Journal of Experimental Botany, Vol. 60, No. 10, pp. 2879–2896, 2009
doi:10.1093/jxb/erp118 Advance Access publication 23 April, 2009
REVIEW PAPER
Exploiting the potential of plants with crassulacean acid
metabolism for bioenergy production on marginal lands
Anne M. Borland1,*, Howard Griffiths2, James Hartwell3 and J. Andrew C. Smith4
1
2
3
4
Institute for Research on the Environment and Sustainability, Newcastle University, Newcastle Upon Tyne, NE1 7RU, UK
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK
School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, UK
Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK
Received 23 December 2008; Revised 17 March 2009; Accepted 18 March 2009
Abstract
Crassulacean acid metabolism (CAM) is a photosynthetic adaptation that facilitates the uptake of CO2 at night and
thereby optimizes the water-use efficiency of carbon assimilation in plants growing in arid habitats. A number of
CAM species have been exploited agronomically in marginal habitats, displaying annual above-ground productivities
comparable with those of the most water-use efficient C3 or C4 crops but with only 20% of the water required for
cultivation. Such attributes highlight the potential of CAM plants for carbon sequestration and as feed stocks for
bioenergy production on marginal and degraded lands. This review highlights the metabolic and morphological
features of CAM that contribute towards high biomass production in water-limited environments. The temporal
separation of carboxylation processes that underpins CAM provides flexibility for modulating carbon gain over the
day and night, and poses fundamental questions in terms of circadian control of metabolism, growth, and
productivity. The advantages conferred by a high water-storage capacitance, which translate into an ability to buffer
fluctuations in environmental water availability, must be traded against diffusive (stomatal plus internal) constraints
imposed by succulent CAM tissues on CO2 supply to the cellular sites of carbon assimilation. The practicalities for
maximizing CAM biomass and carbon sequestration need to be informed by underlying molecular, physiological,
and ecological processes. Recent progress in developing genetic models for CAM are outlined and discussed in
light of the need to achieve a systems-level understanding that spans the molecular controls over the pathway
through to the agronomic performance of CAM and provision of ecosystem services on marginal lands.
Key words: Biomass, CAM, carbon sequestration, circadian control, marginal lands, productivity.
Introduction
The photosynthetic specialization of crassulacean acid
metabolism (CAM) permits the net uptake of CO2 at night
and thereby dramatically improves the water-use efficiency
(WUE) of carbon assimilation in plants growing in arid
habitats. CAM is estimated to be expressed in ;7% of
vascular plant species (Winter and Smith, 1996a), many of
which dominate the plant biomass of arid, marginal regions
of the world. Typically, the water use-efficiency of CAM
plants, expressed as CO2 fixed per unit water lost, may be 3fold higher than that of C4 plants and at least 6-fold higher
than for C3 species. Examples of cultivated CAM species
include Ananas comosus (pineapple), Opuntia ficus-indica,
Agave sisalana, and A. tequilana, all of which can achieve
near-maximal productivity over areas in which precipitation
is inadequate or evapotranspiration so great that rainfall is
insufficient for the cultivation of many C3 and C4 crops. Of
these examples, pineapple (A. comosus: Fig. 1A) is cultivated over 60 of latitude and produces up to 86 Mg fruit
ha1; the international trade value of the fresh produce, not
including processed fruit, was recorded as US$1.9 billion
year1 in 2003 (FAOSTAT, 2005). The high yielding
potential of Opuntia achieved notoriety in the 1900s when
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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2880 | Borland et al.
Fig. 1. (A) Plantation of pineapple [Ananas comosus (L.) Merr.] at
Rollingstone, Queensland, Australia (1902#S, 14623#E). In 2003,
worldwide production of pineapple was 15 3 106 Mg, with an
international trade value of the fresh produce of US$1.9 billion
year1, or ;US$3.0 billion year1 including the processed fruit
(http://www.fao.org/es/esc/en/15/217/highlight_218.html, Current
Situation and Medium-Term Outlook for Tropical Fruits). (B)
Plantation of sisal (Agave sisalana Perrine) at Berenty, southern
Madagascar (2501#S, 4618#E). Madagascar is the world’s sixth
largest producer of sisal. In 2006, worldwide production of fibre
from sisal was 246 3 103 Mg, with a further 22 3 103 Mg being
produced from henequen (Agave fourcroydes Lem.), representing
a combined export value of ;US$200 million (http://www.fao.org/
es/esc/en/15/320/highlight_323.html, Statistical Bulletin June
2008). Production of sisal fibre has gradually declined over the last
40 years to about one-third of its level in the mid-1960s, primarily
because of replacement by synthetic products made from polypropylene. It remains to be seen whether demand for the natural
fibre will increase again in the future as part of the trend to seek
alternatives to petroleum-derived synthetic products. Photographs:
JAC Smith.
an O. stricta monoculture grew to occupy >25 million
hectares in central eastern Australia and produced a total
biomass of ;1.5 billion Mg in ;80 years (Osmond et al.,
2008). Today, Opuntia species are part of natural and
agronomic ecosystems in many parts of the world, with
commercial cultivation (primarily for fodder and forage)
occupying over 1 million hectares and annual dry biomass
productivity for O. ficus-indica of 47–50 Mg ha1 year1
(Nobel, 1996; where 1 ha ¼ 10 000 m2). The current
worldwide cultivation of Agave is >500 000 ha (Nobel
et al., 2002), mostly for fibre (primarily sisal: Fig. 1B) and
fodder, but also for the production of alcohol, either in the
form of tequila (produced from the double distillation of
fermented sugars from the stems and attached leaf bases of
A. tequilana), or as mezcal (a singly distilled beverage
extracted from ;10 other species).
The potential for Agave as an economically viable source
of bioethanol with a zero-waste platform has recently been
highlighted in Mexico as well as for the eroded lands of the
Great Karoo in South East Africa where the climate and
soil are not suitable for the cultivation of other crops
(Boguslavsky et al., 2007; Burger, 2008). The high annual
productivity of A. tequilana (50 Mg dry biomass ha1
year1 on semi-arid land) and high total sugar content (27–
38%) in leaves/stems/fruits (cf. sugar cane 15–22%) have led
to reports that distilled ethanol yields of 14 000 l ha1 can
be obtained from some cultivars, with predictions of
a further 33 650 l ethanol ha1 from cellulose digestion
(Burger 2008). It is recognized that economic and environmental sustainability in the cultivation of dedicated bioenergy crops will require greater emphasis on the application
and diversification of low-input agriculture on marginal
land. This article will highlight the key attributes of CAM
that contribute towards high biomass and bioenergy production in marginal habitats. Areas of current and future
research are outlined for elucidating the causes and
consequences of CAM and for providing a knowledge base
that might inform and improve the potential of CAM plants
for carbon sequestration and bioenergy production on
marginal and degraded lands.
Biochemistry and regulation of CAM
CAM, like C4, employs the enzyme phosphoenolpyruvate
carboxylase (PEPC) for the capture of atmospheric and
respiratory CO2, thereby providing a means of ‘turbo
charging’ Rubisco-mediated C3 photosynthesis and reducing photorespiration for much of the photoperiod. However, whilst the C4 pathway functions through spatial
separation of PEPC and Rubisco between the mesophyll
and bundle sheath cells, the complete CAM pathway occurs
in each photosynthetic mesophyll cell and relies on strict
temporal regulation of C4 and C3 carboxylation processes.
CAM plants open their stomata and perform PEPCmediated CO2 uptake in the dark to form malic acid, which
is subsequently broken down to release CO2 that is fixed by
Rubisco during the following day behind closed stomata.
The closure of stomata in the light period and concomitant
almost complete cessation of transpiration from the shoot
surface underpins the high WUE of CAM plants. In
addition, the temporal separation of metabolism in CAM
provides plasticity for optimizing carbon gain in response to
changing environmental conditions via adjustments in both
the magnitude and relative proportions of direct C3- and
CAM plants and bioenergy | 2881
C4-mediated CO2 uptake. Whilst the enzymatic machinery
required for these carboxylation and decarboxylation reactions is present in all higher plants, evolution of the
pathway required a change in the regulation of key enzymes
and transporters in order to sustain the temporal separation
of the two carboxylation processes that are central to CAM.
CAM biochemistry
The 24 h day/night cycle is the only meaningful unit of time
within which to consider the biochemical processes of CAM
which may be delineated into four phases (Osmond, 1978).
Starting from the end of the photoperiod, the CAM cycle
proceeds with phase I and the metabolic steps are illustrated
in Fig. 2. In the cytosol, PEPC uses phosphoenolpyruvate
(PEP) and HCO–3 (HCO–3 resulting from the action of
carbonic anhydrase on CO2) to generate oxaloacetate,
which is rapidly converted to malate by malate dehydroge-
Fig. 2. The pathway of crassulacean acid metabolism in a mesophyll cell of an NADP-ME (malic enzyme) species such as
Mesembryanthemum crystallinum. Filled arrows represent dark
reactions whilst open arrows represent light reactions. The dashed
line running across the centre separates dark at the top from light
at the bottom. The black-filled boxes on the left of the diagram
represent the leaf epidermis, with the gap representing a stomatal
pore. The enzymes that catalyse the reactions are as follows: (1)
nocturnal starch breakdown possibly via chloroplastic starch
phosphorylase and the export of G6P via the glucose 6phosphate:phosphate translocator (GPT) followed by glycolytic
conversion of G6P to PEP which is then used by phosphoenolpyruvate carboxylase (PEPC) as a substrate for CO2 fixation; (2)
carbonic anhydrase; (3) PEPC; (4) malate dehydrogenase; (5)
voltage-gated malate channel, possibly a tonoplast membranetargeted aluminium-activated malate transporter; (6) vacuolar H+
ATPase; (7) unknown protein proposed to mediate malate efflux,
possibly the tonoplast dicarboxylate transporter; (8) NADP-ME; (9)
ribulose bisphosphate carboxylase oxygenase; (10) unknown
pyruvate transporter on the inner envelope membrane of the
chloroplast; (11) pyruvate orthophosphate dikinase; (12) phosphoenolpyruvate:phosphate translocator; (13) gluconeogenesis;
(14) GPT; (15) starch synthesis beginning with ADP-glucose
pyrophosphorylase.
nase. Malate is transported into the vacuole through
a voltage-gated, inward-rectifying anion channel (Hafke
et al., 2003). The current best candidate for the molecular
identity of this channel protein is the CAM orthologue of
the Arabidopsis thaliana protein ALMT9 (aluminiumactivated malate transporter 9: Kovermann et al., 2007),
although the ALMT family of proteins has yet to be
characterized in a CAM species. Inside the vacuole, malate
accumulates as malic acid due to the high concentration of
H+ generated by the vacuolar H+-ATPase and/or H+-PPiase
(Bartholomew et al., 1996; Tsiantis et al., 1996). Indeed, it is
the electrical component of the H+ electrochemical difference established by these two H+ pumps that maintains the
inside-positive potential needed to drive the influx of
malate2 anions across the tonoplast through the vacuolar
malate channel (Hafke et al., 2003). CO2 uptake and malate
accumulation continue for most of the dark period, such
that the concentration of vacuolar malic acid can reach
upwards of ;200 mM by dawn. PEPC is activated during
the dark period due to phosphorylation by a dedicated
PEPC kinase (PPCK), which mediates a 10-fold increase in
the Ki of PEPC for its feedback inhibitor, malate (Nimmo
et al., 1984, 1987 Carter et al., 1991). The post-translational
activation of PEPC in the dark period is hypothesized to
draw down the internal partial pressure of CO2 inside the
leaf, and it is further hypothesized that this signals stomatal
opening in the dark, thus sustaining the supply of CO2 to
PEPC.
In the few hours prior to dawn, PPCK is degraded and
PEPC is dephosphorylated, rendering the enzyme 10 times
more sensitive to inhibition by malate. The malate-mediated
shutdown of PEPC at the start of the photoperiod may be
considered a critical step for curtailing futile cycling at the
start of the photoperiod in CAM plants (Borland et al.,
1999). The export of malate from the vacuole to the cytosol
(the site of PEPC activity) has long been considered
a passive process, coupled in some way to the stoichiometric
efflux of 2 H+ per malate (Smith et al., 1996), but the
molecular identity of this transport system has also not been
identified. An intriguing possibility is that the CAM
orthologue of the tonoplast dicarboxylate transporter
identified in A. thaliana (Emmerlich et al., 2003; Hurth
et al., 2005) could mediate malate efflux from the vacuole at
dawn. Rubisco activation, mediated via Rubisco activase, is
believed to commence at the start of the photoperiod, and
for a brief period CO2 may be fixed by both PEPC and
Rubisco, a period referred to as phase II of CAM (Fig. 3).
However, it is noteworthy that a study on Kalanchoe¨
daigremontiana found that the Rubisco activation state was
low in phase II and increased gradually throughout the
photoperiod, peaking some 3–4 h before the end of the
photoperiod (Griffiths et al., 2002). This correlated with
a peak in transcript and protein abundance of Rubisco
activase, which occurred around the middle of the light
period, in contrast to the peak of Rubisco activase
transcript abundance that occurs shortly after dawn in C3
species (Griffiths et al., 2002). Additionally, in Kalanchoe¨,
Rubisco has kinetic properties which are intermediate
2882 | Borland et al.
between C3 and C4, with a reduced specificity (compared
with C3) but lower Km for CO2 (compared with C4),
perhaps reflecting the daily switch in carboxylase dominance that occurs over the CAM cycle (Griffiths et al.,
2008).
The decarboxylation of malate (phase III of CAM) may
be catalysed by: mitochondrial NAD+-ME (malic enzyme),
cytosolic NADP+-ME, chloroplastic NADP+-ME, or cytosolic PEPCK, depending on the plant species (Dittrich
et al., 1973; Dittrich, 1976; Holtum et al., 2005; the
decarboxylation of malate via ME is shown in Fig. 2). In
some CAM species, there is correlative evidence that malate
is decarboxylated by more than one of these enzymatic
routes due to high activities of more than one decarboxylase
(Dittrich et al., 1973). Decarboxylation by ME generates
pyruvate. In order for this pyruvate to be recycled through
gluconeogenesis to starch, it must be converted to PEP by
pyruvate, orthophosphate dikinase (PPDK; Fig. 2). PPDK
has long been assumed to be a chloroplastic enzyme, and
this was shown to be the case in Mesembryanthemum
crystallinum (Kondo et al., 1998). In cytosolic NADP-ME
and mitochondrial NAD-ME CAM species, PPDK in the
chloroplast requires a pyruvate transporter on the chloroplast inner envelope membrane (Kore-eda et al., 1996). The
molecular identity of this chloroplast membrane pyruvate
transporter remains to be elucidated. However, in contrast
to M. crystallinum, other CAM species, including K.
daigremontiana and K. pinnata, have isoforms of PPDK
localized to both the cytosol and chloroplast (Kondo et al.,
2000, 2001). These observations are consistent with data
reported for the C3 species A. thaliana, in which a single
PPDK gene was found to have two promoters that produced two different transcripts, one of which was translated
into a chloroplastic PPDK, whilst the other transcript
produced a cytosolic isoform of PPDK (Parsley and
Hibberd, 2006). The regulation and localization of ME
isoforms, PEPCK, and PPDK have received very limited
attention in CAM species, and the function of the PPDKregulatory protein (Chastain et al., 2008)—a homologue of
which has been identified in M. crystallinum—remains to be
established in terms of CAM operation.
The high internal concentration of CO2 generated in the
intercellular spaces by malate decarboxylation in phase III
while stomata are closed effectively saturates the carboxylase activity and suppresses the oxygenase function of
Rubisco, even though internal O2 concentrations are also
elevated at this time. In well-watered CAM plants, stomata
may open once the supply of malate is exhausted and
internal CO2 concentrations drop. Direct fixation of atmospheric CO2 by Rubisco can then proceed for the remainder
of the light period, a period known as phase IV (Fig. 3;
Osmond, 1978). The magnitude and duration of each phase
of the CAM cycle is highly plastic and varies (i) between
species; (ii) in response to the environment; and (iii) with
leaf development (Winter et al., 2008). As illustrated for
Kalanchoe¨ fedtshenkoi (Fig. 3), the magnitude and duration
of phases II– IV is inversely related to the magnitude and
duration of CO2 taken up at night (phase I). The older leaf
pair 5 showed most net CO2 uptake at night, and this was
reflected in a diminished phase II and IV and extended
phase III (the latter probably due to the time taken to
decarboxylate the larger pool of malate present in leaf pair
5 compared with leaf pair 4). These different gas exchange
patterns can also be attributed to the contrasting degree of
succulence and commitment to CAM seen in young and old
leaves of K. fedtshenkoi, which is equivalent to the
difference between mature leaves of K. daigremontiana and
K. pinnata (Griffiths et al., 2008; von Caemmerer and
Griffiths, 2009).
Energetically, the CAM cycle incurs an additional cost
when compared with the standard C3 mode of carbon
fixation. This arises from two sources: first, the cost of
transporting malic acid into the vacuole at night, driven by
one or both of the tonoplast H+ pumps; and secondly, the
cost of converting the C3 residue resulting from malate
decarboxylation during the daytime back to the level of
storage carbohydrate via gluconeogenesis, this being needed
to provide the substrate for PEPC during the following
night-time. In aggregate, these processes probably represent
an additional metabolic cost of ;10% compared with the
standard C3 pathway (Winter and Smith, 1996b), which is
relatively minor when considering that CAM plants typically grow in high-light environments in which photon
supply is not normally limiting for growth. In compensation, however, CAM plants benefit during phase III from
the suppression of photorespiration, which in C3 plants can
increase the cost of net CO2 fixation by a minimum of 25%.
Even under optimal temperature conditions, therefore, the
energetic cost of net CO2 fixation is significantly lower in
CAM plants than in C3 plants (Nobel, 1996; Winter and
Smith, 1996b). This advantage increases with higher ambient temperatures, at which photorespiration increases
steeply in C3 plants, which is relevant when considering the
temperature regimes characteristic of semi-arid habitats at
tropical and subtropical latitudes.
Temporal control of CAM
The complete 24 h cycle of CAM occurs within individual
leaf mesophyll cells, and strict temporal regulation of the
various metabolic and transport components of the pathway is required to avoid the futile cycling that would result
from uncontrolled, simultaneous CO2 fixation and malate
decarboxylation. The coordination and optimization of
CO2 fixation and subsequent metabolism during CAM is
controlled by the endogenous circadian clock (Hartwell,
2005a). In particular, the leaves of CAM species perform
a persistent circadian rhythm of CO2 fixation at constant,
permissive temperatures in continuous light, normal air,
and continuous darkness under CO2-free air (Wilkins, 1992;
Hartwell, 2005a, b). Effective circadian control is probably
critical to the optimal functioning of the CAM pathway,
and has been covered in detail elsewhere (Borland and
Taybi, 2004; Hartwell, 2005a, b). However, it is important
to highlight some key features here, as circadian control
may be a major yield constraint in highly productive CAM
CAM plants and bioenergy | 2883
Fig. 3. The phases of the 24 h CAM cycle. The phases proposed by Osmond (1978) are superimposed onto gas exchange data from:
(A) leaf pair 4 and (B) leaf pair 5 of Kalanchoë fedtschenkoi. Phase I: stomata open, primary CO2 fixation by PEPC in the dark leading to
malic acid accumulation. Phase II: stomata open, simultaneous CO2 fixation by PEPC and Rubisco during the early hours of the light
period. Phase III: stomata closed, refixation of CO2 from decarboxylated malate by Rubisco. Phase IV: stomata reopen under wellwatered conditions, direct fixation of atmospheric CO2 by Rubisco.
species such as Agave and Opuntia, and thus have relevance
in the selection of cultivars as feed stocks for bioenergy
production.
The control of carbon flux through PEPC in CAM
species is known to be mediated via circadian control of the
synthesis and degradation of the dedicated regulatory
kinase PPCK (Nimmo et al., 1984; Carter et al., 1991;
Hartwell et al., 1996, 1999, 2002). The nature of the
underlying circadian oscillator that provides the temporal
signals to optimize the CAM cycle as a whole, however,
remains rather more elusive. Orthologues for many of the
A. thaliana central clock genes have been cloned and
characterized from the facultative CAM plant, M. crystallinum (Boxall et al., 2005). This work has demonstrated that
CAM species possess a multigene loop oscillator very
similar to that in A. thaliana (Hotta et al., 2007; McClung,
2008), supporting the hypothesis that the convergent
evolution of CAM exploited an existing C3 oscillator for
the coordination and optimization of C3 and C4 carboxylation processes. Moreover, phase advancing the M. crystallinum multigene loop oscillator using a temperature pulse in
the late dark period resulted in a highly correlated phase
advance of circadian-regulated processes associated with
CAM, including PPCK gene transcript abundance and
malate oscillations (Hartwell, 2005b; SE Boxall, JM Foster,
and J Hartwell, unpublished data). However, the most
convincing data linking the multigene loop oscillator in the
nucleus to the circadian regulation of CAM comes from
transgenic lines of K. fedtschenkoi overexpressing the central
circadian clock gene, TIMING OF CAB EXPRESSION1
(TOC1). In these TOC1-overexpressor lines, the circadian
rhythm of CO2 fixation collapses rapidly to arrhythmia
following the onset of constant conditions (Hartwell, 2005b;
C Dall’omo, SE Boxall, JM Foster and J Hartwell,
2884 | Borland et al.
unpublished data). This is consistent with the phenotype of
output rhythms in lines of A. thaliana that strongly overexpress TOC1 (Mas et al., 2003), and thus suggests that the
multigene loop oscillator does coordinate CAM. The K.
fedtschenkoi TOC1-overexpressor lines grow less rapidly
and have smaller leaves, indicating that circadian control of
CAM is critical to the growth and development of the plant
(C Dall’omo and J Hartwell, unpublished). The detailed
characterization of the phenotype of these K. fedtschenkoi
TOC1-overexpressor lines will be the subject of a future
publication.
In the light of the growth penalties associated with
incorrect circadian control of CAM in TOC1-overexpressor
lines, it is clear that detailed understanding of circadian
control will be a key element in dissecting yield constraints
in potential CAM biofuel species such as Agave and
Opuntia. The importance of circadian control to growth
and reproductive success has previously been demonstrated
in A. thaliana, where complete arrhythmia of the central
circadian oscillator was accompanied by an ;50% decrease
in net carbon gain from photosynthesis (Dodd et al., 2005).
Furthermore, altered circadian control has recently been
shown to regulate growth vigour in allotetraploids and F1
hybrids of Arabidopsis (Ni et al., 2009). It is generally
considered that CAM evolved via genome duplication or
polyploidy, followed by evolution of the CAM-specific role
for the extra redundant copies of relevant genes such as PPC
and PPCK (Cushman and Bohnert, 1999). It is intriguing to
speculate that the clock-dependent up-regulation of output
genes may predispose a newly formed allopolyploid to the
evolution of the CAM pathway under certain environmental conditions. Incorrect timing of biological activity in arid
habitats tends to have more rapid negative consequences
simply by virtue of the greater environmental extremes (e.g.
temperature) that occur in such habitats. Thus, it can be
argued that accurate circadian control tends to be even
more important in arid-zone species. It remains to be
established if the clock in CAM species has an even more
critical role in reproductive success than that in C3 species.
Certainly, it seems likely that the clock is central to the
photoperiodic induction of CAM and onset of flowering in
Kalanchoe¨ blossfeldiana (Taybi et al., 2002), and may be
associated with the onset of the dry season (Kluge and
Brulfert, 1996).
Carbohydrate economy and
growth processes
Whilst the circadian clock plays a cardinal role in
establishing and maintaining the characteristic phases of
CAM, the day/night turnover of carbohydrate is a key
component in determining the magnitude of CAM expression. The nocturnal uptake of CO2 is sustained by
degradation of carbohydrate reserves to PEP as substrate
for PEPC-mediated carboxylation (Fig. 2). Up to 20% of
leaf dry biomass can be allocated to carbohydrates for
CAM, and these reserves may be accumulated in the form
of soluble, low molecular weight sugars in the vacuole or
as insoluble, polymeric glucan (starch) in the chloroplast
(Kenyon et al., 1985; Christopher and Holtum, 1996,
1998). The typically high content of non-structural carbohydrates in CAM leaves can be viewed as a desirable trait
for bioethanol production (Smith, 2008). Moreover, despite the requirement to bank reserves for dark CO2
uptake, in many CAM species (e.g. pineapple, Agave,
Opuntia) the potential for high biomass productivity is not
compromised. CAM allows metabolic flexibility in the use
of different carbohydrate sources for nocturnal substrate
provision, as demonstrated by the restoration of nocturnal
acidification by feeding glucose or sucrose to leaves of
a starch-deficient mutant of M. crystallinum (Cushman
et al., 2008a). Clearly, the partitioning of carbohydrate
for dark carboxylation must be modulated in line with
the requirements of other competing sinks that include
dark respiration, export, and growth (Borland and Dodd,
2002).
The phasing of leaf expansion growth over the diel
CAM cycle has important implications for the mechanisms
that regulate assimilate partitioning over a 24 h period.
Using high-resolution digital image sequence processing to
examine diel patterns of leaf growth, Gouws et al. (2005)
showed that leaf growth accelerated at night and decelerated during the day in C3-performing M. crystallinum.
In contrast, leaves of the CAM species Kalanchoe¨
beharensis and cladodes of Opuntia engelmanii and O. oricola
showed accelerated growth in the middle part of the day
(phase III of CAM) and little or no growth at night
(Gouws et al., 2005). It was suggested that the markedly
different diel growth patterns in CAM species as compared
with C3 can be explained in terms of both the distinctive
turgor relations and carbon supply of CAM plants (Gouws
et al., 2005). It has been reported for a range of C3 species
that leaf growth occurs predominantly at night, and
studies on Arabidopsis have indicated a close coupling
between the rate of growth and the rate of nocturnal starch
degradation (Walter and Schurr 2005; Smith and Stitt,
2007). In contrast, nocturnal stomatal opening and transpiration in CAM plants can cause leaf turgor to be low for
much of the dark period, with maximum turgor not
occurring until stomata are closed in phase III (Smith and
Lüttge, 1985). The uncoupling of leaf expansion growth
from nocturnal carbohydrate degradation could provide
a means of reconciling potential conflicts of demand
between accumulation of carbohydrate reserves for CAM
(or bioethanol production) and partitioning of resources
for growth.
Starch degradation in CAM plants
The enzymatic route by which starch is degraded at night
in CAM plants could have important implications for
understanding the mechanisms that uncouple nocturnal
starch breakdown from the synthesis of sucrose for
growth. In leaves of Arabidopsis, the hydrolytic pathway
has been shown to be of prime importance for the
CAM plants and bioenergy | 2885
nocturnal conversion of starch to sucrose, with maltose
being the major export product from chloroplasts degrading starch at night (Niittyla et al., 2004). In contrast, the
phosphorolytic pathway of starch degradation has been
proposed to supply carbon for metabolism inside the
chloroplast of A. thaliana, with starch phosphorylase
providing glucose-6 P as substrate for the oxidative
pentose phosphate pathway, particularly under conditions
of stress and when photorespiration is elevated (Zeeman
et al., 2004; Weise et al., 2006).
The induction of CAM in M. crystallinum by exposure to
salinity is accompanied by increased activities of a range of
starch-degrading enzymes implicated in both the hydrolytic
and phosphorolytic pathways (Paul et al., 1993). However,
a change in transport activities across the chloroplast
envelope has been reported with the switch to CAM in M.
crystallinum. Chloroplasts isolated from C3 M. crystallinum
exported mainly maltose, whilst chloroplasts isolated from
plants in the CAM mode exported mainly glucose-6 P
(Neuhaus and Schulte, 1996). Two isogenes encoding
glucose-6 P/Pi translocators have been isolated from M.
crystallinum (McGPT1 and McGPT2); CAM induction is
accompanied by an increase in transcript abundance of both
isogenes, which also show robust circadian patterns of
abundance, implying a key role for these transporters in
CAM (Kore-eda et al., 2005). The nocturnal export of
glucose-6 P from the chloroplast to the cytosol in CAM
plants might be predicted to cause allosteric activation of
PEPC (Osmond and Holtum, 1982), whilst glycolytic
conversion of glucose-6 P in the cytosol would provide
ATP by substrate-level phosphorylation and thus help to
energize the nocturnal accumulation of malate in the
vacuole (Holtum et al., 2005). Further indications that the
phosphorolytic route may predominate over the hydrolytic
route for starch degradation in CAM plants is provided by
the very high extractable activities of chloroplastic starch
phosphorylase from leaves of M. crystallinum, K. fedstchenkoi,
and A. comosus, with activities in the CAM species
;10-fold higher than that in Arabidopsis (T Taybi and AM
Borland, unpublished observation). In contrast, glucanotransferase (DPE2), which in Arabidopsis acts on cytosolic
maltose to provide a substrate for sucrose synthesis (Smith
et al., 2005), shows very low activity in CAM-performing
M. crystallinum (AM Borland, unpublished observation).
Interrogation of an expressed sequence tag (EST) database
for M. crystallinum containing 25 000 ESTs failed to
identify any candidate chloroplastic maltose transporters (J
Hartwell, unpublished observation), which might suggest
that maltose export from the chloroplast in M. crystallinum
(and indeed other CAM species) is quantitatively less
important than that in Arabidopsis.
Lüttge et al. (1981) first pointed out that use of the
phosphorolytic pathway for starch breakdown would reduce the energetic cost of nocturnal malic acid accumulation in CAM plants compared with the hydrolytic pathway.
A full understanding of the relevance of utilizing the
phosphorolytic pathway for directing carbon skeletons
towards the synthesis of PEP rather than sucrose will
require genetic manipulation of key enzymes and transporters implicated in these pathways in a CAM species.
Day/night turnover and storage of sugars in CAM plants
For CAM species such as pineapple and agave in which
soluble sugars are the major storage carbohydrate and the
source of PEP for nocturnal carboxylation, the vacuole is
likely to play a key role in regulating partitioning of sugars
between growth and CAM. The photosynthetically active
cells of CAM plants are typically dominated by a large
central vacuole that occupies ;95% of the cell volume.
Isolated vacuoles of A. comosus (pineapple) contain mainly
glucose and fructose (Kenyon et al., 1985; Christopher and
Holtum, 1998), whilst whole-leaf extracts contain substantial amounts of sucrose (Kenyon et al., 1985). Given the
very high extractable activities of acid invertase reported for
pineapple leaves (Black et al., 1996), it has been proposed
that sucrose is synthesized in the cytoplasm during the day,
transported across the tonoplast into the vacuole, and
hydrolysed in the vacuolar lumen by acid invertase (Smith
and Bryce, 1992). To avoid futile cycling, the hexoses thus
produced would need to be stored in the vacuole and not
released to the cytoplasm until the following dark period,
when they would be metabolized via glycolysis to provide
the C3 substrate (PEP) for nocturnal malate synthesis. This
model is supported by the finding that the tonoplast of
pineapple possesses a sucrose transport system with kinetics
appropriate to catalyse sucrose fluxes of the required
magnitude. McRae et al. (2002) demonstrated that sucrose
uptake by isolated tonoplast-enriched vesicles prepared
from pineapple leaves exhibits concentration-dependent
saturation kinetics, ATP independence and trans stimulation by internal sucrose, characteristics that are consistent
with the operation of a carrier type of sucrose transporter.
Recently, a specific hexose transport system has also been
observed in isolated pineapple tonoplast vesicles (D Haines
and JAM Holtum, personal communication). Thus, sugar
transporters located at the tonoplast could play a strategic
role in controlling the supply and demand for carbon over
the day–night cycle in sugar-accumulating CAM plants
such as pineapple (Antony and Borland, 2008).
The mechanisms that control import and export of
photoassimilates from the vacuole during the day–night
cycle are largely unexplored for any plant species, and the
first plant tonoplast sugar carriers have only recently been
identified at the molecular level. Currently, the only
vacuolar sucrose transporters to be identified at the
molecular level (HvSUT2 from barley and AtSUC4 from
Arabidopsis; Endler et al., 2006) are believed to facilitate
sucrose export from the acidic lumen of the vacuole
(Neuhaus, 2007). The first tonoplast monosaccharide transporters were recently identified from Arabidopsis (AtTMT)
and are believed to operate via an H+-coupled antiport
mechanism that would allow import of glucose and fructose
in the vacuole by a seconday active transport mechanism,
possibly in response to stimuli (i.e. cold, drought, salinity),
that promotes sugar accumulation in Arabidopsis (Wormit
2886 | Borland et al.
et al., 2006). The model proposed for vacuolar sugar
transport in the leaves of A. comosus (Smith and Bryce,
1992; McRae et al., 2002) implies the existence of a tonoplast hexose transport system to permit efflux of glucose
and fructose at night to provide substrates for dark CO2
uptake. A putative hexose transporter (AcMST1) recently
identified from pineapple leaves that localized to the
tonoplast of tobacco epidermal cells is a potential candidate
for the energy-independent export of hexoses from the
vacuole (Antony et al., 2008). However, the crucial kinetic
mechanism that restricts efflux of vacuolar hexose to the
dark period (thus avoiding futile cycling during the light
period) is still completely unknown. There is no evidence for
diurnal or circadian control of transcript abundance of this
pineapple tonoplast hexose transporter, nor was there any
difference in transcript abundance of AcMST1 between
pineapple cultivars that differed in the magnitude of CAM
(Antony et al., 2008). Recent examinations of the phosphoproteome of the tonoplast from rice and Arabidopsis suggest
that phosphorylation of sugar transporters could be an
important mechanism for controlling the day/night loading
and unloading of sugars across the vacuolar membrane
(Whiteman et al., 2008a, b).
In Agave, fructans stored in the stems and leaf bases are
the major source of ethanol from this plant and as such
represent an important vacuolar sink for photoassimilate.
In mature leaves of Agave deserti, fructan biosynthesis was
restricted to the vascular tissue (Wang and Nobel, 1998).
Long-distance transport in the phloem of fructans with
a low degree of polymerization has been reported for
a number of plant species, but the mechanisms whereby
fructans are unloaded into sink tissues and subsequently
accumulated in the vacuole are unknown. Generally, it is
considered that fructans are synthesized in the vacuole via
the enzyme fructosyl transferase using imported sucrose as
substrate (Valluru and Van den Ende, 2008). Fructans are
believed to protect membranes in plants under abiotic stress
and may also participate in the scavenging of reactive
oxygen species in the vacuole (Van den Ende and Valluru,
2009). Substantial variation in sugar and fructan content
has been reported for different cultivars of Agave (VargasPonce et al., 2007) which could provide opportunities for
selecting varieties for enhanced bioethanol production and
stress tolerance. To maximize such potential will require
a better understanding of the biochemical and molecular
basis of carbohydrate partitioning in Agave alongside
a systems-level approach to identify control points for
sugar partitioning. Genes that encode vacuolar sugar
transporters could be appropriate candidates for increasing sugar content in plants grown for bioethanol production. Indeed, metabolic control analysis of the kinetics
of sucrose accumulation in the maturing culm tissue of
sugar cane identified the rate of sucrose uptake by the
vacuole as a key control element in the magnitude of
sucrose accumulation (Uys et al., 2007). Given that leaf
succulence and vacuolar capacity are particularly high in
CAM species, knowledge of the tonoplast proteome in
a sugar-accumulating CAM species could offer potential
for increasing leaf sugar content whilst maintaining
photosynthetic carbon assimilation by avoiding feedback
repression of primary carbon metabolism and providing
substrate for nocturnal carboxylation.
Genetic models for CAM
An important step towards maximizing the yield potential
of CAM species as feed stock for biofuel production will be
the development of a systems-level understanding of the
molecular and metabolic controls over the pathway. Tractable model CAM species will be key to dissecting the
pathway at molecular and biochemical levels. Early
attempts to develop a molecular–genetic model for the
study of CAM centred on M. crystallinum (Bohnert and
Cushman, 2000). CAM may be induced on a C3 background via the imposition of salinity or drought in M.
crystallinum (Winter and Holtum 2007), and this metabolic
switch has proved a very attractive system for identifying
CAM-associated genes and proteins (Cushman and
Bohnert, 1989, 1999; Cushman, 1992; Boxall et al., 2005).
Other beneficial attributes of M. crystallinum include:
a relatively rapid life cycle (7–14 weeks depending on
growth conditions); a relatively small genome, perhaps the
smallest known amongst CAM species measured to date
(;390 Mbp; De Rocher et al., 1990); and the setting of
thousands of small seeds which are ideal for mutagenesis,
allowing traditional forward genetic screens to be undertaken. Mutant populations of M. crystallinum have been
developed and screened to identify CAM-deficient mutants
(Cushman et al., 2008a). A database of M. crystallinum
ESTs containing some 27 000 sequences (Kore-eda et al.,
2004) remains the largest readily accessible gene index for
a CAM species. This gene index has been used to generate
a Nimblegen oligonucleotide microarray with representation of 8455 unique genes, which was used to investigate
both the induction and circadian regulation of CAM
(Cushman et al., 2008b).
The further development of M. crystallinum as a model
CAM system is presently hampered by the lack of an
efficient and stable transformation system to facilitate in
vivo testing of gene function using both overexpression,
and gene silencing/RNAi (RNA interference) approaches.
In addition, the study of CAM in M. crystallinum is
complicated by the requirement for drought or salt stress
to induce CAM, and it is therefore very difficult to
separate CAM genes from those genes that are more
directly involved in resisting the osmotic stress imposed by
the drought or salt. Given such constraints, attention is now
shifting towards the Madagascan endemic K. fedtschenkoi as
the next major genetic model for CAM based on the
following rationale. (i) K. fedtschenkoi is an obligate CAM
species that displays a clear developmental progression from
C3 to CAM, even under well-watered conditions. The
complication of the drought or salt stress response is
therefore avoided, and it is thus regarded as a much
‘cleaner’ model for the study of CAM. (ii) K. fedtschenkoi
CAM plants and bioenergy | 2887
has the key advantage of a simple and efficient stable
transformation system, permitting transgenic approaches to
deciphering gene function. (iii) K. fedtschenkoi has a reasonably small genome at ;858 Mbp (De Rocher et al., 1990),
which places it well within the capabilities of the latest
DNA sequencing technologies. (iv) K. fedtschenkoi is very
easy to grow and develops rapidly from cuttings or leaf
plantlets, reaching a size suitable for CAM experiments
within 2 months. The ease of clonal propagation means that
large quantities of developmentally coordinated plants can
be generated very quickly from individual transformants.
(v) K. fedtschenkoi has the further advantage that a wealth
of background literature exists providing detailed characterization of the physiology and biochemistry of the circadian
control of CAM (Wilkins, 1992; Hartwell et al., 2002;
Hartwell, 2005b).
The characteristics of K. fedtschenkoi described above
have led to a new project in the Hartwell laboratory
(University of Liverpool, UK) to perform in-depth sequencing of the transcriptome of this species. The project will also
establish a large-insert bacterial artificial chromosome
(BAC) library and perform pilot BAC sequencing in
readiness for whole genome sequencing. Digital transcriptomics (the use of the number of sequence reads per gene as
a quantitative digital read-out of transcript abundance in
the original RNA sample) is being employed to identify
CAM-associated genes whose transcript abundance is upregulated in CAM leaves relative to C3 leaves. Candidate
CAM genes are subjected to more detailed real-time reverse
transcription-PCR (RT-PCR) analysis to test for circadian
regulation in CAM leaves, and the most interesting
candidates will be overexpressed and silenced in transgenic
K. fedtschenkoi to test the in vivo function of each candidate
CAM gene. Thus, this project aims to identify all of the
genes that K. fedtschenkoi uses to perform CAM, and set
the stage for future proteomic and metabolomic analysis of
CAM in K. fedtschenkoi. The ultimate goal is to combine
data concerning transcripts, proteins, and metabolites into
predictive models, which can be used to identify the key
control points for optimal performance and yield associated
with CAM.
The phylogenetic position of K. fedtschenkoi within the
family Crassulaceae and order Saxifragales means that this
species shared its last common ancestor with M. crystallinum
(Aizoaceae, Caryophyllales) some ;80–90 milllion years
ago, and thus evolved the CAM pathway completely
independently. Ongoing genomics projects with M. crystallinum and K. fedtschenkoi will thus permit comparative
analysis of the functional genomics of CAM in divergent
species that evolved the CAM pathway independently. Such
an approach may reveal differences in the regulatory and
enzymatic steps that have been co-opted into a CAMspecific role during the evolution of this pathway in
different taxonomic groups. This is important in light of
the fact that M. crystallinum and K. fedtschenkoi are not
closely related to the high-yielding CAM species of cacti
and agaves that appear to be suited to bioenergy production
on marginal lands.
Carbon uptake and sequestration—from leaf
to canopy
Carbon uptake by leaf or stem succulent CAM plants
represents a classic example of conflict resolution in
physiological ecology, whereby the potential for carbon
gain across a 24 h cycle is traded against diffusive constraints imposed on CO2 supply by succulent tissues (Dodd
et al., 2002; Pierce et al., 2002; Griffiths et al., 2007). The
growth characteristics of CAM plants, as summarized in
Table 1, present a number of favourable attributes when
considering the cultivation of dedicated bioenergy crops in
semi-arid regions of the world. However, the practicalities
for maximizing CAM biomass and carbon sequestration
need to be informed by underlying molecular, physiological,
and ecological processes. Understanding the integration of
molecular and cellular signals and their regulation by
environmental conditions will allow chemical and structural
composition to be targeted towards a specific end-product,
provided that any potential market is sustainable and the
local community supports the initiative (Cowling et al.,
2008). Additionally, there are uncertainties over the maintenance of potential productivity for the future, because
a changing climate will lead to altered precipitation patterns
and tensions between food and fuel production, which are
current even in today’s marginal habitats. However, it
should be noted that the exceptional degree of stress
tolerance of typical CAM plants towards restricted water
availability, high temperatures, and high light intensities
(Table 1) would be expected to make them relatively robust
to the impact of future climate change.
Stomatal versus mesophyll (internal) constraints to
carbon gain
At the scale of individual photosynthetic organs, stomata
occur in relatively low densities and have low conductances
to water vapour in CAM plants, reflecting the high waterstorage capacity, low external surface area:volume ratio,
and high WUEs, for leaves, cladodes, and stems of CAM
plants (Osmond, 1978; Nobel, 1988). The compromise
between maximizing day- and night-time uptake is exemplified in comparisons of two Kalanchoe¨ species (K. daigremontiana
and K. pinnata) which contrast in the degree of leaf
succulence (Griffiths et al., 2008; von Caemmerer and
Griffiths, 2009). Stomatal densities were lower in the more
succulent species (K. daigremontiana), which was more
committed to the conventional CAM cycle, with higher
rates of acid accumulation, CO2 uptake, and higher
stomatal conductance at night. In contrast, the less succulent K. pinnata showed the more C3-like expression of the
CAM phases by day, with a higher proportion of integrated
24 h net CO2 uptake mediated directly by Rubisco during
phases II and IV. Overall, the ratios of internal:external
CO2 concentration were similar at night for the two species,
and actually higher during phase IV for K. daigremontiana.
However, the sensitivity of stomata to transient changes in
external CO2 concentration during the day–night cycle
2888 | Borland et al.
Table 1. Growth characteristics of CAM plants favourable for cultivation as a bioenergy crop in semi-arid regions
Examples taken from Nobel (1988, 1994), Day (1993), and Winter and Smith (1996c).
Trait
Example
Comment
High water-use efficiency
High drought tolerance
5–16 mmol CO2 per mol H2O on an annual basis
Can grow in areas with as little as 25 mm year1
precipitation
Up to 70 C, based on 50% loss of cell viability after
1 h; can survive exposure to 74 C
Can tolerate >1000 lmol m2 s1
(or >40 mol m2 d1) without photoinhibition
Only 1% incident UV-B transmitted through
epidermis of Yucca filamentosa (Agavaceae)
Whole shoot photosynthetic in both leaf- and
stem-succulent species; limited bark formation
even on stems of arborescent cacti
Shoot:root ratio as high as 10:1; above-ground
biomass readily harvested
Effective physical defences (stem succulents) and
chemical defences (leaf succulents)
Especially monocotyledons (;20% dry weigth);
ready conversion of soluble sugars to bioethanol
Weak secondary thickening and lack of true wood formation
Typically 4- to 10-fold higher than C3 plants
Tissues can tolerate up to 90% loss of water content (cacti)
Tolerance of high temperatures
Tolerance of high PPFD
Tolerance of UV-B radiation
Entire shoot surface typically
photosynthetic
High shoot:root ratio and
harvest index
High resistance to herbivores
High content of non-structural
carbohydrate
Low lignin content
suggested that internal CO2 concentration is not the only
effector regulating the CAM diel stomatal cycle (von
Caemmerer and Griffiths 2009).
These data help to explain the compromise between the
constraints imposed by succulent tissues, and the potential
for internal metabolism facilitated via high PEPC and
Rubisco carboxylation capacities as a means of overcoming
external diffusion limitation (Griffiths et al., 2002). However, with the succulent photosynthetic tissues of typical
CAM plants containing only ;5% airspace (Smith and
Heuer, 1981; Maxwell et al., 1997), there are significant
constraints imposed on the internal diffusive supply of CO2
(outside the ‘regenerative’ phase III, when internal CO2
concentrations are likely to saturate Rubisco). Thus, at
night, the carbon isotope signals associated with PEPC were
strongly suggestive of diffusion limitation in K. daigremontiana
(Griffiths et al., 2007). Additionally, mesophyll conductances derived during phase IV of gas exchange in the light
(and for a range of Clusia species) reflected the degree of
succulence, with internal CO2 supply at Rubisco potentially
as low as 110 lmol mol1 (Griffiths et al., 1999).
Given such diffusive constraints, the interpretation of
carbon isotope composition of bulk organic material as
being representative of the balance between daytime (C3)
and night-time (C4) carboxylation processes is complicated.
When carbon isotope composition is predicted by stomatal
conductance and carboxylase fractionations, lower discrimination is likely to be associated with CO2 uptake during
phase IV in succulent tissues by day, and higher discrimination may be predicted at night (Griffiths et al., 1990, 2007,
2008; Roberts et al., 1997). The use of carbon isotope
composition to predict ‘furtive’ CAM species (or C3–CAM
intermediates) in a given population may be confounded
both by internal constraints to diffusion and by the extent
Typically upper limit of 50–55 C in C3 plants
Generally more tolerant of high PPFD than agronomically
important C3 plants
Generally thick epidermis and high foliar concentrations of
phenolics in CAM plants
Many C3 species deciduous (shedding photosynthetic
organs for part of year) or woody (limited stem photosynthesis)
of carbon gain actually undertaken for the short period that
CAM may be active during extreme conditions (Pierce
et al., 2002; Winter and Holtum, 2002; Silvera et al., 2005).
Additionally, the isotopic composition of CAM plants tends
to be fairly conservative across a wide range of habitats
(Griffiths, 1992), and may not always be indicative of
precipitation gradients (Amundson et al., 1994). This may
limit the use of carbon isotopes as a tool in selecting
improved productivity and water use of bioenergy crop
cultivars, which are mostly as yet unimproved. However,
when combined with oxygen isotopes, which provide
a marker for precipitation inputs and water sources within
the soil profile, carbon isotope composition may be more
revealing. It has recently been shown that oxygen isotopes
can resolve water vapour exchanges by epiphytic CAM
plants (Helliker and Griffiths, 2007; Reyes-Garcia et al.,
2008) and act as a means of climate reconstruction from
saguaro spines (English et al., 2007).
Tolerance of water deficits, water use and productivity
Despite the metabolic limitations of high gas-phase resistances associated with succulent tissues, high productivities
can be achieved by CAM plants in habitats where precipitation is intermittent, but regular (i.e. seasonal) and
reasonably predictable on an annual basis (Ellenberg, 1981).
With their relatively shallow root systems, CAM plants are
able to exploit efficiently the small amounts of water
available even from intermittent rainfall events delivering
<10 mm, which may be sufficient to moisten only the
uppermost soil layers. Coupled with this efficient harvesting
of incident precipitation, leaf- and stem-succulent CAM
plants also have very high water-storage capacitance in their
above-ground tissues, meaning that they are able to make
CAM plants and bioenergy | 2889
maximum benefit of the water absorbed by their root
systems in improving tissue water relations. At the cellular
level, this high storage capacitance is a consequence of two
important characteristics of CAM plants. First, compared
with other drought-tolerant plants, they typically have
rather dilute cell sap, with osmotic pressures almost invariably <1.0 MPa (Walter, 1960; Smith and Lüttge, 1985;
Smith et al., 1986). Secondly, the large parenchymatous
cells in the shoot tend to have thin cell walls, and
consequently are relatively elastic (technically, they have
a low volumetric elastic modulus: Steudle et al., 1980; Smith
et al., 1987). These properties combine to confer on the
tissues of the shoot a high water-storage capacitance, as
capacitance is inversely proportional to both cell osmotic
pressure and elastic modulus (Steudle et al., 1980; Smith
et al., 1987). Furthermore, at the level of the whole organ,
CAM plants can withstand large changes in relative volume:
whereas loss of one-fifth of their relative water content is
lethal for many plants species, some CAM plants can
tolerate loss of 80–90% of their water content and still
survive, as may occur in exceptional periods of several years
without rainfall (Nobel, 1988).
There is an evident contrast between tolerance of water
deficits at cellular and ecological levels, whereby CAM
water and solute potentials are close to –1 MPa, whilst
nearby C3 shrubs may approach –4 MPa or lower (Smith
et al., 1986). Avoidance of soil water deficits seems to be
a convergent property of CAM in many phylogenetic
groups, and the classic work by Park Nobel and colleagues
has revealed the mechanisms which allow roots to become
effectively isolated from the soil (Nobel, 1988). Shrinkage of
the root cortex occurs even at modest soil water deficits
(approximately –0.1 MPa), which, together with cavitation
of root xylem, helps to protect any reverse flux of water
from storage tissues to soil (Nobel, 1988; North et al.,
2004). In older roots, a sclerified exodermis also helps to
prevent water loss, and aquaporins in the cortex and
endodermis provide short-term control of root hydraulic
conductance (North et al., 2004). In contrast, in younger
roots (and presumably ‘rain’ roots, which may take 3–4 d to
reach maximum hydraulic conductance (Nobel 1988), aquaporins in the epidermis are quantitatively more significant in
regulating water fluxes (North et al., 2004).
By controlling connectivity with the soil, the advantages
conferred by a high water-storage capacitance in CAM
plants translate into an ability to buffer fluctuations in
environmental water availability. This is manifested in high
WUEs for growth compared with other plant life forms
and, in some species, a considerable degree of flexibility in
the partitioning of gas exchange and CO2 uptake between
the day and night. Seasonal measurements of 24 h gas
exchange for six CAM species that were cultivated in semiarid plantations in Mexico without addition of water or
nutrients indicated that 75–97% of total daily net CO2
uptake occurred at night and that total daily net CO2
uptake averaged 823 mmol m2 d1 (Nobel et al., 2002).
The highest values of daily net CO2 uptake reported for
these CAM species exceeds that of nearly all productive C3
and C4 crops and occurred under rain-fed as well as dry
conditions when moderate day/night temperatures prevailed
(Nobel et al., 2002). Thus, CAM plants that have been
exploited agronomically in marginal habitats can display
annual productivities close to those found in the most
productive C3 or C4 agronomic systems (Nobel, 1988,
1991a). For instance, maximal above-ground dry-biomass
productivities of 47–50 Mg ha1 year1 have been recorded
for platyopuntias in Chile and Mexico, and of 42 Mg ha1
year1 for agaves in Mexico (Nobel, 1996). For pineapple,
the most commercially important CAM plant, a maximal
dry-biomass productivity of 35 Mg ha1 year1 has been
observed in Hawaii. These values compare with average
above-ground productivities of ;35 Mg ha1 year1 and
50 Mg ha1 year1 for the most productive C3 and C4
crops, respectively (Nobel, 1991a).
To put this in an agronomic context, we can convert the
standard physiological term WUE (CO2 assimilated per mol
H2O transpired) and figures for above-ground carbon
sequestration into crop water demand (as g H2O required
per g C sequestered in biomass, thereby ignoring water
evaporated directly from soils). Using the crop productivities identified above and typical ranges of WUE reported
for crops with different photosynthetic pathways, Table 2
allows a comparison of calculated values of crop water
demand for CAM, C3 and C4 crops. Thus, CAM crops use
;20% of the water required for cultivation of the most
water-use efficient C3 or C4 crops (although it should be
noted that WUE values may be somewhat higher for CAM
plants growing under cultivation compared with native
CAM species growing in their natural habitats; Nobel,
1994). Agronomists calculate WUE as the combined
evaporative loss from the crop and soil, and hence these
figures only equate to the minimum water use, but the
Table 2. A comparison of typical agronomic traits relating to above-ground dry biomass productivity, water-use efficiency (integrated
over 24 h), and crop water demand for cultivated crops belonging to the different photosynthetic pathways.
Data for average above-ground productivity are taken from Nobel (1991a).
Agronomic traits
Average above-ground productivity [Mg (tonnes) ha
Water use efficiency (mmol CO2 per mol H2O)
Crop water demand (Mg H2O ha1 year1)
Photosynthetic pathway
1
1
year ]
CAM
C3
C4
43
4–10
2580–6450
35
0.5–1.5
14 000–42 000
49
1–2
14 000–28 000
2890 | Borland et al.
precipitation input from a 100 mm rain event equates to
1000 Mg H2O ha1. Since precipitation in the Sonoran
Desert—where agaves and platyopuntias grow abundantly
in their natural habitat—varies between ;200 mm and
400 mm per year, the observations summarized in Table 2
help to illustrate how the budgets for hydrology and carbon
sequestration are interlinked. For marginal semi-arid landscapes, it is clear that the water demand of typical C3 and
C4 crops would exceed the water available from natural
precipitation.
increases in productivity may be facilitated by the high
succulence of CAM species which can accommodate large
increases in chlorenchyma thickness and accumulation of
photosynthate without feedback inhibition of photosynthesis (Nobel, 2000). The succulent nature of the photosynthetic organs of CAM plants and the associated diffusional
constraints to CO2 imposed by densely packed cells could
also be a key factor underpinning the various reports of
stimulation of diurnal and nocturnal CO2 uptake by
elevated [CO2].
Climate change and CAM productivity
Carbon sequestration and ecosystem services
Formulating best agronomic practice and selection of
appropriate cultivars of CAM plants such as cacti and
agaves for biofuel production on marginal land in the 21st
century will require an understanding of how atmospheric
[CO2] may modify plant responses to precipitation variability/
abundance and increases in temperature. For C3 plants,
rising CO2 offers the potential to stimulate crop productivity and offset crop losses caused by greater water and
temperature stress, since Rubisco is not saturated at current
atmospheric [CO2] and photorespiration can result in
significant reductions in plant yield (Bowes, 1991). Whilst
C4 photosynthesis is saturated under ambient [CO2],
stomatal conductance may be reduced by up to 20% under
elevated [CO2], and yield improvements in maize grown
under 550–600 ppm CO2 were achieved through an amelioration of short-term drought stress via the conservation of
soil moisture (Leakey et al., 2004). Given that CAM is
considered to suppress photorespiration (at least for the
middle/hottest part of the day) and is an adaptation to
conserving plant water status under arid and semi-arid
conditions, it might be predicted that elevated CO2 would
have negligible impact on carbon gain and biomass productivity in plants with this photosynthetic pathway. In
reality, a wide and seemingly contradictory range of
responses to elevated [CO2] have been reported in terms of
day/night patterns of gas exchange and WUE in CAM
species. Responses to elevated [CO2] include: decreased
nocturnal CO2 uptake (e.g. Portulacaria afra, Clusia
uvitana: Huerta and Ting, 1988; Winter et al., 1992); no
change in 24 h carbon gain (e.g. K. daigremontiana: Holtum
et al., 1983); an increase in daytime CO2 uptake (e.g. K.
pinnata, Aechmea maya: Winter et al., 1997; Ceusters et al.,
2008); an increase in nocturnal CO2 uptake (A. deserti,
A. salmiana: Graham and Nobel, 1996; Nobel et al., 1996);
and an increase in both day and nocturnal CO2 uptake (e.g.
A. comosus, O. ficus-indica: Nobel and Israel, 1994; Zhu
et al., 1999). The contrasting responses to elevated [CO2] are
a reflection of the inherent photosynthetic plasticity of
CAM. However, for a range of desert CAM succulents,
Drennan and Nobel (2000) demonstrated that alongside
increases in temperature and drought, the daily net CO2
uptake increased by 1% as the atmospheric [CO2] increased
by 10 ppm, and dry biomass production of O. ficus-indica
was stimulated by 40% over a 1-year period of exposure to
750 ppm [CO2] (Nobel and Israel, 1994). Sustained
From a climate change perspective and for the sustainable
management of ecosystem services, detailed budgets comparing above-ground productivity, water use, and soil
organic carbon are urgently required for potential bioenergy crops and their contrasting photosynthetic pathways.
This is particularly relevant for the dense canopies of C4
bioenergy crops, for which water use as well as light
limitation needs to be taken into consideration (Kromdijk
et al., 2008), and for the sustainability of any irrigation
needed to maximize CAM productivity. Carbon sequestration below ground by perennial crops is also an important
positive gain from a climate change perspective, and many
desert soils have a low soil organic matter (SOM) (Amundson
et al., 1994).
To date, there has been limited use of eddy covariance
techniques to evaluate carbon sequestration of bioenergy
crops (but see Kromdijk et al., 2008). However, one highly
innovative study has characterized the carbon balance and
net ecosystem productivity for a pineapple crop (San José
et al., 2007a, b). Here, it was evident that rates of carbon
uptake matched those predicted by Nobel (1991a), whilst
carbon sequestration was affected by both climatic extremes
associated with ENSO (El Niño Southern Oscillation)
events and the seasonality of rainfall. Thus, the eddy flux
system could distinguish the balance between day- and
night-time carbon uptake on a daily and seasonal basis, as
well as resolving crop and soil water use as a function of
soil water availability, stomatal sensitivity, and atmospheric
water deficit (San José et al., 2007a, b). For future studies,
the use of carbon isotopes could help to partition plant and
soil respiration (Kromdijk et al., 2008), as well as determining the contribution that CAM or C4 pathways make
to SOM.
The contribution of CAM to soil carbon sequestration
can be inferred from the isotope composition of SOM, as
shown for sites along the Californian Baja peninsula
(Amundson et al., 1994). The SOM d13C values ranged
from –23.3& to –20.2& along a north to south transect,
which represented a change in vegetation dominance from
27% to 49% CAM inputs, as inferred from isotopic mass
balances. There seem to be no other isotopic analyses of
SOM from CAM-dominated habitats: it would be interesting to know how much carbon from the saguaro-dominated
landscapes of the Sonoran Desert is captured in the soil.
Whilst Carnegia gigantea provides such a striking visual
CAM plants and bioenergy | 2891
dominance of this landscape, it is probable that soil carbon
sequestration in such habitats is mainly derived from the C3
shrubs that dominate the understorey. Habitats supporting
a higher proportion of succulents in the above-ground
vegetation in arid-zone regions of southern Africa and
Madagascar, for example, might provide much more compelling examples of soil carbon storage being dominated
by CAM plants. Indeed, the distinctive semi-deciduous
thicket, or ‘spiny forest’, of southern and south-western
Madagascar (rainfall <600 mm year1) probably represents
the highest standing biomass of CAM vegetation on earth,
dominated by woody members of the endemic family
Didiereaceae, together with numerous species of CAM
plants from the genera Aloe¨, Euphorbia, and Kalanchoe¨,
amongst others (Rauh, 1973; Winter 1979; Grubb, 2003;
Cowling et al., 2005).
In the Eastern Cape of South Africa, the natural
vegetation is dominated by the C3–CAM intermediate
Portulacaria afra, with leaf carbon isotope ratios ranging
from –17.4& to –20.5& (Mills et al., 2005), suggesting that
CAM is more important under natural conditions than was
inferred from early laboratory studies (Guralnick et al.,
1994). The vegetation in this area of South Africa, formerly
known as ‘valley bush veldt’ or more recently as ‘subtropical thicket’, is another striking example of a habitat
consisting predominantly of stem- and leaf-succulent CAM
plants. The subtropical thicket occupies 17% of the land
area, equivalent to some 1.4 M ha1 (or 14 000 km2) of
which 45% is degraded to grassland savanna, and another
35% subject to degradation due to overgrazing (Mills et al.,
2005; Mills and Cowling, 2006). In pristine thicket, the
succulents make up nearly half of the above-ground dry
biomass, with the inducible CAM plant P. afra forming
a dense impenetrable matrix, and acting (probably) as
a nurse plant for a variety of CAM succulents, which all
then help to protect the ;5% of species which are C3.
However, P. afra is particularly palatable, previously
sustaining the diverse native megafauna, but is readily
overgrazed by goats, leading to the conversion into
grassland savanna (Mills et al., 2005).
The ecosystem carbon storage in pristine examples of this
biome can be partitioned into 76 Mg (C) ha1 (biomass)
and 133 Mg (C) ha1 (upper soil profile), and because P.
afra can be readily regenerated from cuttings, it has been
suggested that up to 80 Mg (C) ha1 could be sequestered
by restored thicket (Mills et al., 2005). The focus for this
innovative programme of work undertaken in South Africa
has partly been to provide a scientific basis for evaluating
biodiversity and conservation of natural vegetation (Rouget
et al., 2006). An additional framework has been the
opportunities provided by the Clean Development Mechanism of the Kyoto protocol, which allows such biomes to
receive carbon trading credits (Mills et al., 2005; Mills and
Cowling, 2006). The need to evaluate such developments in
terms of ‘ecosystem services’ has also been pioneered by
these researchers (Cowling et al., 2008; Rouget et al., 2008).
Cowling et al. (2008) show the importance of including
all stakeholders in decision support systems (users, land-
owners, researchers, and policy makers, as well as the
benefits for local communities) during the assessment,
planning/implementation, and management of developments that may have implications for land and water use at
regional scales. Carbon sequestration by this subtropical
thicket biome also has potential for biofuels and bioenergy,
particularly when local solutions in energy generation are
made available for local people. Bioethanol generation
plants, or combined heat and power plants, could be allied
with bioenergy crop plantations to ensure the involvement
(literally the empowerment!) of local communities. Thus,
notions of ecosystem services are equally relevant for
developments involving biomass crops and bioenergy generation, particularly for marginal landscapes where the
tensions between food, fuel, and water availability may be
all too tangible for the future.
Conclusions
To exploit the yield potential of CAM species as feedstock
for biofuel production, a systems-level understanding is
required that spans the molecular and metabolic controls
over the pathway through to the agronomic performance of
CAM in marginal ecosystems. Whilst M. crystallinum and
K. fedtschenkoi are tractable model systems that will allow
great advances in our understanding of CAM, they are not
closely related to CAM species such as Agave that could
potentially achieve very high yields for bioethanol production. Genetic diversity for bioethanol production is
reported to exist within A. tequilana, but the relationship
between such quality-related aspects and photosynthetic
performance is not known for Agave or indeed for any
CAM species. Comprehensive transcriptome sequencing,
proteomic and metabolomic analysis of high-yielding CAM
species would be an important step towards maximizing the
potential of such species as biofuel feedstocks through
selection and breeding.
The application of predictive models of biomass production as part of a whole-system approach to land use and
natural resource management will also play a crucial role in
exploiting the potential of CAM for carbon sequestration
and biofuel production on marginal lands. The power of
predictive biology was harnessed >20 years ago for CAM
plants by Nobel (1988; 1991b) who developed an environmental productivity index (EPI) to inform and improve
agronomic practice for CAM cultivation, as well as to
predict the geographical regions that CAM plants might
successfully exploit in a changing environment. Using EPI,
it was recognized not only that the global range of CAM
cultivation could be extended but also that CAM plants
could play a significant role in terrestrial sequestration of
atmospheric CO2 in arid and semi-arid regions of the world.
Desertification is a worldwide problem directly affecting 250
million people and a third of the earth’s land surface (i.e. >4
billion hectares). In addition, the livelihoods of some 1
billion people who depend on land for most of their needs
and usually the world’s poorest in >100 countries are
2892 | Borland et al.
threatened by land degradation. Such regions are poorly
suited to C3 and C4 crops without irrigation. The substantial biomass increases reported for CAM species under
elevated CO2 on marginal lands indicate that serious
consideration should be directed towards exploring the
potential of CAM plants as a low-input source of bioenergy
and as a means of stimulating sustainable economic growth
in developing countries.
Acknowledgements
We are grateful to our many colleagues within the CAM
research community for providing the platform upon which
this review is built. The Kalanchoe¨ fedtschenkoi sequencing
project is supported by BBSRC (BB/F009313/1 awarded to
JH).
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