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Mechanistic and Functional Studies of Proteins The co-ordination of central plant metabolism by the circadian clock J. Hartwell1 School of Biological Sciences, University of Liverpool, Biosciences Building, Crown Street, Liverpool L69 7ZB, U.K. Abstract A circadian clock optimizes many aspects of plant biology relative to the light/dark cycle. One example is the circadian control of primary metabolism and CO2 fixation in plants that carry out a metabolic adaptation of photosynthesis called CAM (crassulacean acid metabolism). These plants perform primary CO2 fixation at night using the enzyme phosphoenolpyruvate carboxylase and exhibit a robust rhythm of CO2 fixation under constant conditions. Transcriptomic analysis has revealed that many genes encoding enzymes in primary metabolic pathways such as glycolysis and starch metabolism are under the control of the circadian clock in CAM plants. These transcript changes are accompanied by changes in metabolite levels associated with flux through these pathways. The molecular basis for the circadian control of CAM remains to be elucidated. Current research is focusing on the identity of the CAM central oscillator and the output pathway that links the central oscillator to the control of plant metabolism. Circadian rhythms of CO2 fixation and metabolism A wide range of fundamental processes in plant biology are under the control of an endogenous circadian clock. Examples of plant circadian rhythms include leaf movements, hypocotyl elongation, gene transcript and protein abundance rhythms and the photoperiodic control of flowering time (for a review, see [1]). Another classic plant circadian rhythm is the rhythm of CO2 fixation observed in plants that perform a metabolic adaptation of photosynthesis known as CAM (crassulacean acid metabolism) [2]. CAM plants perform CO2 fixation in two steps that are separated temporally within a single photosynthetic cell [3]. Primary CO2 fixation occurs during the dark period catalysed by PEPc (phosphoenolpyruvate carboxylase). Malic acid, the product of dark CO2 fixation, accumulates in the vacuole. After dawn, malic acid is released from the vacuole and is decarboxylated by either malic enzyme or PEP carboxykinase, depending on the species. CO2 builds up to high partial pressures inside the leaf and this signals stomatal closure. Secondary CO2 fixation is catalysed by RuBisCO (ribulose bisphosphate carboxylase oxygenase) and carbon is assimilated via the reductive pentose phosphate pathway (Calvin–Benson cycle) using the energy provided by photosynthetic light harvesting in the chloroplast. CAM plants possess a number of key adaptations that bestow on them a selective advantage in arid and semi-arid environments. First, they open their stomatal pores and perKey words: Arabidopsis, circadian clock, CO2 fixation, crassulacean acid metabolism, glycolysis, plant metabolism. Abbreviations used: CAM, crassulacean acid metabolism; CCG, clock-controlled gene; EST, expressed sequence tag; LD cycle, light/dark cycle; PEPc, phosphoenolpyruvate carboxylase; PP2A, protein phosphatase type 2A; PPCK, PEPc kinase; RT, reverse transcriptase; RuBisCO, ribulose bisphosphate carboxylase oxygenase. 1 email [email protected] form primary CO2 fixation at night when evapotranspiration is minimized by low air temperatures. Secondly, they close their stomata during the day, preventing water loss and promoting the build up of CO2 inside the leaf due to malate decarboxylation. Thirdly, the accumulating CO2 favours the carboxylase over the oxygenase activity of RuBisCO and thus reduces the loss of energy and carbon via the photorespiratory pathway. These remarkable adaptations endow CAM plants with up to 6-fold greater water-use efficiency relative to plants that perform C3 photosynthesis [4]. Well-known examples of CAM plants are the cacti and succulents, but CAM has evolved many times and is found in 33 families representing 6–7% of the higher plants [5]. The CAM pathway can function efficiently only if there is a strict temporal control of primary and secondary CO2 fixation. Without tight regulation, CO2 fixation and malate decarboxylation would occur simultaneously in single cells and this futile cycle would waste large amounts of ATP and NADPH. In the few CAM species for which the regulation of the pathway has been studied in some detail, a circadian oscillator co-ordinates the pathway. Following a period of entrainment in 24 h LD (light/dark) cycles, rhythms of CO2 fixation persist both in constant darkness, CO2 -free air and constant temperature (DD) and constant light, normal air and constant temperature (LL) [6]. Maximum CO2 fixation coincides with the subjective dark period, that is the period when dark occurred during entrainment in LD cycles (light/ dark cycles). This rhythm of CO2 fixation has all the features of a bona fide circadian rhythm, including persistence in constant conditions, significant temperature compensation and phase resetting by light and temperature [6]. Experiments feeding 14 CO2 to leaves of the CAM plant Kalanchoë fedtschenkoi demonstrated that the product of periods of CO2 fixation under constant conditions was malate [2]. This confirmed that the nocturnal activation of PEPc was under C 2005 Biochemical Society 945 946 Biochemical Society Transactions (2005) Volume 33, part 5 circadian control and thus the search for the underlying regulatory mechanism began. Biochemical aspects of metabolic rhythms Early work led to the discovery that the apparent K i of PEPc for its feedback inhibitor L-malate varied between day and night in CAM plants [7]. The enzyme existed in two forms. During the dark period, PEPc had a high K i for malate and, in the light, it had a low K i for malate. This meant that the enzyme was relatively insensitive to feedback inhibition by malate during the dark period, making it more active in vivo. Investigations into the biochemical basis for this daily change in the K i of PEPc for malate revealed that the night form of the enzyme is phosphorylated on a serine residue, whilst the day form is dephosphorylated [8]. Furthermore, the phosphorylated form of the purified enzyme had a high K i for malate and the dephosphorylated form had a low K i for malate [9]. PEPc was dephosphorylated by a PP2A (protein phosphatase type 2A) and phosphorylated by a specific protein kinase, termed PPCK (PEPc kinase) [10]. This protein kinase could be assayed in desalted extracts of CAM leaves [11]. The PP2A activity in leaf extracts remained relatively constant throughout a 24 h LD cycle, but the PPCK activity varied greatly with high activity in the dark and no activity in the light [10,11]. In DD, the rhythm in the phosphorylation state of PEPc persisted: high phosphorylation and a high K i coincided with the subjective dark period and a trough occurred during the subjective light period [12]. Thus all the evidence pointed to PPCK as a key point for the circadian control of the CAM pathway. Owing to its extremely low abundance, the PPCK protein proved very difficult to purify to homogeneity. Cloning the corresponding gene therefore required a novel approach. Two groups identified the first PPCK genes from unrelated CAM plants. Hartwell et al. [13] cloned the CAM PPCK gene from K. fedtschenkoi using an in vitro transcription–translationcoupled PPCK assay on pools and subpools of clones from a dark leaf cDNA library. This approach allowed the identification of cDNA clones that encoded proteins capable of phosphorylating PEPc following in vitro transcription and translation. Several rounds of screening subpools of the original library allowed the identification of the PPCK gene. A second group identified a PPCK gene from the inducible-CAM species Mesembryanthemum crystallinum [14]. This species represents a very powerful system for the study of CAM, because it grows as a C3 plant when well watered, but switches to CAM in response to drought stress or salt stress. Taybi et al. [14] adopted a protein kinase-targeted differential display RT (reverse transcriptase)–PCR approach and compared C3 and CAM leaves in the light and dark. By comparing these four conditions, they were able to identify protein kinases that were only expressed in the dark in CAMinduced leaves. One dark and CAM-expressed band on their differential display gels encoded the M. crystallinum PPCK gene. C 2005 Biochemical Society The PPCK gene consisted of a minimal protein kinase domain; one of the smallest protein kinases at only 30– 32 kDa [13,14]. It was most closely related to a large family of plant calcium-dependent protein kinases, but lacked the Nterminal variable domain and the C-terminal EF hands that confer calcium sensitivity on this group of proteins. Instead, PPCK was regulated by synthesis and degradation [13–15]. The transcript abundance of the PPCK gene oscillated under constant conditions, revealing that rhythms of PPCK activity were mediated through circadian control of gene expression [13]. PPCK was the first CCG (clock-controlled gene) to be identified in CAM plants. Detailed characterization of the regulation of PPCK expression and activity revealed that preventing nocturnal CO2 fixation and malate accumulation in Kalanchoë daigremontiana lead to prolonged expression of PPCK into the light period [16]. This suggested that the circadian control of PPCK could be overridden by metabolic signals, possibly the cytosolic concentration of malate. Molecular basis for nocturnal CO2 fixation rhythms The circadian control of PEPc activity by PPCK was established as one of the key points of circadian control of the CAM pathway. However, the identification of the PPCK gene gave fresh impetus to several important questions. First, it was important to ascertain whether PPCK was the only point of circadian control of the CAM pathway or whether the pathway was subject to circadian control at multiple points. Secondly, the identity of the circadian oscillator that regulates the rhythm of PPCK expression had not been determined, and finally, the components of the clock-output signalling pathway that links CAM control to a circadian oscillator were unknown. To investigate the extent of circadian control of the CAM pathway, we initiated a transcriptomic analysis of CAM using high-throughput RT–PCR on M. crystallinum leaf RNA samples. Analysis of the transcript abundance profiles for 180 genes in C3 and CAM leaves under both LD cycles and LL free-running conditions has provided a wealth of information about the pervasiveness of circadian control within the CAM pathway. The genes that encode the enzymes and metabolite transporters that catalyse many of the major metabolic steps of the CAM cycle are under circadian control (S.F. Boxall and J. Hartwell, unpublished work). Interestingly, a number of these genes are only subject to circadian regulation in CAM-induced leaves. At least 25 genes qualify as CAM-associated CCGs, including genes that encode cytosolic isoforms of every enzyme in glycolysis and numerous enzymes and plastidic metabolite transporters with roles in starch metabolism. In C3 Arabidopsis, the only cytosolic glycolysis CCGs are fructose bisphosphate aldolase, pyrophosphate-dependent phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase [17]. A number of genes associated with starch metabolism are also under circadian and diurnal control in Arabidopsis [17,18]. To briefly summarize our transcriptomic Mechanistic and Functional Studies of Proteins work on CAM, circadian control of CAM is not simply imposed by the regulation of dark CO2 fixation by PEPc due to the circadian control of PPCK. Instead, circadian control is spread throughout the entire 24 h cycle of CAM from fixation to decarboxylation and back again via starch synthesis and degradation. We also determined that malate levels oscillate under LL conditions, indicating that, in M. crystallinum, the rhythms of CAM CCGs are associated with rhythms in the metabolism they control (J.M. Foster and J. Hartwell, unpublished work). These results reveal a very important fact about the evolution of CAM, namely that there must have been a strong selective advantage to be gained by placing multiple steps of the pathway under circadian control. In Arabidopsis, a number of other pathways in plant metabolism are subject to broad circadian control of steadystate transcript abundance. For example, 23 genes encoding enzymes with a role in the phenylpropanoid pathway were found to be under circadian control using Affymetrix gene chip analysis [17]. This pathway synthesizes photoprotective pigments and it was remarkable that all of the genes shared peak expression before dawn. This led to the hypothesis that Arabidopsis can protect itself with a ‘phenolic sunscreen’ by co-ordinately activating the phenylpropanoid pathway in anticipation of dawn [17]. The discovery of almost global circadian control of the transcript abundance of CAM-associated genes in M. crystallinum raised new questions, not least of which is whether CAM species that have evolved CAM independently from M. crystallinum also have multiple points of circadian control in the CAM pathway. K. fedtschenkoi, M. crystallinum and several Clusia species have CAM-associated circadian-regulated isoforms of PPCK [13,14,19]. However, M. crystallinum is currently the only species for which circadian control has been identified at multiple steps in the CAM pathway. It will be very interesting to discover whether CAM and circadian control are inseparable, or whether some species have evolved CAM without coupling it with circadian control. The identity of the CAM circadian oscillator? Two possible hypotheses have been proposed to explain the CAM circadian oscillator. The first hypothesis consists of a biophysical oscillator in which changes in vacuolar turgor controlled tension and relaxation of the tonoplast and the time was set by the subcellular localization of malate [20]. In this model, the cytosolic malate concentration controlled CO2 fixation due to its inhibitory effect on PEPc activity. A computer model of this biophysical oscillator has been generated and this model was able to support all of the experimental data until recently [20]. Wyka et al. [21] perturbed the CO2 fixation rhythm in K. daigremontiana leaves by removing CO2 from the air supplied to leaves in LL. This prevented nocturnal CO2 fixation, allowing Wyka et al. [21] to determine whether stored malate was providing the time-keeping information responsible for the observed rhythms of CO2 fixation. The computer model predicted phase delays under these conditions, with peak CO2 fixation occurring later after the CO2 -free air treatment. The model predicted a phase delay due to the time taken for the leaf to ‘catch-up’ and synthesize the malate that it was unable to make during the CO2 -free air treatment. However, the experimental data did not show any change in phase upon release of the leaves from CO2 -free air [21]. This demonstrated that the circadian clock co-ordinating the CAM cycle continued to maintain its phase, even when malate synthesis was prevented. This represents compelling evidence that the biophysical oscillator is not the underlying circadian clock that co-ordinates rhythms of CO2 fixation in CAM plants. The second hypothesis that could explain the underlying circadian oscillator that co-ordinates CAM involves the pathway being coupled with the autoregulatory negativefeedback gene loops that form a circadian oscillator in the nucleus of every plant cell. The components of this oscillator are beginning to be defined in the model C3 plant Arabidopsis thaliana and it is now clear that a relatively large number of genes are likely to be involved in creating the interlocking autoregulatory gene loops that constitute a robust plant oscillator [22–25]. However, Arabidopsis is not a CAM plant and does not perform circadian rhythms of dark CO2 fixation. It was therefore important to determine whether CAM species possess the genes of the Arabidopsis clock and if so, whether they are regulated in a similar way such that they could generate a circadian oscillator. To achieve this goal, work was focused on the model CAM plant M. crystallinum because a large EST (expressed sequence tag) database has been established for this species. The ESTs revealed M. crystallinum orthologues of several circadian clock components and degenerate PCR allowed the cloning of other components to the extent that seven circadian clockassociated genes were identified and characterized [26]. This work achieved a number of key steps forward. First, it revealed that a CAM species possessed a circadian oscillator very similar to the oscillator in Arabidopsis. In particular, the single MYB repeat transcription factor CCA1/LHY and the pseudo-response regulator protein TOC1 were under reciprocal circadian control in a manner entirely consistent with their roles as components of the autoregulatory loop at the core of the plant clock [26]. Most importantly, this finding demonstrated that a CAM plant possessed a molecular oscillator similar to the oscillator in Arabidopsis, and thus that the CCA1/LHY-TOC1 oscillator could be responsible for co-ordinating rhythms of dark CO2 fixation in CAM species. Secondly, one clock component, ZEITLUPE (ZTL), was found to be under circadian control at the level of its relative transcript abundance in M. crystallinum, whilst the transcript abundance of the Arabidopsis gene does not oscillate [26]. This suggested that the development of subtly different modes of circadian regulation has occurred within the core components of the plant clock during evolution. The CAM clock genes provide the opportunity to manipulate the clock in planta by allowing us to generate C 2005 Biochemical Society 947 948 Biochemical Society Transactions (2005) Volume 33, part 5 transgenic CAM plants that overexpress a clock gene and therefore have an arrhythmic clock [27]. Conversely, the expression of the CAM clock genes can be knocked down using RNAi (RNA interference) constructs. These transgenic plants will permit the question of a link between the CCA1/LHY-TOC1 clock and CAM to be answered unequivocally. A transgenic CAM plant with an arrhythmic central clock should be arrhythmic for dark CO2 fixation rhythms only if the two are coupled. If the CAM CO2 fixation rhythm persists in arrhythmic plants then a novel clock must be operating. My group has generated these transgenic lines using the CAM plant K. fedtschenkoi and they are currently under preliminary analysis. Early results suggest that the CCA1/LHY-TOC1 oscillator does control CAM. The next major challenge will be to determine the components of the clock output pathway that links the central clock to CAM. To this end, we have cloned the promoters of a number of clock-controlled, CAM-associated genes and are in the process of defining the motifs required for their circadian control and cloning the transcription factors responsible for mediating their circadian control. This knowledge can then inform metabolic engineering efforts aimed at optimizing the production of novel metabolites in plants. References 1 2 3 4 Eriksson, M.E. and Millar, A. (2003) Plant Physiol. 132, 732–738 Warren, D.M. and Wilkins, M.B. (1961) Nature (London) 191, 686–688 Borland, A.M. and Taybi, T. (2004) J. Exp. Bot. 55, 1255–1265 Nobel, P.S. (1996) in Crassulacean Acid Metabolism. Biochemistry, Ecophysiology and Evolution, vol. 114 (Winter, K. and Smith, J.A.C., eds.), pp. 255–265, Springer-Verlag, Berlin 5 Winter, K. and Smith, J.A.C. (1996) in Crassulacean Acid Metabolism. Biochemistry, Ecophysiology and Evolution, vol. 114 (Winter, K. and Smith, J.A.C., eds.), pp. 1–13, Springer-Verlag, Berlin C 2005 Biochemical Society 6 Wilkins, M.B. (1992) New Phytol. 121, 347–375 7 Winter, K. (1980) Plant Physiol. 65, 792–796 8 Nimmo, G.A., Nimmo, H.G., Fewson, C.A. and Wilkins, M.B. (1984) FEBS Lett. 178, 199–203 9 Nimmo, G.A., Nimmo, H.G., Hamilton, I.D., Fewson, C.A. and Wilkins, M.B. (1986) Biochem. J. 239, 213–220 10 Carter, P.J., Nimmo, H.G., Fewson, C.A. and Wilkins, M.B. (1990) FEBS Lett. 263, 233–236 11 Carter, P.J., Nimmo, H.G., Fewson, C.A. and Wilkins, M.B. (1991) EMBO J. 10, 2063–2068 12 Nimmo, G.A., Wilkins, M.B., Fewson, C.A. and Nimmo, H.G. (1987) Planta 170, 408–415 13 Hartwell, J., Gill, A., Nimmo, G.A., Wilkins, M.B., Jenkins, G.I. and Nimmo, H.G. (1999) Plant J. 20, 333–342 14 Taybi, T., Patil, S., Chollet, R. and Cushman, J.C. (2000) Plant Physiol. 123, 1471–1481 15 Hartwell, J., Smith, L.H., Wilkins, M.B., Jenkins, G.I. and Nimmo, H.G. (1996) Plant J. 10, 1071–1078 16 Borland, A.M., Hartwell, J., Jenkins, G.I., Wilkins, M.B. and Nimmo, H.G. (1999) Plant Physiol. 121, 889–896 17 Harmer, S.L., Hogenesch, J.B., Straume, M., Chang, H.-S., Han, B., Zhu, T., Wang, X., Kreps, J.A. and Kay, S.A. (2000) Science 290, 2110–2113 18 Smith, S.M., Fulton, D.C., Chia, T., Thorneycroft, D., Chapple, A., Dunstan, H., Hylton, C., Zeeman, S.C. and Smith, A.M. (2004) Plant Physiol. 136, 2687–2699 19 Taybi, T., Nimmo, H.G. and Borland, A.M. (2004) Plant Physiol. 135, 587–598 20 Lüttge, U. (2000) Planta 211, 761–769 21 Wyka, T.P., Bohn, A., Duarte, H.M., Kaiser, F. and Lüttge, U.E. (2004) Planta 219, 705–713 22 Salomé, P.A. and McClung, C.R. (2005) Plant Cell 17, 791–803 23 Farré, E.M., Harmer, S.L., Harmon, F.G., Yanovsky, M.J. and Kay, S.A. (2005) Curr. Biol. 15, 47–54 24 Alabadi, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Más, P. and Kay, S.A. (2001) Science 293, 880–883 25 Mizuno, T. and Nakamichi, N. (2005) Plant Cell Physiol. 46, 677–685 26 Boxall, S.F., Foster, J.M., Bohnert, H.J., Cushman, J.C., Nimmo, H.G. and Hartwell, J. (2005) Plant Physiol. 137, 969–982 27 Wang, Z.-Y. and Tobin, E.M. (1998) Cell (Cambridge, Mass.) 93, 1207–1217 Received 20 June 2005