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TheOrganizationandRegulationofPlantGlycolysis
ARTICLEinANNUALREVIEWOFPLANTBIOLOGY·JULY1996
ImpactFactor:18.71·DOI:10.1146/annurev.arplant.47.1.185·Source:PubMed
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Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996. 47:185–214Copyright © 1996 by Annual Reviews Inc. All rights reserved
THE ORGANIZATION ANDREGULATION OF PLANTGLYCOLYSIS
William C. PlaxtonDepartments of Biology and Biochemistry, Queen’s University, Kingston, Ontario K7L3N6, Canada
KEY WORDS: compartmentation, metabolic regulation, isozymes, pyrophosphate, gluconeo-genesis, respiration
ABSTRACT
This review discusses the organization and regulation of the glycolytic pathwayin plants and compares and contrasts plant and nonplant glycolysis. Plant glyco-lysis exists both in the cytosol and plastid, and the parallel reactions are cata-lyzed by distinct nuclear-encoded isozymes. Cytosolic glycolysis is a complexnetwork containing alternative enzymatic reactions. Two alternate cytosolic re-actions enhance the pathway’s ATP yield through the use of pyrophosphate inplace of ATP. The cytosolic glycolytic network may provide an essential meta-bolic flexibility that facilitates plant development and acclimation to environ-mental stress. The regulation of plant glycolytic flux is assessed, with a focus onthe fine control of enzymes involved in the metabolism of fructose-6-phosphateand phosphoenolpyruvate. Plant and nonplant glycolysis are regulated from the“bottom up” and “top down,” respectively. Research on tissue- anddevelopmental-specific isozymes of plant glycolytic enzymes is summarized.Potential pitfalls associated with studies of glycolytic enzymes are considered.Some glycolytic enzymes may be multifunctional proteins involved in processesother than carbohydrate metabolism.
CONTENTSINTRODUCTION ......................................... ..........................................................................186
The Functions of Glycolysis..................... .......................................................................... 186THE ORGANIZATION OF PLANT GLYCOLYSIS ............................................................. 187
Compartmentation of Glycolysis............. .......................................................................... 187The Glycolytic Network of the Plant Cytosol...................................................................... 190
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THE REGULATION OF PLANT GLYCOLYSIS ..................................................................195Coarse Metabolic Control of Plant Glycolysis................................................................... 196Fine Metabolic Control of Plant Glycolysis....................................................................... 196
PRACTICAL ASPECTS OF PLANT GLYCOLYTIC ENZYMOLOGY ..............................205Errors and Artifacts in Assays of Glycolytic Enzymes........................................................ 205Protease and Dilation Problems.............. .......................................................................... 206
OTHER FUNCTIONS FOR GLYCOLYTIC ENZYMES.......................................................207CONCLUDING REMARKS......................... ..........................................................................208
INTRODUCTION
In about 1940, glycolysis became the first major metabolic pathway to befully elucidated, an achievement that was instrumental in the development ofmany experimental and conceptual aspects of modern biochemistry. Subse-quent studies of glycolysis have shown that it is the “central” metabolic path-way that is present, at least in part, in all organisms. Glycolysis is an excellentexample of how (a) a ubiquitous metabolic pathway can show significant dif-ferences in terms of its roles, structure, regulation, and compartmentation indifferent phyla, and even within different cells of the same species; (b) thefunction of the ATP/ADP system in biological energy transduction can be re-placed or augmented by the pyrophosphate/orthophosphate (PPi/Pi) system;and (c) the various mechanisms of metabolic regulation apply to a specificpathway in vivo. Glycolysis is also directly involved in many biochemical ad-aptations of plant and nonplant species to environmental stresses such as nu-trient limitation, osmotic stress, drought, cold/freezing, and anoxia.
Several reviews concerning plant glycolysis have appeared in the past dec-ade (3, 4, 12, 14, 24, 32, 37, 39, 40, 68, 92, 108, 111, 135, 136, 140). This re-view considers the organization and regulation of the glycolytic pathway inplants and compares and contrasts plant glycolysis with its counterpart in non-plant systems.
The Functions of GlycolysisGlycolysis evolved as a catabolic anaerobic pathway to fulfil two fundamentalroles. It oxidizes hexoses to generate ATP, reductant, and pyruvate, and it pro-duces building blocks for anabolism. Glycolysis is also an amphibolic path-way because it can function in reverse to generate hexoses from various low-molecular-weight compounds in energy-dependent gluconeogenesis. Muchattention has been devoted to determining the mechanisms by which the op-posing processes of glycolysis and gluconeogenesis are reciprocally regulatedin vivo.
In contrast with animal mitochondria, which frequently respire fatty acidsand glycolytically derived pyruvate, plant mitochondria rarely respire fatty ac-ids (5). Glycolysis is thus of crucial importance in plants because it is the pre-dominant pathway that “fuels” plant respiration. Moreover, a significant pro-
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portion of the carbon that enters the plant glycolytic and tricarboxylic acid(TCA) cycle pathways is not oxidized to CO2 but is utilized in the biosynthe-sis of numerous compounds such as secondary metabolites, isoprenoids, aminoacids, nucleic acids, and fatty acids. The biosynthetic role of glycolysis and res-piration is particularly important in actively growing autotrophic tissues (5).
THE ORGANIZATION OF PLANT GLYCOLYSIS
So-called classical glycolysis, which occurs in most nonplant organisms (Fig-ure 1a), is the cytosolic linear sequence of 10 enzymatic reactions that cata-lyze the net reaction:
glucose + 2 ADP + 2 Pi + 2 NAD+ → 2 pyruvate + 2 ATP + 2 NADH.
Although higher plants use sucrose and starch as the principal substratesfor glycolysis, they are known to metabolize the immediate products of su-crose and starch breakdown via the classic intermediates of glycolysis (Figure1b) (3, 4, 32, 136). Nevertheless, as outlined in Figure 1, there are several pro-found differences in structural and associated bioenergetic features of the gly-colytic pathway in plant vs nonplant organisms.
Compartmentation of GlycolysisThe sequential conversion of hexoses to pyruvate in plants can occur inde-pendently in either of two subcellular compartments, the cytosol and plastid(Figure 1b). Compartmentation concentrates enzymes of a pathway and theirassociated metabolites and prevents the simultaneous occurrence of poten-tially incompatible metabolic processes (38). The integration of cellular me-tabolism necessitates controlled interactions between pathways sequestered invarious subcellular compartments. Thus, plastidic and cytosolic glycolysiscan interact through the action of highly selective transporters present in theinner plastid envelope (Figure 1b) (46). The prime functions of glycolysis inchloroplasts in the dark and in nonphotosynthetic plastids are to participate inthe breakdown of starch as well as to generate carbon skeletons, reductant,and ATP for anabolic pathways such as fatty acid synthesis (14, 37, 40, 46).Although plastids from several nonphotosynthetic tissues, including develop-ing wheat and castor seeds, have been found to possess all the enzymes of gly-colysis from glucose to pyruvate, some chloroplasts may lack one or severalof the enzymes of the lower half of glycolysis (e.g. enolase and phosphoglyc-eromutase) (40, 46, 92). In contrast, the cytosol of many unicellular green al-gae appears to lack a complete glycolytic pathway (and is generally deficientin the enzymes of the upper half of glycolysis), whereas their chloroplastsseem to contain the entire suite of glycolytic enzymes (68, 128). This is be-cause starch is the dominant respiratory fuel for algae, and sucrose is rela-tively unimportant in algal carbon metabolism (68).
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188 PLAXTON
Figure 1 A comparison of the organization of nonplant (A) vs plant (B) glycolysis. The enzymesthat catalyze the numbered reactions are as follows: 1, hexokinase; 2, phosphorylase; 3, phospho-glucomutase; 4, phosphoglucose isomerase; 5, PFK; 6, ALD; 7, triose phosphate isomerase; 8,NAD-dependent GAPDH (phosphorylating); 9, 3-PGA kinase; 10, phosphoglyceromutase; 11,enolase; 12, PK; 13, invertase; 14, sucrose synthase; 15, UDP-glucose pyrophosphorylase; 16, nu-cleoside diphosphate kinase; 17,α- andβ-amylases; 18, PFP; 19, NADP-dependent GAPDH (non-phosphorylating); 20, PEPase; 21, PEPC; 22, MDH; 23, ME. Abbreviations are as in the text or asfollows: Glu-1-P, glucose-1-phosphate; DHAP, dihydroxyacetone phosphate; G3P,glyceraldehyde-3-phosphate; 1,3-DPGA, 1,3-diphosphoglycerate; 2-PGA, 2-phosphoglycerate;OAA, oxaloacetate.→, indicates physiologically irreversible reactions;3 or ↔ indicate physio-logically reversible reactions. Note that the number of substrate and product molecules in all reac-tions from G3P to pyruvate should be doubled because two molecules of G3P are formed from onemolecule of hexose.
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PLANT GLYCOLYSIS 189
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The parallel plastidic and cytosolic glycolytic reactions are believed to becatalyzed by isozymes encoded by distinct nuclear genes (14, 32, 57, 58, 61,91, 111). The relative proportions of the isozymes can vary, not only accord-ing to the type of tissue and its developmental stage but also as a result of al-terations in the plant’s environmental or nutritional status (24, 37, 38, 40, 52,93, 126). The cytosolic and plastidic isozymes can be nearly identical, exceptfor differences in charge, or, in the case of key regulatory enzymes such asATP-dependent phosphofructokinase (PFK) (22, 29, 39, 78) and pyruvate ki-nase (PK) (52, 85, 86, 106, 110a, 111, 126), the differences in physical, im-munological, and kinetic properties can be profound. Plastidic glycolytic en-zymes are thought to be synthesized as inactive precursors on ribosomes inthe cytosol, followed by their import into the organelle with concomitantcleavage of anN-terminal transit peptide (15, 48).
EVOLUTION OF CYTOSOLIC AND PLASTIDIC GLYCOLYTIC ISOZYMES Genepairsencoding plastidic and cytosolic isozymes could have evolved from dupli-cations of common ancestral nuclear genes, or the genes encoding plastidic iso-zymes may have been transferred to the nucleus from the genome of theprokaryotic symbiont thought to have given rise to plastids (46). The formerpossibility appears to be the case for plastidic and cytosolic fructose bisphos-phate aldolases (ALDs), which both appear to belong to the eukaryotic family ofclass I ALDs (128). Likewise, DNA sequence analyses of triose phosphateisomerase and NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase(NAD-GAPDH) demonstrated that the plastidic isozymes arose via duplicationof the preexisting nuclear counterparts for the corresponding cytosolic isozyme(61, 91). In contrast, the gene and/or derived amino acid sequences encoding cy-tosolic and plastidic isozymes of PK (PKc and PKp, respectively) and hexose-phosphate isomerase show closer respective homologies to their eukaryotic andprokaryotic counterparts (16, 17, 57, 58). Similarly, immunoblot and peptidemapping analyses demonstrated that PKp from the green algaSelenastrum mi-nutumis more closely related to a bacterial PK than it is toS. minutumPKc orrabbit muscle PK (76).
The Glycolytic Network of the Plant CytosolThe cytosolic glycolytic pathway is a complex network containing parallel en-zymatic reactions at the level of sucrose, fructose-6-phosphate (Fru-6-P),glyceraldehyde-3-phosphate, and phosphoenolpyruvate (PEP) metabolism(136, 140) (Figure 1b). An ongoing and challenging problem has been to elu-cidate the respective role(s), regulation, and relative importance of the variousalternative reactions of plant cytosolic glycolysis. The cytosolic glycolyticnetwork is proposed to furnish plants with the requisite metabolic optionsneeded to facilitate their development and to acclimate to unavoidable envi-ronmental stresses such as anoxia and Pi starvation (90, 136, 140). A funda-
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mental characteristic of cytosolic glycolysis is the existence of two alternativereactions that can utilize PPi rather than nucleoside triphosphates (NTPs) as aphosphoryl donor (Figure 1b).
PPi: AN AUTONOMOUS ENERGY DONOR OF THE PLANT CYTOSOL PPi is a by-product of ahost of reactions involved in macromolecule biosynthesis. Onedogma of cellular bioenergetics is that the anhydride bond of PPi is never util-ized and that PPi produced in anabolism is always removed by the hydrolytic ac-tion of an inorganic alkaline pyrophosphatase (PPiase), thereby providing athermodynamic “pull” for biosynthetic processes. Macromolecule biosynthe-sis, however, remains thermodynamically favorable no matter how the low con-centration of PPi is maintained, whether by hydrolysis or by some other means,including the utilization of the high energy of the PPi bond (148).
The importance of PPi in the glycolytic metabolism of some organismswas discovered in research on so-called energy-poor anaerobic microorgan-isms such as the bacteriaPriopionibacterium shermaniiand parasitic amoebaEntamoeba histolytica(148). These species have no PFK but instead convertFru-6-P to fructose-1,6-bisphosphate (Fru-1,6-P2) via a PPi-dependent phos-phofructokinase (PFP). They also lack PK but employ PPi to convert PEP andAMP into pyruvate, ATP and Pi via pyruvate, Pi dikinase (PPDK), therebyconverting the bond energy of PPi into a high-energy phosphate of ATP. Ow-ing to their use of PFP and PPDK, these organisms are theoretically able toyield 5 ATP instead of 2 per glucose degraded to pyruvate. This obviouslyrepresents a considerable energetic advantage for obligate anaerobes such asP. shermaniiandE. histolytica.
The discovery in 1979 of the strictly cytosolic PFP in plants (27) and thesubsequent observation of its potent activation byµM levels of the regulatorymetabolite fructose-2,6-bisphosphate (Fru-2,6-P2) (125) led to a surge of re-search on the role of PPi in plant sugar-phosphate metabolism. Because PFPcatalyzes a reaction that is readily reversible (Keq = 3.3) and is close to equi-librium in vivo (24, 82, 135), it could theoretically catalyze a net flux in thedirection of glycolysis or gluconeogenesis. Possible functions of PFP as a gly-colytic or gluconeogenic enzyme have been reviewed (12, 14, 24, 39, 135,136, 140). PFP activity and molecular composition depend on a variety of en-vironmental, developmental, species- and tissue-specific cues. Recent reportsdescribe the production of transgenic potato plants exhibiting significantlylower expression of PFP (56) or overexpression of mammalian 6-phosphofructo-2-kinase (81). Metabolite studies of the transgenic plants ledboth groups to conclude that PFP catalyzes a net glycolytic flux in potato tu-bers.
The plant cytosol lacks soluble inorganic alkaline PPiase and consequentlycontains PPi concentrations of up to 0.3 mM (3, 4, 135, 145). Moreover, PPilevels of plant cells are remarkably insensitive to environmental perturbations
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that elicit significant decreases in NTP pools (35, 44, 94, 140). The signifi-cance of PPi in plant metabolism was recently demonstrated by the introduc-tion of the Escherichia coliinorganic PPiase gene into tobacco and potatoplants under control of a constitutive promoter (70). Expression of theE. coliPPiase in the cytosol reduced PPi levels by up to threefold and led to a dra-matic inhibition of plant growth (70). To assess the relative importance of PPivs ATP as an energy donor in the plant cytosol, Davies et al (36) computedthe standard free energy changes for PPi and ATP hydrolysis under a varietyof cytosolic conditions. The results indicate that PPi would be particularly fa-vored as a phosphoryl donor, relative to ATP, under cytosolic conditionsknown to accompany stresses such as anoxia or nutritional Pi deprivation.These results underscore the importance of PPi as an autonomous energy do-nor of the plant cytosol. In support, tolerance of acclimated maize root tips toanoxia is not critically dependent upon high energy charge (150).
Apart from PFP, PPi could be employed as an energy donor for two othercytosolic reactions: (a) the conversion of UDP-glucose to UTP and glucose-1-phosphate catalyzed by UDP-glucose pyrophosphorylase (Figure 1b), and (b)the PPi-dependent H+-pump of the tonoplast. The H+-PPiase is one of two pri-mary H+-pumps residing at the tonoplast; the other is an H+-ATPase (36).That PPi-powered processes may be a crucial facet of the metabolic adapta-tions of plants to environmental stresses that cause depressed NTP (but notPPi) pools is indicated by the significant induction of: (a) sucrose synthase(55, 122), PFP (90), and the tonoplast H+-PPiase (28) by anoxia in rice seed-lings; and (b) PFP ofBrassica nigra, B. napus,and related crucifers by Pi star-vation (44, 137, 139, 140; VL Murley, CC Carswell & WC Plaxton, unpub-lished observations). Recent evidence (55) indicates that sucrose metabolismof anoxic rice seedlings occurs mainly through sucrose synthase with nucleo-side diphosphate kinase facilitating the cycling of uridilates needed for opera-tion of this pathway (Figure 1b). Assuming that PPi is a by-product of anabo-lism, no ATP is needed for the conversion of sucrose to hexose-Ps via the su-crose synthase pathway, whereas 2 ATPs are needed for the invertasepathway. Mertens (90) argued that PFP functions in glycolysis in anoxic riceseedlings because both PFP activity and the level of Fru-2,6-P2 increase,while PFK activity declines. Thus, the net yield of ATP obtained during gly-colytic fermentation of sucrose is increased from 4 to 8 if sucrose is metabo-lized via the sucrose synthase and PFP bypasses, relative to the invertase andPFK pathways (Figure 1b).
THE UNIQUE FLEXIBILITY OF PLANT PHOSPHOENOLPYRUVATE METABOLISM
PEP is able to generate a large amount of energy for anabolism (it occupies thehighest position on the thermodynamic scale of known phosphorylated metabo-lites) and participates in a wide range of reactions by enzymatic cleavage on ei-
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ther side of its enol oxygen atom (111). It is also both a key allosteric effector ofa number of plant enzymes as well as a major branchpoint leading into a varietyof primary and secondary metabolic pathways.
Plant cells have been proposed to employ two alternative metabolic routesto indirectly or directly circumvent the reaction catalyzed by PKc (Figure 1b).PEP carboxylase (PEPC), a ubiquitous plant cytosolic enzyme, plays the im-portant role in C3 plants and nonphotosynthetic tissues of C4 and Crassu-lacean acid metabolism plants of replenishing TCA cycle intermediates con-sumed in biosynthesis (30, 67, 68). However, together with cytosolic malatedehydrogenase (MDH) and the mitochondrial NAD-dependent malic enzyme(ME), PEPC can also function as a glycolytic enzyme by indirectly bypassingthe reaction catalyzed by PKc (Figure 1b). On the basis of pulse-chase radiola-beling experiments using NaH14CO3, it was concluded that this bypass oper-ates in roots ofPisum sativum(26). The PEPC-MDH-ME bypass of PKc isthought to be important during nutritional Pi deprivation when PKc activitymay become ADP limited (102, 138, 140). Compared to nutrient-sufficientcontrols, PEPC activity was five- and threefold greater in extracts of Pi-deficientB. nigraandCatharanthus roseussuspension cells, respectively (44,102). Metabolite determinations and kinetic studies of PKc and PEPC inC.roseussuggested that the contribution of PEPC to the metabolism of PEP in-creased in Pi-starved cells in vivo (102). Further evidence for the operation ofthis PKc bypass in Pi starvedC. roseuswas provided by the rapid release of14CO2 from organic compounds derived from fixed NaH14CO3 and from [4-14C]malate (102). In addition, dark CO2 fixation rates ofS. minutumand pro-teoid roots ofLupinus albuswere found to increase with Pi limitation, whilerespiration declined (71, 138). This suggested that the Pi-starvation-dependentin vitro and in vivo elevations ofC. roseus, S. minutum,andL. albusPEPCactivities were in response to increased demands for pyruvate and/or Pi recy-cling.
A variation on this scheme occurs in developing oil seeds where PEPC, inconcert with cytosolic MDH and plastidic NADP-ME, may provide an alter-native to PKp to supply pyruvate for fatty acid biosynthesis (111, 127, 133)(Figure 1b). Malate is an excellent exogenous substrate for fatty acid biosyn-thesis by isolated leucoplasts of developingRicinus communisseeds (133).Relative to most nonphotosynthetic tissues, the PEPC activity in developingR. communisandB. napusseeds is substantial (127). Furthermore, the mostsignificant increase in the activity and concentration of PEPC in developingR.communisendosperm coincides with the tissue’s most active phase of storageoil accumulation (127). All of the reductant required for carbon incorporationinto fatty acids is produced as a consequence of the metabolism of malate toacetyl-CoA through the leucoplast-localized NADP-ME and pyruvate dehy-drogenase complex (133). However, fatty acid synthesis also requires a source
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of ATP (for acetyl-CoA carboxylase), and serving as this source may be an in-dispensable function for the 3-phosphoglycerate kinase and PKp of leucoplas-tic glycolysis (Figure 1b). The respective contributions of the leucoplastic gly-colysis vs PEPC-MDH-ME pathways for supporting oilseed fatty acid synthe-sis require clarification. It has been suggested that the relative importance ofeach route varies diurnally according to the rate of photosynthate import fromsource tissues (111).
PEP phosphatase: a glycolytic enzyme?It has long been recognized that aPEP phosphatase (PEPase) activity often interferes with the determination ofplant PK activity (3, 108, 111). PEPase activity was generally attributed to anonspecific acid phosphatase (APase). However, two dissimilar plant phos-phatases exhibiting exceptional selectivity for PEP have been isolated (42, 88).Although a PEP-specific alkaline phosphatase was purified and characterizedfrom germinating mung beans (88), a subsequent study provided immunologi-cal and kinetic evidence suggesting that this activity arose from PKc during itspurification (114). In contrast, a PEP-specific APase that is physically and im-munologically distinct from PKc was purified to homogeneity from hetero-trophic B. nigra suspension cells (42). Although its substrate specificity wasnonabsolute, this APase was designated as a PEPase because of its extremelylow Km for PEP of about 50µM (a value equivalent to those reported for variousplant PKcs) (86, 115, 116, 119, 149) and because its specificity constant(Vmax/Km) for PEP was at least sixfold higher than that obtained for any of theother of 14 nonsynthetic substrates identified (42).
B. nigraPEPase demonstrated potent inhibition by Pi (42), and its specificactivity was increased more than ten-fold following Pi-deprivation (44). Simi-lar results have been reported for the PEPase of Pi-limitedS. minutum(138).PEPase may function to directly bypass the ADP-limited PKc and to recycleintracellular Pi during Pi starvation ofB. nigraandS. minutum(44, 138, 140).Immunoquantification studies have revealed that the significant induction ofB. nigraPEPase activity during Pi stress arises from de novo synthesis of PE-Pase protein (45). According to the “glycolytic bypass” theory for PEPase, asB. nigraPEPase is localized in the cell vacuole (43) Pi-depletedB. nigrasus-pension cells transport PEP into—and pyruvate out of—it (Figure 1b). Futurestudies must evaluate whether the tonoplast of Pi-starved plant cells is perme-able to PEP and pyruvate. However, movement of PEP into the vacuole iselectrogenically feasible, because at the alkaline pH of the cytosol it wouldcarry three net negative charges (E Blumwald, personal communication).
Transgenic tobacco plants lacking cytosolic pyruvate kinase in their leavesTransformation of tobacco (Nicotiana tabacum) with a vector that was designedto examine the impact of overexpressing potato PKcunexpectedly led to the pro-
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duction of plants that specifically lacked PKc in their leaves as a result of cosup-pression (53). The primary tobacco transformants showed no alteration in shootgrowth, morphology, photosynthesis, or respiration (53). Analysis of the homo-zygous offspring of these transformants, which either lacked PKc in their leaves(PKc−) or were genetically equivalent to wild type (PKc+), led to these major(77) results and conclusions:
1. The absence of leaf PKc had a particularly deleterious effect on root devel-opment, which may be the first example where a specific “knockout” of anenzyme in one tissue exerts a maximal detrimental effect in a different tis-sue where the same enzyme is expressed normally.
2. Partially purified tobacco leaf PKc displayed regulatory properties consis-tent with PKc’s involvement in controlling respiratory C-flow to supportN-assimilation by glutamine synthetase/glutamate-oxoglutarate amino-transferase. That the regulation of N-metabolism was impaired in eight-week-old PKc− plants was indicated by the elevated free amino acid con-tent of their leaves. The PKc− plants also had significantly reduced starch,glucose, and hexose-P levels in leaves and roots accompanied by enhancedrates of photosynthesis and dark respiration in leaves. Although the meta-bolic basis for these observations remains to be determined, the analysisproves that plants contain metabolic bypasses to PKc. In contrast, PK by-passes are absent in yeast,E. coli,and animals (cited in 53).
3. Leaves of the PKc− plants contained twofold more PEPase activity thandid the PKc+ controls, whereas activities of PEPC, PFK, and PFP were un-affected. This indicates that PEPase might be acting to circumvent the PKcreaction in the PKc− leaves.
Overall, these results illustrate the remarkable flexibility of plant PEP me-tabolism. This flexibility is probably an evolutionary adaptation to the stressesthat plants are exposed to in a fluctuating environment.
THE REGULATION OF PLANT GLYCOLYSIS
The magnitude of metabolite flux through any metabolic pathway dependsupon the activities of the individual enzymes involved. “Coarse” and/or “fine”metabolic controls can vary the reaction velocity of a particular enzyme invivo (32, 107). Coarse control is achieved through varying the total popula-tion of enzyme molecules via alterations in the rates of enzyme biosynthesisor proteolysis; it most frequently comes into play during tissue differentiationor long-term environmental (adaptive) changes (107). By modulating the ac-tivity of preexisting enzymes, fine controls function as “metabolic transduc-ers” that sense the momentary metabolic requirements of the cell and adjustthe rate of metabolite flux through the various pathways accordingly.
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Coarse Metabolic Control of Plant Glycolysis
Some of the best evidence for coarse control of plant glycolytic enzymescomes from studies of developing and germinating seeds (9, 13, 18–21, 23,24, 40, 52, 66, 93, 106, 109, 118, 127), cell and tissue cultures (8a, 44, 101,126, 134, 137), and anaerobically induced proteins such as cytosolic ALD(ALDc) and enolase (49, 73, 92). Although an understanding of the transcrip-tional and translational regulation of genes encoding enzymes such as PFP,phosphoglyceromutase, enolase, PKc, PKp, and PEPC is beginning to emerge(13, 15, 30, 47, 49, 52, 66, 83), very little is known about how the proteolyticturnover of plant glycolytic enzymes is regulated. Analysis of PKc and PKp atthe mRNA and protein levels in developing tobacco seeds demonstrated thatthe expression of these isozymes may be controlled by independent transcrip-tional and posttranscriptional mechanisms, and that PKp has a much greaterrate of turnover than PKc (52). An asparginyl endopeptidase has been sug-gested to be involved in the turnover of PKp in developingR. communisseeds(34, 109), whereasVicia fabia PEPC appears to be degraded via ubiquitin-dependent proteolysis (131).
Fine Metabolic Control of Plant GlycolysisDiverse fine control mechanisms have evolved to regulate the interplay ofglycolysis with its associated pathways and to coordinate glycolytic flux withcellular needs for energy and anabolic precursors. Quantitative and quali-tativeapproaches have been used to identify these control mechanisms (6, 32,48).
CONTROL ANALYSIS One important approach to metabolic regulation is thecontrol analysis theory and the analytical tools used to implement it (6). Thegoal of control analysis is to elucidate key points of regulation in vivo withoutrelying on preconceived notions as to which pathway enzymes are “rate-determining-steps.” Control analysis of nonplant glycolysis clearly shows thatthe contribution of the various enzymes to the overall flux control is highly de-pendent upon the organism, tissue, and physiological condition (48). Changes inphysiological conditions may cause a redistribution of the control that each en-zyme exerts on glycolytic flux, and this undoubtedly applies to plant glycolysis.However, because plant glycolysis is a complex network rather than a strictlycytosolic linear pathway, the practical use of control analysis for analyzing gly-colytic pathway regulation in plants may prove difficult. Readers are referred toa recent review (6) for further insights concerning the application of controlanalysis to plant metabolism.
KEY REGULATORY ENZYMES OF GLYCOLYSIS The traditional (or qualitative)approach to metabolic regulation involves identifying the so-called key regula-
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tory enzymes of a pathway and the mechanisms whereby their activities are con-trolled in vivo. These reactions are often characterized by their strategic positionin a pathway—by being greatly displaced from equilibrium in vivo—and theywere classically recognized by showing that their substrate(s) to product(s) con-centration ratio changed in the opposite direction to the flux when the latterwas varied (6, 32). Elucidation of the fine control of plantglycolysis is com-plicated by the alternative glycolytic reactions that exist in the plant cyto-sol (Figure 1b). Flexibility in the structure of cytosolic glycolysisnecessar-ily implies flexibility in glycolytic regulation, and the regulation will vary ac-cording to the specific tissue, its developmental stage, and the externalenvironment. Nevertheless, various approaches have demonstrated that, as innonplant systems, fine control of plant glycolysis is primarily exerted by thoseenzymes that catalyze reactions involved in the conversion of hexose to hexose-P, Fru-6-P to Fru-1,6-P2, and PEP to pyruvate (32, 82, 92). It has been arguedthat the first committed step of plant glycolysis is the conversion of Fru-6-P toFru-1,6-P2 (39). I concentrate on the fine control of the glycolytic sequencefrom Fru-6-P to pyruvate. The study of plant glycolytic regulation has been fa-cilitated by advances in protein purification technologies that have made it pos-sible to fully purify—and thus more thoroughly characterize—PFP (87, 98,104), PEPC (30, 83, 84), cytosolic PFK (PFKc) (29, 60, 143, 146), plastidic PFK(PFKp) (25, 78), PKc (65, 105), and PKp (76, 103, 110a) from various plantsources.
SPECIFIC MECHANISMS OF FINE CONTROL AS APPLIED TO PLANT GLYCOLYTIC
REGULATION At least six mechanisms of fine control can modulate the ac-tivities of preexisting enzymes: variation in substrate(s) and cofactor concen-trations, variation in pH, metabolite effectors, subunit association-disassociation, reversible covalent modification, and reversible associations ofmetabolically sequential enzymes (107). These fine controls often interact withor may actually be dependent upon one another. Examples of how each mecha-nism may apply to the regulation of plant glycolysis are briefly considered be-low.
Fine control #1: variation in substrate(s) or cofactor concentrationChangesin substrate concentrations that normally occur in vivo are generally not sig-nificant in metabolic regulation (107). Nevertheless, regulation of severalplant glycolytic enzymes may be achieved, in part, through changes in a sub-strate. For example, the initial activation of PKc that accompanies N- or P-resupply to nutrient-limitedS. minutumhas been proposed to arise from a re-lease of ADP-limitation of the enzyme (50, 68). Similarly, the low affinity ofsoybean nodule cytosolic NAD-GAPDH for its cosubstrate Pi (Km = 9 mM) has
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been postulated to make it responsive to changes in Pi levels that could occur invivo (33).
During long-term Pi deprivation, cellular pools of adenylates and Pi be-come severely depressed (8b, 35, 43, 138, 140). This process has been pro-posed to restrict the activities of the adenylate-dependent (e.g. PFK, 3-phosphoglycerate kinase, and PK) and Pi-dependent (e.g. NAD-GAPDH) gly-colytic enzymes as well as the phosphorylating (cytochrome) pathway of mi-tochondrial respiration. As a consequence, the flux of hexose-P through in-ducible adenylate- and Pi-independent cytosolic glycolytic bypasses (i.e. PFP,irreversible nonphosphorylating NADP-GAPDH, PEPase, and PEPC) (Figure1b) may be promoted, concomitant with an increased participation of the non-phosphorylating cyanide- and rotenone-insensitive alternative pathways ofmitochondrial electron transport (44, 102, 137–140).
Fine control #2: variation in pH The pH dependence of enzyme activity maybe an important aspect of the fine control of plant glycolysis because the pH ofthe cytosolic and plastidic compartments can change in response to a variety ofenvironmental factors. Inhibition of leaf PFKp activity may arise from alkalini-zation of the stroma that follows illumination (32). The shift from lactate to etha-nol production that occurs during fermentation in many plant cells ishypothesized to be mediated by anoxia-induced reductions in cytosolic pH,which lead to stimulation of pyruvate decarboxylase (73, 92). PEPC activity andresponse to metabolite effectors are well known to be sensitive to cytosolic pHchanges that may occur in vivo. PEPCs from various sources includingS. minu-tum(J Rivoal, R Dunford, WC Plaxton & DH Turpin, unpublished data), soy-bean nodules (130), maize leaves (83, 112), banana fruit (84), and cotyledons ofgerminatingR. communisseeds (119) demonstrate a greater activity and weakerresponse to various metabolite inhibitors as assay pH is increased from about pH7 to 8. This has given rise to the proposed “pH stat” function for PEPC becauseits activity should increase in response to cytosolic alkalinization owing to di-rect pH effects on its activity as well as the enzyme’s desensitization to inhibi-tory metabolites. It was recently reported that light-dependent alkalinizationand dark-acidification of cytosol-enriched cell sap are respectively correlatedwith the light-activation and dark-inactivation of leaf PEPC activity, particu-larly in C4plants (120). The regulation of the PKc isoforms from endosperm andcotyledons of germinatingR. communisseeds may also arise from pH-dependent alterations in the enzyme’s response to a variety of metabolite inhibi-tors (115, 119). It was proposed that an enhancement in PKc activity of germi-natingR. communisendosperms occurs during anaerobiosis through concerteddecreases in cytosolic pH and concentrations of several key inhibitors (115), andthat an enhancement in PKc activity of germinatingR. communiscotyledonswill arise from reduced cytosolic pH and ATP levels caused by operation of a
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plasmalemma H+-symport that powers the uptake of endosperm-derived su-crose and amino acids from the apoplast (119).
Fine control #3: metabolite effectorsTissue-specific expression of kineticallydistinct isoforms of key enzymes such as PEPC and PKc(see below) gives rise toseveral important tissue-specific differences with respect to what metabolitesare key effectors of plant glycolytic enzymes. Two fundamental facets of thisregulation emerge. First, adenine nucleosides (and hence energy charge) do notalways play as prominent a role in regulating plant glycolysis as they do in manynonplant systems. Recall that PPi can function as an autonomous energy donorin the plant cytosol, and glycolysis in many plant tissues is key to supplying bio-synthetic precursors, rather than ATP production per se. Thus, ATP and AMPare usually not considered to be critical effectors of PFKc or PFKp (39, 78, 143),PFP (87, 104, 135), PEPC (68, 83, 119, 129, 130), and PKc or PKp(65, 69a, 86,119, 149). One exception to this view may be in anoxia-tolerant tissues such asgerminating seeds. For example, MgATP is a key inhibitor of the PFKc fromgerminatingCucumis sativusseeds (22). Similarly, the switch from gluconeo-genesis to glycolysis that follows anoxia stress in the endosperm of germinatingR. communisseeds is thought to arise in part from the release of inhibition of theendosperm-specific PKc isoform by MgATP (115, 117).
A second notable attribute of plant glycolytic regulation by metabolite ef-fectors is the ubiquitous role played by PEP, Pi, Fru-2,6-P2, and TCA/ glyoxy-late cycle intermediates. Virtually all PFKcs and PFKps examined to dateshow potent inhibition by PEP, and this inhibition is relieved by the activatorPi. It is thus the concentration ratio of Pi:PEP that is believed to be critical inregulating PFK activity in vivo (4, 32, 39, 60, 143). PEP may also allosteri-cally inhibit ALDc, because it is a mixed-type inhibitor of the enzyme fromcarrot storage roots (97). In contrast with PFK, Pi is a potent inhibitor of PFPin the forward direction (87, 135) and PEPase (42). The large (up to 50-fold)reductions in intracellular Pi levels that follow long-term Pi starvation have,therefore, been proposed to promote the activity of PFP and PEPase whilecurtailing the activity of PFKc (42, 140).
The cytosolic regulatory metabolite Fru-2,6-P2 reciprocally regulates liverglycolysis and gluconeogenesis owing to its potent activation and inhibitionof PFK and FBPase, respectively (Figure 2a) (48). In plants, Fru-2,6-P2 acti-vates and inhibits PFP and cytosolic FBPase, respectively, but has no effecton PFK (Figure 2b). The metabolism and possible functions of Fru-2,6-P2 inplants have been reviewed (135). Although the roles of Fru-2,6-P2 have notbeen fully resolved, in at least some instances it probably operates to regulatethe opposing processes of gluconeogenesis and glycolysis in the plant cytosol.A rise in the cytosolic concentration ratio of Pi:3-phosphoglycerate (3-PGA)
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favors Fru-2,6-P2 synthesis owing to reciprocal effects of Pi (activator) and 3-PGA (inhibitor) on 6-phosphofructo-2-kinase (135).
The TCA cycle intermediates citrate, 2-oxoglutarate, succinate, and/or ma-late are effective feedback inhibitors of many plant PKcs (10, 65, 86, 115,119, 149) and PEPCs (68, 83, 84, 112, 119, 124, 129, 130). Glycolytic fluxrises whenever TCA cycle intermediates are consumed via anabolism or res-piration. All PEPCs examined to date display varying degrees of inhibition bymalate, and this is usually relieved by the activator glucose-6-phosphate (30,84, 112, 119, 129, 130) or through protein kinase-mediated phosphorylation(30, 67, 83).
The amino acids aspartate (Asp) and/or glutamate (Glu) are importanttissue-specific effectors of several PEPCs and PKcs. Potent inhibition by Aspand Glu has been reported for PEPC from the green algaS. minutum(129),soybean root nodules (130), cotyledons of germinatedR. communisseeds(119), and ripened banana fruit (84). Likewise, Glu is a potent allosteric in-hibitor of PKc from the green algaeS. minutum(86) andChlamydomonas re-inhardtii (149), cotyledons of germinatedR. communisseeds (119), and spin-ach andR. communisleaves (10, 65). In contrast, the activity of PKc from ger-minating endosperm ofR. communisshows no response to any amino acid(115). The regulatory differences in germinatingR. communisseed PKc iso-forms are consistent with the role of the endosperm as a substrate exporter andthe cotyledons as a biosynthetic tissue active in sucrose and amino acid im-port. The inhibition of some PEPCs and PKcs by Asp and/or Glu provides atight feedback control that could closely balance their overall activity with theproduction of carbon skeletons (e.g. oxaloacetate and 2-oxoglutarate) requiredfor NH4
+ assimilation and transamination reactions in tissues active in aminoacid and protein synthesis (68).
Fine control #4: subunit association-dissociationRegulatory enzymes in-variably exist as oligomers. Many can reversibly dissociate, usually in responseto effector binding. Because dissociation is often accompanied by a change inenzyme activity, it provides a mechanism for regulation (107, 142). A recent re-view (142) summarized evidence indicating that this form of fine control may beimportant in vivo for at least 13 enzymes of nonplant carbohydrate metabolism,including mammalian PFK and PK. Likewise, subunit association-disassociation has been suggested to regulate plant PFP (12, 39, 104, 135), PFKc(25, 39, 69b, 79, 146), PEPC (83, 112), and PKc (79, 124). If an enzyme can beshown to reversibly dissociate in vitro, no assumption should be made that thesame process will occur in vivo until appropriate experiments indicate that theconcentrations of enzyme and effectors are physiological. Although such rigor-ous proof is generally lacking for the aforementioned plant glycolytic enzymes,
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it appears likely that subunit association-disassociation will prove to be an im-portant facet of the fine control of plant glycolytic flux in vivo.
Fine control #5: reversible covalent modificationEnzyme regulation by re-versible covalent modification is the major mechanism whereby extracellularstimuli such as hormones or light coordinate the regulation of intermediary me-tabolism. Disulfide-dithiol and phosphorylation-dephosphorylation intercon-versions are the most important types of reversible covalent modification usedin higher eukaryote enzyme regulation (107).
Covalent modification by disulfide-dithiol exchange links photosyntheticelectron transport flow to the light regulation of several key enzymes of thechloroplast stroma via the thioredoxin system (46, 107). This process is criti-cal for the light-dependent activation and inhibition of the Calvin cycle andoxidative pentose phosphate pathways, respectively (46, 68). That disulfide-dithiol interconversion may directly participate in the fine control of plantplastidic glycolysis was suggested by the observation that pea leaf PFKp islight inactivated, an effect that could be mimicked by the addition of dithio-threitol to darkened chloroplasts (62). The discovery of a cytosolic-specificthioredoxinh suggests that this form of covalent modification could poten-tially regulate cytosolic glycolytic enzymes (107). Of note are reports that re-duced thiol groups (a) cause a sixfold activation of cytosolic NAD-GAPDHfrom roots ofMesembyanthemum crystallinum(2b), (b) elicit maximal activa-tion of tomato fruit and wheat endosperm PFP by Fru-2,6-P2 (75), and (c) pro-tect potato tuber PFP from dilution-dependent declines in intrinsic fluores-cence and activity through stabilization of the enzyme’s native 460-kDa hete-rooctameric form (113).
Protein phosphorylation-dephosphorylation plays a central role in regula-tion of cellular metabolism in all cells (67, 107). Of relevance here is the hor-monal regulation of animal glycolytic enzymes such as PK by phosphoryla-tion (48). Moreover, at least ten plant enzymes, including the cytosolic-localized sucrose phosphate synthase and PEP carboxykinase have beenshown, or strongly suggested, to be controlled by reversible phosphorylationin vivo (67, 144). As summarized in this volume (30) and elsewhere (67, 83),this mechanism of regulation appears to apply to all higher plant PEPCs ex-amined to date. Phosphorylation has also been invoked as a possible explana-tion for the disparity between enolase protein levels and activities found in de-velopingR. communisseeds andM. crystallinumroots (47, 93). Although theanaerobically induced enolase fromEchinochloa phyllopogonhas been phos-phorylated in vitro by an endogenous protein kinase, it remains to be estab-lished whether this process also occurs in vivo (100).32P-labeling and kineticstudies have determined that PKc is not phosphorylated in vivo in aerobic oranoxic germinatingR. communisendosperms (115). This is consistent with
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the absence in potato PKc of a phosphorylation consensus sequence that isfound in yeast PK (16). Although the phosphorylation site of the yeast PKdoes align with a similar motif on the deduced amino acid sequence for aR.communisdeveloping seed PKp (15), attempts to phosphorylateR. communisPKp by incubating isolated intact leucoplasts or leucoplast lysates with [γ-32P]-ATP have proved unsuccessful (FB Negm & WC Plaxton, unpublishedobservations). Nevertheless, these results do not eliminate the possibility ofplant PKc or PKp being phosphorylated in other tissues or under other physio-logical conditions. Relatively little research has been done with regard tophosphorylation of other plant glycolytic enzymes, particularly PFK and PFP.
Fine control #6: reversible associations of metabolically sequential enzymesAssociation of glycolytic enzymes into multienzyme complexes (or “meta bo-lons”) has also been proposed as a mechanism to control glycolytic flux (48, 92,107). In particular, glycolytic enzymes in muscle cells are thought to form tran-sient complexes on contractile proteins during contraction-induced stimulationof glycolysis (48, 107). Thus, restricted diffusion or even direct transfer (or“channeling”) of intermediates can occur between active sites of sequential en-zymes. In addition to the possible kinetic advantages, channeling may reducethe concentration of the channeled intermediates in the bulk solution, thus spar-ing the limited solvent capacity of the cell. Channeling may also alter enzymekinetic properties because of conformation changes occurring during binding(64, 107). Although the existence of a complete glycolytic metabolon is doubt-ful, it seems evident that physical interactions between groups of sequential gly-colytic enzymes such as PFK, ALD, and/or NAD-GAPDH can occur in vivo inanimal cells (48). The recent development of a metabolic control theory formuscle concluded that flux control coefficients for enzymes of channeled path-ways are usually larger than those in the corresponding nonchanneled pathway(74).
This mechanism of regulation also appears to be involved in the control ofseveral plant metabolic pathways, including the Calvin cycle and aromaticbiosynthesis (2a, 64, 107). Yet, few researchers have considered how it mightapply to plant glycolysis. Kinetic and physical studies of homogeneous en-zymes indicated that NADP-GAPDH, triose phosphate isomerase, and ALDinteract during glycolysis in the chloroplast ofPisum sativum(2a) and thatNAD-GAPDH and 3-PGA kinase form a specific complex in the cytosol ofgerminating mung beans (89). Furthermore, the well-characterized stimula-tion of respiration that accompanies aging of carrot and sugar beet storageroot slices was suggested to arise, in part, from an interaction between cyto-solic glycolytic enzymes (96). A subsequent study applied the techniques ofimmunoaffinity chromatography and immunoblotting to demonstrate thatALDc may specifically interact with the metabolically sequential PFKc and
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PFP in carrot storage roots (99). Evidence also indicates that ALDc specifi-cally associates with cytosolic FBPase in the gluconeogenic endosperm ofgerminatingR. communisseeds (95). Whether interactions between plant gly-colytic and gluconeogenic enzymes occur in vivo remains to be established.
OVERVIEW OF THE FINE METABOLIC CONTROL OF NONPLANT VS PLANT GLYCO-
LYSIS A notable discrepancy in the fine control of nonplant vs plant glycolysisis that the overall regulation exerted by respective enzymes involved in Fru-6-Pand PEP metabolism appears to be reversed (Figure 2). In nonplant systems suchas mammalian liver (Figure 2a), primary control of glycolytic flux to pyruvate isbelieved to be mediated by PFK, with secondary control at PK (48, 111). Activa-
PLANT GLYCOLYSIS 203
Figure 2 A comparison of the metabolite regulation of glycolytic flux from hexose-monophosphates to pyruvate in mammalian liver (A) vs the plant cytosol (B).⊕ and, denote acti-vation and inhibition, respectively. The abbreviations are as in the text.
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tion of PFK enhances the level of its product, Fru-1,6-P2, which is a potent feed-forward allosteric activator of the majority of nonplant PKsexamined to date(Figure 2a). In contrast, quantification of changes in levels ofglycolytic in-termediates that occur following stimulation of glycolytic flux in green algae(50, 68), ripening fruit (11), aged storage root slices (1),R. communiscotyle-dons (51), andChenopodium rubrumsuspension cell cultures(59) consis-tently demonstrate that plant glycolysis is controlled from the “bottom up”with primary and secondary regulation exerted at the levels of PEP and Fru-6-P utilization, respectively (Figure 2b). These findings are compatible with thepotent allosteric inhibition by PEP of many isolated plant PFKps and PFKcs(32, 39, 60, 143). Enhancement in the activity of PK or PEPC (or other en-zymes that metabolize PEP) relieves the PEP inhibition of PFK and thereby al-lows the glycolysis of hexose-P to proceed (Figure 2b). Reduced cytosolic PEPlevels also cause elevated Fru-2,6-P2 levels (and thus possible PFP activation)because a drop in PEP results in a fall in 3-PGA (these metabolites are at equi-librium in vivo) (51, 59). 3-PGA is a potent inhibitor of 6-phosphofructo-2-kinase (135). This regulatory scenario is consistent with the fact that, unlikemany nonplant PKs, no plant PK has ever been found to be activated by Fru-1,6-P2 (10, 69a, 86, 111, 115, 119, 149). In fact, homogeneous PKc from en-dosperm of germinatingR. communisseeds exhibits a kinetic reaction mecha-nism similar to that described for the Fru-1,6-P2-activated yeast PK (116). Oneconceivable benefit of bottom up regulation of glycolysis is that it permits plantsto regulate net glycolytic flux to pyruvate independent of related metabolic pro-cesses such as the Calvin cycle and sucrose-triose phosphate-starch interconver-sion.
TISSUE- AND DEVELOPMENTAL-SPECIFIC ISOZYMES OF KEY GLYCOLYTIC EN-
ZYMES In animals, glycolytic isozymes are often expressed in a tissue- and/ordevelopmental-specific manner. Such isozymes are frequently characterized byunique kinetic and regulatory properties, closely matching the cell’s metabolicrequirements in each situation. For example, different isozymes of mammalianhexokinase, PFK, and PK are separately expressed in tissues such as brain, kid-ney, liver, and muscle (48). These isozymes can be encoded by independentgenes or by one gene. In the latter case the different isozymes arise via posttran-scriptional or posttranslational processes (48).
Relatively little is known about the existence, functions, or genetic basisfor tissue- or developmental-specific isozymes of plant glycolytic enzymes.Several lines of evidence suggest that these isozymes could be important incontributing to cell-specific metabolism in higher plants. For example, organ-and developmental-specific changes in the proportion of several hexokinaseisozymes have been proposed to contribute to the regulation of hexose me-tabolism in the potato plant (121). Similarly, PEPC is encoded by a small mul-
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tigene family; the expression of each member is controlled by exogenousand/or endogenous stimuli in a tissue-specific fashion (30, 83). In addition,endosperm-, cotyledon-, and leaf-specific isozymes ofR. communisPKc thatexhibit marked differences in their respective physical and kinetic/regulatorycharacteristics have been purified and characterized (65, 115, 116, 119).These results are consistent with Southern blot studies indicating the existenceof at least six PKc genes in potato (31). Likewise, kinetic and/or immunologi-cal studies suggested that distinct isozymes of PFKp and PKp are expressed inleaves and developing seeds ofR. communis(76, 78, 110a). Different iso-forms of PFP showing unique physical and kinetic characteristics are ex-pressed during seed germination (19, 21, 24, 118), fruit ripening (147), or fol-lowing exposure to environmental stresses such as anoxia (18), or Pi starva-tion (137, 139, 140). Evidently, elucidation of plant glycolytic regulationrequires the purification and characterization of the various control enzymeson a tissue-by-tissue basis.
PRACTICAL ASPECTS OF PLANT GLYCOLYTICENZYMOLOGY
Formulation of models for glycolytic control has traditionally been based oncombining quantification of in vivo concentrations of enzymes, substrates,products, and effectors with in vitro studies of the kinetic and regulatory prop-erties of the purified key enzymes. The need to ensure that measurements ofmetabolites and enzymes are reliable and authenticated, particularly if thesevalues are to be used in any application of metabolic control analysis, was re-cently stressed (6). Nevertheless, determining what glycolytic enzymes char-acterized in vitro actually do in situ remains a problem.
Errors and Artifacts in Assays of Glycolytic Enzymes
The in vitro activities of most glycolytic enzymes are usually assessed withcoupled spectrophotometric assays based upon the differential absorbanceof NAD(P) and NAD(P)H. These assays are fraught with potential artifactsthat may arise from contamination of substrates, cofactors, and/or couplingenzymes. As a result, erroneous biochemical and physiological conclusionsconcerning plant enzymes such as PFKc, PFKp, and PFP (80) have been pub-lished. Kruger (80) recently summarized important criteria for establishingoptimal assay conditions, ensuring that the coupled assay accurately reflectsenzyme activity, and identifying artifacts resulting from contaminants that ex-ist in coupled assay components.
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Protease and Dilution Problems
Large multimeric regulatory proteins such as PFK, PFP, PK, and PEPC aresusceptible to artifactual posttranslation modifications such as proteolysis aswell as dilution-dependent alterations in their oligomeric structure. Very mi-nor proteolysis can have profound effects on the allosteric properties of aplant regulatory enzyme (110b), indicating that kinetic data obtained with par-tially degraded enzymes are of dubious significance. Moreover, many differ-ent protease inhibitors may need to be screened to identify one that effectivelysuppresses proteolysis of a specific enzyme in vitro (109, 110b). Plant glyco-lytic enzymes that have been shown to be vulnerable to partial degradation byendogenous proteases during their extraction, purification at 4°C, and/or stor-age at−20°C include developingR. communisseed PKp (103, 109) and potatotuber andB. nigra suspension cell PFP (113; ME Theodorou & WC Plaxton,unpublished data). Similarly, theN-terminal phosphorylation domain of vari-ous PEPCs is very prone to proteolysis during the enzyme’s purification in theabsence of specific protease inhibitors (30, 83). Advice concerning the diag-nosis and prevention of artifactual proteolysis of plant enzymes and the prepa-ration and use of protease inhibitors was provided by Gray (54). Monitoringenzyme subunit size is facilitated by the availability of monospecific antibod-ies against the enzyme of interest. Such antibodies allow verification of theenzyme’s subunit composition via immunoblotting of extracts prepared undercompletely denaturing conditions (84, 95, 109, 110b).
The effect of protein concentration must also be considered because en-zymes are present in vivo at far higher concentrations than they are during invitro assays. Concentration-dependence is thought to be particularly signifi-cant for enzymes important in metabolic regulation, because their structure,and hence their kinetic properties, may be affected by protein-protein interac-tions (7, 63, 113, 117, 142). The interactions between enzyme subunits thatnormally exist at the high protein concentrations prevailing in vivo can bespecifically promoted in vitro by the addition of compatible solutes such asglycerol or polyethylene glycol (PEG) to the reaction mixture. The mecha-nism involves exclusion of the protein from the binary solvent, thus increasingthe local enzyme concentration and favoring protein:protein interactions (7).The in vitro activities of rat liver PFK and PK, for instance, are enhanced bythe presence of PEG (7). Few studies have examined the influence of enzymeconcentration on the oligomeric and catalytic properties of plant glycolytic orgluconeogenic enzymes. Protein concentration does markedly influence theactivity and/or aggregation state of various PEPCs (83) andChlorella pyren-soidosaPFK (72). The presence of 10% (v/v) glycerol, a compound that stabi-lizes the native homotetrameric structure of maize leaf PEPC (112), activatedhomogeneous banana fruit PEPC by decreasing theKm(PEP) by threefold andthe Ka values for hexose-Ps by up to 7.5-fold, while greatly amplifying the
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ability of glucose-6-phosphate to relieve malate inhibition (84). Similarly, theaddition of 5% (w/v) PEG stabilized the native heterotetrameric structure ofhomogeneousR. communisendosperm PKc in dilute solutions, caused a 2.6-fold increase inVmax and 12.5- and twofold reductions inKm values for PEPand ADP, respectively, and enhanced the enzyme’s inhibition by MgATP(117). It was concluded thatR. communisPKc activity and regulation aremodified by extreme dilution in the assay medium lacking PEG as a result ofpartial dissociation of the native tetrameric enzyme (117). A rapid decline inthe intrinsic fluorescence of homogeneous potato tuber PFP occurred in re-sponse to dilution. This was paralleled by a loss in activity and a concomitantdisassociation of the nativeα4β4 heterooctamer into the inactive free subunits;dissociation was followed by random aggregation of the subunits into an inac-tive, high-molecular-weight conglomerate (113). These dilution-dependentprocesses were prevented by the presence of 5% (w/v) PEG (113). The addi-tion of PEG has also been shown to increase the substrate affinity of homoge-neous cytosolic FBPase from germinatingR. communisendosperm, throughstabilization of the enzyme’s native tetrameric structure (63). Thus, compati-ble solutes appear to aid examination of catalytic properties of regulatoryoligomers in an in vitro environment that may be closer to the conditions pre-vailing in vivo.
OTHER FUNCTIONS FOR GLYCOLYTIC ENZYMES
Several animal proteins with functions not related to glycolysis have turnedout to be encoded by genes identical to those for glycolytic enzymes. For ex-ample, (a) yeast hexokinase exhibits protein kinase activity; (b) the sequenceof the growth factor neuroleukin is identical to that of mouse hexose-phosphate isomerase; (c) enolase is a structural protein of the eye lens and inyeast is a heat shock protein that may confer thermotolerance; (d) themonomeric form of human NAD-GAPDH is a uracil DNA glycosylase, an en-zyme involved in DNA repair; (e) yeast PK is involved in cell cycle control;and (f) the monomeric form of mammalian muscle-type PK is a thyroidhormone-binding protein, and the conversion of inactive PK monomer to ac-tive PK tetramer is promoted by its allosteric activator, Fru-1,6-P2 (48). Thussome plant glycolytic enzymes may also have nonglycolytic functions in vivo.Enolase fromE. phyllopogonis induced by anoxia as well as by cold and heatshock and may act as a general stress protein that protects cellular componentsat the structural level (49, 132). Furthermore, immunolocalization studies in-dicated that in addition to their cytosolic and chloroplastic compartments, 3-PGA kinase, NAD-GAPDH, and ALD are located in the nucleus of leaf meso-phyll cells ofP. sativum(2c). It was postulated that these enzymes are nuclear
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proteins with secondary roles not directly related to their enzymatic functionsin carbohydrate metabolism.
CONCLUDING REMARKS
Despite the remarkable progress in analysis of the organization and regulationof plant glycolysis, many complex issues remain. Developments in moleculargenetics have led to exciting new strategies. Five years ago, little was knownabout the primary structure of the key regulatory enzymes. Since then, the pri-mary structures of the subunit(s) of PFP, PKc, PKp, and PEPC have been de-duced from the respective cDNA sequences. This information in turn has pro-vided new insights into molecular mechanisms of catalysis and regulation ofthese proteins. Isolation of cDNAs for enzymes has also permitted the quanti-fication of specific mRNAs, analyses of their gene expression, and elucidationof requirements for import into plastids. Nuclear genes encoding several ofthese enzymes have been sequenced, and their promoter regions are beingcharacterized. Transformation of plants with sense or antisense cDNA con-structs provides new information on the functions and control of these en-zymes in vivo. In addition, there is a practical interest in genetically modify-ing crops to over- or underexpress glycolytic enzymes in order to redirect theflux of photosynthate into economically important endproducts such as starch,triglycerides, and protein.
Although significant advances in our understanding of plant glycolysis arefacilitated by molecular genetics, it is imperative that future workers continueto examine physiological aspects of glycolysis. We also require further purifi-cation and characterization of the cell- and/or developmental-specific iso-zymes that regulate cytosolic and plastidic glycolytic flux. Not only does en-zyme purification lead to the production of crucial tools for the molecular bi-ologist (e.g. antibodies), but together with the appropriate kinetic andphysiological studies it also serves to establish the control mechanisms thatregulate glycolysis in vivo. Only when these basic mechanisms are under-stood will the ongoing efforts of plant molecular biologists be fully realized.
ACKNOWLEDGMENTS
Research in my laboratory is supported by grants from The Natural Sciencesand Research Council of Canada. I also thank Drs. H. Ashihara, D. T. Dennis,N. J. Kruger, T. C. Fox, B. L. Miki, and M. E. Rumpho for sending me theirunpublished manuscripts and preprints. I am indebted to the current membersof my laboratory as well as to Drs. S. D. Blakeley, M. Kuzma, and J. Rivoalfor critical reading of the manuscript. The many contributions and collabora-tions provided by my fellow plant biology colleagues at Queen’s Universityare gratefully acknowledged.
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Any Annual Reviewchapter, as well as any article cited in anAnnual Reviewchapter,may be purchased from the Annual Reviews Preprints and Reprints service.
1-800-347-8007; 415-259-5017; email: [email protected]
PLANT GLYCOLYSIS 209
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Annual Review of Plant Physiology and Plant Molecular Biology Volume 47, 1996
CONTENTS
REFLECTIONS OF A BIO-ORGANIC CHEMIST, Jake MacMillan 1HOMOLOGY-DEPENDENT GENE SILENCING IN PLANTS, P. Meyer, H. Saedler 23
14-3-3 PROTEINS AND SIGNAL TRANSDUCTION, Robert J. Ferl 49DNA DAMAGE AND REPAIR IN PLANTS, Anne B. Britt 75
PLANT PROTEIN PHOSPHATASES, Robert D. Smith, John C. Walker 101THE FUNCTIONS AND REGULATION OF GLUTATHIONE S-TRANSFERASES IN PLANTS, Kathleen A. Marrs 127
PHYSIOLOGY OF ION TRANSPORT ACROSS THE TONOPLAST OF HIGHER PLANTS, Bronwyn J. Barkla, Omar Pantoja 159THE ORGANIZATION AND REGULATION OF PLANT GLYCOLYSIS, William C. Plaxton 185LIGHT CONTROL OF SEEDLING DEVELOPMENT, Albrecht von Arnim, Xing-Wang Deng 215DIOXYGENASES: Molecular Structure and Role in Plant Metabolism, Andy G. Prescott, Philip John 245PHOSPHOENOL PYRUVATE CARBOXYLASE: A Ubiquitous, Highly Regulated Enzyme in Plants, Raymond Chollet, Jean Vidal, Marion H. O'Leary 273XYLOGENESIS: INITIATION, PROGRESSION, AND CELL DEATH, Hiroo Fukuda 299
COMPARTMENTATION OF PROTEINS IN THE ENDOMEMBRANE SYSTEM OF PLANT CELLS, Thomas W. Okita, John C. Rogers 327WHAT CHIMERAS CAN TELL US ABOUT PLANT DEVELOPMENT, Eugene J. Szymkowiak, Ian M. Sussex 351THE MOLECULAR BASIS OF DEHYDRATION TOLERANCE IN PLANTS, J. Ingram, D. Bartels 377BIOCHEMISTRY AND MOLECULAR BIOLOGY OF WAX PRODUCTION IN PLANTS, Dusty Post-Beittenmiller 405
ROLE AND REGULATION OF SUCROSE-PHOSPHATE SYNTHASE IN HIGHER PLANTS, Steven C. Huber, Joan L. Huber 431STRUCTURE AND BIOGENESIS OF THE CELL WALLS OF GRASSES, Nicholas C. Carpita 445
SOME NEW STRUCTURAL ASPECTS AND OLD CONTROVERSIES CONCERNING THE CYTOCHROME b 6f COMPLEX OF OXYGENIC PHOTOSYNTHESIS, W. A. Cramer, G. M. Soriano, M. Ponomarev, D. Huang, H. Zhang, S. E. Martinez, J. L. Smith 477CARBOHYDRATE-MODULATED GENE EXPRESSION IN PLANTS, K. E. Koch 509
CHILLING SENSITIVITY IN PLANTS AND CYANOBACTERIA: The Crucial Contribution of Membrane Lipids, I. Nishida, N. Murata 541
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THE MOLECULAR-GENETICS OF NITROGEN ASSIMILATION INTO AMINO ACIDS IN HIGHER PLANTS, H.-M. Lam, K. T. Coschigano, I. C. Oliveira, R. Melo-Oliveira, G. M. Coruzzi 569MEMBRANE TRANSPORT CARRIERS, W. Tanner, T. Caspari 595LIPID-TRANSFER PROTEINS IN PLANTS, Jean-Claude Kader 627REGULATION OF LIGHT HARVESTING IN GREEN PLANTS, P. Horton, A. V. Ruban, R. G. Walters 655THE CHLOROPHYLL-CAROTENOID PROTEINS OF OXYGENIC PHOTOSYNTHESIS, B. R. Green, D. G. Durnford 685
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