C₃ and C₄ Pathways

Photosynthesis is the process by which plants, algae, and some bacteria convert light energy into chemical energy. A key part of this process is carbon fixation, in which atmospheric carbon dioxide (CO₂) is converted into organic compounds. The two main pathways of carbon fixation are the C₃ and C₄ pathways. These pathways differ in their efficiency and the environmental conditions they are best suited for.

The C₃ Pathway (Calvin Cycle)

• In C₃ plants, the Calvin cycle occurs in the stroma of mesophyll chloroplasts.
• The first stable compound of the Calvin cycle is a 3-carbon compound–PGA (Phosphoglyceric acid or phosphoglycerate), thus the Calvin cycle is called the C₃ cycle. (First compound is unstable, 6C keto acid–carboxy ketoribitol bisphosphate).
• Calvin studied the dark reaction in green algae Chlorella & Scenedesmus. During his experiment, he used radioisotopy (14C) and chromatography techniques to identify and separate intermediates of the C₃ cycle.
• RuBisCO (Ribulose-1,5-bisphosphate carboxylase-oxygenase) is the main enzyme in the C₃ cycle, which is present in the stroma. RuBisCO is the most abundant enzyme and protein on earth.

For ease of understanding, the Calvin cycle can be described under three stages: carboxylation, reduction and regeneration.

• Carboxylation – Carboxylation is the fixation of CO₂ into a stable organic intermediate. Carboxylation is the most crucial step in the Calvin cycle, in which CO₂ is used to carboxylate RuBP. This reaction is catalysed by the enzyme RuBP carboxylase, which results in the formation of two molecules of 3-PGA. Since this enzyme also has oxygenase activity, it is more correct to call it RuBP carboxylase-oxygenase, or RuBisCO.
• Reduction – A series of reactions that lead to the formation of glucose. The steps involve the utilisation of 2 molecules of ATP for phosphorylation and two of NADPH for reduction per CO₂ molecule fixed. The fixation of six molecules of CO₂ and 6 turns of the cycle are required for the formation of one molecule of glucose from the pathway.
• Regeneration – Regeneration of the CO₂ acceptor molecule RuBP is crucial if the cycle is to continue uninterrupted. The regeneration steps require one ATP for phosphorylation to form RuBP.
• Hence, for every CO₂ molecule entering the Calvin cycle, 3 molecules of ATP and 2 of NADPH are required. It is probably because of the difference in the amounts of ATP and NADPH used in the dark reaction that cyclic phosphorylation occurs.
• 6 turns of the Calvin cycle are required for the formation of one glucose, as 6 CO₂ are required for the synthesis of one hexose.
• 12 NADPH + H+ & 18 ATP are required as assimilatory power to produce one Glucose in the dark reaction in C₃ cycle.

C₄-PATHWAY/CO₂ CONCENTRATING MECHANISM/CO-OPERATIVE PHOTOSYNTHESIS /DICARBOXYLIC ACID CYCLE (DCA CYCLE)/HATCH & SLACK PATHWAY

• Hatch & Slack (1967) studied it in detail in sugarcane and maize leaves and proposed a new pathway for dark reactions.
• The first stable product of this pathway is OAA, a 4C, DCA (Dicarboxylic Acid); thus, the Hatch & Slack pathway is also called the C₄ pathway or the DCA cycle.
• Most C₄ plants are monocots (Tropical grasses) belonging to the Gramineae & Cyperaceae families. C₄ plants are adapted to hot and dry environments. e.g. of C₄ plants – Sugarcane, Maize, Sorghum. Wheat, Rice and Barley (monocot) are C₃ species.
• Kranz (Wreath) Anatomy – Present in leaves of C₄ plants. In these plants, large green cells are found around the vascular bundles in leaves; these are called bundle sheath cells, and leaves with this anatomy are said to have 'Kranz anatomy'. 'Kranz' means 'wreath' and is a reflection of the arrangement of cells.
• The bundle sheath cells may form several layers around the vascular bundles; they are characterised by a large number of chloroplasts, thick walls impervious to gaseous exchange, and the absence of intercellular spaces.
• Dimorphic chloroplasts are present in leaf cells of C₄ plants. Chloroplasts of bundle sheath cells or Kranz cells are large and without grana (Agrana, i.e. the thylakoids are present only as stroma lamellae). Mesophyll chloroplasts are small and with grana (Granal chloroplasts, i.e. both grana and stroma thylakoid are present).

• First CO₂ acceptor in C₄ plants is PEP (Phosphoenol Pyruvate) (3C–compound) in mesophyll cells, while the second CO₂ acceptor is RuBP (5C–compound) in bundle sheath cells.
• Released CO₂ in bundle sheath cells is accepted by RuBisCO, which catalyses its fixation. The C₃ cycle/Calvin cycle operates in bundle sheath cells, utilising assimilatory power (18 ATP & 12 NADPH2) to form glucose.
• Pyruvic acid produced in bundle sheath cells is returned to mesophyll cells. It regenerates the PEP (primary CO₂ acceptor) using 12 ATP, catalysed by the enzyme PPDK (Pyruvate phosphate dikinase). So, in C₄ plants, a total of 30 ATP and 12 NADPH₂ are utilised for the synthesis of one glucose.
• C₄ plants are special because:

(i) They have a special type of leaf anatomy (Kranz anatomy).

(ii) They tolerate higher temperatures.

(iii) They show a response to high light intensities.

(iv) They lack a process called photorespiration, so they have greater productivity of biomass.

• In C₃ plants, if O₂ bind to RuBisCO, then RuBP form one molecule of phosphoglycerate (3 carbon) and one molecule of phosphoglycolate (2 Carbon) in the Chloroplast.
• Photorespiration, or the C₂ cycle, occurs in chloroplasts, peroxisomes, and mitochondria.
• In photorespiration, neither the synthesis of sugar nor of ATP and NADPH occurs.
• In C₄ plants photorespiration does not occur. This is because they have a mechanism that increases CO₂ concentration at the RuBisCO enzyme site. This happens when the C4 acid (malic or aspartic acid) from the mesophyll cells is broken down in the bundle sheath cells, releasing CO₂ (CO₂ pumping) and thereby increasing the intracellular concentration of CO₂. In turn, this ensures that the RuBisCO functions as a carboxylase, minimising the oxygenase activity.
• In addition, in C₄ plants, the site of O₂ evolution (mesophyll cell) and the site of RuBisCO activity (Bundle sheath cell) are different.
• The evolution of the C₄ photosynthetic system is one of the strategies for maximising CO₂ availability while minimising water loss. C₄ plants are twice as efficient as C₃ plants in terms of fixing carbon dioxide (making sugar). However, a C₄ plant loses only half as much water as a C₃ plant for the same amount of CO₂ fixed.

Key Differences: C3 vs. C4 Plants