The Remarkable Diversity of Plant Phosphoenolpyruvate Carboxylase

Biochem. J. (2011) 436, 15–34 (Printed in Great Britain) doi:10.1042/BJ20110078 15

REVIEW ARTICLEThe remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase):recent insights into the physiological functions and post-translationalcontrols of non-photosynthetic PEPCsBrendan O’LEARY*, Joonho PARK* and William C. PLAXTON*†1

*Department of Biology, Queen’s University, Kingston, ON, Canada, K7L 3N6, and †Department of Biochemistry, Queen’s University, Kingston, ON, Canada, K7L 3N6

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightlycontrolled enzyme located at the core of plant C-metabolismthat catalyses the irreversible β-carboxylation of PEP to formoxaloacetate and Pi. The critical role of PEPC in assi-milating atmospheric CO2 during C4 and Crassulacean acidmetabolism photosynthesis has been studied extensively. PEPCalso fulfils a broad spectrum of non-photosynthetic functions,particularly the anaplerotic replenishment of tricarboxylic acidcycle intermediates consumed during biosynthesis and nitrogenassimilation. An impressive array of strategies has evolved toco-ordinate in vivo PEPC activity with cellular demands forC4–C6 carboxylic acids. To achieve its diverse roles andcomplex regulation, PEPC belongs to a small multigene familyencoding several closely related PTPCs (plant-type PEPCs), alongwith a distantly related BTPC (bacterial-type PEPC). PTPCgenes encode ∼110-kDa polypeptides containing conservedserine-phosphorylation and lysine-mono-ubiquitination sites, andtypically exist as homotetrameric Class-1 PEPCs. In contrast,BTPC genes encode larger ∼117-kDa polypeptides owing to a

unique intrinsically disordered domain that mediates BTPC’s tightinteraction with co-expressed PTPC subunits. This associationresults in the formation of unusual ∼900-kDa Class-2 PEPChetero-octameric complexes that are desensitized to allostericeffectors. BTPC is a catalytic and regulatory subunit of Class-2 PEPC that is subject to multi-site regulatory phosphorylationin vivo. The interaction between divergent PEPC polypeptideswithin Class-2 PEPCs adds another layer of complexity tothe evolution, physiological functions and metabolic controlof this essential CO2-fixing plant enzyme. The present reviewsummarizes exciting developments concerning the functions,post-translational controls and subcellular location of plant PTPCand BTPC isoenzymes.

Key words: 14-3-3 protein, mono-ubiquitination, phosphoen-olpyruvate carboxylase, protein phosphorylation, protein–proteininteraction.

INTRODUCTION

PEPC [PEP (phosphoenolpyruvate) carboxylase] (EC 4.1.1.31)is an important enzyme situated at a crucial branch pointin plant carbohydrate metabolism. It catalyses the irreversibleβ-carboxylation of PEP in the presence of HCO3

− to yieldOAA (oxaloacetate) and Pi using Mg2 + as a cofactor. Theenzyme is present in all plants, green algae and cyanobacteria,most archaea and non-photosynthetic bacteria, but is absent fromanimals and fungi [1,2]. Plant PEPCs belong to a small multigenefamily encoding several PTPCs (plant-type PEPCs), along withat least one distantly related BTPC (bacterial-type PEPC) [3–7].PTPC genes encode closely related 105–110-kDa polypeptidesthat have a conserved N-terminal serine-phosphorylation domainand typically exist as homotetrameric Class-1 PEPCs [8,9].PTPCs have been categorized as being either photosynthetic[C4 and CAM (Crassulacean acid metabolism) PEPCs] or non-photosynthetic (C3) isoenzymes. All PTPCs evolved from acommon ancestral gene and display a high degree of conservationat the genetic level [10]. Katsura Izui and colleagues solved the

crystal structures for Zea mays (maize) C4 PTPC and Escherichiacoli PEPC, which, in combination with site-directed mutagenesis,have uncovered many of the structure–function relationships ofPEPC catalysis, allosteric control and regulatory phosphorylation[8,9]. Vascular plant BTPC genes encode larger 116–118-kDapolypeptides exhibiting low (<40%) sequence identity withPTPCs [3,4,6,7,11]. BTPCs lack the distinctive N-terminal serine-phosphorylation motif of PTPCs, and appear to only exist ascatalytic and regulatory subunits of extraordinary Class-2 PEPCheteromeric complexes whose novel structure, properties, control,expression patterns and putative functions are discussed below.

Extensive biochemical and genetic studies of photosyntheticPTPC isoenzymes of C4 and CAM leaves have elucidated catalyticand regulatory features that are well suited for their functionas an atmospheric CO2-concentrating mechanism that reducesphotorespiration to improve overall photosynthetic efficiency upto 2-fold relative to C3 leaves [10,12–14]. The post-translationalcontrol of photosynthetic PEPCs appears to be largely achievedby allosteric activation and inhibition by glucose 6-phosphateand malate respectively, as well as phosphorylation catalysed

Abbreviations used: BTPC, bacterial-type phosphoenolpyruvate carboxylase; CAM, Crassulacean acid metabolism; co-IP, co-immunopurification; COS,castor (Ricinus communis) oil seed; FHA, forkhead-associated; FP, fluorescent protein; GOGAT, glutamate 2-oxoglutarate aminotransferase; GS, glutaminesynthetase; MDH, malate dehydrogenase; ME, malic enzyme; NLS, nuclear localization signal; OAA, oxaloacetate; 2-OG, 2-oxoglutarate; PA, phosphatidicacid; PDHp, plastidic isoenzyme of the pyruvate dehydrogenase complex; PEP, phosphoenolpyruvate; PEPC, PEP carboxylase; PFK, phosphofructokinase;PKc, cytosolic pyruvate kinase; PP2A, protein phosphatase 2A; PPCK, PEPC protein kinase; PTM, post-translational modification; PTPC, plant-type PEPC;RT, reverse transcription; TCA, tricarboxylic acid; USP-2, ubiquitin-specific protease-2.

1 To whom correspondence should be addressed (email [email protected]).

c© The Authors Journal compilation c© 2011 Biochemical Society

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16 B. O’Leary, J. Park and W. C. Plaxton

Figure 1 The diverse functions of plant PEPC

by a specific Ca2 + -independent serine/threonine kinase knownas PPCK (PEPC protein kinase), and dephosphorylation by aPP2A (protein phosphatase 2A) [8,13,15,16]. Phosphorylation atthe conserved N-terminal serine residue activates the enzyme bydecreasing inhibition by malate while increasing activation by glu-cose 6-phosphate. A number of excellent reviews and bookchapters concerning various aspects of the evolution, molecularand biochemical characteristics, and physiological functions andregulation of photosynthetic and non-photosynthetic PEPCsand their corresponding PPCKs have appeared over the last15 years [8–10,12,13,15–21]. Development of a more completeaccount of PEPC’s diverse and ubiquitous involvement in plantmetabolism has recently made considerable progress owing to theuse of non-photosynthetic model plant systems such as developingseeds and heterotrophic suspension cell cultures, togetherwith increased access to state-of-the-art genomic, bioinformaticand proteomic tools. The first part of the present reviewsummarizes established and proposed metabolic roles for non-photosynthetic plant PEPCs. The second section outlines recentadvancements in our understanding of the post-translationalcontrol, protein–protein interactions and subcellular location ofnon-photosynthetic PEPCs.

DIVERSE METABOLIC FUNCTIONS FOR NON-PHOTOSYNTHETICPLANT PEPC

PEPC and primary carbon metabolism

Besides its fundamental role in the initial fixation of atmosphericCO2 during C4 and CAM photosynthesis, PEPC has a wide rangeof non-photosynthetic roles including supporting carbon–nitrogeninteractions, seed formation and germination, fruit ripening,guard cell metabolism during stomatal opening and provisionof malate as a respiratory substrate for symbiotic N2-fixingbacteroids of legume root nodules (Figure 1). The traditionalview of non-photosynthetic PEPC function is its ubiquitousanaplerotic carboxylation of PEP needed to replenish TCA

(tricarboxylic acid) cycle carbon skeletons that are withdrawn tosupport anabolism. Although certainly valid, this oversimplifiesthe contribution of PEPC to primary metabolism for severalreasons. First, the TCA ‘cycle’ does not always operate as acontinuous cycle in certain plant cells. Flux through one part ofthe pathway may be negligible or disproportionately low (e.g.as occurs to various extents in illuminated and senescing leavesand developing seed embryos) [22–25]. The partial TCA ‘cycle’thus resembles a linear pathway with PEPC as a componentenzyme, and the notion of anaplerosis is unnecessary as themaintenance of OAA concentrations is of no more importancethan any other metabolite [22]. Also, when combined withMDH (malate dehydrogenase) and NAD-ME (NAD+ -dependentmalic enzyme), PEPC forms an alternative flux mode whichcan function in place of PKc (cytosolic pyruvate kinase) togenerate pyruvate (Figure 2A) [22,26,27]. This endows plantswith the ability to respire OAA generated from PEPC, againnot an anaplerotic function. Secondly, as discussed below, theorganic acids generated by PEPC have diverse roles outsidethe TCA cycle [28]. Thirdly, when coupled with the cytosolicMDH reaction, the metabolism of PEP to malate and otherorganic acids has important implications in the redox balanceof NAD(P)H/NAD(P)+ [29]. Malate transported from thecytosol and oxidized to pyruvate within ‘sink’ organelles (e.g.mitochondria or plastids) by ME isoenzymes functions as both aNAD(P)H shuttle and a carbon source (Figure 2).

A distinguishing feature of the anaplerotic PEPC reaction is thatit represents a highly flexible aspect of primary plant metabolism.Several studies have quantified in vivo flux changes throughprimary metabolic pathways in isolated plant cells acclimatizedto environmental perturbations such as a change in oxygenconcentrations [30], temperature [31,32], glucose availability[33] or nitrogen source [24]. As the overall rates of flux andbiosynthesis changed, the ratio of carbon flux through mostnodes within primary metabolism was relatively rigid, whereasanaplerotic fluxes were relatively plastic. In other words, changesin carbon partitioning at the PEP branchpoint (Figure 2A) are


Functional control of non-photosynthetic plant PEPC 17

Figure 2 Models illustrating several metabolic functions of plant PEPC

(A) Interactions between carbon and nitrogen metabolism involves three compartments inplants. This scheme highlights the important role of the two terminal enzymes of plantcytosolic glycolysis, PEPC and PKc, in controlling the provision of the mitochondria withrespiratory substrates, as well as for generating the 2-OG and OAA respectively required forNH4

+ -assimilation via GS/GOGAT in plastids and aspartate amino transferase in the cytosol. Theco-ordinate control of PEPC and PKc by allosteric effectors, particularly glutamate and aspartate,provides a mechanism for the regulation of cytosolic glycolytic flux and PEP partitioning duringand following NH4

+-assimilation as discussed in the text. The central role of PEP in providing‘bottom-up’ control of glycolysis is also illustrated. Broken arrows with a circled plus and minussign indicate enzyme activation and inhibition respectively by allosteric effectors. (B) Alternativemetabolic routes for the conversion of sucrose into fatty acids in developing oil seeds. Thismodel illustrates the role of PEPC in controlling PEP partitioning to malate as a source of carbonskeletons and reducing power for leucoplast fatty acid synthesis. Abbreviations are as definedin the text, in addition to the following: AAT, Asp aminotransferase; E.T.C., electron transportchain; MEm/PDHm and MEp/PDHp, mitochondrial and plastidic isoenzymes of ME and PDHrespectively.

crucial in allowing plant cells to synchronize their metabolismwith changing environmental conditions. Control of PEPCactivity clearly occurs at both the transcriptional/translational andpost-translational levels. Transcript and enzymological studieshave shown that the supply of exogenous sugars, Pi and/ornitrogen influences both the amount and phosphorylation status ofPEPC in a wide range of tissues, including developing Hordeumvulgare (barley) seeds and COS [castor oil (Ricinus communis)seeds] [34–36], Solanum lycopersicum (tomato) cell cultures [33],Arabidopsis thaliana (thale cress) cell cultures and seedlings[37,38], Lotus japonicus and Glycine max (soya bean) rootnodules [39,40], Solanum tuberosum (potato) seedlings [41] andTriticum aestivum (wheat) leaves [42].

PEPC plays a central role in the control of plant glycolysis andrespiration

In animals, primary control of glycolytic flux of hexosephosphates to pyruvate is mediated by ATP-dependent PFK(phosphofructokinase), with secondary control at PK (pyruvatekinase). Activation of PFK increases the level of its product,fructose 1,6-bisphosphate, which is a potent feed-forwardallosteric activator of many non-plant PKs [27]. In contrast,quantification of changes in levels of glycolytic intermediatesthat occur immediately following stimulation of respiration in awide variety of plant systems (including green algae, ripeningfruit, aged storage root slices, germinating seeds and suspensioncell cultures) consistently demonstrated that plant glycolysis iscontrolled from the ‘bottom up’ with primary and secondaryregulation exerted at the levels of PEP and fructose 6-phosphateutilization respectively (Figure 2A) [27]. These findings arecompatible with the observation that plant ATP-PFKs demonstratepotent allosteric feedback inhibition by PEP. It follows that anyenhancement in the activity of PEPC or PKc will relieve the PEPinhibition of ATP-PFK, thereby elevating glycolytic flux fromhexose phosphate (Figure 2A). Reduced cytosolic PEP levelsalso cause elevated levels of the signal metabolite fructose 2,6-bisphosphate (and thus activation of the PPi-dependent PFK)since PEP is a potent inhibitor of plant 6-phosphofructo-2-kinase [27]. A possible advantage of bottom-up regulation ofglycolysis is that it permits plants to control net glycolytic fluxindependent of related plant-specific metabolic processes suchas the Calvin–Benson cycle and sucrose–starch interconversion.The hypothesis that plant glycolysis is regulated from the bottomup was corroborated by the application of metabolic controlanalysis to assess the distribution of respiratory flux control intubers of transgenic potato plants expressing different amountsof E. coli ATP-PFK [43]. It was determined that ATP-PFKexerts a low flux control coefficient over both glycolysis andrespiration, whereas far more flux control was exerted in themetabolism of PEP. The relatively low flux control coefficient ofATP-PFK was explained as a consequence of its potent inhibitionby PEP. In this way, PEPC and PKc play a central role in theoverall regulation of plant respiration, since the control of theiractivities in vivo will ultimately dictate the rate of mobilization ofsucrose or starch for respiration, while simultaneously controllingthe provision of: (i) respiratory substrates (e.g. pyruvate and/ormalate) for ATP production via oxidative phosphorylation, and(ii) TCA cycle carbon skeletons needed for nitrogen assimilationor as biosynthetic precursors. It is obvious that PEPC andPKc represent promising targets for metabolic engineering ofplant respiration and photosynthate partitioning. For example,transgenic bean plants were generated that expressed a bacterialmalate-insensitive PEPC in a seed-specific manner [44]. Analysis



of the transgenic seeds indicated enhanced PEP partitioningthrough the PEPC anaplerotic pathway, resulting in a shift ofphotosynthate partitioning from sugars/starch into organic acidsand amino acids. Consequently, the transgenic seeds accumulatedup to 20% more storage protein [44].

PEPC produces malate as an alternative respiratory substrate forplant mitochondria

Malate has been frequently proposed to be an importantrespiratory substrate for plant mitochondria, such that a significantfraction of pyruvate enters the TCA cycle via the combinedreactions of PEPC, MDH and NAD-ME, rather than PKc

(Figure 2A). However, in vivo flux studies of plant cells incubatedwith 13C-labelled substrates demonstrated that the flux of malate topyruvate via mitochondrial NAD-ME is usually very low, relativeto the flux of PEP to pyruvate via PKc [22,27]. Thus a markedreduction in mitochondrial NAD-ME in transgenic potatoes andArabidopsis had little impact on respiratory metabolism [45,46].It was concluded that the import of PEPC-derived malate intothe mitochondria generally serves an anaplerotic role to supportbiosynthesis and nitrogen assimilation, whereas pyruvate derivedfrom the PKc reaction is the most significant substrate forrespiration [45,46]. Nevertheless, there are specific situations,such as prolonged Pi starvation [47] or the light-enhanced darkrespiration peak that occurs immediately after C3 leaf darkening[48], when the oxidation of malate as a respiratory substratefor mitochondrial ATP production appears to be of particularsignificance. Similarly, overexpression of PEPC in several plantspecies resulted in a stimulation of respiration when illuminatedleaves were darkened, and an increase in extractable NAD-MEand NADP-ME activities [19,49–51]. Future studies are requiredto determine the full suite of physiological and developmentalconditions that stimulate intramitochondrial pyruvate productionvia the NAD-ME-dependent oxidative decarboxylation of malate(Figure 2A).

Carbon–nitrogen interactions

PEPC plays a crucial role regulating respiratory carbon flux invascular plant tissues and green algae that are actively assimilatingnitrogen [27,52–54]. The organic acids supplied by PEPC haveseveral distinct roles within nitrogen metabolism. α-Oxo acids,especially OAA and 2-OG (2-oxoglutarate), are obligate carbonskeletons on to which NH4

+ is assimilated and exported to othertissues (Figure 2A). In nodules, malate is also the predominantrespiratory substrate supplied to symbiotic rhizobia bacteria tosupport the huge energy demand of reducing N2 into NH4

+

[55,56]. Malate and citrate may also act as counterions to replacenitrate and maintain cytosolic pH [57]. The tight integration andcritical importance of PEPC at the interface of carbon and nitrogenmetabolism has been continuously elaborated by numerousstudies of green algae and vascular plants. Legume speciesexpress a specific nodule-enhanced PTPC isoenzyme that is up-regulated and activated by in vivo phosphorylation during activenitrogen assimilation by N2 fixing root nodules [39,55,58–60], andwhose suppression results in greatly diminished rates of nitrogenassimilation [55,56]. In leaves and non-nodular root tissues,PTPC transcripts, activity and phosphorylation status are also up-regulated when metabolism is switched to a nitrogen-assimilatingstate following the addition of NO3

− or NH4+ [42,53,61–66].

PEPC is also up-regulated during nitrogen remobilization fromsenescing leaves [25].

A key study compared metabolic fluxes of autotrophiccanola (Brassica napus) embryos supplied with organic orinorganic nitrogen (amino acids compared with NH4

+) [24]. Thedifferences in primary metabolism were mainly a restructuringof flow through the TCA cycle caused by the reciprocalchanges in PEPC compared with mitochondrial NAD-MEflux. The dynamic response of PEPC to different nitrogensources has been particularly well studied in chemostats ofunicellular green algae where nitrogen availability can beprecisely manipulated [52]. NO3

− or NH4+ resupply to N-limited

green algae leads to an immediate and massive stimulation of: (i)respiratory carbon flux in order to generate TCA carbon skeletonsrequired by GS (glutamine synthetase)/GOGAT (glutamate 2-OGaminotransferase), and (ii) dark CO2 fixation catalysed by PEPC(Figure 2A) [52]. During transient nitrogen assimilation, over50% of net plant carbon may be committed to the productionof carbon skeletons and energy (ATP and reductant) required forthe GS/GOGAT system [27,52]. Green algal (Chlamydomonasreinhardtii) PTPC and BTPC transcript and protein levels are bothup-regulated upon nitrogen starvation, presumably to facilitaterapid provision of OAA and 2-OG when a nitrogen sourcebecomes available [5,67]. As discussed below, the control ofgreen algal and vascular plant PEPCs by allosteric effectors,particularly glutamate and aspartate, has clearly evolved to bein tune with carbon–nitrogen interactions [68], and to co-operatewith other enzymes involved in nitrogen assimilation, particularlyPKc, nitrate reductase and GS (Figure 2A) [53,62,69].

Regulatory phosphorylation represents a second level ofpost-translational PEPC control in plant cells engaged inactive nitrogen assimilation [27,54]. Nodule-specific PPCKshave been reported for several legume species whose activitymirrors PEPC’s phosphorylation status and the presence ofphotosynthate supply [6,39,40,70,71]. PEPC phosphorylationin roots and leaves also responds to the carbon/nitrogenbalance due to the action of PPCK [16,37,42,66,72]. Theimportance of post-translational PEPC control in mediatingcarbon–nitrogen interactions has been elegantly demonstratedby a series of metabolic engineering efforts. Various forms ofPEPC with diminished feedback inhibition have been expressedin transgenic potatoes, Arabidopsis and Vicia narbonsis (vetch)seeds [44,51,73,74]. These alterations increased carbon fluxinto organic acids and amino acids, and boosted the overallorganic nitrogen content at the expense of starch and solublesugars. The mutant potato plants had diminished growth ratesand tuber yield, whereas the PEPC-overexpressing Arabidopsisplants suffered severe stunting owing to reduced PEP levels thatlimited aromatic amino acid production via the shikimate pathway[51]. Conversely, overexpression of PEPC without diminishedfeedback inhibition has little impact on overall metabolism, evenwhen large increases in extractable PEPC specific activity wereachieved [19,49–51]. As a further tier of integration into nitrogenmetabolism, PEPC has been shown to be a major target of theplant-specific Dof1 transcription factor which is implicated inorganic acid metabolism [75]. Increased expression of Dof1 intransgenic Arabidopsis caused an increase in nitrogen contentand improved growth under low nitrogen that was correlated withincreased PEPC and PKc expression and activity [75].

PEPC participates in photosynthate partitioning in developingseeds and organic acid metabolism of germinating seeds

Developing seeds

Developing seeds are crucial metabolic sinks that import largeamounts of sugars and amino acids from leaves which must be



efficiently metabolized into starch, triacylglycerols and storageproteins. Owing to the agronomic significance of these storage re-serves, a detailed understanding of developing seed metabolismhas obvious practical applications in crop breeding andmetabolic engineering. PEP metabolism via PEPC, PKc and PKp

(plastidic pyruvate kinase) plays a prominent role in partitioningimported photosynthate towards leucoplast fatty acid biosynthesiscompared with the mitochondrial production of carbon skeletonsand ATP needed for amino acid interconversion in support ofstorage protein biosynthesis. PEPC may also aid in refixingrespired CO2 to improve overall seed carbon economy [76–80].Increased PEPC and PPCK activity and PTPC phosphorylationstatus during seed development coincides with the main stagesof storage oil and protein biosynthesis [35,78,79,81–84], andhave been shown to be controlled by photosynthate supply indeveloping COS and barley seeds [34–36]. Seed storage proteinsynthesis has been traditionally linked to PEPC activity owing tothe obvious requirement for OAA and 2-OG needed to assimilatenitrogen during formation of amino acids (Figure 2A) [44,74,85].Seeds import nitrogen in the form of amino acids, but stillrequire additional carbon skeletons because: (i) glutamine, a majorcomponent of the supplied amino acids contains two amino groupsper 2-OG skeleton; and (ii) amino acids such as alanine can bedeaminated and respired through the TCA cycle, yielding NH4

+

that must be rapidly assimilated into glycine and glutamate viaGS/GOGAT (Figure 2A).

PEPC’s role in generating metabolite precursors and reducingpower to support triacylglycerol synthesis in developing oil seedshas also received considerable attention. De novo fatty acidsynthesis occurs within the leucoplast, is ATP- and NAD(P)H-dependent, and uses acetyl-CoA as an immediate precursor.However, the pathway leading from sucrose-derived cytosolichexose phosphates to plastidial acetyl-CoA is highly flexibleand certainly varies within and between species (Figure 2B)[86,87]. Studies of isolated leucoplasts from developing COSand Helianthus annuus (sunflower) seeds showed that, relativeto other precursors, exogenous malate supported maximal ratesof fatty acid synthesis [88,89]. As a consequence of malate’soxidation into acetyl-CoA via the plastidial isoenzymes of NADP-ME and PDH (pyruvate dehydrogenase complex), all of thereductant required for carbon incorporation into fatty acids isproduced (Figure 2B). Both PEPC and plastidial NADP-MEare abundant in developing COS endosperm [81,90,91], and aspecific malate/Pi antiporter exists in the COS and maize embryoleucoplast envelope [92,93]. These results are compatible withthe hypothesis that PEPC has an important role to control sucrosepartitioning at the level of the cytosolic PEP branchpoint for gene-ration of carbon skeletons and reducing power needed forfatty acid biosynthesis by developing oil seeds such as COS(Figure 2B). Consistent with this view are the high rates of darkCO2 fixation exhibited by intact pods of developing COS [94].

Metabolic flux studies of developing oil seeds have reporteda spectrum of values regarding anaplerotic PEP carboxylationto support biosynthetic pathways [24,26,32,95–99]. The fluxfrom malate to plastidial acetyl-CoA appears to be negligible indeveloping green oil seeds such as canola [97], despite the strongcorrelation that exists between PEPC expression and rates of oilsynthesis by developing canola embryos [83]. However, fatty acidsynthesis by green oilseed rape/canola and soya bean embryos islight-dependent [86,100,101], since most of the reducing power[NAD(P)H] they need to assemble fatty acids from acetyl-CoA isgenerated via the light reactions of leucoplast photosynthesis.In the non-green developing maize and sunflower embryos,however, 30% and 10% of carbon flux into fatty acids wasderived from PEPC-generated malate respectively [95,96]. One

caveat of metabolic flux analyses is that they require artificiallystatic conditions, such as constant light and sucrose supply, andnon-physiological levels of oxygen [102]. Conversely, theactivities of the enzymes surrounding the PEP node and theirsupport of fatty acid synthesis are likely to be tightly controlledto be in tune with changing environmental conditions, includingthe diurnal cycle.

Germinating seeds

Research with starch-storing cereal seeds such as barley andwheat, and oil-storing dicotyledon seeds such as COS haveconsistently implied an important anaplerotic function for PEPCduring germination [36,81,103–107]. Cristina Echevarria andcolleagues have performed pioneering studies with PEPC ofgerminated cereal seeds such as wheat and barley [105,106], inwhich the aleurone layer synthesizes and secretes various acidhydrolases needed to mobilize starch and protein reserves inthe underlying endosperm. Aleurone PEPC is thought to playan important role in regulating the production of organic acids,chiefly malic acid, that are excreted for the acidification ofthe starchy endosperm, or transported to the growing embryoto replenish pools of TCA cycle intermediates consumedduring biosynthesis and transamination reactions. Althoughbarley and wheat seed PTPC becomes progressively activatedby phosphorylation following imbibition, PPCK was alreadypresent in the dry seeds and was apparently not up-regulatedduring germination [36,105]. The phytohormone abscisic acidappears to play a role in triggering PPCK accumulation duringthe desiccation stage of maturing barely seeds [36]. Also, theprotein synthesis inhibitor cycloheximide, as well as otherpharmacological agents known to block the signal-inducedphosphorylation of PEPC in C4 leaf mesophyll cells, had noinfluence on PEPC’s phosphorylation in germinated cereal seeds,suggesting that their PPCK may be subject to a post-translationalcontrol mechanism [36,105].

The carbon metabolism of germinating oil seeds such as COSis dominated by the mobilization of storage lipid and proteinreserves to support the needs of the growing hypocotyls androots. In germinating COS endosperm, this process includesthe massive conversion of reserve triacylglycerols into sucrose,which is absorbed by the cotyledons of the growing seedling[108,109]. PEPC has been suggested to fulfil a crucial functionvery early in the germination process to build up cellular pools ofC4 acids needed to trigger subsequent TCA and glyoxylate cycleactivity [81]. An ensuing role for PEPC in germinating oil seedsis to replenish dicarboxylic acids required as substrates for thesubstantial transamination reactions that follow storage proteinhydrolysis [108], as well as for GS’s reassimilation of NH4

+

released during the oxidative deamination of glutamate to 2-OGvia glutamate dehydrogenase [110]. The labelling of metabolicintermediates to isotopic steady state and the modelling of theTCA cycle in germinating lettuce seeds indicated that 70% ofglycolytic flux from carbohydrate oxidation enters the TCA cyclevia the PEPC reaction [111]. At the same time, PEP carboxykinasecatalyses the initial step in the gluconeogenic conversion ofstorage lipids into sucrose, as it uses ATP to decarboxylate andphosphorylate OAA derived from the glyoxylate cycle into PEP[108]. PEP carboxykinase is highly abundant in the cytosol ofgerminating COS endosperm [112], but is incompatible withPEPC, since their combined reactions result in a futile cycle,i.e. the wasteful hydrolysis of ATP to ADP and Pi. In theirclassic study of gluconeogenesis in germinating COS endosperm,Kobr and Beevers [109] determined that the maximum rate



of glycolysis is about one-tenth that of gluconeogenesis andthat the pools of glycolytic and gluconeogenic intermediatesprobably occur in separate intracellular regions and appear to beregulated independently. Several studies have provided evidencefor glycolytic or gluconeogenic multi-enzyme complexes(metabolons) in the plant cytosol [27], including the cytosolicisoenzymes of fructose-1,6-bisphosphate aldolase and fructose-1,6-bisphosphatase in germinated COS [113]. It will thereforebe important to establish whether PEPC mono-ubiquitination(see below) possibly contributes to formation of a glycolyticmetabolon while minimizing its futile cycling with PEPcarboxykinase in the cytosol of the germinating oil seeds suchas COS.

PEPC mediates organic acid accumulation by developing fruit andexpanding cells

Many developing fruits accumulate substantial concentrations ofmalate and citrate (>300 mM) in their vacuoles. This stockpile oforganic acids is believed to sustain respiration and act as carbonsource during ripening when sugars are accumulated [114,115].A fruit-specific PTPC isoenzyme has been identified in tomatowhose transcripts and activity increase throughout development,simultaneous with enhanced cellular malate and citrate levels[115,116]. PEPC from tomato and Citrus sinensis (orange) fruitsdisplayed a significantly decreased sensitivity to malate inhibitionrelative to many other PTPCs [116,117]. Whether this is theresult of specific isoenzyme properties or control by a PTM(post-translational modification) such as phosphorylation remainsunknown. Adding to this uncertainty, a PPCK transcript steadilyincreases in tomato fruit from early development until ripening,but does not correlate with PEPC activity in this tissue [118]. AClass-1 PEPC has also been purified and characterized from Musacavendishii (banana) fruit [119]. The PEPC of ripening bananas issubject to regulatory phosphorylation, and was suggested to playan anaplerotic role to replenish carbon skeletons consumed duringnitrogen assimilation and/or transamination reactions [120].

The tomato fruit PTPC is located predominantly in largeor expanding cells of the pericarp, where malate productionvia PEPC and MDH has been suggested to sustain osmoticpotential to allow rapid cell expansion [116]. A similar role formalate has been suggested for other expanding cells as well.For example, malate accumulation in the guard cell vacuoleis involved in stomatal opening. Several studies analysing thegenetic or pharmacological alteration of PEPC activity in guardcells have correlated stomatal opening with PEPC activity andPTPC phosphorylation status [19,121,122]. Cotton fibres are hair-like single cells that undergo a stage of rapid elongation to severalcentimetres at anthesis. PEPC activity and PTPC transcripts, andthe accumulation of malate were all correlated with the rate, extentand developmental profile of fibre elongation [123]. Treatment offibres with LiCl, an inhibitor of PPCK activity, reduced fibrelength and implicated regulatory PTPC phosphorylation in thistissue as well. Aside from acting as an osmolyte to increaseturgor pressure, the malate generated by cotton fibre PEPC mayalso support fatty acid synthesis (Figure 2B) needed for the rapidextension of the plasma membrane and tonoplast [123].

PEPC helps plants to acclimatize to abiotic and biotic stresses

PEPC has been widely implicated in plant stress tolerance in twomain ways. First, as discussed above, PEPC provides metabolicflexibility that allows PEP metabolism to adjust to environmentalconditions. Generally, this involves either increased anaplerosis

to support specifically up-regulated biosynthetic processes and/ora glycolytic bypass of PKc as an adjustment to differing Pi orreductant levels. Secondly, some roots excrete huge amounts oforganic acids into the soil (rhizosphere) to acidify the soil andchelate cations as a response to nutrient deprivation and/or toxicmetal stress. PEPC up-regulation, as documented by increasedtranscripts, protein, extractable activity and phosphorylationstatus appears to be an integral component of these stressresponses.

Drought, salt and ozone stress

An up-regulated PEPC and PPCK response to salinity or droughtstress has been well documented in C3, C4 and CAM plants[11,84,124–133]. Consistent with these reports, RNAi (RNAinterference) knockdown of a canola PTPC isoenzyme resultedin increased sensitivity to poly(ethylene glycol)-induced osmoticstress [134]. The adaptive value of increased PEPC activityupon salt or drought stress requires further clarification, butcould potentially improve carbon metabolism during periods ofreduced stomatal conductance by reassimilating respired CO2

and/or increasing rates of CO2 fixation at night when stomataare open, as in CAM photosynthesis [126,129,132]. PEPC mayalso support the biosynthesis of biocompatible osmolytes such asproline that rapidly accumulate upon drought or salinity stress inmany plant species [134].

Exposure to elevated ozone causes oxidative stress and leads toa major rearrangement in primary carbon metabolism involving asuppression of photosynthesis and an increase in respiration [135].Specifically, a major up-regulation of PEPC and concomitantdown-regulation of ribulose-1,5-bisphosphate carboxylase istypical of this response [135,136]. The up-regulation of PEPCalong with ME is hypothesized to contribute to the increasedproduction of NAD(P)H necessary to detoxify reactive oxygenspecies, as well as the increased anaplerotic flux to support proteinsynthesis for defence and repair processes [137].

Nutritional phosphate and iron deficiency

Phosphorus is an essential element for growth and metabolismthat plays a central role in virtually all metabolic processes.Plants preferentially absorb phosphorus from the soil in its fullyoxidized anionic form, Pi. Despite its importance, Pi is one ofthe least available macronutrients in many terrestrial and aquaticenvironments. In soil, Pi is frequently complexed with Al3 + orCa2 + cations and thus exists as insoluble mineral forms thatrender it unavailable for plant uptake. PEPC was suggested toprovide a metabolic bypass (with MDH and NAD-ME) to theADP-limited PKc to facilitate continued pyruvate supply tothe TCA cycle, while concurrently recycling the PEPC by-productPi for its reassimilation into the metabolism of the Pi-deficientcells [27]. PEPC induction and its regulatory phosphorylationduring Pi stress has also been correlated with the synthesis andconsequent excretion of large amounts of malic and citric acidsby roots [27,38,138,139]. This increases Pi availability to theroots by acidifying the rhizosphere to: (i) solubilize otherwiseinaccessible sources of mineralized soil Pi [138], while (ii) makingphosphorus esters more accessible to hydrolysis by secretedacid phosphatases [140]. An excellent correlation of organicacid excretion and PEPC up-regulation with enhanced ratesof dark CO2 fixation occurs in proteoid (cluster) roots of Pi-deficient Lupinus albus (white lupin) [138]. Recent developmentshave clarified the molecular basis of the PEPC response to Pi

stress in Arabidopsis cell cultures and seedlings [37,38,141,142],



Oryza sativa (rice) leaves [72] and white lupin proteoid roots[143], which involves the up-regulation of specific PTPC andPPCK isoenzymes under the control of multiple transcriptionfactors. The simultaneous induction and in vivo phosphorylationactivation of the PTPC isoenzyme AtPPC1 (Arabidopsis PEPC 1)at its conserved Ser11 phosphorylation site (Figure 3) contributesto the metabolic adaptations of Pi-starved Arabidopsis [38]. Thisis consistent with the observation that the PPCK-encoding genesAtPPCK1 and AtPPCK2 are among the most strongly inducedgenes when Arabidopsis is subjected to nutritional Pi deprivation[141,144,145].

Iron deficiency also up-regulates PEPC activity [up to 60-fold in Beta vulgaris (sugar beet)], specifically in the corticalcells of root apical meristem [146]. This was hypothesized tosupport organic acid excretion to the soil for increased iron uptakeand organic acid accumulation within the plant for increasedvascular transport [146–150]. The rising intracellular citrateconcentrations that occur may inhibit PKc and thus promote fluxthrough PEPC, MDH and NAD-ME as a glycolytic bypass [148].

Heavy metal toxicity

The exudation of PEPC-derived organic acids also functions tochelate metals in the rhizosphere to alleviate heavy metal toxicityby preventing their uptake into the cell [138,151]. Al3 + toxicityis the most prevalent form of plant heavy metal stress. Althoughelevated soil Al3 + levels result in root efflux of organic acids, thereappears to be no corresponding induction of root PEPC [152,153].However, a recent study demonstrated that overexpression of a C4

PTPC isoenzyme in rice led to increased oxalate exudation andAl3 + tolerance [154]. In contrast with Al3 + , Cd2 + toxicity leadsto PEPC up-regulation [155,156]. This is believed to support thesynthesis of the phytochelatins, intracellular metal chelators thatrapidly accumulate during heavy metal stress. Phytochelatins arepolymers of the tripeptide glutathione whose synthesis requiresde novo amino acid synthesis and therefore increased anapleroticPEP carboxylation via PEPC.

Biotic stress

PEPC is also up-regulated during infection of tobacco (Nicotianatabacum) plants by the potato virus [157,158]. The up-regulationmay be a response to: (i) closed stomata and the ensuing alterationof metabolism, and/or (ii) increased anaplerotic flux needed tosustain the rapid synthesis of viral defence proteins.

THE MULTIFACETED POST-TRANSLATIONAL CONTROL OF PLANTPEPC

Allosteric effectors and regulatory phosphorylation: new tricks forold dogs

Allosteric effectors

The basic features of post-translational control of plant Class-1PEPCs have been known since the early 1980s. MostClass-1 PEPCs show allosteric inhibition by malate and activationby glucose 6-phosphate. However, the enzyme’s specific kineticand allosteric properties are quite variable and appear to bewell adapted to the physiological role(s) of specific PEPCisoenzymes and the nature of their cellular environment. A goodexample is the increased Km (PEP) and IC50 (malate) values ofC4 Class-1 PEPC relative to typical C3 Class-1 PEPCs [10].The C3 Class-1 PEPCs from developing fruit, the chloroplasticClass-1 PEPC isoenzyme of rice leaves and the Class-2 PEPC

heteromeric complexes composed of tightly associated PTPCand BTPC subunits (discussed below) also display distinctivekinetic properties [116,117,159,160]. Furthermore, aspartate andglutamate are important feedback effectors of Class-1 PEPCand PKc isoenzymes in green algae and plant tissues activein nitrogen assimilation and/or transamination reactions. Thisprovides a regulatory link between nitrogen metabolism and thecontrol of respiratory carbon metabolism (Figure 2A). Feedbackinhibition of specific Class-1 PEPC and PKc isoenzymes byglutamate provides a rationale for the known activation of thetwo enzymes that occurs in vivo during periods of enhancednitrogen assimilation when intracellular glutamate concentrationsbecome transiently reduced [27,52]. In contrast with PEPC,aspartate functions as an allosteric activator of various plant PKcsby effectively relieving the enzyme’s inhibition by glutamate(Figure 2A) [27,69,161–164]. Reciprocal control of Class-1 PEPC and PKc by aspartate has been documented in awide range of plant cells and tissues involved in nitrogenassimilation and/or transamination reactions including: greenalgae, Spinacia oleracea (spinach) and castor oil plant leaves,ripening banana fruit, canola cell cultures and endosperm ofdeveloping COS [52,69,82,161–164]. This was suggested toprovide a mechanism for decreasing flux from PEP to aspartate(via PEPC and aspartate aminotransferase) while promoting PKc

activity whenever cytosolic aspartate levels become elevated. Thismay occur when the cell’s demands for nitrogen are satisfied, andthe overall rate of protein synthesis becomes more dependent onATP availability, rather than the supply of amino acids. In thisinstance, respiration may assume a more significant role in termsof satisfying a large ATP demand, rather than the anapleroticgeneration of biosynthetic precursors.

PA (phosphatidic acid) is a lipid second messenger thattransiently accumulates in plant cells within minutes of applyinga wide array of stress conditions [165]. Several C3 and C4

PTPC isoenzymes were recently identified as PA-binding proteins[166,167]. Moreover, a low concentration (50 μM) of PA andother anionic phospholipids resulted in significant inhibition(>50%) of C4 Class-1 PEPC activity [167]. Little or no effectwas observed with neutral, zwitterionic or positively chargedphospholipids, ruling out a non-specific effect of the fatty acylchains (which activate E. coli PEPC). Interestingly, hypo-osmotictreatment of Arabidopsis cells increased PEPC’s affinity for PAthrough an unknown mechanism [166]. Although most of the C4

PTPC of Sorghum bicolor (sorghum) leaves was in the solublefraction, a subfraction was membrane-associated [167]. Anionicphospholipids represent novel regulators that may inhibit theactivity and/or mediate membrane association of certain PEPCs.How PEPC activity and/or cellular location is influenced bydynamic changes in anionic membrane phospholipids such asPA will be an interesting area for further research.

Regulatory phosphorylation

In the early 1980s, Hugh Nimmo (University of Glasgow)and Raymond Chollet (University of Nebraska) respectivelydocumented the regulatory phosphorylation of CAM and C4

photosynthetic Class-1 PEPC isoenzymes at their single highlyconserved serine residue close to the N-terminus of the protein.Since their seminal discoveries, this PTM has been implicated inthe in vivo control of a wide array of non-photosynthetic PTPCs[34,36,38,42,106,120,168–172]. This research area has beenelaborated in several excellent reviews that have appeared withinthe last decade [8,16,17]. In brief, Class-1 PEPC phosphorylationuniformly results in enzyme activation at physiological pH



Figure 3 Amino acid sequence alignment of Arabidopsis and castor oil plant PEPC isoenzymes

Experimentally verified in vivo phosphorylation sites of castor oil plant and Arabidopsis PTPCs (RcPPC3 and AtPPC1 respectively) and castor oil plant BTPC (RcPPC4), are highlighted in green[34,38,191,201], whereas RcPPC3’s conserved Lys628 mono-ubiquitination site is marked with a red font [103]. BTPC’s highly divergent ∼10-kDa domain that was predicted to exist largely as anintrinsically disordered region [191] is enclosed in a red rectangle. The proteolytically susceptible peptide bond (Lys446–Ile447) within RcPPC4’s disordered region [4] is marked with an X. BoxesI–III denote conserved subdomains essential for PEPC catalysis [9]. The predicted pI, molecular mass and sequence identity (%) of the various PEPCs are shown. The deduced PEPC sequenceswere aligned using ClustalW software (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Semi-colons and asterisks indicate identical and conserved amino acids respectively.


http://www.ebi.ac.uk/Tools/clustalw2/index.html


by decreasing PEPC’s sensitivity to allosteric inhibitors whileincreasing its affinity for PEP and sensitivity to allostericactivators. Hence, much attention has also been directed to theCa2 + -independent PPCK and, to a far lesser extent, PP2A,that mediate Class-1 PEPC’s reversible phosphorylation in vivo.Detailed PPCK studies, including its cloning from CAM, C4

and C3 plants, continues to provide one of the best understoodexamples of regulatory enzyme phosphorylation in the plantkingdom [16,17]. There is also compelling evidence fromseveral systems that Class-1 PEPC phosphorylation correlateswith increased flux through the enzyme in vivo [16]. Withmolecular masses of 31–33 kDa, the PPCKs are the smallestprotein kinases yet reported, consisting of only a core kinasedomain [8,17]. Class-1 PEPC phosphorylation status appears tobe generally controlled by changes in rates of PPCK synthesiscompared with degradation (with the notable exception ofgerminating cereal seeds; see above) [8,16]. This is a novel meansof control, since most protein kinases are post-translationallycontrolled by second messengers such as Ca2 + ions, intracellularsignals such as AMP, and/or phosphorylation by other proteinkinases.

PPCK is up-regulated in response to a range of differentsignals, including light in C4 leaves, a circadian rhythm in CAMleaves, light and nitrogen supply in C3 leaves, nutritional Pi

deprivation in C3 plants and cell cultures, and photosynthatesupply in legume root nodules and developing seeds [16,17,35–37,72,127,173,174]. Endogenous PPCK activity and PTPCsubunit phosphorylation of developing COS and soya bean rootnodules were both eliminated following prolonged darkness ofintact plants [6,34,35,40,70]. Both effects were fully reversedfollowing reillumination of darkened plants. These results implya direct relationship between the up-regulation of COS orsoya bean nodule PPCK and PTPC phosphorylation during therecommencement of photosynthate delivery from illuminatedleaves to these non-photosynthetic sink tissues. The abilityof plant cells to sense sugars plays a crucial role in carbonpartitioning and allocation in source and sink tissues. Theseprocesses are modulated as a consequence of the plant’s sugarstatus, and sugar signals function both at the transcriptional andtranslational levels in tight co-ordination with light and otherenvironmental stimuli [84]. PPCK of non-green sink tissues suchas soya bean root nodules or developing oil seeds appears to beone of the many proteins whose expression is markedly influencedby sucrose supply. Developing seed and legume nodule geneexpression, respiration, storage product synthesis and carbon–nitrogen interactions are highly dependent upon the supply ofrecent photosynthate [85]. A key area for future studies willbe to establish signalling pathway(s) that link sucrose supplyto enhanced PPCK expression and PTPC phosphorylation inheterotrophic sinks such as legume root nodules or developingseeds.

A variety of post-translational PPCK controls have also beenproposed including: (i) malate inhibition of PPCK activity,possibly by interacting with PEPC [35,120,175], (ii) thiol–disulfide interconversion [35,176,177], and (iii) a proteinaceousinhibitor [178]. The mechanisms and in vivo relevance of thesecontrols requires further evaluation. Functional genomic studieshave evaluated the impact of in vivo PTPC phosphorylation onthe growth and development of transgenic C4 (Flaveria bidentis)and C3 (Arabidopsis) plants in which PPCK expression and thusin vivo PEPC phosphorylation was disrupted [171,179]. Althoughperturbation of growth and/or metabolite levels were observed,the results of both studies led to the surprising conclusionthat inhibition of PTPC phosphorylation has little impact onmetabolic flux through PEPC (albeit, in non-stressed plants).

In Arabidopsis, however, several pathways were perturbed,and it was hypothesized that leaf PPCK may have severaltargets in addition to PEPC [171]. The physiological roles ofPTPC phosphorylation, and the substrate specificity of PPCKrequire further clarification. A PP2A appears to dephosphorylatephotosynthetic and non-photosynthetic PTPCs [8,15,16,20].Additional research is needed to assess the possibility that theactivity of PP2A catalytic subunits are themselves controlled byregulatory subunits with which they might be associated.

Protein phosphorylation can not only control enzymatic activitydirectly, but also generate specific docking sites for otherproteins, particularly the 14-3-3 proteins, a family of highly con-served and abundant proteins that play a central regulatoryrole in eukaryotic cells [27]. The 14-3-3 homodimers bind tospecific phospho-sites on diverse target proteins, thereby forcingconformational changes or influencing interactions betweentheir targets and other molecules. In these ways, 14-3-3s‘finish the job’ when phosphorylation alone lacks the powerto drive changes in the activities of intracellular enzymes. Forexample, the phosphorylated forms of plant nitrate reductase andsucrose-phosphate synthase are both potently inhibited by 14-3-3 binding [27]. Proteomic profiling of tandem-affinity-purified14-3-3 protein complexes from transgenic Arabidopsis seedlingsrecently identified the PTPC isoenzyme AtPPC1 as being a14-3-3-binding protein [180]. However, plant PEPCs, includingAtPPC1, do not contain any established 14-3-3-binding motifs,and recent attempts to demonstrate 14-3-3 binding by the purifiedphosphorylated or dephosphorylated forms of native AtPPC1 [38]using 14-3-3 far-Western overlay assays have proven unsuccessful(C. MacKintosh and W.C. Plaxton, unpublished work). It ispossible that AtPPC1 binds to (i) 14-3-3 proteins, but doesnot perform well in the overlay assay which requires proteinrefolding, or (ii) another 14-3-3-interacting phosphoprotein andthus appeared in the tandem-affinity-purified 14-3-3 complexes.It would be of interest to employ 14-3-3 pull-down assays coupledwith PEPC immunoblotting to further assess putative 14-3-3binding by PTPCs, and/or whether 14-3-3s exert any influenceon the kinetic and regulatory properties of AtPPC1 or other plantPEPCs.

PEPC mono-ubiquitination: the new kid in town

Ubiquitin is a highly conserved globular protein of eukaryoticcells that modifies target proteins via its covalent attachmentthrough an isopeptide bond between the C-terminal glycineresidue of ubiquitin and the ε-amino group of a lysine residue ona target protein. A multi-enzyme system consisting of activating(E1), conjugating (E2) and ligating (E3) enzymes attach ubiquitinto cellular proteins. Polyubiquitination is a well-known PTMthat tags many proteins for their proteolytic elimination bythe 26S proteasome. Indeed, degradation of both the PTPCand PPCK by the polyubiquitin–proteasome pathway has beenreported [181,182]. However, protein mono-ubiquitination hasbeen demonstrated recently to play a variety of crucial non-destructive functions in yeast and mammalian cells [183,184].Mono-ubiquitination is a reversible PTM that mediates protein–protein interactions (by recruiting ubiquitin-binding domainclient proteins) and localization to help control processes suchas endocytosis, DNA repair, transcription and translation, andsignal transduction [183,184]. Ubiquitin-related pathways arebelieved to be of widespread importance in the plant kingdom.Genomic analyses indicated that the ubiquitin-related pathwayalone comprises over 6% of the Arabidopsis or rice proteomeswith thousands of different proteins being probable targets [185].



Recent studies on Class-1 PEPC from germinated COSendosperm provided the first example of regulatory mono-ubiquitination of a metabolic enzyme [103]. Extracts of fully ma-ture and germinating COS endosperm had been shown to containan immunoreactive PTPC polypeptide that co-migrated with the107-kDa subunit of the developing COS Class-1 PEPChomotetramer [4,77,81]. However, an additional immunoreactivePTPC polypeptide of approximately 110 kDa appearedimmediately following COS imbibition and persisted throughoutgermination at an equivalent ratio with the 107-kDa subunit. Inorder to clarify the molecular basis for this observation, a 440-kDa Class-1 PEPC heterotetramer composed of an equivalentratio of non-phosphorylated 110- and 107-kDa subunits waspurified from 3-day-old germinated COS endosperm [103]. N-terminal microsequencing, MS and immunoblotting establishedthat both subunits arise from the same PTPC gene (RcPpc3)(Figure 3) encoding the phosphorylated 410-kDa Class-1PEPC homotetramer of developing COS, but that the 110-kDasubunit is a mono-ubiquitinated form of the 107-kDa subunit.Tandem MS sequencing of tryptic peptides identified Lys628

as PEPC’s mono-ubiquitination site [103]. Lys628 is absolutelyconserved in all PTPCs and BTPCs, and is proximal to a PEP-binding/catalytic domain (Figure 3). Incubation of the purifiedgerminated COS Class-1 PEPC with a recombinant humandeubiquitinating enzyme [USP-2 (ubiquitin-specific protease-2)core] cleaved ubiquitin from its 110-kDa subunits (Figure 4) whilesignificantly reducing the enzyme’s Km (PEP) and sensitivity toallosteric activators (hexose phosphates, glycerol 3-phosphate)and inhibitors (malate, aspartate) [103]. It is also notable thatelimination of photosynthate supply to developing COS bydetaching intact developing COS pods from the plant causedthe 107-kDa PTPC subunits of the endosperm and cotyledonto become dephosphorylated [34,35], and then subsequentlymono-ubiquitinated in vivo (B. O’Leary and W. C. Plaxton,unpublished work), concomitant with the disappearance ofPPCK activity. Because depodding abolishes photosynthatedelivery to COS, it is conceivable that the metabolism ofthe depodded developing endosperm is rearranged to mimicthat of the gluconeogenic germinating COS. PTPC mono-ubiquitination has since been demonstrated in Lilium longiflorum(lily) pollen [7] and Arabidopsis seedlings (B. O’Leary andW. C. Plaxton, unpublished work). An immunoreactive PTPC‘doublet’ highly reminiscent of the mono-ubiquitinated Class-1PEPC of germinated COS has been frequently observed on PTPCimmunoblots of clarified extracts from a broad variety of plants,including Hydrilla verticillata (hydrilla) leaves [186], Viciafaba (broad bean) stomata [187], Cucumis sativus (cucumber)roots [188], germinating seeds of wheat, barley and sorghum[104,105,107], and ripening banana fruit [119]. In contrast withthe COS system, the Class-1 PEPC of cereal seeds such asbarley and wheat is phosphorylated during germination, whereasthat of fully mature maize seeds appears to be maximallyphosphorylated before germination [36,106,168]. We recentlycompleted a survey of tissue-specific PTMs of PTPC polypeptidesin various photosynthetic and non-photosynthetic tissues of thecastor oil plant. Immunoblotting using PTPC (anti-RcPPC3)and phospho-site-specific PTPC (anti-pS11) antibodies followingpre-incubation of clarified extracts with USP-2 demonstratedthat PTPC mono-ubiquitination rather than phosphorylationappears to be the predominant PTM of Class-1 PEPCs thatoccurs in non-stressed castor oil plants (B. O’Leary andW. C. Plaxton, unpublished work). Moreover, the distinctivedevelopmental patterns of PTPC phosphorylation compared withmono-ubiquitination in the castor oil plant indicated that thesePTMs may be mutually exclusive. As discussed above, Class-1

Figure 4 A human deubiquitinating enzyme (USP-2 core) cleavedubiquitin from the 110-kDa subunits of the germinating castor bean PEPCheterotetramer

Native PEPC purified from endosperm of 4-day-old germinated COS endosperm was incubatedwith or without 1 μM USP-2 core. Aliquots were removed at various times and subjectedto immunoblot analysis using (A) anti-(bovine ubiquitin IgG) (1 μg of PEPC/lane), or (B)anti-[developing COS Class-1 PEPC (RcPPC3) IgG] (0.1 μg of PEPC/lane). Figure modifiedfrom [103] with permission. Molecular masses are indicated in kDa.

PEPC phosphorylation activation was mainly observed in planttissues in which a high and tightly controlled flux of PEP tomalate has an obvious metabolic role; e.g. as occurs duringatmospheric CO2 assimilation in C4 or CAM leaves, photosynthatepartitioning to storage end-products by developing COS, rapidnitrogen assimilation following NH4

+ or NO3− resupply

to nitrogen-limited cells, or during nutritional Pi starvation[16,17,34,38,42,54,169]. Clearly, additional research is warrantedto assess the interplay between and metabolic functions ofPTPC phosphorylation and mono-ubiquitination. Future researchalso needs to characterize the: (i) ubiquitin-binding domainproteins that might interact with the ubiquitin-‘docking site’ ofmono-ubiquitinated PEPCs, as well as the possible influenceof this PTM on their subcellular location, and (ii) signallingpathways and specific E3 ligase that mediate tissue-specificPTPC mono-ubiquitination. High-throughput proteomic screenshave identified numerous ubiquitinated metabolic enzymesin Arabidopsis, including PTPC [189,190]. However, it wasnot determined whether the various targets were poly- ormono-ubiquitinated. This discrimination is crucial for futurestudies of the plant ubiquitome since polyubiquitination andmono-ubiquitination are destructive and non-destructive PTMsrespectively that mediate entirely different effects on target proteinfunction [183,184].

BTPC functions as a catalytic and regulatory subunit of Class-2PEPC complexes of vascular plants and green algae

BTPC genes are unique and specifically expressed

In 2003, interrogation of Arabidopsis and rice PEPC gene familiesled to the surprising discovery that, alongside their various PTPCgenes, both genomes also encode and express an enigmatic BTPCgene whose deduced amino acid sequence shares a slightly highersimilarity with PEPCs from proteobacteria than with other plantPEPCs (Figure 5) [3]. These novel PEPC isoenzymes werethus named BTPCs, whereas all remaining C3, C4 and CAMPEPCs were classified as PTPCs. All plant genomes sequencedto date, including that of ancestral green algae, contain at leastone BTPC gene. The BTPCs constitute a monophyletic group,separate from either the PTPCs or bacterial and archaeal PEPCs,and appear to have evolved in green algae (Figure 5). PTPCgenes have a highly conserved genomic structure composed ofapproximately ten exons, whereas BTPC genes have a verydifferent and more complex structure with approximately 20exons [8]. Deduced BTPC polypeptides of vascular plants rangefrom ∼116 to 118 kDa (or approximately 130 kDa in green algae)



Figure 5 Phylogenetic analysis of vascular plant, green algal, bacterial and archaeal PEPCs using a neighbour-joining consensus tree

Protein accession numbers from GenBank®, Phytozome and Maizesequence databases were used to construct the phylogenetic tree and are shown in square brackets. Bootstrap analysis was carriedout with 100 replicates. Numbers at the branches correspond to percentage bootstrap frequencies for each branch. Only values >50 are shown.



compared with ∼105–110 kDa for the corresponding PTPCs [8].All BTPC sequences deduced contain residues critical for PEPCcatalysis (Figure 3), and heterologous expression of green algal(C. reinhardtii) and castor oil plant BTPCs yielded active PEPCs[5,160,191]. Deduced PEPC polypeptides are readily classifiedas a BTPC or PTPC by three main criteria: (i) their C-terminaltetrapeptide is either (R/K)NTG for BTPCs or QNTG for PTPCs,(ii) BTPCs lack the distinctive N-terminal serine phosphorylationmotif (acid-base-XXSIDAQLR) characteristic of PTPCs [3–5],and (iii) all BTPCs contain a unique and highly divergent insertionof approximately 10 kDa (corresponding to residues 325–467of the COS BTPC, RcPPC4) that was predicted to exist in alargely unstructured and highly flexible conformation, known asan intrinsically disordered region (Figure 3) [191]. Disordered-region-containing proteins are ubiquitous in all organisms. Theirdisordered region typically exists as a flexible linker that connectstwo globular domains to mediate protein–protein interactions.The flexible linker allows the connected domains to freely twistand rotate through space to recruit their binding partners. Linkersequences can vary greatly in length and amino acid sequence,but tend to be susceptible to proteolytic cleavage while exhibitinga low content of bulky hydrophobic amino acids and a higherproportion of polar and electrically charged amino acids [192].Indeed, a recent imaging study of transiently expressed wild-type and truncated mutants of castor oil plant BTPC (RcPPC4)and PTPC (RcPPC3) fluorescent fusion proteins in tobacco BY2suspension cells demonstrated that RcPPC4’s disordered regionmediates its in vivo interaction with RcPPC3 (e.g. as a Class-2PEPC complex, see below) [193].

The tissue-specific expression of BTPC transcripts has beeninvestigated in several species. Although expression of BTPCtranscripts tends to be quite low relative to PTPC transcripts, andis dependent upon the developmental stage or metabolic statusof the tissue being examined, no obvious pattern of expressionhas emerged [3,4,6,7,11,194]. Arabidopsis BTPC transcriptswere initially detected at low levels in siliques and flowers[3], but were shown subsequently to be specifically expressedin the late stages of pollen development [7] and induced inroots by salt and drought stress [11]. BTPC transcripts andpolypeptides are co-ordinately and highly expressed during COSdevelopment; their developmental profile coincided with stages ofrapid endosperm growth and oil accumulation [4]. Nevertheless,our current understanding of plant/algal BTPC biochemistrywas initially built upon a foundation of integrating classicalenzyme biochemistry with modern tools of MS and associatedbioinformatic databases.

Native BTPCs interact with PTPCs as subunits of heteromeric Class-2 PEPCcomplexes

The study of plant BTPC biochemistry began unknowingly inthe 1990s with a series of papers describing the purification andcharacterization of two distinct PEPC classes from unicellulargreen algae (Selenastrum minutum and C. reinhardtii) thatexhibited highly dissimilar physical and kinetic properties, butthat shared an identical PTPC subunit [195–199]. From thesepreparations, typical 400-kDa PEPC homotetramers composedsolely of PTPC subunits were classified as a Class-1 PEPC,whereas high-molecular-mass complexes of PTPC and animmunologically unrelated 130-kDa subunit (subsequently shownto be a BTPC) were termed Class-2 PEPCs. Relative to theClass-1 PEPC, the algal Class-2 PEPCs displayed enhancedthermal stability, a broader pH-activity profile, biphasic PEPsaturation kinetics and a markedly reduced sensitivity to allosteric

effectors. This work was corroborated and significantly extendedby Raymond Chollet and co-workers who cloned and analysedthe expression of C. reinhardtii PTPC and BTPC genes [5,67].The PTPC cDNA expressed as an active ∼110-kDa subunitof both Class-1 PEPC and Class-2 PEPC, whereas the BTPCcDNA expressed as an active ∼130-kDa subunit found onlyin Class-2 PEPC. At the level of gene and protein expression,both algal PEPC subunits responded to inorganic nitrogen levels.However, the effect was more pronounced with the PTPCsubunits, consistent with their proposed anaplerotic functionduring transient nitrogen assimilation [5,67].

Low- and high-molecular-mass PEPC isoforms weresubsequently purified and characterized from the triacylglycerol-rich endosperm of developing COS whose respective physicaland kinetic/regulatory properties were remarkably analogous tothe highly distinctive Class-1 and Class-2 PEPCs of unicellulargreen algae [82]. Thus native COS Class-1 PEPC is a classic410-kDa homotetramer of 107-kDa PTPC subunits. In contrast,the allosterically desensitized COS Class-2 PEPC 910-kDahetero-octameric complex consists of the same Class-1 PEPChomotetrameric core tightly associated with four 118-kDa BTPCsubunits (Figures 6 and 7) [4,82]. The COS Class-2 PEPCcomplex has been documented by additional in vitro techniquesincluding co-IP (co-immunopurification) and non-denaturingPAGE of clarified extracts coupled with in-gel PEPC activitystaining and parallel immunoblotting using BTPC- and PTPC-specific antibodies [4,34,77]. Both approaches were recentlyemployed to demonstrate comparable Class-1 and Class-2 PEPCisoforms in extracts of developing lily pollen [7].

Upon extraction, the BTPC subunits of green algal and vascularplant Class-2 PEPCs are extremely susceptible to rapid in vitroproteolytic cleavage at a specific site within their disorderedregion (e.g. see Figure 3) by an endogenous thiol endopeptidase[4,196,197]. This in vitro proteolysis has prevented purificationof native COS Class-2 PEPC containing non-truncated BTPCsubunits, even in the presence of a wide array of proteaseinhibitors and cocktails. However, limited protection, suitablefor BTPC immunoblotting and co-IP from clarified extracts,was afforded by a combination of PMSF and the ProteCEASE100 Cocktail marketed by G-Biosciences [4,77]. Two additionaltechniques were central in allowing further characterization ofnon-proteolytically degraded native BTPC from developing COS.First, co-IP of highly enriched native BTPC from developingCOS endosperm extracts was achieved using an anti-(castor oilplant PTPC IgG) immunoaffinity column [77]. Relatively largequantities (>5 mg) of purified non-proteolysed 118-kDa BTPCsubunits were eluted from this column using Pierce’s GentleAg/Ab Elution Buffer and used for detailed MS characterizationof its in vivo phosphorylation sites [77,191]. Secondly, E. colilysates containing heterologously expressed wild-type and mutantversions of castor oil plant BTPC (RcPPC4) were mixed with arecombinant Arabidopsis PTPC (AtPPC3) to create intact stablechimaeric Class-2 PEPCs in vitro, which were then purified andcharacterized [160,191]. Both PTPC and BTPC subunits werecatalytically active and consequently the chimaeric Class-2 PEPCdisplayed biphasic PEP saturation kinetics [160], as documentedpreviously for the purified non-proteolysed native Class-2 PEPCfrom the green alga S. minutum [197]. BTPC is a low-affinityallosterically desensitized Class-2 PEPC subunit that exhibits Km

(PEP) and IC50 (malate) values approximately 10- and 15-foldhigher respectively than those of the PTPC subunits [160,191].The BTPC subunits appear to have an additional regulatory rolebecause the PTPC subunits of Class-2 PEPC were far less sensitiveto allosteric inhibitors compared with the same subunits within aClass-1 PEPC (Figure 7) [160,191]. It is also notable that the



Figure 6 Model illustrating the biochemical complexity of castor bean PEPC

In developing COS endosperm, the PTPC RcPPC3 exists: (i) as a typical Class-1 PEPC homotetramer (PEPC1) which is activated in vivo by sucrose-dependent phosphorylation of its 107-kDasubunit (p107) at Ser11 [34,35], and (ii) tightly associated with 118-kDa BTPC (RcPPC4) subunits (p118) to form the allosterically desensitized Class-2 PEPC hetero-octameric complex (PEPC2).The RcPPC4 subunits are subject to in vivo multi-site phosphorylation at Thr4, Ser425 and Ser451 [77,191,201]. COS maturation is accompanied by disappearance of the Class-2 PEPC complex andRcPPC4 polypeptides and transcripts, a marked reduction in the amount of Class-1 PEPC coupled with dephosphorylation of its p107-PTPC subunits [4,34,82]. COS imbibition and germinationis accompanied by increased RcPpc3 gene expression, PEPC activity and amount, and ubiquitination of 50 % of RcPPC3’s subunits at Lys628 to form the mono-ubiquitinated Class-1 PEPCheterotetramer. UB, ubiquitin. Figure modified from [103] with permission.

Figure 7 Differential influence of glucose 6-phosphate and malate on theactivity of native Class-1 and Class-2 PEPC (PEPC1 and PEPC2 respectively)isoforms purified from developing castor beans

A 0.5 and IC50 denote the concentration of glucose 6-phosphate (Glc-6-P/G6P) and malaterequired for half maximal activation and inhibition respectively. Assays were conducted at pH 7.3with subsaturating PEP concentrations (0.2 mM). All values represent the means ( +− S.E.M.) ofthree separate determinations. Figure modified from [82] with permission.

recombinant COS BTPC readily formed insoluble aggregateswhen extracted from E. coli cells in which it was overexpressed.However, when mixed with native or recombinant Class-1 PEPCsfrom various plant sources, the recombinant COS BTPC subunitsspontaneously rearranged to form stable soluble Class-2 PEPCcomplexes [160]. Although plant and algal BTPCs exhibit PEPCactivity, several additional lines of evidence strongly suggest thatPTPC subunits are essential binding partners for BTPC subunits:(i) native BTPC subunits have only been observed in associationwith PTPC subunits as a Class-2 PEPC [4,67,82,196,197], (ii)

the BTPC subunits tightly associate with the PTPC subunitsduring purification [77,82], and (iii) FP (fluorescent protein)-tagged castor oil plant PTPC and BTPC subunits interact invivo during their transient co-expression in tobacco BY2 cells(see below) [193]. PTPCs have thus been hypothesized to becompulsory BTPC-binding partners that play an indispensablerole in maintaining BTPCs in their proper structural and functionalstate in Class-2 PEPCs [160].

Significant levels of Class-2 PEPC have been observed indeveloping COS endosperm and lily pollen. Both are reproductivesink tissues in which mitosis has been completed and thecells are rapidly expanding while simultaneously metabolizinglarge amounts of imported photosynthate into storage lipids andproteins [7,82,160]. The unique kinetic and regulatory propertiesof Class-2 PEPC have been hypothesized to function as a‘metabolic overflow’ mechanism capable of sustaining significantflux from PEP to malate under in vivo conditions where thecorresponding Class-1 PEPC activity would become largelysuppressed by feedback allosteric inhibitors [7,160]. For example,malate levels in developing COS endosperm have been measuredat 5 mM, a value ∼80-fold higher than the IC50 (malate) of COSClass-1 PEPC at physiological pH (Figure 7), but within the rangeof the IC50 (malate) of the BTPC subunit of Class-2 PEPC [82,89].

BTPC of developing castor beans is subject to multi-sitephosphorylation in vivo

As plant BTPCs lack the conserved N-terminal serine phosphory-lation motif characteristic of PTPCs (Figure 3), they weresuggested to be non-phosphorylatable [11,37]. However, the useof Pro-Q Diamond phosphoprotein staining, immunoblotting withcommercially available phospho-(serine/threonine) Akt substrate-specific antibodies, and Pi-affinity PAGE using Phos-TAGacrylamide [200] demonstrated that co-immunopurified native



BTPC from developing COS endosperm was phosphorylatedat multiple sites in vivo [77]. Detailed analyses of co-immunopurified COS BTPC using Fourier-transform MS (>93%sequence coverage) identified three novel phosphorylation sites:Thr4 at the N-terminus, and Ser425 and Ser451 within itsdisordered region (respectively corresponding to acidophilic,proline-directed and basophilic protein kinase recognition motifs)(Figure 3) [77,201]. The Thr4 and Ser451 phosphorylation sitesare conserved in other BTPC orthologues, but the Ser425 siteis only partially conserved (e.g. see Figure 3) [191,201].Phosphomimetic mutagensis of the Ser425 and Ser451 sites haveshown them to be regulatory in nature, with both causing markedinhibition of the BTPC subunits within a Class-2 PEPC byincreasing their Km (PEP) and sensitivity to feedback inhibitionby malate and aspartate [191,201]. The developmental patternsof BTPC phosphorylation at Ser425 and Ser451 were examinedby immunoblotting clarified COS extracts with the respectivephosphosite-specific antibodies, and shown to be very distinctfrom the in vivo phosphorylation activation of COS Class-1PEPC’s PTPC subunits at Ser11, implying control by separateprotein kinases and signalling pathways [191,201]. In greenalgae, BTPC phosphorylation: (i) also appears to be inhibitoryin nature, and (ii) may be involved in mediating BTPC/PTPCsubunit stoichiometry within algal Class-2 PEPC complexes[198]. The function of the N-terminal phosphorylation site ofdeveloping COS BTPC (Thr4) is unknown. Kinetic analysis ofa T4D phosphomimetic RcPPC4 mutant indicated that it is notregulatory in nature [201]. However, it is intriguing that Thr4 ofplant BTPC exists in a conserved FHA (forkhead-associated)-binding domain (pTXXD) (Figure 3) and also corresponds to anacidophilic protein kinase CK2 motif. As FHA domains havegained considerable prominence as phosphothreonine-dependentprotein interaction modules [202], it will be necessary to establishthe role that BTPC phosphorylation at Thr4 might play inmediating the interaction of Class-2 PEPCs with FHA domain-containing proteins. Characterization of BTPC phosphorylationfrom additional vascular plant and algal sources, alongsideidentification of the responsible protein kinases and relatedsignalling pathways, will also be essential to further validate andextend the role of BTPC phosphorylation in Class-2 PEPCs.

SUBCELLULAR LOCALIZATION AND PROTEIN–PROTEININTERACTIONS OF PLANT PEPC

Class-2 PEPC appears to associate with the mitochondrial outerenvelope

The subcellular location and in vivo interaction of COS PTPC(RcPPC3) and BTPC (RcPPC4) were assessed recently byimaging FP–PEPC fusion proteins that had been transientlyexpressed in heterotrophic tobacco suspension cells [193]. FP–PTPC sorted to the diffuse cytosol, whereas NLS (nuclearlocalization signal)–FP–PTPC sorted to the nucleus [193]. Incontrast, BTPC-FP localized to discrete punctate structures,tentatively identified as mitochondria by immunostaining ofendogenous mitochondrial cytochrome oxidase. However, whenNLS–FP–PTPC or FP–PTPC was co-expressed with BTPC–FP, fluorescent PTPC signals co-localized with BTPC–FP.Transmission electron microscopy of immunogold-labelleddeveloping COS endosperm and cotyledon using monospecificCOS BTPC and PTPC antibodies indicated that both proteinstended to cluster together into discrete regions of themitochondrial outer envelope. The overall results indicate that: (i)COS BTPC and PTPC interact in vivo as a Class-2 PEPC complex,

(ii) BTPC’s unique and divergent intrinsically disordered regionmediates its tight interaction with PTPC, (iii) the BTPC-containing Class-2 PEPC may be located on the mitochondrialouter membrane, whereas (iv) the PTPC-containing Class-1 PEPCis uniformly distributed throughout the cytosol. Mitochondrial-associated cytosolic glycolytic isoenzymes have also beenreported in several studies. Lee Sweetlove and colleagues showedthat seven different glycolytic enzymes formed a metabolonon the mitochondrial surface of Arabidopsis suspension cellsduring periods of increased respiration so as to channel carbonfrom cytosolic metabolite pools into the mitochondria whilerestricting substrate use by competing metabolic pathways[203,204]. Furthermore, FP-tagged aldolase, enolase, ATP-PFK and hexokinase have shown a dual mitochondrial (outerenvelope) and cytosolic localization [203]. We therefore need todetermine the prevalence, mechanism and metabolic role(s) ofmitochondrial-associated Class-2 PEPC complexes. In addition,it will be important to identify any Class-2 PEPC-interactingproteins that may form a metabolon to facilitate respiratoryCO2 refixation (e.g. carbonic anhydrase) and/or anaplerotic PEPpartitioning to metabolic end-products such as storage lipids andproteins.

Class-1 PEPC may interact with a cytosolic-targeted PDH

Biochemical analyses of several purified plant PTPC isoenzymesindicated that they form a complex with the metabolicallysequential MDH and ME in C4 leaves [9], its own PPCK inripening banana fruit [120], as well as the BTPC leading tothe formation of Class-2 PEPC hetero-oligomeric complexes ofgreen algae and vascular plants [4,5,67,82,197]. Multienzymecomplexes containing PEPC, MDH and acetyl-CoA carboxylasewere also isolated from the unicellular photosynthetic protistEuglena gracilis [205]. A surprising observation of the recentco-IP proteomics study of the Class-1 PEPC interactome ofdeveloping COS endosperm was the identification of the PDHp

(plastidial PDH) as a putative PTPC interactor [77]. PDH catalysesthe irreversible oxidative decarboxylation of pyruvate into acetyl-CoA (Figure 2). Immunoblotting using monospecific antibodiesraised against E1α, E1β, E2 and E3 subunits of ArabidopsisPDHp verified the presence of all four PDHp subunits in eluatesfrom the anti-[COS Class-1 PEPC (RcPPC3)] immunoaffinitycolumn [77]. PDHp appears to specifically associate with thePTPC subunits of COS Class-1 PEPC, since a parallel co-IP studyof the PEPC interactome of germinating COS endosperm (whichcontains no detectable BTPC or Class-2 PEPC) also identifiedPDHp as a PTPC-interacting protein [193]. Although COS Class-1PEPC and PDHp are believed to be localized in different metaboliccompartments, there are only two enzymatic steps between them(MDH and ME), and PEPC and PDHp have both been implicatedin playing an important role in supporting fatty acid synthesis indeveloping COS [81,89,206]. Plant cells are unique in containingtwo different PDH isoenzymes: mitochondrial PDH which linkscytosolic glycolysis with the TCA cycle, and PDHp whichproduces acetyl-CoA and NADH for plastidic fatty acid synthesis(Figure 2) [206,207]. The structure and post-translational controlof PDHp is very different from that of mitochondrial PDH, andboth isoenzymes can be readily discriminated by immunoblotting.Immunoreactive PDHp subunits were markedly enriched whenclarified COS extracts were subjected to co-IP on the anti-(COS Class-1 PEPC) immunoaffinity column. In contrast, noimmunoreactive bands were evident when parallel immunoblotsof the same co-IP column eluates were probed with anti-(plantmitochondrial PDH) antibodies [77]. A previous report indicatedthat PDHp may not be exclusively plastidic in developing COS



endosperm [206]. In this study, the plastid marker enzyme acetyl-CoA carboxylase demonstrated 98% of its total activity to beleucoplast-localized, whereas only 62% of total PDH activity wasin the same fraction. Conversely, 2 % and 38% of total acetyl-CoA carboxylase and PDH activity were respectively measuredin the corresponding cytosolic fraction [206]. Further studies arerequired to establish PDHp’s distribution in the plastid comparedwith cytosol of non-green plant cells, such as developing andgerminating COS endosperm. Nevertheless, the aforementionedfindings support the hypothesis that a specific PEPC–PDHp

interaction might exist in developing COS. This interaction couldfacilitate CO2 recycling from PDH to PEPC and/or occur as part ofa metabolon that channels PEP to cytosolic acetyl-CoA requiredfor the biosynthesis of isoprenoids, flavonoids and malonatedderivatives, in addition to the elongation of C16 and C18 fatty acids.Cytosolic acetyl-CoA is believed to be generated from citrateand CoA by ATP-citrate lyase [208]. However, ATP-citrate lyaseactivity was barely detectable in developing COS extracts [208].PDHp could provide an alternative metabolic route for acetyl-CoA production within the COS cytosol. It will be interestingto determine whether the observed in vitro interaction betweena Class-1 PEPC and PDHp exists in vivo, and, if so, the rolethat it plays in carbohydrate partitioning and CO2 recycling indeveloping seeds.

The chloroplast Class-1 PEPC isoenzyme of rice leaves

Until recently, PEPC had been described as being exclusivelycytosolic, so it was a major surprise when a chloroplasticPTPC isoenzyme was identified in rice leaves [159]. This PTPCisoenzyme contains a transit peptide and its expression waslargely confined to leaf mesophyll cells where it was estimated toaccount for a third of the total PEPC protein. It exhibited a loweraffinity for both PEP and allosteric effectors compared withtypical C3 PTPC isoenzymes, and gene knockouts produceda phenotype which implied its involvement in leaf nitrogenassimilation [159]. Earlier immunogold imaging studies reportedthat PTPC may associate with the chloroplast outer envelopein C3, C4 and CAM leaves [209,210], whereas PTPC partiallypelleted with the total membrane fraction from sorghum leaves[167]. However, this is the first well-documented instance of aplant PEPC being targeted to the plastid stroma. It is unknownwhether plastidic PEPC isoenzymes exist in other genera, asorthologous genes were not detected. Nevertheless, the existenceof a chloroplastic PTPC isoenzyme endows rice mesophyll cellswith an alternative route of organic acid metabolism to supportnitrogen assimilation that had not previously been considered inplant primary metabolism [159].

CONCLUDING REMARKS

Although the central importance of PEPC in plant metabolism andphysiology has been appreciated for many years, recent researchhas provided numerous insights into the complex biochemicaland molecular mechanisms that underpin the control of PEPCactivity, and the influence that PEPC and PPCK exert on a widearray of cellular processes. Integrative analyses at the genomic,transcriptomic, enzymological/biochemical and cellular levelshave revealed PEPC’s biochemical complexity (e.g. see Figure 6),whereas the application of metabolic flux analysis and functionalgenomic tools have consistently indicated the importance of post-translational PEPC control in ensuring the optimal regulation andplasticity of anaplerotic PEP metabolism. Owing to its multiplefunctions and location at a pivotal branchpoint of plant primary

metabolism (Figures 1 and 2A), an impressive array of strategieshas evolved to post-translationally control the activity of plantPEPCs. Regulatory phosphorylation and mono-ubiquitination,14-3-3 proteins, as well as changes in intracellular pH andallosteric effector levels have all been described as mechanismsto control Class-1 PEPC activity in different plant tissues undervarious physiological conditions. The discovery of allostericallydesensitized Class-2 PEPC hetero-octameric complexes in greenalgae and vascular plants that arise from a tight physicalassociation between a Class-1 PEPC with co-expressed BTPCsubunits adds another layer of complexity to plant PEPC functionsand control. A major challenge will be to link the multifacetedpost-translational controls and associated PTMs of Class-1 andClass-2 PEPCs with the cell-specific partitioning of PEP intodiscrete metabolic pathways.

Many metabolic functions have been attributed to malateand other TCA cycle intermediates within different cell types[28], and the versatility of these metabolites is undoubtedlylinked to the evolved diversity of plant PEPC (Figure 1).A comprehensive understanding of the linkages between theexpression of individual PEPC isoenzymes, their specificphysiological/metabolic functions, and the synchronous regula-tion of PEPC with other key enzymes of the PEP branchpoint(e.g. PKc, PEP carboxykinase, pyruvate orthophosphate dikinase,the shikimate pathway) represents a major challenge. Only withthe concerted efforts of plant physiologists, biochemists andenzymologists and molecular biologists, will the cell-specificfunctions and control of plant PEPC be fully comprehended.It is anticipated that the further characterization of green algaland vascular plant Class-1 and Class-2 PEPC, their in vivoPTMs (including the requisite interconverting enzymes such asPPCK or E3 ubiquitin ligases, and relevant signalling pathways),subcellular localization and protein–protein interactions willremain a fruitful research area for the foreseeable future. Itwill also be important to establish a structural model for plantand algal Class-2 PEPC subunit architecture. Lastly, patternsof Class-1 compared with Class-2 PEPC expression need tobe re-evaluated together with the continued application offunctional genomic tools to assess the impact of altered PTPC andBTPC expression on anaplerotic PEP-partitioning to storage end-products in developing seeds, as well as plant carbon–nitrogeninteractions and acclimatization to environmental stress.

ACKNOWLEDGEMENTS

W.C.P. is greatly indebted to past and present members of his laboratory who madeinteresting discoveries concerning the molecular and biochemical characteristics of non-photosynthetic plant PEPCs. W.C.P. is also very grateful to collaborators who havecontributed to our PEPC research, particularly Dr David Turpin, Dr Florencio Podesta,Dr Jean Rivoal, Dr Wayne Snedden, Dr Chao-fu Lu, Dr Yi-min She, Dr Craig Leach,Dr Alexander Valentine and Dr Robert Mullen.

FUNDING

Research in this laboratory is generously supported by grants from the Natural Sciencesand Engineering Research Council of Canada (NSERC) and the Queen’s Research Chair’sprogram (to W.C.P.), an NSERC post-graduate scholarship (to B.O.’L.) and a Queen’sUniversity-Province of Ontario postdoctoral fellowship (to J.P.).

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Received 14 January 2011/18 February 2011; accepted 22 February 2011Published on the Internet 27 April 2011, doi:10.1042/BJ20110078


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The Remarkable Diversity of Plant Phosphoenolpyruvate Carboxylase