In most plant species, CO₂ is fixed by Ribulose Bisphosphate Carboxylase/Oxygenase (Rubisco) in the Calvin cycle to generate a three-carbon compound (Figure 1A). These plants are referred to as C₃. As the name of the enzyme suggests, Rubisco also reacts with O₂, and when this happens a toxic compound is produced that has to be eliminated by the plant. Because the detoxification process uses energy, the result is an overall decrease in the efficiency of photosynthesis. Species that use the C₄ pathway (Figure 1B) evolved to overcome this inefficiency. CO₂ is initially fixed by the oxygen-insensitive carboxylase phosphoenolpyruvate carboxylase (PEPCase) into a four-carbon compound. The four-carbon compound is then decarboxylated, and the released CO₂ is refixed in the Calvin cycle into a three-carbon compound.

Figure 1. Schematic of C3 (A) and C4 (B) pathways

Most C₄ plants, and certainly all known C₄ grasses, compartmentalize photosynthetic reactions between two morphologically distinct cell-types that are arranged in concentric circles around veins. This so-called ‘Kranz’ anatomy comprises enlarged bundle sheath (BS) cells immediately adjacent to the vein that are surrounded by mesophyll (M) cells (Figure 2). This arrangement generates a consistent interveinal distance of four cells (vein-BS-M-M-BS-vein). Metabolic reactions are divided between BS and M cells, with CO₂ being initially fixed in the M cells. C₄ acids are then transported to the BS for decarboxylation and the released CO₂ is refixed in the Calvin cycle. The C₄ carbon shuttle thus concentrates CO₂ at the site of Rubisco, reducing the likelihood of the oxygenation reaction occuring.

Figure 2. Rice (C3) (left) and maize (C4) l(right) leaf anatomy. Note different vein spacing patterns.