Photosynthesis: Regulation of Carbon fixation reactions

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In photosynthetic organisms, the carbon fixation reactions are regulated by light to ensure that the production of carbohydrates is synchronized with the availability of light energy. This regulation involves several mechanisms, including the activation of key enzymes, changes in the concentration of metabolic intermediates, and the formation of supramolecular complexes.

Induction Period in Carbon Fixation

After the onset of illumination, there is an induction period during which the rate of CO2​ fixation increases gradually before reaching a steady state. This initial lag is because the photosynthetic enzymes need time to be activated by light, and the concentration of Calvin-Benson cycle intermediates needs to increase.

• During the induction period, six triose phosphates produced in the Calvin-Benson cycle are used for the regeneration of ribulose 1,5-bisphosphate (RuBP).

• At the steady state, five of the six triose phosphates regenerate RuBP, while the remaining one is either used for starch formation in the chloroplast or sucrose synthesis in the cytosol.

Regulation of the Calvin-Benson Cycle

The regulation of the Calvin-Benson cycle involves both long-term and short-term mechanisms.

Long-term regulation involves changes in the amount of enzymes, which is controlled by the expression of nuclear and chloroplast genes. Regulatory signaling between nucleus and plastids is mostly anterograde — that is, the products of nuclear genes control the transcription and translation of plastid genes. 

Short-term regulation involves post-translational modifications that can rapidly alter the activity of enzymes.

Light-dependent mechanisms quickly regulate the specific activity of five key enzymes:

1. Rubisco

2. Fructose 1,6-bisphosphatase

3. Sedoheptulose 1,7-bisphosphatase

4. Phosphoribulokinase

5. NADP–glyceraldehyde-3-phosphate dehydrogenase

Rubisco: Activation and Catalysis

Rubisco, a crucial enzyme for carbon fixation, is regulated by Rubisco-activase. 

A CO2​ molecule has a dual role in Rubisco's activity: it acts as an activator and a substrate.

• Activation: The activation process begins when an activator CO2​ molecule forms a lysyl-carbamate with the enzyme. The subsequent binding of Mg2+ stabilizes this complex, making Rubisco catalytically active. In the light, an increase in both the stroma's pH and Mg2+ concentration facilitates this activation.

• Catalysis: A separate CO2​ molecule, the substrate CO2​, then reacts with RuBP at the active site, yielding two molecules of 3-phosphoglycerate.

Activation of RuBisCo by CO2 and Mg2+

Rubisco-activase overcomes several types of inhibition. Sugar phosphates like xylulose 1,5-bisphosphate and 2-carboxyarabinitol 1-phosphate (naturally occurring inhibitor) can bind tightly to uncarbamylated Rubisco, preventing its activation and inhibiting catalysis. The substrate RuBP can also bind to the carbamylated enzyme, preventing activation. Rubisco-activase removes these inhibitors, allowing the enzyme to be activated by activator CO2 and Mg2+. 

Formation of the Dead-End Complex

• A Rubisco active site that has lost its activating CO2​ molecule (a process called decarbamylation) can bind to RuBP.

• This binding leads to a conformational change in Rubisco, which creates an inactive, dead-end complex where RuBP is tightly bound to the enzyme.

How Rubisco-Activase Restores Activity

Rubisco-activase removes the tightly bound RuBP in an ATP dependent process. The steps are as follows: 

1. Activase Priming: ATP hydrolysis by Rubisco-activase is required for the conformational changes that prime the activase.

2. Physical Interaction: The primed Rubisco-activase physically interacts with the Rubisco-RuBP dead-end complex.

3. Conformational Change: This interaction changes the conformation of Rubisco, reducing its affinity for RuBP.

4. RuBP Dissociation: With a lower affinity, the tightly bound RuBP dissociates from the active site of Rubisco.

5. Reactivation: Once the active site is free, Rubisco-activase likely dissociates, allowing the site to be reactivated through carbamylation by CO2​. This allows the enzyme to resume its role in carbon fixation.

At high temperatures, Rubisco deactivates faster than Rubisco-activase can reactivate it, leading to a loss of function.

Activation of RuBisCo by RuBisCo activase (adapted from Jensen (2000) PNAS)

The Ferredoxin-Thioredoxin System

The ferredoxin-thioredoxin system links the light-dependent reactions of photosynthesis to the regulation of Calvin-Benson cycle enzymes. Light transfers electrons from water to ferredoxin. This reduced ferredoxin is then used to reduce ferredoxin-thioredoxin reductase, which in turn reduces thioredoxin (Trx). The reduced thioredoxin activates target enzymes by reducing their regulatory disulfide bonds.

This system regulates the activity of several enzymes, including:

• Fructose 1,6-bisphosphatase

• Sedoheptulose 1,7-bisphosphatase

• Phosphoribulokinase

• NADP–glyceraldehyde-3-phosphate dehydrogenase

When it's dark, electron flow from ferredoxin stops, and thioredoxin becomes oxidized, ultimately inactivating the target enzymes.

Regulation via ferredoxin-thioredoxin system

Light-Dependent Ion Movements and Enzyme Activity

Light also regulates Calvin-Benson cycle enzymes through the movement of ions. When illuminated, protons flow from the stroma into the thylakoid lumen, while Mg2+ is released from the thylakoid space into the stroma. This increases the stromal pH and Mg2+ concentration, which activates enzymes like Rubisco, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and phosphoribulokinase. These ionic changes are reversed in the dark, deactivating the enzymes.

Regulation of Chloroplast Enzymes via Supramolecular Complexes

Another mechanism for light-dependent regulation of the Calvin cycle involves the formation of supramolecular complexes. This process primarily centers on a small regulatory protein called CP12 and involves regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK).

A2B2 and A4 Forms of GAPDH

The regulation of GAPDH is complex, involving two major isoforms in higher plants: 

• the heterotetramer A2B2
• the homotetramer A4

Both are assembled from different subunits and play distinct roles in the light-dark regulation of photosynthesis. 

A4 Homotetramer

• Composition: The A4 form consists of four identical GapA subunits. It is the catalytically active homotetramer.

• Regulation: The A4 homotetramer is regulated by its interaction with the small protein CP12.

A2B2 Heterotetramer

• Composition: The A2B2 form is made of two GapA subunits and two GapB subunits. This is the most abundant and active form of GAPDH in land plants.

• Regulation: Its regulation is largely autonomous, primarily controlled by the C-terminal extension (CTE) of the GapB subunit. 

A4-GAPDH Regulation

The CP12-Mediated Complex:

In the dark, when there is no need for active carbon fixation, the regulatory protein CP12, which is oxidized by the absence of light, binds to two key Calvin cycle enzymes:

• A4 isoform of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

• Phosphoribulokinase (PRK)

This binding forms a ternary supramolecular complex (GAPDH/CP12/PRK). The formation of this complex effectively sequesters and inactivates both GAPDH and PRK, switching off the Calvin-Benson cycle and preventing the wasteful consumption of ATP and NADPH that would otherwise occur in the dark.

Dissociation in the Light:

When light becomes available, the thylakoid electron transport chain produces reducing power in the form of NADPH. This is used to reduce ferredoxin, which then reduces thioredoxin. The reduced thioredoxin acts on the CP12-mediated complex.

Reduced thioredoxin cleaves the disulfide bonds within the oxidized CP12. This reduction of CP12 causes the entire supramolecular complex to dissociate. The dissociation releases the A4 isoform of GAPDH and PRK, which become catalytically active.

This mechanism ensures that the Calvin cycle is tightly coupled to the availability of light, providing a quick and efficient way to regulate carbon fixation in response to light-dark transitions.

Regulation by CP12-Mediated Complex (adapted from Plant Physiology and Development by Taiz, Zeiger, Møller, and Murphy)

A2B2-GAPDH Regulation

The A2B2 isoform is a heterotetramer made of A and B polypeptides. The B subunit contains a C-terminal extension with two cysteines that can form a disulfide bridge. The A2B2 form can also assemble into a larger, inactive A8B8 oligomer.

• In Darkness: The A2B2-GAPDH forms the inactive A8B8 oligomer.

• In Light: Reduced thioredoxin (Trx) cleaves the disulfide bond in the B subunit of the A8B8 oligomer. This reduction converts the inactive oligomer into the active A2B2-GAPDH. This autonomous redox-modulation is particularly suited for conditions of very active photosynthesis.

Regulation by C-terminal extension (adapted from Plant Physiology and Development by Taiz, Zeiger, Møller, and Murphy)

GAPDH Isoform	Composition	Regulation Mechanism	Active State	Inactive State
A4	A4 homotetramer	Forms a ternary complex with CP12 and PRK in the dark.	Free, active enzymes.	Inactive ternary complex.
A2B2	A2B2 heterotetramer	Forms an A8B8 oligomer in the dark; regulated by disulfide bond in subunit B.	Active A2B2 tetramer.	Inactive A8B8 oligomer.

Regulation of C4 Enzymes

In C4 plants, light also regulates key enzymes through thiol-disulfide exchange and phosphorylation-dephosphorylation.

• NADP–malate dehydrogenase (MDH) is activated in the light by the ferredoxin-thioredoxin system (reduced by thioredoxin) and deactivated in the dark (oxidized).

Regulation of MDH (Carr et al. 1999, Cell)

• Phosphoenolpyruvate (PEP) carboxylase is regulated by diurnal phosphorylation. Phosphorylation makes it active at night and insensitive to inhibition by malate. Dephosphorylation by a protein phosphatase in the day brings the enzyme to low activity.

Regulation of PEP

• Pyruvate–phosphate dikinase is regulated by a bifunctional kinase-phosphatase enzyme. In the dark, ADP-dependent phosphorylation deactivates the enzyme, while in the light, Pi-dependent dephosphorylation restores its activity.

Photosynthesis: Bioenergetics

 

Energy consumption of photosynthesis

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

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

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

• 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

• 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.

Photosynthetic efficiency = 673/2016 x 100 = 33%

C3 cycle

Fixation of 1 molecule of CO2 requires: 3 ATP and 2 NADPH

               1 ATP = 7.3 Kcal, 1 NADPH = 52.6 Kcal

Energy requirement to fix 6 molecules of CO2: 

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

                                

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

Total: 131.4 Kcal + 631.2 Kcal = 762.6 Kcal

Internal efficiency = 673/762.6 x 100 = 88%

C4 cycle

Fixation of 1 molecule of CO2 requires: 5 ATP and 2 NADPH

            1 ATP = 7.3 Kcal, 1 NADPH = 52.6 Kcal

Energy requirement to fix 6 molecules of CO2:

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

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

Total: 219 Kcal + 631.2 Kcal = 850.2 Kcal

Internal efficiency = 673/850.2 x 100 = 79%

Photosynthesis: Photoprotection of the Photosynthetic Apparatus

 

Photoprotection of the Photosynthetic Apparatus

Photoprotection is the process by which photosynthetic organisms protect themselves from the damaging effects of excess light energy, a condition known as photoinhibition. 

The photosynthetic membrane can be easily damaged if the large amount of energy absorbed by pigments cannot be stored through photochemistry. Photoprotection acts as a safety valve, dissipating this excess energy before it can cause harm.

The Role of Carotenoids

Carotenoids are essential accessory pigments that play a vital role in photoprotection in addition to their light-harvesting function. They protect the photosynthetic apparatus by a process called quenching, where they rapidly dissipate the excess energy from excited chlorophyll molecules.

If an excited chlorophyll molecule is not quickly quenched by energy transfer or photochemistry, it can react with molecular oxygen to form highly reactive singlet oxygen. Singlet oxygen can cause oxidative damage to various cellular components, particularly the lipids within the thylakoid membranes. 

Carotenoids prevent this by accepting the excess energy from excited chlorophyll and returning to their ground state by releasing the energy as harmless heat. The excited state of carotenoids does not have enough energy to form singlet oxygen, making them an effective shield against this damage.

Non-photochemical Quenching (NPQ) and the Xanthophyll Cycle

A major process of photoprotection is non-photochemical quenching (NPQ), which involves dissipating excess light energy as heat. It is often described as a "volume knob" that adjusts the flow of excitations to the Photosystem II (PSII) reaction center to a manageable level.

A specific group of carotenoids called xanthophylls are involved in NPQ. The xanthophyll cycle is a key regulatory mechanism that involves the interconversion of three xanthophylls: violaxanthin, antheraxanthin, and zeaxanthin. Under high light conditions, the enzyme violaxanthin de-epoxidase converts violaxanthin to zeaxanthin via antheraxanthin. The binding of protons and zeaxanthin to specific light-harvesting antenna proteins (like the PsbS protein) is believed to cause conformational changes that promote the quenching of chlorophyll and heat dissipation.

Xanthophyll cycle

Photoinhibition and Repair Mechanisms

Even with these protective mechanisms, excess light can lead to photoinhibition, which can inactivate and damage the PSII reaction center. When this happens, a multi-level defense system is activated:

1. Quenching: The first line of defense is the dissipation of excess energy as heat.

2. Scavenging: If quenching is insufficient, the formation of toxic photoproducts is followed by the action of various scavenging systems that eliminate these reactive molecules.

3. Repair: If the damage persists, the main target is the D1 protein of the PSII reaction center. The damaged D1 protein is removed from the membrane and a newly synthesized D1 protein is inserted to restore the function of the PSII reaction center. This ensures that the other undamaged components of the PSII complex are recycled.

Photosynthesis: Carbon reactions (Updated)

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Carbon Reactions of Photosynthesis

The carbon reactions, formerly known as "dark reactions," occur in the stroma of the chloroplast. They are responsible for reducing atmospheric carbon dioxide into carbohydrates using the energy stored in ATP and NADPH, which are products of the light-driven reactions. Although these reactions can occur without light, they depend on the products of the light reactions to provide substrates for enzymes and regulate the catalytic rate.

C3 Cycle

The primary pathway for autotrophic CO2 fixation is the Calvin-Benson cycle, also referred to as the reductive pentose phosphate cycle or photosynthetic carbon reduction cycle. This cycle decreases the oxidation state of carbon from +4 in CO2, to +2 in keto groups and 0 in secondary alcohols found in sugars. 

The Calvin-Benson cycle proceeds in three main phases:

1. Carboxylation

This phase is where atmospheric CO2 is covalently bonded to a carbon skeleton.

• One molecule of CO2 and one molecule of water react with one molecule of ribulose 1,5-bisphosphate (RuBP).

• This reaction yields two molecules of 3-phosphoglycerate.

• The enzyme that catalyzes this reaction is ribulose 1,5-bisphosphate carboxylase/oxygenase, commonly known as rubisco.

The Calvin cycle is also called the C₃ cycle because the first stable intermediate compound formed during carbon fixation is a three-carbon molecule, 3-PGA.

2. Reduction

In this phase, the carbon from the 3-phosphoglycerate is reduced to form a carbohydrate. This step requires the ATP and NADPH generated during the light reactions.

• ATP phosphorylates 3-phosphoglycerate, producing 1,3-bisphosphoglycerate. This reaction is catalyzed by the enzyme 3-phosphoglycerate kinase.

• NADPH then reduces the 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate. The enzyme involved is NADP-glyceraldehyde-3-phosphate dehydrogenase.

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

3. Regeneration

This phase is crucial for the continuous operation of the cycle, as it restores the CO2 acceptor, ribulose 1,5-bisphosphate (RuBP).

• For every three molecules of CO2 that enter the cycle, six molecules of glyceraldehyde 3-phosphate are formed.

• Five of these glyceraldehyde 3-phosphate molecules (5x3C) are used to regenerate three molecules of RuBP (3x5C).

• The remaining one molecule of glyceraldehyde 3-phosphate represents the net assimilation of three CO2 molecules and becomes available for the plant's metabolism.

• The glyceraldehyde 3-phosphate can be used to synthesize starch in the chloroplast or be transported to the cytosol for sucrose synthesis. Sucrose is then transported to other parts of the plant for growth or storage.

Steps:

• One molecule of glyceraldehyde-3-phosphate (G3P) is isomerized to dihydroxyacetone phosphate (DHAP) by the enzyme triose phosphate isomerase.

• A second G3P molecule and the DHAP molecule are joined together to form fructose-1,6-bisphosphate (FBP). This reaction is catalyzed by the enzyme aldolase.

• Fructose-1,6-bisphosphate is dephosphorylated to fructose-6-phosphate (F6P) by the enzyme fructose-1,6-bisphosphatase.

• A third G3P molecule reacts with F6P. This reaction, catalyzed by transketolase, produces erythrose-4-phosphate (E4P) (a four-carbon sugar) and xylulose-5-phosphate (Xu5P) (a five-carbon sugar).

• Erythrose-4-phosphate (E4P) and a fourth G3P molecule (converted to DHAP by triose phosphate isomerase) are combined by aldolase to form sedoheptulose-1,7-bisphosphate (SBP).

• Sedoheptulose-1,7-bisphosphate is dephosphorylated to sedoheptulose-7-phosphate (S7P) by sedoheptulose-1,7-bisphosphatase.

• Sedoheptulose-7-phosphate and the remaining G3P molecule are converted by transketolase to ribose-5-phosphate (R5P) and another molecule of xylulose-5-phosphate (Xu5P).

• The two molecules of xylulose-5-phosphate (Xu5P) are isomerized to ribulose-5-phosphate (Ru5P) by the enzyme phosphopentose epimerase.

• The molecule of ribose-5-phosphate (R5P) is isomerized to ribulose-5-phosphate (Ru5P) by the enzyme phosphopentose isomerase.

• Finally, the three molecules of ribulose-5-phosphate are phosphorylated by phosphoribulokinase, using three molecules of ATP, to regenerate the three molecules of ribulose-1,5-bisphosphate (RuBP).

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

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

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

The C₂ Photorespiratory Cycle

The C₂ photorespiratory cycle, or C₂ oxidative photosynthetic carbon cycle, is a metabolic pathway that occurs in plants, primarily as a consequence of the dual activity of the enzyme rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase).

Role of Rubisco

While rubisco typically catalyzes the addition of carbon dioxide to ribulose 1,5-bisphosphate during the Calvin cycle, it can also bind with oxygen. This oxygenase activity leads to the loss of some of the carbon that was fixed in the Calvin cycle.

When rubisco reacts with oxygen instead of carbon dioxide, it produces two molecules:

• One molecule of 3-phosphoglycerate, which can be used in the Calvin cycle.

• One molecule of 2-phosphoglycolate.

The 2-phosphoglycolate molecule is an inhibitor of important chloroplast enzymes like triose phosphate isomerase and phosphofructokinase. To prevent this inhibition, the plant metabolizes 2-phosphoglycolate through the C₂ photorespiratory cycle.

Location of the Cycle

This cycle is unique because it involves three different organelles in the plant cell:

• Chloroplasts

• Leaf peroxisomes

• Mitochondria

Key Steps of the C₂ Cycle

1. In the Chloroplast

• Step 1: Rubisco oxygenates ribulose 1,5-bisphosphate using a molecule of O₂, which then splits into 3-phosphoglycerate and 2-phosphoglycolate.

  Step 2: The 2-phosphoglycolate is quickly converted into glycolate by the enzyme 2-phosphoglycolate phosphatase.

2. In the Peroxisome

• Step 3: The glycolate from the chloroplast moves into the peroxisome. Here, the enzyme glycolate oxidase oxidizes it using O₂, producing glyoxylate and hydrogen peroxide (H2​O2​).

• Step 4: The hydrogen peroxide is immediately broken down into water and oxygen by peroxisomal catalase.

• Step 5: Glyoxylate undergoes transamination with glutamate, producing the amino acid glycine and 2-oxoglutarate, a reaction catalyzed by glutamate:glyoxylate aminotransferase.

3. In the Mitochondria

• Step 6: Glycine travels from the peroxisome into the mitochondria. Two molecules of glycine undergo oxidative decarboxylation, which uses one molecule of NAD⁺. This reaction, catalyzed by glycine decarboxylase, releases a molecule each of NADH, NH4+​, and CO2​. The CO2​ released here is the primary reason photorespiration is considered a wasteful process, as it represents a loss of fixed carbon.

• Step 7: A one-carbon unit from the first glycine molecule is added to a second glycine molecule, forming the amino acid serine. The enzyme responsible for this reaction is serine hydroxymethyl transferase.

4. Back to the Peroxisome

• Step 8: The newly formed serine diffuses from the mitochondria back to the peroxisome.

• Step 9: Serine donates its amino group to 2-oxoglutarate, forming glutamate and hydroxypyruvate. The reaction is catalyzed by serine:2-oxoglutarate aminotransferase.

• Step 10: Hydroxypyruvate is converted to glycerate by the enzyme NADH-dependent reductase.

5. Return to the Chloroplast

• Step 11: Finally, the glycerate re-enters the chloroplast, where it is phosphorylated by ATP to regenerate 3-phosphoglycerate. The reaction is catalyzed by glycerate kinase. This 3-phosphoglycerate can then re-enter the Calvin cycle, completing the pathway.

The ammonia (NH4+​) released in the mitochondria is also re-assimilated in the chloroplast by the GS-GOGAT system, which restores the glutamate needed for the cycle to continue.

Photorespiratory cycle

The C₄ Carbon Cycle

The C₄ carbon cycle is a specialized photosynthetic pathway used by certain plants, such as corn, sugarcane, and sorghum, to concentrate carbon dioxide and overcome the limitations of the C₃ cycle. This mechanism enhances the efficiency of the enzyme rubisco, which is central to photosynthesis.

Key Features and Anatomy

Unlike C₃ plants, C₄ plants have a unique leaf structure known as Kranz anatomy. This structure consists of a ring of bundle sheath cells surrounding the vascular tissues, with an outer layer of mesophyll cells. This arrangement creates a physical separation, allowing for the initial capture of atmospheric carbon in the mesophyll cells and its assimilation by rubisco in the bundle sheath cells. This separation also minimizes the leakage of carbon dioxide from the bundle sheath cells back to the mesophyll cells.

Steps

The C₄ cycle operates across two different cell types: mesophyll and bundle sheath cells.

1. Carboxylation in Mesophyll Cells: In the cytoplasm of the mesophyll cell, the enzyme phosphoenolpyruvate carboxylase (PEPCase) catalyzes the initial fixation of atmospheric carbon dioxide. PEPCase adds a molecule of carbon dioxide to the three-carbon compound phosphoenolpyruvate (PEP), forming the four-carbon acid, oxaloacetate.

2. Transport and Decarboxylation: The oxaloacetate is then either reduced to malate or transaminated to aspartate and then transported to the adjacent bundle sheath cell. Once inside the bundle sheath cell, the four-carbon acid is decarboxylated, releasing a molecule of carbon dioxide.

3. Calvin Cycle: The released carbon dioxide is refixed by rubisco and enters the Calvin cycle within the bundle sheath cell. This process maintains a high concentration of carbon dioxide around rubisco, which reduces the wasteful process of photorespiration.

4. Regeneration of PEP: The three-carbon acid (pyruvate or alanine) that remains after decarboxylation is transported back to the mesophyll cell. In the mesophyll cell, this acid is phosphorylated using ATP to regenerate PEP, allowing the cycle to continue. This regeneration step is catalyzed by the enzyme pyruvate phosphate dikinase and requires two ATP molecules.

Variations of C4 cycle

Comparison of three variations of C4 cycle:

Feature	NADP-Malic Enzyme (NADP-ME) Type	NAD-Malic Enzyme (NAD-ME) Type	PEP-Carboxykinase (PEP-CK) Type
Primary Decarboxylation Enzyme	NADP-Malic Enzyme	NAD-Malic Enzyme	Phosphoenolpyruvate Carboxykinase (PEP-CK)
Location of Decarboxylation	Chloroplast of bundle sheath cell	Mitochondrion of bundle sheath cell	Cytosol of bundle sheath cell
Primary C₄ Acid Transported	Malate	Aspartate	Malate
C₃ Compound Returned to Mesophyll	Pyruvate	Alanine	PEP or Alanine
Electron Source for Regeneration	NADPH in the mesophyll chloroplasts is used for the reduction of oxaloacetate to malate.	NADH produced during the decarboxylation of malate in the bundle sheath mitochondria is used.	ATP is required for the regeneration of PEP.
Examples of Plants	Maize, Sugarcane, Sorghum	Amaranthus, Panicum miliaceum, Portulaca oleracea	Panicum maximum, Sporobolus, Urochloa
Anatomy of Bundle Sheath Cells	Bundle sheath chloroplasts are deficient in grana and are positioned centrifugally (towards the mesophyll cells).	Bundle sheath chloroplasts have well-developed grana and are positioned centripetally (towards the vascular tissue).	Bundle sheath chloroplasts are similar to NAD-ME types with well-developed grana and centripetal positioning.

Energy Requirements

The C₄ cycle is more energy-intensive than the C₃ cycle. For every molecule of carbon dioxide fixed, two additional ATP molecules are required for the regeneration of PEP. This is in addition to the ATP and NADPH needed for the Calvin cycle itself.

Significance of the C₄ Cycle

High temperature limits the rate of carbon dioxide assimilation in C3 plants by decreasing solubility of carbon dioxide. This reduces the ratio of carboxylation to oxygenation reactions of RuBisCO. As a result, energy demand for photorespiration increases. The C₄ pathway is a successful adaptation for plants in hot and dry climates. In these conditions, elevated temperatures can decrease the solubility of carbon dioxide, and the ratio of carboxylation to oxygenation by rubisco is negatively affected, increasing the rate of photorespiration. The C₄ cycle's ability to concentrate carbon dioxide around rubisco minimizes photorespiration. Furthermore, PEPCase has a very high affinity for carbon dioxide, allowing C₄ plants to reduce their stomatal openings at high temperatures to conserve water without significantly compromising their rate of photosynthesis. The optimal temperature range for C₄ plants is 25–35°C, compared to 20–25°C for C₃ plants.

Crassulacean Acid Metabolism (CAM)

Crassulacean Acid Metabolism (CAM) is a carbon-concentrating mechanism found in plants adapted to arid environments with limited water availability, such as cacti, pineapples, and orchids. CAM plants have specific leaf characteristics that minimize water loss, including thick cuticles, large vacuoles, and stomata with small apertures. The tightly packed mesophyll cells also help restrict CO2 loss during the day, enhancing the plant's CAM performance.

The CAM Process

The CAM process separates the initial capture of CO2 and its final fixation into carbon skeletons by performing them at different times of the day.

1. Nighttime (Phase I) 🌙

At night, the plant's stomata open to take in atmospheric CO2. Cytosolic phosphoenolpyruvate carboxylase (PEPCase) fixes this CO2, as well as respiratory CO2, into oxaloacetate. This reaction uses phosphoenolpyruvate (PEP), which is formed from the breakdown of stored carbohydrates. 

A cytosolic NADP-malate dehydrogenase then converts the oxaloacetate into malate, which is stored in the plant's large vacuoles.

2. Daytime (Phase III) ☀️

During the day, the stomata close to prevent water loss through transpiration. The stored malate is released from the vacuoles and undergoes decarboxylation. This process, which is similar to the mechanisms in C4 plants, releases CO2, which is then fixed by rubisco in the chloroplasts through the Calvin-Benson cycle. 

The high concentration of CO2 created around rubisco during this time helps to minimize photorespiration, a process that can be detrimental to photosynthesis. The three-carbon acid that is co-produced is converted to triose phosphates and then to starch or sucrose via gluconeogenesis.

CAM pathway

Phases of CAM

The CAM cycle has four distinct phases that regulate the temporal separation of carbon fixation:

• Phase I (Night): The stomata are open, and CO2 is taken in and stored as malate in the vacuole.

• Phase II (Early Morning): This is a transitional phase where rubisco activity increases and PEPCase activity decreases.

• Phase III (Daytime): The stomata are closed, and the stored malate is decarboxylated, releasing CO2 for the Calvin-Benson cycle.

• Phase IV (Late Afternoon): This is another transitional phase where rubisco activity decreases and PEPCase activity increases in preparation for the nighttime CO2 uptake.

 Comparison between C4 cycle and CAM:

Feature	C4 Cycle	CAM (Crassulacean Acid Metabolism)
Plants	Maize, sugarcane, sorghum, millet	Cacti, succulents, pineapples, orchids
Separation of CO₂ Fixation	Spatially separated in different cells (mesophyll and bundle sheath cells).	Temporally separated at different times (night and day).
Stomata Activity	Partially open during the day.	Open at night, closed during the day.
Initial CO₂ Fixation	Occurs in mesophyll cells during the day.	Occurs in mesophyll cells at night.
CO₂-Fixing Enzyme	PEP carboxylase in mesophyll cells, followed by RuBisCO in bundle sheath cells.	PEP carboxylase at night, followed by RuBisCO during the day.
First Stable Product	A four-carbon compound, oxaloacetate.	A four-carbon compound, malic acid.
Photorespiration	Minimized due to spatial separation, which concentrates CO₂ around RuBisCO.	Minimized due to temporal separation, which concentrates CO₂ around RuBisCO during the day.
Water Efficiency	High water-use efficiency.	Very high water-use efficiency.
Best Adapted to	Hot and sunny environments.	Very hot and dry, arid environments.