C4 carbon fixation

C₄ carbon fixation or the Hatch–Slack pathway is one of three known photosynthetic processes of carbon fixation in plants. It owes the names to the 1960s discovery by Marshall Davidson Hatch and Charles Roger Slack.

C₄ fixation is an addition to the ancestral and more common C₃ carbon fixation. The main carboxylating enzyme in C₃ photosynthesis is called RuBisCO, which catalyses two distinct reactions using either CO₂ (carboxylation) or oxygen (oxygenation) as a substrate. RuBisCO oxygenation gives rise to phosphoglycolate, which is toxic and requires the expenditure of energy to recycle through photorespiration. C₄ photosynthesis reduces photorespiration by concentrating CO₂ around RuBisCO.

To enable RuBisCO to work in a cellular environment where there is a lot of carbon dioxide and very little oxygen, C₄ leaves generally contain two partially isolated compartments called mesophyll cells and bundle-sheath cells. CO₂ is initially fixed in the mesophyll cells in a reaction catalysed by the enzyme PEP carboxylase in which the three-carbon phosphoenolpyruvate (PEP) reacts with CO₂ to form the four-carbon oxaloacetic acid (OAA). OAA can then be reduced to malate or transaminated to aspartate. These intermediates diffuse to the bundle sheath cells, where they are decarboxylated, creating a CO₂-rich environment around RuBisCO and thereby suppressing photorespiration. The resulting pyruvate (PYR), together with about half of the phosphoglycerate (PGA) produced by RuBisCO, diffuses back to the mesophyll. PGA is then chemically reduced and diffuses back to the bundle sheath to complete the reductive pentose phosphate cycle (RPP). This exchange of metabolites is essential for C₄ photosynthesis to work.

Additional biochemical steps require more energy in the form of ATP to regenerate PEP, but concentrating CO₂ allows high rates of photosynthesis at higher temperatures. Higher CO₂ concentration overcomes the reduction of gas solubility with temperature (Henry's law). The CO₂ concentrating mechanism also maintains high gradients of CO₂ concentration across the stomatal pores. This means that C₄ plants have generally lower stomatal conductance, reduced water losses and have generally higher water-use efficiency. C₄ plants are also more efficient in using nitrogen, since PEP carboxylase is cheaper to make than RuBisCO. However, since the C₃ pathway does not require extra energy for the regeneration of PEP, it is more efficient in conditions where photorespiration is limited, typically at low temperatures and in the shade.

The first experiments indicating that some plants do not use C₃ carbon fixation but instead produce malate and aspartate in the first step of carbon fixation were done in the 1950s and early 1960s by Hugo Peter Kortschak and Yuri Karpilov. The C₄ pathway was elucidated by Marshall Davidson Hatch and Charles Roger Slack, in Australia, in 1966. While Hatch and Slack originally referred to the pathway as the "C₄ dicarboxylic acid pathway", it is sometimes called the Hatch–Slack pathway.

C₄ plants often possess a characteristic leaf anatomy called kranz anatomy, from the German word for wreath. Their vascular bundles are surrounded by two rings of cells; the inner ring, called bundle sheath cells, contains starch-rich chloroplasts lacking grana, which differ from those in mesophyll cells present as the outer ring. Hence, the chloroplasts are called dimorphic. The primary function of kranz anatomy is to provide a site in which CO₂ can be concentrated around RuBisCO, thereby avoiding photorespiration. Mesophyll and bundle sheath cells are connected through numerous cytoplasmic sleeves called plasmodesmata whose permeability at leaf level is called bundle sheath conductance. A layer of suberin is often deposed at the level of the middle lamella (tangential interface between mesophyll and bundle sheath) in order to reduce the apoplastic diffusion of CO₂ (called leakage). The carbon concentration mechanism in C₄ plants distinguishes their isotopic signature from other photosynthetic organisms.

Although most C₄ plants exhibit kranz anatomy, there are, however, a few species that operate a limited C₄ cycle without any distinct bundle sheath tissue. Suaeda aralocaspica, Bienertia cycloptera, Bienertia sinuspersici and Bienertia kavirense (all chenopods) are terrestrial plants that inhabit dry, salty depressions in the deserts of the Middle East. These plants have been shown to operate single-cell C₄ CO₂-concentrating mechanisms, which are unique among the known C₄ mechanisms. Although the cytology of both genera differs slightly, the basic principle is that fluid-filled vacuoles are employed to divide the cell into two separate areas. Carboxylation enzymes in the cytosol are separated from decarboxylase enzymes and RuBisCO in the chloroplasts. A diffusive barrier is 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 C₄ cycle to operate, it is relatively inefficient. Much leakage of CO₂ from around RuBisCO occurs.

There is also evidence of inducible C₄ photosynthesis by non-kranz aquatic macrophyte Hydrilla verticillata under warm conditions, although the mechanism by which CO₂ leakage from around RuBisCO is minimised is currently uncertain.