Download The co-ordination of central plant metabolism by the circadian clock

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
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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.
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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
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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.
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Received 20 June 2005