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C4 carbon fixation
C4 carbon fixation
C4 carbon fixation
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Leaf anatomy in most C4 plants.
A: Mesophyll cell
B: Chloroplast
C: Vascular tissue
D: Bundle sheath cell
E: Stoma
F: Vascular tissue
1. CO2 is fixed to produce a four-carbon molecule (malate or aspartate).
2. The molecule exits the cell and enters the bundle sheath cells.
3. It is then broken down into CO2 and pyruvate. CO2 enters the Calvin cycle to produce carbohydrates.
4. Pyruvate reenters the mesophyll cell, where it is reused to produce malate or aspartate.
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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.[1]

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.[2] C4 plants are also more efficient in using nitrogen, since PEP carboxylase is cheaper to make than RuBisCO.[3] 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.[4]

Discovery

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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.[5][6] The C4 pathway was elucidated by Marshall Davidson Hatch and Charles Roger Slack, in Australia, in 1966.[1] While Hatch and Slack originally referred to the pathway as the "C4 dicarboxylic acid pathway", it is sometimes called the Hatch–Slack pathway.[6]

Anatomy

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Cross section of a maize leaf, a C4 plant. Kranz anatomy (rings of cells) shown

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[7] 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.[8][9][10][11] 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.[12]

Biochemistry

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In C3 plants, the first step in the light-independent reactions of photosynthesis is the fixation of CO2 by the enzyme RuBisCO to form 3-phosphoglycerate. However, RuBisCo has a dual carboxylase and oxygenase activity. Oxygenation results in part of the substrate being oxidized rather than carboxylated, resulting in loss of substrate and consumption of energy, in what is known as photorespiration. Oxygenation and carboxylation are competitive, meaning that the rate of the reactions depends on the relative concentration of oxygen and CO2.

In order to reduce the rate of photorespiration, C4 plants increase the concentration of CO2 around RuBisCO. Often, to facilitate this, two partially isolated compartments differentiate within leaves: the mesophyll and the bundle sheath. Instead of direct fixation by RuBisCO, CO2 is initially incorporated into a four-carbon organic acid (either malate or aspartate) in the mesophyll. The organic acids then diffuse through plasmodesmata into the bundle sheath cells. There, they are decarboxylated creating a CO2-rich environment. The chloroplasts of the bundle sheath cells convert this CO2 into carbohydrates by the conventional C3 pathway.

There is large variability in the biochemical features of C4 assimilation, and it is generally grouped in three subtypes, differentiated by the main enzyme used for decarboxylation ( NADP-malic enzyme, NADP-ME; NAD-malic enzyme, NAD-ME; and PEP carboxykinase, PEPCK). Since PEPCK is often recruited atop NADP-ME or NAD-ME it was proposed to classify the biochemical variability in two subtypes. For instance, maize and sugarcane use a combination of NADP-ME and PEPCK, millet uses preferentially NAD-ME and Megathyrsus maximus, uses preferentially PEPCK.

NADP-ME

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NADP-ME subtype

The first step in the NADP-ME type C4 pathway is the conversion of pyruvate (Pyr) to phosphoenolpyruvate (PEP), by the enzyme Pyruvate phosphate dikinase (PPDK). This reaction requires inorganic phosphate and ATP plus pyruvate, producing PEP, AMP, and inorganic pyrophosphate (PPi). The next step is the carboxylation of PEP by the PEP carboxylase enzyme (PEPC) producing oxaloacetate. Both of these steps occur in the mesophyll cells:

pyruvate + Pi + ATP → PEP + AMP + PPi
PEP + CO2 → oxaloacetate

PEPC has a low KM for HCO−
3 — and, hence, high affinity, and is not confounded by O2 thus it will work even at low concentrations of CO2.

The product is usually converted to malate (M), which diffuses to the bundle-sheath cells surrounding a nearby vein. Here, it is decarboxylated by the NADP-malic enzyme (NADP-ME) to produce CO2 and pyruvate. The CO2 is fixed by RuBisCo to produce phosphoglycerate (PGA) while the pyruvate is transported back to the mesophyll cell, together with about half of the phosphoglycerate (PGA). This PGA is chemically reduced in the mesophyll and diffuses back to the bundle sheath where it enters the conversion phase of the Calvin cycle. For each CO2 molecule exported to the bundle sheath the malate shuttle transfers two electrons, and therefore reduces the demand of reducing power in the bundle sheath.

NAD-ME

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NAD-ME subtype

Here, the OAA produced by PEPC is transaminated by aspartate aminotransferase to aspartate (ASP) which is the metabolite diffusing to the bundle sheath. In the bundle sheath ASP is transaminated again to OAA and then undergoes a futile reduction and oxidative decarboxylation to release CO2. The resulting Pyruvate is transaminated to alanine, diffusing to the mesophyll. Alanine is finally transaminated to pyruvate (PYR) which can be regenerated to PEP by PPDK in the mesophyll chloroplasts. This cycle bypasses the reaction of malate dehydrogenase in the mesophyll and therefore does not transfer reducing equivalents to the bundle sheath.

PEPCK

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PEPCK subtype

In this variant the OAA produced by aspartate aminotransferase in the bundle sheath is decarboxylated to PEP by PEPCK. The fate of PEP is still debated. The simplest explanation is that PEP would diffuse back to the mesophyll to serve as a substrate for PEPC. Because PEPCK uses only one ATP molecule, the regeneration of PEP through PEPCK would theoretically increase photosynthetic efficiency of this subtype, however this has never been measured. An increase in relative expression of PEPCK has been observed under low light, and it has been proposed to play a role in facilitating balancing energy requirements between mesophyll and bundle sheath.

Metabolite exchange

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While in C3 photosynthesis each chloroplast is capable of completing light reactions and dark reactions, C4 chloroplasts differentiate in two populations, contained in the mesophyll and bundle sheath cells. The division of the photosynthetic work between two types of chloroplasts results inevitably in a prolific exchange of intermediates between them. The fluxes are large and can be up to ten times the rate of gross assimilation.[13] The type of metabolite exchanged and the overall rate will depend on the subtype. To reduce product inhibition of photosynthetic enzymes (for instance PECP) concentration gradients need to be as low as possible. This requires increasing the conductance of metabolites between mesophyll and bundle sheath, but this would also increase the retro-diffusion of CO2 out of the bundle sheath, resulting in an inherent and inevitable trade off in the optimisation of the CO2 concentrating mechanism.

Light harvesting and light reactions

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To meet the NADPH and ATP demands in the mesophyll and bundle sheath, light needs to be harvested and shared between two distinct electron transfer chains. ATP may be produced in the bundle sheath mainly through cyclic electron flow around Photosystem I, or in the mesophyll mainly through linear electron flow, depending on the light available in the bundle sheath or in the mesophyll. The relative requirement of ATP and NADPH in each type of cell will depend on the photosynthetic subtype.[13] The apportioning of excitation energy between the two cell types will influence the availability of ATP and NADPH in the mesophyll and bundle sheath. For instance, green light is not strongly adsorbed by mesophyll cells and can preferentially excite bundle sheath cells, or vice versa for blue light.[14] Because bundle sheaths are surrounded by mesophyll, light harvesting in the mesophyll will reduce the light available to reach bundle sheath cells. Also, the bundle sheath size limits the amount of light that can be harvested.[15]

Efficiency

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Different formulations of efficiency are possible depending on which outputs and inputs are considered. For instance, average quantum efficiency is the ratio between gross assimilation and either absorbed or incident light intensity. Large variability of measured quantum efficiency is reported in the literature between plants grown in different conditions and classified in different subtypes but the underpinnings are still unclear. One of the components of quantum efficiency is the efficiency of dark reactions, biochemical efficiency, which is generally expressed in reciprocal terms as ATP cost of gross assimilation (ATP/GA).

In C3 photosynthesis ATP/GA depends mainly on CO2 and O2 concentration at the carboxylating sites of RuBisCO. When CO2 concentration is high and O2 concentration is low photorespiration is suppressed and C3 assimilation is fast and efficient, with ATP/GA approaching the theoretical minimum of 3.

In C4 photosynthesis CO2 concentration at the RuBisCO carboxylating sites is mainly the result of the operation of the CO2 concentrating mechanisms, which cost circa an additional 2 ATP/GA but makes efficiency relatively insensitive of external CO2 concentration in a broad range of conditions.

Biochemical efficiency depends mainly on the speed of CO2 delivery to the bundle sheath, and will generally decrease under low light when PEP carboxylation rate decreases, lowering the ratio of CO2/O2 concentration at the carboxylating sites of RuBisCO. The key parameter defining how much efficiency will decrease under low light is bundle sheath conductance. Plants with higher bundle sheath conductance will be facilitated in the exchange of metabolites between the mesophyll and bundle sheath and will be capable of high rates of assimilation under high light. However, they will also have high rates of CO2 retro-diffusion from the bundle sheath (called leakage) which will increase photorespiration and decrease biochemical efficiency under dim light. This represents an inherent and inevitable trade off in the operation of C4 photosynthesis. C4 plants have an outstanding capacity to attune bundle sheath conductance. Interestingly, bundle sheath conductance is downregulated in plants grown under low light[16] and in plants grown under high light subsequently transferred to low light as it occurs in crop canopies where older leaves are shaded by new growth.[17]

Evolution and advantages

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Further information: Evolutionary history of plants § Evolution of photosynthetic pathways

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.[18]

C4 carbon fixation has evolved in at least 62 independent occasions in 19 different families of plants, making it a prime example of convergent evolution.[19][20] 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.[21] C4 plants arose around 35 million years ago[20] during the Oligocene (precisely when is difficult to determine) and were becoming ecologically significant in the early Miocene around 21 million years ago.[22] C4 metabolism in grasses originated when their habitat migrated from the shady forest undercanopy to more open environments,[23] where the high sunlight gave it an advantage over the C3 pathway.[24] Drought was not necessary for its innovation; rather, the increased parsimony in water use was a byproduct of the pathway and allowed C4 plants to more readily colonize arid environments.[24]

Today, C4 plants represent about 5% of Earth's plant biomass and 3% of its known plant species.[18][25] Despite this scarcity, they account for about 23% of terrestrial carbon fixation.[26][27] 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 degrees) where the high air temperature increases rates of photorespiration in C3 plants.

Plants that use C4 carbon fixation

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See also: List of C4 plants

About 8,100 plant species use C4 carbon fixation, which represents about 3% of all terrestrial species of plants.[27][28] All these 8,100 species are angiosperms. C4 carbon fixation is more common in monocots compared with dicots, with 40% of monocots using the C4 pathway[clarification needed], compared with only 4.5% of dicots. Despite this, only three families of monocots use 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. 46% of grasses are C4 and together account for 61% of C4 species. C4 has arisen independently in the grass family some twenty or more times, in various subfamilies, tribes, and genera,[29] including the Andropogoneae tribe which contains the food crops maize, sugar cane, and sorghum. Teff is also C4, as are various kinds of millet.[30][31][32] 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 1,000 species of the related Amaranthaceae also use C4.[18][33]

Members of the sedge family Cyperaceae, and members of numerous families of eudicots – including Asteraceae (the daisy family), Brassicaceae (the cabbage family), and Euphorbiaceae (the spurge family) – also use C4.

No large trees (above 15 m in height) use C4,[34] however a number of small trees or shrubs smaller than 10 m exist which do: six species of Euphorbiaceae all native to Hawaii and two species of Amaranthaceae growing in deserts of the Middle-East and Asia.[35]

Genetic engineering C4 rice project

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Given the advantages of C4, a group of scientists from institutions around the world are working on the C4 Rice Project to produce a strain of rice, naturally a C3 plant, that uses the C4 pathway by studying the C4 plants maize and Brachypodium.[36] 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 claims C4 rice could produce up to 50% more grain—and be able to do it with less water and nutrients.[37][38][39]

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 US$14 million over three years towards the C4 Rice Project at the International Rice Research Institute.[40] In 2019, the Bill & Melinda Gates Foundation granted another US$15 million to the Oxford-University-led C4 Rice Project. The goal of the 5-year project is to have experimental field plots up and running in Taiwan by 2024.[41]

C2 photosynthesis, an intermediate step between C3 and Kranz C4, may be preferred over C4 for rice conversion. The simpler system is less optimized for high light and high temperature conditions than C4, but has the advantage of requiring fewer steps of genetic engineering and performing better than C3 under all temperatures and light levels.[42] In 2021, the UK Government provided £1.2 million on studying C2 engineering.[43]

See also

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References

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

C4 carbon fixation

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from Grokipedia
C4 carbon fixation, also known as the Hatch–Slack pathway, is a variant of in which atmospheric (CO₂) is initially captured in mesophyll cells to form a four-carbon , such as oxaloacetate or malate, via the enzyme (PEPC), before being shuttled to bundle sheath cells for and ultimate incorporation into the three-carbon compounds of the Calvin–Benson cycle by ribulose-1,5-bisphosphate carboxylase/oxygenase (). This CO₂-concentrating mechanism minimizes the oxygenase activity of , thereby suppressing and enhancing carbon assimilation efficiency under conditions of high temperature, intense light, and low atmospheric CO₂. The pathway relies on specialized Kranz anatomy in leaves, characterized by a wreath-like arrangement of bundle sheath cells—enlarged and often chlorophyll-rich—surrounding the vascular bundles, which spatially separates initial CO₂ fixation from the to create a high-CO₂ microenvironment around . Biochemically, three main subtypes exist based on the primary decarboxylating and transport: the NADP-malic (NADP-ME) type, predominant in grasses like ; the NAD-malic (NAD-ME) type, common in dicots; and the phosphoenolpyruvate carboxykinase (PEP-CK) type, which uses aspartate as the main transport . Discovered in 1966 through ¹⁴CO₂-labeling experiments on leaves by Marshall Davidson Hatch and Charles Roger Slack, the pathway was initially proposed as an "accessory" CO₂ fixation route but later recognized as a complete adaptation enhancing photosynthetic rates by up to 50% compared to the ancestral C3 pathway. C4 plants represent about 3% of all species (roughly 7,500–8,000 angiosperms, primarily in the family) yet account for approximately 23% of global terrestrial primary productivity, dominating tropical and subtropical ecosystems and underpinning key staple crops such as , , millet, and . Compared to C3 plants, C4 species exhibit superior (up to twofold higher due to reduced ), (from concentrating CO₂ around fewer molecules), and , making them resilient in arid, high-irradiance environments where in C3 plants can consume 20–30% of fixed carbon. These advantages stem from the C4 cycle's ability to pump CO₂, effectively raising its concentration at Rubisco to 10–20 times ambient levels, which curtails wasteful oxygenation reactions. Evolutionarily, C4 photosynthesis has arisen independently at least 61 times across 19 angiosperm lineages since the late (around 30 million years ago), driven by declining atmospheric CO₂ levels and rising temperatures that favored the pathway's suppression of . This underscores its adaptive significance, with ongoing research exploring bioengineering of C4 traits into C3 crops like to boost yields amid .

Introduction and History

Overview of C4 Photosynthesis

C4 carbon fixation is a CO2-concentrating mechanism in that spatially separates the initial fixation of CO2 from the , enabling more efficient carbon assimilation in certain . In this pathway, atmospheric CO2 is first incorporated into four-carbon (C4) organic acids in mesophyll cells through a reaction catalyzed by , which has a high affinity for CO2. These C4 acids are then transported to bundle sheath cells, where they are decarboxylated to release CO2 at elevated concentrations around the enzyme , allowing the to proceed with reduced interference from oxygen. This spatial organization relies on Kranz anatomy, characterized by distinct mesophyll and bundle sheath cell layers. Although C4 photosynthesis occurs in only about 3% of species, these contribute approximately 23% of global terrestrial carbon fixation due to their superior efficiency under certain conditions. It is particularly prevalent in tropical and subtropical regions with high light intensity, elevated temperatures, and water-limited environments, where it supports higher rates of productivity compared to other photosynthetic pathways. Unlike the C3 pathway, in which directly fixes CO2 but can also react with O2 to cause —leading to carbon loss especially at high temperatures and low atmospheric CO2—the C4 mechanism suppresses by maintaining CO2 levels around at 10- to 20-fold higher than in the atmosphere, thus optimizing efficiency.

Discovery and Historical Development

The discovery of C4 carbon fixation began with early experiments in the 1950s that revealed unexpected patterns of carbon labeling in tropical plants. In 1954, researchers at the Hawaiian Sugar Planters' Association, including Hugo P. Kortschak, Constance E. Hartt, and George O. Burr, conducted 14CO2 feeding studies on leaves and observed that the initial products of photosynthesis were primarily the four-carbon compounds aspartate and malate, rather than the three-carbon phosphoglyceric acid (PGA) typical of the Calvin-Benson cycle. These findings, initially reported in proceedings and later detailed in a 1965 publication, challenged the prevailing understanding of but were not widely recognized or mechanistically explained at the time. Similarly, in 1960, Yuri S. Karpilov at the Kazan Agricultural Institute in the performed 14CO2 labeling experiments on leaves, noting a predominance of C4 dicarboxylic acids as early photosynthetic products, which further hinted at an alternative fixation mechanism. The key breakthrough came in the mid-1960s through systematic work by Marshall D. (Hal) Hatch and Charles R. Slack at the Commonwealth Scientific and Industrial Research Organisation (CSIRO) in . Building on the earlier observations, Hatch and Slack initiated 14CO2 pulse-chase experiments on and other tropical grasses in 1965–1966, consistently demonstrating that C4 acids such as malate and aspartate were rapidly labeled within seconds of exposure, followed by the appearance of intermediates like PGA after a short delay. In their seminal 1966 paper published in the Biochemical Journal, they proposed a novel "dicarboxylic acid pathway" in which CO2 is initially fixed into oxaloacetate by an enzyme other than ribulose-1,5-bisphosphate carboxylase/oxygenase (), forming C4 compounds that are then decarboxylated to release CO2 for the standard C3 cycle. This model, refined in subsequent 1967 publications, explained the anomalous labeling and highlighted the pathway's role in enhancing CO2 concentration around . By the early 1970s, biochemical studies confirmed the core components of the C4 pathway, solidifying its acceptance in research. Researchers identified (PEPC) as the primary enzyme for initial CO2 fixation into oxaloacetate, with high activity in C4 plants like and . Decarboxylating enzymes, such as NADP-malic enzyme and , were purified and characterized, verifying the metabolite shuttling between cell types. The term "C4 pathway" was formally adopted during this period to denote the four-carbon intermediates, distinguishing it from the C3 cycle, and it gained widespread recognition for its implications in adaptation to hot, arid environments.

Anatomical Adaptations

Kranz Anatomy

Kranz anatomy is the characteristic leaf structure in many C4 plants, named after the German word for "" due to the concentric arrangement of bundle sheath cells encircling the vascular bundles in a ring-like pattern. These bundle sheath cells are enlarged and contain numerous chloroplasts, distinguishing them from the surrounding mesophyll cells and enabling spatial separation of photosynthetic processes. This anatomy, first described by Haberlandt in 1882, is a key adaptation that supports the C4 pathway by facilitating metabolite shuttling between cell types. Mesophyll cells form the outer layer of this dimorphic arrangement, positioned peripherally around the bundle sheath. They feature thin walls, transparent , and extensive intercellular air spaces, which promote light penetration to deeper tissues and gas . In C4 like , mesophyll cells are arranged in a single layer or loosely packed to maximize exposure, serving as the primary site for initial carbon assimilation. Bundle sheath cells, in contrast, possess thick, often suberized walls that limit gas permeability and contain large, centripetally positioned chloroplasts with reduced grana thylakoids. This structural specialization confines the enzymes, such as , within these cells, where CO2 levels are elevated through the C4 mechanism. The wreath-like configuration ensures close proximity to veins, optimizing transport via plasmodesmata connecting the two cell types. The development of Kranz anatomy originates from the ground meristem during leaf primordia formation, where procambial signals induce differentiation into bundle sheath and mesophyll lineages. Genetic regulators, including the SCARECROW/SHORT-ROOT pathway, coordinate this process, extending vascular patterning cues to establish the 1:1 cell ratio typical of C4 leaves. Evolutionarily, Kranz anatomy correlates with increased vein density—often 1.9 to 2.1 times higher than in C3 plants—which reduces interveinal distances to 60–150 μm, enhancing the efficiency of the CO2-concentrating system across multiple independent origins in angiosperms.

Variations in C4 Anatomy

While the classic Kranz anatomy defines most C4 plants through a dimorphic arrangement of mesophyll and bundle sheath cells, certain species exhibit single-cell C4 photosynthesis, where CO2 fixation and concentration occur within individual chlorenchyma cells via intracellular compartmentalization. In these plants, dimorphic chloroplasts are spatially separated: peripheral chloroplasts positioned against the plasma membrane perform initial PEP carboxylase-mediated CO2 fixation, while central or proximal chloroplasts, enriched with , handle decarboxylation and reactions. This arrangement mimics the intercellular metabolite shuttling of Kranz anatomy but relies on cytoskeletal partitioning and vacuolar development to maintain domain separation within the cell. Prominent examples of single-cell C4 include species in the family, such as Bienertia sinuspersici and aralocaspica, which display bienertioid anatomy characterized by elongated, succulent leaves adapted to arid environments. In B. sinuspersici, the peripheral chloroplasts form a cytoplasmic sleeve around the central compartment, facilitating efficient CO2 pumping without cell walls as barriers. Similarly, S. aralocaspica partitions chloroplasts longitudinally, with distal ones for fixation and proximal for activity, supported by distinct thylakoid arrangements and mechanisms. These succulent adaptations enhance water-use efficiency in saline or dry habitats. Beyond fully dimorphic single-cell forms, intermediate or partial Kranz anatomies occur in evolutionary transitional species, such as those in the genus Flaveria (Asteraceae), which bridge C3 and C4 . These intermediates feature proto-Kranz traits, including enlarged bundle sheath cells with increased density and reduced interveinal distance, but lack the full dimorphic separation or partitioning of mature C4 plants. For instance, Flaveria pringlei and related C3-C4 hybrids show heightened glycine decarboxylase activity in bundle sheath cells, correlating with partial suppression of photorespiration and vein patterns that prefigure Kranz structure. These anatomical variations underscore the evolutionary flexibility of C4 photosynthesis, suggesting multiple convergent pathways for its emergence independent of strict Kranz requirements. Single-cell forms demonstrate that intracellular partitioning can achieve CO2 concentration without multicellular specialization, potentially representing an alternative route in lineages like . In Flaveria, partial Kranz intermediates highlight incremental anatomical changes—such as vein density increases and cell size modifications—that facilitate the transition from C3 ancestors, implying that C4 evolvability is enabled by pre-existing traits rather than de novo inventions. Such diversity indicates that C4 anatomy adapts to ecological pressures like or herbivory through modular developmental shifts.

Biochemical Mechanisms

Initial CO2 Fixation

In C4 plants, the initial fixation of atmospheric CO₂ occurs in the mesophyll cells, primarily catalyzed by the phosphoenolpyruvate carboxylase (PEPC), which is localized in the . This efficiently captures CO₂ after its hydration to (HCO₃⁻) by , initiating the C4 pathway before CO₂ is concentrated for the . The core reaction involves the β-carboxylation of phosphoenolpyruvate (PEP), a three-carbon phosphoglycolytic intermediate, to produce the four-carbon compound oxaloacetate (OAA) and inorganic phosphate (Pᵢ): \cePEP+HCO3−−>[PEPC]OAA+Pi\ce{PEP + HCO3^- ->[PEPC] OAA + P_i} This step was first characterized in leaves, where short-term exposure to ¹⁴CO₂ revealed rapid labeling of OAA and related acids. The OAA product is then quickly metabolized: it is either reduced to malate by NADP⁺- in the chloroplasts or transaminated to aspartate by aspartate aminotransferase in the or mitochondria, depending on the C4 subtype (NADP-ME or NAD-ME). These C4 acids serve as stable carriers for CO₂ transport to bundle sheath cells. A key advantage of PEPC is its affinity for HCO₃⁻, with a Kₘ value (approximately 20–50 μM) comparable to the effective concentration derived from ambient CO₂ via , compared to 's Kₘ for CO₂ (around 10–15 μM). Moreover, PEPC exhibits no oxygenase activity, unlike , which avoids wasteful oxygenation reactions and in the oxygen-exposed mesophyll environment. Additionally, PEPC shows reduced discrimination against ¹³C compared to Rubisco, resulting in C4 plants having δ¹³C values of approximately -9 to -13‰, less negative than the -23 to -30‰ typical of C3 plants. This isotopic signature is used to distinguish photosynthetic pathways in ecological and dietary analyses. PEPC activity is tightly regulated to align with photosynthetic conditions, primarily through reversible at a conserved N-terminal serine residue, which increases catalytic efficiency and reduces sensitivity to inhibitors like malate during exposure. This -dependent is mediated by a dedicated PEPC (PEPC-PK), stimulated by factors such as and elevated photosynthetic metabolites, ensuring rapid response to diurnal cycles.

Decarboxylation Pathways

In C4 photosynthesis, the decarboxylation step occurs in the bundle sheath cells, where C4 acids are converted back to CO2 and a three-carbon compound, concentrating CO2 for the Calvin cycle. This process varies among subtypes, classified based on the primary decarboxylating enzyme, cellular compartment, and associated metabolites. The three main subtypes are the NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEPCK) types, each adapted to specific anatomical and environmental contexts. The NADP-ME subtype is the most prevalent, accounting for the majority of C4 plants, particularly in monocot families such as . In this pathway, malate, produced in the mesophyll cells via (PEPC), is transported to the bundle sheath chloroplasts. There, NADP-dependent malic enzyme catalyzes the : Malate+NADP+→Pyruvate+CO2+NADPH\text{Malate} + \text{NADP}^+ \rightarrow \text{Pyruvate} + \text{CO}_2 + \text{NADPH} The released CO2 elevates concentrations in the bundle sheath, while NADPH supports the , and pyruvate returns to the mesophyll for regeneration. This subtype is characteristic of crops like (Zea mays) and (Sorghum bicolor), where it enhances efficiency under high light and temperature. In the NAD-ME subtype, common in dicotyledonous families like , aspartate serves as the primary transport from mesophyll to bundle sheath. Within the bundle sheath , aspartate is converted to malate, which is then imported into mitochondria for by NAD-dependent malic : Malate+NAD+→Pyruvate+CO2+NADH\text{Malate} + \text{NAD}^+ \rightarrow \text{Pyruvate} + \text{CO}_2 + \text{NADH} The NADH generated is shuttled to chloroplasts via the malate-aspartate shuttle to provide reducing power. This subtype predominates in species such as Amaranthus and , often in more arid environments, and is linked to larger mitochondrial volumes in bundle sheath cells. The PEPCK subtype relies on phosphoenolpyruvate carboxykinase for decarboxylation in the bundle sheath cytosol, using oxaloacetate derived from aspartate transport. The reaction is: Oxaloacetate+ATP→PEP+CO2+ADP\text{Oxaloacetate} + \text{ATP} \rightarrow \text{PEP} + \text{CO}_2 + \text{ADP} This pathway generates PEP directly, which can re-enter the C4 cycle or feed into the Calvin cycle after phosphorylation, and is energy-intensive due to ATP consumption. Pure PEPCK types are rare, but the enzyme supplements NADP-ME or NAD-ME pathways in grasses like Setaria and Panicum maximum, contributing to metabolic flexibility under varying light conditions. The subtypes' distribution correlates with leaf anatomy: NADP-ME with centrifugal chloroplasts, NAD-ME with granal chloroplasts and mitochondria, and PEPCK with large chloroplasts and mitochondria.

Metabolite Transport

In C4 photosynthesis, the transport of metabolites between mesophyll (M) and bundle sheath (BS) cells is essential for the spatial separation of initial CO2 fixation and the . The primary shuttle involves the export of C4 acids—malate in the NADP-malic (NADP-ME) subtype or aspartate in the NAD-malic (NAD-ME) and (PEPCK) subtypes—from M cells to BS cells. This movement occurs predominantly via passive through plasmodesmata, facilitated by steep concentration gradients maintained by enzymatic activities in each cell type; for instance, in (a NADP-ME ), malate concentrations are approximately 11 mM in M cells versus 5 mM in BS cells, and aspartate is about 1.2 mM in M cells versus 0.2 mM in BS cells. The return path completes the cycle by shuttling the three-carbon carbon skeletons back to M cells for regeneration of the CO2 acceptor phosphoenolpyruvate (PEP). In the NADP-ME subtype, pyruvate produced from malate in BS cells diffuses back to M cells, where it is converted to PEP by pyruvate, phosphate dikinase (PPDK) in the chloroplasts. In NAD-ME plants, serves as the return metabolite, while PEPCK subtypes directly produce PEP in BS cells for potential back-transport. These intercellular exchanges rely on the high density of plasmodesmata at the M-BS interface, which supports efficient flux without identified specific channel proteins, though gradients for return metabolites like pyruvate can be subtle or reversed, suggesting rapid consumption in M cells maintains directionality. Intracellular transport within Kranz anatomy cells involves specialized envelope transporters to support the C4 cycle. In M cell chloroplasts, the dicarboxylate transporter DiT1 (also known as ZmDiT1 in ) exports malate (or exchanges it with oxaloacetate) to the for subsequent plasmodesmatal transfer, while plastidic pyruvate transporters such as MEP1 facilitate pyruvate import for PPDK activity. In BS cells, metabolite channels like the / translocator (TPT) enable export of products, and potential malate-pyruvate antiporters (e.g., MEP2/MEP4 isoforms) aid product handling. Additionally, ATP/ADP carriers, such as AATP1 (a translocator), supply nucleotides to M cell chloroplasts to power PPDK, ensuring energy availability for PEP regeneration despite the lack of proton gradients in these agranal plastids. This metabolite shuttling incurs an additional energy cost beyond C3 photosynthesis, primarily from in the regeneration step. PPDK in M cells consumes two ATP equivalents per pyruvate-to-PEP conversion (one for the dikinase reaction producing AMP and another for AMP recycling to ATP), while the PEPCK subtype requires one ATP per in BS cell . These costs, totaling about two extra ATP per CO2 fixed, are met by enhanced photosynthetic electron transport and supported by efficient ATP/ADP exchange across membranes. in BS cells yields pyruvate or other products that feed directly into this return transport.

Integration with Photosynthesis

Light Harvesting and Energy Use

In C4 plants, chloroplast dimorphism plays a central role in optimizing light harvesting and energy allocation between mesophyll and bundle sheath cells. In the NADP-malic enzyme (NADP-ME) subtype predominant in grasses like , mesophyll s are grana-rich, featuring stacked thylakoids that support (PSII) activity for efficient light capture and linear electron transport, generating both ATP and NADPH to drive initial CO₂ fixation. In contrast, bundle sheath s are predominantly agranal, with reduced grana stacks and a focus on (PSI) for cyclic electron flow, which primarily produces ATP needed for CO₂ without significant NADPH generation. This structural specialization ensures that light is partitioned to match the metabolic demands of the C4 pathway, minimizing waste. Antenna complexes in C4 plants are adapted to enhance capture efficiency, with larger light-harvesting complexes (LHCII) associated with PSII in mesophyll chloroplasts to maximize photon absorption under varying intensities. State transitions further facilitate energy balancing: mesophyll chloroplasts undergo dynamic shifts between State 1 (LHCII associated with PSII) and State 2 (LHCII with PSI) in response to redox signals, optimizing electron distribution. Bundle sheath chloroplasts, however, remain in a permanent State 2, where LHCII primarily serves PSI to support cyclic flow and prevent over-reduction. These mechanisms allow C4 plants to fine-tune energy allocation across cell types. Under high light conditions, the CO₂-concentrating mechanism in C4 plants sustains higher quantum yields of PSII compared to C3 plants under ambient CO₂, as reduced prevents downregulation of electron transport. The operating efficiency of PSII (ΦPSII\Phi_{PSII}) can reach up to approximately 0.8 in C4 species under ambient CO₂ and high light, versus about 0.5–0.7 in C3 species, reflecting more effective use of absorbed light for productive due to minimized by O₂. At saturating CO₂ levels, both C4 and C3 plants approach similar maximum ΦPSII\Phi_{PSII} values of around 0.8. C4 plants adapt to fluctuating through metabolite signaling that coordinates rapid adjustments in photosynthetic rates between mesophyll and bundle sheath cells. Intermediates like malate and aspartate serve as signals to balance electron transport and carbon , enabling quicker recovery of CO₂ assimilation during light transitions compared to C3 plants under similar conditions. This signaling enhances overall energy use efficiency in dynamic environments, such as shaded canopies.

Coupling to Calvin Cycle

In C4 photosynthesis, the CO₂-concentrating mechanism delivers elevated levels of CO₂ to the bundle sheath cells, where ribulose-1,5-bisphosphate carboxylase/oxygenase () catalyzes its fixation via the . The C4 acids, primarily malate or aspartate, are transported from mesophyll cells to the bundle sheath, where releases CO₂ directly in proximity to , ensuring efficient integration with the reductive . This spatial separation and biochemical coupling allow the to operate under non-limiting CO₂ conditions, with the three-carbon products of recycled back to the mesophyll to sustain the C4 shuttle. The CO₂ concentration in bundle sheath cells is typically 10-20 times higher than in mesophyll cells, reaching up to 1000-2000 ppm during active , which optimizes 's carboxylase activity. This elevation enables C4 plants to utilize isoforms with lower CO₂/O₂ specificity factors, which are faster in but less selective than those in C3 plants, without compromising due to the minimized oxygenation risk. For each oxaloacetate molecule processed through the C4 cycle, one CO₂ is released for fixation, and the resulting three-carbon compound (such as pyruvate) is returned to the mesophyll for regeneration into phosphoenolpyruvate, maintaining a balanced carbon flow that aligns with the Calvin cycle's requirement for three CO₂ molecules to produce one net glyceraldehyde-3-phosphate. This integration yields a net carbon gain by preventing losses associated with alternative reactions, enhancing overall photosynthetic productivity. In the NADP-malic enzyme subtype of C4 photosynthesis, decarboxylation of malate in bundle sheath chloroplasts generates NADPH alongside CO₂ release, providing reducing power that directly supports the reduction phase of the . Although the requires two NADPH per CO₂ fixed for 3-phosphoglycerate reduction, the one NADPH produced per decarboxylation event supplements the NADPH generated via bundle sheath , facilitating coordinated energy use and minimizing reliance on mesophyll-derived metabolites. This feedback mechanism ensures seamless coupling between the C4 pump and reductions, particularly under varying light conditions.

Efficiency and Advantages

Photorespiration Suppression

In C3 plants, photorespiration arises when the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the oxygenation of ribulose-1,5-bisphosphate (RuBP) instead of its carboxylation, yielding one molecule of 3-phosphoglycerate and one of 2-phosphoglycolate (2-PG). The toxic 2-PG is then dephosphorylated to glycolate, which enters the photorespiratory pathway: glycolate is oxidized in peroxisomes to glyoxylate and then aminated to glycine, which is transported to mitochondria where two glycine molecules are converted to serine, releasing CO2 and NH3 in the process. This cycle consumes ATP and NADPH without net carbon gain, resulting in a substantial loss of fixed carbon—typically 25–30% under ambient atmospheric conditions at 25°C. C4 photosynthesis suppresses by employing a CO2-concentrating mechanism that delivers high concentrations of CO2 to in bundle sheath cells, elevating the local CO2/O2 ratio by 10- to 60-fold compared to the mesophyll or ambient air. This elevated ratio overwhelmingly favors the carboxylase reaction over the oxygenase, effectively minimizing oxygenation rates to near negligible levels. Consequently, the specificity factor τ of —which measures its relative affinity for CO2 versus O2—becomes largely irrelevant in the C4 context, as the environmental conditions at the enzyme site dominate reaction kinetics regardless of intrinsic τ variations between C3 and C4 Rubiscos. The suppression of photorespiration provides C4 plants with a pronounced advantage at elevated temperatures, where the oxygenase activity of in C3 plants accelerates due to decreased CO2/O2 solubility ratios and reduced τ values. At 35°C under ambient conditions, photorespiration can account for approximately 50% carbon loss in C3 plants, whereas it remains below 5% in C4 plants owing to the persistent high CO2 environment. Despite this efficiency, the C4 CO2 pump is not perfectly sealed; bundle sheath leakiness (φ), the proportion of decarboxylated CO2 that diffuses back to mesophyll cells without fixation, introduces a small inefficiency, with typical φ values ranging from 5% to 20% across C4 species depending on anatomy, light, and environmental factors.

Resource Use Efficiency

C4 plants demonstrate superior use compared to C3 plants, primarily through reduced (gs) while maintaining high net CO2 assimilation rates (A), facilitated by the CO2-concentrating mechanism that minimizes loss via . Intrinsic use (iWUE = A/gs) in C4 species is approximately twice that of C3 species under warm conditions, with representative short-term values of about 100 μmol CO₂ mol⁻¹ H₂O for C3 plants versus 200 μmol CO₂ mol⁻¹ H₂O for C4 plants. This advantage is particularly pronounced in hot, dry environments, where lower gs in C4 plants (often 0.1-0.3 mol m⁻² s⁻¹) compared to C3 (0.4-0.6 mol m⁻² s⁻¹) allows for sustained with less evaporative cooling demand. Nitrogen use efficiency (NUE) is also enhanced in C4 plants due to the lower requirement for , the nitrogen-intensive enzyme that comprises only 5-9% of leaf organic nitrogen in C4 species versus 10-27% in C3 species, as the CO2 pump elevates CO2 concentrations around to boost its carboxylation efficiency. This reduced investment in results in photosynthetic NUE that is 40-50% higher in C4 plants, allowing greater biomass production per unit of nitrogen applied, as evidenced by comparisons in model species like (C4) and (C3). The efficiency stems from the CO2 concentrating mechanism, which decreases the need for excess to compensate for oxygenation, thereby optimizing nitrogen allocation to other photosynthetic components. In tropical environments, C4 plants exhibit photosynthetic productivity rates up to 50% higher than C3 plants, driven by efficient resource utilization and minimal under high light and temperature, leading to greater overall accumulation. However, this advantage diminishes under low light or cool temperatures (below 20°C), where the additional energy costs of the C4 cycle reduce efficiency relative to C3 plants. Recent research highlights C4 plants' superior performance in fluctuating light, sustaining CO2 assimilation during low-light phases and providing approximately 20% greater yield stability compared to C3 species, which aids resilience in variable field conditions.

Evolutionary Origins

Timeline of Evolution

C4 photosynthesis is believed to have first emerged around 30–35 million years ago during the late Eocene to early Oligocene epochs, coinciding with a significant decline in atmospheric CO2 concentrations to below 600 ppm at the Eocene-Oligocene transition. This environmental shift, including reduced CO2 levels that favored carbon-concentrating mechanisms, likely provided the selective pressure for the initial evolution of the C4 pathway, particularly in grasses. Isotopic analyses of fossil grass pollen from southwestern Europe indicate the presence of C4 grasses as early as the early Oligocene, around 30 million years ago, supporting molecular clock estimates for these origins. The C4 pathway has evolved independently more than 62 times across at least 19 families of angiosperms, with the majority of origins occurring in the grass family (over 20 independent lineages) and the amaranth family (formerly Chenopodiaceae, with at least 10 origins). These convergent evolutions highlight the pathway's adaptive value under low CO2 conditions, though the precise timing varies, with many clustered in the and . The oldest direct evidence comes from carbon isotopic signatures in grass pollen grains dated to approximately 27–30 million years ago in late deposits. By the early , around 20 million years ago, C4 grasses had spread and become locally dominant in certain ecosystems, contributing to habitat heterogeneity in regions transitioning to more open environments. This expansion is evidenced by increasing proportions of C4 signatures in records from 21–16 million years ago, marking a key phase in the rise of C4-dominated biomes. Recent genomic and -integrated studies, including those from 2023, correlate rapid diversification of C4 lineages with post-Oligocene environmental changes, showing accelerated rates after 25 million years ago in response to and CO2 fluctuations.

Genetic Mechanisms

The evolution of C4 carbon fixation involved the coordinated upregulation and cell-specific expression of several key genes encoding enzymes central to the pathway, alongside the relocation of others to specific cell types. (PEPC), pyruvate, phosphate dikinase (PPDK), and malic enzyme (ME) are upregulated in mesophyll cells to facilitate initial CO2 fixation and its transport as C4 acids, while the small subunit of (RbcS) is predominantly expressed and relocated to bundle sheath cells to concentrate CO2 around the carboxylase. These changes ensure spatial separation of photosynthetic reactions, minimizing . In C4 species, PEPC and PPDK transcripts are enriched in mesophyll cells, with PEPC activity often 10- to 100-fold higher than in C3 plants, driven by and promoter modifications. Similarly, NADP-ME type C4 plants show elevated ME expression in bundle sheath cells, supporting . 's confinement to bundle sheath cells reduces its exposure to oxygen, enhancing efficiency. Regulatory networks governing these expression patterns rely on cis-regulatory elements in gene promoters that confer cell-specificity, often through the binding of transcription factors. In mesophyll cells, promoters of PEPC and PPDK contain light-responsive and tissue-specific motifs, such as G-box and T-box elements, that drive high-level, Kranz anatomy-dependent expression. Bundle sheath-specific expression of genes like RbcS and ME involves conserved cis-elements that respond to developmental cues. A pivotal mechanism is the exaptation of ancestral vein-identity gene networks, where pre-existing cis-regulatory codes from C3 leaves—originally associated with vascular development—were conscripted to enhance bundle sheath identity and gene expression in C4 species. This 2024 study in Nature demonstrated that C4 bundle sheath cells gained a combinatorial cis-code from C3 progenitors, involving motifs bound by DOF transcription factors, enabling the recruitment of photosynthetic genes without de novo evolution of regulatory elements. Such exaptation facilitated the anatomical changes supporting C4 photosynthesis, like enlarged bundle sheath cells surrounding veins. Convergent evolution across C4 lineages highlights parallel genetic adaptations, with independent mutations yielding similar regulatory outcomes. In grasses, the Golden2-like (GLK) transcription factors exemplify this, where GLK1 and GLK2 paralogs evolved distinct roles: GLK1 drives chloroplast biogenesis in mesophyll cells, while GLK2 is upregulated in bundle sheath cells of C4 species like maize and sorghum, promoting photosynthetic gene expression. This pattern arose convergently in Andropogoneae and Panicoideae subfamilies, with cis-regulatory changes in GLK2 enhancers enabling bundle sheath specificity. Comparative transcriptomics across C4 grasses revealed that 82% of bundle sheath-enriched transcription factors, including GLK2 and members of the C2H2 family, show conserved differential expression in independent lineages, underscoring regulatory convergence despite phylogenetic distance. Recent genomic advances have illuminated the stepwise of genes during C4 evolution, particularly through surveys in model systems like Flaveria. High-resolution mRNA-Seq and chromatin accessibility profiling in Flaveria species spanning C3 to C4 transitions show that C4 pathway genes were recruited progressively: initial photorespiratory modifications in C3-C4 intermediates upregulated glycine decarboxylase in bundle sheath cells, followed by full C4 enzyme expression via enhancer evolution. A 2023 study revealed dynamic co-regulation networks, with various transcription factors conscripted early for cell-type differentiation. Complementing this, a 2025 Trends in Plant Science review synthesizes how pre-existing cis-regulatory codes—such as those for vein patterning—were exapted across angiosperms, enabling rapid C4 emergence without wholesale rewiring. These insights from Flaveria underscore a modular, gene-by-gene process, with over 50 C4-related genes showing stepwise expression shifts correlated to gains.

C4 Plants and Applications

Diversity and Examples

C4 photosynthesis has independently evolved in at least 61 lineages across 19 families of angiosperms, resulting in approximately 8,100 that employ this pathway. The vast majority of these species—around 80%—are monocots, primarily in the (grasses) and (sedges) families, with alone accounting for over half of all C4 . Representative examples in include economically important tropical grasses such as (Zea mays), (Sorghum bicolor), and (Saccharum officinarum), which utilize the NADP-malic (NADP-ME) subtype predominant in this family. In contrast, the family, which includes about 17% of C4 , features dicots like Amaranthus (grain amaranths) that often employ the NAD-malic (NAD-ME) subtype, especially in arid-adapted lineages. The distribution of C4 subtypes varies by taxonomic group, reflecting adaptations to specific environmental pressures. The NADP-ME subtype dominates in monocot families like , particularly tropical grasses that thrive in high-light, warm conditions, while the NAD-ME subtype is more common in eudicot families such as and Chenopodiaceae (now subsumed under Amaranthaceae sensu lato), often in arid dicots like species. A third subtype, (PEPCK), occurs less frequently and is scattered across families, including some grasses and . This biochemical diversity underscores the of C4 traits across disparate lineages, with no single subtype monopolizing any family. C4 plants predominantly occupy open, high-irradiance habitats such as tropical savannas, grasslands, and hot deserts, where elevated temperatures and low CO₂ diffusion favor their efficiency over C3 photosynthesis. They are scarce in shaded forest understories or aquatic environments, which typically support C3-dominated flora due to lower light and temperature demands. In Southwest Asia, for instance, C4 species form psammophytic communities in sandy deserts and coastal dunes, as well as halophytic assemblages in saline habitats. While C4 carbon fixation is a hallmark of certain angiosperms, analogous carbon-concentrating mechanisms exist in non-vascular organisms like , which use carboxysomes to enhance CO₂ fixation efficiency in low-carbon environments; however, true C4 is confined to vascular plants. This focus on angiosperms highlights the pathway's role in terrestrial , particularly in dominating biomes like savannas where C4 grasses can comprise up to 50% of species diversity.

Agricultural Importance

C4 plants are pivotal in global agriculture, serving as foundational crops for , feed, and . Among the most prominent are (Zea mays), (Saccharum officinarum), and (Sorghum bicolor), which leverage the C4 pathway to achieve high productivity in warm, often resource-limited environments. As of 2024, global maize production reached approximately 1.22 billion metric tons, sugarcane ~1.95 billion metric tons, and sorghum ~60 million metric tons. These crops, particularly the C4 cereals like maize and sorghum, contribute about 40% of world cereal production, providing a substantial share of caloric intake through direct consumption and . The agricultural advantages of C4 plants stem from their superior and capacity for high accumulation, enabling reliable yields under heat and water stress that challenge C3 counterparts. For instance, their CO2-concentrating mechanism minimizes water loss via , supporting cultivation on marginal lands with limited . This trait is particularly valuable for applications, as seen with switchgrass (), a C4 grass that yields high for production while requiring minimal inputs. In 2024, C4 crops demonstrated yield stability amid increased drought frequency linked to . Despite these strengths, C4 crops face limitations in cold tolerance, which restricts their expansion into temperate zones where chilling temperatures impair and growth. Most C4 species exhibit sensitivity to temperatures below 10–15°C, leading to reduced activity and frost damage, thereby confining their primary use to tropical and subtropical regions. In the context of climate change, C4 crops are poised to enhance global food security by adapting well to projected increases in temperature and drought frequency, potentially expanding viable arable land for their cultivation in currently marginal areas. Models indicate moderate yield stability for C4 systems under warming scenarios, contrasting with greater vulnerabilities in C3-dominated agriculture, thus underscoring their role in sustaining production amid environmental shifts.

Engineering Efforts

C4 Rice Project

The C4 Rice Project is an international collaborative initiative led by the (IRRI) in partnership with over 20 research groups from more than 15 institutions across eight countries, aimed at engineering C4 photosynthetic traits into rice to boost yield and resource efficiency. Conceived in the late 1990s by IRRI's John Sheehy, the project gained momentum following a 2006 workshop at IRRI and received initial funding from the Bill & Melinda Gates Foundation in 2008, marking the start of Phase I. Subsequent phases have been supported by additional grants, including a $14 million investment from the Gates Foundation, IRRI, and the government for Phase II in 2018, and a $15 million renewal in 2019 for Phase IV, extending through mid-2026. The project's primary goal is to introduce the biochemical and anatomical features of C4 photosynthesis into , a C3 crop, to overcome yield plateaus from traditional breeding and address global challenges for a projected of 9.7 billion by 2050. This involves developing Kranz-like in rice leaves, similar to that in natural C4 , alongside enhanced expression and metabolic pathways. The approach encompasses three main pillars: anatomical modifications to increase vein density and bundle sheath cell development using regulators like SHR and G2 genes; biochemical enhancements through overexpression of key C4 enzymes such as (PEPC) and NADP-malic enzyme (ME), demonstrated by elevated malate levels in transgenics; and genetic strategies including modeling, CRISPR-based , and multi-gene constructs to optimize regulatory networks. Key milestones include the adoption of as a grass for mutant screening and transformation protocols in Phases I and II (2008–2015), enabling rapid testing of rice-applicable traits. In Phase III (2016–2019), researchers successfully introduced a partial C4 biochemical pathway into using a five-gene construct, achieving detectable PEP carboxylase activity and improved stomatal density, while anatomical studies reduced candidate vein regulators from 60 to about 10 genes. By 2020, field experiments with G2-overexpressing lines showed over 20% yield gains under irrigated conditions. Phase IV (2019–2026) has advanced prototypes with up to 15 transgenes for photosynthetic evaluation, gene-edited lines relocating to the to mimic C4 function, and enhanced bundle sheath volume via tml1 editing, with ongoing assessments in informing iterations; as of November 2025, the phase continues with extensions supporting further development. A 2025 consortium meeting in highlighted integration with related efforts, though large-scale field trials remain in development. Predicted outcomes from modeling and early prototypes include a 50% increase in and grain yield relative to 2010 baselines, alongside improved nitrogen use efficiency and doubled water use efficiency, potentially benefiting smallholder farmers in and where supports over 3 billion people. These gains would stem from suppressed and optimized CO2 concentration in bundle sheath cells, without requiring extensive new infrastructure.

Recent Advances and Challenges

Recent research has advanced the understanding of C4 photosynthesis engineering by elucidating the genetic foundations that could simplify its introduction into C3 crops. In 2024, scientists at the Salk Institute demonstrated that C4 photosynthesis evolved through the of pre-existing cis-regulatory codes in bundle sheath cells of C3 ancestors, enabling more efficient CO2 concentration without requiring entirely new genetic networks. This discovery suggests that targeted modifications to existing regulatory elements in non-C4 plants could mimic C4-like efficiency, potentially reducing the complexity of engineering efforts. Building on this, 2025 studies have employed -based to refine bundle sheath , a critical step for establishing the Kranz essential to C4 pathways. For instance, in-context promoter editing in identified key regulatory regions that control C4-specific , offering a blueprint for precise edits in C3 species like and . Similarly, research on ensembles in revealed how multiple regulators orchestrate bundle sheath differentiation, with CRISPR disruptions confirming their role in potential C4 conversion. Efforts have extended to other crops, including , where engineering C4 pathways offers potential improvements in photosynthetic rates under conditions, though full integration remains elusive. In , transgenic expression of C4 enzymes like PEPC has enhanced CO2 fixation and stress tolerance, highlighting applicability beyond grasses. A 2025 study on the Ottelia alismoides revealed a novel single-cell NAD-ME C4 subtype integrated with CAM and use, offering a model for engineering C4 mechanisms at the cellular level to minimize anatomical changes. Despite these gains, engineering C4 photosynthesis faces significant hurdles, including the coordination of over 50 genes across metabolic, anatomical, and regulatory networks. Energy costs associated with metabolite shuttling in the C4 cycle can offset efficiency benefits if not optimized, particularly in cooler climates where is less pronounced. Field verification remains challenging, as engineered plants often exhibit CO2 leakiness exceeding 20%, which diminishes the CO2 concentrating mechanism's effectiveness and requires extensive testing under real-world conditions. In 2025, key updates underscored the potential impacts of these advances. The C4 Rice Project's meeting in facilitated collaboration between the core team and the Gates-funded C4 initiative, emphasizing accelerated progress toward field trials despite ongoing anatomical barriers. Concurrently, a PNAS study modeled CO2 adaptation in C4 crops like and , showing that elevating content could yield 20-30% productivity gains under elevated atmospheric CO2 by alleviating limitations. These models predict substantial benefits for engineered C3 crops in future climates, provided regulatory and energetic challenges are addressed.

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