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Understanding trait diversity associated with
crassulacean acid metabolism (CAM)
Nicholas A Niechayev1, Paula N Pereira1 and John C Cushman
Crassulacean acid metabolism (CAM) is a specialized mode of
photosynthesis that exploits a temporal CO2 pump with
nocturnal CO2 uptake and concentration to reduce
photorespiration, improve water-use efficiency (WUE), and
optimize the adaptability of plants to climates with seasonal or
intermittent water limitations. CAM plants display a plastic
continuum in the extent to which species engage in net
nocturnal CO2 uptake that ranges from 0 to 100%. CAM plants
also display diverse enzyme and organic acid and
carbohydrate storage systems, which likely reflect the multiple,
independent evolutionary origins of CAM. CAM is often
accompanied by a diverse set of anatomical traits, such as
tissue succulence and water-storage and water-capture
strategies to attenuate drought. Other co-adaptive traits, such
as thick cuticles, epicuticular wax, low stomatal density, high
stomatal responsiveness, and shallow rectifier-like roots limit
water loss under conditions of water deficit. Recommendations
for future research efforts to better explore and understand the
diversity of traits associated with CAM and CAM Biodesign
efforts are presented.
Address
Department of Biochemistry and Molecular Biology, University of
Nevada, Reno, Reno, NV 89557-0330, United States
Corresponding author: Cushman, John C (jcushman@unr.edu)
1
Joint first-coauthors.
Current Opinion in Plant Biology 2019, 49:74–85
This review comes from a themed issue on Physiology and
metabolism
Edited by Elizabeth A Ainsworth and Elizabete Carmo Silva
https://doi.org/10.1016/j.pbi.2019.06.004
1369-5266/ã 2019 Elsevier Ltd. All rights reserved.
Introduction
Crassulacean acid metabolism (CAM) is a temporally
controlled, inorganic carbon-concentrating, photosyn-
thetic adaptation that improves water-use efficiency
(WUE; a measure of the unit of CO2 fixed per unit of
water lost) by rescheduling part or all CO2 uptake from
the day, as is typical of C3 and C4 photosynthesis plants, to
the night when air:leaf water vapor pressure deficits are
lower compared with the day [1]. The primary, nocturnal
Current Opinion in Plant Biology 2019, 49:74–85
carbon fixation phase by phosphoenolpyruvate (PEP)
carboxylase (PEPC) using HCO3 as a substrate is tem-
porally separated from the secondary, daytime phase of
CO2 (re)fixation by ribulose-1, 5-bisphosphate carboxyl-
ase/oxygenase (RUBISCO). Nocturnal carbon uptake by
PEPC is made possible by inverse stomatal behavior, in
which stomata are open at night, but closed during all or
part of the day, thereby reducing water loss from the plant
and increasing WUE. The two phases are linked by
C4 acid carbon storage intermediates, typically malic acid,
which are stored in the vacuole and then decarboxylated
during the day to supply CO2, which is (re)fixed by
RUBISCO, leading to carbohydrate production via the
C3 photosynthesis Calvin–Benson cycle. This intracellu-
lar release of CO2 in the vicinity of RUBISCO elevates
CO2 concentrations within the leaf up to 60-fold com-
pared with atmospheric CO2 levels [2]. This ‘CO2 pump’
favors RUBISCO’s carboxylase rather than oxygenase
activity, which reduces photorespiration, a process that
reduces the efficiency of photosynthesis up to 40% in C3
photosynthesis plants [3]. The net result of the CAM
cycle is up to a threefold to sixfold improvement in WUE
compared with C4 and C3 photosynthesis species, respec-
tively, and a potential enhancement in net CO2 uptake
over a 24-hour cycle in habitats with limited water or
nutrient resources [4–6].
Global climate change threatens to increase the fre-
quency, duration, and geographical distribution of
droughts over the global landmass during the 21st century
[7]. Interest in expanding the use of CAM species as food,
feed, and fuel feedstocks has increased in recent years
due mainly to their high WUE, low demand for water, and
ability to be grown in abandoned lands with marginal soils
poorly suited for traditional crops [4,8,9,10–13]. The
WUE advantages of CAM over C3 and C4 photosynthesis
species has extremely practical consequences. Global-
scale geographic information system (GIS)-based
productivity modeling has revealed that climate-resilient,
high biomass producing CAM species, such as
Opuntia ficus-indica, have the capacity to meet ‘extreme’
biofuel demand scenarios using low-grade arid lands
while avoiding significant conflicts with food production
or contributing to deforestation [14,15]. Alternatively,
the possibility of engineering CAM into C3 and C4
photosynthetic species as a means of improving
plant WUE has received increasing attention
[10,16,17,18,19]. Here we explore the diversity of
CAM itself and key traits often associated with CAM
to improve our understanding of this key photosynthetic
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adaptation to water-limited environments and to high-
light how some of these adaptive traits might be har-
nessed to improve the climate resiliency of agricultural
crops in a warming and drying global climate.
CAM species diversity
As a trait, CAM has evolved in multiple lineages and more
often than C4 photosynthesis across the plant kingdom
[20]. CAM occurs in more than 6% of all higher plant
species across more than 38 families that include over
400 genera [21,22]. In some families, such as the Cacta-
ceae and Didiereaceae, CAM is most likely present in all
genera [23,24]. In the Orchidaceae, more than 10% of
species exhibit CAM [25,26], whereas in the Bromelia-
ceae 57% of species perform CAM [27,28]. However,
these estimates, which are based on d13C values, are
unlikely to reveal weak CAM activity, which can be
readily detected by titratable acidity measurements
[29]. CAM has also been documented in basal plant
lineages such as lycophytes, which include submerged
aquatic macrophytes of the Isoetes genus [30,31], gneto-
phytes such as Welwitschia mirabilis (Welwitschiacea) [32],
cycads, such as Dioon edule (Zamacieae) [33], and several
families of epiphytic and lithophytic ferns [34–36]. The
need to maximize the exploitation of limited inorganic
carbon in oligotrophic lakes or seasonal pools while plants
are submerged likely led to the appearance of CAM in
Isoetes [37]. For cycad lineages, global aridification during
the Miocene has been suggested to have been a driver for
the diversification of weak CAM among several traits
likely to assist in exploiting drier environmental niches
[38]. CAM has been associated with the large-scale adap-
tive radiation in lineages such as the Aizoaceae, Cacta-
ceae, and the Orchidaceae, suggesting that CAM, among
other adaptations, likely contributed to success in colo-
nizing more arid niches [25,39–41]. In addition to
increased global aridity during the late Miocene, a decline
in atmospheric CO2 levels during this same time frame
would likely have improved the competitiveness of (pre-
sumably CAM-performing) drought-adapted succulents
within the Aizoaceae, Cactaceae, and Euphorbiacea, and
thus, contributed to their ability to expand into more arid
habitats [42,43]. Recent analysis of anatomical changes
within the Agavoideae (Asparagaceae) suggests that suc-
culent leaf anatomy predates the appearance of CAM
[44]. Large-scale transcriptome sequencing revealed that
CAM-related genes were among many genes associated
with the evolution of several plant lineages within the
Portulacineae (Caryophyllales), such as the Cactaceae,
Didiereaceae, and Montiaceae, associated with adapta-
tion to harsh or extreme environments [45].
In addition to seasonally warm or dry environments
within semi-tropical to tropical zones, CAM species can
occupy cooler climates defined by higher altitudes or
latitudes, but are less predominant [25]. Nonetheless,
CAM species can occupy environments that regularly
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CAM trait diversity Niechayev, Pereira and Cushman 75
experience seasonally freezing temperatures [46,47].
For example, some CAM Opuntia species can survive
temperatures down to 50 C when cold acclimated [48].
The mechanistic basis of this extreme freezing tolerance
likely involves partial water loss and accumulation of
cryoprotectant sugars and sugar alcohols (e.g. mannitol)
to prevent intracellular ice formation and dehydration and
extracellular nucleation of ice by extracellular
polysaccharides [47]. However, the basis of such low-
temperature adaptation deserves further investigation.
CAM continues to be discovered as new genera are
investigated with both isotopic and titratable acidity
measurements. For example, CAM has now been docu-
mented in all four subfamilies of the Aizoaceae, including
the Sesuvioideae [49]. CAM is also widespread within
the ‘portullugo’ (Portulacineae plus Molluginaceae) in
the Caryophyllales together with all four clades of the
Aizoaceae, including recently described new species
within the Bassellaceae [50]. Closely related lineages
within the Caryophyllales, Aizoaceae, and Portulacaceae
contain both CAM and C4 photosynthesis species [51,52].
Notably, at least six species within the genus Portulaca
express both CAM and C4 photosynthesis in their leaves
[53,54]. Weak CAM with a pronounced facultative com-
ponent is prevalent within terrestrial species endemic to
Australia [55]. However, more extensive surveys should
be performed across a greater number of plant families
using both d13C and titratable acidity measurements to
reveal the true prevalence of weak CAM. A focus on
known hotbeds of CAM species radiations will be useful
to provide a more detailed mechanistic understanding of
how CAM evolved within discrete plant lineages and to
determine if the same mechanistic steps occur in support
of parallel evolution [20,56].
CAM plasticity
CAM can be described as a continuous trait due to the great
variability with which plants engage in net nocturnal CO2
uptake and fixation [22]. The extent to which plants engage
in nocturnal CO2 fixation to total 24-hour carbon gain may
range from close to 0% to 100% depending upon the evolu-
tionary history, developmental status, and proximate envi-
ronmental conditions that influence the relative water status
of the plant [22,57] (Figure 1). Along this continuum or
gradient, plants can display CAM cycling, in which detect-
able organic acid fluctuations are observed without detect-
able net nocturnal CO2 fixation [58]. CAM cycling is thought
to play an important role in reducing CO2 loss during
nocturnal respiration, maintaining a positive carbon balance
to promote plant growth and improve reproductive fitness,
and protecting against photooxidative stress damage [59].
Weak CAM (or C3-CAM) species are defined as those that
display clearly detectable nocturnal organic acid accumula-
tion, but have d13C values of 26m to 20m [60]. In contrast,
strong CAM species display pronounced nocturnal organic
acid accumulation with d13C values ranging from 20m
Current Opinion in Plant Biology 2019, 49:74–85
76 Physiology and metabolism

Figure 1

Current Opinion in Plant Biology

A schematic distribution of C3 photosynthesis to crassulacean acid metabolism (CAM) along a continuous gradient derived from large-scale
surveys of d13C values (m). Relative placement of CAM variations including CAM cycling, the transitional ‘Winter–Holtum’ zone (WHZ), indicated in
yellow, containing weak-CAM (or C3-CAM) revealed by surveys of nocturnal titratable acidity assays, strong-CAM, and CAM-idling species [22].
Inducible or facultative CAM is observed for species located within the WHZ subjected to water-deficit stress (or other signals such as ionic,
osmotic, dehydration, low-humidity, and high light intensity treatments that increasing water-vapor pressure deficit in roots or leaves), abscisic
acid (ABA) treatments, and alterations in nutrient status (left triangle). Removal of water-deficit stress under well-watered conditions results in
partially or fully reversible CAM in many facultative CAM species (right triangle).

to 8m. Based upon large d13C surveys of several plant
families with mixed C3 photosynthesis and CAM species
distributions, the C3 photosynthesis and CAM species dis-
play discrete bimodal distributions with a minimum fre-
quency distribution centered around 20m, reflecting an
approximately 40% contribution of net nocturnal CO2 fixa-
tion to total daily carbon gain [60]. The transitional zone
referred to as the Winter–Holtum zone [61], which contains
weak-CAM species that display d13C values spanning  23
to 19m, separates the larger distributions of C3 photosyn-
thesis species ( 33 to 22m) and mainly strong CAM
species with less negative d13C values [22]. Lastly, CAM
idling, in which low-level, but sustained diel fluctuations in
organic acids occur with essentially all of the CO2 fixed into
malate being derived from internally recycled respiratory
CO2 typically occurs under extreme water-deficit stress
conditions with stomata closed day and night [62].
Constitutive or obligate CAM species, such as tropical
platyopuntia species Opuntia elatior, can exhibit a
developmental progression from C3 to CAM as tissues
mature [63]. However, species with d13C values span-
ning the transitional Winter–Holtum zone can exhibit
facultative CAM when subjected to water-deficit stress
[63,64] (Figure 1). Mesembryanthemum crystallinum (Aizoa-
ceae), a halophytic species in which facultative CAM was
first described, has been studied extensively and is
capable of switching from C3 to CAM under salinity
or drought stresses [57,65,66]. An increasing number of
facultative CAM species have been documented to
display inducible and reversible CAM depending upon
prevailing conditions of water availability including
Calandrinia spp. [53,67], Clusia spp. (Clusiaceae), the
epiphytic fern, Vittaria lineata [68], Talinum triangulare
[69,70], Yucca gloriosa, and related species within the
Asparagaceae [71,72] among others. Recently, faculta-
tive CAM has been described in a C3-C4-photosynthesis
intermediate species within the Portulaceae (e.g.
Portulaca cryptopetala, P. molokiniensis) suggesting that
CAM can co-exist in C3, C4, and C3-C4 photosynthesis
species and is likely ancestral to C4 photosynthesis [73].
Clearly, additional surveys of putative facultative CAM
species are needed along with detailed investigations
into possible changes in gene family structure and
expression patterns to better elucidate the mechanistic
basis for CAM evolution (and devolution) within mixed
C3-CAM, C4-CAM, and C3-C4-CAM lineages.
Metabolic plasticity
Coincident with the extent of CAM performance along
the C3-CAM continuum is the apparent diversity of the
enzymatic machinery and carbohydrate storage forms
necessary to perform CAM itself. During nocturnal car-
boxylation, malate is the predominant organic acid stored

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as malic acid within the vacuole; however, citrate also
accumulates in many species [74–76]. Other organic acids
such as isocitrate, fumarate, and succinate also accumu-
late in CAM species [77–79]. Their accumulation might
help reduce mitochondrial respiration or photorespiration
[77,78], promote carbon retention via the tricarboxylic
acid cycle [79], or facilitate water uptake [75]. However,
the precise role for these compounds, aside from malate,
in net nocturnal carbon gain deserves further study.
CAM species can be divided into two general groups at
the family level depending upon the predominant
enzymes employed during the daytime decarboxylation
of malate leading to intracellular CO2 release (phase
III). NAD(P)-malic enzyme (ME) type species pre-
dominate within the Agavaceae, Aizoaceae, Cactaceae,
Crassulaceae, Nolindoideae, and Orchidaceae, which
decarboxylate malate to release CO2 and pyruvate with
pyruvate being regenerated to PEP by pyruvate ortho-
phosphate dikinase (PPDK) [80–82]. In contrast, PEP
carboxykinase (PEPCK) type species predominate
within the Apocynaceae, Asphodelaceae, Bromeliaceae,
Clusiaceae, Euphorbiaceae, and Liliaceae, which
employ NAD(P)-malate dehydrogenase (MDH) to con-
vert malate to oxaloacetate, which is then decarboxy-
lated by PEPCK to release CO2 and regenerate PEP,
predominate [80–82]. Furthermore, the subcellular
localization of key CAM enzymes, such as PPDK,
can differ depending on the CAM species investigated
[83,84]. More detailed surveys of the taxonomic distri-
bution and subcellular localization of the decarboxyl-
ation enzymes are necessary to better understand the
evolutionary histories of CAM species within these
diverse plant lineages.
CAM species also show considerable diversity in the types of
storage carbohydrates used to supply PEP for nocturnal CO2
fixation as HCO3 by PEPC. Eudicot CAM families, where
starch is the major daytime carbohydrate storage form,
include the Apocynaceae, Aizoaceae, Cactaceae, Crassula-
ceae, Cuburbitaceae, and Orchidaceae [82,85]. However,
some eudicot families, such as the Clusiaceae, contain species
that accumulate soluble sugars (Fru/Glu). Monocot CAM
families, such as the Agavaceae, contain species that accu-
mulate fructans; whereas the Bromeliaceae, contain species
that accumulate either starch or soluble sugars (Fru/Glu/Suc)
or both [82,85]. Other monocot CAM families, such as the
Asphodelaceae contain species that accumulate either starch/
Glu or glucomannan; whereas the Nolindoideae, contain
species that accumulate Suc [82]. These diverse patterns
of carbohydrate storage are thought to reflect the evolutionary
histories of each lineage. Nonetheless, sampling of carbohy-
drate storage capacities within CAM taxa remains limited and
more surveys should be conducted to elucidate the metabolic
and energetic constraints that these different storage forms
might impose on CAM performance. The availability of
complete genome and transcriptome sequencing of several
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CAM trait diversity Niechayev, Pereira and Cushman 77
CAM species will help to decipher the specific modes of
carbohydrate biosynthesis, transport, and degradation oper-
ating in these species [86–90]. Additional information regard-
ing the metabolic diversity of CAM will also inform more
versatile strategies for CAM Biodesign.
Anatomical diversity of CAM species
The biochemical process of CAM requires only single,
chloroplast-containing cells to function, along with precise
temporal (and presumably circadian) control of both the
carboxylation and decarboxylation reactions to avoid or
minimize futile cycles of carbon fixation [5]. Evidence that
both the carboxylation and decarboxylation reactions are
controlled, in part, by the circadian clock comes from
RNAi-mediated knock-down experiments of key CAM
enzymes (e.g. phosphoenolpyruvate carboxylase kinase
(PPCK), mitochondrial NAD-ME, cytosolic/plastidic
PPDK), which not only impairs CAM performance, but
also disrupts or dampens the rhythmic expression of core
circadian clock genes [91,92]. Despite this apparent ana-
tomical simplicity, CAM plants occupy typically warm and
seasonally dry terrestrial and epiphytic habitats and display
an enormous diversity of life forms that aid in plant survival
and reproduction that includes photosynthetic leaves,
stems, roots, and even flowers that perform CAM [93,94].
Tissue succulence
Tissue succulence or the ability to store water within
living tissues to allow plants to be temporarily indepen-
dent of external water supply is found in 3–5% of flower-
ing plants and among C3 and C4 photosynthesis and CAM
species [95,96]. However, some degree of succulence, as
measured by greater leaf (or stem) thickness, chloren-
chyma thickness, and cell size, and reduced intracellular
air spaces (IAS) or aerenchyma area is typically associated
with CAM species (Figure 2) [61,96], but is less well
correlated with weak or C3-CAM species [22,29,61,97].
Tissue succulence provides the ability of plants to avoid
the harmful effects of extended periods of water deficit
typical of epiphytic, epilithic, and semi-arid environ-
ments and provides a valuable resource for horticultural,
carbon sequestration, and bioenergy [98]. Obligate CAM
columnar stem succulents are able to survive extended
periods under extremely hot and dry conditions [99].
Increased succulence of photosynthetic tissues might
have served as a progenitor trait for CAM
[44,61,100,101]. Larger cell size and associated increases
in vacuolar malic acid storage capacity of CAM species
might also aid in osmotically driven water uptake and
remobilization within the chlorenchyma [102,103]. Leaf
and stem succulence provides a mechanism to avoid
drought stress through the gradual metering of water
via transpiration during periods of prolonged water defi-
cit. However, the three-dimensional venation patterns of
highly succulent species also serves to maintain moderate
hydraulic path lengths, which would likely facilitate rapid
Current Opinion in Plant Biology 2019, 49:74–85
78 Physiology and metabolism

Figure 2

Current Opinion in Plant Biology

Diverse co-adaptive traits found in crassulacean acid metabolism (CAM) plants. Adaptive traits to attenuate water deficits include tissue
succulence and water-storage or water-capture strategies. Adaptive traits to limit water loss include thick cuticles, epicuticular wax, low stomatal
density, and high stomatal responsiveness to low humidity conditions. Shallow rectifier-like roots limit water loss to dry soils and maximize water
uptake upon rewatering. Image copyrights: Succulent plant (Crassula portulacea) ã scisettialfio, Bromeliad tank ã Jojoo64, Orchid pseudobulb ã
Notpossible, Tillandsia plant ã Mark Brandon, Thick cuticle ã Rattiya Thongdumhyu, Prickly pear cuticle ã Pavlovska Yevheniia, Stomata ã imv,
Guard cells ã Kazakova Maryia, plant roots ã enterphoto.

uptake of water following periods of rehydration
[104,105].
CAM species display a great diversity of tissue succulence
anatomy ranging from ‘all-cell succulents’ defined by
water storage within an expanded chlorenchyma, to
‘storage succulents’ with defined zones of chlorenchyma
and centralized water-storing hydrenchyma, as well as
various configurations of these two anatomies [96,106].
For CAM species, the low surface area to volume (SA:V)
ratio created by the large cells facilitates nocturnal malic
acid storage within the vacuole [107]. However, large

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cell volumes also reduce IAS within the leaf and meso-
phyll cell surface area exposed to IASs thereby reducing
mesophyll conductance of CO2 (gm) and improving its
recapture and thereby optimizing CAM performance
[108,109]. While advantageous for CAM, the reduced
gm due to reduced IAS can also restrict access of CO2
limiting the performance of C3 photosynthesis [109],
which might explain the observed bimodal distribution
of C3 photosynthesis and CAM species noted earlier with
a frequency distribution centered around 20m [22].
While mesophyll conductance of CO2 (gm) constrains
maximal rates of photosynthesis in ‘all-cell succulents’,
leaf anatomy configurations with less densely packed and
well-aerated spongy mesophyll cells have been suggested
to optimize direct C3-mediated CO2 fixation, whereas
enlarged and densely packed palisade mesophyll cells
might accommodate the potential for C4 carboxylation
and nocturnal storage capacity of organic acids [107].
Many succulents must also tolerate high tissue tempera-
tures during the day, but this is offset by the high thermal
capacity of succulent tissues to slow radiative heat loss
during the night to buffer the plant against cold nighttime
conditions and protect sensitive tissues from chilling or
freezing damage [96,106]. Expanding investigations into
the diversity of succulent leaf anatomies will help inform
strategies for generating optimal succulent leaf anatomy
for bioengineered CAM [16,93], biomass production
[110], or improved tolerance to salinity [111], drought,
and temperature extremes.
Water-storage organs
In addition to succulence, many epiphytic CAM species
within the Orchidaceae and the Bromeliaceae have
evolved-specific anatomical structures for water storage
and conservation. Epiphytic orchids possess highly suc-
culent, water-storing pseudobulbs (enlarged succulent
internodal stem segments) (Figure 2), comprised of large
tightly packed chlorenchyma cells and aerenchyma, and
aerial roots, which are capable of performing weak-CAM
under drought conditions by recycling respiratory CO2
[112,113]. Pseudobulbs lack stomata and have a lignified
exodermis to curtail water loss. Roots are enveloped by a
velamen, a spongy epidermis that limits water loss under
drought conditions. The velamen acts as a sponge and is
capable of accumulating water and minerals in its internal
spaces. The velamen also likely protects roots from
UV radiation damage due to the expression of
UV-B-absorbing flavonoids [114].
Within the Bromeliaceae, overlapping basal portions of
the leaves form tank structures that function to accumu-
late water and nutrients, which are absorbed by epidermal
trichomes present at a higher density towards the base
compared with the apical portion (Figure 2) [115]. How-
ever, the trichomes can also play a hydrophobic role in
repelling water serving to provide for water retention
within the tank. In the CAM facultative epiphytic tank
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CAM trait diversity Niechayev, Pereira and Cushman 79
bromeliad species, Guzmania monostachia, the base of the
leaf also contains more hydrenchymal cells to help buffer
the water status in the apical regions of the leaf. In
contrast, the apical portions of leaves display a greater
stomatal density and performed more nocturnal CO2
fixation as well as organic acid accumulation associated
with CAM under drought stress [116].
Water-capture strategies
In order to aid survival in epiphytic habitats with transient
water availability, many ‘atmospheric’ or ‘fog-catching’
nebulophytic CAM epiphytes, particularly within the
Tillandsia genus, display dense epidermal trichomes to
maximize water capture and absorption, which are often
accompanied by a reduction or complete loss of roots
(Figure 2) [115]. A dense trichome layer coated in water
can reduce CO2 exchange, suggesting that CAM can
result in more CO2 uptake beyond the photoperiod
and recycle CO2 from respiration, while also acting as a
water-conserving mechanism during dry periods [117].
Leaf trichomes also play a clear role in reflecting and
reducing light absorption, particularly under conditions of
damagingly high photosynthetic photon flux densities,
and protecting against the damaging effects of UV-B
radiation from the accumulation of flavonoid compounds
within trichomes and the leaf cuticle [118].
Thick cuticles
The plant cuticle is a structure present between aerial
organs and the atmosphere that plays important roles in
the prevention of water loss, defense against insect pests
and microbial pathogens, xenobiotics, and damage from
UV radiation [119]. CAM species, such as O. ficus-indica,
possess cuticles that are two orders of magnitude thicker
than those found in Arabidopsis thaliana [120]. To illus-
trate the critical role of the cuticle in preventing water
loss, four Dendrobium species with different cuticle thick-
nesses were evaluated for differing levels of water loss
[113]. The thicker upper leaf cuticle in D. chrysotoxum
(C3 species) and D. officinale (facultative CAM species)
helped to limit water loss, whereas two other species,
D. chrysanthum and D. crystallinum (C3 species) with thin-
ner leaf cuticles, displayed greater rates of water loss. The
CAM species had the thickest cuticle among the four
species, which would limit water loss under conditions of
low humidity (Figure 2). However, species with thinner
cuticles tended to have pseudobulbs, presumably to
compensate for more rapid water loss [113]. In addition
to preventing water loss, the presence of flavonoids within
the cuticular matrix or the epicuticular wax layer serves to
attenuate and reflect UV radiation, which is critical to
CAM plants living in semi-arid or arid environment with
high insolation rates [121,122]. The polymers that make
up plant cuticles also display a higher specific heat than
other plant polymers, meaning that a greater amount of
heat is required to raise the temperature of the plant
surface [122]. The ability to thermoregulate these
Current Opinion in Plant Biology 2019, 49:74–85
80 Physiology and metabolism
surfaces would be an important adaptive trait for CAM
plants occupying hot and dry environments. Given our
lack of fundamental knowledge about plant cuticles,
more studies are needed to understand the relevance
of cuticle thickness and composition in CAM species.
Epicuticular wax
In addition to a thickened cuticle, many CAM plants,
particularly those in semi-arid and arid environments
accumulate intracuticular and epicuticular wax, which
provide the main barrier against transpirational water loss
(Figure 2). Recent evidence suggests that the intracuti-
cular wax and not the epicuticular wax layer forms the
transpirational barrier against water loss from the leaf
[123]. Highly hydrophobic epicuticular surfaces are also
typically non-wetting and self-cleaning, which afford
protection against pathogens and insect predation
[121]. Analysis of the wettability of the Opuntia surface
demonstrated that water moves quickly off of juvenile
pads down to mature base pads [124]. The superhydro-
phobicity of younger and more distal pads would facilitate
rainfall movement to the basal pads, whereas wetting of
older pads would assist in movement of precipitation to
the roots. Considering the limited precipitation available
to many CAM species, rapid movement of water to
surface roots would serve to maximum water storage
and accumulation. Overall, the role of epicuticular waxes
is understudied in CAM species and deserves greater
attention to better understand how this key anatomical
trait facilitates survival under warm and dry conditions.
Stomata guard cells
Most CAM species display stomata on leaves and stems
depending on their life form. Species within the genus
Pereskias are particularly interesting in that they perform
C3 photosynthesis in their leaves and weak CAM on the
surface of stems [94]. These species are thought to be an
example of an evolutionary transition, as the majority of
closely related Opuntioideae have small areolar leaves on
young pads that perform C3 photosynthesis before senes-
cing, leaving behind only the stems to perform CAM [94].
However, some aquatic CAM species [30] and leafless
epiphytic orchids with chlorophyllous roots [125] are
‘astomatous’, yet display 24-hour gas exchange typical
of conventional CAM species.
Xerophytic and epiphytic CAM species typically display
lower rates of stomatal conductance than mesophytic
species due in part to lower stomatal densities (Figure 2)
[107,113,126,127]. Lower stomatal densities are corre-
lated with larger stomatal pore size. Fewer stomata are
thought to be a consequence of resource limitations for
plants growing under water-limited conditions combined
with the added energetic costs of performing CAM
[93,128]. Current surveys of stomatal density and form
among CAM species are limited and expansion of such
surveys across known CAM families would be highly
Current Opinion in Plant Biology 2019, 49:74–85
informative to understand if the co-evolution of CAM
is tightly correlated to observed decreases in stomatal
densities and size of stomatal pores. CAM plants display
large variations in stomatal complex morphology reflec-
tive of the evolutionary histories of CAM lineages.
Whereas some C3 monocot families display ‘anomocytic’
(absence of subsidiary cells) stomata, none of the monocot
families that perform CAM display such stomatal com-
plexes [127]. Furthermore, tetracytic stomata (four sub-
sidiary cells) are twice as common in CAM lineages
compared with C3 photosynthesis families. Within eudi-
cot families, anomocytic stomata are less common in
CAM lineages, whereas paracytic stomata (two subsidiary
cells) are more common [127]. In addition to variations
stomatal complex morphologies, several CAM plant fam-
ilies, such as the Agavaceae, display deeply sunken sto-
mata at the base of hypostomatal cavities to reduce water
vapor loss [13]. Despite recent developments in under-
standing the function of subsidiary cells and stomatal
movement in C3 photosynthesis plants [129], additional
studies are needed that detail the roles of the subsidiary
cells and the influence of stomatal size in influencing
kinetics of stomatal responsiveness in CAM plants.
Stomatal responsiveness
Nocturnal stomata opening in CAM plants is regulated
via reductions in leaf intracellular [CO2] (Ci) driven by
PEPC activation and formation of malate (Figure 2)
[127,130,131]. Overexpression of individual carboxyla-
tion module enzymes in A. thaliana provided evidence
suggesting that lower Ci appears to drive stomatal open-
ing along with nocturnal malate accumulation [18].
Conversely, overexpression of individual malate decar-
boxylating enzymes presumably resulting in increased
Ci appeared to drive stomatal closing along with a
reduction in malate accumulation. In contrast to
C3 photosynthesis plants, the guard cells of CAM plants
also appear to not respond to white-light-activated or
blue-light-activated opening [132,133]. mRNA expres-
sion analysis in Agave americana suggest that key
blue-light photoreceptors (e.g. CRY1, PHOT1) might
not be involved in stomatal opening in this obligate
CAM species [134]. However, other genes involved in
CO2 and abscisic acid (ABA) perception and stomatal
closing and opening exhibited rescheduled patterns of
expression relative to A. thaliana suggesting roles in
reprogramming stomatal behavior in CAM plants.
Successful CAM Biodesign will required C3 photosyn-
thesis hosts whose stomatal guard cells are fully respon-
sive to low Ci for nocturnal opening at night and high
Ci for complete daytime stomatal closure [93]. However,
defining the distinctive traits of CAM plant guard cell
signaling and responsiveness to environmental signals
clearly deserved greater attention. Although surveys have
been limited, stomatal guard cells are thought to be more
sensitive to leaf-air vapor pressure differences and
www.sciencedirect.com

possibly ABA than C3 photosynthesis species [80]. Such
exquisite responsiveness is consistent with the idea that
succulent plants prefer to avoid water loss and attenuate
water-deficit stress [127]. Stomata conductance also
changes in response to temperature in CAM plants.
Temperatures around 15–25 C are associated with opti-
mal operation of CAM, which coincides with the
nocturnal temperature when stomata open [135].
Shallow rectifier-like roots
Many CAM species utilize shallow root systems to opti-
mize the use of limited, but seasonal or intermittent
rainfall. Rectifier-like root hydraulic conductivity
responses allow these species to capitalize on fluctuating
soil water potentials by preventing water loss during
drought, but facilitating water uptake after precipitation
events [136]. For example, old and young nodal roots and
lateral or ‘rain’ roots of Agave deserti rapidly decline in
hydraulic conductivity upon drought episodes to limit
water loss to dry soil [137]. However, young nodal roots of
A. deserti recover 50% of their hydraulic conductivity after
just two days following rewatering [137]. This rapid
recovery allows this species to completely restore leaf
water storage to pre-drought levels after just seven days of
rewatering the entire root system [138]. Shallow-rooted
members of the Aizoaceae (and Crassulaceae) along the
western coast of South Africa and Namibia have adapted
to capture shallow surface water to minimize competition
with non-Aizoaceae species when rainfall is limiting
[139]. Shallow rooting systems allow the plants to maxi-
mize water uptake and storage during the wet season,
coupled with tissue succulence to extend their survival
during the dry season, which might help to explain the
spectacular diversification of these species [139].
Conclusions and future directions
CAM-associated traits represent remarkable examples of
phenotypic convergence across a wide range of phyloge-
netic and functional diversity. While cellular or tissue
succulence provides one key unifying feature, the addi-
tional traits highlighted above reveal how these morpho-
logical and biochemical specializations have been
co-opted for adaptive advantage. Combined with CAM,
these traits provide integrated solutions to a wide range of
harsh climatic conditions and offer an expanded scope for
future research coupling ecophysiology with molecular
and synthetic biology approaches. While future efforts to
introduce facultative CAM into C3 and C4 photosynthesis
crops will likely enhance WUE, leveraging these
CAM-associated traits is expected to provide comple-
mentary adaptive advantages to plants under hotter
and drier environments. Therefore, greater investment
in research efforts to better understand these traits, such
as water storage, cuticular and epicuticular interfaces,
stomatal behavior, and root hydraulic properties will be
essential to improve our ability to engineer more climate-
resilient crops in the future.
www.sciencedirect.com
CAM trait diversity Niechayev, Pereira and Cushman 81
Conflict of interest statement
Nothing declared.
Acknowledgements
This work was supported, in part, by the U.S. Department of Energy, Office
of Science, Genomic Science Program under award number DE-
SC0008834, the U.S. National Institute of Food and Agriculture (NIFA)/U.
S. Department of Agriculture (USDA) under award number 2018-68005-
27924, and the Nevada Agricultural Experiment Station (NEV-00377 and
NEV-00380). The authors would like to thank Lisa Petrusa for critical
reading of the manuscript.
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