Despite some apparent benefits of photorespiration, several taxa of plants have evolved strategies that minimize photorespiration. These additional pathways for carbon fixation evolved in plants growing under high light conditions with limited water resources and took advantage of proteins from other primary metabolic processes, co-opting them for a new purpose. These pathways exist in addition to the process of photosynthetic carbon reduction already described, serving to concentrate CO2 for incorporation by Rubisco rather than operating instead of Rubsico.
One additional pathway that concentrates CO2 for Rubisco is known as the C4 pathway, and it is found in some members of the grass (Poaceae) family such as maize, sorghum, and sugar cane. These grass species display a specialized anatomical adaptation consisting of a ring of cells surrounding leaf vascular bundles called the bundle-sheath cells. The expression of Rubisco and other genes encoding enzymes of the Calvin-Benson pathway are limited to the bundle-sheath cells, resulting in the spatial separation of initial carbon fixation and Rubisco activity. The initial fixation of CO2 happens in the mesophyll cells of the leaf, and is carried out by an enzyme usually associated with glycolysis called phosphoenolpyruvate (PEP) carboxylase. Following carbon fixation, the resulting 4-carbon molecule (malate) is transported into a bundle-sheath cell and decarboxylated, giving off a CO2 that will be incorporated by Rubisco into the Calvin-Benson cycle.
The formation of a gradient never occurs without the input of energy, and this process of concentrating CO2 in the bundle-sheath is no exception. C4 plants invest heavily in the form of ATP and NADPH to sustain the gradient, but because this pathway is found in plants in high light environments, this cost is easily absorbed. Ha.
Unlike C4 plants, CAM plants do not have any particular anatomical adaptations related to carbon fixation. CAM plants employ the same alternate enzymes as the C4 plants for initial carbon fixation, but place them under the control of circadian regulation, restricting carbon fixation to the night, when stomata are open. This arrangement allows the plant to exchange gas with the environment when temperatures are much lower, resulting in significant water savings. As CO2 is fixed by PEP carboxylase, the product (malate) is imported to the vacuole and stored as malic acid. During the daytime, when stomata are closed, this malate is exported from the vacuole and decarboxylated, increasing CO2 concentrations and favoring its use over O2 by Rubisco.
PEP carboxylase activity is regulated by two main mechanisms. High concentrations of malate cause a form of enzyme inhibition known as feedback inhibition, in which the product of a pathway exerts an influence over an enzyme upstream of its production. Levels of malate are kept low in the vicinity of PEP carboxylase by exporting it to the bundle-sheath cells in the case of C4 pathway plants or importing it into the vacuole in the case of CAM plants. Another means of regulation over these alternative carbon fixation pathways involves the phosphorylation of PEP carboxylase, which occurs in the light. Upon phosphorylation, the affinity of PEP carboxylase for the substrate phosphoenolpyruvate increases. The affinity of an enzyme for its substrate, known as the Michaelis constant (Km), can be empirically determined in a carefully-controlled in vitro experiment. A decreasing Km is indicative of greater substrate binding at a lower substrate concentration. Conversely, the phosphorylation of PEP carboxylase causes an increase in the inhibition constant (Ki) for malate, meaning that the same amount of malate would inhibit the enzyme less under phosphorylated conditions.