Plant Physiol. (1991) 95, 981-9850032-0889/91/95/0981/05/$01 .00/0

Received for publication August 24, 1990Accepted November 15,1990

Review

Posttranslational Regulation of PhosphoenolpyruvateCarboxylase in C4 and Crassulacean Acid

Metabolism Plants1

Jin-an Jiao and Raymond Chollet*

Department of Biochemistry, University of Nebraska-Lincoln, East Campus, Lincoln, Nebraska 68583-0718

ABSTRACT

Control of C4 photosynthesis and Crassulacean acid metabo-lism (CAM) is, in part, mediated by the diel regulation of phos-phoenolpyruvate carboxylase (PEPC) activity. The nature of thisregulation of PEPC in the leaf cell cytoplasm of C4 and CAM plantsis both metabolite-related and posttranslational. Specifically, theregulatory properties of the enzyme vary in accord with thephysiological activity of C4 photosynthesis and CAM: PEPC isless sensitive to feedback inhibition by L-malate under light (C4plants) or at night (CAM plants) than in darkness (C4) or duringthe day (CAM). While the view that a light-induced change in theaggregation state of the holoenzyme is a general mechanism forthe diel regulation of PEPC activity in CAM plants is currentiy indispute, there is no supportive in vivo evidence for such a tetra-mer/dimer interconversion in C4 plants. In contrast, a wealth of invitro and in vivo data has accumulated in support of the view thatthe reversible phosphorylation of a specific, N-terminal regulatoryserine residue in PEPC (e.g. Ser-15 or Ser-8 in the maize orsorghum enzymes, respectively) plays a key, if not cardinal, rolein the posttranslational regulation of the carboxylase by light/dark or day/night transitions in both C4 and CAM plants,respectively.

Phosphoenolpyruvate carboxylase (PEPC2, EC 4.1.1.31)catalyzes the irreversible ,B-carboxylation of PEP in the pres-ence of bicarbonate and Me2", (e.g. Mg2", Mn2") to yieldoxalacetate and Pi, a reaction that serves a variety of physio-logical functions in plants. This cytoplasmic enzyme com-prises four identical subunits with monomeric molecular massof about 110 kD. During both C4 photosynthesis and CAM,PEPC is the initial carboxylating enzyme that fixes atmos-pheric CO2 into C4-dicarboxylic acids (oxalacetate, malate,and aspartate), from which CO2 is subsequently releasedinternally by various decarboxylating enzymes and photosyn-thetically reassimilated by the Calvin cycle (7, 21). This initial

'The research described herein from this laboratory was supportedin part by grant DMB-8704237 from the National Science Founda-tion. This review is published as Paper No. 9323, Journal Series,Nebraska Agricultural Research Division.

2Abbreviations: PEPC, phosphoenolpyruvate carboxylase; PEP,phosphoenolpyruvate; PPDK, pyruvate,Pi dikinase; PP, protein phos-phatase; PK, protein kinase.

carboxylation reaction is compartmented either spatially (inmesophyll cells in the light) or temporally (in the dark) in C4or CAM plants, respectively.Based on physiological and biochemical considerations, it

is evident that PEPC activity must be regulated by light +

dark transitions in C4 and CAM species in order to: (a)coordinate C4- and C3-photosynthetic carbon metabolism inresponse to light intensity in C4 plants (7, 10); (b) avoid futiledecarboxylation/carboxylation cycling during the day inCAM plants (21); and (c) minimize uncontrolled utilizationof glycolytic PEP during the dark in C4 plants (2, 7). Whilelight-induced changes in the cytoplasmic levels of knownmetabolite effectors (e.g. glucose 6-P, L-malate, triose-P) likelycontribute to the overall regulation of PEPC activity (5, 7,21), recent attention has focused on the diel regulation of thisenzyme's activity in C4 and CAM species by posttranslationalmodification. In this review we summarize the recent devel-opments in this latter area of research and discuss the molec-ular mechanism(s) involved in this regulatory process.

GENERAL PROPERTIES OF THE REGULATORYPROCESS

The initial interest in the posttranslational modulation ofPEPC activity came from work on the diel regulation ofCAM. The enzyme responsible for the initial fixation ofatmospheric CO2 by CAM plants in darkness is PEPC, which,like the C4 isoform, is activated allosterically by glucose 6-Pand feedback inhibited by L-malate (7, 21). A related, phys-iologically important feature of CAM PEPC is its day/nightfluctuation in enzymatic properties (29); the rapidly prepared,desalted enzyme extracted from night leaf tissue has lowerKm(PEP) and higher Ki(malate) values than those from thecorresponding day leaf tissue. These day/night changes in theproperties of PEPC lead to a higher enzymatic activity atnight and a much lower activity during the day (e.g. the night/day activity ratio is about 8 [15, 29]) which are paralleled bythe classical physiological changes in the activity ofCAM (e.g.external C02 fixation, titratable acidity [21]). Further detailedstudy of several CAM plants under continuous dark or lightconditions indicated that CAM physiology, as well as themalate sensitivity of PEPC, are controlled by an endogenouscircadian rhythm rather than by light or dark signals per se(20, 29).

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With respect to C4 PEPC, intensive investigation of its light/dark regulation was initiated only recently. Despite this limi-tation, it is now established unequivocally that light reversiblyinduces a two- to threefold increase in catalytic activity anddecrease in malate sensitivity of the enzyme from a variety ofC4 plants when assayed at suboptimal, but physiological levelsof pH and PEP and in the presence of L-malate. Typically,such assay conditions result in a light/dark activity ratio ofabout 5 (8-10, 19, and references therein). Darkness reversesthese effects of illumination on C4-leaf PEPC activity. More-over, light activation of PEPC in C4 plants is related, eitherdirectly or indirectly, to photosynthetic electron transportand/or photophosphorylation and is modulated by severalphotosynthesis-related environmental factors, including lightintensity, CO2 concentration, and temperature (19, 23, 24and references therein). It is notable that when compared tothe light induction of C4 photosynthesis and the activation ofphotoregulated C4 mesophyll-chloroplast stromal enzymes(NADPH-malate dehydrogenase, PPDK), light activation ofthis cytoplasmic enzyme is relatively slow, taking 30 to 60min for completion (9, 19, 24).

In contrast to the situation in CAM and C4 plants, recentreports indicate that there is no such light/dark modulationof PEPC activity in C3 leaf tissue (4) and guard cells (25).

MOLECULAR MECHANISMS OF PEPCPOSTTRANSLATIONAL REGULATION

CAM-PEPC

Wu and Wedding (30) first reported the purification ofCAM PEPC from day- and night-adapted Crassula argentealeaves and found that these two enzyme-forms existed askinetically distinct, but thermodynamically interconvertible,oligomers. The day enzyme was mainly a malate-sensitivehomodimer (a2 [Ki, 1.6 mM]) and the night enzyme a malate-insensitive homotetramer (a4 [Ki, 3.9 mM]). The results fromin vitro studies with the purified day enzyme (30, 31) furthershowed that the substrate PEP and Mg2+, a bivalent cationrequired for catalysis, favor the conversion of a2 to a4, whereasL-malate, a feedback inhibitor, shifts the dimer-tetramer equi-librium toward the dimeric enzyme-form. These in vitrofindings suggested that interconversion between the malate-sensitive dimer and malate-insensitive tetramer of PEPCmight be the molecular mechanism for the diel regulation ofPEPC activity in CAM plants. However, this hypothesis wasnot consistent with subsequent results obtained with otherCAM species in which both the day and night enzyme-formswere found to be in the same aggregation state but stilldisplayed the characteristic differential sensitivity to feedbackinhibition by L-malate (15, 18). In this context, it is notablethat the reported in vitro dimer-tetramer interconversion wasinfluenced by protein concentration (30), with a higher PEPCconcentration favoring tetramer formation and vice versa.Based on our calculations from the PEPC protein- and yield-related data reported by Nimmo et al. (18) and the assump-tions that 1 g fresh weight of leaf tissue is roughly equivalentto 1 mL in total volume and that the cytoplasmic matrixrepresents about 5% of the total cell volume (21), the in vivo

concentration ofCAM PEPC would be about 1.6 mg/mL (orabout 0.1 mg PEPC per g fresh weight of leaf tissue). Thisestimate is manyfold higher than the PEPC concentrationsused in vitro for the size-exclusion studies (e.g. less than 0.05mg/mL after the HPLC chromatography step [calculationsfrom ref. 31]). Thus, whether a dimer-tetramer interconver-sion of PEPC takes place in vivo during day/night transitionsof CAM leaf tissue and, if so, whether it is in any wayinfluenced by other posttranslational modifications of theenzyme (see below) are questions certainly worthy of furtherinvestigation.An alternative mechanism for the diel regulation of CAM

PEPC activity was proposed initially by Nimmo's group (3,17, 18, 20). By a combination of feeding Bryophyllum fedt-schenkoi leaf tissue with 32Pi and the subsequent SDS-PAGEanalysis of PEPC immunoprecipitates, they found (17) thatPEPC was seryl-phosphorylated in vivo at night when theenzyme is relatively malate insensitive [apparent Ki(malate),3 mm] and dephosphorylated during the day when it is highlymalate sensitive [Ki(malate), 0.3 mM]. Furthermore, when thepurified, malate-insensitive night form of 32P-labeled PEPCwas preincubated withl exogenous alkaline phosphatase invitro, dephosphorylation was correlated with a marked in-crease in malate sensitivity [i.e. a 10-fold decrease in theapparent Ki(malate)] of the enzyme (18). Notably, undervarious in vitro conditions (e.g. high or low protein concen-tration, in the absence or presence of malate or Mg2+), boththe phosphorylated, malate-insensitive PEPC and the dephos-phorylated, malate-sensitive enzyme-form from B. fedtschen-koi existed as a tetramer (cf. 30, 31), suggesting no directrelationship between the enzyme's aggregation state and itsmalate sensitivity/phosphorylation status in this particularCAM species (18).

Additional biochemical evidence that reversible regulatoryseryl-phosphorylation of PEPC in CAM plants contributes tothe day/night changes in properties of the target enzymecomes from the recent identification of a okadaic acid-sensi-tive type-2A protein phosphatase (PP) that dephosphorylatesPEPC from B. fedtschenkoi (3). Incubation of the in vivo 32p_labeled PEPC with exogenous mammalian type-2A PP resultsin a direct correlation between 32P-release from the targetenzyme and the concomitant decrease in apparent Ki(malate).In contrast, mammalian type-1 PP did not have any sucheffect on CAM PEPC. Notably, a partially purified type-2Aprotein phosphatase preparation from B. fedtschenkoi leavesconverted the malate-insensitive, night-form PEPC to a ma-late-sensitive form. This diel modulation of CAM PEPCproperties by reversible regulatory phosphorylation has beenconfirmed by both in vivo and in vitro studies with otherCAM species(l, 14).

C4-PEPC

The first observation that suggested a possible relationshipbetween the light-induced changes in the properties of the C4enzyme and its reversible phosphorylation status came fromexperiments with an in vitro 32P-phosphorylation system (2).This work demonstrated that PEPC from maize and sugar-cane can, indeed, be phosphorylated in vitro exclusively onserine residues by an ATP-dependent, soluble protein ki-

982 JIAO AND CHOLLET

POSTTRANSLATIONAL REGULATION OF PEP CARBOXYLASE

nase(s) in desalted extracts prepared from illuminated greenleaf tissue. Subsequent in vivo 32Pi-labeling studies with maize(13, 19) and sorghum (13, 26) leaf tissue indicated that C4PEPC is more sery phosphorylated in the light when theenzyme is less malate sensitive than in darkness when it ismore malate sensitive. Using rapidly purified PEPC frommaize leaves, Jiao and Chollet (9) reported that the enzymeisolated from light-adapted plants had a higher catalytic activ-ity and lower malate sensitivity than the corresponding darkenzyme-form. Although both light and dark enzymes con-tained P-serine, the degree of phosphorylation in the lightform was greater than that in the dark enzyme. As in the caseof phosphorylated CAM PEPC (18), preincubation of themaize light-form enzyme with exogenous alkaline phospha-tase converted the more active, less malate-sensitive PEPC toa less active, more malate-sensitive enzyme with propertiessimilar to those of the control or phosphatase-preincubateddark enzyme (9). It is interesting to note that exogenousalkaline phosphatase had little effect on the properties ofdark-form PEPC (9) even though it is partially phosphorylated (9,13, 19). Similar results were later obtained with sorghum leafPEPC (26).To assess critically the effects of phosphorylation on the

properties of C4 PEPC, in vitro experiments were performed(10) with an homologous reconstituted phosphorylation sys-tem comprised ofpurified, dark-form maize PEPC, a partiallypurified protein kinase(s) from light-adapted leaves, and ATP.Mg. The results from this phosphorylation system unequivo-cally established that the PK-mediated changes in the catalyticactivity and malate sensitivity of C4 PEPC are directly corre-lated with the concomitant changes in the seryl-phosphoryl-ation status of the target enzyme in vitro. Moreover, thesechanges in the enzymatic properties of dark-form PEPCcaused by in vitro phosphorylation were in quantitative agree-ment with those induced by light in vivo (9, 19). These invitro results, which have been subsequently confirmed byNimmo's group with maize (see pp. 357-364 in ref. 26) andVidal's group with sorghum (26), clearly demonstrate that theregulatory seryl phosphorylation ofPEPC by an ATP-depend-ent, soluble leaf protein kinase(s) is a key component in theposttranslational regulation ofC4 PEPC activity by light/darktransitions in vivo.Deduced primary sequences of various isoforms of PEPC

have been reported from diverse organisms, including Esch-erichia coli, Anacystis nidulans, and C3, C4, and CAM plants(1 1, 12 and references therein). Comparative analysis of thesesequences has revealed that the C-terminal region ofthe - 10-kD polypeptide is relatively conserved, perhaps encompassingthe active-site domain, whereas the N-terminal region is morevariable. Along these lines, the regulatory phosphorylationsite from in vitro 32P-phosphorylated/activated dark-formmaize PEPC (10) has recently been isolated and sequenced(1 1). The amino acid sequence ofthis regulatory phosphopep-tide is His-His-Ser(P)-Ile-Asp-Ala-Gln-Leu-Arg, which cor-responds exactly to residues 13 to 21 in the deduced primarystructure of the maize leaf enzyme. This N-terminal regula-tory site (Ser- 15) is far removed from a recently identified,species-invariant, active-site lysine (Lys-606) in the C-termi-nal region of the maize primary structure (12). Extension ofthe regulatory phosphorylation site sequence to the N-termi-

nal methionine reveals that (a) the phosphorylation site (Ser-15) is two residues removed from a basic amino acid (Lys-12)in the primary structure, a feature similar to that in variousprotein-serine/threonine kinase substrates; (b) C4 PEPC fromsorghum leaves and CAM PEPC from salt-stressed Mesem-bryanthemum crystallinum leaves contain N-terminal se-quences that are highly homologous to this regulatory phos-phorylation site, including the structural motif of Lys/Arg-X-X-Ser; and (c) no such homology is present in the bacterial,cyanobacterial or C3 (noninduced) M. crystallinum enzymes(1 1, 12, and references therein).More recently, comparative sequence analyses of the phos-

phopeptides isolated from in vivo 32P-labeled, purified dark-and light-form PEPC from maize or sorghum indicate thatSer- 15 or its structural homolog in the sorghum enzyme, Ser-8, is the only residue to be 32P-phosphorylated in vivo uponillumination and dephosphorylated in the dark (13). Theseresults, which are in complete agreement with the previous invitro studies (1 1), unequivocally establish that Ser- 15 is theregulatory site that undergoes light/dark changes in phos-phorylation status and, thus, contributes to the regulation ofmaize PEPC activity in vivo.Other available data also support the view that Ser-15 in

maize leaf PEPC is the regulatory phosphorylation site that isdirectly involved in the light/dark regulation of this enzymein vivo. Along these lines, McNaughton et al. (16) recentlyreported that dark-form maize PEPC that had been partiallydegraded by an endogenous protease(s) sensitive to chymosta-tin, a chymotrypsin inhibitor, had not only lost a -4-kDpeptide fragment from its N- or C-terminal, but also its malatesensitivity and the ability to be phosphorylated in vitro.

Vidal et al. (26) have recently isolated a calcium-dependentprotein kinase(s) from sorghum leaves that phosphorylatesdark-form PEPC in vitro but yet does not change the proper-ties of the target enzyme. In contrast, the soluble leaf PK(s)that phosphorylates (activates) maize PEPC at Ser- 15 (10, 1 1)is not affected by either calcium/calmodulin, EGTA, fructose2,6-bisP, or reduced cytoplasmic thioredoxin h from spinach.However, this kinase preparation is inhibited by NaCl (KC1),L-malate, and glucose 6-P. Such metabolite-mediated inhibi-tion may be a result of a direct interaction of the PK withthese carbon compounds or a result of a conformationalchange in the protein substrate, PEPC, induced by these twoknown effectors. It is not clear at present whether the calcium-dependent C4-leaf protein kinase(s) (26) phosphorylates thespecific, N-terminal regulatory seryl-phosphorylation siteidentified previously (1 1, 13) and, if so, whether the molarstoichiometry (i.e. mol Ser-P per 110-kD subunit) is suffi-ciently low that changes in catalytic activity and malate-sensitivity of the target enzyme can not be detected.With regard to the possible involvement of changes in the

aggregation state of PEPC in the reversible light regulation ofthe C4 enzyme, no supportive in vivo evidence has yet beenreported. In fact, both the light and dark enzyme-forms existas tetramers under normal conditions (2, 8, 16). However,the in vitro dissociation of the active C4 tetramer into dimersand monomers induced by dilution, NaCl, malate or lowtemperature (16, 22, 28, 32) leads to loss of enzyme activity,but with no change in malate sensitivity (16). Once again, itmust be emphasized that the dissociation of C4 PEPC is

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Plant Physiol. Vol. 95, 1991

Light (C4) or Night/ Rhythm (CAM)

Protein kinase - ' Protein kinase(Less active) - A

A (More active)

Dark (C )IOr Day/ Rhythm (CAM)

A TrJ A,DPATP VSE_<SER PK eSERP

[ ~~~~~~PEPC] PCu/-Tpe-2A'pi PP H20

toss active and/or More active and/ormore malate-sensitive less malate-sensitive

Figure 1. Proposed mechanism for the light/dark regulation of PEPCactivity in C4 and CAM plants in which reversible seryl-phosphoryla-tion plays a key, if not cardinal, role. The circle and square representtwo putative conformational states of the 1 1 0-kD PEPC subunitinduced by phosphorylation/dephosphorylation of a specific N-termi-nal serine residue (e.g. Ser-1 5 or Ser-8 in maize or sorghum PEPC,respectively [1 1, 13]). PK, light/dark-regulated protein kinase (6); PP,type 2A protein phosphatase (3).

inversely related to its concentration, and that the proteinconcentrations used in the above in vitro studies (0.01 toabout 0.5 mg/mL) are far below the estimated concentrationof C4 PEPC in vivo (about 15 mg/mL or 0.75 mg PEPC per

g fresh weight of leaf tissue based on the protein- and yield-related data in refs. 16 and 28 and the same volume-relatedassumptions mentioned before). At PEPC concentrations inthe range of 0.5 to 1 mg/mL, the in vitro dissociation of thenative tetrameric enzyme is minimized, even in the presence

of L-malate (22, 28 [Fig. 1], 32 [Fig. 6]). These findingseffectively argue against a significant regulation of C4 PEPCactivity in vivo by changes in its aggregation state.A related question is whether the way in which PEPC is

purified influences the enzymatic and dissociation propertiesof the enzyme in vitro. Along these lines, it is known thatPEPC from both C4 and CAM plants loses its sensitivity tomalate when the protein is proteolyzed at its N- or C-terminalduring purification (e.g. ifno malate/glycerol and/or proteaseinhibitor(s) are included in the isolation buffers or the purifi-cation protocol is too slow) (8, 16, 18, 29, and referencestherein). Based on these observations, it is not surprising thatmaize PEPC purified by conventional procedures (22) or

obtained from a commercial source (27) is much less sensitiveto feedback inhibition by L-malate than the same enzymepurified by more rapid protocols (9, 10, 13, 16). We are ofthe opinion that a markedly reduced malate-sensitivity ofpurified light-form maize PEPC is likely related to the prote-olytic loss of a -4-kD N-terminal fragment containing theregulatory phosphorylation site, Ser- 15 (11, 13, 16). It wouldbe of interest to determine whether the maize PEPC used byPodesta and Andreo (22) and Wedding's group (27) is missingthe N-terminal peptide fragment encompassing Ser- 15 and, ifso, whether this loss accounts for the ready dissociation of theholoenzyme in vitro (22, 28).

CONCLUDING REMARKS

It is becoming increasingly evident that the reversible serylphosphorylation of PEPC plays a key, if not cardinal, role in

the light/dark regulation of the C4- and CAM-leafenzymes invivo (Fig. 1). Future work on the regulatory properties of theprotein-senne kinase(s) (10) and type-2A protein phospha-tase(s) (3) involved in this cytoplasmic phosphorylation/de-phosphorylation cycle should provide much-needed insightinto the specific nature of the photosynthesis-related lightsignal (23, 24) and its transduction pathway in the C4 meso-phyll cytoplasm and of the putative factor that controls theendogenous rhythm (20, 29) of PEPC phosphorylation inCAM plants. Along these lines, recent work by Chollet's (6)and Nimmo's (see pp. 357-364 in ref. 26) groups has revealedthat the PEPC protein kinase activity in desalted crude ex-tracts of light-adapted maize leaves (or dark-adapted CAMtissue) is severalfold greater than that from the correspondingdark (light) tissue when using either endogenous or purifieddephosphorylated PEPC as substrate, in the absence or pres-ence of okadaic acid, a potent type 1 and 2A PP inhibitor (3).These exciting observations indicate that the protein-serinekinase(s) per se is highly regulated by either protein turnover,covalent modification or a tight-binding effector and implythat the light (rhythmic) signal and ensuing phosphorylation/activation of PEPC in C4 (CAM) plants involves a bicyclicregulatory cascade (Fig. 1).

LITERATURE CITED

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2. Budde RJA, Chollet R (1986) In vitro phosphorylation of maizeleafphosphoenolpyruvate carboxylase. Plant Physiol 82: 1107-1114

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12. Jiao JA, Podesta FE, Chollet R, O'Leary MH, Andreo CS (1990)Isolation and sequence of an active-site peptide from maizeleafphosphoenolpyruvate carboxylase inactivated by pyridoxal5'-phosphate. Biochim Biophys Acta 1041: 291-295

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POSTTRANSLATIONAL REGULATION OF PEP CARBOXYLASE

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19. Nimmo GA, McNaughton GAL, Fewson CA, Wilkins MB,Nimmo HG (1987) Changes in the kinetic properties andphosphorylation state of phosphoenolpyruvate carboxylase inZea mays leaves in response to light and dark. FEBS Lett 213:18-22

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29. Winter K (1982) Properties of phosphoenolpyruvate carboxylasein rapidly prepared, desalted leaf extracts of the Crassulaceanacid metabolism plant Mesembryanthemum crystallinum L.Planta 154: 298-308

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