Tansley insight
Crassulacean acid metabolism: a continuous or
discrete trait?
Author for correspondence:
Klaus Winter
Tel: +507 212 8131
Email: winterk@si.edu
Received: 21 January 2015
Accepted: 22 March 2015
Klaus Winter1, Joseph A. M. Holtum2 and J. Andrew C. Smith3
1SmithsonianTropical Research Institute, POBox 0843-03092, Balboa, Anc
on,Republic of Panama; 2Centre for Tropical Biodiversity
and Climate Change, James CookUniversity, Townsville, QLD 4811, Australia; 3Department of Plant Sciences, University of Oxford,
South Parks Road, Oxford, OX1 3RB, UK
Contents
Summary 1
I. Introduction 1
II. Phenotypic diversity 2
III. Ecological context 2
IV. Structure–function context 3
V. Biochemical–genomics context 4
VI. Transitional states? 4
VII. Conclusions – what is a CAM plant? 5
Acknowledgements 5
References 5
New Phytologist (2015)
doi: 10.1111/nph.13446
Key words: C3 photosynthesis, CAM
photosynthesis, carbon-isotope ratio d13C,
crassulacean acid metabolism (CAM),
evolution, phosphoenolpyruvate carboxylase.
Summary
The key components of crassulacean acid metabolism (CAM) – nocturnal fixation of
atmospheric CO2 and its processing via Rubisco in the subsequent light period – are now
reasonably well understood in terms of the biochemical reactions defining this water-saving
mode of carbon assimilation. Phenotypically, however, the degree to which plants engage
in the CAM cycle relative to regular C3 photosynthesis is highly variable. Depending upon
species, ontogeny and environment, the contribution of nocturnal CO2 fixation to 24-h
carbon gain can range continuously from close to 0% to 100%. Nevertheless, not all
possible combinations of light and dark CO2 fixation appear equally common. Large-scale
surveys of carbon-isotope ratios typically show a strongly bimodal frequency distribution,
with relatively few intermediate values. Recent research has revealed that many species
capable of low-level CAM activity are nested within the peak of C3-type isotope signatures.
While questions remain concerning the adaptive significance of dark CO2 fixation in such
species, plants with low-level CAM should prove valuable models for investigating the
discrete changes in genetic architecture and gene expression that have enabled the
evolutionary transition from C3 to CAM.
I. Introduction
Crassulacean acid metabolism (CAM) is a water-conserving mode
of photosynthesis and one of three photosynthetic pathways in
vascular plants. CAM and C4 are modifications of the basic C3
pathway and represent CO2-concentrating mechanisms that
elevate [CO2] around Rubisco and suppress photorespiration. In
CAM, this is achieved in two principal phases separated in time. At
night, atmospheric CO2 is incorporated by phosphoenolpyruvate
carboxylase (PEPC) via oxaloacetate into malic acid, which
accumulates in the large vacuoles of chloroplast-containing
mesophyll cells. During the following light period, malic acid is
released from the vacuoles and decarboxylated, and the CO2 thus
liberated is refixed by Rubisco and reduced in the Calvin cycle
(Osmond, 1978; Winter & Smith, 1996). Decarboxylation of
malate generates high intercellular [CO2] and is associated with
stomatal closure, minimizing water loss in the middle of the day
when evaporative demand is highest.
In common garden experiments to compare growth rates under
identical conditions, plant biomass production per unit water
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utilized was 6 or 2 times higher for CAM than for C3 or C4,
respectively (Winter et al., 2005). MaximumCO2 uptake rates per
unit surface area are generally lower in CAM plants than in C3 and
C4 plants, but this is partially offset by the fact that almost the entire
shoot surface is photosynthetic in typical CAM plants, as in agaves
and cacti. Furthermore, in terms of the overall energetics of carbon
assimilation, CAM is estimated to represent a fairly small marginal
cost of c. 10% relative to the C3 pathway (Winter & Smith, 1996),
and indeed CAM plants typically grow in open or exposed habitats
in which incident light energy is not the primary factor limiting
growth. Thus, annual productivities of CAM plants can be
considerable (Nobel, 1988), and agaves, platyopuntias and other
CAM species have been proposed as potential biofuel crops on land
not suitable for conventionalC3 andC4 crops (Borland et al., 2009;
Davis et al., 2011; Holtum et al., 2011). The effect of leaf or
photosynthetic–stem area ratio on the relative growth rate of CAM
species has never been rigorously quantified. A comparison of
agaves and platyopuntias with arborescent CAM species that
allocate substantial biomass to a woody, nonphotosynthetic stem
(e.g. in the genus Clusia ; L€uttge, 2006) would be informative.
Amongst the estimated 350 000 species of vascular plants, c. 6%
are believed capable of CAM photosynthesis, belonging to at least
35 families and over 400 genera, and outnumbering C4 species
approximately two-fold (Winter & Smith, 1996; Yang et al.,
2015). Most of the longer lived stem-succulent CAM species of
large stature inhabit warm, seasonally dry habitats such as semi-
deserts with little, but relatively predictable, seasonal rainfall
(Ellenberg, 1981). The extant diversity of these and other major
terrestrial CAM lineages seems to have arisen largely during the
global expansion of arid environments in the lateMiocene (Arakaki
et al., 2011; Horn et al., 2014). Among epiphytic lineages, CAM is
considered to have enabled diversification of the more extreme
epiphytic life-forms occupying arid microhabitats in forest cano-
pies, notably in the tropical Bromeliaceae and Orchidaceae (Crayn
et al., 2004, 2015; Silvera et al., 2009).
CAM is not onlymultifaceted in terms of diversity of species and
habitats. There is also continuous variation in the extent to which
species engage in CAM relative to C3 photosynthesis. The fact that
the engagement of CAM is not an all-or-nothing phenomenon
raises fundamental questions about both the ecological significance
and evolutionary origins of CAM.
II. Phenotypic diversity
CAM comes in many variants, differing, for example, in the
enzymes of malate decarboxylation, and the type and compart-
mentation of storage carbohydrates that fluctuate reciprocally with
malic acid (Holtum et al., 2005). Above all, CAMprovides some of
the best examples of phenotypic diversity and plasticity in the plant
kingdom, for example in the form of facultative CAM species such
as the annuals Mesembryanthemum crystallinum (Aizoaceae) and
Calandrinia polyandra (Montiaceae), or some perennial species of
Clusia (Clusiaceae), in all of which CAM is drought-inducible
(Winter et al., 2008; Winter & Holtum, 2014). Over a relatively
short period, these plants have the ability to transition progressively
from full C3 to full CAM and vice versa.
Even in obligate CAM species such as desert cacti, there is an
ontogenetic progression fromC3 toCAMas tissuesmature (Winter
et al., 2008, 2011). Indeed, most species regarded as constitutive
CAMplants also incorporate, to varying degrees, atmospheric CO2
directly in the light via Rubisco. Depending on species, environ-
mental conditions and developmental status, the contribution of
nocturnal CO2 gain to total daily carbon gain may range from
100% to close to 0% (Winter & Holtum, 2002, 2014). In its
weakest form, CAM is merely evident as a small nocturnal increase
in tissue titratable acidity. Nocturnal H+ increases of
c. 1 mmol kg
1 fresh mass 12 h
1 currently represent the limit of
detection for low-level CAM. They may correspond to average
CO2 fixation rates of < 0.05 lmol m

2 s
1, which are challenging
to resolve with conventional gas-exchange systems against respira-
tory background CO2 effluxes of typically c. 1 lmol m

2 s
1.
CO2 fixation via PEPC at night is by no means exclusive to
CAM. All C3 plants presumably have the capacity to fix CO2 in
the dark, as can be detected in 14CO2 tracer studies (Kunitake
et al., 1959). While the nocturnal fixation of CO2 and the linked
stoichiometric accumulation of malic acid (Medina & Osmond,
1981) may be unique to CAM, PEP carboxylation and accumu-
lation and decarboxylation cycles of the malate anion are not; these
are integral to the maintenance of charge balance during processes
such as NO3

 reduction and changes in stomatal aperture (Smith
et al., 1996; Martinoia et al., 2012). Moreover, the essential
enzyme and transport reactions needed for CAM are all seemingly
present in C3 plants. Given the fundamental nature of these
metabolic processes, the questions arise: where does C3 end and
CAM begin; are the differences between C3 and CAM of a
qualitative or merely quantitative nature; is CAM a continuous or
a discrete trait?
III. Ecological context
On the basis of the wide spectrum of CAM phenotypes recogniz-
able in CO2 exchange studies, it is tempting to conclude that CAM
is a continuous trait. However, it is notable that in large-scale
surveys of d13C values (a measure of the ratio of 13C : 12C), which
serve as integrated long-term proxies of the ratio of CO2 fixed
during the day to that fixed during the night by plants in their
natural habitat (Cernusak et al., 2013), d13C values are not equally
distributed over the whole range of possible values (Fig. 1). Rather,
their distribution is typically bimodal, with a minimum in the
frequency distribution at c.
20&. Based on the linear relationship
observed between the d13C value and the proportion of CO2 fixed
at night, this value would correspond to a c. 40% contribution of
dark CO2 fixation to total carbon gain (Winter &Holtum, 2002).
As yet, the ecological significance of this minimum is not fully
understood, but it might somehow reflect a fitness cost of
intermediate phenotypes, or a paucity of habitats in which such
an intermediate phenotype is favoured. Ongoing research,
especially in the Orchidaceae, is revealing the occurrence of
low-level CAM in many species with C3-type d
13C values,
possibly indicating a second frequency peak of species capable of
CAM nested within the C3 cluster of isotope values (Silvera et al.,
2005).
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IV. Structure–function context
CAM is in essence a single-cell phenomenon. This contrasts
with the greater structural complexity of the dual-cell anatomy
typical of C4 plants, which necessitates close metabolic coor-
dination between mesophyll and bundle-sheath cells and has
been linked to as many as 25 forms of Kranz anatomy (Aubry
et al., 2014). But by virtue of being confined to individual
chlorenchyma cells, operation of the CAM cycle must be
underpinned by strict temporal control of the carboxylation and
decarboxylation reactions if carbon assimilation is to be
optimized and futile cycling kept to a minimum (Smith &
Bryce, 1992; Borland et al., 2011).
Further structural hallmarks of CAM are manifested at the
higher morphological levels of tissues and organs, most character-
istically in the succulent leaves and stems that endow the shoot with
a low surface area : volume ratio and high water-storage capaci-
tance. The tightly packed, thin-walled, highly vacuolated cells that
make up the chlorenchyma tissuemaximize the storage capacity for
malic acid per unit surface area of shoot across which uptake of
atmospheric CO2 occurs (Smith et al., 1996). In fact, CAM plants
in general are characterized by relatively low stomatal densities on
their shoot surfaces and low maximal stomatal conductances
(Pfeffer, 1897; Kluge & Ting, 1978; Gibson, 1982; Zambrano
et al., 2014), which although helping to restrict water loss in
transpiration also act as a partial constraint on maximum daily
carbon gain.
Increased succulence of photosynthetic tissues may be one of the
key preconditioning traits for CAM (Ogburn & Edwards, 2010;
Edwards & Ogburn, 2012). This is convincingly seen in clades
possessing a full spectrum of intermediate phenotypes such as the
Orchidaceae, inwhich the trend fromC3 throughC3–CAMspecies
to strongly expressed CAM is associated with progressively
increasing succulence (Fig. 2). A similar relationship is also seen
in other families in which a wide range of leaf morphologies have
been studied, such as the Polypodiaceae (Winter et al., 1983),
Bromeliaceae (Baresch et al., 2011), Clusiaceae (Zambrano et al.,
2014) and Crassulaceae (Teeri et al., 1981; Kluge et al., 1993).
Another correlate of succulence and tight cell packing in the
chlorenchyma is the reduction in intercellular air spaces and
internal conductance to CO2, which it has been argued favours
PEPC-mediated nighttime (phase I) fixation relative to daytime
(principally phase IV) fixation directly via Rubisco, as the latter is
strongly diffusion-limited (Maxwell et al., 1999; Nelson & Sage,
2008). The transition between the C3 and CAM pathways is thus
associated with a complex suite of biochemical and structural
tradeoffs that may determine the optimal niches for these plants in
their natural environments (cf. Fig. 1).
CAM plants are not unique in having their stomata open at
night, as many C3 plants maintain stomata partially open in the
dark (Darwin, 1898; Caird et al., 2007). In CAMplants, nocturnal
stomatal opening is largely mediated by the decline in CO2
concentration in the intercellular air spaces caused by activation of
PEPC (Griffiths et al., 2007; von Caemmerer & Griffiths, 2009).
Thus, CAM plants do not fix CO2 at night because their stomata
open at night; instead, stomatal opening is driven by nocturnal
CO2 fixation. The pattern of diel CO2 exchange of Kalancho€e
daigremontiana leaves with the lower epidermis removed still
resembles that of fully intact leaves (Kluge & Fischer, 1967).
Furthermore, net CO2 exchange of roots of leafless CAM orchids
exhibits all four phases ofCAMgas exchange as definedbyOsmond
(1978), even though the roots lack stomata (Winter et al., 1985). It
remains to be seen whether or not stomata of CAM plants have
acquired CAM-specific features that help optimize the physiology
of CAM.
Similarly, there has been considerable discussion about the role
played by endogenous circadian rhythms in CAM plants and the
requirement for a CAM-specific oscillator for a functional CAM
cycle. When studied under constant conditions, many biological
processes including CO2 fixation show circadian behaviour related
to the action of endogenous oscillators, which are formed from a
series of interlocked transcription/translation feedback loops
(Dodd et al., 2014). While certain key features of the CAM cycle
have long been known to show endogenous circadian rhythmicity
under constant conditions (Wilkins, 1959; Hartwell, 2006), the
extent to which the operation of the CAM cycle and the growth of
δ13C (‰)
–32 –28 –24 –20
N
um
be
r o
f s
pe
ci
es
200
160
120
100
60
20
0
180
140
80
40
–36 –16 –12 –8
C3– CAM
CAMC3
Fig. 1 Frequency histogram of d13C values of species of Bromeliaceae
plotted in class intervals of 1& (re-plotted from data in Crayn et al., 2015;
copyright © 2015, John Wiley & Sons), showing the strongly bimodal
distribution of isotope ratios with a frequency minimum at c. 
20&, as is
typically observed in large-scale surveys of crassulacean acid metabolism
(CAM) species. The two clusters of d13C values more negative and less
negative than 
20& are principally composed of C3 species and CAM
species, respectively. However, nested within the C3 cluster are species that
exhibit some degree of CAM activity, based on measurements of nocturnal
increases in tissue titratable acidity and nocturnal net CO2 uptake. Thesewe
define as C3–CAM species, although their exact number is not yet known
accurately because, to date, nocturnal acidification has been tested in
relatively few species of Bromeliaceae with d13C values more negative than

20& (Pierceet al., 2002).While this representationof the frequencyofC3–
CAMspecies is schematic, it illustrates that theymay be part of a progressive
trendof increasingcontributionofdarkCO2fixation to total carbongainwith
increasing d13C value. At d13C values above 
20&, this trend merges with
the cluster of CAM plants that show nocturnal fixation as their dominant
mode of carbon assimilation. Analysis of the original data for goodness of fit
(G-test) shows that the C3 cluster of d
13C values does not differ significantly
from a normal frequency distribution, whereas the CAM cluster does
(P < 0.05), reflecting the slightly higher than expected abundance of species
with values in the range
20 to 
17&.
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 2015 New Phytologist Trust
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Phytologist Tansley insight Review 3
CAMplants under natural day : night cycles is dependent upon, or
optimized by, aCAM-specific oscillatorwill be a key issue for future
research.
V. Biochemical–genomics context
The enzymes and transporters involved in the CAM cycle appear to
be homologues of proteins ubiquitous in C3 species. For instance,
pyruvate, orthophosphate dikinase, first discovered in the plant
kingdom in C4 and CAM plants (Hatch & Slack, 1968; Kluge &
Osmond, 1971), was briefly considered a C4- and CAM-specific
novelty, but the enzyme was soon shown to be present in C3 plants
as well (Aoyagi & Bassham, 1984).
There is some evidence that the gain of CAM is associated
with gene duplication and neofunctionalization, allowing the
novel isoforms to fulfil CAM-specific functions. Discrete
changes may include alterations in expression patterns involving
changes in cis-regulatory elements and transcription factors,
and/or changes in kinetic properties of key enzymes resulting
from adaptive amino acid substitutions. For example, analysis of
PEPC genes in the Caryophyllales, an order containing multiple
C4 and CAM lineages, indicates that an early genome-wide
duplication event before the emergence of land plants was
followed by another whole-genome duplication that gave rise to
two PEPC gene lineages now found in most eudicots; one of
these (ppc-1E1) was then repeatedly duplicated, leading to
several gene lineages containing CAM-specific isoforms of
PEPC in Aizoaceae, Cactaceae, Portulacaceae and Didiereaceae
(Christin et al., 2014). Gene duplication events have also been
implicated in the evolution of CAM-specific PEPC isoforms in
the monocot family Orchidaceae (Silvera et al., 2014). CAM-
specific posttranslational regulation optimizes CAM functioning
through diel control of the kinetic properties of CAM PEPC,
stimulating dark CO2 fixation and minimizing the futile cycling
of CO2 in the light (Winter, 1982; Nimmo, 2000). These day–
night changes in PEPC kinetics are brought about through
reversible phosphorylation of the enzyme by a specific protein
kinase (Hartwell et al., 1996). In principle, these and other
changes to CAM could have been initiated by random de novo
mutation, or by exploiting the standing genetic variation already
present in populations (West-Eberhard et al., 2011).
VI. Transitional states?
Thus far, it is not known whether weakly expressed CAM and
facultative CAM represent transitional states along an ordered
stepwise evolutionary trajectory fromC3 to strong CAM (Hancock
& Edwards, 2014). Low-level and facultative CAM species are
frequently found in the same lineages as species with fully expressed
CAM, suggesting common ecological, anatomical and genomic
predispositions. However, compared with the extensive carbon-
isotope surveys of herbarium material, relatively few species have
been tested physiologically for their mode of photosynthesis, so our
knowledge of the true extent of low-level CAM is probably very
incomplete. The most extensive and systematic information of this
sort to date comes from theOrchidaceae, inwhich livingmaterial of
173 species has been studied for day–night changes in titratable
acidity (Fig. 2; Silvera et al., 2005).
Another challenging question is whether an adaptive benefit
or fitness advantage of low-level CAM activity can be convinc-
ingly demonstrated. Even if CAM suffices only to minimize the
loss of respiratory CO2 at night, as is the case for plants
displaying ‘CAM cycling’ (Harris & Martin, 1991; Herrera,
2009), mortality could be reduced during drought stress.
However, persuasive evidence for the adaptive significance of
low-level CAM is still missing. Some species of Oncidium
(Orchidaceae) with C3-type d
13C values, yet showing small and
significant rates of net dark CO2 fixation under well-watered
and droughted conditions, are highly tolerant to water deficit
stress (Katia Silvera, personal communication). An adaptive
advantage is more clearly evident for the facultative CAM
expressed in species such as M. crystallinum (Winter et al., 1978)
and C. polyandra (Winter & Holtum, 2011). Facultative CAM
in these annuals combines C3-driven growth after germination
in the wet season with prolonged, CAM-based carbon gain at
low water cost during the subsequent dry season, thereby aiding
reproduction. Drought-stressed plants of M. crystallinum
exposed to CO2-free air at night have drastically reduced seed
set compared with drought-stressed plants that can take full
advantage of CAM (Winter & Ziegler, 1992). Facultative CAM
may be an optimal strategy for these annuals in their
characteristic habitats, and it is difficult to envision that these
plants would be merely transitional forms on their way to a
perennial life-style with full CAM.
Leaf thickness (mm)
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pe
ci
es
50
40
30
0
20
10
0.40 0.8 1.2 1.6 2.0 >2.0
C3
C3– CAM
CAM
Fig. 2 Frequency histogram of leaf thickness, plotted in class intervals of
0.2mm,of largelyPanamanian speciesofOrchidaceae forwhichd13Cvalues
and nocturnal changes in leaf titratable acidity were determined (based on
data in Silvera et al., 2005). Following the nomenclature introduced in Fig. 1,
species with d13C values less negative than 
20& were considered
crassulacean acidmetabolism (CAM) species, while species with d13C values
more negative than 
20& were considered C3 species or C3–CAM species
on thebasisof theabsenceorpresence, respectively, ofnocturnal increases in
tissue acidity.
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VII. Conclusions – what is a CAM plant?
Attempts to reconstruct the evolutionary origins of CAM photo-
synthesis necessarily involve decisions about the most appropriate
character or trait to map onto the phylogenetic trees. A restrictive
approach would be to code the presence or absence of strongly
expressed CAM as a binary character state, for example when
surveys of the study group reveal a clear bimodal distribution of
d13C values (cf. Fig. 1). A more inclusive approach would be to
code for the occurrence in a taxon of any degree of CAM activity,
however small, as detected by measurements of nocturnal CO2
fixation or associated diel acid fluctuations. In itsmostminimalistic
form, a complete CAM cycle could theoretically operate with just a
single molecule of atmospheric CO2 being fixed by PEPC at night,
leading to the storage of a single molecule of malic acid, and
generating 1 CO2 during the following day for refixation via
Rubisco. This highlights the need for systematic collection of living
material to obtain a much more complete picture of lineages
possessing the capacity for nocturnal CO2 fixation via the CAM
cycle. Furthermore, if low-level CAM is only facultatively
expressed, its detection may depend on investigating the species
under the precise conditions (e.g. of water deficit stress) that induce
this activity.
Based on the distinct bimodal distribution of d13C values in taxa
where CAM is present (Fig. 1), we propose that the terminology in
this field can be rationalized by reserving the simple, unqualified
designation ‘CAM species’ or ‘CAM plant’ for taxa that are part of
the strong CAM cluster in the frequency histogram of d13C values.
These plants will have isotopic signatures less negative than c.

20& and will correspond to species such as agaves and
platyopuntias in which CAM makes a substantial, and typically
the major, contribution to carbon acquisition. We propose
classifying as ‘C3–CAM species’ all taxa in the C3 cluster (d
13C
values more negative than 
20&) for which some capacity to
engage in CAM has been demonstrated, as determined by CO2
exchange and/or nocturnal H+ increase. In these species the CAM
cycle is demonstrably present, but C3 photosynthesis clearly
remains the principal mechanism of carbon gain (e.g. the
gymnosperm Welwitschia mirabilis; von Willert et al., 2005).
Future research will show whether C3–CAM species are transi-
tional intermediates (phylogenetically and/or in the metabolic
complexity of the CAM cycle) along the evolutionary trajectory
from C3 to full CAM.
Acknowledgements
This work was supported by the Smithsonian Tropical Research
Institute.
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