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OHLROGGE& JAWORSKIRegulationof Fatty Acid SynthesisAnnu.Rev. Plant Physiol. Plant Mol. Biol. 1997. 48:109–136Copyright© 1997by AnnualReviewsInc. All rightsreserved

REGULATION OFFATTY ACIDSYNTHESIS

John B. OhlroggeDepartment of Botany andPlantPathology, MichiganState University, East Lansing,Michigan48824

JanG. JaworskiChemistry Department, Miami University, Oxford, Ohio45056

KEY WORDS: acetyl-CoA carboxylase,oil seeds,triacylglycerol, feedback,metabolic control

ABSTRACT

All plant cells produce fatty acids from acetyl-CoA by a common pathwaylocalized in plastids. Although the biochemistry of this pathway is now wellunderstood, much less is known about how plants control the very differentamounts and types of lipids produced in different tissues. Thus, a centralchallengefor plant lipid researchis toprovideamolecular understandingof howplantsregulatethemajor differencesin lipid metabolismfound, for example, inmesophyll, epidermal, ordevelopingseedcells.Acetyl-CoA carboxylase(AC-Case) is onecontrol point that regulates ratesof fatty acid synthesis.However,the biochemical modulators that act on ACCaseand the factors that in turncontrol thesemodulatorsarepoorly understood.In addition, li ttleisknownabouthowtheexpressionof genesinvolved in fatty acid synthesisis controlled.Thisreviewevaluatescurrent knowledgeof regulationof plantfatty metabolismandattemptsto identify themajor unanswered questions.

CONTENTSINTRODUCTION..................................................................................................................... 110

Compartmentalizatonand theNeedfor Interorganelle Communication............................ 111OVERVIEW OF FATTY ACID SYNTHESIS:THE ENZYME SYSTEMS......................... 112BIOCHEMICAL CONTROLS:WHICH ENZYMESREGULATE FATTY ACID

SYNTHESIS?................................................................................................................... 115Analysis ofRate-Limiting Steps........................................................................................... 115

Is Acetyl-CoA CarboxylaseRate-Limiting? ........................................................................ 117Other Potential Rate-Limiting Steps.................................................................................... 118

FEEDBACK REGULATION ................................................................................................... 119WhatIs the Feedback System?............................................................................................ 120Control of Substrateand Cofactor Supply .......................................................................... 121

WHAT DETERMINESHOW MUCH OIL IS PRODUCED BY A SEED?........................... 122Control byFatty AcidSupply (Source)............................................................................... 123Control byDemand (Sink)................................................................................................... 124

WHAT HAVE WE LEARNED FROMTRANSGENIC PLANTS AND MUTANTS?......... 126Fatty Acid Composition of SeedsCan Be MoreRadically AlteredThan Other Tissues..... 126Manipulationof Oil Quantity .............................................................................................. 126ManyEnzymesof Fatty AcidSynthesis ArePresentin Excess........................................... 127

CONTROL OFGENEEXPRESSION..................................................................................... 128MultigeneFamilies.............................................................................................................. 128Promoter Analysis............................................................................................................... 128WhatControls Promoter Activity of FASGenes?............................................................... 130

SUMMARY AND PERSPECTIVES....................................................................................... 131

INTRODUCTION

All cells in a plantmustproducefatty acids,andthis synthesismustbetightlycontrolledto balancesupplyanddemandfor acyl chains.For mostplantcells,this meansmatchingthe level of fatty acidsynthesisto membranebiogenesisandrepair.Dependingon thestageof development, time of theday,or rateofgrowth, theseneedscanbe highly variable,and thereforeratesof fatty acidbiosynthesis must be closely regulatedto meet thesechanges.In somecelltypes,the demandsfor fatty acid synthesis aresubstantially greater.Obviousexamplesareoil seeds,which duringdevelopmentcanaccumulateasmuchas60% of their weight as triacylglycerol.Another exampleis epidermalcells,which traffic substantial amounts of fatty acidsinto surfacewax andcuticularlipid biosynthesis.In leek, eventhoughthe epidermisis lessthan4% of thetotal freshweight of the leaf, asmuchas15% of the leaf lipid is found in asingle wax component,a C31 ketone(52). How do cells regulatefatty acidsynthesisto meet thesediverseand changeabledemandsfor their essentiallipid components?We are only beginningto understandthe answerto thisquestion.

In this review,we focuson questionsaboutregulationof fatty acidsynthe-sis.Severalreviewsin recentyearsprovideexcellentoverviewsof fatty acidsynthesis,and we do not duplicatethoseefforts exceptwherenecessaryforclarity. An excellentandcomprehensivereviewof plantfatty acidmetabolismhasrecentlybeenpublished(33), and severalother recentreviewscoveringplant lipid metabolism, molecularbiology and biotechnological aspectsofplantfatty acidshavealsoappeared(10,44,57,64,97,105).Becausethereismuchyet to be learnedaboutregulationof this essentialandubiquitouspath-

110OHLROGGE &JAWORSKI

way,weoftendwell onwhatis unknown. Thisapproachis intendedto providethereaderwith a clearersenseof themajorquestionsof fatty acidmetabolismthat remainto be answeredbeforea reasonableunderstandingof this regula-tion is achieved.

Compartmentalizaton andtheNeedfor InterorganelleCommunication

Overall fatty acid synthesis,and consequentlyits regulation, may be morecomplicatedin plantsthanin any otherorganism(Figure1). Unlike in otherorganisms,plant fatty acid synthesisis not localizedwithin the cytosol butoccursin anorganelle,theplastid. Althoughaportionof thenewlysynthesizedacyl chainsis thenusedfor lipid synthesis within theplastid (theprokaryoticpathway),a majorportion is exportedinto thecytosolfor glycerolipidassem-bly at theendoplasmicreticulum(ER) or othersites(theeukaryoticpathway)

Figure 1 Simpli fied schematic of overall flow of carbon through fatty acid andlipid metabolismin ageneralizedplantcell. Becauseacyl chainsareused ineverysubcellular compartmentbut areproducedalmostexclusively in theplastid, interorganellarcommunicationmustbalancetheproduc-tionanduseof these acylchains.

REGULATION OFFATTY ACID SYNTHESISI 111

(82, 100). In addition,someof the extraplastidialglycerolipidsreturn to theplastid,which resultsin considerableintermixing betweentheplastidandERlipid pools.Both the compartmentalizationof lipid metabolismandthe inter-mixing of lipid intermediatesin thesepoolspresentspecialrequirementsfortheregulationof plantfatty acidsynthesis.Foremostis theneedfor regulatorysignalsto crossorganellarboundaries.Becausefatty acidsareproducedin theplastid,but areprincipally esterifiedoutsidethis organelle,a systemfor com-municatingbetweenthesourceandthesinksfor fatty acidutilization is essen-tial. The natureof this communication and the signal moleculesinvolvedremain anunsolvedmystery.

In addition to havingregulatorymechanismsthat control overall levelsofindividual lipids, plantsmustalsoregulateratesof fatty acid synthesis undercircumstances in which therearelargeshifts inthedemandby majorpathwaysof lipid metabolismlocated both in and out of the plastid.For example,considerthe consequenceof the Arabidopsismutantact1,which hasa muta-tion in the plastidialacyl transferasethat directsnewly synthesizedfatty acidinto thylakoid glycerolipids (49). This mutanthas the remarkableability tocompensatefor lossof theprokaryoticpathwayby divertingnearlyall thefattyacidsinto thephospholipidsof theeukaryoticpathway.It thenfunnelsunsatu-rateddiacylglycerolfrom theER phospholipids backinto theplastidial lipids,with only minor changein the overall composition of either of thesemem-branes.

OVERVIEW OFFATTY ACID SYNTHESIS:THE ENZYMESYSTEMS

The simplestdescription of the plastidial pathwayof fatty acid biosynthesisconsistsof two enzymesystems:acetyl-CoAcarboxylase(ACCase)andfattyacid synthase(FAS). ACCasecatalyzesthe formationof malonyl-CoA fromacetyl-CoA, and FAS transfersthe malonyl moiety to acyl carrier protein(ACP) and catalyzesthe extensionof the growing acyl chainwith malonyl-ACP.In nature,ACCaseoccursin two structurally distinct forms:amultifunc-tional homodimeric proteinwith subunits >200kDa, anda multisubunitAC-Caseconsisting of four easilydissociatedproteins.Ideas aboutthe structureofplant ACCaseshaveundergoneconsiderableevolution in the pastfew years.Until 1992, most researchershad concludedthat plant ACCasewas a large(>200 kDa) multifunctional proteinsimilar to that of animalandyeast.Thistype ofenzyme had beenpurified fromseveral dicotand monocotspecies, andpartial cDNAclones wereavailable. However,in 1993, Sasakiandco-workersdemonstratedthat the chloroplastgenomeof pea encodesa subunit of an


ACCasewith structurerelated to the β subunit of the carboxyltransferasefoundin themultisubunitACCase ofEscherichiacoli (88).A flurry of interestin this topic hasresultedin rapidextensionof theseinitial studies.It hasnowbeenclarified that dicotsandmostmonocots haveboth forms of ACCase,a>200-kDa homodimeric ACCase(probably localized in the cytosol) and aheteromericACCasewith at leastfour subunitsin theplastid(2, 48, 80). It isthe heteromericplastid form of the ACCasethat providesmalonyl-CoA forfatty acidsynthesis.

In addition to the β-carboxyltransferasesubunit characterizedby Sasaki,clonesarenowavailablefor thebiotin carboxylase(94),biotin carboxylcarrierprotein(BCCP)(17), andα-subunitof thecarboxyltransferase(95). The foursubunitsareassembledinto a complexby gel filtration with a sizeof 650–700kDa. However, thesubunitseasily dissociate such that ACCase activity is lost,which accountsfor thefailure to identify the multisubunitform of ACCaseformany years.Becausethe β–carboxyltransferase(β-CT) subunit is plastomeencoded,whereastheotherthreesubunitsarenuclearencoded,assemblyof acompletecomplexrequirescoordination of cytosolicandplastid productionofthe subunits. Little is know aboutthis coordinationandassembly.However,several-foldoverexpressionand antisenseof the biotin carboxylasesubunitdoes notalter the expressionof BCCP, whichsuggests thata strict stoi-chiometricproductionof subunitsmay notbe essential(92).

The structureof ACCasein Gramineaespeciesis different in that thesespecieslacktheheteromericform of ACCaseandinsteadhavetwo typesof thehomodimeric enzyme(89). An herbicide-sensitive form is localizedin plas-tids,anda resistantform is extraplastidial. BecausebothGramineaeanddicotplastidFASareregulatedby light anddependentonACCaseactivity, it will beof considerableinterestto discoverwhetherthetwo structurallyvery differentforms ofACCasearesubjectto the sameor different modesof regulation.

The structureof FAS hasmanyanalogiesto ACCasestructure.In nature,bothmultifunctionalandmultisubunit formsof theFASarefound.In addition,asin thecaseof ACCase,theplastidial FAS foundin plantsis very similar totheE. coli FAS andis theeasilydissociatedmultisubunitform of theenzyme.It is now well establishedin both plants and bacteriathat the initial FASreactionis catalyzedby 3-ketoacyl-ACPIII (KAS III), which resultsin thecondensationof acetyl-CoAandmalonyl-ACP (37,106).Subsequentconden-sationsarecatalyzedby KAS I andKAS II. Beforea subsequentcycleof fattyacidsynthesis begins,the3-ketoacyl-ACPintermediateis reducedto thesatu-ratedacyl-ACPin theremainingFAS reactions,catalyzedsequentiallyby the3-ketoacyl-ACP reductase, 3-hydroxyacyl-ACP dehydrase,and the enoyl-ACP reductase.


In additionto ACCaseandFAS, discussionof the regulationof fatty acidsynthesismustalsoconsiderthosereactionsthatprecedeandfollow thesetwoenzymesystems.It is not fully understoodwhich reactionsareresponsible forproviding acetyl-CoAto ACCase,but extensiveexperimentswith leaf tissueindicatethatacetyl-CoAsynthetasecanrapidly convertacetateto acetyl-CoA,andthereforefree acetatemay be an importantcarbonsource(83–85).Otherpossiblesourcesof acetyl-CoAincludesynthesisfrom pyruvateby a plastidialor mitochondrial pyruvatedehydrogenase(14, 51) or citrate lyase(61). Be-causeacetyl-CoAis a centralmetabolite requiredfor synthesisof isoprenoids,amino acids, andmany other structures, it is likelythat morethan one pathwayprovidesacetyl-CoAfor fatty acid synthesis,and the sourcemay dependontissueand developmental stage(40).

The final productsof FAS areusually16:0- and18:0-ACP,and the finalfatty acidcomposition of aplant cellis in largepartdeterminedby activitiesofseveralenzymesthatusetheseacyl-ACPsat theterminationphaseof fatty acidsynthesis.Therelativeactivitiesof theseenzymesthereforeregulatetheprod-uctsof fatty acidsynthesis.Stearoyl-ACPdesaturasemodifiesthe final prod-uct of FAS by insertion of a cis doublebondat the 9 position of the C18:0-ACP. Reactionsof fatty acidsynthesisare terminatedby hydrolysisor transferof theacyl chainfrom theACP. Hydrolysis is catalyzedby acyl-ACPthioes-

Figure 2 Overexpression of the California Bay medium-chain thioesterase(MCTE) results inactivation of β-oxidationaswell asincreasedexpressionof theenzymesof fatty acidbiosynthesis.


terases,of which therearetwo maintypes:onethioesteraserelativelyspecificfor 18:1-ACPand a secondmore specific for saturatedacyl-ACPs(24, 39).Fatty acids that have beenreleasedfrom ACPs by thioesterasesleave theplastid andenterinto theeukaryoticlipid pathway(82),wheretheyareprimar-ily esterifiedto glycerolipids on the ER. In someplants,thioesteraseswithspecificity for shorter chainlength acyl-ACPs arecapableof prematurelyterminatingfatty acid synthesis,therebyregulatingthe chain length of theproductsincorporatedinto lipids. For example,thioesterasesspecificfor me-dium-chainfatty acidsare responsiblefor high levels of C10 and C12 fattyacidsin triacylglycerolsof California Bay,Cuphea,andotherspecies(19,23,71).Acyl transferasesin theplastid, incontrastto thioesterases,terminatefattyacid synthesis by transesterifyingacyl moietiesfrom ACP to glycerol, andthey arean essentialpart of the prokaryoticlipid pathwayleadingto plastidglycerolipidassembly(82, 100).

BIOCHEMICAL CONTROLS: WHICH ENZYMESREGULATE FATTY ACID SYNTHESIS?

Control in metabolic pathways is not a dictatorship, nor is it an Atheniandemocracy; rather, it is a halfway housein which (to quote Orwell) “all pigsareequal butsomearemoreequal thanothers” (101).

A numberof approacheshavebeenusedby biochemists to identify whereregulationoccursin a pathway.Oneapproachdependson examination of thein vitro propertiesof enzymesof a pathway.Enzymesthat havelow activityrelativeto othermembersof thepathwayarefrequentlyconsideredaspoten-tially ratelimit ing. Otherproperties,suchaschangingactivitiesduringdevel-opmentalregulationof thepathway,or influence(activationor inhibition) onthe enzymeby intermediatesof the pathwaycanprovideadditional evidencetoward identification ofcontrolpoints.

Analysis of Rate-Limiting Steps

ACCaseis frequently consideredthe first committed step in the fatty acidbiosynthetic pathway.In animals(30) andin yeast(111),thereis evidenceforACCaseasa majorregulatoryenzymein fatty acidproduction. Therefore,forsome time this enzymewasalsoproposedto beratedetermining forplant fattyacidbiosynthesis.Severallinesof evidencesupportedthis suggestion: Acetateor pyruvatewereincorporatedinto acetyl-CoAin thedarkby isolatedchloro-plasts,but malonyl-CoA and fatty acidswere formed only in the light (58).Thus, the light-dependentstepof fatty acid synthesisappearedto be at the


ACCasereaction.Eastwell& Stumpf (26) found that chloroplastandwheatgerm ACCasewere inhibited by ADP and suggestedthis may accountforlight-dark regulationof theenzyme.Nikolau & Hawke(63) characterizedthepH, Mg, ATP, andADP dependenceof maizeACCaseactivity andconcludedthat changesin theseparametersbetweendark and light conditions couldaccountfor increasedACCaseactivity upon illuminationof chloroplasts.Fi-nally, ACCaseactivity and protein levels are coincidentwith increasesanddecreasesin oil biosynthesis indevelopingseeds (e.g. 80,107;but see 40a).

However,in vitro approachesarelimi ted becausethey showonly that theenzymehasin vitro propertiesconsistentwith control,not thatit actuallydoescontrol in vivo metabolism. Therearenumerousexamplesof enzymeswhosebiochemicalpropertiesimply controlbutwhich havesubsequentlybeenfoundto havelittl e role in controlling flux in vivo (96). Thus,thereis no obligatorylink betweenenzymecharacteristicsobservedin vitro andregulatoryproper-tiesin vivo.

Thesitesof metabolic controlof a pathwaycanbemorereliably identifiedby examinationof in vivo propertiesof enzymes.Although it is often techni-cally difficult, examiningtheconcentrationsof thesubstratesandproductsofeachenzymaticstepin a pathwayprovidesinformationon which reactionsareat equilibrium andwhich aredisplacedfrom equilibrium. This information isimportantbecauseanessentialfeatureof almostall regulatoryenzymesis thatthe reaction that they catalyzeis displaced farfrom its thermodynamic equilib-rium. This is a requirementfor regulationbecauseif anenzymehassufficientactivity in vivo to bring its reactionto or nearequilibrium, thenchangesin theactivity of theenzymewill leadto no netchangein flux throughthepathway(81). On thebasisof this criterion, it is possibleto classifyenzymeswithin apathwayaseithernonregulatoryor potentially regulatorybasedsimply on anexaminationof thepoolsof substratesandproductsfor eachreaction.Furtherevidenceof actualcontrolcanbeobtainedby examiningchangesin pool sizeswhenflux throughthe pathwayis altered.It canbe shownboth theoreticallyand experimentallythat when flux through the pathway is reducedat theregulatoryreaction,thesubstratepool for theregulatorystepwill increaseandtheproduct poolwill usually decrease.Thus,animportant experimentalproce-durefor identifying regulatorystepsis to examinechangesin thepool sizesofintermediateswhenflux throughthe pathwayis altered.

How canthese principles be applied to evaluating control of plantfatty acidbiosynthesis? Most of the substratesand intermediatesof plant FAS are at-tachedto acyl carrierprotein(ACP). Analysisof acyl-ACPsis aidedbecausethe chain length of fatty acids attachedto ACP alters the mobility of theproteinin nativeor ureaPAGEgels.Becauseof thesealterationsin mobility,


most of the acyl-ACP intermediates of fatty acid synthesiscan be resolved,andwhentransferredto nitrocellulose,antibodiesto ACP canprovidesensi-tive detectionatnanogramlevels.Althoughtheacyl-ACPintermediateshaveahalf life in vivo of only a few seconds(37, 99), by rapidly freezingtissuesinliquid nitrogenit hasbeenpossibleto determinetherelativeconcentrationsoffree, nonacylatedACPs and of the individual acyl-ACPs.Analysis of acyl-ACP poolshasbeenusedto studyregulationof FAS in spinachleaf andseed(72) in chloroplasts (74) in developing castorseeds(73) and in tobaccosus-pensioncultures(93).

Theinitial examinationof thecomposition of theacyl-ACPpoolsprovidedinformationaboutthepotential regulatoryreactionsin plantfatty acidbiosyn-thesis.The varioussaturatedacyl-ACP intermediatesbetween4:0 and 14:0occur in approximatelyequalconcentrations.Becausethe 3-ketoacyl-ACPs,enoyl-ACPs,or 3-hydroxyacyl-ACPs,which aresubstratesfor thetwo reduc-tasesanddehydrasereactions,werenot detected,it is likely that thesereac-tions arecloseto equilibriumandthat the in vivo activitiesof theseenzymesare in excess.Thus it is not likely that theseenzymesare regulatory. Incontrast,the concentrationof acetyl-ACPwas considerablyabove thatofmalonyl-ACP. This resultsuggeststhat the acetyl-CoAcarboxylasereaction,which hasan equilibrium constantslightly favoring malonyl-CoA formation,is significantlydisplaced from equilibriumand thereforepotentially regulatory(37, 72, 74). Thecondensingenzymescanalsobeconsidereddisplacedfromequilibrium becauseof the concentrationof malonyl-ACP and the saturatedacyl-ACPs.

Is Acetyl-CoACarboxylaseRate-Limiting?

To obtainmore information on sitesof regulation,the changesin pool sizeswhen flux throughthe fatty acid biosynthetic pathwaychangeswere exam-ined. The rateof spinachleaf fatty acid biosynthesisin the dark is approxi-mately one sixththerateobservedin thelight (9). In thelight, thepredominantform of ACP wasthefree,nonacylatedform, whereasacetyl-ACPrepresentedabout 5–6% of the total ACP (72). In the dark, the level of acetyl-ACPincreasedsubstantially with a correspondingdecreasein free ACP, suchthatacetyl-ACPwasnow the predominantform of ACP. In similar experiments,when chloroplastsare shifted to the dark, malonyl-ACP and malonyl-CoAdisappearwithin a few seconds,andacetyl-ACPlevelsincreaseovera periodof severalminutes. The rapid decreasein malonyl-ACP and malonyl-CoAwhenfatty acidsynthesisslows,togetherwith the increasein acetyl-ACPandlack of changein otherintermediate acyl-ACPpoolsall leadto theconclusion


that ACCaseactivity is themajordeterminantof light/dark control over FASrates inleaves.

The aboveexperimentson acyl-ACPandacyl-CoApoolshavebeencarriedout with dicot plants.Gramineaespeciessuch as maize and wheat have asubstantially different (homodimeric)structureof ACCase(describedabove).Is thishomomericenzymealsoinvolvedin regulatingleaf fatty acidsynthesis?This questionwasaddressedby a differentapproachtowardevaluatingmeta-bolic control.Pageet al (69) took advantageof thesusceptibility of maizeandbarleyplastidACCaseto theherbicidesfluaxifop andsethoxidim. Whenchlo-roplastsor leaveswere incubatedwith herbicidesandradiolabeledacetate,aflux control coefficientof 0.5 to 0.6 wascalculatedfor acetateincorporationinto lipids. Flux control coefficientsof this magnitude indicatestrongcontrolby the ACCasereactionover fatty acid synthesis (18, 41). Thus, in a widevariety of speciesandtissues,both in vivo andin vitro experimentspoint toACCaseas a majorregulatorypoint for plantfatty acid synthesis.

OtherPotential Rate-Limiting Steps

Although the bestevidenceavailableso far implicatesACCaseactivity asaprimary determinantof fatty acid synthesis rates,the conceptof a singlerate-determiningreaction is clearlyan over-simplification. Control over flux isfrequentlysharedby morethanoneenzymein apathway,andfurthermore,therelativecontributions of enzymesto control arevariable.For example,underlow irradience,only ADP-glucosepyrophosphorylaseexertedstrongcontrolover CO2 incorporationinto starch,whereasunderhigh irradience,phospho-glucoisomerase and phosphoglucomutasealsoexerted control(101).A similarsituationmay apply to fatty acid synthesis.For example,chloroplastsincu-batedin thelight havehigh ratesof fatty acidbiosynthesis,but theseratescanbe further stimulated by addition of Triton-X-100. Under theseconditions,malonyl-CoA and malonyl-ACP levels increasefivefold or greater,but theincreasein fatty acidsynthesisis only 30–80%(74).Onepossibleimplicationof this observationis that underhigh flux conditions,biochemicalregulationof ACCasemaybemosteffectivein decreasingfatty acidsynthesisrates,anda large increase in ACCaseactivity mayyield a relativelysmallincrease intheoverallflux throughthepathway.Theseresultsfurther suggestthatunder highflux conditions through ACCase,thecondensingenzymesor otherfactorsmaybeginto limit FAS rates.All thesaturatedacyl-ACPintermediatesof fatty acidsynthesisfrom 4:0 to 14:0 aredetectedin approximatelyequalquantities inextracts fromleaf orseed. This suggests that activitiesof all the KASisoformsareapproximatelyequal.Furthermore,it suggeststhat if therateof anyoneof


theKAS isoformswasto increase,theeffectwould besmall,becausetheotherKAS isoformswould belimit ing. Evidenceto supportthis hascomefrom theincreasedexpressionof spinachKAS III in tobaccounderthe control of theCaMV 35S promoter(104). KAS III activity was increased20- to 40-fold,with no effecton either thequantity or compositionof thefatty acids.Analysisof theACP andacyl-ACPintermediatesrevealedthat themajor form of ACPin the transgenicleaveswas4:0-ACP,which suggestedthat KAS I wasnowthe limi ting condensingenzymein this tissue,andpreventedany significantchange inthe overallflux throughthe pathway.

FEEDBACK REGULATION

Most biochemicalpathwaysarecontrolledin part by a feedbackmechanismwhich fine-tunesthe flux of metabolitesthroughthe pathway.Whenevertheproductof a pathwaybuildsup in thecell to levelsin excessof needs,theendproduct inhibits the activity of the pathway. In most casesthis inhibitionoccursat a regulatoryenzymewhich is often the first committed stepof thepathway.When the activity of the regulatoryenzymeis reduced,all sub-sequentreactionsare also slowed as theirsubstratesbecomedepletedbymass-action.Becauseenzymeactivity can be rapidly changedby allostericmodulators, feedbackinhibition of regulatoryenzymesprovidesalmostinstan-taneouscontrolof the fluxthroughthepathway.

Evidencefrom leaves,isolatedchloroplasts, and suspension culture cellsstronglyimplicatesACCaseasa majorpoint of flux control.If ACCaseis thevalvethatdeterminesflow of carbontowardfatty acids,thekey questionnowbecomes“How is this enzyme’s activity regulated?”In animalsandyeast,ithas long been considered that fatty acidsynthesis is partly controlled byfeedbackon ACCaseby long-chainacyl-CoAs.Sinceacyl-CoAsareoneendproductof the FAS pathway,they weretestedin vitro andfound to stronglyinhibit ACCaseatsubmicromolar concentrations(29).Althoughthis inhibitionseemslogical, it has beencalled into question by the discoverythat acyl-CoA–binding proteinsexist at high concentrationsin the cytosol of animals(76), yeast(46), and plants(28, 34). Becausetheseproteinshaveextremelyhigh affinity for acyl-CoAs,the concentrationof free acyl-CoA in the cyto-plasm may be only nanomolar, a level unlikely to inhibit ACCase.Thesestudiesfurtheremphasizethedifficulty in extrapolating in vitro enzymestud-ies toin vivo conditions.

The in vitro analysesof plant ACCasehaveindicatedthat pH, Mg, andATP/ADP canexplainmuchof thelight-darkmodificationsof ACCaseactiv-ity observedin leaves(63). However,other modifications in the enzyme’s


activity are apparentlyinvolved. For example,light activationof fatty acidsynthesisin chloroplasts doesnot requireATP synthesis by light (58) andhasbeenreportedto be stimulated by photosystem(PS) I but not PS II activity(70). In addition,Sauer& Heise (90)reportedthatACCaseactivity washigherafter rapid lysis and assayof light-incubatedchloroplaststhan after similartreatmentof dark-incubatedchloroplasts. The initial ACCaseactivity fromchloroplastsincubatedin thelight is three-to fourfold higherthanfrom eitherchloroplastsincubated5 min in darkor from chloroplasts incubatedin thelightfollowed by 2 min in dark(K Nakahira& JB Ohlrogge,unpublishedinforma-tion). After lysis, ACCaseactivity from the dark-incubatedchloroplasts in-creaseduntil it reachedlight levels within 3–4 min. Thus, ACCaseactivityappearsto be transiently inactivated or inhibited in dark-incubatedchloro-plasts.Becausethelysisof chloroplasts into assaybufferwouldresultin overa100-fold dilution of stromalcontents,any differencesin the ACCaseactivitycannot be attributedto contributions by the stromacontents(suchas ATP)during the assay.At presentthere is not an explanationfor this transientinactivation. Animal ACCaseis known to be inactivatedby phosphorylation(43). However,additionof proteinkinaseandphosphataseinhibitors to chlo-roplastlysatesdid not substantially alter thepatterns.

In an effort to examineunderin vivo conditionswhetherfeedbackinhibi-tion of fatty acid synthesis occursin plants,Shintani& Ohlrogge(93) addedexogenouslipids to tobaccosuspension cultures.The incorporation of 14C-acetateinto fatty acids(but not sterols)wasrapidly decreasedby theadditionof lipids to the cultures.Thus, the cells apparentlyhavea mechanismthatsensesthe supply of fatty acids and responds bydecreasingthe de novoproductionof morefatty acid.Furthermore,examination of thepoolsof acyl-ACP intermediatesindicatedincreasesin acetyl-ACP,decreasesin long-chainacyl-ACPs,andno changein medium-chainacyl-ACPs.Theseresponsesareidenticalto thoseobservedwhenfatty acidsynthesis is decreasedby shiftingleavesor chloroplaststo the dark and are expectedif ACCaseactivity isdecreased.Therefore,ACCaseregulationcanalsoaccountfor thedecreaseoffatty acid production in responseto exogenouslipids. Immunoblot analysisindicated that ACCaseprotein levels did not changeduring the feedbackinhibition, indicating that biochemicalcontrolsare primarily responsible forthe reduced ACCaseandfatty acidsynthesisactivity.

What Is theFeedbackSystem?

The first endproductsof plastid fatty acid synthesisarethe long-chainacyl-ACPs,andthereforetheseseemlogical candidatesfor feedbackregulatorsof


fatty acidsynthesis.However,whenfatty acidsynthesisslows inthedarkor inresponseto exogenouslipids, the long-chainacyl-ACP pools drop signifi-cantly. Therefore,thesemoleculeshavethe opposite concentrationresponseexpectedfrom a feedbackinhibitor. Furthermore,in vitro assayshave failedtoreveal substantialinhibition of ACCaseby acyl-ACPs(80).

Acyl-ACPs may play a role in the regulationof KAS III, however.WhenKAS III in seedhomogenatesof Cupheawaschallengedwith acyl-ACPs,aslittle as 0.5 µM 10:0-ACP was able to cause50% inhibition (12). Similarresultswere obtainedusing 1 µM 10:0-ACPwith homogenates of Brassicaseedor spinachleaf. In eachcase,maximum inhibition wasobservedwith the10:0-ACP,compared withlonger-chainacyl-ACPs,and thesource ofthe ACPwasE. coli. We haveincubateda purifiedpreparationof spinachKAS III with10:0-ACP(ACP source:spinach)at concentrationsup to 0.6µM andfailed toobserveany inhibition (B Hinneberg-Wolf& J Jaworski,unpublished infor-mation).This suggeststhatinhibition of KAS III activity observedin the planthomogenateswas indirect. A correspondingstudy carriedout using purifiedpreparationsof E. coli KAS III and100-µM 16:0- or 18:1-ACPresultedin a50% inhibition (77). Becausetheselevels far exceedthe intracellularlevelsobservedin E. coli, thephysiologicalsignificanceof this inhibition remainstobe demonstrated.

Severalother potential feedbackinhibitors such as acyl-CoA, free fattyacids,andglycerolipids alsofail to stronglyinhibit theplantACCaseat physi-ological concentrations(80). BecauseFAS occursinside the plastid but themajor utili zation of the productsof fatty acid synthesisis at the ER mem-branes,it is likely that feedbackregulationmustallow communicationacrosstheplastidenvelope.At this time we do not haveanyclearindications of whatmoleculesareinvolved in feedbackregulationof plastid fatty acid synthesis,and theirdiscoveryremains a majorchallengefor plantbiochemists.

Control of Substrate and Cofactor Supply

Another way to control flux through a pathway is to regulatedelivery ofsubstratesandcofactorsto thepathway.In animals(30) andsomeoleaginousyeast (7), there is evidencethat the accumulation of acetyl-CoA via theATP:citratelyasereactionis a majordeterminantof fatty acidsynthesis rates.Although conclusiveevidenceis not yet availablefor plants,indicationsarethatacetyl-CoAsupplydoesnot usuallylimit plastidfatty acidproduction.Ifacetyl-CoAlevelswere limi ting ratesof fatty acid production, a decreaseinacetyl-CoAlevel would beexpectedduring high ratesof fatty acidsynthesis.However,almostall CoA found in chloroplasts is in the form of acetyl-CoA,


and despitevery largedifferencesin ratesof fatty acid synthesis in light ordark, acetyl-CoA levels in chloroplastsremain almost unchanged(74). Indevelopingseeds,levelsof acetyl-ACParehigherthanin light-grown leaves,and the level doesnot changesubstantially throughout seeddevelopment,despitemajor changesin ratesof FAS (73). Again, theseresultssuggestthatacetyl-CoAconcentrationsin seedplastids arehigh anddo not changeduringdevelopment.

Although we tentatively concludethat carbonsupply doesnot limit fattyacidproductionin leavesandmostseedsin normalplants,thereareexamplesof transgenicplants that may give someindication of what is requiredforcarbonlimi tation to occur.The targetingof theenzymesof thepolyhydroxy-butyrate(PHB) pathwayinto thechloroplasts of Arabidopsisthalianaresultedin anaccumulationof PHB of up to 14%dry weightof theplant(59).Synthe-sis of this largecarbonsink alsorequiresthe plastidialacetyl-CoApool, andyet therewasno detectabledeleteriouseffecton fatty acidbiosynthesis.Thus,it wasconcluded that amechanismmust existthat allows theplastidto synthe-sizethe requiredacetyl-CoAin responseto additional metabolicdemandforPHB production. In anotherexample,over-expressionof theE. coli ADP:glu-cosepyrophosphorylasein developing Brassicanapusseedsleadsto a largeincreasein starchcontentof seedsanda 50%decreasein oil content(6). Oneinterpretationof this result is that in this case,diverting carbon to starchstoragewassufficient to “starve” the fatty acid pathwayfor availablecarbonprecursors.

As in the caseof substrates,considerationshouldbe given to cofactorsorenergyaspotentially limi ting undercertainconditions.Fattyacidsynthesisisanenergy-demandingpathwaythat requiresat least7 ATP and14 NAD(P)Hto assemblean 18-carbonfatty acid.However,energyis usuallynot a limita-tion in overallplantgrowth.In chloroplasts,ATP andNADPH canbederivedfrom photophosphorylationandelectrontransport.In thedarkor in nongreentissues,glycolysisandthe oxidative pentosephosphate pathwaycanprovidethe ATP andreductant (1,21).

WHAT DETERMINES HOWMUCH OIL IS PRODUCEDBYA SEED?

Theoil contentof seedsof differentplantspeciesvariesfrom under4% of dryweight (e.g.Triticum sativum) to over60%(e.g.Ricinuscommunis). Further-more,whereasleaves,roots,andothervegetativetissuesusuallycontainlessthan10% lipid by dry weight,mostof which is polar membranelipids,seedlipids usuallycontainover 95% neutralstoragelipids in the form of triacyl-


glycerol (TAG). An understandingof how plantsregulatefatty acid metabo-lism to achievethesemajordifferencesin lipid contentandcomposition is notyet available.

The only enzymeuniqueto TAG biosynthesisis diacylglycerol acyltrans-ferase(DAGAT). Severallinesof evidencesuggestthat tissue-specific expres-sionof DAGAT is not sufficientto explaineitherthehigh proportionof TAGin seedsor the highlevelof oil in someseeds. 1.DAGAT activity is foundnotonly in seedsbut also in leaves(15, 55). 2. Certaintypesof stresssuchasozonecauseleavesto producehigh proportionsof TAG (87). Evendetachingleavesandfloating themin buffer plus0.5 M sorbitol is sufficient to increasetheaccumulationof neutrallipids severalfold (11). 3. Addition of fatty acidsto thesurfaceof spinachleavesresultsin a substantial proportionbeingincor-poratedinto TAG (86). Clearly, leavesaswell asseedshavethecapacityforTAG synthesis,and thereforespecificexpressionof DAGAT seemsinsuffi-cient to explain abundantTAG synthesis in seeds.To understandthe highproportionof TAG in seeds,it maybeusefulto considerwhetheroil synthesisis controlled by thesupply(source) of fattyacids or fattyacid precursorsor bythe demand(sink) for fatty acids.

Control byFatty Acid Supply (Source)

Control of oil productionin seedsmay reflect a responseto the high rateoffatty acidsynthesisin this tissue.If DAGAT is presentatsimilar levelsin mosttissues,thenhigh levelsof TAG synthesis may occur in seedsbecausehighlevelsof fatty acid areproduced.Datathat supportthis conceptareavailablefor CupheaandChlamydomonas.Addition of excess exogenousfatty acid andglycerol to developingCupheacotyledonsrapidly resultedin ratesof TAGaccumulationseveral-foldhigherthan observedon the plant (5). InChlamydo-monas,additionof exogenouslipids (PCliposomes)to culturesresultedin upto 10-fold increasesin TAG accumulation(32). Thus,it appears tobe thefattyacidsubstrates,not theutilizationenzymesthatlimit TAG synthesis in Cupheaand Chlamydomonas,and thecapacity for TAG synthesis is greater thanactually used.Furtherexamplessupporting this theory comefrom compari-sonsof oleaginous(oil-accumulating) versusnonoleaginousyeast.Extensivestudiesby Ratledgeandcoworkershaveled to theconclusionthatdifferencesbetweenthesetwo typesof speciesarecontrolledby theproductionratherthanutilization of fatty acids (7). In particular, the production of acetyl-CoAthrough the action of ATP:citrate lyase is consideredto control the flux ofcarbon intostoragelipids.


Control byDemand (Sink)

Evidence that utilization of fatty acids canincreaserates offatty acidsynthesisis availablefrom experimentswith E. coli (38, 65, 108). Whena plant l2:0-ACP thioesteraseis overexpressedin E. coli cells, fatty acid synthesis isincreasedsubstantially. Suchculturesaccumulateat least10-fold more totalfatty acid (mostin the form of free lauric acid) thancontrolcultures.In theseexperiments,the removalof the productsof fatty acid synthesisto a metabo-lically inert endproduct(free fatty acid)appearedto releasefeedbackinhibi-tion on acetyl-CoAcarboxylase,resulting in higher malonyl-CoA and fattyacid production(65). In a completely different type of experiment,massiveoverexpressionof cloned membraneproteins resultedin no changeof themembrane’s phospholipid to protein ratio,but rathermore fatty acid wasproducedto accommodatethe excessproteins(110,112).Therefore,the rateof fatty acid synthesis can apparentlybe regulatedby the demandfor fattyacids neededfor membranesynthesis.Extrapolating this concept to plantseeds:Perhapsfatty acid synthesisincreasesin responseto the high demandfor acyl chainsbroughton by the depletion(or increase)of a key metabolicintermediate.A hypotheticalexampleto illustrateonepossibility might bethathigh expressionof DAGAT (or someotheracyl transferase)leadsto a deple-tion of acyl-CoAs.This could in turn lead to releaseof feedbackinhibition(probablyindirectly) of ACCaseby acyl-CoA andthusprovidea mechanismwherebyremovalof acyl-CoA by the acyltransferasescould stimulate fattyacid production.

Additional recentevidencesuggeststhat both of the mechanisms abovemay be involved in determiningseedoil content.When a transit peptideisaddedto the cytosolic ACCaseof Arabidopsisand this chimeric gene isexpressedin B. napusundercontrol of the napinpromoter,the oil contentofthe seedsis increasedapproximately5% (79). Thus,increasingthe supplyoffatty acidsat the first stepin thepathwayleadsto increasedoil content.Evenlarger increases(>20%) in Arabidopsisand B. napusseedoil contentwereachievedwhen a yeastlysophosphatidicacid acyltransferase(LPAAT) wasexpressedin developing seeds(114).Becauseoverexpressionof coconut(60)or Limnanthes(50) LPAAT did not alter oil content,the ability of the yeastenzyme toincrease oilmayinvolve its lack of regulationin theplanthost.

Analysisof developingB. napusseedsexpressinghigh levelsof the Cali-fornia Bay 12:0-ACPthioesterase(MCTE) providedintriguing andsurprisingresultsthat emphasizehow much we needto learn beforewe havea goodunderstandingof what determinesthe amountof oil in a seed(seeFigure2).Voelker et al (109) found that increasedexpressionof the MCTE in seeds


resultedin linear increasesof lauricacid (12:0) in TAGup to∼35%.However,for quantities beyond35%, the correlationwith MCTE beganto reach aplateau.To achieve60mol% 12:0,a further10-fold increasein MCTE expres-sionwasrequired.At high levelsof MCTE, expressionof fatty acidβ-oxida-tion aswell asenzymesof theglyoxylate cycle were induced (68).Thus,theseseedsappearto producemore 12:0 than canbe metabolized to TAG by theBrassicaacyltransferasesor otherenzymeswhosenormalsubstratesareC16andC18fatty acids,andtheexcess12:0signalstheinduction of thecatabolicpathway.Thus,a portionof thefatty acidsynthesisin theseseedsis involvedin a futile cycle of synthesisand breakdownof 12:0. Surprisingly, despiteinductionof fatty acidβ-oxidation, the total amountof TAG in theseMCTE-expressingseedsis not substantially reduced.How is oil contentmaintainedifa significantamountof thelauricacidproducedby FAS isbeingbrokendownin a futile cycle?Analysisof thefatty acidbiosyntheticenzymesrevealedthatthe levelsof ACP andseveralof the fatty acid biosynthetic enzymes(acetyl-CoA carboxylase,18:0-ACPdesaturase,KAS III, etc) had increasedtwo- tothreefold at midstage development of high MCTE-expressingseeds.Theseresultshaveseveralimportantimplications. First, acoordinate inductionof theenzymesof thefatty acidpathwayhadoccurred,presumablyto compensateforthe lauric acid lost through β-oxidation or the shortageof long-chainfattyacidsin theseseeds.This suggeststhat the enzymesfor the entireFAS path-way may be subjectto a systemof global regulationperhapssimilar to lipidbiosynthesis genesof yeast(16, 91). Second,theseresultsindicate that al-thoughB. napusseedsarerelativelyhigh in oil content(ca 40%) theexpres-sion of the FAS enzymesis not at a maximumandcanbe induceda furthertwo- to threefold over the levels found at midstageof seeddevelopment.Finally, the resultssuggestthat theseseedsmight be preprogrammedto pro-ducea particularamountof oil, and the levels of expressionthe fatty acidpathway mayadjustto meetthe prescribed demandfor TAG synthesis.

What determinesthe level of TAG in thesecells andwhat arethe signalsthat result in increasedexpressionof theFAS pathway?Onemajordifficultywith interpretingthe abovedataoccursif we attemptto fit it to the minimalmodelcomprisedof the currentlyunderstoodrolesof the pathwayenzymes.However,recentresultsclearly indicatethat therearestill major gapsin ourunderstandingof lipid and TAG biosynthesis.Analysis of the pathwayforpetroselinicacid (18:1∆6) from 16:0 in corianderindicatedthat this planthasevolved a specialized3-ketoacyl-ACPsynthase(KAS), not found in otherplants,that hashigh activity with 16:1∆4-ACP (13). Similarly, a KAS fromCupheawrightii thathashomology to plantKAS II wascoexpressedwith theC. wrightii MCTE in Arabidopsis,andthis resultedin dramaticincreasein the


levels of 10:0 and 12:0 in the seedscomparedwith seedsexpressingtheMCTE alone.Both studiessuggestthat thereare additional specializeden-zymes for seed lipidmetabolismthat were not predicted on the basis ofpreviousbiochemicalunderstanding.At this time thereis no clear ideahowtheseenzymesinteractor whatadditionalspecializedfunctionstheymayhavein oil synthesis. Presumablyotherenzymesor regulatoryinteractionsareyet tobediscoveredthathavea role in determiningthequantityaswell asquality ofthe oil.

WHAT HAVE WE LEARNED FROM TRANSGENICPLANTSAND MUTANTS?

FattyAcid Compositionof SeedsCan Be MoreRadicallyAlteredThanOtherTissues

Thecomposition of fatty acidsproducedin plantsis primarily determinedbythioesterases,condensingenzymes,anddesaturases.Manipulation of thethio-esterasesand desaturasesin transgenicplantshasbeenhighly successfulinproducingmajormodificationsof thechainlengthandlevel of unsaturationofplant seedoils (for reviews,see44, 57, 66, 105). An additional new insightinto regulation offatty acidmetabolism wasrecentlyobtainedfrom expressionof enzymesproducingunusualfatty acidsin othernonseedtissues.For exam-ple, transgenicexpressionof the 12:0-ACP thioesterasein B. napusundercontrol of the constitutive 35Spromoterresultedin lauric acid productionintheseeds but notin leavesor othertissues (27).However,chloroplasts isolatedfrom the B. napus leavesproduced upto 35% lauric acid. These resultsindicatethat sometissuesexpressingMCTE producelauric acidbut that it issubsequentlydegraded.In supportof this hypothesis,theactivity of isocitratelyaseof theglyoxylatecycle wasinducedin leavesexpressingtheCaliforniaBay thioesterase.A similar mechanismmay apply to the productionof hy-droxy fatty acidswhich accumulatein seedsbut not leavesof Arabidopsistransformedwith the castoroleatehydroxylaseundercontrol of the 35Spro-moter(8). Together,theseresultsimply thatnonseedtissuesmayhavegeneralmechanismsto degradeunusualor excessfatty acidsandtherebypreventtheirincorporationinto membranes.

Manipulationof Oil Quantity

Plant breeding and mutation studies have demonstrated that the amount ofoil in aseedcanbevariedoverawiderange.A classicexampleis theselectionfor high andlow oil maizethat over a periodof almost100 yearsresultedin


lines ranging from 0.5% to 20% lipid (25). Arabidopsismutants with bothincreasedseedoil (36) andreducedtriacylglycerol(42) havebeenreported.Inthe latter example,not only was oil content reducedbut 18:3 levels weredoubled,18:1 and 20:1 levels were reduced,and severalenzymesof lipidmetabolismhad alteredactivity. Thesepleiotropic effects of a single genemutationillustratethecomplexities and inter-relationships oflipid metabolismwhich aredifficult to explainbasedon our currentmodelsof seedoil biosyn-thesis.

Many Enzymesof Fatty AcidSynthesisAre Presentin Excess

Although therehasbeenmuchsuccessin manipulating chainlengthandun-saturationof plant seedoils in transgenicplants,directedalterationsin oilquantityarejust beginningto be achieved.A numberof thecoreenzymesoffatty acidsynthesishavebeenoverexpressedor underexpressedin transgenicsoybeanor B. napusseeds(45). Overexpressionof ACP, KAS III, KAS I,KAS II, oleoyl-ACP thiosterase(FatA), or saturate-preferringacyl-ACPthio-esterase(FatB) individually hasnot resultedin increasedseedoil content.It isnot known whetherincreased expression ofcombinations ofthese componentsmight be moreeffective.As discussedabove,increasedACCaseanda yeastacyltransferase have been reported toincrease oilin B. napusseeds.

Complete suppression of any of the core enzymesof FAS would be ex-pectedto reducefatty acid synthesisandseedoil content.In supportof this,cosuppressionof FatA or KAS I in soybeanresultedin reducedembryooilcontent(45),andantisenseof enoyl-reductasein B. napusgaveshrunkenseeds(113). Antisenseof the tobaccobiotin carboxylaseundercontrol of the 35Spromoterwas found to result in stuntedplantswith a 26% reductionin leaffatty acid content(92). However,theseeffectswereonly observedwhenthereduction in BC level was 80% or greater.At 50% reductionsin BC, nophenotypecould be detected.Similar resultswereobtainedwith antisenseofstearoyl-ACPdesaturases(47). Many other experimentson antisenseof en-zymesof plant carbohydratemetabolismhavealsofound that phenotypes(ifobservedat all) only occurwhenenzymelevel is reduced80%or more(102).Furthermore,the impact of an enzyme’s reductionis usually dependentongrowthconditions.Theseresultssuggestthatundermostconditions,manyoftheenzymesof plant metabolismarepresentin functionalexcess.Additionalevidencethatenzymesof lipid metabolism areexpressedin excesscomesfromcrossesof mutants. For severalmutantsof Arabidopsisfatty aciddesaturation(fad2,fad5,andfad6), crosseswith wild-type givea nearwild-type phenotyperatherthana fatty acid composition intermediatebetweenparents.Thus, for


severaldesaturasesandotherenzymes,genedosageis not theprimary deter-minant of the enzymesactivity, andmorecomplexcontrolsmust operateinvivo. In the caseof highly regulatedenzymessuchas ACCase,a variety ofmechanisms,suchasincreasedenzymeactivation, maycompensatefor reduc-tionsin expressionbroughton byantisenseor mutation.

CONTROLOF GENEEXPRESSION

MultigeneFamilies

A puzzlingaspectof themolecularbiology of plant fatty acidsynthesis is theroleof multiple genes.Evenwithin Arabidopsiswith its smallgenome,thereisconsiderablerangein the numberof genesencodingthe different proteinsofplant fatty acid synthesis. Acyl carrierproteinandthe 18:0-ACPdesaturasesare eachencodedby at least five genes,whereasmany other enzymesareencodedby a singlegene.Theexpressionof theACP geneshasbeenstudiedin somedetail,andbothconstitutive andtissue-specificpatternsof expressionhavebeenobserved (3, 35).In severalotherplant species,with genomeslargerthanArabidopsis, tissue-specificdesaturasesandotherenzymesareknowntocontrol seedfatty acid composition (67). Although it might be consideredadvantageousto have multiple genesto allow fine tuning of expressionindifferent tissues,it is clearthat this is not essentialfor manyof the genesoffatty acidmetabolismasseveralof theproteinsin thepathwayareencodedbya singlegene.In thecaseof theglycerolipid desaturases,onegeneencodesaplastid isozyme,whereasa secondgeneencodesa presumablyER localizedisozyme.However,two genesencodethe 18:2 desaturaseof plastids,oneofwhich istemperatureregulated (10).

Promoter Analysis

Currently,thepromoterof acyl carrierproteingeneshasbeenexaminedin thegreatestdetail. de Silva et al (22) fused 1.4 kB of a B. napusACP gene(ACP05)to β-glucuronidase(GUS) and determinedexpressionlevelsin trans-genic tobacco.GUS activity increasedduring seeddevelopment,concurrentwith lipid synthesisandat its maximum was100-fold higher than in leaves.Surprisingly,althoughACP is not an abundantprotein, the activity of theACP/GUS constructwas comparableto that obtainedfrom the strong 35Spromoter.Severalconstructsof a promoterfrom anotherArabidopsisACPgene,Acl1.2,havebeenfusedto GUSandexaminedaftertransformation intotobacco(4). Fluorometricanalysisindicatedstrongestexpressionin develop-ing seeds.However the promoterwas active at lower levels in all organs


(approx.50 fold lower in leaves).HistochemicalanalysisindicatedhighestAcl1.2 expressionin the apical/meristematic regions ofvegetativetissues.During initial flower development, Acl1.2promoteractivity wasdetectedin allcell types,but as the flower matured,GUS stainingwas lost in the sepals,epidermisof thestyle,andmostcellsof theanther.Intensestainingremainedin the ovary, stigma, stylar transmitting tissue,and tapetalandpollen of theanther.Thus,the expressionpatternof this particulargeneappearscomplex.Similar analysisof otherpromotersis neededto determinewhethercommonsignalsare responsiblefor suchpatterns.

Six deletionsof the Acl1.2 promoterrevealeddistinct regionsof the pro-moter involved in vegetativeand reproductivedevelopment. ExpressionofAcl1.2 in youngleavesdroppedto a basallevel whenan 85-bpdomainfrom−320 to −236 was removed,but expressionin seedswas not alteredby thisdeletion.A protein factor was detectedin leavesand roots, but not seeds,which bindsto the−320to −236domain.Seedexpressionwasreduced∼100-fold when the −235 to −55 regionwas removed.This sameregionwasalsoessentialfor highexpressionin the flower tissuesdescribed above.

2.2 kB of a B. napusstearoyl-ACPdesaturasepromoter wasfusedto GUS(98). Expressionwasapproximately2.5-fold higherin developing seedsthanin youngleavesandthusdid notshowthedramaticdifferencesreportedfor theACPpromotersabove.However,similar to theACPpromoters,strongactivitywasalsoobservedin tissuesundergoingrapiddevelopment, including imma-ture flowers,tapetum,and pollengrains.

Recentlytheenoyl-ACPreductasepromoterof Arabidopsis hasundergonesimilar GUSfusionanddeletionanalysis(20).Unlike ACP andstearoyl-ACPdesaturase,thereappearsto beonly a singlegenein Arabidopsisencodingtheenoyl-ACP reductase.High expressionof enoyl-ACP reductase promoter-GUSfusionswasagainobservedin youngestleaf tissues,with vasculartissueandshootor root apicalmeristems highest.As eachnew leaf matured,GUSactivity faded.Threedomainsof thepromoterwereidentified.Seedexpressionwasunchangedby deletionto −47 bp of the transcriptionstartsite,indicatingthat all elementsneededfor high level seedexpressionare presentin thisrelatively small region. Removalof an intron in the 5′ untranslatedregionresultedin increasedexpressionin roots,suggesting the presenceof negativeregulatoryelementsin this region.

Although the studiesdescribedabovesuggestthat quantitativedifferencesoccur betweenrelative expressionlevels of ACP, stearoyl-ACPdesaturase,andenoyl-ACPreductasepromotersin different tissues,all of theanalysesofFAS promotersso far havereachedsimilar conclusionsabouthighestexpres-sion in apicalmeristems,developingseeds,andflowers.Sucha patternis not


surprising,becausethesetissuesarethemost rapidlygrowing orareproducinglipids in high amountsfor storage.Furthermore,expressionof themRNA formostcomponents of FAS probablyis undercoordinantcontrol.For example,in situ hybridization of the mRNA for biotin carboxylase,BCCP, and car-boxyltransferasesubunits of ArabidopsisACCaseindicatesclosecoordinationof thesethreesubunits, which is coincidentwith oil deposition (62). It shouldalsobeemphasizedthatpromotersof lipid biosynthetic proteinsarelikely notunusualin theseexpressionpatterns.Highestexpressionin rapidly growingcells andcoordinantregulationwould be expectedfor promotersinvolved inmostprimarybiosynthetic pathways.

What Controls Promoter Activity of FASGenes?

A majorchallengefor thefuture is to discoverhow the level of expressionofgenesof lipid synthesis iscontrolled. Theinitial studiesof promotersreviewedabovehaveidentified domainsinvolved in control of geneexpressionlevels.Efforts areunderway to identify transcription factorsthat may bind to theseelements.In addition, geneticapproachesto searchfor mutantswith alteredregulationof fatty acid metabolism may allow identification of additionalcontrols.By analogyto thestudyof otherorganisms,thecontrolof plantFASgenesis likely to involve a complexarrayof cis andtrans actingfactors.Forexample,promotersof animalfatty acid biosynthetic genesarecontrolled byhormonessuchas insulin (56), by dietary fatty acids(78), by glucoselevels(75), and by differentiation [particularly to adipocytes(54)]. Repressionoftranscriptionby negativeregulatoryelementshas beensuggestedfor bothyeast(16) andanimal lipid biosynthetic genes(103), andin Saccharomyces,commonDNA sequenceshave been identifiedin thepromotersof many genesinvolved in lipid metabolism (16, 91). As with genesof the glyoxylate andmanyotherpathways,transcriptionof plantFAS genesis likely dependentonboth developmental and metabolicsignals(31). Becauseall indications arethat the enzymesof fatty acid synthesisarecoordinatelyregulated,it seemsprobablethatglobal transcriptionalsignalsmaycontrolexpressionof manyorall genesof the pathway[perhapssimilar to the R andC locusproductsthatcontrol transcriptionof theanthocyanin biosynthetic pathway(53)]. Identifica-tion of such global controls may provide the most effective meanstowardmanipulating the amountof fatty acidproducedin transgenicplants.If meta-bolic controloveroil synthesis isshared amongseveral enzymes,there willbelimits to how much the flux through this pathwaycan be manipulatedintransgenicplants using overexpressionof one or a few genes.Thus, more


completecontrol may come from identifying transcriptionfactors that canincrease expressionof the entirepathway.

SUMMARY AND PERSPECTIVES

Fattyacidsynthesisis a primarymetabolicpathwayessentialfor the functionof everyplantcell. Its productsserveasthecentralcoreof membranesin everyplant cell, and in specializedcells, fatty acidsor fatty acid derivativesact assignalor hormonemolecules,ascarbonandenergystorage,andasa surfacelayer protectingthe plant from environmental and biological stress.Despitethesevery diversefunctions, essentially all fatty acidsin a cell areproducedfrom a singlesetof enzymeslocalizedin theplastid. Understandinghow cellsregulatetheproduction of these fattyacids anddirect themtowardtheir differ-ent functions is thus central to understandinga large rangeof fundamentalquestions inplantbiology. In addition, muchinteresthasrecentlydevelopedingeneticengineeringof the fatty acidbiosynthetic pathwayto producenew orimproved vegetableoils and industrial chemicals.Therefore,knowledgeofhow cells control the amountof fatty acid they producemay be essentialforoptimalcommercialproductionof fatty acids.

Our understanding of regulationof fatty acid metabolismis much lessdevelopedthanthatof carbohydrateor aminoacidbiosynthetic pathways.Wenow haveconvincingevidencethatACCaseis oneenzymethat is involvedinregulatingfatty acidsynthesisrates,andthereareindicationsfor othercontrolpoints.However,this is only thebeginning.ACCasemight beconsideredone“slave” enzyme that controls flux into fattyacidsbutwhoseactivity isdepend-ent uponhigherlevel mastercontrol systemsin the cell. But what moleculesregulateACCaseby feedbackor othermechanismsandwhatmetabolicsignalsor mechanismscontrol those molecules?How is the globalregulation ofdozensof genesfor lipid synthesisaccomplished? As discussedin this review,we have only fragmentaryinformation about the natureof thesecontrols.Thus,understandingregulationof fatty acid synthesisis a rich andrelativelyunexploredfield with muchwork left to be done.

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REGULATION OF FATTYACID SYNTHESIS - UFVarquivo.ufv.br › DBV › PGFVG › BVE684 › htms › pdfs_revisao › metabo… · synthesis. The relative activities of these enzymes therefore