C4 carbon fixation



C4 carbon fixation is one of three biochemical mechanisms, along with C3 and CAM photosynthesis, functioning in land plants to "fix" carbon dioxide (binding the gaseous molecules to dissolved compounds inside the plant) for sugar production through photosynthesis. Along with CAM photosynthesis, C4 fixation is considered an advancement over the simpler and more ancient C3 carbon fixation mechanism operating in most plants. Both mechanisms overcome the tendency of RuBisCO (the first enzyme in the Calvin cycle) to photorespire, or waste energy by using oxygen to break down carbon compounds to CO2. However C4 fixation requires more energy input than C3 in the form of ATP. C4 plants separate rubisco from atmospheric oxygen, fixing carbon in the mesophyll cells and using oxaloacetate and malate to ferry the fixed carbon to rubisco and the rest of the Calvin cycle enzymes isolated in the bundle-sheath cells. The intermediate compounds both contain four carbon atoms, hence the name C4.

The pathway
The C4 pathway was discovered by M. D. Hatch and C. R. Slack, in Australia, in 1966, so it is sometimes called the Hatch-Slack pathway.

In C3 plants, the first step in the light-independent reactions of photosynthesis involves the fixation of CO2 by the enzyme RuBisCo into 3-phosphoglycerate. However, due to the dual carboxylase / oxygenase activity of RuBisCo, an amount of the substrate is oxidized rather than carboxylated resulting in loss of substrate and consumption of energy, in what is known as photorespiration. In order to bypass the photorespiration pathway, C4 plants have developed a mechanism to efficiently deliver CO2 to the RuBisCO enzyme. They utilize their specific leaf anatomy where chloroplasts exist not only in the mesophyll cells in the outer part of their leaves but in the bundle sheath cells as well. Instead of direct fixation in the Calvin cycle, CO2 is converted to a 4-carbon organic acid which has the ability to regenerate CO2 in the chloroplasts of the bundle sheath cells. Bundle sheath cells can then utilize this CO2 to generate carbohydrates by the conventional C3 pathway.

The first step in the pathway is the conversion of pyruvate to PEP by the enzyme pyruvate-phosphate dikinase (pyruvate, orthophosphate dikinase); this reaction requires inorganic phosphate and ATP plus pyruvate, giving phosphoenolpyruvate, AMP, and PPi (inorganic pyrophosphate) as products. The next step is the fixation of CO2 by the enzyme phosphoenolpyruvate carboxylase. Both of these steps occur in the mesophyll cells:
 * pyruvate + Pi + ATP → PEP + AMP + PPi
 * PEP carboxylase + PEP + CO2 → oxaloacetate

PEP carboxylase has a lower Km for CO2—and hence higher affinity—than Rubisco. Furthermore, O2 is a very poor substrate for this enzyme. Thus, at relatively low concentrations of CO2, most CO2 will be fixed by this pathway.

The product is usually converted to malate, a simple organic compound that is transported to the bundle-sheath cells surrounding a nearby vein, where it is decarboxylated to release CO2, which enters Calvin cycle. The decarboxylation leaves pyruvate, which is transported back to the mesophyll cell.

Since every CO2 molecule has to be fixed twice, the C4 pathway is more energy-consuming than the C3 pathway. The C3 pathway requires 18 ATP for the synthesis of one molecule of glucose while the C4 pathway requires 30 ATP. But since otherwise tropical plants lose more than half of photosynthetic carbon in photorespiration, the C4 pathway is an adaptive mechanism for minimizing the loss.

There are several variants of this pathway:
 * 1) The 4-carbon acid transported from mesophyll cells may be malate as above, or may be aspartate.
 * 2) The 3-carbon acid transported back from bundle-sheath cells may be pyruvate as above, or alanine.
 * 3) The enzyme which catalyses decarboxylation in bundle-sheath cells differs. In maize and sugarcane, the enzyme is NADP-malic enzyme, in millet, it is NAD-malic enzyme, and in Panicum maximum it is PEP carboxykinase.

C4 Leaf Anatomy
The C4 plants possess a characteristic leaf anatomy. Their vascular bundles are surrounded by two rings of cells. The inner ring, called Bundle Sheath Cells, contain starch-rich chloroplasts lacking grana which differ from those in mesophyll cells present as the outer ring. Hence, the chloroplasts are called dimorphic. This peculiar anatomy is called Kranz Anatomy (Kranz-Crown/Halo). The primary function of the Kranz is to provide a site in which carbon dioxide can be concentrated around RuBisCO, thus reducing photorespiration. In order to facilitate the maintenance of a significantly higher carbon dioxide concentration in the bundle sheath compared to the mesophyll, the boundary layer of the Kranz has a low conductance to carbon dioxide, a property which may be enhanced by the presence of suberin.

Although most C4 plants exhibit Kranz anatomy, there are a number of species which operate a limited C4 cycle without any distinct bundle sheath tissue. Suaeda aralocaspica (formerly known as Borszczowia aralocaspica), Bienertia cycloptera and Bienertia sinuspersici (all chenopods) are terrestrial plants which inhabit dry, salty depressions in the deserts of south-east Asia. These plants have been shown to operate single-cell C4 carbon dioxide concentrating mechanisms which are unique amongst the known C4 mechanisms. Although the cytology of both species differ slightly, the basic principle is that fluid filled vacuoles are employed to divide the cell into to separate areas. Carboxylation enzymes in the cytosol can therefore be kept separate from decarboxylase enzymes and RuBisCo in the chloroplasts, and a diffusive barrier can be established between the chloroplasts (which contain RuBisCO) and the cytosol. This enables a bundle-sheath type area and a mesophyll type area to be established within a single cell. Although this does allow a limited C3 cycle to operate, it is relatively inefficient, with much leakage of CO2 from around RuBisCO occurring. There is also evidence for the non-Kranz aquatic macrophyte Hydrilla verticillata exhibiting inducible C4 photosynthesis under warm conditions, although the mechanism by which CO2 leakage from around RuBisCO is minimised is currently uncertain.

The Evolution and Advantages of the C4 Pathway
C4 plants have a competitive advantage over plants possessing the more common C3 carbon fixation pathway under conditions of drought, high temperatures and nitrogen or carbon dioxide limitation. 97% of the water taken up by plants is lost through transpiration, Today they represent about 5% of Earth's plant biomass and 1% of its known plant species. However, they account for around 30% of terrestrial carbon fixation. These species are concentrated in the tropics (below latitudes of 45°) where the high air temperature contributes to higher possible levels of oxygenase activity by RuBisCO, which increases rates of photorespiration in C3 plants.