Various experiments and investigations regarding the fixation of CO₂ and the path of carbon during the dark reaction have indicated that there are a number of different pathways for CO₂ fixation in green plants. These are

1. C₃ Pathway (Calvin cycle)
2. C₄ Pathway (H-S pathway or Hatch-Slack cycle)
3. CAM (Crassulacean acid metabolism)

1. The C₃ pathway or Calvin cycle is the main pathway and is present in most green plants. Plants which follow the Calvin cycle are called C₃ plants.

2. C₄ Pathway (or Hatch-Slack pathway) : In some plants, the first stable product, after the fixation of CO₂, is a 4-C dicarboxylic acid called oxaloacetic acid (OAA). Such plants are called C₄ plants and the path of carbon (dark reaction) is the C₄ pathway. It was first noticed by Kortschak (1964) in the photosynthesis of sugarcane leaves. However, details of the C₄ pathway (i.e. the first CO₂ acceptor, the first stable product, the complete carbon pathway, etc.) were worked out by Hatch and Slack (1966). Therefore, it is known as Hatch-Slack Pathway. C₄ pathway is observed in many plants of family Gramineae (e.g. sugarcane, maize, some other monocot and some dicot plants).

Anatomical peculiarities of C₄ plants : Most of the C₄ plants have a characteristic leaf anatomy and dimorphic chloroplasts. For example,

(1) The leaf mesophyll consists of more or less compactly arranged cells.

(2) It is not differentiated into palisade and spongy mesophyll as it is in C3 plants.

(3) The vascular bundles (veins) in the leaf are surrounded by a distinct bundle sheath of radial enlarged parenchyma cells.

(4) The chloroplasts in leaf cells are dimorphic( i.e., of two types):
(a) Chloroplasts in the mesophyll cells are smaller and possess grana.
(b) Chloroplasts in the bundle sheath cells are larger and without grana.

This type of leaf anatomy in C₄ plants is described as Kranz anatomy.

The various steps in C₄ pathway are completed in two parts and in two different regions in the leaves. (a) First part reactions are completed in the stroma of the chloroplasts in mesophyll cells, and (b) Second part reactions are completed in the stroma of chloroplasts in bundle sheath cells.

(i) First CO₂ Fixation: In the pathway, the first CO₂ acceptor is the 3-C phosphoenol pyruvate acid (PEP). CO₂ first combines with 3-C PEP to form 4-C OAA (oxaloacetic acid). As OAA is a dicarboxylic acid, this is also known as the dicarboxylic acid pathway.

(ii) 4-C OAA may be converted into 4-C malic acid or 4-C aspartic acid and transported to bundle sheath cells.

(i) In the chloroplasts of the bundle sheath cells, 4-C malic acid undergoes decarboxylation to form CO₂ and 3-C pyruvic acid.

(ii) Second CO₂ fixation : The CO₂ released in decarboxylation of malic acid combines with 5-C RUDP (ribulose diphosphate) to form 2 molecules of 3-C PGA as in the Calvin cycle. Further conversion of PGA to sugars is the same as in the Calvin cycle.

(iii) The pyruvic acid produced in decarboxylation of malic acid is transported back to the mesophyll cells. Here it is converted into PEPC and again made available for the C₄ pathway.

These steps in the Hatch-Slack pathway in the mesophyll and the bundle sheath cells are schematically shown in the figure below.

Thus, in the C₄ plants, the initial few steps are different (typical of C₄ pathway). However, later on the reactions are similar to the Calvin cycle (i.e., C₃ pathway). Hence, C₄ plants have both C₄ and C₃ pathways.

Moreover, in C₄ plants, CO₂ fixation (i.e. carboxylation) occurs twice: first in the mesophyll cells (PEP + CO₂) and then again in the bundle sheath cells (RUDP + CO₂). For this reason, the C₄ pathway is also called a dicarboxylation pathway.

Chapter 5