C4 – Dicarboxylic Acid Pathway (Hatch–Slack Pathway) | Photosynthesis

In this article, we will discuss about the C₄ – Dicarboxylic Acid Pathway (Hatch–Slack Pathway).

For considerable period of time the Calvin cycle as described earlier was thought to be the only photosynthetic reaction sequence operating in higher plants and algae. But in 1965 Kortschak, Hartt and Burr reported that 4-C containing dicarboxylic acids, malate and aspartate were the major labelled products when sugarcane leaves were allowed to photo­synthesize for short periods in ¹⁴CO₂.

This finding was confirmed and greatly elaborated by Hatch and Slack (1966, 70) who observed heavy labeling of oxaloacetate, malate, and asparate only when sugarcane leaves were exposed to ¹⁴CO₂ for 1 sec. Longer exposures resulted in decrease of radio-activity in these acids with simultaneous increase in 3-phosphoglyceric acid, hexose monophosphates and sucrose. This and further studies by these workers led to the establishment of yet another CO₂ reduction pathway which is called as Hatch-Slack pathway and because C₄-dicarboxylic acids are the earliest products it is also called as C₄-dicarboxylic acid pathway.

Besides sugarcane leaves this pathway has been found to operate in many plant spe­cies of the family Gramineae e.g., maize, sorghum etc. which are grouped together as ‘tropical grasses’ and other plants e.g., Atriplex, Amaranthus etc.

These are all known as C₄ Plants and are distinguished by:

(i) Absence of photo-respiration and,

(ii) Anatomical similarities of leaf (‘cane type’).

In the leaves of these plants the vascular bundles are surrounded by bundle sheath of larger parenchymatous cells which in turn are surrounded by smaller mesophyll cells. Moreover, the chloroplasts in cells of bundle sheath are larger and usu­ally lack grana; the chloroplasts in mesophyll cells are smaller and always contain grana. These two types of chloroplasts may even be separated from one another by density gradi­ent centrifugation. In maize and sugarcane plasmodesmata have been observed which con­nect cells of bundle sheath with adjacent mesophyll cells.

(PEP = Phosphoenol pyruvic acid; OAA = Oxaloacetate;

RuBP = Ribulose bis-phosphate; PGA = Phosphoglyceric acid).

(The anatomy of the leaf of C₄ plants is also called as ‘Krantz’ anatomy because of the presence of radially arranged cells of bundle sheath around their vascular bundles which look like a ring or wreath. ‘Krantz’ in German means wreath. The cells of bundle sheath in such plants, are therefore, also called as Krantz cells).

Various steps of Hatch-Slack pathway (Fig. 11.22) which involves two carboxylation reactions, one taking place in chloroplasts of mesophyll cells and another in chloroplasts of cells of bundle sheath are as follows:

(i) The first step involves the carboxylation of phosphoenol pyruvic acid in chloroplasts of mesophyll cells to form C-4 dicarboxylic acid, oxaloacetic acid. This reaction is catalysed by phosphoenol pyruvate carboxylase.

(The CO₂ is first dissolved in water in cytoplasm and ionised into HCO₃^– probably under the influence of carbonic anhydrase. The resulting HCO^–₃ (bicarbonate ion) is then used in carboxylating phosphoenol pyruvic acid).

(ii) Oxaloacetic acid readily equilibrates with other C₄-dicarboxylic acids, aspartic acid and malic acid in the presence of enzymes transaminase and NADP⁺ specific malate dehydrogenase respectively.

(iii) From chloroplasts of mesophyll cells the malic acid is transferred to the chloro­plasts of bundle sheath cells where it is decarboxylated to form CO₂ and pyruvic acid in the presence of a NADP⁺ specific malic enzyme.

(iv) Now, second carboxylation occurs in chloroplast of bundle sheath cells. Ribulose- bisphosphate accepts CO₂ produced in step (iii) in the presence of Rubisco (i.e., RuBP-car- boxylase) and ultimately yields 3-phosphoglyceric acid as in case of Calvin cycle. Some of the 3-phosphoglyceric acid is utilised in the formation of hexose monophosphates, sucrose and starch, while rest regenerates ribulose-bisphosphate in the system (see Calvin cycle).

(v) The pyruvic acid produced in step (iii) is transferred to chloroplasts of mesophyll cells where it is phosphorylated to regenerate phosphoenol pyruvic acid. This reaction is catalysed by pyruvate Pi kinase.

The AMP is phosphorylated by ATP under the catalytic influence of the enzyme ade­nylate kinase to form 2 ADP molecules.

(These ADPs may be converted back into ATPs in the light reaction of photosynthesis).

i. The enzymes catalysing reactions in chloroplasts of mesophyll cells are not found in chloroplasts of bundle sheath cells and vice versa.

ii. Hatch-Slack pathway begins with the carboxylation of phosphoenol pyruvate and not of ribulose-bisphosphate. It is because the former has great affinities with CO₂ than the latter.

iii. In contrast to the C₄ plants, the other higher plants lack Hatch-Slack pathway and have only Calvin cycle (C₃-pathway) for the fixation of CO₂ in photosynthesis. These are called as C₃ plants and have only one type of chloroplasts.

The C₄ mode of photosynthesis (Hatch-Slack pathway) is less efficient in itself in com­parison to the C₃ mode (Calvin cycle in C₃ plants). It is because the fixation of 1 CO₂ mol. in C₃ mode of photosynthesis requires 2NADPH + 3ATP molecules while in C₄ mode of photosynthesis 2NADPH + 5ATP molecules are required (additional 2 ATPs are needed in reaction no. v) for the fixation of 1CO₂ mol.

However, C₄ plants are more efficient photosynthetically than C₃ plants because of the absence (or negligible presence) of photorespiration in C₄ plants. Thus, net requirement of ATP + NADPH per CO₂ mol. fixed (i.e. resultant of photosynthesis minus photorespiration) is considerably lower in C₄ plants than in C₃ plants.

Variations in the Mechanism of C₄– Pathway:

In recent years minor variations in the mechanism of C₄ mode of photosynthesis (Hatch- Slack pathway) have been observed in different C₄ plants. The main difference is in the way the 4C-dicarboxylic acid is decarboxylated in the bundle sheath cells. On the basis of this, 3 categories of C₄ plants and also of C₄ -pathway are recognised:

(i) Some C₄-plants such as Zea mays, Saccharum officinarum, Sorghum sudanense utilise NADP⁺ specific malic enzyme for this decarboxylation. The mechanism of C₄ path­way in such plants is said to be of ‘NADP⁺ – Me-Type’.

(ii) Secondz category of plants such as Atriplex spongiosa, Portulaca oleracea, Amaranthus edulis etc., utilise NAD⁺ specific malic enzyme for this purpose. The mecha­nism of C₄-pathway in these plants is said to be of ‘NAD⁺ -Me-Type’.

(iii) Some C₄-plants such as Panicum maximum Chloris gayana etc., utilise PEP- carboxykinase enzyme. The mechanism of C₄-pathway in such plants is known as ‘PCK- Me-Type’.

i. The mechanism of C₄-pathway described earlier is of ‘NADP⁺-Me-Type’ but it shows generalised picture of the C₄-pathway too. (See Fig. 11.22 also)

ii. In ‘NAD⁺-Me-Type’ pathway (Fig. 11.23A) the oxaloacetic acid in cytoplasm of mesophyll cells is transaminated by the enzyme aspartate aminotransferase which is present in the cytoplasm and requires glutamic acid as the donor of amino group.

The aspartic acid thus produced is transferred to mitochondria of bundle sheath cells (i.e., Krantz cells).

In the mitochondria of Krantz cells the aspartic acid is converted back into oxaloacetic acid by reversal of above transmination reaction. Oxaloacetic acid is now reduced by NAD⁺ specific malate dehydrogenase into malic acid.

The malic acid is now decarboxylated by mitochondrial NAD⁺ specific malic enzyme to form pyruvic acid and CO₂.

The CO₂ now diffuses from mitochondria to chloroplasts where it is reduced to carbo­hydrate through Calvin cycle.

The pyruvic acid diffuses from mitochondria to cytoplasm where it is transaminated to yield alanine. The enzyme involved is alanine amino-transferase. Glutamic acid acts as amino donor.

The alanine now diffuses from cytoplasm of bundle sheath cells (Krantz cells) to the cytoplasm of mesophyll cells where it is converted back into pyruvic acid by the reverse of above reaction. Pyruvic acid now passes from cytoplasm of mesophyll cells to chloroplasts of meso­phyll cells where it is converted into phosphoenol pyruvic acid (PEP) utilizing 2ATP mol­ecules (as in NADP⁺-Me-Type).

The PEP diffuses into cytoplasm of the mesophyll cells, thus completing the cycle NAD⁺-Me-Type. The main difference is that oxaloacetic acid in Krantz cells is di­rectly decarboxylated (without being converted into malic acid) in PCK-Type. Secondly, mi­tochondria are not involved in this PCK-Type pathway.

(In the C₄ species of the plants of the family gramineae there appears to be correlation between anatomy, ultrastructure of Krantz cell chloroplasts and the decarboxylating mecha­nism. When viewed in cross section NADP⁺-Me-Type species have agranal and centrifugal chloroplasts (situated towards the outer part of the Krantz cell). In PCK-type species, the chlo­roplasts have grana and are centrifugal in position. In NAD⁺-Me-type species, the chloroplast have grana but the chloroplasts are centripetal in position being situated towards the inner side of the Krantz cell).

Significance of Hatch Slack Pathway:

The significance of this pathway is not clearly understood.

But it has been suggested that:

(i) This pathway is a modification of Calvin cycle and is advantageous to plants growing in dense stands of tropical vegetation where the CO₂ concentration may be very much reduced.

(ii) There has been a reduction of atm. CO₂ concentration since the evolution of photosynthe­sis and this might have prompted C₄ plants to select this pathway.

(iii) The discovery of this pathway has indicated the existence of yet undiscovered photosynthetic reactions other than the conventional Calvin cycle.