Although CAM and C₄ photosynthesis share common enzyme machineries, the physiological bases of spatially-separated and time-separated CCMs are very different and involve complex suites of distinctive regulatory processes ranging from allosteric modulation of enzyme activities, through cell and organelle membrane metabolite transport systems, to long-term responses to stress. The resulting metabolism is rarely at steady state. It is thus helpful to reference the principal biochemical interacting components of CAM to the CO₂ exchange patterns and the pool sizes of acidity and carbohydrates in the archetypical Kalanchoë daigremontiana as outlined in Figure 2.33.

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Figure 2.33 Schematic outline of the phases of CAM, showing net CO₂ exchange and malic acid and carbohydrate (glucan) metabolism in Kalanchoë daigremontiana leaves. (Diagram courtesy B. Osmond)

The four phases of CAM metabolism are:

• Phase I - acidification in the dark (PEPC active and stomata open)
• Phase II - a transitional phase with  stomata open and both carboxylases active
• Phase III - deacidification (PEPC inhibited, Rubisco active and stomata closed)
• Phase IV - C₃ photosynthesis (stomata open, Rubisco active and PEPC inhibited)

Within these four phases, the distinctive underlying biochemistry of CAM involves the up-regulation of cytoplasmic PEPC activity during phase I in the dark. Up-regulation is catalysed by PEPC kinase which phosphorylates PEPC making it less sensitive to inhibition by malic acid as it accumulates in the vacuole. Towards night’s end, CO₂ fixation by PEPC declines as its carbohydrate substrates are exhausted (Figure 2.33). PEPC kinase is degraded during phase II and PEPC becomes increasingly sensitive to malic acid (declining K_i malate; Figure 2.34). It remains inhibited throughout phases III and IV.

CAM also involves the up-regulation of Rubisco in the light by ATP-dependent Rubisco activase as photosynthetic electron transport (ETR) increases in phase II and is maintained throughout phases III and IV (Figure 2.34).

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Figure 2.34 Regulation of enzyme activities in deacidification phases of CAM as photosynthetic ETR increases in the light. (Diagram based on K. Maxwell et al. Plant Physiol 121: 849-856, 1999)

Partitioning of carbohydrate metabolism occurs in the light to retain chloroplast starch or vacuolar sugars as substrates for the next nocturnal acidification phase (phase I) while diverting sugars for phloem transport and growth. In pineapple, for example, degradation of starch in the chloroplast may provide the substrate for PEPC despite the large diel turnover of soluble sugars. The complexity of this “conflict of interest” (Borland and Dodd 2002) in carbohydrate metabolism varies between CAM plants with different deacidification pathways.

Sophisticated interactions occur between metabolite transporters in membrane systems of the vacuole, mitochondria and chloroplasts. Many of these are unique to CAM but of 48 such transporters required to support known variations of CAM (including Clusia spp. that also accumulate citric acid) up until 2005, only 8 had been demonstrated in at least one species (Holtum et al. 2005).

When studied under constant conditions, many of the above distinctive biochemical processes in CAM exhibit circadian rhythms. The extent to which endogenous oscillators orchestrate the clearly interacting biochemical, physiological and environmental controls seems likely to remain a challenging area of research.

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Figure 2.35 Diel expansion growth of Opuntia oricola cladodes during drought in the desert biome of the Biosphere 2 Laboratory in Oracle Arizona USA measured by time-lapse photography. Heterogeneity of growth rate throughout the day is colour coded as red = 2.0% to blue = 0.5% per hour (Diagram by B. Osmond based on Gouws et al. Funct Plant Biol 32: 421-428, 2005)

Compared to the photosynthetic biochemistry and physiology in leaves of C₃ and C₄ plants, the 6% of taxa estimated to exhibit CAM (in at least 35 families and >400 genera) express it with staggering variety (Winter et al. 2015). That is, the distinctive biochemical attributes of CAM outlined above, derived from a handful of research-compliant leafy model species, are but the tip of an iceberg of what really qualifies as a CAM plant (Borland et al. 2011).

The following summary of some distinctive physiological attributes of CAM underscores this conundrum:

Biochemical and physiological determinants of stable isotopic composition of plants with CAM. Fixation of CO₂ by PEPC and Rubisco in vitro show clearly different discriminations against the heavier, naturally occurring, non-radioactive (stable) ¹³C isotope of carbon when expressed as a \(\delta\)¹³C value. Thus total carbon in C₄ plants reflects a small discrimination against ¹³C resulting in \(\delta\)¹³C values of about –12.5 ‰, with more negative values in C₃ plants (about –27 ‰). It is therefore not surprising that CAM plants tend to fall between these values depending on the balance between total carbon assimilated by PEPC in phase I and that added by Rubisco in phase IV. Partial closure of stomata adds a diffusional discrimination to the biochemical discrimination associated with Rubisco, so \(\delta\)¹³C values in C₃ plants (and CAM plants) become less negative under water stress (Griffiths et al. 2007). Recently it has been suggested that unequivocal identification of CAM can be assigned on the basis of net nocturnal CO₂ assimilation, acidification and \(\delta\)¹³C values less negative than -20 ‰. If some dark CO₂ uptake and net acidification is detectable, but \(\delta\)¹³C is more negative than -20 ‰, these plants would be designated as C₃-CAM species, indicating that CAM is present but the contribution of the CAM pathway to net 24h carbon gain is small in comparison to the contribution of daytime CO₂ uptake (Winter et al. 2015).

1. Stomatal opening in the dark is a fundamental physiological feature of CAM. Dark CO₂ fixation lowers internal [CO₂] and promotes stomatal opening. Stomata retain their responsiveness to external CO₂ in phases I and IV, opening further when external CO₂ is reduced. They do not respond to low external CO₂ during phase III, when closure occurs in response to high internal [CO₂] from deacidification of malic acid but do seem to sense the completion of deacidification itself.
2. CAM is essentially a single cell phenomenon and succulence (low surface to volume ratio) is a feature of many but not all CAM plants. Succulence makes at least two important contributions to the physiology of CAM: large vacuoles for malic acid storage in mesophyll cells and tight packing of cells with small intercellular spaces. The latter means that CO₂ diffusion internally is largely confined to wet cell walls and is thus 3 to 4 orders of magnitude slower than in the gas phase, potentially mitigating CO₂ fixation by Rubisco in phase IV.  It remains to be seen whether Clusia rosea may have resolved this trade-off by anatomical/physiological differentiation. Enlarged, tightly-packed PEPC-enriched upper palisade cells have a potential for nocturnal CO₂ fixation and acidification whereas lower spongy mesophyll cells exhibit predominantly C₃ metabolism (Zambrano et al. 2014).  
3. Whereas leaf expansion growth of C₃ plants in the hot dry desert usually occurs at night, leaves and cladodes of some CAM plants grow in the light during phase III (Figure 2.35). This is not surprising because this phase coincides with reliable availability of CHO, maximum temperature and highest cytoplasmic acidity required for growth (Gouws et al. 2005). On the other hand Mesembryanthemum crystallinum (a facultative CAM plant) shows maximum growth in the dark.

Appreciation of the remarkable plasticity in expression of CAM in response to development and environment has greatly advanced the understanding of the ecological attributes of this photosynthetic pathway. One attempt to bring order to the complexity of CAM expression was the designation of constitutive and facultative categories of CAM. Assignation of these terms requires close monitoring of CAM attributes throughout the life-cycle in response to stochastic environmental events such as water availability.

Constitutive CAM seems securely associated with many massive succulents such as the emblematic columnar cacti in the desert South Western USA but current research also shows it to be prevalent in tropical orchids and bromeliads. Young photosynthetic tissues of constitutive CAM plants are often C₃ but CAM is always present at maturity, when the magnitude of the phases of CAM nevertheless remains responsive to stress, light and temperature.

Facultative CAM describes the reversible up-regulation of CAM in response to drought or salinity stress in plants that are otherwise C₃ or display low-level CAM. In these, the up-regulated CAM activity is reversible, being reduced (or lost) on removal of stress (Winter et al. 2008; Winter and Holtum 2014). Facultative CAM has been demonstrated in annual plants of seasonally arid environments (e.g. Australia’s desert Calandrinia; Winter and Holtum 2011) as well as in tropical trees of the genus Clusia. The diel patterns of growth in facultative CAM Clusia minor shift from night when in C₃ mode to phase III when in CAM mode (Walter et al. 2008).

Slow incremental increase in biomass through vegetative reproduction is a feature of CAM-dominated ecosystems. In CAM plants such as Agave and Opuntia, essentially all of the aboveground tissues are photosynthetic, and this partially compensates for lower rates of CO₂ fixation on an area basis. With the noted exception of pineapple and Agave, few CAM species are domesticated, but others have been proposed as potential low-input biofuel crops on land not arable for C₃ and C₄ crops (Borland et al. 2011; Yang et al. 2015). There is no doubt that communities dominated by CAM plants can attain high biomass (Figure 2.36) and nowhere was this more obvious than during the invasion of 25 million hectares of central eastern Australia during 1846-1926 by prickly pear (Opuntia stricta).

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Figure 2.36 High biomass of invasive Opuntia stricta (left) at the Chinchilla, Queensland site C27 before and after release of Cactoblastis cactorum larvae (photos courtesy Queensland Department of Lands 1980) and (right) heritage listed Cactoblastis memorial hall at nearby Boonarga, perhaps the only memorial building to commemorate the achievements of an insect. (Photograph courtesy B. Osmond)

After 2 decades of heroic chemical warfare (hand to hand stabbing or spraying with 10-15% arsenic pentoxide in sulphuric acid at close quarters) failed to restrain the “incubus”, an estimated 1.5 billion tonnes of prickly pear succumbed to trillions of larvae of the diminutive moth Cactoblastis cactorum in about 3 years. Eighty years later, this biological control system remains functionally intact thanks to the remarkably sensitive CO₂ detectors in the mouth parts of the female moth that identifies the CAM plant as a target for oviposition by its distinctive nocturnal, inwardly directed CO₂ flux in Australian ecosystems (Osmond et al. 2008). The hunger of emerging larvae does the rest. Nevertheless, around 27 species of opuntioid cacti remain naturalised across a range of soil types and climatic zones in the mainland states of Australia. It is not known why Cactoblastis cactorum does not attack a broad range of other feral opuntioid cacti.  In South Australia, with an estimated 1,000,000 ha affected (Chinnock 2015), a control management plan has been enacted (Harvey 2009).

Until the 1980s it was thought that the Australian native flora possessed few CAM plants and that prickly pear had occupied an “empty niche”. Field and laboratory studies by Klaus Winter using acid titration and \(\delta\)¹³C values demonstrated CAM in the desert succulent Sarcostemma australe as well as drought and salinity induced facultative CAM in Dysphyma clavellatum and Carpobrotus aequilaterus. He also found CAM in rainforest epiphytes and in a diminutive succulent Calandrinia polyandra from sandy and rocky desert habitats. The latter was recently shown to display one of the most overt transitions from C₃ photosynthesis when well watered to classic CAM when drought stressed (Figure 2.37).  The impression persists that the warm, dry continent of Australia is either CAM-depauperate or ripe for CAM exploration. On the basis of the size of the Australian flora one might predict around 1,300 Australian CAM species, only about 80 have been documented.

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Figure 2.37 Small is beautiful. Diminutive Calandrinia polyandra seems set to become the Arabidopsis analog for CAM research. The diel CO₂ exchange patterns on the right were obtained from the same plant well watered for 33 days (top) and 46 days after water had been withheld (bottom). (Photograph courtesy K. Winter; data from K. Winter and J.A.M. Holtum, Funct Plant Biol 38: 576-582, 2011)

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Figure 2.38 A clump of Isoetes andicola symbolizes the extraordinary functional biodiversity of CAM. (Photograph courtesy J. E. Keeley)

From the above it will be clear that tracking the origins of CAM autotrophy in plants will involve no mean feat (“a laudable triumph of great difficulty”). From a holistic perspective, CAM tests the extremities of most aspects of the physiology and ecology of terrestrial plants, as testified in a comprehensive recent collection of reviews and research papers over-viewed by Sage (2014).  With all the emphasis on water-use efficiency in arid environments as a dominant selective pressure for CAM it is often overlooked (and perhaps ironic) that this pathway today is found in aquatic plants, including the fern-ally Isoetes. The origins of Isoetes, though not the present-day taxa themselves, are Triassic, some 100 x10⁶ years before the commonly imagined emergence of CAM in terrestrial plants (Keeley 2014).

The selective pressure for nocturnal storage of CO₂ in malic acid by CAM in terrestrial plants may well be closure of stomata to conserve water loss in a dry atmosphere in daylight. In aquatic plants the selective pressure may be the slow diffusion of CO₂ in water and its depletion from solution by photosynthesis. In between we have Isoetes andicola from the high Andes of Peru, in which non-functional stoma-like epidermal structures seem literally stitched up (Figure 2.38).  

Clumps of I. andicola are embedded in mounds of peat, with the tips of leaf-like structures forming small rosettes (~5 cm diam.) on the surface. These contain chloroplast-containing cells surrounding large air spaces that evidently maintain gas-phase connections through their large “drinking straw-like” roots to high [CO₂] in the peat (~ 4%). The green tips can’t fix CO₂ from the air, but when ¹⁴CO₂ is supplied to the peat it is fixed within leaves into malic acid in the dark and metabolized to photosynthetic products in the light. Understanding how the habit of I. andicola manages to “CAMpeat” in these high elevation ecosytems remains a challenge.          

Any comment on the ecological and evolutionary attributes of CAM must acknowledge the often remarkable features of sexual reproduction, especially in orchids so highly prized in horticultural and gardening contexts. It is also fair to observe that this popular zoocentric fascination pays little or no heed to the distinctive autotrophic metabolism that supports such ecological exotica. One must concede that nocturnal pollination of saguaro by bats is not very amenable to experiment, so plant ecophysiologists might be excused their preference to focus on the resilience of these organisms in the face of environmental stress. 

However, few would deny that the cameo performances of night-blooming cacti are an astonishingly beautiful reward for the nightshift efforts that have unraveled our current understanding of CAM (Figure 2.39).

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Figure 2.39 A standing ovation for several centuries of CAM research? The spectacular night blooming cactus Epiphyllum oxypetalum. (Photograph courtesy B. Osmond)

The chapter is dedicated to the memory of Thomas Neales (1929-2010) who pioneered Australian research on CAM with Opuntia stricta in the Botany Department, University of Melbourne.

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