Roles and regulation of phosphoenolpyruvate carboxylase kinase in higher plants
Regulation of phosphoenolpyruvate carboxylase in CAM plants
Crassulacean acid metabolism, shown here, is a metabolic adaptation to arid environments. Stomata are open at night, and phosphoenolpyruvate carboxylase (PEPCase) catalyses the fixation of atmospheric carbon dioxide. This yields malate, which is stored as malic acid in the vacuole. During the following day,the stomata are shut but malate is released from the vacuole and decarboxylated. The resulting carbon dioxide is re-fixed via the Calvin cycle and starch, which is broken down to provide phosphoenolpyruvate at night, is re-formed.
Hugh Nimmos main area of interest is the regulation of PEPCase in higher plants, and particularly in CAM plants. The huge diurnal change in flux through PEPCase in CAM plants is achieved via reversible phosphorylation. The enzyme is dephosphorylated and strongly inhibited by malate during the day, while at night it is phosphorylated and much less sensitive to malate. The phosphorylation state of PEPCase, and thereby its activity, is controlled mainly by the activity of a novel protein kinase termed PEPCase kinase. Hughs main interest is the structure and regulation of this protein kinase, which is unique in being controlled at the level of synthesis / degradation.
Control of phosphoenolpyruvate carboxylase kinase
The great majority of protein kinases are controlled either by a second messenger or by a phosphorylation cascade. However Hughs group have cloned PEPCase kinase genes from several plant species and have shown that PEPCase kinase is controlled directly by synthesis / degradation. In Crassulacean acid metabolism plants, this process responds to a circadian rhythm. In C4 plants, PEPCase catalyses the primary fixation of carbon dioxide during the day. In these plants (e.g. maize), PEPCase kinase is also controlled by synthesis / degradation, but synthesis is directly induced by light. In C3 and most non-photosynthetic plant tissues, PEPCase catalyses the major anaplerotic reaction and thereby plays a key role in carbon allocation. In C3 plants PEPCase kinase is induced in response to light and/or nitrogen supply. In guard cells, PEPCase supplies malate during stomatal opening; the kinase is induced by opening stimuli. In legume root nodules, PEPCase provides malate as a source of carbon skeletons and energy; the kinase is induced by photosynthate. Hughs group are investigating the molecular mechanisms by which these different stimuli control expression of the PEPcase kinase gene in different plant tissues.
Circadian regulation of PEPCase kinase in CAM plants
Several CAM plants, including Kalanchoë (Bryophyllum)fedtschenkoi exhibit circadian rhythms of carbon dioxide metabolism. The rhythm observed in constant darkness is directly attributable to circadian changes in the flux through PEPCase. These changes are caused by circadian control of the phosphorylation state of PEPcase. Thus this circadian rhythm of carbon dioxide metabolism offers an excellent system for a biochemical analysis of the output pathway that connects the central oscillator to a well-defined physiological rhythm. Some time ago we showed that this results from circadian appearance and disappearance of PEPCase kinase activity, and that appearance of the kinase activity requires protein synthesis. Cloning of the PEPCase kinase gene (see below) has allowed us to show that PEPCase kinase is indeed controlled directly by expression. The PEPCase kinase gene seems to be the first known clock-controlled gene encoding a protein kinase.
Cloning of PEPCase kinase
PEPCase kinase is a very low abundance enzyme, comprising less than 0.001% of CAM leaf soluble protein. Consequently it has not been purified in sufficient quantities to obtain peptide sequence or to raise an antiserum. Our cloning strategy depended on our development of an assay for PEPCase kinase mRNA.
Measurement of PEPCase kinase mRNA
The method involves translation of total RNA, followed by direct assay of the translation products for PEPCase kinase activity. Incorporation of 32P into PEPCase is quantified by immunoprecipitation of the PEPCase, SDS gel electrophoresis and phosphoimaging. Control experiments show that this gives a valid assay for the kinase mRNA. Measurement of the amounts of protein synthesised using [35S]Met allows us to correct for differences in the efficiency of translation of different RNA samples. This illustration shows SDS gels of both the translation products and the PEPCase kinase assays from three samples:
1 a control with no RNA,
2 RNA from K. fedtschenkoi during the middle of the night,
3 RNA from K. fedtschenkoi during the middle of the day.
The arrow indicates the main PEPCase band. A flow diagram for this assay is shown here and full details are given in Hartwell et al., Plant J. 10, 1071-1078 (1996). This assay can, in principle, be used for any protein kinase for which there is both a specific substrate and an antibody to that substrate.
The assay for PEPCase kinase mRNA was used to assess its level in pools and sub-pools of a cDNA library. The top panel shows [35S]Met-labelled translation products and the bottom panel shows the incorporation of 32P into PEPCase in PEPCase kinase assays.
A, lane1 - a control with no RNA, lane 2 - transcribed RNA from the original library, lane 3 - total RNA from K. fedtschenkoi during the middle of the night,
B, lanes 1-3 - transcribed RNA from three different pools after three rounds of screening, lane 4 - no RNA, lane 5 - total RNA from K. fedtschenkoi during the middle of the night,
C, lanes 1-2 - transcribed RNA from two unidentified K. fedtschenkoi protein kinase clones unrelated to PEPCase kinase, lane 3 - no RNA, lane 4 - total RNA from K. fedtschenkoi during the middle of the night,
D, lane 1 - transcribed RNA from Arabidopsis thaliana PEPCase kinase, lane 2 - transcribed RNA from K. fedtschenkoi PEPCase kinase, lane 3 - no RNA, lane 4 - total RNA from K. fedtschenkoi during the middle of the night.
PEPCase kinase is a novel protein kinase
The sequence of PEPCase kinase shows that it is the smallest protein kinase yet found. It comprises a protein kinase catalytic domain with very few additional residues at the N- and C-terminal ends. The catalytic domain is similar to the catalytic domain of plant calcium-dependent protein kinases but PEPCase kinase does not have the autoinhibitory and calmodulin-like regions of these enzymes. Thus PEPCase kinase is a member of the CaMK family, though its activity is independent of calcium ions. A phylogenetic tree of PEPCase kinases and other members of the CaMK family is shown here. The GenBank files for the cDNA sequences of PEPCase kinase from K. fedtschenkoi and Arabidopsis thaliana are AF162662 and AF162660 respectively. We have recently identified a further Arabidopsis PEPCase kinase, see gene T27C4.19 in AC022287.
PEPCase kinase is controlled at the level of expression
During the normal diurnal cycle in the CAM plant K. fedtschenkoi, the levels of PEPCase kinase translatable mRNA and transcripts clearly determine the activity of PEPCase kinase and the phosphorylation state of PEPCase, as judged by its sensitivity to inhibition by malate.
Metabolite and circadian regulation of PEPCase kinase expression in CAM plants
Recently (Borland et al., Plant Physiol. 121, 889-896, 1999), we showed that metabolites (probably cytosolic malate) could over-ride the circadian control of PEPCase kinase. We have now extended this idea to suggest that, as shown here, the primary effect of the circadian oscillator (1) is on a switch (2) which controls malate uptake (3) and release (4). This leads to a circadian rhythm in cytosolic malate which controls an unknown nuclear signal inhibiting transcription of PEPCase kinase. There may be feedback from malate to the oscillator (5) and/or some direct connection between the oscillator and the kinase gene (6). This model is now being tested.
Control of PEPCase kinase in other systems
PEPCase also catalyses the primary fixation of carbon dioxide in C4 plants. In these plants, PEPCase is found exclusively in the mesophyll. PEPCase kinase activity appears and PEPCase becomes phosphorylated in the light. The role of this is to prevent inhibition of PEPCase by the build-up of malate in the mesophyll that sustains operation of the C4 pathway. In C3 plants, PEPCase is also phosphorylated in response to light, probably to coordinate the provision of carbon skeletons for aminoacid biosynthesis. By using our assay for PEPCase kinase RNA, we have shown that light stimulates the expression of the PEPCase kinase gene in both C4 and C3 plants.
The signalling pathway that leads to PEPCase kinase expression
In almost all systems studied so far, increases in PEPCase kinase require RNA and protein synthesis. Our ability to measure changes in kinase mRNA has allowed us to show that the signalling pathways that lead to PEPCase kinase expression in both CAM and C4 plants involves both a calcium/calmodulin - like interaction and protein dephosphorylation. Here is a colourful summary of our current view of the regulation and roles of the phosphorylation of PEPCase.