Photosynthesis: Biosynthetic Pathway (Dark reaction) and Difference Between C3 and C4 Plants

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The process by which carbon dioxide is reduced to carbohydrates by making use of the products of light reaction (ATP and NADPH2) is called carbon dioxide fixation or biosynthesis phase. These series of reactions which catalyse the reduction of CO2 to carbohydrates (also called fixation of CO2) take place in the stroma of the chloroplast. These reactions are independent of light i.e. light is not necessary but can continue in light as well if products of the light reaction are available. Thus, it is also called dark reaction. Depending on whether the first product of photosynthesis is a 3-carbon compound or a 4-carbon compound, there are two pathways.

C3 Cycle (Also Called Calvin Cycle After Its Discoverer, Melvin Calvin)

A CO2 molecule combines with a five-carbon acceptor molecule, ribulose-1, 5-bisphosphate (RuBP). This step makes a six-carbon compound that splits into two molecules of a three-carbon compound, 3-phosphoglyceric acid (3-PGA). This reaction is catalyzed by the enzyme RuBP carboxylase/oxygenase or rubisco. This enzyme is probably the most abundant protein on earth. In reduction stage, ATP and NADPH are used to convert the 3-PGA molecules into molecules of a three-carbon sugar, glyceraldehyde 3-phosphate (G3P). In the next step, PGA is reduced to 3-carbon carbohydrate called triose phosphate using NADPH2 and ATP (from light reaction). Much of these molecules are then diverted from the C3 cycle and used for synthesis of other carbohydrates such as glucose and sucrose. To complete the cycle, the initial 5-carbon acceptor molecule, RuBP is regenerated from the triose phosphates using ATP molecule thus the C3 cycle continues to regenerate the CO2-acceptor (RuBP).

Image showing the Calvin cycle.

Image Showing the Calvin Cycle.

Image showing the Calvin cycle.

C4 Cycle (Or Hatch and Slack Cycle)

  • The C4 plants are generally found in the tropical and subtropical regions and they have evolved C4 pathway as a mechanism to avoid the occurrence of photorespiration, which is considered a wasteful process. All those plants which exhibit C4 pathway of photosynthesis are called C4 plants, e.g. Zea maize (maize), Saccharum (sugar cane), Amaranthus, Digitalis, etc. Photorespiration (oxidation of RuBP in presence of O2) is absent in these plants. So, the photosynthetic rate is high.

  • The bundle sheath cells are large Chlorenchyma/parenchyma cells, arranged in a single layer around the vascular bundles like a wreath; this type of arrangement is known as Kranz anatomy. These are they have dimorphic chloroplasts, light reactions and the carbon cycle occur separately in two different types of cells, the former taking place in mesophyll cells and the latter occurring in bundle sheath cells. This type of cellular arrangement does not allow the oxygen to come in contact with the Rubisco in the bundle sheath cells, hence oxygenation of RuBP is completely avoided. Added to this, they have evolved a carbon dioxide concentrating mechanism, called C4 pathway.

Image showing transverse section of maize leaf.

Image Showing Transverse Section of Maize Leaf.

Image showing transverse section of maize leaf.

  • The CO2 acceptor in C4 plants is phosphoenolpyruvate (PEP). PEP reacts with CO2 to form oxaloacetic acid which is reduced by NADPH to form malic acid. The malic acid then reacts with RuBP to form pyruvic acid and PGA. The pyruvic acid is then phosphorylated by ATP to regenerate PEP while PGA is converted to triose phosphate as far as C3 plants. It combines with CO2 in presence of an enzyme Phosphoenolpyruvate carboxylase (PEPCase) and forms a C4 acid, oxaloacetic acid (OAA). OAA fixation of CO2 occurs in the cytosol of the mesophyll cells of the leaf. OAA is the first stable product of this cycle and hence the name C4 pathway. OAA then travels from mesophyll cells to the chloroplasts of bundle sheath cell where it releases the fixed CO2. C3 cycle operates within these cells and this CO2 immediately combines with RuBP and C3 cycle continues producing sugars.

Image showing C4 photosynthetic carbon cycle.

Image Showing C4 Photosynthetic Carbon Cycle.

Image showing C4 photosynthetic carbon cycle.

Difference between C3 and C4 Plants

Table Showing C3 and C4 Plants Differences.
Table showing C3 and C4 plants differences.

C3 Plants

C4 Plants

Carbon dioxide fixation

Occurs once

Occurs twice, first in mesophyll cells, then in bundle sheath cells

Carbon dioxide acceptor

RuBP, a 5-C-compound

Mesophyll cells. PEP (phosphoenolPyruvic acid), a 5C- compound in the bundle sheath cells-RuBP

Carbon dioxide fixing enzymes

RuBP carboxylase, which is inefficient

PEP carboxylase which is very efficient.

RuBP carboxylase, works efficiently because carbon dioxide concentration is high

First product of photosynthesis

A C3 acid, PGA

A C4 acid, e.g. oxaloacetic acid

Leaf anatomy

Only one type of chloroplast

Kranz anatomy, i.e. two types of cell, each with its own type of chloroplast


Occurs; oxygen is an inhibitor of photosynthesis

Is inhibited by high carbon dioxide concentration. Therefore, atmosphere oxygen is not an inhibitor of photosynthesis


Less efficient photosynthesis than C4 plants. Yields usually much lower

More efficient photosynthesis than C3 plants. Yields usually much higher.


Monomorphic-one type

Dimorphic-two types


Bean, Rice, potato etc.

Maize, sugar cane, sorghum, etc.

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