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Synchronization of metabolic processes in plants with Crassulacean acid
metabolism
Article in Journal of Experimental Botany · June 2004
DOI: 10.1093/jxb/erh105 · Source: PubMed
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Advance Access published April 8, 2004
Journal of Experimental Botany
Understanding Photosynthetic Performance Special Issue, Page 1 of 11
DOI: 10.1093/jxb/erh105
Synchronization of metabolic processes in plants with
Crassulacean acid metabolism
Anne M. Borland* and Tahar Taybi
Environmental and Molecular Plant Physiology, School of Biology, King George VI Building,
University of Newcastle, Newcastle Upon Tyne NE1 7RU, UK
Received 28 October 2003; Accepted 19 January 2004
In plants with Crassulacean acid metabolism, a diel
separation of carboxylation processes mediated by
phosphoenolpyruvate
carboxylase
(PEPC)
and
Rubisco optimizes photosynthetic performance and
carbon gain in potentially limiting environments. This
review considers the mechanisms that synchronize
the supply and demand for carbon whilst maintaining
photosynthetic plasticity over the 24 h CAM cycle.
The circadian clock plays a central role in controlling
many of the metabolic, transport and physiological
components of CAM. The level of control exerted by
the clock can range from transcriptional through to
post-translational regulation, depending on the
genes, proteins, and even plant species under consideration. A further layer of control is provided by
metabolites, including organic acids and carbohydrates, which show substantial reciprocal ¯uctuations in content over the diel cycle. Mechanisms
responsible for the sensing of metabolite contents
are discussed, together with signalling requirements
for the co-ordination of carbon ¯uxes. Evolutionary
implications are considered in terms of how circadian
and metabolic control of the CAM cycle may have
been derived from C3 plants.
Key words: CAM, circadian control, metabolite partitioning,
PEPC.
Introduction
Crassulacean acid metabolism is a specialized mode of
photosynthetic carbon assimilation that has evolved in
response to exceptional environmental conditions. To date,
approximately 7% of plant species, encompassing 33
families and 328 genera are known to possess a capacity
for CAM (Winter and Smith, 1996a). Such taxonomic
diversity is re¯ected by the range of habitats favoured by
CAM plants which range from arid deserts, through
tropical rainforests to aquatic ecosystems. CAM is a CO2
concentrating mechanism that employs the enzyme
phosphoenolpyruvate carboxylase (PEPC) for the capture
of respiratory and atmospheric CO2 at night. The physiological signi®cance of CAM is to conserve carbon and
water in plants growing in environments that restrict the
availability of either or both resources on an intermittent or
longer-term basis. Whilst the enzymatic machinery
required for CAM is present in all higher plants, evolution
of the pathway required a change in the regulatory capacity
of key enzymes and transporters in order to sustain the
temporal separation of carboxylation processes that are
central to CAM. The aim of this review is to consider the
mechanisms that might synchronize the supply and
demand for carbon over the 24 h CAM cycle. Molecular
approaches and emerging genomic resources provide an
unprecedented potential for exploiting the diel CAM cycle
to elucidate components of circadian and metabolite
control that optimize photosynthetic performance in
potentially limiting and extreme environments.
In essence, CAM may be expressed on a background of
C3 photosynthesis via the deployment of nocturnal
carboxylation and subsequent day-time decarboxylation
processes (Fig. 1a). At night, when evapotranspiration
rates are low, atmospheric CO2 and/or respiratory CO2 are
®xed in the cytosol by the enzyme phosphoenolpyruvate
carboxylase (PEPC). The 3-C substrate, phosphoenolpyruvate (PEP), is produced via the glycolytic breakdown
of carbohydrate formed during the previous day. The ®nal
4-C product, malic acid, is stored in a large central vacuole.
* To whom correspondence should be addressed. Fax: +44 (0)191 222 5228. E-mail: a.m.borland@ncl.ac.uk
Journal of Experimental Botany, ã Society for Experimental Biology 2004; all rights reserved
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Abstract
2 of 11 Borland and Taybi
A circadian clock sets the diel phases of CAM
The well-documented rhythms of gas exchange in species
of the genus KalanchoeÈ, grown under continuous darkness
and CO2-free air, have indicated that an endogenous
circadian clock plays a cardinal role in establishing and
synchronizing the temporally separated metabolic
components of CAM (Wilkins, 1992; LuÈttge, 2000).
Circadian control of carbon ¯ux through PEPC is generally
regarded as a key component underpinning the day/night
Fig. 1. The major metabolic components of the CAM cycle.
Schematic outline (a) illustrating the day (light background) and night
(dark background) separation of the major metabolic and transport
processes for carbon. The partitioning of carbohydrates between
growth and storage (whether as soluble sugars in the vacuole or as
starch in the chloroplast) is an important point of control for the diel
cycle. The net results of this diel separation of metabolism (b) are
reciprocating levels of organic acids (mainly malate) and carbohydates
which can represent up to 20% of leaf dry weight. The solid bar on
the x-axis indicates the dark period and metabolite data were collected
from Clusia ¯uminensis (A Borland, unpublished results).
separation of carboxylation processes that de®ne CAM
(Nimmo, 2000). PEPC is activated at night via phosphorylation of a serine residue near the N-terminus of the
protein which renders the enzyme more sensitive to PEP
and the positive effectors, glucose-6-P and triose-P and
less sensitive to the allosteric inhibitor, malate (Fig. 2;
Nimmo et al., 1986, 1987; Chollet et al., 1996). The timeframe over which PEPC remains active, as indicated by
patterns of nocturnal malate accumulation, is thus mirrored
by diel changes in the apparent phosphorylation state of the
enzyme, as indicated by [malate] required for 50%
inhibition of enzyme (i.e. Ki malate). Moreover, the degree
of PEPC phosphorylation is a major determinant of the
amount of CO2 taken up and stored as malate overnight in
different CAM species, where the extractable activity of
PEPC varied by no more than 10% (Fig. 3).
The phosphorylation state of PEPC is determined by the
presence or absence of a dedicated Ca2+-independent Ser/
Thr protein kinase, which, in turn, is regulated at the level
of gene expression by a circadian oscillator (Fig. 2; Carter
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During the day, malate exits the vacuole and decarboxylation may occur through the single or combined action of
three carboxylases (depending on plant species): NADPmalic enzyme (NADP-ME), NAD-ME, and phosphoenolpyruvate carboxykinase (PEPCK). In addition to the 3-C
products PEP or pyruvate, CO2 is released at a high
internal partial pressure (pCO2) which is often suf®cient to
result in stomatal closure and thus conserve water. The
high pCO2 generated via decarboxylation also suppresses
photorespiration. The recovery of carbohydrate via
gluconeogenesis imposes a high energetic cost on the
pathway, but ensures the production of substrate for
subsequent nocturnal carboxylation and partitioning for
growth. The carbohydrates that will provide substrates
for the nocturnal reactions are transported either into the
chloroplast and stored as starch or transported into the
vacuole and stored as sucrose and/or hexose, depending on
species (Christopher and Holtum, 1996). In terms of net
carbon ¯ux, the result of the reactions outlined in Fig. 1a
are substantial diel and reciprocating ¯uctuations in malate
and carbohydrate contents (Fig. 1b) which can represent up
to 20% of leaf dry weight.
Despite the energetic costs associated with CAM, in
many cases the potential for high productivity is not
compromised. Agronomically important CAM species
including pineapple (Ananas comosus) and several of the
Agaves, can show productivities rivalling that of sugar
cane (Bartholomew and Kadzimin, 1977; Nobel, 1996).
Such desirable attributes are a consequence of the plasticity by which CAM may be engaged or disengaged in
response to intermittent or longer-term (seasonal) environmental perturbations. Developmental and environmental
factors (e.g. water availability, light intensity) strongly
in¯uence the proportion of CO2 taken up at night via PEPC
and directly during the day via Rubisco in CAM plants
(Cushman and Borland, 2002; Dodd et al., 2002). Growth
and productivity of most CAM plants are maximal when
direct daytime ®xation of CO2 via Rubisco (Phase IV gas
exchange) predominates. As a de®ning feature of CAM,
photosynthetic plasticity must be achieved whilst
maintaining synchronization between the carboxylation,
decarboxylation, and transport processes outlined in Fig. 1a
in order to minimize futile cycles of carbon turnover over
the diel cycle.
Metabolic synchronization of CAM 3 of 11
et al., 1991; Hartwell et al., 1996, 1999; Taybi et al.,
2000). Theoretically, the control of PEPC kinase (PPCK)
activity by an endogenous clock should enable an anticipation of the photoperiod and ensure a rapid inactivation of
PEPC at the start of the day, thereby avoiding futile cycling
of malate synthesis and decarboxylation. However, the
ecological advantage of such circadian control of CO2
uptake is less clear, given the inherent plasticity of CAM
plants for modulating carbon ¯ux in response to changing
environmental conditions. Field-based measurements of
instantaneous carbon isotope discrimination on hemiepiphytic stranglers of the genus Clusia have indicated
that PEPc can remain active for 4±5 h after dawn (Borland
et al., 1993; Roberts et al., 1997). Figure 3 illustrates that
the timing of PEPC inactivation can vary between different
CAM species even when grown under identical
environmental conditions.
Metabolites provide a further layer of control
over the diel CAM cycle
Experimental observations now indicate that PPCK
expression and activity can be modi®ed by leaf metabolic
status. In leaves of KalanchoeÈ daigremontiana that were
prevented from accumulating malate by enclosure in an
atmosphere of N2 for part or all of the night, dramatic shifts
in the amplitude and duration of CO2 uptake by PEPC were
observed after transfer to ambient air (Fig. 4a; Borland
et al., 1999). The stimulation of CO2 uptake via PEPC at
night and the start of the day were attributed to shifts in the
magnitude of PPCK activity (Fig. 4b) which are controlled
at the level of gene expression (Hartwell et al., 1999;
Fig. 3. Diel regulation and apparent phosphorylation status of PEPC
in contrasting CAM species. The malate sensitivity of PEPC (a) and
leaf malate content (b) in leaves of KalanchoeÈ daigremontiana (®lled
circles), Clusia minor (open circles) and Tillandsia usneoides (open
diamonds) grown under identical controlled conditions with the solid
bar on the x-axis indicating the period of darkness. Graphs have been
compiled from data presented by Borland and Grif®ths (1997) and
Haslam et al. (2002).
Fig. 4. Metabolite control of PEPC phosphorylation and net CO2
uptake. Diel patterns of net CO2 uptake (a) in leaves of KalanchoeÈ
daigremontiana under ambient air (control) or maintained in an
atmosphere of N2 for the ®rst 6 h (half N2) or the entire 12 h (full N2)
of the dark period (indicated by the solid bar on the x-axis). Enclosure
in N2 prevents the accumulation of malate and subsequent release
from N2 results in a doubling of net CO2 uptake via PEPC compared
with controls. The increase in net CO2 uptake in N2-treated leaves is
accompanied by an increase in the activity (b) and expression (data
not shown) of PEPC kinase, the protein responsible for the
phosphorylation of PEPC. Data have been re-drawn from Borland et al.
(1999).
Borland et al., 1999). The observations are consistent with
the view that cytoplasmic malate (or a related metabolite)
elicits feedback inhibition of PPCK gene expression. Thus,
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Fig. 2. Regulation of phosphoenolpyruvate carboxylase in CAM
plants. The Ppc gene can be rapidly induced by environmental factors
(water stress, salt stress, etc) or as a long-term response to the change
in the photoperiod through control by a circadian oscillator. Ppc
transcipts also oscillate on a circadian basis, but this change does not
translate into changes in PEPC protein amounts over the 24 h cycle.
Diel regulation of PEPC is controlled via nocturnal phosphorylation
catalysed by a dedicated kinase (PEPC-kinase). The Ppck gene can be
induced by salt-stress in some species (Mesembryanthemum
crystallinum, sorghum) and is controlled by the circadian oscillator
and/or metabolites (organic acids and sugars). Ppck transcripts
accumulate mainly during the night in CAM plants resulting in
nocturnal PEPC-kinase accumulation and PEPC phosphorylation.
Phosphorylated PEPC is more active, more sensitive to activation by
glucose-6-phosphate and triose phosphate, and less sensitive to
inhibition by malate.
4 of 11 Borland and Taybi
Which components of CAM are regulated by the
clock?
Clock-controlled genes
Analyses of approximately 40 genes in the inducible CAM
plant, Mesembryanthemum crystallinum have indicated
rhythmic changes in transcript abundance of more than 30
of the selected genes with expression peaks at various
phases throughout the diel cycle (Boxall et al., 2001,
2002). Moreover, more genes show rhythmic changes in
transcript abundance in plants expressing CAM compared
with plants in the C3 state (Boxall et al., 2001, 2002). One
way in which the clock could synchronize the metabolic
components of CAM would be to phase the transcription of
particular genes to speci®c times in the day/night cycle,
thus ensuring that appropriate enzymes and transporters
are most abundant when required. Genes found to be
controlled by the clock in CAM-performing M. crystallinum encode enzymes involved in photosynthesis, glycolysis, nocturnal CO2 uptake, decarboxylation, sucrose and
starch metabolism (Dodd et al., 2003), chloroplast
metabolite transport, and the vacuolar ATPase (Boxall
et al., 2001, 2002). Thus, the major metabolic components
of CAM, as outlined in Fig. 1a, appear to be subject to an
element of control exerted by the circadian clock. The ongoing analyses of M. crystallinum gene chips, containing
8400 genes, will undoubtedly give a fascinating insight on
which genes fall under the control of the clock as CAM is
induced (J Cushman, personal communication).
Although it is tempting to speculate that a key event in
the evolution of CAM was the coupling of more/different
genes to a central oscillator, caution is required in
interpreting the functional signi®cance of rhythmic
changes in transcript abundance. In the C3 plant,
Arabidopsis thaliana, microarray experiments have
shown that at least 6% of genes are rhythmically expressed
(Harmer et al., 2000; Schaffer et al., 2001). However,
more recent approaches using in vivo enhancer trapping
have indicated that 36% of the Arabidopsis genome is
potentially under transcriptional control by the circadian
clock (Michael and McClung, 2003). The discrepancy
between these reports may be attributed to differences in
transcript stability between genes. Thus, for oscillations in
transcription to yield oscillations in mRNA abundance
requires that the transcript be suf®ciently unstable to turn
over within a circadian cycle. The induction of CAM
in M. crystallinum is accompanied by an increase in
transcript stability of the CAM-speci®c isoform of PEPC
but a decrease in stability of transcripts encoding the small
sub-unit of Rubisco (Cushman et al., 1990; DeRocher and
Bohnert, 1993). Thus, regulation of a complex metabolic
pathway like CAM by the circadian clock is likely to
involve several layers of control, from transcription
through to post-translational protein modi®cation,
operating on any number of enzymes or transporters.
Post-transcriptional control by the clock
In order to determine the physiological relevance of
circadian oscillations in transcript abundance, levels of the
amount/activity of the corresponding enzymes must also
be assessed. For example, whilst PPCK activity is reported
to be regulated at the level of gene expression (Hartwell
et al., 1999; Taybi et al., 2000), transcript abundance of the
CAM-speci®c isoform of PEPC also displays diurnal and
circadian oscillations in M. crystallinum (peaking towards
the end of the photoperiod), although PEPC protein and
extractable activity remain relatively constant (J Hartwell,
personal communication; Boxall et al., 2001). Increased
day-time transcript abundance of PEPC has also been
noted in CAM-performing species of Clusia but not in the
C3 species C. multi¯ora (T Taybi, unpublished observations). In all of these CAM species, one important role for
the clock may be to control the turnover of PEPC protein,
and refresh the level of enzyme each night. A further layer
of circadian control is then provided by rhythmic shifts in
the expression and activity of PPCK, which, in turn, will
activate the PEPC protein at night. As more data on
rhythmic gene expression becomes available from microarray comparisons of C3 and CAM-performing M. crystallinum, it will be important to link such studies with
conventional biochemical measurements of the amount
and activities of corresponding enzymes and transporters.
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in plants curtailed from accumulating malate, PPCK
activity and expression is elevated over that in controls
and there is a delay in the down-regulation of PPCK in
plants with reduced malate content (Fig. 4; Borland et al.,
1999). Consequently, it has been suggested that the
circadian control of PPCK gene expression in CAM is a
secondary response to circadian changes in malate transport across the tonoplast membrane of the vacuole
(Nimmo, 2000). Independent evidence in support of the
tonoplast membrane as the `master switch' for circadian
regulation of CAM has been provided from computer
simulations of CAM rhythms based on osmotic considerations of malate turnover and a tonoplast tension/relaxation mechanism (LuÈttge, 2000, 2002a). Since shifts in
day/night changes in malate content are a distinguishing
feature of the plasticity of CAM, metabolite control of
PEPC phosphorylation would provide an effective means
of ®ne-tuning CO2 uptake over the day/night cycle to
changes in environmental conditions. Such integration of
circadian and environmental signals could provide the
basis for the synchronization and plasticity of metabolism
that is inherent to CAM. However, such an hypothesis
raises further questions in terms of how many metabolic
components of the CAM cycle are directly connected to
the circadian oscillator and how many are contingent upon
the day/night ¯uxes of metabolites across the tonoplast?
Metabolic synchronization of CAM 5 of 11
Such information will help to determine if the expression
of CAM requires not only a change in the complement of
genes that are linked to the clock, but also necessitates
a shift in the level of control (transcriptional or
post-transcriptional) that is exerted by the clock.
Metabolite control of CAM
A number of genes show rhythmic changes in transcript
abundance only after CAM is induced in M. crystallinum
(Boxall et al., 2001, 2002), and it is possible that many of
these rhythms might arise as a downstream consequence of
Fig. 5. Diel expression of PEPC kinase in Clusia species. Day (3 h
and 6 h) and night (15 h and 18 h) steady-state levels of Ppck1 and
Ubq1 (control) transcripts in leaves of Clusia multi¯ora (C3 species),
C. aripoensis (C3), C. minor (CAM), and C. rosea (CAM) grown
under identical controlled conditions.
the metabolite cycling associated with CAM. The issue of
metabolite control is also pertinent for understanding how
individual oscillators, as proposed above, might be integrated in space and time to co-ordinate diel CO2 exchange
under diverse environmental conditions. Substantial reciprocal cycling of organic acids and carbohydrates occurs
over the 24 h CAM cycle (Fig. 1b) and both categories of
metabolites are known to affect expression of a diverse
range of genes (Smeekens, 2000; Stitt et al., 2002).
Organic acid sensing and signalling
Recent evidence in support of metabolite-induced cycling
of existing C3 genes is provided by comparisons of PEPC
kinase (PPCK) expression in a range of Clusia species that
show marked differences in the capacity for CAM. The
capacity for CAM in Clusia appears to be determined by
the amount of PEPC protein (Borland et al., 1998), which,
in turn, is regulated at the level of PEPC transcript
abundance (Taybi et al., 2004). However, the gene that
encodes PPCK, the protein responsible for activating
PEPC, is expressed at comparable levels in leaves of both
C3 and CAM-performing species of Clusia and day/night
changes in the expression of PPCK appear to be a
consequence of the diel cycling of organic acids and
soluble sugars (Fig. 5; Taybi et al., 2004). Moreover, diel
changes in PPCK transcript abundance appear to be
controlled through a down-regulation of the gene during
the day rather than an up-regulation during the night
(Fig. 5). These ®ndings contrast with results obtained with
M. crystallinum in which PPCK transcripts are present in
low abundance in C3 leaves and where the induction of
CAM is accompanied by an up-regulation of PPCK
transcript abundance at night (Taybi et al., 2000). The
day-time ef¯ux of malate (or some other metabolite) from
the vacuole could be a primary signal for the downregulation of PPCK expression during the photoperiod in
Clusia species once CAM is induced. The subsequent
decarboxylation of organic acids will be a key factor
in¯uencing the increase in PPCK expression for the
following night. It is interesting to note that Clusia species
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Physiological processes controlled by the clock
A complementary approach to tackling the question of
which components of the CAM pathway are regulated by
the clock, has been to exploit the overt circadian rhythms
of leaf gas exchange found in some species in order to
examine the higher levels of metabolic organization that
fall under the control of a circadian oscillator. Recent
analyses of the stoichiometry of CO2 uptake and malate
accumulation in M. crystallinum and K. daigremontiana
during free-running rhythms of gas exchange indicate that
C3 carboxylation makes a major contribution to the
generation of rhythmic CO2 uptake under continuous
light (Dodd et al., 2002, 2003; Wyka and LuÈttge, 2003).
Under extended periods of continuous light, the dampening of PEPC activity appears to be more rapid than the
dampening of the overt rhythm in net CO2 uptake. This
indicates that whilst endogenous control of PEPC phosphorylation is not sustainable under continuous light,
another and possibly separate C3-related oscillator maintains the rhythms in net CO2 uptake (Wyka and LuÈttge,
2003). It is presently unclear whether this rhythmic activity
in C3 carboxylation can be directly attributed to circadian
control of Rubisco activation/activity or to stomatal
conductance. Both of these aspects of metabolism are
known to be subject to at least some element of circadian
control in C3 spp (Liu et al., 1996; Webb, 2003). The
concept that overall control of overt rhythms in CO2
uptake in CAM plants is mediated through oscillations in
stomatal aperture has been discussed elsewhere (LuÈttge,
2002a; Wyka and LuÈttge, 2003). Within this framework,
the circadian oscillators that directly control PEPC and
Rubisco might constitute other components of a network
that stabilize and co-ordinate diel CO2 uptake in response
to metabolite feedback loops and environmental cues
(Dodd et al., 2003). Such considerations are consistent
with the idea that sustained rhythms of leaf gas exchange
are not necessarily controlled by one central clock
mechanism that might operate at the level of gene
transcription. Rather, free-running oscillations in net CO2
uptake appear to be directed by complex regulatory
networks that are integrated in time and space (LuÈttge,
2000, 2002a; Rascher et al., 2001).
6 of 11 Borland and Taybi
Carbohydrate sensing and signalling
Carbohydrate availability is a major limiting factor for
dark CO2 uptake in CAM plants (Borland and Dodd,
2002). In leaves of M. crystallinum that were depleted in
carbohydrates by 50% via exposure to CO2-free air for
24 h, subsequent net dark CO2 uptake in ambient air was
reduced by 50% relative to controls, despite a marked
increase in PEPC-kinase expression (Dodd et al., 2003).
Evidence for sensing of carbohydrate (and possibly
organic acid) de®cits in plants curtailed in nocturnal CO2
uptake is also indicated by shifts in subsequent day-time
net CO2 uptake and carbohydrate partitioning, which can
compensate for the previous nocturnal shortfall in carbon
gain (Roberts et al., 1997; Borland and Grif®ths, 1997;
Dodd et al., 2002). Thus, carbohydrate status appears to be
a key element in synchronizing metabolic ¯uxes over the
diel CAM cycle in line with changes in environmental
conditions.
Carbohydrate status is determined by an interplay of
circadian and metabolite control which regulate turnover
and subcellular partitioning over the diel cycle. The beststudied CAM models (i.e. M. crystallinum, K. daigremontiana) produce predominantly starch in the chloroplast
as the transitory carbon reserve to support nocturnal
carboxylation and malate synthesis. In C3 plants it has been
suggested previously that diurnal starch accumulation is
under circadian control (Li et al., 1992; Geiger et al.,
1995). For CAM-performing M. crystallinum, circadian
control over the major rate-controlling step in starch
biosynthesis catalysed by ADP-glucose pyrophosphorylase has been suggested (Boxall et al., 2001, 2002;
J Hartwell, personal communication). A key role for
chloroplast transporters in regulating carbohydrate turnover over the diel cycle is indicated by the ®nding that
chloroplasts of CAM-performing leaves of M. crystallinum
are unique in containing three classes of phosphate
translocators (i.e. PEP, triose-P, and glucose-6-P translocators), in addition to a chloroplast glucose transporter
(HaÈusler et al., 2000). Moreover, dynamic diel and
circadian regulation of the transcript abundance and
activity of these chloroplast translocators is apparent
when CAM is induced (HaÈusler et al., 2000; Boxall et al.,
2001, 2002). The PEP, triose-P, glucose-6-P, and glucose
chloroplast transporters may play key roles in sensing
carbohydrate and associated metabolite levels and, consequently, modulating transport activities in a manner that
maintains the reciprocating metabolic ¯uxes during the
CAM cycle (HaÈusler et al., 2000).
Rhythmic changes in transcript abundance of several
genes encoding enzymes implicated in starch degradation
have also been reported in M. crystallinum, with transcript
abundance peaking before the end of the (subjective)
photoperiod (Boxall et al., 2001, 2002; Dodd et al., 2003).
Circadian control of starch degradation in M. crystallinum
could provide a means of anticipating the substrate
requirements of nocturnal carboxylation by ensuring the
retention of adequate carbohydrate reserves during the
photoperiod. However, it is also likely that mechanisms
exist for `sensing' carbohydrate availability throughout the
night since the shift from C3 to CAM results in sustained
`metering' of carbohydrate reserves, which maintain
substrate availability for the duration of the dark period
(Dodd et al., 2003). It is currently unclear if this apparent
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can accumulate high concentrations of both malic and
citric acids and background concentrations of both organic
acids can be relatively high in plants performing C3
photosynthesis (Borland et al., 1996). Again, this contrasts
with the situation in M. crystallinum where both malate
and citrate contents are low in the C3 mode. Thus, in
Clusia, it would appear that day-time mobilization of
organic acids is a key factor regulating both the expression
of CAM and PEPC-kinase (Borland et al., 1996; Roberts
et al., 1997). The marked phenotypic plasticity of plants
such as C. minor for switching rapidly and reversibly
between C3 photosynthesis and CAM may thus be
attributed to the transport and/or enzymatic processes
that regulate partitioning of metabolites, particularly
organic acids, between vacuole and cytosol.
The transport steps that mediate the in¯ux and ef¯ux of
malic and citric acids across the tonoplast membrane will
be a central component in organic acid sensing and
signalling in CAM plants. Nocturnal malate accumulation
involves the stoichiometric transport of 2H+ per malate
with H+ transport driven by a tonoplast H+-ATPase and,
additionally by an H+-PPiase (Smith et al., 1996).
Circadian ¯uctuations in transcript abundance of the
V-ATPase subunit C have been described in M. crystallinum (Rockel et al., 1997), but it is not known if the
activity of the V-ATPase is subject to circadian regulation.
Although the actual organic acid transporters have not yet
been identi®ed at the molecular level, it seems likely that
distinct in¯ux and ef¯ux systems exist on the tonoplast of
CAM plants (Smith et al., 1996). The in¯ux of malate
across the tonoplast appears to be mediated by a voltagedependent anion-selective channel (Hafke et al., 2003).
This channel responds to cytoplasmic pH in a manner that
is consistent with nocturnal uptake of the malate anion and
which helps to minimize futile cycling of malate back into
the vacuole during the day-time phase of de-acidi®cation.
A tonoplast carboxylate transporter that appears capable of
transporting both malate and citrate has recently been
cloned from A. thaliana (Emmerlich et al., 2003).
Moreover, the transcript abundance of this transporter
can be modulated in response to malate concentration
(Emmerlich et al., 2003). A CAM-orthologue of the
Arabidopsis carboxylate transporter could potentially act
as a key sensor for organic-acid mediated signalling over
the diel CAM cycle by regulating ¯ux across the tonoplast
in response to malate concentration.
Metabolic synchronization of CAM 7 of 11
CO2 sensing and signalling
The large, highly vacuolated and densely packed cells of
the leaves of many CAM plants, present a substantial
constraint to the diffusion of CO2 both into and out of the
leaf. This can potentially result in a 450-fold drop in pCO2
in a matter of a few hours from the time of maximal
decarboxylation in Phase III to the time when direct uptake
of atmospheric CO2 occurs during Phase IV (Maxwell
et al., 1997). Thus, over the diel CAM cycle, the shifts in
C3 and C4 modes of carboxylation result in ¯uctuations in
internal CO2 concentration that can range from 0.011% to
5%. The increase in pCO2 is believed to be a major internal
factor for controlling stomatal closure in Phase III (Bohn
et al., 2001). Sensing of pCO2 during the shift from Phase
III to Phase IV is also indicated at a metabolic level by an
increase in the activation state of Rubisco, which has been
suggested to maintain the drawdown of CO2 during Phase
IV (Maxwell et al., 1999: Maxwell, 2002; Grif®ths et al.,
2002). LuÈttge (2002b) has also considered the role of pCO2
as a signal for co-ordinating the rate of CO2 consumption
by Rubisco and malic acid remobilization from the vacuole
during Phase III of CAM. Thus pCO2 sensing and
signalling may represent yet another integral component
of the mechanisms that synchronize carbon ¯uxes over the
diel cycle in CAM plants.
Integration of environmental and metabolic
signals with the circadian clock
The mechanism underpinning circadian clocks in all
organisms is based on delayed negative loops regulating
genes involved in the core of the oscillator (Dunlap, 1999;
Harmer et al., 2001). These loops involve negative
regulators that feed back to repress their own expression
by blocking positive acting elements (Harmer et al., 2001;
Young and Kay, 2001). Different levels of control
including post-transcriptional regulation and associated
interlocked feedback loops provide the mechanisms to
ensure clock stability and robustness under changing
conditions and to reset the phase of the clock by
environmental signals, particularly light (Shearman et al.,
2000). In Arabidopsis, the circadian clock includes a set of
genes, some of which are well characterized transcription
factors (i.e. the pseudo response regulator TOC1: Timing
of CAB Expression), and factors from the Myb family
(CCA1: Circadian Clock Associated and LHY1: Late
Elongated Hypocotyl). These factors regulate the expression of each other in a negative feedback loop resulting in
the approximate 24 h period. The lack of expression of
TOC1 causes arrhythmia, whilst CCA1 and LHY1 genes,
which have overlapping functions, participate in controlling the period of the clock. Mutants lacking the
expression of CCA1 and LHY1 are unable to maintain
sustained oscillations in either constant light or darkness
(Alabadi et al., 2002). The circadian expression of these
clock genes control the downstream expression of a variety
of genes involved in many vital processes, including
photosynthetic metabolism, leaf movements, and ¯owering. Work is currently in progress to identify homologue
genes of TOC1, CCA1, and LHY1 in the inducible CAM
plant, M. crystallinum (J Hartwell, personal communication). The results obtained will help to elucidate how the
clock-control of metabolic processes is engaged as CAM is
induced. Moreover, studies of such clock genes could
reveal an additional role for the clock in regulating the
long-term induction of CAM. In species such as
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sensing of carbohydrate levels is determined by the pull
from PEPC for 3-C substrate or by direct modulation of the
rate of carbohydrate degradation. However, recent results
obtained with a CAM-de®cient mutant of M. crystallinum
indicate that the activity of starch-degrading enzymes can
be modulated in line with substrate availability. In a
mutant line isolated by John Cushman's group, the failure
to accumulate malate overnight appears to be correlated
with low starch content (Branco et al., 2003). In these
CAM and starch-de®cient plants the activity and diel
¯uctuation of chloroplastic starch phosphorylase is also
reduced, compared with the wild type (A Borland and
J Cushman, unpublished observations). Similarly, the
mutant shows lower activities and reduced diel ¯uctuations
of a number of glucan hydrolases, indicating that starch
degradative activity can be adjusted in line with substrate
availability (A Borland and J Cushman, unpublished
observations). Thus, starch degradation in M. crystallinum
appears to be subject to circadian control at the level of
gene expression and metabolite control at the level of
enzyme activity.
In many CAM plants, including the crop species Ananas
comosus (pineapple), Aloe vera, and Agave species,
vacuolar soluble sugars are the predominant form of
carbohydrate accumulated during the day to support the
CAM cycle (Winter and Smith, 1996b). The day/night
¯uxes of sugars across the tonoplast membrane in pineapple can be substantial (up to 20% of leaf dry biomass)
and are a major determinant of CAM expression. A
putative vacuolar sucrose transporter has been described in
tonoplast vesicles of A. comosus that shows the kinetic
characteristics of a sucrose uniporter and which appears
capable of facilitating substantial ¯uxes of sugars into the
vacuole (McRae et al., 2002). Studies on the plasma
membrane of C3 plants have indicated that the expression
of sugar-transport transcripts can be highly responsive to
metabolic and environmental signals and the gene products
can be involved in functions such as sugar sensing and
signalling in addition to transport (Williams et al., 2000).
Thus, the ¯ux of sugars across the tonoplast may represent
a strategic checkpoint for the integration of circadian and
metabolite control of the diel cycle in CAM species that
accumulate soluble sugars.
8 of 11 Borland and Taybi
Evolutionary implications and conclusions
Two fundamental layers of control preside over the
metabolic components of CAM. Control by a circadian
clock sets up the diel phases of CAM and achieves
appropriate synchronization of metabolic and transport
processes by phasing the transcription of particular genes
to speci®c times in the day/night cycle. This circadian
control is overlain by metabolite control, acting at both the
transcriptional and post-transcriptional levels, which
facilitates photosynthetic plasticity by entraining output
from the clock to ¯uctuations in the environment. The
reciprocating pools of organic acids and carbohydrates are
central to the metabolic control of CAM. Membranelocalized transporters and sensors for organic acids, sugars,
and metabolic intermediates thus occupy strategic checkpoints for integrating the circadian and metabolic signals
that synchronize and modulate the diel phases in response
to the environment. The tonoplast, in particular, plays a
key role in orchestrating metabolism over the day/night
cycle by controlling the uptake and release of organic acids
to the cytosol. Indeed, in some CAM species (e.g. Clusia),
diel ¯uctuations in transcript abundance of genes encoding
key CAM enzymes (i.e. PEPC kinase), appear to be a
downstream consequence of metabolite cycling across the
tonoplast (Fig. 5).
The integrated mechanistic overview of the CAM cycle
described above raises the question as to how the
metabolic control features of CAM evolved from C3
photosynthesis. If CAM evolved by a series of incremental
modi®cations to existing features, it is important to
consider the attributes of CAM-progenitors that presented
a predisposition towards the development of both circadian
and metabolic-control components of the pathway. An
initial step in CAM evolution is thought to encompass the
nocturnal scavenging of respiratory CO2 by PEPC, a
feature that was probably facilitated by the succulent and
large vacuolated leaf anatomy that is typical of CAM
species (Grif®ths, 1989; Sage, 2002). Succulence may
have been a pre-existing trait that improved hydraulic
transport and capacitance in habitats with high transpiration demand (Sage, 2002). The tightly packed cells of
succulent leaves would have minimized leakage of
respiratory CO2 out of the leaf whilst the enlarged vacuole
was essential for storing the ®xed CO2 as organic acids
over the night. Since the vacuole typically occupies c. 97%
of cell volume and the cytoplasm c. 1% in the leaves of
CAM plants, the cytoplasm could easily be ¯ooded with
the vacuolar contents, thereby disrupting metabolic
homeostasis. Thus, transport across the tonoplast must be
tightly regulated, indicating that an early event in terms of
the metabolic control of CAM may have been an increase
in the regulatory capacity of tonoplast transporters. This
could have been achieved by a diversi®cation in the
complement of tonoplast transporters (e.g. separate transporters for in¯ux and ef¯ux of organic acids) together with
a change in the regulatory properties of transporters. At the
molecular level, such qualitative and quantitative changes
in tonoplast transporters could have evolved via gene
duplication followed by speci®c regulation of one or more
isogenes, as generally proposed for several of the major
metabolic components of CAM (Cushman and Bohnert,
1999). The importance of the vacuole in maintaining
cellular homeostasis may also have established the
tonoplast as a key (if not the primary) component of the
circadian oscillator that controls CAM.
In considering the evolutionary origins of the circadian
control of CAM, it is also pertinent to consider if the
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K. blossfeldiana and M. crystallinum, the long-term
developmental control of CAM expression can be correlated with a change in the length of the photoperiod
(Brulfert et al., 1975; Edwards et al., 1996). Whilst this
developmental transition to CAM can be accelerated by a
variety of environmental factors (e.g. drought, salinity,
high light), by sensing a change in the length of the
photoperiod, the circadian clock may establish metabolic
and physiological adjustments in anticipation of a change
in environmental conditions (Taybi et al., 1995, 2002;
Taybi and Cushman, 1999). In both K. blossfeldiana and
M. crystallinum the photoperiodic induction of CAM is
always accompanied by ¯owering and seed production. It
will be of future interest to establish if long-term (seasonal)
responses such as CAM induction and ¯owering and shortterm (diel) responses are controlled through the same
clocks or sets of clock genes.
It is clear that external (e.g. light, temperature) and
internal factors (metabolites, hormones) can act on both
the amplitude and the period of the basic rhythms
generated from the clock in CAM plants. Such modulation
is necessary in order to optimize the use of resources on a
diel time-scale and to ensure co-ordination of the developmental progression of CAM with seasonal change. It is
currently unclear if these external and internal factors act
directly on the clock genes or act downstream to entrain
metabolic output from the clock. However, the tonoplast
membrane remains a strong candidate for the integration of
metabolic and circadian control of the diel CAM pathway.
LuÈttge (2000, 2002a) has proposed that the biophysical
tension/relaxation of the tonoplast represents the primary
oscillator or pacemaker for CAM. Another conceivable
model for the tonoplast oscillator hypothesis involves
the synthesis of a peptide that is incorporated into the
membrane, where it has negative feedback on its own
synthesis (LuÈttge et al., 2002a). Direct circadian control of
a peptide associated with the transport of malate, a peptide
that, in turn, regulates its own expression, would be one
possible mechanism by which the tonoplast could integrate
circadian and metabolic signals for controlling the CAM
cycle.
Metabolic synchronization of CAM 9 of 11
Acknowledgements
We are grateful to the many colleagues who have contributed to the
ideas expressed above, particularly John Cushman, James Hartwell,
Hugh Nimmo, and Andrew Smith. Our research is supported by the
Natural Environment Research Council, UK.
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