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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
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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.
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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).
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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.
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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
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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
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