the remaining molecule of dihydroxyacetone phosphate to yield the seven-carbon sugar sedoheptulose 1,7-bisphosphate

Enzyme: aldolase

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Regeneration phase:

aldolase

Dr. Riddhi Datta

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6. Sedoheptulose 1,7-bisphosphate is then hydrolyzed to sedoheptulose 7-phosphate

Enzyme: chloroplastic sedoheptulose 1,7-bisphosphatase

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7. Sedoheptulose 7-phosphate donates a two-carbon unit (carbons 1 and 2) to the fifth (and last) molecule of glyceraldehyde 3-phosphate producing xylulose 5-phosphate. The remaining five carbons (carbons 3–7) of sedoheptulose 7-phosphate molecule become ribose 5-phosphate.

Enzyme: transketolase

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Regeneration phase:

sedoheptulose 1,7 bisphosphatase

transketolase

Dr. Riddhi Datta

8. Two molecules of xylulose 5-phosphate are converted to two molecules of ribulose 5-phosphate

Enzyme: ribulose 5-phosphate epimerase

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9. A third molecule of ribulose 5-phosphate originates from ribose 5-phosphate

Enzyme: ribose 5-phosphate isomerase

Regeneration phase:

ribulose 5-phosphate epimerase

ribose 5-phosphate isomerase

Dr. Riddhi Datta

10. Finally, three molecules of ribulose 5-phosphate are phosphorylated with ATP thus regenerating the three molecules of ribulose 1,5-bisphosphate needed for restarting the cycle

Enzyme: phosphoribulokinase (also called ribulose 5-phosphate kinase)

Regeneration phase:

phosphoribulokinase

3

3

Dr. Riddhi Datta

C₂ Oxidative Photosynthetic Carbon Cycle

• Rubisco catalyzes both the carboxylation and the oxygenation of ribulose 1,5-bisphosphate.

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• Carboxylation yields two molecules of 3-phosphoglycerate

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• Oxygenation produces one molecule each of 3-phosphoglycerate and 2-phosphoglycolate

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• The oxygenase activity of rubisco causes partial loss of the carbon fixed by the Calvin–Benson cycle and yields 2-phosphoglycolate

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• 2-phosphoglycolate is an inhibitor of two chloroplast enzymes: triose phosphate isomerase and phosphofructokinase.

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• Therefore, 2-phosphoglycolate is metabolized through the C₂ oxidative photosynthetic carbon cycle (also called photorespiration)

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• The C₂ oxidative photosynthetic carbon cycle occurs in chloroplasts, leaf peroxisomes, and mitochondria

Dr. Riddhi Datta

1. The oxygenation of the 2,3-enediol isomer of ribulose 1,5-bisphosphate with one molecule of O₂ yields an unstable intermediate that rapidly splits into one molecule each of 3-phosphoglycerate and 2-phosphoglycolate.

Enzyme: Rubisco

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2. The 2-phosphoglycolate is then rapidly hydrolyzed to glycolate.

Enzyme: 2-phosphoglycolate phosphatase

2-phosphoglycolate phosphatase

In chloroplast

Dr. Riddhi Datta

In peroxisome

Glycolate is oxidised by O₂, producing H₂O₂ and glyoxylate.

Enzyme: Gycolate oxidase

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The H₂O₂ is broken down to O₂ and H₂O.

Enzyme: peroxisomal catalase

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The catalyzes the transamination of Glyoxylate is transaminated with glutamate, yielding the amino acid glycine and 2-oxoglutarate.

Enzyme: glutamate:glyoxylate aminotransferase

Dr. Riddhi Datta

In mitochondria

Glycine exits the peroxisomes and enters the mitochondria where one molecule of glycine undergoes oxidative decarboxylation using one molecule of NAD⁺ and yields one molecule each of NADH, NH₄⁺, and CO₂ and the activated one-carbon unit methylene tetrahydrofolate (THF) bound to GDC (GDC-THF-CH₂ )

Enzyme: Glycine decarboxylase

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Next, the methylene unit is added to a second molecule of glycine, forming serine and regenerating THF

Enzyme: serine hydroxymethyltransferase

Glycine decarboxylase

serine hydroxy methyltransferase

Dr. Riddhi Datta

In peroxisome

The newly formed serine diffuses from the mitochondria back to the peroxisomes and donates its amino group to 2-oxoglutarate via transamination, forming glutamate and hydroxypyruvate .

Enzyme: serine:2-oxoglutarate aminotransferase

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Next, hydroxypyruvate is converted to glycerate.

Enzyme: NADH-dependent reductase

serine:2-oxoglutarate aminotransferase

NADH-dependent reductase

Dr. Riddhi Datta

In chloroplast

Finally, glycerate reenters the chloroplast, where it is phosphorylated by ATP to yield 3-phosphoglycerate and ADP.

Enzyme: glycerate kinase

The NH₄⁺ released in the oxidation of glycine diffuses rapidly from the matrix of the mitochondria to the chloroplasts where it is converted to glutamate by GS-GOGAT system.

The reassimilation NH₄⁺ of into the photorespiratory cycle restores glutamate for the action of the peroxisomal glutamate:glyoxylate aminotransferase in the conversion of glyoxylate to glycine.

glycerate kinase

Dr. Riddhi Datta

Dr. Riddhi Datta

• C4 photosynthesis has evolved as one of the major carbon-concentrating mechanisms used by land plants to compensate for limitations associated with low levels of atmospheric CO₂ .

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• Some of the most productive crops on the planet (e.g., corn; sugarcane, sorghum) use this mechanism to enhance the catalytic capacity of rubisco.

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• In the C4 cycle, the enzyme phosphoenolpyruvate carboxylase (PEPCase), rather than rubisco, catalyzes the initial carboxylation of phosphoenol pyruvate (PEP) in mesophyll.

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• The product is oxaloacetate which is then quickly reduced to malate or transaminated to aspartate and transported to adjacent bundle sheath cell.

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• The four-carbon acids formed in mesophyll cells flow to the bundle sheath cells, where they are decarboxylated, releasing CO₂ that is refixed by rubisco via the Calvin–Benson cycle.

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• The C3 acid (pyruvate or alanine) that remains in the bundle sheath is then transported to the mesophyll cell. Here alanine is converted to pyruvate. Pyruvate is then phosphorylated to regenetate the PEP. This reaction is catalyzed by pyruvate phosphate dikinase and requires two ATPs.

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• Although all C4 plants share primary carboxylation via PEPCase, there are three major variations among different C4 species.

Inorganic Carbon–Concentrating Mechanisms: The C4 Carbon Cycle

Dr. Riddhi Datta

• Here, oxaloacetate is first converted to malate which is transported to bundle sheath cell.
• Here it is decarboxylated to yield pyruvate and CO2.
• Pyruvate is transported to mesophyll cell and phosphorylated to PEP.

• Here, oxaloacetate is transaminated to aspartate which is transported to bundle sheath cell mitochondrion.
• Here it is converted to malate which is decarboxylated to yield pyruvate and CO2.
• Pyruvate is converted to alanine which is transported to mesophyll cell.
• Here alanine is again converted to pyruvate which is phosphorylated to PEP.

• Similar to NAD-ME type except that decarboxylation occurs in chloroplast.

NADP-ME type: Maize

NAD-ME type: Millet

PEPCK type: Guinea grass

Dr. Riddhi Datta

Energy requirement:

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For every molecule of CO₂ fixed, 2 ATP must be expended in regeneration of PEP in addition to the requirement of Calvin cycle.

Dr. Riddhi Datta

• The C4 cycle has been associated with a particular leaf structure, called Kranz anatomy.

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• Typical Kranz anatomy exhibits an inner ring of bundle sheath cells around vascular tissues and an outer layer of mesophyll cells.

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• This particular leaf anatomy generates a diffusion barrier that
• separates the uptake of atmospheric carbon in mesophyll cells from CO2 assimilation by rubisco in bundle sheath cells
• limits the leakage of CO2 from bundle sheath to mesophyll cells.

Dr. Riddhi Datta

Significance of C4 Carbon Cycle

• Elevated temperatures limit the rate of photosynthetic CO₂ assimilation in C3 plants by decreasing the solubility of CO₂ , and the ratio of the carboxylation to oygenation reactions of rubisco.

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• As a result, the energy demands associated with photorespiration increases.

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• In C4 plants, two features contribute to overcome the deleterious effects of high temperature:

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• Atmospheric CO₂ enters the mesophyll cell cytoplasm where carbonic anhydrase converts CO₂ into bicarbonate. Warm climates decrease the levels of CO₂, but the low concentrations of cytosolic HCO₃⁻ saturate PEPCase because the affinity of the enzyme for its substrate is sufficiently high. Thus, the high activity of PEPCase enables C4 plants to reduce their stomatal aperture at high temperatures and thereby conserve water while fixing CO₂ at rates equal to or greater than those of C3 plants.

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• The high concentration of CO₂ in bundle sheath chloroplasts minimizes the operation of the C2 oxidative photosynthetic carbon cycle.

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• Optimal photosynthetic efficiency:
• C3 plants: 20–25°C
• C4 plants: 25–35°C

Dr. Riddhi Datta

• 2 quanta of light (1 quantum for PSI and PSII each) are required for transfer of 1 electron from H₂O to NADP⁺.

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• 4 electrons are required for reduction of H₂O and evolution of 1 molecule of O₂.

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• Therefore, 8 quanta of light are required for evolution of 1 molecule of O₂ and fixation of 1 molecule of CO₂.

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• This means that 48 quanta (8 x 6) of light are required to fix 6 molecules of CO₂ and generate 1 molecule of glucose (6C).

Quantum yield =48 quanta of red light

• 1 quantum = 42 Kcal, 48 quanta = 2016 Kcal

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• The standard Gibbs free energy change for the synthesis of one mole of glucose from CO₂ and water is approximately 673 kcal/mol. This is the chemical energy that plants store.

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Energy efficiency of photosynthesis

Photosynthetic efficiency = 673/2016 x 100 = 33%

Dr. Riddhi Datta

Fixation of 1 molecule of CO2 requires:

3 ATP and 2 NADPH

1 ATP = 7.3 Kcal, 1 NADPH = 52.6 Kcal

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Energy requirement to fix 6 molecules of CO2:

18 ATP (3 x 6) = 18 x 7.3 Kcal = 131.4 Kcal

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12 NADPH (2 x 6) = 12 x 52.6 Kcal = 631.2 Kcal

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Total: 762.6 Kcal

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Internal efficiency = 673/762.6 x 100 = 88%

Fixation of 1 molecule of CO2 requires:

5 ATP and 2 NADPH

1 ATP = 7.3 Kcal, 1 NADPH = 52.6 Kcal

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Energy requirement to fix 6 molecules of CO2:

30 ATP (5 x 6) = 30 x 7.3 Kcal = 219 Kcal

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12 NADPH (2 x 6) = 12 x 52.6 Kcal = 631.2 Kcal

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Total: 850.2 Kcal

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Internal efficiency = 673/850.2 x 100 = 79%

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C3 cycle

C4 cycle

Energy consumption of photosynthesis

The standard Gibbs free energy change for the synthesis of one mole of glucose from CO₂ and water is approximately 673 kcal/mol. This is the chemical energy that plants store.

Dr. Riddhi Datta

Inorganic Carbon–Concentrating Mechanisms: Crassulacean Acid Metabolism (CAM)

• Present in many plants that inhabit arid environments with seasonal water availability.

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• Example: pineapple (Ananas comosus), agave (Agave spp.), cacti (Cactaceae), and orchids (Orchidaceae)

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• First identified in Bryophyllum calycinum, a succulent member of the Crassulaceae

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• The leaves of CAM plants have traits that minimize water loss, such as thick cuticles, large vacuoles, and stomata with small apertures.

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• Tight packing of the mesophyll cells enhances CAM performance by restricting CO2 loss during the day.

Dr. Riddhi Datta

Inorganic Carbon–Concentrating Mechanisms: Crassulacean Acid Metabolism (CAM)

• Initial capture of CO₂ into four-carbon acids takes place at night, and fixation of CO₂ into carbon skeletons occurs during the day.

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• At night, cytosolic PEPCase fixes atmospheric and respiratory CO₂ into oxaloacetate using phosphoenolpyruvate formed via the glycolytic breakdown of stored carbohydrates.

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• A cytosolic NADP–malate dehydrogenase converts the oxaloacetate to malate, which is stored in vacuoles.

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• During the day, the stored malate exits the vacuole for decarboxylation. The mechanisms is similar a cytosolic NADP–ME or mitochondrial NAD–ME C4 plants.

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• The released CO₂ is fixed in chloroplasts via rubisco.

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• The coproduced three-carbon acid is converted to triose phosphates and subsequently to starch or sucrose via gluconeogenesis.

Dr. Riddhi Datta

Dr. Riddhi Datta

Four distinct phases encompass the temporal control of C4 and C3 carboxylations within the same cellular environment:

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• Phase I (night): stomata are open, CO₂ is captured and stored as malate in the vacuole

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• Phase II (early morning): transient phase, rubisco activity increases. PEPCase activity decreases

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• Phase III (daytime): stomata are closed and leaves are photosynthesizing, stored malate is decarboxylated. This results in high concentrations of CO₂ around rubisco, thereby alleviating the adverse effects of photorespiration

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• Phase IV (late afternoon): transient phase, rubisco activity decreases. PEPCase activity increases

Phases of CAM