C4 carbon fixation

C4 pathway

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 P. Kortschak and Yuri Karpilov. The C₄ pathway was elucidated by Marshall Davidson Hatch and C. R. Slack, in Australia, in 1966; 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 and oxygenase activity of RuBisCo, some part 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 to RuBisCO in the Calvin cycle, CO₂ is incorporated into 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 phosphoenolpyruvate (PEP), by the enzyme pyruvate orthophosphate dikinase. This reaction requires inorganic phosphate and ATP plus pyruvate, producing phosphoenolpyruvate, AMP, and inorganic pyrophosphate (PPi). The next step is the fixation of CO₂ into oxaloacetate by the enzyme PEP carboxylase. Both of these steps occur in the mesophyll cells:

PEP carboxylase has a lower Km for HCO−
3 — 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, which is transported to the bundle-sheath cells surrounding a nearby vein. Here, it is decarboxylated to produce CO₂ and pyruvate. The CO₂ now enters the Calvin cycle and the pyruvate is transported back to the mesophyll cell.

Since every CO₂ molecule has to be fixed twice, first by 4-carbon organic acid and second by RuBisCO, the C₄ pathway uses more energy than the C₃ pathway. The C₃ pathway requires 18 molecules of ATP for the synthesis of one molecule of glucose, whereas the C₄ pathway requires 30 molecules of ATP. This energy debt is more than paid for by avoiding losing more than half of photosynthetic carbon in photorespiration as occurs in some tropical plants, making it 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 aspartate.
2. The 3-carbon acid transported back from bundle-sheath cells may be pyruvate, as above, or alanine.
3. The enzyme that 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 Kranz leaf anatomy

The 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. In order to maintain a significantly higher CO₂ concentration in the bundle sheath compared to the mesophyll, the boundary layer of the kranz has a low conductance to CO₂, a property that may be enhanced by the presence of suberin.

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 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 the occurrence of much leakage of CO₂ from around RuBisCO. There is also evidence for the exhibiting 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.

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 CO₂ limitation. When grown in the same environment, at 30 °C, C₃ grasses lose approximately 833 molecules of water per CO₂ molecule that is fixed, whereas C₄ grasses lose only 277. This increased water use efficiency of C₄ grasses means that soil moisture is conserved, allowing them to grow for longer in arid environments.

C₄ carbon fixation has evolved on up to 61 independent occasions in 19 different families of plants, making it a prime example of convergent evolution. This convergence may have been facilitated by the fact that many potential evolutionary pathways to a C₄ phenotype exist, many of which involve initial evolutionary steps not directly related to photosynthesis. C₄ plants arose around 35 million years ago during the Oligocene (precisely when is difficult to determine) and did not become ecologically significant until around 6 to 7 million years ago, in the Miocene Period. C₄ metabolism originated when grasses migrated from the shady forest undercanopy to more open environments, where the high sunlight gave it an advantage over the C₃ pathway. Drought was not necessary for its innovation; rather, the increased resistance to water stress was a by-product of the pathway and allowed C₄ plants to more readily colonise arid environments.

Today, C₄ plants represent about 5% of Earth's plant biomass and 3% of its known plant species. Despite this scarcity, they account for about 23% of terrestrial carbon fixation. Increasing the proportion of C₄ plants on earth could assist biosequestration of CO₂ and represent an important climate change avoidance strategy. Present-day C₄ plants are concentrated in the tropics and subtropics (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.

Plants that use C4 carbon fixation

About 8,100 plant species use C₄ carbon fixation, which represents about 3% of all terrestrial species of plants. All these 8,100 species are angiosperms. C₄ carbon fixation is less common in dicots than in monocots, with only 4.5% of dicots using the C₄ pathway, compared to 40% of monocots. Despite this, only three families of monocots utilise C₄ carbon fixation compared to 15 dicot families. Of the monocot clades containing C₄ plants, the grass (Poaceae) species use the C₄ photosynthetic pathway most. Forty-six percent of grasses are C₄ and together account for 61% of C₄ species. These include the food crops maize, sugar cane, millet, and sorghum. Of the dicot clades containing C₄ species, the order Caryophyllales contains the most species. Of the families in the Caryophyllales, the Chenopodiaceae use C₄ carbon fixation the most, with 550 out of 1,400 species using it. About 250 of the 1000 species of the related Amaranthaceae also use C₄.

Members of the sedge family Cyperaceae, and numerous families of Eudicots, including the daisies Asteraceae, cabbages Brassicaceae, and spurges Euphorbiaceae also use C₄.

Trees which use C₄ include Paulownia.

Converting C3 plants to C4

Given the advantages of C₄, a group of scientists from institutions around the world are working on the C₄ Rice Project to turn rice, a C₃ plant, into a C₄ plant by studying the C4 plants maize and Brachypodium. As rice is the world's most important human food—it is the staple food for more than half the planet—having rice that is more efficient at converting sunlight into grain could have significant global benefits towards improving food security. The team claim C₄ rice could produce up to 50% more grain—and be able to do it with less water and nutrients.

The researchers have already identified genes needed for C₄ photosynthesis in rice and are now looking towards developing a prototype C₄ rice plant. In 2012, the Government of the United Kingdom along with the Bill & Melinda Gates Foundation provided $14 million over 3 years towards the C₄ Rice Project at the International Rice Research Institute.