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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.