The role of membrane transport in metabolic engineering of plant primary metabolism
Andreas PM Weber and Andrea Bra¨utigam
Plantcellsarehighlycompartmentalizedandsoistheir metabolism.Mostmetabolicpathwaysaredistributedacross severalcellularcompartments,whichrequirestheactivitiesof membranetransporterstocatalyzethefluxofprecursors, intermediates,andendproductsbetweencompartments.
Metabolitessuchassucroseandaminoacidshavetobe transportedbetweencellsandtissuestosupply,forexample, metabolismindevelopingseedsorfruitswithprecursorsand energy.Thus,rationalengineeringofplantprimarymetabolism requiresadetailedandmolecularunderstandingofthe membranetransporters.Thisknowledgehoweverstilllags behindthatofsolubleenzymes.Recentadvancesincludethe molecularidentificationofpyruvatetransportersatthe chloroplastandmitochondrialmembranesandofanewclass oftransporterscalledSWEETthatareinvolvedinthereleaseof sugarstotheapoplast.
Address
InstituteforPlantBiochemistryandCenterofExcellenceonPlant Sciences(CEPLAS),Heinrich-Heine-University,Geb.26.03.01, Universita¨tsstrasse1,D-40225Du¨sseldorf,Germany
Correspondingauthor:Weber,AndreasPM(andreas.weber@hhu.de)
CurrentOpinioninBiotechnology2013,24:256–262
ThisreviewcomesfromathemedissueonPlantbiotechnology EditedbyNataliaDudarevaandDeanDellaPenna
ForacompleteoverviewseetheIssueandtheEditorial Availableonline4thOctober2012
0958-1669/$–seefrontmatter,#2012ElsevierLtd.Allrights reserved.
http://dx.doi.org/10.1016/j.copbio.2012.09.010
Introduction
Metabolicengineeringtargetsthealterationofnetworks ofbiochemicalreactionstomodifythequalityortheyield of desired products. This includes adjustment of the amountand kinetic propertiesof metabolicenzymesas wellastheregulatorynetworksthatgoverntheexpression ofpathwaycomponents,whichinmostcasesisachieved throughgeneticengineering[1,2].Thanksto thedevel- opmentofmathematicalmodelsofmetabolism,thecon- cepts of metabolic engineering have been successfully applied totheengineeringof microbial metabolism,for exampletoincreasetheamountofdesiredaminoacidsor fermentation products [3], as wellas the production of plantspecializedmetabolitesinyeast,suchasartemisinic acid[4].Progressinplantmetabolicengineeringstilllags
behind achievements in the microbial field, which is partly owing to the complexity of plant metabolism, a fact that impedes the development of genome scale metabolic models for important crop and model plants [5,6].Twomajorgoalscanbedefinedforplantmetabolic engineering: (i) Increasing theyield or quality of plant products,suchasoilsandfats,cellwalls,sugarsandstarch, proteinsorspecializedmetabolitesthathavehealthpro- motingpropertiesorserveindefenseagainstorattraction of biotic interactors; (ii) Increasing photosynthetic and resource-useefficiencyto optimizetheamount of plant products that can be achieved with a given amount of water,fertilizer,and land. The latter pointis of special concerngiventheneedtoincreaseplantproductivityto meetthedemandsofagrowinghumanpopulationwith respectto food,feed,fiber,andenergy[7].Thisreview highlightstheimportanceofadetailedunderstandingof themolecularmechanismsofmetabolitetransportwithin andbetweencellsandorganellesforrationalengineering ofplantmetabolism.Giventheimportanceofthechlor- oplast as the site of photosyntheticcarbon assimilation andofmultipleotheranapleroticroutes,specialemphasis willbeputonrecentfindingsonthetransportintoandout ofthechloroplastsandotherplastid subtypes.
Improvingphotosyntheticefficiency– a metabolite transportperspective
Severalprincipalavenuescanbeenvisagedforincreasing plantproductivity,includingthealterationofplantarchi- tecture or life history traits. We focus on improving photosynthetic efficiency through (i) increasing the ef- ficacyofphotosyntheticcarbonassimilationandthrough (ii)optimizingsinkstrength,therebyrelievingfeedback inhibitionofphotosynthesis.
Improvingphotosyntheticcarbonassimilation
Photosyntheticcarbonassimilationproceedsthroughcar- boxylation of the carbon dioxide acceptor ribulose 1,5- bisphosphate(RubP)inthechloroplaststroma,areaction thatiscatalyzedbytheenzymeribulose1,5-bisphosphate carboxylase/oxygenase(Rubisco)aspartofthereductive pentosephosphatepathway(RPPP).Rubiscoisabifunc- tional enzyme that not only catalyzes the productive incorporationof carbondioxide intoorganicmatter,but alsooxygenationofRubPandasaconsequencethelossof carbonfromplantbiomassintheformofcarbondioxide through photorespiration [8]. To minimize the rate of carbon loss through photorespiration and to thereby increase photosynthetic efficiency, theconcentration of carbondioxideattheactive siteoftheRubiscoenzyme
must be increased. Several means to this end have evolved in plants and algae, such as the bicarbonate pumpingsystemsfoundinmanyalgaeandcyanobacteria [9], and the C4 photosynthetic CO2 pump in land plants [10]. Computational approaches have identified additional,potentiallysuperiorcarbonfixationroutesthat donotcurrentlyexistinnaturebutthatmightbeimple- mentedthroughsyntheticbiology[11,12].Majorefforts arecurrentlyunderwaytointroducecarbonconcentrating systemsintoC3cropsplants,suchaswheatandrice,with special emphasis on converting C3 plants to C4 plants [13].Alternativeapproachesinclude shortcuttingphoto- respiration by detoxifying the product of the Rubisco oxygenation reaction, 2-phosphoglycolate(2PG), within the chloroplast stroma [14,15].Interestingly, many cya- nobacteria possess a complete pathway for converting 2PGto3-PGAviatatronate-semialdehyde[16,17],similar totheonethathasrecentlybeenimplementedinplants throughgeneticengineering[18].Apparently, thispath- way was lost during the evolution of chloroplasts from cyanobacteriabyendosymbiosis.Possibly,theformation of glycine from glycolate during photorespiration was of selective advantage, which favored the loss of the tatronate semialdehyde route that does not produce
glycine as a pathway intermediate. While shortcutting photorespirationhasalreadybeendemonstratedtoleadto increased biomass production at least under short day conditionsinengineeredArabidopsisplantsintwoinde- pendentstudies[18,19],aproofofconceptfortransform- ingaC3toaC4plantisstilllacking.Asoutlinedbelow, the implementation of both, shortcutting photorespira- tionandtransformingC3toC4,willbenefitfromabetter understanding ofintracellularsolutetransport.
C3toC4–transportersrequiredforafunctionalpathway ThreedistinctbiochemicalsubtypesofC4photosynthesis have been described and named afterthe enzyme that carriesoutdecarboxylationoftheC4acid:theNAD-malic enzyme (NAD-ME) type, the NADP-malic enzyme (NADP-ME)type,andthephosphoenolpyruvatecarbox- ykinase (PEP-CK) type. Accumulating evidence indicates thatsignificantflexibilityexistsbetweenthese decarboxylationpathwayswithinaparticularC4species, depending on environmental and developmental cues [20,21].TheNADP-ME type isfrequently considered to bethemostefficientsubtype ofC4.However,italso requires thelargestamount oftransmembranetransport steps[22].Fromtheperspectiveofmembranetransport,
Figure1
VB
Asp OAA
PEP CO2
malate
malate
pyruvate NADH NAD+
ATP
NAD-ME
PEPCK
PEP OAA OAA
Asp malate
pyruvate pyruvate
PEP PPDK
NADP-MDH
Calvin cycle
mito
chloro mesophyll cell
(a) (b)
bundle sheath cell
HCO3-
PEPCAsp-AT
Asp-AT
Ala Ala
Ala-AT Ala-AT
DiT1
DiT1
PPT
BASS2/
NHD1
MPC DIC
pyruvate Na+
Na+
H+ BASS2
NHD1
Current Opinion in Biotechnology
(a)Schematicoutlineofaphosphoenolpyruvatecarboxykinase(PEP-CK)/NAD-malicenzyme(NAD-ME)typeC4photosyntheticpathways,including thesixknowntransportersrequiredtorunthepathway:PPT,phosphoenolpyruvate/phosphatetranslocator;DiT1,plastidicoxaloacetate/malate transporter;DIC,mitochondrialdicarboxylatetransporter;MPC,mitochondrialpyruvatecarrier;BASS2,plastidicpyruvate:sodiumsymporter;NHD1, plastidicsodium:protonantiporter.
Additionalabbreviations:Ala,alanine;Ala-AT,alanineaminotransferase;Asp,aspartate;Asp-AT,aspartateaminotransferase;NAD-ME;NAD- dependentmalicenzyme;NADP-MDH,NADP-dependentmalatedehydrogenase;OAA,oxaloacetate;PEP,phosphoenolpyruvate;PEPC;
phosphoenolpyruvatecarboxylase;PPDK,pyruvate,phosphatedikinase;VB,vascularbundle.(b)Functionoftheplastidicpyruvatedouble translocatorsystem.PyruvateisimportedintoplastidstogetherwithsodiumionsbyBASS2;thesodiumionsareexcretedfromtheplastidincounter- exchangewithprotonsbythesodium:protonantiporterNHD1.
this is the subtype that is most difficult to engineer, a processhinderedbythefactthatthetransporterscatalyz- ingtheimportofmalateintoandpyruvateoutofbundle sheathcellchloroplastshavenotyetbeenidentifiedatthe molecularlevel.Thesituation isdifferentfor thePEP- CKandalsofortheNAD-MEsubtypes.Withtherecent discovery of the plastidial pyruvate:sodium symporter [23], all metabolite transporters required for imple- menting a rudimentary PEP-CK C4 are now known.
The PEP-CK mini-cycleitself requires no intracellular transportsteps(Figure1).CarboxylationofPEPbyPEP- carboxylaseyields oxaloacetate (OAA)thatisthen ami- nated to aspartate (Asp) by aspartate aminotransferase (AspAT).Drivenbyaconcentrationgradientfrommeso- phylltobundlesheathcells,Aspistransportedtobundle sheathcellswhereitisconvertedbacktoOAAbyAspAT and decarboxylated to PEP by PEPCK. CO2 resulting fromthisdecarboxylationisentering thebundlesheath cellchloroplastswhereitisfixedbyRubisco.PEPreturns tothemesophyllcellsforanotherroundofcarboxylation.
This mini-cycle would however generate an ammonia imbalancebetweenMCandBSCbecausetheaminoacid AspisimportedintoBSCandPEPisreturnedto MCs, leavingoneaminogroup intheBSCspercycle.Thisis compensated by a parallel-running NAD-ME cycle, whichbringsmalateto theBSCsand returnstheamino acidalanine(Ala),therebyreturningtheaminogroupto theMCs(Figure1).Thecompensatorycyclerequiresthe increased expression of four solute transporters in the chloroplastenvelopemembranesofmesophyllcellchlor- oplasts,threeofwhicharerequiredforPEPrecycling:(i) the pyruvate:sodium symporter (BASS2), and (ii) a sodium:protonantiporter(NHD1)thatmaintainsplasti- dial sodium homeostasis, and (iii) the PEP/phosphate antiporter(PPT).BASS2importspyruvateintothechlor- oplast(jointlywithsodium,whichissubsequentlytrans- portedtothecytosolbyNHD1,incounter-exchangewith protons, Figure 1b) where it is converted to PEP by pyruvate:phosphate dikinase. PEP, the primary CO2
acceptor in C4 photosynthesis, is exported from the chloroplast in counter-exchange with inorganic ortho- phosphate (Pi) by PPT. The NAD-ME cycle also requiresanOAA/malateantiporterattheMCchloroplast
envelope, which imports OAA into MC chloroplasts whereitisreducedtomalatebyNADP-malatedehydro- genase and exports the resulting malateto the cytosol.
This step is catalyzed by the plastidial dicarboxylate transporter 1 (DiT1), which can exchange malate for OAA and 2-oxoglutarate [24]. Malate diffuses along a concentrationgradient to the BSCswhere it entersthe mitochondria,probablybyamitochondrialdicarboxylate translocator [25], and is decarboxylated by NAD-ME, yielding pyruvate, NADH, and CO2. CO2 is entering theBSC chloroplaststo be fixed by Rubisco. Pyruvate leaves the mitochondria and is converted to Ala by AlaAT.Alaisreturnedto MCswhereitisconverted to pyruvateby AlaAT and pyruvateenters the MCchlor- oplasts to be converted to PEP, thereby closing the branched PEP-CK/NAD-ME cycle. Until recently, it was unknown how pyruvate is transported across the mitochondrial membrane. However, mitochondrial pyr- uvatecarriers(MPCs)wererecentlyidentifiedinhumans, yeast, and Drosophila [26,27] and orthologous proteins also exist in plants (with Arabidopsis At5g20090andAt4g14695beingthemostcloselyrelated proteinstomammalianMPC1andMPC2,respectively).
Thus,incontrast totheNADP-ME subtype,candidate genes for all required transporters to implement a PEP-CK/NAD-ME cycle have now been identified (seeTable1foralistingofAGIsofthecandidategenes inArabidopsis).Maintainingionandmetabolitebalance across the plastid envelope as demonstrated by the BASS2/NHD1systemwithregardtosodium mayprove critical for other approaches to increase photosynthetic efficiency, such as adding cyanobacterial bicarbonate transporters to the chloroplast envelope of plants [28].
Candidatetransporters, suchas BicAand SbtA catalyze the co-transport of bicarbonate with sodium ions [29], which require the activity of a plastidial sodium-trans- porter to drive theprocess. NHD1 fulfills this require- ment, although its expression level might need to be adjustedtomeettherequiredflux.
PrimingtheNADP-MEC4pumpbyphotorespiration TheNADP-MEsubtypedepends,atleastinmaizeand sorghum,onshuttlingof 3-PGAandtriosephosphates
Table1
ArabidopsisGeneIdentifiers(AGIs)oftransportersrequiredfortheimplementationofaPEP-CK/NAD-MEC4-typephotosynthesis
AGI Membranelocation Nameoftransporter Acronym Substrate(s)
At5g33320,At3g01550 Chloroplast Phosphoenolpyruvate phosphatetranslocator
PPT1,PPT2 Phosphoenolpyruvate,phosphate
At2g26900 Chloroplast Pyruvatesodiumsymporter BASS2 Pyruvate,sodium
At3g19490 Chloroplast Sodiumprotonsymporter NHD1 Sodium,proton
At5g12860 Chloroplast Dicarboxylatetranslocator1 DiT1 Oxaloacetate,malate,
2-oxoglutarate,fumarate,succinate At2g22500,At4g24570,
At5g09470
Mitochondrion Dicarboxylatecarrier DIC1,DIC2, DIC3
C4acids,phosphate
At5g20090,At4g14695 Mitochondrion – – Pyruvate
(TPs) betweenBSC andMC chloroplasts(see[22]for review). Whiletherequired transportersforthesteady stateoperationofthe3-PGA/TPshuttleareknown,the shuttlemightrequireoneadditionaltransportproteinto getthecyclestartedatthebeginningofthelightphase.
The 3-PGA/TP shuttle operates via the 3-PGA/TP/Pi antiporter TPT, which catalyzes the strict counter- exchange ofTPswith either3-PGA orPi[30]. 3-PGA that cannotbereducedtoTPsintheBSCchloroplasts owing to lack of redox equivalents is exported by the TPTincounter-exchangewithTPsandtransportedto theMC,whereitistakenupintochloroplasts,againby theTPT,incounter-exchangewithTPs.Inessence,3- PGA istransportedfromBSCs tothe MCs,reducedto TPsandthenshuttledbacktoBSCs(Figure2A).How- ever,inthemorning, MCchloroplasts probablydonot contain the required counter-exchange metabolites (either TP,3-PGA,orPi)and thusarenotabletotake up3-PGAviatheTPT.Theyneedtobe‘preloaded’bya metabolite uniporter, similar to priming a pump. As initially suggested byBauwe and co-workers, a photo- respiratory glycerate shuttle between BSCs and MCs mightachievethepriming[31].Withtheexceptionof thechloroplast-localizedenzymeglyceratekinase(GK) thatconvertsglycerateto3-PGA,allenzymesofphoto- respiration are localized in the BSCs of the C4 plant maize. In contrast to all other known GK enzymes in plants, the maize mesophyll GK is regulated by
thioredoxin, being activated in the light [31]. The primingishypothesizedtoworkasfollows:Oxygenation ofRubPintheBSCgeneratesphosphoglycolate,which is converted to glycerate by the photorespiratory enzymesinthe BSCs andthen transportedtothe MC whereitistakenupintothechloroplastsbytheglycerate transporter (Figure2B). Whilethis transporter has not yet been identifiedat themolecular level, it has been demonstratedinbiochemicalstudiestoexchangeglycer- ate for protons [32], hence it would be able to load glycerate into chloroplasts in counter-exchange with protons. Glycerate is then converted by GKto 3-PGA and further to TP by GAPDH, which in maize MC chloroplasts cannot enter the RPPP as in C3 plants butneedstobeexportedtotheBSCs.Hence,through importofglycerateintoMCchloroplastsanditsconver- sionto3-PGAand/orTPs,ametabolitepoolinsideMC chloroplastsisestablishedthatenablestheoperationof the 3-PGA/TP shuttle viathe TPT.It is importantto note that this priming of the NADP-ME 3-PGA/TP shuttle via intercellular transport of photorespiratory intermediates at this point is hypothetical and awaits experimentaltesting.
Optimizingthephotorespiratorybypassthrough modificationofplastidialglycolatetransportcapacity Asnotedabove,biomassproductionofArabidopsisplants could be increased by shortcutting photorespiration
Figure2
malate
CO2 + RubP pyruvate
NADPH 2x 3PGA
DHAP 3PGA + 3PGA
DHAP HCO3- +PEP
OAA malate
(a) (b)
mesophyll bundle sheath
DHAP TPT TPT
PSI + PSII
NADPH + ATP NADPH
O2 RubisCO
P-Glycolate Glycolate Glycerate
3-PGA DHAP
ATP ADP NADPH NADP
mesophyll bundle sheath
RuBP
Glycerate Glycolate
DHAP
Pi GlycT
TPT TPT
GlycT GK
GAPDH
3PGA
Current Opinion in Biotechnology
(a)The3-PGA/triosephosphateshuttlebetweenbundlesheathandmesophyllcellsinNADP-ME-typeC4photosynthesis.Bundlesheathcellsinsome NADP-MEC4plantssuchasmaizeandsorghumhavelittlephotosystemIIactivityinbundlesheathcellsandthusaredependentonredoximportfrom themesophyllcells.Redoxpowerinbundlesheathcellsisnotsufficienttoreduceall3-PGAinthereductivepentosephosphatecycletotriose phosphates(DHAP),hencepartofthe3-PGAmustbeexportedtothemesophyllcellsforreductiontoDHAPbythetriosephosphatetranslocatorTPT.
BecausetheTPTcatalyzesthestrictcounter-exchangeof3-PGAwithDHAP(orortho-phosphate),thisshuttle-systemisdependentontheavailability ofsuitablecounter-exchangesubstratesoncis-siteandtrans-siteofthechloroplastenvelopemembrane.(b)PrimingtheNADP-MEC4redoxshuttle byphotorespiratorymetabolites.Glyceratekinase(GK)localizedinmesophyllcellchloroplastsprovidesameanstobuildupapoolofcounter- exchangesubstratesforTPT.Photorespirationinthebundlesheathgeneratesglyceratethatistransportedtothemesophyllcellsandtakenupintothe mesophyllcellchloroplastsincounter-exchangewithprotonsbytheplastidialglycolate/glyceratetransporter(GlycT).Glycerateisconvertedto3-PGA byGKandfurtherreducedtoDHAPbyGAPDH.Thisbuildsupapooloftriosephosphatesthatcanbeexchangedfor3-PGAcomingfrombundle sheathcells,therebyprimingthe3-PGA/DHAPshuttle.
[19,33].Inbothcurrentlyknownsyntheticphotorespira- tory bypasses, 2-PG, the toxic product of the Rubisco oxygenationreaction,isdetoxifiedwithinthechloroplast, insteadofconvertingitto3-PGAinthecomplexphoto- respiratory cycle that involves peroxisomes, mitochon- dria, and cytosol [15]. Both synthetic shortcuts use glycolateas theinitial substrate,converting iteither to 3-PGA or to CO2 within the chloroplast. Chloroplasts however possess a very active glycolate transporter [34];that is,thetransgenically introducedpathways for glycolate metabolism will compete with the glycolate transporter for substrate. To fully assess the potential ofthephotorespiratorybypassesforincreasingphotosyn- theticefficiency,requiresreducingorblockingthefluxof glycolatefromthechloroplast,for examplebyreducing the activity of the plastidial glycolate transporter. The proteincatalyzingthisfluxishowevercurrentlyunknown.
Optimizingsinkstrengthandsource-to-sinkallocation ofcarbohydrates
Increasing photosynthetic efficiency through reducing photorespiration,asoutlinedabove increases thesource capacity,whichisessentialtoincreasetheproductivityof cropssuchasrice inwhichsink strengthhasapparently outgrown source capacity [13]. In other crops, such as potato,sinkstrengthmightstillrepresentalimitingfactor andincreasingtransportcapacitycanpotentiallycontrib- utetoovercomingthislimitation.Supportiveevidencefor thiscomesfromtheobservationthatincreasingthetrans- portcapacity of potatotuber amyloplasts for glucose 6- phosphateandATPwasassociatedwithalargeincrease instarchcontentsofthetubersandtotaltuberyield[35].
Thiswasachievedbysimultaneousoverexpressionofthe glucose6-phosphate/phosphatetranslocatorGPTandthe ATP/ADPantiporter NTT [35]. Overexpressionof the singletransportersdidnotinfluenceyield,indicatingaco- limitationin tuber starch biosynthesisby energyin the form of ATP and by carbon in the form of glucose 6- phosphate [35,36]. Also the long-distance transport of carbohydrates,suchassucrose,fromsourcetosinktissues might limit sink strength. One of the players in this pathway,thetransportercatalyzingtheeffluxof sucrose from leaf mesophyll cells into the apoplastic space of apoplastic loaders was unknown until recently, which preventedanassessmentoftheroleofsucroseunloading in the source-to-sink translocation. Using an elegant approach,thesucroseunloaderswererecentlyidentified [37].Human cellculture cellswereengineered to co- expressfluorescence–resonanceenergytransfer(FRET) sucrose-specific nano-sensors jointly with candidate proteins (SWEETs) that were identified in a previous similarscreenthatemployedglucosenano-sensors[38].
SeveralcandidategenescouldbeidentifiedfromArabi- dopsis(AtSWEET10–15) andrice (OsSWEET 11 and 14)anditwasshownthatanArabidopsisdoublemutant carrying T-DNA insertions in AtSWEET 11 and 12 showedreducedexportofcarbonfromtheleaf,increased
accumulation of starch, and reduced photosynthetic capacity.Thisclearlydemonstratesthatnotonlyphloem loading, but also unloading of sucrose from the leaf mesophyllisanimportantfactorinsource-to-sinktrans- location of sucrose. Of similar importance is the long- distance transport of amino acids between sources and sinks, although the mechanisms of amino acid export fromsourcecells arenotyetunderstood [39].
Conclusions
Engineeringthetransportcapacityofcellularmembranes isanimportantandfrequentlyunderappreciatedaspectin efforts to engineer plant primary (and probably also specialized)metabolism.Forexample,converting C3to C4 photosynthesisrequires amassivelyincreasedmeta- bolic flux for some metabolites across the chloroplast envelope by at least one order of magnitude [40].
Proteomic and transcriptomic approaches contributed to identifying candidate proteins carrying this flux [23,41,42], which now enables the implementation ofacompleteC4carbonconcentrationmechanism.Still, thecurrent knowledge aboutintracellular and intercel- lular metabolitetransport is rather limited,for example withrespecttotheintracellulartransportofaminoacids [39]. Technological breakthroughs, such as the use of metabolite nano-sensors [43] and large-scale electro- physiological screens [44] are needed to increase the repertoire of membrane transporters that can be employedinengineeringapproaches.
Acknowledgements
Workintheauthors’laboratoryissupportedbytheDeutsche
Forschungsgemeinschaft(grantsWE2231/8-2,IRTG1525,EXC1028),the FederalMinistryofEducationandResearch,andthe7thFrameworkofthe EuropeanUnion(3to4,http://3to4.org).
Referencesand recommendedreading
Papersofparticularinterest,publishedwithintheperiodofreview, havebeenhighlightedas:
ofspecialinterest ofoutstandinginterest
1. NielsenJ:Metabolicengineering:techniquesforanalysisof targetsforgeneticmanipulations.BiotechnolBioeng1998, 58:125-132.
2. SteuerR,KnoopH,Machne´ R:Modellingcyanobacteria:from metabolismtointegrativemodelsofphototrophicgrowth.
JExpBot2012,63:2259-2274.
3. KoffasM,StephanopoulosG:Strainimprovementbymetabolic engineering:lysineproductionasacasestudyforsystems biology.CurrOpinBiotechnol2005,16:361-366.
4. RoD-K,ParadiseEM,OuelletM,FisherKJ,NewmanKL, NdunguJM,HoKA,EachusRA,HamTS,KirbyJetal.:Production oftheantimalarialdrugprecursorartemisinicacidin engineeredyeast.Nature2006,440:940-943.
5. O’GradyJ,SchwenderJ,Shachar-HillY,MorganJA:Metabolic cartography:experimentalquantificationofmetabolicfluxes fromisotopiclabellingstudies.JExpBot2012,63:2293-2308.
6. LibourelIGL,Shachar-HillY:Metabolicfluxanalysisinplants:
fromintelligentdesigntorationalengineering.AnnuRevPlant Biol2008,59:625-650.
7. MaurinoVG,WeberAPM:Engineeringphotosynthesisinplants andsyntheticmicroorganisms.JExpBot2012http://dx.doi.org/
10.1093/jxb/ers263.
8. BauweH,HagemannM,KernR,TimmS:Photorespiration hasadualoriginandmanifoldlinkstocentralmetabolism.
CurrOpinPlantBiol2012,15:269-275.
9. PriceGD:Inorganiccarbontransportersofthecyanobacterial CO2concentratingmechanism.PhotosynRes2011,109:47-57.
10. SageRF,SageTL,KocacinarF:Photorespirationandthe evolutionofC4photosynthesis.AnnuRevPlantBiol2012, 63:19-47.
11.
Bar-EvenA,NoorE,LewisNE,MiloR:Designandanalysisof syntheticcarbonfixationpathways.ProcNatlAcadSciUSA 2010,107:8889-8894.
Thisstudyconductsacomputationalanalysisofpossiblecarbonfixation pathways acrossorganismicdomainsandsuggests severalsynthetic pathwaysthatdonotexistinNaturebutthatmightbesuperiortoextant metabolicroutes.
12. DucatDC,SilverPA:Improvingcarbonfixationpathways.Curr OpinChemBiol2012,16:337-344.
13. CaemmerervonS,QuickWP,FurbankRT:Thedevelopmentof C4rice:currentprogressandfuturechallenges.Science2012, 336:1671-1672.
14. MaurinoVG,Peterha¨nselC:Photorespiration:currentstatus andapproachesformetabolicengineering.CurrOpinPlantBiol 2010,13:249-256.
15. Peterha¨nselC,MaurinoVG:Photorespirationredesigned.
PlantPhysiol2011,155:49-55.
16. EisenhutM,RuthW,HaimovichM,BauweH,KaplanA, HagemannM:Thephotorespiratoryglycolatemetabolismis essentialforcyanobacteriaandmighthavebeenconveyed endosymbionticallytoplants.ProcNatlAcadSciUSA2008, 105:17199-17204.
17. HagemannM,EisenhutM,HackenbergC,BauweH:Pathwayand importanceofphotorespiratory2-phosphoglycolate
metabolismincyanobacteria.AdvExpMedBiol2010,675:91-108.
18. KebeishR,NiessenM,ThiruveedhiK,BariR,HirschH-J, RosenkranzR,Sta¨blerN,Scho¨nfeldB,KreuzalerF,Peterha¨nselC:
Chloroplasticphotorespiratorybypassincreases photosynthesisandbiomassproductioninArabidopsis thaliana.NatBiotechnol2007,25:593-599.
19.
MaierA,FahnenstichH,CaemmerervonS,EngqvistMKM, WeberAPM,Flu¨ggeU-I,MaurinoVG:Transgenicintroductionof aglycolateoxidativecycleintoA.thalianachloroplastsleads togrowthimprovement.FrontPlantSci2012,3:38.
Thismanuscriptreportsanintraplastidialshortcuttophotorespirationin which phosphoglycolateisconvertedtoCO2.TransgenicArabidopsis plants expressing thispathwaydisplay higher biomassaccumulation thanwildtypeplantsundershortdayconditions.
20.
FurbankRT:EvolutionoftheC4photosyntheticmechanism:
aretherereallythreeC4aciddecarboxylationtypes?JExpBot 2011,62:3103-3108.
ThismanuscripthighlightsthefactthatfewC4plantscanbegroupedinto distinct decarboxylation types. More frequently, the decarboxylation biochemistryisdeterminedbyenvironmentalanddevelopmentalcues.
21. SommerM,BrautigamA,WeberAPM:ThedicotyledonousNAD malicenzymeC4plantCleomegynandradisplaysage- dependentplasticityofC4decarboxylationbiochemistry.
PlantBiol(Stuttg)2012,14:621-629.
22. WeberAPM,vonCaemmererS:Plastidtransportand metabolismofC3andC4plants—comparativeanalysisand possiblebiotechnologicalexploitation.CurrOpinPlantBiol 2010,13:257-265.
23.
FurumotoT,YamaguchiT,Ohshima-IchieY,NakamuraM, Tsuchida-IwataY,ShimamuraM,OhnishiJ,HataS,GowikU, WesthoffPetal.:Aplastidialsodium-dependentpyruvate transporter.Nature2011,476:472-475.
This manuscript reports the plastidial pyruvate:sodium cotransporter BASS2.Thisisthefirstdemonstrationofasodium-drivensolutetrans- porterinplants.
24.
KinoshitaH,NagasakiJ,YoshikawaN,YamamotoA, TakitoS,KawasakiM,SugiyamaT,MiyakeH,WeberAPM, TaniguchiM:Thechloroplastic2-oxoglutarate/
malatetransporterhasdualfunctionasthemalate valveandincarbon/nitrogenmetabolism.PlantJ2011, 65:15-26.
Thispapershowsthatthechloroplastidial2-oxoglutarate/malatetrans- porterDiT1alsohasthefunctionofamalate/oxaloacetateantiporter.
25. TaniguchiM,SugiyamaT:Theexpressionof2-oxoglutarate/
malatetranslocatorinthebundle-sheathmitochondriaof Panicummiliaceum,aNAD-malicenzyme-typeC4plant,is regulatedbylightanddevelopment.PlantPhysiol1997, 114:285-293.
26.
BrickerDK,TaylorEB,SchellJC,OrsakT,BoutronA,ChenYC, CoxJE,CardonCM,VanVrankenJG,DephoureNetal.:A mitochondrialpyruvatecarrierrequiredforpyruvate uptakeinyeast,Drosophila,andhumans.Science2012, 337:96-100.
Thispaperreportsforthefirsttimethemitochondrialpyruvatecarrier, whichisrequiredfortheprovisionofthetricarboxlicacidcyclewiththe precursorpyruvate.
27.
HerzigS,RaemyE,MontessuitS,VeutheyJL,ZamboniN, WestermannB,KunjiERS,MartinouJC:Identificationand functionalexpressionofthemitochondrialpyruvatecarrier.
Science(NewYork,NY)2012,337:93-96.
Like[26],thispaperreportsforthefirsttimethemitochondrialpyruvate carrier,whichisrequiredfortheprovisionofthetricarboxlicacidcycle withtheprecursorpyruvate.
28. PriceGD,BadgerMR,vonCaemmererS:Theprospectof usingcyanobacterialbicarbonatetransporterstoimprove leafphotosynthesisinC3cropplants.PlantPhysiol2011, 155:20-26.
29. PriceGD,BadgerMR,WoodgerFJ,LongBM:Advancesin understandingthecyanobacterialCO2-concentrating- mechanism(CCM):functionalcomponentsCitransporters, diversity,geneticregulationandprospectsforengineering intoplants.JExpBot2008,59:1441-1461.
30. FluggeU-I,FischerK,GrossA,SebaldW,LottspeichF, EckerskornC:Thetriosephosphate-3-phosphoglycerate- phosphatetranslocatorfromspinachchloroplasts:nucleotide sequenceofafull-lengthcDNAcloneandimportoftheinvitro synthesizedprecursorproteinintochloroplasts.EMBOJ1989, 8:39-46.
31.
BartschO,MikkatS,HagemannM,BauweH:Anautoinhibitory domainconfersredoxregulationtomaizeglyceratekinase.
PlantPhysiol2010,153:832-840.
Thisstudyreportsforthefirsttimeaplastidialglyceratekinaseenzyme thatisregulatedbythethioredoxinsystem.Theautoinhibitorydomainis apparentlyspecifictotheenzymefromtheC4plantmaize.
32. YoungXK,McCartyRE:Assayofproton-coupledglycolate andD-glyceratetransportintochloroplastinnerenvelope membranevesiclesbystopped-flowfluorescence.
PlantPhysiol1993,101:793-799.
33. BariR,KebeishR,KalamajkaR,RademacherT,PeterhanselC:A glycolatedehydrogenaseinthemitochondriaofArabidopsis thaliana.JExpBot2004,55:623-630.
34. HowitzKT,McCartyRE:D-Glyceratetransportbythepea chloroplastglycolatecarrier:studieson[14-C]D-glycerate uptakeandD-glyceratedependentO2evolution.PlantPhysiol 1986,80:390-395.
35. ZhangL,Ha¨uslerRE,GreitenC,HajirezaeiM-R,HaferkampI, NeuhausHE,Flu¨ggeU-I,LudewigF:Overridingtheco-limiting importofcarbonandenergyintotuberamyloplastsincreases thestarchcontentandyieldoftransgenicpotatoplants.
PlantBiotechnolJ2008,6:453-464.
36. Flu¨ggeU-I,Ha¨uslerRE,LudewigF,GierthM:Theroleof transportersinsupplyingenergytoplantplastids.
JExpBot2011,62:2381-2392.
37.
ChenL-Q,QuX-Q,HouB-H,SossoD,OsorioS,FernieAR, FrommerWB:SucroseeffluxmediatedbySWEET proteinsasakeystepforphloemtransport.Science2012, 335:207-211.
Using the technology reported in [38], Chen et al. identified the elusiveleafmesophyllcellsucroseexporterprotein,whichisrequired forunloadingsucrosefromthephotosyntheticmesophyllintotheapo- plasticspace,whereitistakenupintothephloem forlongdistance transport.
38.
ChenL-Q,HouB-H,LalondeS,TakanagaH,HartungML,QuX-Q, GuoW-J,KimJ-G,UnderwoodW,ChaudhuriBetal.:Sugar transportersforintercellularexchangeandnutritionof pathogens.Nature2010,468:527-532.
Abreakthroughstudythatreportstheapplicationoffluorescentmeta- bolitenano-sensorstotheidentificationofanovelclassoftransporter proteinsthatisinvolvedinthereleaseofsugarsfromplantcellstothe apoplasticspace.
39. TegederM:Transportersforaminoacidsinplantcells:some functionsandmanyunknowns.CurrOpinPlantBiol2012, 15:315-321.
40. Bra¨utigamA,WeberAPM:Dometabolitetransportprocesses limitphotosynthesis? PlantPhysiol2011,155:43-48.
41.
Bra¨utigamA,KajalaK,WullenweberJ,SommerM,GagneulD, WeberKL,CarrKM,GowikU,MaßJ,LercherMJetal.:AnmRNA blueprintforC4photosynthesisderivedfromcomparative transcriptomicsofcloselyrelatedC3andC4species.Plant Physiol2011,155:142-156.
ToourknowledgethefirstmanuscriptemployingmRNA-Seqtotrans- speciesquantitativetranscriptomiccomparison.Severalhundredgenes are identified that are differentially expressed between species that performC3orC4photosynthesis,respectively.
42. Bra¨utigamA,Hoffmann-BenningS,Hofmann-BenningS, WeberAPM:Comparativeproteomicsofchloroplast envelopesfromC3andC4plantsrevealsspecificadaptations oftheplastidenvelopetoC4photosynthesisandcandidate proteinsrequiredformaintainingC4metabolitefluxes.
PlantPhysiol2008,148:568-579.
43. HouB-H,TakanagaH,GrossmannG,ChenL-Q,QuX-Q, JonesAM,LalondeS,SchweissgutO,WiechertW,FrommerWB:
Opticalsensorsformonitoringdynamicchangesof intracellularmetabolitelevelsinmammaliancells.NatProtoc 2011,6:1818-1833.
44.
Nour-EldinHH,AndersenTG,BurowM,MadsenSR, JørgensenME,OlsenCE,DreyerI,HedrichR,GeigerD, HalkierBA:NRT/PTRtransportersareessentialfor
translocationofglucosinolatedefencecompoundstoseeds.
Nature2012,488:531-534.
High-throughput electrophysiologicalscreenswere usedtoidentify a transporterthatisrequiredforthelong-distancetransportofglucosino- latesbetweenthesiteoftheirbiosynthesisandstorage.
