JXB Advance Access originally published online on April 8, 2004
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Journal of Experimental Botany, Vol. 55, No. 400, pp. 1255-1265, May 1, 2004
© 2004 Oxford University Press
Photosynthetic Carbon Fluxes |
Synchronization of metabolic processes in plants with Crassulacean acid metabolism
Received 28 October 2003; Accepted 19 January 2004
Environmental and Molecular Plant Physiology, School of Biology, King George VI Building, University of Newcastle, Newcastle Upon Tyne NE1 7RU, UK
* To whom correspondence should be addressed. Fax: +44 (0)191 222 5228. E-mail: a.m.borland{at}ncl.ac.uk
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Abstract |
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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 fluctuations 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 fluxes. 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.
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Introduction |
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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 reflected 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 significance 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 fixed 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 final 4-C product, malic acid, is stored in a large central vacuole. During the day, malate exits the vacuole and decarboxylation may occur through the single or combined action of three carboxylases (depending on plant species): NADP-malic 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 sufficient 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 flux, the result of the reactions outlined in Fig. 1a are substantial diel and reciprocating fluctuations in malate and carbohydrate contents (Fig. 1b) which can represent up to 20% of leaf dry weight.
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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 influence 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 fixation of CO2 via Rubisco (Phase IV gas exchange) predominates. As a defining 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.
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A circadian clock sets the diel phases of CAM |
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The well-documented rhythms of gas exchange in species of the genus Kalanchoë, 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; Lüttge, 2000). Circadian control of carbon flux through PEPC is generally regarded as a key component underpinning the day/night separation of carboxylation processes that define 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 time-frame 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).
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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 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 flux in response to changing environmental conditions. Field-based measurements of instantaneous carbon isotope discrimination on hemi-epiphytic 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.
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Metabolites provide a further layer of control over the diel CAM cycle |
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Experimental observations now indicate that PPCK expression and activity can be modified by leaf metabolic status. In leaves of Kalanchoë 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; 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, 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 (Lüttge, 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 fine-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 fluxes of metabolites across the tonoplast?
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Which components of CAM are regulated by the clock? |
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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 specific 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 on-going 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 significance 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 sufficiently 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-specific 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 modification, 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-specific 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. multiflora (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. 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.
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 Lüttge, 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 Lüttge, 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 (Lüttge, 2002a; Wyka and Lüttge, 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 (Lüttge, 2000, 2002a; Rascher et al., 2001).
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Metabolite control of CAM |
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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 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 findings 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 efflux of malate (or some other metabolite) from the vacuole could be a primary signal for the down-regulation of PPCK expression during the photoperiod in Clusia species once CAM is induced. The subsequent decarboxylation of organic acids will be a key factor influencing the increase in PPCK expression for the following night. It is interesting to note that Clusia species 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.
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The transport steps that mediate the influx and efflux 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 fluctuations 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 identified at the molecular level, it seems likely that distinct influx and efflux systems exist on the tonoplast of CAM plants (Smith et al., 1996). The influx of malate across the tonoplast appears to be mediated by a voltage-dependent 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-acidification. 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 flux across the tonoplast in response to malate concentration.
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) deficits 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 Griffiths, 1997; Dodd et al., 2002). Thus, carbohydrate status appears to be a key element in synchronizing metabolic fluxes 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 best-studied 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 finding 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 (Häusler 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 (Häusler 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 fluxes during the CAM cycle (Häusler 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 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-deficient 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-deficient plants the activity and diel fluctuation 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 fluctuations 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 fluxes 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 fluxes 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 flux 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.
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 fluctuations 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; Griffiths et al., 2002). Lüttge (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 fluxes over the diel cycle in CAM plants.
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Integration of environmental and metabolic signals with the circadian clock |
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 TopAbstractIntroductionA circadian clock sets...Metabolites provide a further...Which components of CAM...Metabolite control of CAM Integration of environmental and... Evolutionary implications and...References |
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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 flowering. 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 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 flowering and seed production. It will be of future interest to establish if long-term (seasonal) responses such as CAM induction and flowering and short-term (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. Lüttge (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 (Lüttge 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.
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Evolutionary implications and conclusions |
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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 specific 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 fluctuations in the environment. The reciprocating pools of organic acids and carbohydrates are central to the metabolic control of CAM. Membrane-localized 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 fluctuations 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 modifications 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 (Griffiths, 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 fixed 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 flooded 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 diversification in the complement of tonoplast transporters (e.g. separate transporters for influx and efflux 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 specific 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 progenitors of CAM already possessed clocks with a pervasive control over metabolism. Whilst circadian gene expression in the prokaryotic organism, Synechococcus appears to be a universal phenomemmon (Liu et al., 1995), in the higher plant A. thaliana 
36% of the genome is potentially under transcriptional control by the circadian clock (Michael and McClung, 2003). However, of the Arabidopsis genes that are known to show circadian fluctuations in transcript abundance, a large proportion (almost 70%) also respond directly to environmental stress (i.e. low temperature, salt, and drought; Kreps et al., 2002). This observation has prompted the suggestion that rhythmic expression of such stress-related genes in anticipation of predictable environmental changes, might prepare the plant to withstand a stress or make best use of a potentially limiting resource (Eriksson and Millar, 2003). Since CAM is usually considered to have arisen in extreme environments (Raven and Spicer, 1996), it is conceivable that the progenitors of CAM already possessed clocks with a pervasive control over metabolism as a means of maintaining homeostasis in potentially limiting environments. Subsequent evolution of the pathway could have proceeded by either coupling more/different components of metabolic output to a central oscillator and/or through changes in the pathways (and perhaps the mechanisms) which connect the central oscillator(s) to metabolic output. Future approaches for understanding photosynthetic performance in CAM plants will require the application of post-genomic technologies, encompassing transcriptomics, proteomics, and metabolomics, in order to identify the genes and proteins that are controlled by the clock and for determining the role of metabolites (e.g. organic acids and sugars) in synchronizing diel carbon fluxes. Gene-chip technology is currently being applied to dissect out the relative contributions of clock and metabolite control in wild-type and CAM-deficient mutants of M. crystallinum (J Cushman, personal communication). Further progress demands the development of a suitable and amenable transformation system to test hypotheses on the circadian and metabolic signals that control both the expression and the synchronization of the metabolic components that constitute CAM.
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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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