REGULATION OF PHOSPHOENOLPYRUVATE CARBOXYLASE: AN EXAMPLE OF SIGNALTRANSDUCTION VIA PROTEIN PHOSPHORYLATION IN HIGHER PLANTS HUGH G. NIMMO*, PAMELA J. CARTER*t, CHARLES A. FEWSON*, GAVIN A. L. McNAUGHTON*t, GILLIAN A. NIMMO*t and MALCOLM B. WILKINSt Departments of *Biochemistry and tBotany, University of Glasgow, Glasgow G12 8QQ, Scotland
Over the last thirty years it has become clear that protein phosphorylation/dephosphorylation plays a central role in the regulation of many processes and pathways in both eukaryotic and prokaryotic cells (1, 2). For much of this time, relatively little attention was paid to protein phosphorylation in higher plants. However, it is now appreciated that phosphorylation probably plays as important a role in stimulus/response coupling and metabolic control in higher plants as it does in animal systems. The control of metabolism in the photosynthetic tissue of higher plants poses particularly interesting questions of enzyme regulation. In plants that use the C 3 or C4 pathways of CO 2 assimilation, carbon and energy metabolism in leaf tissue is geared towards the fixation of CO 2 during the day. At night, however, energy is obtained via respiration and stored products may be used for oxidation or export. One might therefore expect to observe light-dependent regulation of enzyme activities, and indeed a number of chlorplastic enzymes are regulated by thiol/disulfide interchange in an electron tra~,sport-dependent process (for a review see Ref. 3). However, a number uf key enzymes are regulated by reversible phosphorylation, including pyruvate dehydrogenase (EC 184.108.40.206) (4), pyruvate, phosphate dikinase (EC 220.127.116.11) (5), sucrose phosphate synthase (EC 18.104.22.168) (6) and phosphoenolpyruvate carboxylase (EC 22.214.171.124) (PEPc) (see below). In the latter three cases, the phosphorylation state of the enzyme is light-dependent in at least some plant species. In this article we focus on the control of PEPc by phosphorylation in leaf tissue of C 4 and Crassulacean acid metabolism (CAM) plants. PEPc catalyzes the fixation of CO 2 (as H C O 3 - ) into oxaloacetate. It plays 121
H . G . NIMMO, et al.
an anaplerotic role in C 3 plants and in nonphotosynthetic tissue in C4 and CAM plants (7). However, the leaves of C a and CAM plants contain distinct isoenzymes of PEPc with specialized roles in CO 2 fixation. As illustrated in Figure 1, in C a plants PEPc is located in leaf mesophyll cells and catalyzes the first committed step in the C4 pathway of CO 2 fixation. The pathway ResolYnyll cells
BUndle slleath cells
=-HCO 3- f1=Oxaloacetate~k~2 ~ - Malate
FIG. 1. The C 4 pathway in Zea mays. The numbered steps are catalyzed by the following enzymes: 1, PEPc; 2, NADP-dependent malate dehydrogenase; 3, malic enzyme; 4, pyruvate, phosphate dikinase.
--- HCO3---7---~-Oxaloacetate ~
NAD+ Halate ,~
FIG. 2. The CAM pathway in Bryophyllum fedtschenkoi. Open arrowheads indicate reactions that proceed during the day and closed arrowheads those that proceed at night. The numbered steps are catalyzed by the following enzymes: 1, PEPc; 2, NADP-dependent malate dehydrogenase; 3, malic enzyme; 4, pyruvate, phosphate dikinase.
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of CAM is shown in Figure 2. CAM plants fix external C O 2 into malate at night via PEPc, and during the day the CO 2 is released from malate and refixed photosynthetically. Mechanisms must therefore exist to reduce or eliminate the flux through PEPc at night in C 4 plants and during the day in CAM plants. The PEPcs of both C 4 and CAM plants can be regulated allosterically. Effectors include glucose 6-phosphate, which activates the enzyme, and malate, which inhibits it. It is widely thought that malate may act as a feedback inhibitor that regulates flux through PEPc in vivo. However, some years ago, it became clear from the work of several groups that CAM PEPc must be controlled at an additional level, in that the enzyme is more sensitive to inhibition by malate, or less active, during the day than at night (8-10). Subsequently, several groups showed that illumination of C 4 plants results in a reduction of the sensitivity of leaf PEPc to inhibition by malate (11-15). Here we review the evidence that the malate sensitivity of PEPc is regulated by reversible phosphorylation in C 4 and CAM plants. MATERIALS
Preparation and analysis of leaf extracts. Bryophyllum fedtschenkoi and Zea rnays (maize) plants were grown and illuminated as described previously (13, 16). The procedures for preparation of extracts, assays of PEPc activity and malate content and measurement of the apparent K i of PEPc for malate have been described (13, 16).
Phosphorylation of PEPc. The dephosphorylated 'day' form of PEPc from B. fedtschenkoi was purified as described previously (17). Extracts of leaves in the middle of the dark period were prepared and desalted as in (16). Incubation and analysis conditions are described in the text. Dephosphorylation of PEPc. The [32p]-phosphorylated 'night' form of PEPc was prepared as described previously (17). Rabbit skeletal muscle protein phosphatases 1 and 2A, prepared as described in (18), were kindly given by Dr. C. MacKintosh (Department of Biochemistry, University of Dundee). Incubation conditions are described in the text and the release of 32p i w a s monitored as in (18). RESULTS
Phosphorylation of CAM PEPc in Intact Tissue Using freshly prepared and desalted extracts of the CAM plants
Mesembryanthernum crystallinum (9) and Bryophyllurn fedtschenkoi (16) it was found that the PEPc was significantly more sensitive to inhibition
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by malate (i.e. the K i for malate was lower) during the light period than during the middle of the dark period. For the Mesembryanthemurn enzyme the Km for phosphoenolpyruvate was higher in the light period than in the dark period. Results for the Bryophyllum enzyme are shown in Figure 3. This reveals that the conversions of the 'night' to the 'day' form of PEPc and vice versa coincided with the cessation and onset of malate accumulation, respectively. Both conversions occurred during the dark period, suggesting that they are controlled by a circadian rhythm rather than by light/dark changes. The mechanism responsible for this interconversion of two kinetically distinct forms of PEPc was then investigated using B. fedtschenkoi (16, 17). Immunoprecipitation of PEPc from detached leaves that had been prelabeled with 32p i revealed that the 'night' form of the enzyme contained 32p whereas the 'day' form did not. Purification of the two forms of the enzyme allowed us to show that the 'night' form was phosphorylated on one or more serine residues. The phosphoserine was not a covalent intermediate in the reaction mechanism. Removal of the phosphate group by alkaline phosphatase increased the malate sensitivity of the purified 'night' form to that of the 'day' form. These data showed
I Specific actlvlty (unlt/mg)
2 o o oo
°o o )000
o °°°°cb o
Apparent K 1 (mM)
~rmOles/g, esh w t . )
16 1'8 2;
Time of day (h)
FIG. 3. Diurnal rhythm in the malate sensitivity of PEPc in B. fedtschenkoi. Symbols show the specific activity (open circles) and apparent K i for malate (open triangles) of PEPc and the malate content (closed circles) of leaves throughout the day. The dark and light periods are shown by the closed and open bars, respectively. From Ref. 16 with permission.
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that the conversion of the 'day' to the 'night' form of Bryophyllum PEPc correlated with phosphorylation of the enzyme. A similar conclusion was reported for the PEPc of Kalanchoe species (19). Detached leaves of B. fedtschenkoi can show persistent circadian rhythms of CO 2 metabolism in continuous darkness or continuous light (20, 21). In some conditions, such as in continuous darkness and CO2-free air at 15°C, PEPc exhibited a persistent circadian rhythm of interconversions between a malate sensitive, dephosphorylated form and a less sensitive, phosphorylated form. The changes in the K i of PEPc for malate were in phase with the rhythm of CO 2 output (22). These results strengthen the views that the phosphorylation of Bryophyllum PEPc is controlled by an endogenous rhythm rather than by changes in illumination, and that it plays a significant role in regulation of the flux through PEPc. The 'day' and 'night' forms of Bryophyllum PEPc were indistinguishable by gel filtration or gel electrophoresis in nondenaturing conditions (17). Both forms appeared to be tetramers that could dissociate to dimers. In contrast, the 'day' and 'night' forms of PEPc from Crassula argentea could be resolved by nondenaturing gel electrophoresis (23). The former appeared to be a dimer whereas the latter behaved as a tetramer (24). It remains to be seen whether this represents a species difference and whether phosphorylation of PEPc occurs in C. argentea.
Phosphorylation of C4 PEPc in Intact Tissue The effects of illumination and darkening on the malate sensitivity of maize leaf PEPc are shown in Figure 4. The K i of the maize enzyme for malate changes only some 2- to 3-fold, a much less dramatic effect than the 10-fold change seen for the Bryophyllum enzyme. The change requires light intensities of above 400/zE/m2/sec, is complete in 30-60 min and is reversed in darkness over 30-60 min (13). Blue, red and white light are equally effective in producing the change in K i for malate, and the change is blocked by 3-(3',4'-dichlorophenyl)-l,l-dimethylurea (DCMU), which inhibits noncyclic electron transport at a site near the primary acceptor of photosystem II (data not shown). These results suggest that the photoreceptor involved in this effect is the photosynthetic apparatus itself. The mechanism responsible for the reduction in the malate sensitivity of maize leaf PEPc upon illumination was investigated by immunoprecipitation of the enzyme from [32Pi]-labeled tissue (13). This showed clearly that phosphorylation and dephosphorylation of serine residues of PEPc correlated with the decrease and increase in malate sensitivity observed upon illumination and darkening, respectively. In agreement with this, Jiao and Chollet (15) purified PEPc from darkened and illuminated maize
H. G. NIMMO, et al.
0"6": ~ o 0.4i
60 80 Time (mln)
FIG. 4. Effects of light and darkness on the malate sensitivity of maize leaf PEPc. (a) Leaves were kept in darkness for 10-14 hr at 15°C and then detached and transferred to light (800 p.E/m2/sec) at 27°C at zero time. (b) Detached leaves were illuminated for 1 hr as above and then transferred to darkness at zero time. Circles and triangles refer to extracts prepared in a Waring blender and by grinding under liquid N2, respectively. Taken from Ref. 13 with permission. leaves and f o u n d that the e n z y m e f r o m the latter tissue was significantly m o r e p h o s p h o r y l a t e d than that f r o m the former. T r e a t m e n t of the ' d a y ' f o r m o f the e n z y m e with alkaline p h o s p h a t a s e b o t h d e p h o s p h o r y l a t e d it and increased its malate sensitivity. Maize leaf P E P c can u n d e r g o association/dissociation b e t w e e n a dimer and a t e t r a m e r but this b e h a v i o r is not related to the malate sensitivity of the e n z y m e (25). Thus, like the C A M e n z y m e , P E P c in C 4 plants seems to be controlled by p h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n . In C4 species the interconversions are triggered by light/dark changes and there is no evidence that a circadian r h y t h m is involved. T h e significance of these interconversions i n regulation o f flux t h r o u g h the C 4 p a t h w a y is n o t yet clear.