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Crassulacean Acid Metabolism Secondary article Article Contents . CAM Pathway Characteristics and Metabolic Carbon Flux Ulrich Lüttge, Darmstadt University of Technology, Darmstadt, Germany . Ecophysiological Advantages of CAM Crassulacean acid metabolism (CAM) is a CO2 acquisition, CO2 transient storage and CO2 concentrating mechanism based on organic acid synthesis. In this variant of photosynthesis CO2 can be fixed nocturnally in the dark and is used during the day for assimilation in the light. . Productivity of CAM Plants . Evolution of the CAM Pathway . Constitutive and Inducible CAM: Developmental and Environmental Regulation . CAM Enzymes . Regulation of CAM: Diurnal and Circadian Rhythmicity CAM Pathway Characteristics and Metabolic Carbon Flux Dark period (phase I) of CAM Crassulacean acid metabolism (CAM) is a CO2 acquisition, CO2 transient storage and CO2 concentrating mechanism based on organic acid synthesis. CO2 acquisition occurs in the dark. CO2 is fixed and stored in the form of organic acids. Remobilization of CO2 from the organic acids occurs in the light and leads to massive CO2 concentration inside photosynthesizing plant organs (green leaves or stems, in some cases also aerial roots of epiphytes). No particular biochemical reactions or enzymes participate in CAM. Although special isoforms of enzymes may be involved, basically all reactions have wellknown housekeeping functions in all green plants. It is the special linkage of metabolic elements that makes up the CAM network (Figure 1; Cushman and Bohnert, 1997; Lüttge, 1998). The key enzyme of CO2 dark fixation is phosphoenolpyruvate carboxylase (PEPC, (1) in Figure 1) in the cytosol. It forms oxaloacetate, which is then reduced to malate. This is the only, or at least the dominating, form of nocturnal CO2 storage in most CAM plants. In some CAM plants, however, citrate may also play a considerable role additionally or alternatively to malate. It can be formed in the mitochondria. Protons are generated together with the organic acid anions. To maintain cytosolic pH homeostasis, these are pumped into the cell sap vacuole by tonoplast H 1 pumps, mainly the vacuolar H 1 ATPase (V-ATPase, (2) in Figure 1). Proton pumping energizes vacuolar organic acid accumulation as it establishes an H 1 electrochemical gradient (Dmd) driving organic acid anions across the tonoplast, the vacuolar membrane. Malate must be removed from the cytosol because it effects a feedback inhibition of its own synthesis via PEPC. The central aqueous cell sap vacuole is a suitable storage compartment occupying often 98% or even more of the volume of cells in succulent CAM tissues (leaf or stem succulence). Nocturnally accumulated organic acids typi- cally amount to 100–300 mmol L 2 1 malate but may be as high as 1.4 mol L 2 1 titratable proton equivalents in species of Clusia, which accumulate both malate and citrate with a stoichiometry of 2 and 3 titratable protons, respectively, per malate and citrate (Lüttge, 1998). Light period (phase III) of CAM During the light period, organic acids are remobilized from the vacuole by passive efflux and are decarboxylated. Malate is decarboxylated by NAD(P)-dependent malic enzymes in the cytosol (NADP-ME) and mitochondria (NAD-ME), respectively ((3) in Figure 1) or via PEPcarboxykinase (PEPCK). Citrate could be partially decarboxylated by cytosolic isocitrate dehydrogenase ((4), in Figure 1) and/or metabolized via the tricarboxylic acid cycle (TCA-C), where it can be broken down totally to CO2. The CO2 regenerated is fixed and reduced in the Calvin cycle (PPCred) to carbohydrate, part of which can be used for energy metabolism, maintenance and growth ([CH2O]n in Figure 1). Another part is stored in the form of starch in the plastids or as free sugars (hexoses but also sucrose) in the vacuole to serve as precursor for the glycolytic synthesis of PEP as CO2-acceptor in the subsequent dark period. Gluconeogenesis with the key enzyme pyruvate Pi dikinase (PPDK, (5) in Figure 1) may also contribute to this carbohydrate pool. Organic acid influx and efflux at the tonoplast are mediated by carboxylate carriers and/or anion channels in the membrane. (Details still await clarification.) Ecophysiological Advantages of CAM CAM-phases Nocturnal CO2 acquisition and storage has been named phase I of CAM, during which stomata are normally open and CO2 is taken up from the atmosphere. Daytime CO2 remobilization and refixation via the Calvin cycle in the light, during which stomata are closed and large internal ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 1 Crassulacean Acid Metabolism P Free hexose Starch Free hexose Free hexose Starch Pi ADPG ATP Pi ADP Hexose-P NADP+ ATP ADP ADP Hexose-bis-P Hexose-bis-P Hexose-bis-P Triose-P/3-PGA Triose-P/3-PGA NAD++Pi C Di-PGA ATP PEP AMP+PPi ATP+Pi 2 2H+ ATP ATP ADP 5 Pyr NADH+H+ ADP Pyr Pi Pi Triose-P C ATP Hexose-P Hexose-P Pentose-P 6 NADH+H+ NAD+ CO2 Pyr PPCox NADPH+H+ Hexose-bis-P Triose-P/3-PGA PPi V Hexose-P ATP P PPCred [CH2O]n 2H+ ADP PEP Pyr Pyr M CO2 AcCoA CoASH NADH +H+ NAD+ OAA NADPH +H+ OAA NADH+H+ NADP+ 3 CO2 NADPH +H+ NADP+ 3 CO2 NAD+ Mal M 1 Citr Mal Citr Mal Citr Mal α KGA NADPH CO2 +H+ 4 NADP+ (Iso-) Citr Mal α KGA TCA-C Citr Figure 1 Crassulacean acid metabolism (CAM) – a CO2 acquisition, CO2 transient storage and CO2 concentrating mechanism. Simplified metabolic scheme of carbon flow, metabolic pathway and compartmentation. Left part: dark period; right part: light period. C, cytosol; M, mitochondrion; P, plastid (chloroplast); V, vacuole. The following abbreviations are used: AcCoA, acetyl-coenzyme A; ADPG, adenosine-diphosphate-glucose; [CH2O]n, carbohydrate; Citr, citrate; CoASH, coenzyme A; Di-PGA, 2,3-diphosphoglyceric acid; aKGA, a-ketoglutaric acid; Mal, malate; OAA, oxaloacetate; Pi, inorganic phosphate; PPi, inorganic pyrophosphate; PPCox, oxidative pentose phosphate cycle; PPCred, reductive pentose phosphate cycle (Calvin cycle); PEP, phosphoenolpyruvate; Pyr, pyruvate; TCA-C, tricarboxylic acid cycle. Numbers refer to key enzymes highlighted in the text: (1) PEP-carboxylase (PEPC); (2) H 1 -transporting V-ATPase at the tonoplast; (3) NAD(P)dependent malic enzymes; (4) Isocitrate dehydrogenase; (5) Pyruvate, Pi dikinase (PPDK); (6) NAD:glyceraldehyde-3-phosphate dehydrogenase. CO2 concentrations of up to a few per cent build up in the intercellular airspaces, has been called phase III of CAM. These two phases are separated by a phase II when stomata open widely in the morning and a peak of uptake of atmospheric CO2 is explained by concomitant operation of PEPC and ribulose-bisphosphate carboxylase/oxygenase (Rubisco), the CO2-fixing enzyme of the Calvin cycle, and by a phase IV, when stomata open in the afternoon after organic acid remobilization is completed and again CO2 is taken up from the atmosphere (Figure 2; Osmond, 1978). 2 Ecophysiological roles of diurnal malate rhythms Examination of the CAM phases offers a clue to the evaluation of the ecophysiological significance of CAM (Lüttge, 1998). Stomatal rhythms are evidently associated with this, but they are not the sine qua non of CAM, because CAM may also occur in stomata-less leaves of water plants and in green aerial roots of orchids. In leaves of submerged freshwater plants, nocturnal malate synthesis of CAM facilitates acquisition of CO2 when it is more readily available by nocturnal respiratory activities in the aqueous ecosystem than during the day when other photosynthesizing organisms are competing. ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 100 3 2 1 Malate (µmol gFW–1) — JCO2 (µmol m–2 s–1) — 4 40 80 30 60 20 40 10 20 gH2O (mmol m–2 s–1) — Crassulacean Acid Metabolism 0 0 0 0 4 8 12 16 20 Time of day (h) Figure 2 Diurnal course of net CO2 exchange (JCO2), leaf-conductance for water vapour (gH2O) and malate levels (per gram fresh weight, gFW) in a leaf of the CAM-plant Kalanchoe¨ daigremontiana, with nocturnal (dark bar on top of the graph) stomatal opening, CO2 uptake and malate accumulation (phase I); an early morning peak of CO2 uptake (phase II); daytime malate remobilization and stomatal closure (phase III); and afternoon stomatal opening, CO2 uptake and assimilation in the Calvin cycle (phase IV). However, in land plants with an inverted stomatal rhythm of daytime closure and night-time opening, malate formation via PEPC serves as a water-saving mechanism of CO2 acquisition, since evaporative demand is low when stomata open for CO2 uptake in the night instead of during the day, and hence, water use efficiency (WUE 5 CO2 fixed:H2O transpired) is high. This can be further amplified by variation of the expression of CAM phases. If stress due to reduced availability of water (‘water stress’) is severe, first phases II and IV are abolished. With increased stress, stomatal opening may be delayed after the onset of the night. Under very severe water stress, stomata may remain closed night and day. The CAM pathway then remains cycling while the Calvin cycle is driven by light energy during the day, fixing CO2 that is obtained from malate formed from respiratory CO2 during the night via PEPC. This recycling of CO2 (Griffiths, 1989) does not lead to carbon gain but allows maintenance of metabolism while loss of water is minimized, until water stress is relieved and atmospheric CO2 can be taken up again. When stomata remain partially open in the night-time, both atmospheric and respiratory CO2 may contribute to total malate cycling. Conversely, when there is ample availability of water some CAM plants may open stomata day and night using C3-photosynthesis in the light and PEPC in the dark. High internal daytime CO2 concentrations (phase III) contribute to control of photoinhibition at times of highest irradiation. However, high rates of photosynthesis behind closed stomata also imply high O2 production, and it has been shown that CAM plants may be subject to oxidative stress and photoinhibition in phase III (Lüttge, 1999). As a water-saving variant of photosynthesis, CAM is frequent among succulent plants of deserts and tropical savannas and dry forests (e.g. stem succulent Cactaceae, Euphorbiaceae and others, leaf succulent Agavaceae, Liliaceae, Bromeliaceae, Crassulaceae and others) as well as among epiphytes in exposed canopy habitats of wet tropical forests (mainly Bromeliaceae, Orchidaceae, Piperaceae, Cactaceae, but also others). Ecophysiological roles of diurnal citrate rhythms In contrast to malate, nocturnal citrate accumulation does not provide a net gain of carbon, because for each hexose unit used as precursor (C6) only one citrate (C6) is formed as one CO2 is fixed but also one CO2 is lost in the pathway; with malate accumulation, two malates (2 C4) are formed for each hexose unit used, because two CO2 are fixed (Figure 1). However, citrate can contribute significantly to night/day carbon cycling, which becomes important under severe water stress. Citrate is also known to be an effective buffer substance. Its vacuolar accumulation therefore allows considerable total acid accumulation (titratable acidity) while control of vacuolar pH is maintained. Indeed, the highest nocturnal accumulation of titratable protons ( 1.4 mol L 2 1 with vacuolar pH still close to 3) was observed in the genus Clusia, where citrate accumulation is very important (Lüttge, 1998). Productivity of CAM Plants Metabolic schemes such as that of Figure 1 can be used to work out stoichiometric energy budgets. While this may be a gratifying exercise on paper, the fact that in ecophysiological reality various meshes of the metabolic network may be used simultaneously and switches may also occur make this a more bewildering task. What becomes clear at a glance, though, is that CAM is energetically more costly than straightforward C3-photosynthesis (Calvin cycle). In fact, productivity of CAM plants in natural ecosystems is normally much lower than that of C3 plants. CAM appears as an adaptation for ecological survival but not for maximum productivity. Conversely, in agroecosystems with the appropriate maintenance, CAM plants can achieve a productivity close to or even better than that of C3 or also C4 plants. This is always based on a considerable contribution of phase IV to total carbon gain with direct CO2 fixation via Rubisco (Nobel, 1996). Major CAM crops are pineapple (Ananas, Bromeliaceae), Opuntia (Cactaceae) and Agave (Agavaceae). ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 3 Crassulacean Acid Metabolism Evolution of the CAM Pathway Since enzymologically there is nothing particular in CAM (Figure 1), it is no surprise that CAM evolved polyphyletically. CAM plants emerged in very many taxa dispersed over all three classes of the subdivision Angiospermae in the division of Spermatophyta, namely the Magnoliopsida (primitive dicots), the Rosopsida (‘eudicots’) and Liliopsida (monocots). Polyphyletic evolution of CAM has also been traced within a given family, i.e. the Bromeliaceae (Smith, 1989). The occurrence of CAM plants scattered over very different phylogenetically advanced taxa of the angiosperms has supported the contention that emergence of CAM is a relatively recent event in plant evolution. It ought to be noticed, however, that CAM plants also occur in the division of the Pteridophyta, i.e. in the classes of the Lycopodiopsida (order Isoëtales: Isoe¨tes and Stylites) and the Pteridopsida (order Polypodiales: Pyrrosia and Drymoglossum), which emerged much earlier in evolution ( 3 108 years ago) than the Angiospermae ( 1.5 108 years ago). Constitutive and Inducible CAM: Developmental and Environmental Regulation While looking for the traces of CAM plants in the evolutionary tree of the Spermatophyta gives us some ideas about phylogeny, we can also observe development of CAM expression in ontogeny. In the genus of so called obligate or constitutive CAM plants Kalanchoe¨ (Crassulaceae) the youngest leaves have a more C3-like metabolism and CAM is only fully expressed in mature leaves. In Kalanchoe¨ blossfeldiana cv. Tom Thumb, CAM expression is dependent on developmental regulation elicited by shortday photoperiod conditions that also elicit flowering and this, hence, is considered a C3/CAM intermediate species. Another extensively studied C3/CAM intermediate species is the annual facultative halophyte Mesembryanthemum crystallinum (Aizoaceae) (Cushman and Bohnert, 1997). With increasing age this plant exhibits some degree of CAM. However, this is considerably amplified by stress, i.e. salinity, osmotic or water stress, and is reversible depending on plant age. Hence, in this case both developmental and environmental regulation determine the degree of CAM expression, where phytohormones, such as abscisic acid and cytokinins, and secondary messengers, such as Ca2 1 , may be involved in signal transduction. Other C3/CAM intermediate taxa are the bromeliad Guzmania monostachia and in the genera Sedum, Peperomia and Clusia (Lüttge, 1998). In the latter, a genus of neotropical woody plants, C3 –CAM switches are very rapid, occurring within days or even several hours, 4 and are governed by environmental rather than developmental signals, such as availability of water, light intensity and day–night temperature regimes. In perennial tropical plants using their leaves for more than one season, it appears sensible that environmental control should dominate developmental regulation. It is also noted that such phenotypic plasticity allows Clusia to cover a wide ecological amplitude rather than being a straightforward adaptation to a given type of stress (Lüttge, 1999). CAM Enzymes More readily than analysis of evolutionary relations of enzymes and their genes, C3/CAM intermediates draw our attention to CAM enzymes. It has been noted above that there are no really typical CAM enzymes in the CAM pathway (Figure 1). Nevertheless, PEPC ((1) in Figure 1) certainly has a key role. Its phylogenetic analysis does not give any evidence that it falls into a class of its own in CAM plants. However, study of steady-state mRNA levels shows that during the C3 –CAM transition in M. crystallinum there is transcriptional activation of a CAM-specific isogene of PEPC. Similarly PPDK ((5) in Figure 1), the glycolytic enzyme NAD-glyceraldehyde-3-phosphate dehydrogenase ((6) in Figure 1), and an NADP-dependent malic enzyme ((3) in Figure 1) are products of transcriptionally activated specific CAM genes (Cushman and Bohnert, 1997). The H 1 -pumping V-ATPase, a multisubunit enzyme of the tonoplast with a membrane integral proteolipid (V0-domain) and a head and stalk structure (V1-domain) is also affected transcriptionally (subunit mRNA levels) and posttranslationally (protein processing), leading to changes in activity and ATP/H 1 coupling during C3 –CAM transitions (Lüttge and Ratajczak, 1997). Regulation of CAM: Diurnal and Circadian Rhythmicity The key enzyme of carbon acquisition in CAM, PEPC, must be regulated during the normal night/day (diurnal) rhythm of CAM. In its active state it has an 6-fold higher affinity for CO2 than does Rubisco, and this would imply futile cycling of CO2 via PEPC in the light (phase III). Activity is regulated posttranslationally by phosphorylation (PEPC-kinase) and dephosphorylation (phosphorylase) (Carter et al., 1991; Cushman and Bohnert, 1997). The active phosphorylated night-form of PEPC has high CO2 affinity and low malate sensitivity (malate feedback inhibition). Dephosphorylation downregulates CO2 affinity and increases malate sensitivity during the light period, and thus, minimizes futile CO2 refixation. ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Crassulacean Acid Metabolism CAM also shows free running (circadian) rhythmicity under constant external conditions in continuous light. PEPC activity follows these endogenous oscillations due to phosphorylation/dephosphorylation oscillations associated with endogenous oscillations of PEPC-kinase (Carter et al., 1991). The endogenous rhythmicity of CAM has been studied theoretically and by establishing ‘skeleton models’ for computer simulations, where the degrees of freedom were reduced as much as possible. It turns out that the PEPC-phosphorylation rhythm makes the model more stable against perturbations but can be eliminated without destroying rhythmicity of simulations. It is also confirmed experimentally that CAM can oscillate without PEPC-kinase oscillations. Conversely, simulations break down if a tonoplast-located beat oscillator (hysteresis switch) is removed from the model. The decisive regulation of CAM must therefore lie in malate influx/ efflux at the tonoplast (Grams et al., 1997). References Carter PJ, Nimmo HG, Fewson CA and Wilkins MB (1991) Circadian rhythms in the activity of a plant protein kinase. EMBO Journal 10: 2063–2068. Cushman JC and Bohnert HJ (1997) Molecular genetics of crassulacean acid metabolism. Plant Physiology 113: 667–676. Grams TEE, Borland AM, Roberts A, Griffiths H, Beck F and Lüttge U (1997) On the mechanism of reinitiation of endogenous CAM-rhythm by temperature changes. Plant Physiology 113: 1309–1317. Griffiths H (1989) Carbon dioxide concentrating mechanisms and the evolution of CAM in vascular epiphytes. In: Lüttge U (ed.) Vascular Plants as Epiphytes. Evolution and Ecophysiology, pp. 42–86. Berlin: Springer-Verlag. Lüttge U (1998) Crassulacean acid metabolism. In: Raghavendra AS (ed.) Photosynthesis, pp. 136–149. Cambridge: Cambridge University Press. Lüttge U (1999) One morphotype three physiotypes: sympatric species of Clusia with obligate C3-photosynthesis, obligate CAM and C3-CAM intermediate behaviour. Plant Biology 1: 138–148. Lüttge U and Ratajczak R (1997) The physiology, biochemistry and molecular biology of the plant vacuolar ATPase. In: Leigh R and Sanders D (eds) Advances in Botany: The Plant Vacuole, pp. 253–296. London: Academic Press. Nobel PS (1996) High productivity of certain agronomic CAM species. In: Winter K and Smith JAC (eds.) Crassulacean Acid Metabolism. Biochemistry, Ecophysiology and Evolution, pp. 255–265. Berlin: Springer-Verlag. Osmond CB (1978) Crassulacean acid metabolism: a curiosity in context. Annual Review of Plant Physiology 29: 379–414. Smith JAC (1989) Epiphytic bromeliads. In: Lüttge U (ed.) Vascular Plants as Epiphytes. Evolution and Ecophysiology, pp. 109–138. Berlin: Springer-Verlag. Further Reading Adams P, Nelsen DE, Yamada S et al. (1998) Growth and development of Mesembryanthemum crystallinum (Aizoaceae). New Phytologist 138: 171–190. Cockburn W (1985) Variation in photosynthetic acid metabolism in vascular plants: CAM and related phenomena. New Phytologist 101: 3–24. Kluge M and Ting IP (1978) Crassulacean Acid Metabolism. Analysis of an Ecological Adaptation. Berlin: Springer-Verlag. Lüttge U (1987) Carbon dioxide and water demand: crassulacean acid metabolism (CAM), a versatile ecological adaptation exemplifying the need for integration in ecophysiological work. New Phytologist 106: 593–629. Lüttge U (1988) Day night changes of citric acid levels in CAM: phenomenon and ecophysiological significance. Plant, Cell and Environment 11: 445–451. Lüttge U (ed.) (1989) Vascular Plants as Epiphytes. Evolution and Ecophysiology. Berlin: Springer-Verlag. Lüttge U (1993) The role of crassulacean acid metabolism (CAM) in the adaptation of plants to salinity. New Phytologist 125: 59–71. Ting IP (1985) Crassulacean acid metabolism. Annual Review of Plant Physiology 36: 595–622. Wilkins MB (1992) Circadian rhythms: their origin and control. New Phytologist 121: 347–375. Winter K and Smith JAC (eds) (1996) Crassulacean Acid Metabolism. Biochemistry, Ecophysiology and Evolution. Berlin: Springer-Verlag. ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 5