Many terrestrial plants, adapted to hot and arid regions where water is scarce, are stressed by the severe effects of full sunlight, particularly drought and high temperature. To avoid high rates of water transpiration, the stomata of these species are tightly closed during the hot diurnal hours and perform most of their gas exchange with the surrounding atmosphere at night, when the air is relatively cool and humid. On the other hand, the vital process of photosynthesis in which CO₂ is incorporated into carbohydrates takes place during the day. To cope with the lack of an efficient diurnal influx of atmospheric CO₂, these species have evolved a different photosynthetic mechanism, called “crassulacean acid metabolism” (CAM). This unusual process takes place within single leaf cells in which the uptake and subsequent assimilation of atmospheric CO₂ involves three compartments: cytosol, vacuole, and chloroplast. In general, CAM plants are common among desert succulents (e.g., Cactaceae) exposed to daytime high temperatures and low relative humidity, or among tropical forest epiphytes (Bromeliaceae) which are exposed to physiological drought under humid conditions because of their aerial growth habit. Remarkably, CAM also occurs in aquatic species (Isoetaceae) found worldwide in marshy and shallow habitats that exhibit a diurnal limitation of CO₂.

During the cool night-time hours, the open stomata facilitate the uptake of atmospheric CO₂ which diffuses into the cell cytosol, generating HCO₃^– (see textbook Chapter 8). At this stage, phosphoenolpyruvate-carboxylase (PEPCase) catalyzes the incorporation of HCO₃^– into phosphoenolpyruvate, yielding oxaloacetic acid and related four-carbon organic acids (particularly malic acid), that accumulate in the vacuole. During the hot hours of the day, the stomatal closure characteristic of terrestrial CAM plants reduces water loss but causes an internal CO₂ shortage. To overcome this deficiency, the vacuole releases malic acid into the cytosol where the action of decarboxylating enzymes, NADP–malic enzyme or PEP-carboxykinase, produces pyruvate and CO₂. Inside the stroma of chloroplasts, Rubisco incorporates the released CO₂ into 3-phosphoglycerate which, in concert with the ATP and NADPH synthesized in the light via photophosphorylation, initiates a series of Benson–Calvin cycle reactions that result in the accumulation of starch. The temporal separation of the nocturnal formation and storage of malic acid, and the diurnal decarboxylation and the accompanying accumulation of plastid starch, proceeds over the course of a 24-hour photoperiod. On this temporal basis, CAM consists of four metabolic phases that encompass the modulation of C₄ (PEPCase) and C₃ (Rubisco) carboxylation within the same cellular environment (Web Figure 8.11.A). The nocturnal uptake of atmospheric CO₂ via PEPCase to form C₄ acids (phase I) and the diurnal decarboxylation of malic acid to elicit an elevated internal concentration of CO₂ (phase III) are interspersed with transitional periods of net CO₂ uptake in early morning (phase II) and in late afternoon (phase IV), when both the PEPCase- and the Rubisco-mediated carboxylations contribute to the assimilation of CO₂. In this circadian rhythm, the proportion of CO₂ taken up via PEPCase at night or via Rubisco during the day is controlled by:

• stomatal function
• fluctuations in the content of organic acids and storage carbohydrates
• the activity of enzymes involved in the primary (PEPCase) and the secondary (Rubisco) carboxylations, and in the decarboxylation of organic acids (e.g., malic enzyme or PEP-carboxykinase)

Web Figure 8.12.A   CAM Circadian rhythm. Metabolite fluxes [atmospheric CO₂, vacuolar malate, sugar mobilization to form PEP] and enzyme activities [PEPCase, Rubisco] are indicated on the four major temporal phases of CAM [I, II, III, IV]. Phase I comprises the nocturnal PEPCase-driven uptake of atmospheric CO₂ and malic acid accumulation. Phase II is the transitional phase wherein an accelerated burst of CO₂ uptake takes place due to both the PEPCase-mediated and the Rubisco-mediated fixation of CO₂. During diurnal Phase III, the uptake of atmospheric CO₂ is diminished and the decarboxylation of the vacuolar malic acid generates the stromal concentration of CO₂ needed for Rubisco-mediated CO₂ fixation via the Benson–Calvin cycle. Phase IV may include CO₂ uptake that is fixed directly by Rubisco.

In diurnal phase III, the closed stomata preclude gas exchange with the external atmosphere, but photosynthesis and other vital processes continue. At this stage, the internal levels of CO₂ fall precipitously due to an active Benson–Calvin cycle; however, this decline is offset by mitochondrial respiration. The closure of the stomata also leads to the accumulation of O₂ in leaf cells because the rate of photosynthetic O₂ evolution is higher than mitochondrial respiration and other O₂-consuming reactions. At this stage, the oxygenase activity of Rubisco activates the photorespiratory cycle by which the Benson–Calvin cycle recovers 75% of the carbon diverted to glycolate, with the remaining 25% released as CO₂ by the mitochondrial glycine decarboxylase complex (see textbook Chapter 8). Thus, the closed stomata provide an adaptive advantage that minimizes water loss from the leaves by transpiration, and keeps the concentration of stromal CO₂ adequate for carbon assimilation. However, the concurrent accumulation of O₂ is a major adverse consequence because, as a powerful oxidant, O₂ reacts with other molecules and generates harmful free radicals (e.g., superoxide, hydroxyl ion) which may initiate uncontrolled adverse reactions on DNA, proteins, and lipids (see textbook Chapter 7).

In this scenario, photorespiration helps alleviate the undesirable consequences of O₂ accumulation by consuming both stromal and peroxisomal O₂. The release of one mole CO₂ in a single round of the photorespiration cycle requires the successive utilization of: (a) two moles O₂ by the oxygenase activity of Rubisco in the chloroplast stroma (see textbook Figure 8.8, reaction 2.1), and (b) one mole O₂ for the concerted action of glycolate oxidase and catalase in the small oxygen cycle in the peroxisome (see textbook Figure 8.8, reactions 2.3 and 2.4). The photorespiratory cycle driven by ribulose-1,5-bisphosphate {2 Ribulose-1,5-bisphosphate + 3 O₂ + H₂O + ATP → 3 3-phosphoglycerate + CO₂ + 2 P_i + ADP} requires substantially more O₂ than mitochondrial respiration operating with carbohydrates {C₆H₁₂O₆ (e.g., glucose) + 6 O₂ → 6 CO₂ + 6 H₂O}. Thus, three versus one moles O₂ are consumed, respectively, per mole CO₂ released. It is therefore evident that the beneficial consequence of CAM photorespiration in Phase III involves not only the avoidance of carbon loss due to the formation of glycolate, but also the amelioration of oxidative stress due to removal of reactive oxygen species. This mitigation of oxidative stress has been proposed to be a major driving force in the evolution of CAM.