C4 carbon fixation
C4 carbon fixation is one of three biochemical mechanisms, along with C3 and CAM photosynthesis, used in carbon fixation. It is named for the 4-carbon molecule present in the first product of carbon fixation in the small subset of plants known as C4 plants, in contrast to the 3-carbon molecule products in C3 plants.
C4 fixation is an elaboration of the more common C3 carbon fixation and is believed to have evolved more recently. C4 and CAM overcome the tendency of the enzyme RuBisCO to wastefully fix oxygen rather than carbon dioxide in what is called photorespiration. This is achieved by using a more efficient enzyme to fix CO2 in mesophyll cells and shuttling this fixed carbon via malate or aspartate to bundle-sheath cells. In these bundle-sheath cells, RuBisCO is isolated from atmospheric oxygen and saturated with the CO2 released by decarboxylation of the malate or oxaloacetate. These additional steps, however, require more energy in the form of ATP. Because of this extra energy requirement, C4 plants are able to more efficiently fix carbon in only certain conditions, with the more common C3 pathway being more efficient in other conditions.
The first experiments indicating that some plants do not use the established C3 carbon fixation but produce malate and aspartate in the first step were done in the 1950s and early 1960s by Hugo P. Kortschak and Yuri Karpilov. The C4 pathway was finally discovered by Marshall Davidson 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 and 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 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:
- pyruvate + Pi + ATP → PEP + AMP + PPi
- 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, 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:
- The 4-carbon acid transported from mesophyll cells may be malate, as above, or aspartate
- The 3-carbon acid transported back from bundle-sheath cells may be pyruvate, as above, or alanine
- 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 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 40 independent occasions in 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  during the Oligocene (precisely when is difficult to determine) and did not become ecologically significant until around , 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 30% 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 7,600 plant species use C4 carbon fixation, which represents about 3% of all terrestrial species of plants. All these 7,600 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.
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. 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.
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