Control of the phosphorylation of phosphoenolpyruvate carboxylase in higher plants

Control of the phosphorylation of phosphoenolpyruvate carboxylase in higher plants

ABB Archives of Biochemistry and Biophysics 414 (2003) 189–196 www.elsevier.com/locate/yabbi Minireview Control of the phosphorylation of phosphoeno...

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ABB Archives of Biochemistry and Biophysics 414 (2003) 189–196 www.elsevier.com/locate/yabbi

Minireview

Control of the phosphorylation of phosphoenolpyruvate carboxylase in higher plantsq Hugh G. Nimmo* Plant Molecular Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Davidson Building, University of Glasgow, Glasgow, Scotland G12 8QQ, UK Received 2 December 2002, and in revised form 13 February 2003

Abstract Phosphoenolpyruvate (PEP) carboxylase is regulated by reversible phosphorylation in higher plants. Recently several genes encoding PEP carboxylase kinase have been cloned. The purpose of this article is to assess the contribution that information on the structure and expression of these genes is making to our understanding of the posttranslational control of PEP carboxylase activity. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Crassulacean acid metabolism; Gene expression; Phosphoenolpyruvate carboxylase; Protein kinase; Transcriptional control

Phosphoenolpyruvate (PEP)1 carboxylase (PEPC, EC 4.1.1.31) catalyzes the carboxylation of PEP to yield oxaloacetate and Pi . It plays a wide range of metabolic roles in higher plants: in the leaves of Crassulaceanacid-metabolism (CAM) and C4 plants it catalyzes the primary fixation of atmospheric CO2 , in most nonphotosynthetic tissues and in the leaves of C3 plants it is the major anaplerotic enzyme with a key role in the coordination of C and N metabolism, and in guard cells and legume root nodules it is involved in pH regulation and in the provision of malate (e.g., [1–3]). To fulfill these diverse roles, PEPC activity is encoded by a small gene family [2,4]. PEPC is a homotetramer, comprising identical subunits of Mr  110; 000. The crystal structures of the Escherichia coli and maize enzymes have been established [5–7]. Early work showed that PEPC from higher plants is an allosteric enzyme, inhibited by L -malate and activated by glucose 6-phosphate [1–3]. Superimposed on this, its activity is controlled by q

Work form this laboratory has been supported by AFRC and BBSRC. * Fax: +44-141-330-4620. E-mail address: [email protected] 1 Abbreviations used: PEP, phosphoenolpyruvate; PEPC, PEP carboxylase; CAM, Crassulacean acid metabolism; PPCK, PEPC kinase genes; CDPK, calcium-dependent protein kinase.

reversible phosphorylation of a single, highly conserved Ser residue close to the N-terminal end of the polypeptide [2,3,8]. Phosphorylation of this residue results in a decline in the sensitivity of the enzyme to inhibition by malate and an increase in its sensitivity to activation by glucose 6-phosphate. There is now strong evidence from several systems that phosphorylation of PEPC correlates with increased flux through the enzyme in vivo [9–12]. In consequence, much attention has been focused on the factors that control the phosphorylation state of PEPC and the ‘‘machinery’’ involved. This area of research has been well reviewed recently [2,3,8]. In brief, PEPC is phosphorylated by a small, specific Ser/Thr kinase termed PEPC kinase and dephosphorylated by protein phosphatase 2A. The phosphorylation state of PEPC is largely controlled by the activity of PEPC kinase. The cloning and characterization of PEPC kinase genes (hereafter termed PPCK genes) was first reported in 1999, for the CAM species Kalancho€e fedtschenkoi and the model C3 plant Arabidopsis thaliana [13]. Following this, many other PPCK genes have been identified ([14–16] and below). The main objective of this minireview is to assess recent progress in our understanding of the control of PEPC in higher plants in the light of information on the structure and expression of PPCK genes.

0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00115-2

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Structure and expression of PPCK PEPC kinase is a very low-abundance protein; for example, a purification factor of greater than 106 was reported for a relatively rich source, field-grown maize leaves [17]. Hence the first successful cloning of a PPCK cDNA involved an indirect strategy. The abundance of PPCK transcripts could be measured by translation of mRNA in vitro followed by direct assay of the kinase activity [18]. This method was used to screen pools and subpools of a cDNA library from K. fedtschenkoi [13]. Other PPCK cDNAs have been cloned since, either by identifying or employing conserved sequences [15,16 and below] or by a protein kinase-targeted differential display approach [14]. PPCK genes have been obtained either by searching genomic databases or by using PCR ([13,16] and below). This work has led to the following conclusions: (a) PEPC kinase is the smallest known protein kinase. It comprises a protein kinase catalytic domain with minimal insertions or extensions at the N- or C-terminal ends. It is most closely related to the catalytic domains of plant calcium-dependent protein kinases (CDPKs) [19] but lacks the N-terminal extension, the C-terminal autoinhibitory region, and the EF hands of these enzymes. It does not contain the phosphorylatable residues in the activation loop or the ATP-binding loop that have been implicated in the regulation of some other protein kinases [20]. Biochemical evidence suggests that PEPC kinase exists as a monomer [2,3,13]; hence from the sequence data there is no obvious mechanism to control PEPC kinase activity other than by synthesis/degradation. Indeed, it is clear that the CAM PEPC kinase turns over rapidly [13]. (b) Most PPCK genes contain a single intron very close to the 30 end of the coding sequence. This intron usually interrupts the codon for a conserved Arg residue immediately prior to the His.X.aromatic.hydrophobic sequence that is often taken to denote the end of the protein kinase catalytic domain. However, one exception to this rule is described below. The PPCK sequences currently in GenBank (as of January 2003) are summarized in Table 1. Several points emerge from this table. First, plants seem to contain a PPCK gene family: A. thaliana has two PPCK genes, soybean at least three, and tomato at least two. Second, for one gene (sorghum PPCK), the deduced amino acid sequence contains a short, acidic insertion relative to other PEPC kinases. Structural predictions suggest that this additional sequence is located at the surface of the protein on the side opposite to that of the active site (H.G. Nimmo, unpublished). Hence it may have very little effect on the catalytic properties of the enzyme. Third, one gene (tomato PPCK2) contains two introns. One of these is located at the same position as that of the single intron in other PPCK genes. The other lies

upstream, in the middle of the coding sequence, and is subject to alternative splicing (J.T. Marsh, J. Hartwell, and H.G. Nimmo, unpublished). This is illustrated in Fig. 1. Intron 2 (close to the 30 end of the coding sequence) is correctly removed from transcripts. However, alternative splicing of intron 1 gives rise to three transcripts—unspliced, incorrectly spliced, and correctly spliced—all of which are detectable in tissues. Since intron 1 contains an in-frame stop codon, the unspliced and incorrectly spliced transcripts encode a prematurely truncated protein devoid of PEPC kinase activity. The function of this alternative splicing is currently unknown. The existence of a small PPCK gene family in at least some species raises questions about the expression pattern and functions of the different family members. Some progress toward answering these questions has been made with A. thaliana [16]. In this C3 species, PPCK1 is most heavily expressed in rosette leaves; expression is also detectable in roots and flowers but only very low levels of transcript are found in stems, cauline leaves, and siliques. In contrast, PPCK2 is most heavily expressed in roots and flowers; it is moderately expressed in cauline leaves but only very low levels of transcript are found in rosette leaves and stems. These data imply that the two genes play somewhat different functional roles. This conclusion is reinforced by recent studies of the expression of PPCK1 and PPCK2 in an Arabidopsis cell culture. This work has shown that expression of PPCK1 is strongly up-regulated by increases in cytosolic pH, whereas the expression of PPCK2 is unaffected (Z-H. Chen, G.I. Jenkins, and H.G. Nimmo, unpublished). Interestingly, expression of both PPCK1 in rosette leaves and PPCK2 in flowers is increased by light [16]. RT-PCR experiments with other plant species have confirmed that expression of PPCK genes differs between organs but does not appear to show absolute specificity. For example, soybean PPCK2 and PPCK3 are most heavily expressed in root nodules, but transcripts are detectable in other organs; PPCK1 transcripts are detectable in nodules even though this gene is more heavily expressed elsewhere (J.S. Sullivan, G.I. Jenkins, and H.G. Nimmo, unpublished). The expression-pattern experiments carried out to date do not allow us to ascribe organ- and/or tissue-specific functions to particular genes. It will be very important to perform expression studies with greater spatial resolution, to define precisely the cell types that express particular genes; it will also be essential to extend these by studying the accumulation of both the proteins and the transcripts. This will prove technically difficult, given the low abundance of PPCK proteins and the similarities in amino acid sequence in family members. Indeed, to address this issue it may prove necessary to express tagged PPCK constructs behind their native promoters.

Table 1 Compilation of full-length PPCK genes and cDNAs Gene name and alternatives

GenBank Accession No.a

Number of residues

Deduced Mr

Number of introns

Comments

Arabidopsis thaliana

PPCK1 At1g08650 F22O13.13 PPCK2 At3g04530 T27C4.19

gDNA AC003981 cDNA AF162660

284

31832

1

AC003981 is annotated incorrectly; see [13]

gDNA AC022287 (ecotype Columbia) gDNA AY040830 cDNA AF358915 (ecotype Landsberg erecta) cDNA AJ309171 cDNA AB065100

278

31259

1

AC022287 is annotated incorrectly; see [16]

282 231

31995 31785

Not available Not available

282

31577

1

274

30992

1

274

30892

1

274

30695

1

277

30731

1

279

31415

1

278

31100

2

Shows alternative splicing; see text and Fig. 1

279

31835

Not available

289

30602

1

307

32524

Not available

One of a small gene family; see [14] AC065100 is annotated incorrectly Contains acidic insert; see text

Arabidopsis thaliana

Beta vulgaris (sugar beet) Flaveria trinervia

PPCK PPCK

Glycine max (soybean)

PPCK1

Glycine max (soybean)

PPCK2 NE-PPCK

Glycine max (soybean)

PPCK3

Kalancho€e fedtschenkoi

PPCK

Lotus japonicus

PPCK

Lycopersicon esculentum (tomato)

PPCK1

Lycopersicon esculentum (tomato)

PPCK2

gDNA AY144181 cDNA AY144180 GDNA AY144183 cDNA AY143660 cDNA AY144182 gDNA AY144185 cDNA AY144184 gDNA AF162661 cDNA AF162662 gDNA AB092819 cDNA AB092818 gDNAAY190084 cDNA AF203481 gDNA AY188444

Mesembryanthemum crystallinum

PPCK

cDNA AY187634 CDNA AF158091

Oryza sativa (rice)

PPCK

Sorghum bicolor

PPCK

a

gDNA AC065100 (strain Nipponbare) cDNA AF399915

One of a small gene family; see [15]

H.G. Nimmo / Archives of Biochemistry and Biophysics 414 (2003) 189–196

Plant species

Full-length ORFs (many EST sequences are also available); gDNA, sequence of genomic DNA.

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Fig. 1. Structure of the tomato PPCK2 gene. Dark blue, exon; light blue, intron; yellow, splice start site; green, splice end site; red, in-frame stop codon. Intron 1 can be correctly spliced from a, incorrectly spliced from b, or unspliced.

Control of the phosphorylation state of PEPC Early studies showed that the phosphorylation state of PEPC correlated with the activity of PEPC kinase, which is regulated in CAM and C4 leaves by a circadian oscillator and light, respectively [2,3,8]. The availability of PPCK genes allowed a much more detailed analysis, particularly in the constitutive CAM species K. fedtschenkoi. Hartwell et al. [13] demonstrated a good correlation between the level of PPCK transcripts as judged by Northern analysis and the level of functional PEPC kinase-translatable mRNA, with expression peaking in the middle of the night. Both the PEPC kinase activity and the phosphorylation state of PEPC tracked these parameters after a short time lag. These data imply that the major factor controlling the phosphorylation of CAM PEPC, and hence nocturnal CO2 fixation, is the abundance of PEPC kinase transcripts. The data of Taybi et al. [14] on the facultative CAM species Mesembryanthemum crystallinum are compatible with this. However, in at least some systems additional factors may be involved. In germinating cereal seeds, changes in the phosphorylation state of PEPC do not correlate with changes in the activity of PEPC kinase. Osuna et al. [21] showed that PEPC in barley seed becomes phosphorylated following imbibition. However, PEPC kinase activity was already present in the dry seed and was not apparently increased by imbibition. Furthermore, various pharmacological agents that are known to block the signalinduced phosphorylation of PEPC in other systems (cycloheximide, TMB-8, W7, e.g., [2,3,8]) had no effect on the phosphorylation state of PEPC in the seed. This suggests the existence of physical or other factors that prevent the phosphorylation of PEPC in dry seed and implies that the PEPC kinase in these seeds does not exhibit the rapid turnover that is characteristic of other PEPC kinases. It should also be noted that changes in the apparent phosphorylation state of PEPC in barley leaf protoplasts (as judged by the enzymeÕs malate sensitivity) did not always correlate with changes in PEPC kinase activity [22]. Recently, two complicating factors which could account for a lack of correlation between the PEPC kinase activity measured in vitro and the phosphorylation state

of PEPC assessed in vivo have emerged. Nimmo et al. [23] demonstrated the existence in several plant tissues of a protein that reversibly inhibits PEPC kinase. In the CAM species K. fedtschenkoi, PEPC kinase activity during the day is low but finite. There was sufficient inhibitor present during the day to inhibit completely this residual amount of PEPC kinase activity. However, the amount of inhibitor present at night had little effect on the much larger amount of kinase present during this period. This led to the hypothesis that the role of the inhibitor may be to mask in vivo the amount of PEPC kinase activity remaining during periods in which PEPC is dephosphorylated (i.e., the day in CAM plants, the night in C4 plants). The inhibitor protein may be involved in the control of PEPC kinase in dry cereal seeds. However, it is important to stress that the identity, mechanism, and physiological importance of this inhibitory protein remain to be resolved. Saze et al. [17] showed that maize PEPC kinase was rapidly inactivated by incubation in the presence of oxidized glutathione (GSSG). This effect could be reversed by subsequent incubation of the enzyme with reducing agents, and dithiothreitol-dependent reactivation was greatly enhanced in the presence of thioredoxin. Similar, but less extensive, data have been reported for a recombinant PEPC kinase from the C4 dicot Flaveria trinervia [15]. It has been suggested that, in addition to control at the level of expression, the activity of C4 PEPC kinase may be light-activated via the cytoplasmic NADPH/NADPþ ratio and thioredoxin-h [17]. This pattern is clearly the opposite of that expected for PEPC kinase in CAM species. Hence if C4 PEPC kinases are indeed regulated by thiol–disulfide exchange, one might expect them to contain redox-modulated Cys residues that are not present in CAM PEPC kinases. However the sequences available to date do not reveal such C4 -specific residues. Nevertheless, it would be dangerous to assume that PEPC kinase activity is controlled only by synthesis/ destruction of the kinase protein. It will be important to assay the activity of the kinase in different organs and tissues under conditions which could reveal control by an inhibitor protein (e.g., assays at a range of dilutions) or by thiol–disulfide exchange (e.g., assays plus and minus dithiothreitol). Such experiments have the potential to reveal conditions under which PEPC kinase is controlled by mechanisms in addition to its turnover. A further factor that could account for a lack of correlation between the PEPC kinase activity and the phosphorylation state of PEPC is, of course, control of the dephosphorylation of PEPC [2,10,24]. Recently Dong et al. [25] reported the purification and characterization from maize leaves of a PP2A holoenzyme with PEPC phosphatase activity. This activity appeared to be that of a heterotrimer, similar to PP2A holoenzymes from other eukaryotic species. However the purified enzyme could dephosphorylate substrates other than

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PEPC, even though the final purification step involved affinity-chromatography on an immobilized thio-phosphorylated peptide corresponding to the phosphorylation site in C4 PEPC. Hence the additional A and B subunits in the heterotrimer do not render the catalytic C subunit absolutely specific for PEPC. Dong et al. [25] noted that several anionic metabolites known to affect PEPC activity (glucose 6-phosphate, L -malate, PEP) all inhibited the purified heterotrimeric PP2A; these effects were observed using either PEPC or rabbit glycogen phosphorylase a as a substrate, suggesting that inhibition resulted from interaction with the phosphatase and not the substrate. However, similar effects were observed with a mammalian PP2A heterodimer. This suggests that the inhibitory effects of these anionic metabolites may not be physiologically significant. The amount of PEPC phosphatase activity and subunits purified from illuminated leaves was modestly greater than the amount obtained from darkened leaves [25]. However these changes are much less dramatic than those observed for PEPC kinase activity. Further work on PEPC phosphatase is required but at this stage there is little evidence to suggest that it plays a major role in controlling changes in the phosphorylation state of PEPC.

Signaling mechanisms that control expression of PPCK genes Earlier work established the general pattern that PEPC kinase activity is regulated in C4 leaves by illumination/darkness, in CAM leaves by a circadian oscillator, in C3 leaves by a combination of light and N supply, and in legume root nodules by photosynthate supply from the shoots [2,3,8]. The evidence available suggests that these stimuli target expression of PPCK genes. In dissection of the signaling pathways, most progress has been made in the C4 system. Studies with isolated mesophyll–cell protoplasts from Digitaria sanguinalis have allowed major advances [26,27]. First, both light and increases in cytoplasmic pH are required for the induction of PPCK activity; furthermore this induction could be blocked by certain pharmacological agents that perturb Ca2þ signaling [26]. Then more detailed studies [27] showed that the signaling pathway includes transient activation of a phosphoinositide-dependent phospholipase C and production of inositol 1,4,5-trisphosphate which is thought to open tonoplast calcium channels. The resulting increase in cytoplasmic [Ca2þ ] presumably activates a calcium-dependent enzyme, perhaps a protein kinase such as CDPK. Analysis of this system, which has many advantages, has been restricted by the lack of cloned PPCK genes from this C4 weed species. Pharmacological agents have also been used in studies of the induction of PPCK gene expression and PEPC

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kinase activity in detached CAM leaves [28,29]. These experiments showed that the nocturnal appearance of kinase activity could be blocked by inhibitors of Ca2þ / calmodulin-like interactions, phosphoinositide-dependent phospholipase C, and tonoplast calcium channels. Bakrim et al. [28] suggested that increases in cytoplasmic pH resulting from the accumulation of malic acid in the vacuole at night might initiate the signaling pathway leading to PPCK gene expression in CAM plants. However, the timing of the pH changes that have been observed in a CAM species [30] differs significantly from the timing of the changes in PPCK transcripts in Kalancho€e species [13,31]. Moreover, Paterson and Nimmo [32] found that treatment of K. fedtschenkoi leaf disks with NH4 Cl to increase cytoplasmic pH had no effect on the nocturnal phosphorylation of PEPC. Hence the involvement of pH changes in the CAM system remains uncertain. Several lines of evidence have suggested that metabolite regulation may play an important role in the control of PPCK gene expression, at least in CAM plants. The effects of treatments that affect accumulation of malate, either by temperature manipulations or by enclosing leaves in an atmosphere of N2 [31], are consistent with the view that cytoplasmic malate (or a related metabolite) causes feedback inhibition of PPCK gene expression. This work led to the hypothesis that the circadian control of PPCK gene expression in CAM is actually a secondary process, consequent on a primary effect on malate transport across the tonoplast [8]. Hartwell et al. [33] reported that the effects of temperature changes on PPCK expression are ‘‘gated’’ by the circadian clock; they argued that this observation is compatible with indirect control of expression, for example via metabolites, rather than direct control by the central oscillator. However, our direct attempts to monitor the cytoplasmic concentration of malate in CAM leaves using nonaqueous fractionation have proved unsuccessful to date [33]. Metabolite signaling might involve feedforward activation of PPCK gene expression, instead of or in addition to feedback inhibition. It is clearly important to develop techniques for measurement of metabolite distribution and subcellular concentrations in CAM species. Metabolic signaling via malate may also be involved in related systems. Stitt and co-workers [34,35] have studied the regulation of nitrate reductase expression extensively in tobacco. For example, direct feeding of malate to detached leaves reduced the levels of nitrate reductase activity and transcripts [35]. The expression of PEPC is tightly coordinated with that of nitrate reductase [34], and the expression of PPCK genes in the same system clearly warrants investigation. Metabolite control may also be implicated in the induction of PPCK expression in legume root nodules. Studies of the phosphorylation of PEPC suggested that the primary

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signal is metabolic—translocation of photosynthate from the shoots [36]. Consistent with this, the level of a PPCK transcript expressed in nodules of the model legume Lotus japonicus declines during extended darkness but recovers upon reillumination of the shoots [37]. Interestingly, pharmacological experiments showed that this recovery does not require phospholipase C activity. In C4 leaves the primary signal that induces PPCK expression is light, and PPCK is expressed in the face of a high malate content in leaf mesophyll cells [3,11,12] but it remains possible that metabolic control of C4 PPCK expression plays a secondary, coordinating role. Several recent studies have shown that guard-cell PEPC can be phosphorylated (e.g., [38–40]), and the advantages of the guard-cell system for signaling work are now being exploited. Fusicoccin, which activates the plasma membrane Hþ -pumping ATPase and stimulates stomatal opening, can bring about phosphorylation of guard-cell PEPC as judged both directly from 32 P-labeling of tissue and indirectly from activity measurements [38,40,41]. However, the phosphorylation of PEPC correlates with malate accumulation rather than stomatal opening as such; little phosphorylation is observed in the absence of Kþ or in the presence of Cl (which reduces accumulation of malate by acting as an alternative counter-ion to balance Kþ uptake during stomatal opening) [41]. When Kþ was replaced as osmolyte by sucrose, fusicoccin stimulated stomatal opening but not phosphorylation of PEPC. Outlaw et al. [41] found that both fusicoccin and NHþ 4 ions caused an increase in the cytoplasmic pH in guard cells, albeit with different time courses; however, only fusicoccin caused phosphorylation of PEPC. They concluded that activation of the plasma membrane Hþ -ATPase is essential, but not sufficient, to cause phosphorylation of guard-cell PEPC, presumably via the expression of PPCK genes. Since fusicoccin activates the guard-cell Hþ -extruding plasma membrane ATPase [42], one obvious hypothesis is that elevated cytoplasmic pH is part of the signaltransduction pathway leading to the phosphorylation of PEPC. There is, however, controversy as to the effects of fusicoccin on cytoplasmic pH, with both increases and decreases being reported (see [41] and references therein). Indeed, Meinhard and Schnabl [40] showed that treatment of guard-cell protoplasts with butyrate caused activation of PEPC; they interpreted this as evidence that a reduction in cytoplasmic pH is part of the pathway leading to phosphorylation of PEPC. Thus, several uncertainties about the control of PEPC phosphorylation in guard cells remain to be resolved. One concerns the role of PPCK gene expression. The effects of fusicoccin on PEPC in guard cells are relatively rapid and are not blocked by cycloheximide [40]. No PPCK gene expressed preferentially in guard cells has been reported as yet. It remains possible that some of the effects that have been observed result from modulation of the ac-

tivity of existing PEPC kinase rather than de novo PPCK expression. However the broad conclusion of Outlaw et al. [41], that the phosphorylation of guard-cell PEPC is observed only under conditions in which malate accumulates, is striking; this surely implies that the phosphorylation of PEPC is contingent upon metabolism rather than being ‘‘hard-wired’’ to a signaling pathway.

PEPC kinases in C4 plants The importance of the phosphorylation of PEPC for operation of the C4 pathway is well established [2,3], and the C4 system has provided much information about the signaling pathway leading to the phosphorylation of PEPC [3,26,27]. Hartwell et al. [18,43] showed that light induces an increase in the amount of PEPC kinase-translatable mRNA in maize, but more detailed analysis of the effect of light on C4 monocots has proven difficult because of a delay in cloning PPCK genes from these species. However IzuiÕs group [44] has made several important contributions recently. Using an affinitypurified antibody specific to phosphorylated maize C4 PEPC, they noted that phosphorylation of maize leaf PEPC is not wholly dependent on light. Although they noted some differences according to cultivar and plant age, they observed increases in the phosphorylation of PEPC prior to dawn in both field-grown and chambergrown plants. Predawn increases in PEPC kinase activity were also detected in field-grown leaves [44]. Furthermore, both the PEPC kinase activity and the phosphorylation state of PEPC declined to a low level well before dark in field-grown plants. This suggests that, contrary to earlier supposition, PEPC kinase activity in maize is not controlled solely by light but may exhibit some elements of circadian and/or metabolic control. Clearly it would be interesting to know whether the PEPC kinase activity detectable predawn is due to the same protein as that expressed in high light. We have recently identified three maize PPCK cDNAs, all of which are expressed in leaves. Only one of these transcripts is up-regulated by light (M. Shenton, G.I. Jenkins, and H.G. Nimmo, unpublished). However, it will also be necessary to assess the levels of protein and activity of the three PEPC kinase isoforms in maize in response to different environmental signals. A PPCK cDNA from the C4 dicot F. trinervia has recently been identified and cloned by RT-PCR using an illuminated-leaf library [15]. This gene is expressed preferentially in leaves and very strongly up-regulated by light. Like the enzyme purified from maize leaves [17], the activity of the recombinant form of this PEPC kinase (expressed and assayed as an intact NusA fusion protein) was controlled by redox state [15]. Southern analysis indicated that F. trinervia contains several

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PPCK genes [15]. Whether any of the other genes are expressed in leaves and how their expression is controlled remains to be seen. In addition, detailed studies of the activity of PEPC kinase and the phosphorylation state of PEPC in F. trinervia leaves across the diurnal cycle are needed. However, it seems certain that the PPCK gene identified by Tsuchida et al. [15] plays a significant role in the light regulation of C4 PEPC. It has recently been shown that when the C4 plant sorghum is grown under salt stress, the amount of PEPC kinase activity and the phosphorylation state of PEPC in illuminated leaves are markedly increased [45,46]. Control experiments using mannitol and fusicoccin demonstrated that these effects are due to ion toxicity rather than water stress per se; furthermore, they are not mimicked by application of abscisic acid. The PEPC kinase activity in darkened leaves was also significantly increased by salt stress [46]. In salt-stressed plants, but not in control plants, the leaf malate content increased during the night, apparently owing to recycling of respiratory CO2 [46]. Hence the increase in PEPC kinase activity in salt-stressed tissue seems to have physiological consequences. One PPCK gene has been identified to date in sorghum (see Table 1) but whether light and salt stress affect the same or different PEPC kinases remains to be seen. Clearly the roles and regulation of PPCK genes in C4 plants are far from being fully understood.

Some unresolved problems Several studies have made use of ‘‘in-gel’’ analysis of PEPC kinase polypeptides in which proteins are renatured after SDS gel electrophoresis and PEPC kinase activity is specifically visualized by the transfer of 32 P from [c-32 P]ATP to PEPC included in the separating gel (e.g., [36,45]). This work has consistently resolved two PEPC kinase polypeptides with Mr values of 30,000 to 33,000 and 37,000 to 39,000 [2,3 and references therein] in a range of organs including C3 , C4 , and CAM leaves and legume root nodules. However, none of the PPCK genes that have been cloned to date (see Table 1) encode a 37- to 39-kDa polypeptide. This raises the question of whether the higher Mr form of PEPC kinase may represent a posttranslationally modified polypeptide. Another notable feature of PEPC kinase is that, in many cases, both the protein and its message must turn over rapidly. This is best seen in the work of Hartwell et al. [13] on the CAM system, which showed that both kinase activity and PPCK transcripts disappear almost completely within a period of some 3 h. This raises two questions about the turnover of PEPC kinase protein: first, what is the mechanism that underlies this rapid turnover and, second, is it controlled? An attractive possibility which should be investigated is that PEPC kinase might be turned over via the ubiquitin/protea-

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some system. Such a mechanism might account for the higher Mr form of PEPC kinase, which could be a monoubiquitinated species. The slow-turnover form of PEPC kinase in cereal seeds (see [21] and above) could represent an isoform resistant to ubiquitination. Taybi et al. [14] noted that the 30 untranslated region of the M. crystallinium PPCK cDNA contained elements that might contribute to the instability of the mRNA. It is important to test whether this region does, in fact, contribute to transcript instability.

Concluding remarks Following the cloning of PPCK genes, it can be argued that the phosphorylation of PEPC represents the best understood example of metabolic control via protein phosphorylation in plants. This is largely because of the simplicity (at least as it appears at present) of the system: control is vested largely in the expression of one (or more) PPCK genes which encode a simple and specific Ser/Thr kinase with no other known substrates. However, the availability of PPCK clones has itself raised many new questions, particularly concerning the roles of different isoforms and the mechanisms underlying their expression, regulation and turnover. Some experimental approaches are precluded by the low abundance of the PEPC kinase protein. In consequence, other approaches and tools required to answer these questions, for example transgenic plants expressing promoter:reporter fusions and tagged PEPC kinases, are now being developed.

Acknowledgments I gratefully acknowledge the contribution of many co-workers over 20 years, particularly Malcolm Wilkins, Gareth Jenkins, Jill Nimmo, and James Hartwell. I also thank many colleagues who provided information prior to publication.

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