Understanding metabolite transport and metabolism in C-4 plants through RNA-seq

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Understanding metabolite transport and metabolism in C

4

plants through RNA-seq

Urte Schlu¨ter

1

, Alisandra K Denton

2

and Andrea Bra¨utigam

1,3

RNA-seq,themeasurementofsteady-stateRNAlevelsbynext generationsequencing,hasenabledquantitative

transcriptomeanalysesofcomplextraitsinmanyspecies withoutrequiringtheparallelsequencingoftheirgenomes.The complextraitofC4photosynthesis,whichincreases

photosyntheticefficiencyviaabiochemicalpumpthat concentratesCO2aroundRubisCO,hasevolvedconvergently multipletimes.Duetotheseinterestingproperties,C4

photosynthesishasbeenanalyzedinaseriesofcomparative RNA-seqprojects.Theseprojectscomparedbothspecieswith andwithouttheC4traitanddifferenttissuesororganswithina C4plant.TheRNA-seqstudieswereevaluatedbycomparingto earliersinglegenestudies.Thestudiesconfirmedthemarked changesexpectedforC4signaturegenes,butalsorevealed numerousnewplayersinC4metabolismshowingthattheC4

cycleismorecomplexthanpreviouslythought,andsuggesting modesofintegrationintotheunderlyingC3metabolism.

Addresses

1InstituteofPlantBiochemistryandClusterofExcellenceonPlant Science(CEPLAS),Heinrich-Heine-University,Universita¨tsstrasse1, D-40225Du¨sseldorf,Germany

2InstituteforBiologyI,RWTHAachenUniversity,WorringerWeg3, 52074Aachen,Germany

Correspondingauthor:Bra¨utigam,Andrea (braeutigam@ipk-gatersleben.de)

3Presentaddress:NetworkAnalysisandModelingGroup,IPK Gatersleben,Corrensstrasse3,D-06466StadtSeeland,Germany.

CurrentOpinioninPlantBiology2016,31:83–90 ThisreviewcomesfromathemedissueonPhysiologyand metabolism

EditedbyRobertTFurbankandRowanFSage ForacompleteoverviewseetheIssueandtheEditorial Availableonline12thApril2016

http://dx.doi.org/10.1016/j.pbi.2016.03.007 1369-5266/#2016ElsevierLtd.Allrightsreserved.

Introduction

ThecomplextraitofC4photosynthesisrequiresnumer- ous intra-cellular and inter-cellular transport processes [1,2].Species which haveevolved thetraitsupercharge photosynthesis bypre-fixing CO2through phosphoenol- pyruvatecarboxylase(PEPC)inthemesophyll,transport of C4metabolites to the bundle sheath, and decarbox- ylating and enrichingCO2atthesite of RubisCO.The higheraffinity ofPEPC forCO2comparedto RubisCO

lowers theCO2compensation point of theplants. This allowsC4plantstothriveinenvironmentsthatarecarbon limited, thatis,environmentswithhighlight,hightem- perature,andlow orseasonal wateravailability[3].

Bycomparingsisterspecieswithandwithoutthetraitona global scale, RNA-seq can identify trait-related differ- ences in transcript abundance. The global perspective, which tests all genes at the same time, identifies both changesthatareexpectedbasedonpreviouslypublished data(i.e.enzymeassaysorWesternBlots;summarizedin [4]) and changes that are not anticipated. Unlike the targeted approaches that existed before, RNA-seq is not limited to measuring abundance of known or expected transcripts.The downside isthatany changes happeningaftertranscriptabundancesuchastranslation efficiency,proteinstabilityandmodificationareinvisible totheapproach.TheinitialcomparativeRNA-seqpaper found that the abundance of all known C4 cycle tran- scriptsexceptmalatedehydrogenaseisindeedincreased in the C4 plant compared to a closely related C3plant [5].ThisindicatesthatthecomplextraitC4photosyn- thesisistraceableatthetranscriptabundancelevel[5].

AftertheinitialcomparisoninthegenusCleomaceae[5], theC4traithasalsobeentracedintheFlaveriaceae[6,7], the (X=9) Panicoideae [8], and in comparison of grass specieswithwiderphylogeneticspacing,rice(Ehrhartoi- deae)vsEchinocloa(X=9Panicoideae)[9]andrice(Ehr- hartoideae)vs maize(Andropogonae)[10].

C4 photosynthesis is a complex trait which consists of changesincellularandleaftissuearchitecture,changesin regulation, and changes in metabolism. The metabolic aspectof thetraitcanbedeconstructedin theC4cycle itselfconsistingofcarboxylation,transferacidgeneration, decarboxylationand regenerationmodulesand theirin- tegration in theunderlying leaf metabolism which pre- sumably resembles that of non-C4 plants (Figure 1, Table1).Originally,threetypesofC4cycleswererecog- nized,theNADP-malicenzyme(NADP-ME),theNAD- malicenzyme(NAD-ME)andthephosphoenolpyruvate (PEP) carboxykinase(PEP-CK)type [4] andthey were named according to the presence of decarboxylation enzymes[4].Thissimplisticviewwastheoreticallyques- tioned[11]atthesametimeasmultiplelinesofevidence showedamorecomplexcyclethanpreviouslyenvisioned [12–14]. As the C4 cycle changes with developmental status[13]andpossiblyalsowithbioticandabioticcon- ditions [11], it seems prudent to no longer focus on historicalprototypesbuttodescribethecycleasaseries

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84 Physiologyandmetabolism

Figure1

pyruvate malate

unknown DiT2

NADP-ME

NADPH NADP

CO2

malate

pyruvate

pyruvate malate

SO42-/Pi unknown

DIC

OAA Asp

NAD-ME NADH

NAD

CO2 SO42-/Pi

malate OAA Asp

pyruvate

+

PEP OAA

PEP-CK ADP ATP

CO2

AAC ATP

ADP

? ?

NADPH malate

MDH DIT1

OAA NADP+

amino acid Asp

AspAT DIT2

OAA oxo acid

malate

Asp

N assimilation

OAA

OAA unknown

amino acid (Ala?)

amino acid

AspAT unknown

OAA N assimilation

OAA unknown

amino acid (Ala?)

Asp Asp

oxo acid CO2

CO2

carboxylation

regeneration

decarb- oxylation C4

PEPC compartment RubisCO compartment

transfer acid generation PEPC

RubisCO (a)

pyruvate

PEP PEP

ATP Pi AMP

PPi

ATP 2 ADP

2 Pi

PPDK PPase

AMK Pi

Na+ H+

BASS2 NHD

PPT

pyruvate pyruvate

PEP

ATP Pi AMP

PPi

ATP 2 ADP

2 Pi

PPDK PPase

AMK Pi

H+

unknown PPT

PEP

pyruvate

(b) (c)

(d) (e) (f)

(g) (h)

CO2

C3

Current Opinion in Plant Biology

C4metabolismiscarriedoutbyacombinationofcarboxylation,transferacidgeneration,decarboxylationandregenerationmodules.(a)Contribution ofdifferentmodulestoC4metabolism;(b,c)regenerationofPEPbytheplastidicenzymePPDKinthemesophyll,transportofthereactionproductis performedbyPPT,theimportofpyruvatecanbemediatedbysodiumcoupledBASS2/NHDsystem(b)orbyanunknownpyruvatetransporterinthe Andropogonae(c);(d)–(f)decarboxylationofthetransfermetaboliteinthebundlesheath,NADP-MEdecarboxylation(d)islocalizedinthechloroplast withDiT2alikelymalateimportercandidateinmaizeandthepyruvateexporterunknown(d);NAD-MEdecarboxylationtakesplaceinthe

mitochondria,importofmalateispredictedtobecarriedoutbytheDICtranslocator,thepyruvateexporterisunknown(e),DecarboxylationbyPEP- CKinthecytosolislikelytodependontransportofATPfromthemitochondriabyAAC;(g,h)transfermetabolitegenerationinthemesophyll.Malate andaspartateareproducedintheplastidofthemesophyllespeciallyinspecieswithNADP-MEdecarboxylation,theirexportismediatedbyDiT1and DiT2respectively,aspartatecanalsobesynthesizedinthemitochondria,butacooperatingtransporterisstillunknown.Transportofaspartateoutof theplastidwouldrequirerecyclingoftheaminogroup,thiscouldbesupplieddirectlyfromNassimilationorbyimportofaminoacidviaanunknown mechanism.Forabbreviations,seetext.

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TranscriptomicsandC4photosynthesisSchlu¨ter,DentonandBra¨utigam85 VT_13,G. gynandravalues remapped from Kulahogluet al.(2014), leaf stage 4,F. trinerviavalues averaged from Mallmannet al.(2014),Z. maysmesopyll and bundle sheath (BS) data

from Changet al.(2012); action: E = enzyme; T = transporter; compartment: cp = chloroplast, cy = cytosol, mi = mitochondria

Module Gene

name

Compartment Action Arabidopsis IDb

Reaction P. maximum G. gynandra F. trinervia Z. mays

whole leaf

Z. mays mesophyll

Z. mays BS Regeneration

module

PPDK cp E AT4G15530 Pyruvate + 2 ATP + P!

PEP + AMP + PP

13,380 20,941 25,638 11,092 35,421 7810

AMK cp E At5g47840 AMP + ATP!2 ADP 986 2328 1916 527 4739 1018

Ppase cp E At5g09650 PP!2 P 451 1828 1138 371 579 286

BASS2 cp T At2g26900 Pyruvate (out) + Na+

(out)!pyruvate (in) + Na+(in)

2797 4298 5790 nd nd nd

NHD cp T At3g19490 H+(out) + Na+(in)!H+(in) + Na+(out) 838 1081 1592 5 7 3

PPT cp T At5g33320 PEP (in) + H+(in) + P

(out)!PEP (out) + H+(out) + P (in)

405 1633 2225 562 1498 243

Proton pyruvate transporter

cp T Unknown pyruvate (out) + H+

(out)!pyruvate (in) + H+(in)

Not needed

Not needed

Not needed

? ? ?

Carboxylation PEPC cy E At2g42600 PEP + HCO3!OAA + P 18,393 19,879 15,712 5053 9485 432

cytCA cy E Multiple

solutions

CO2+ H2O!HCO3+ OH 28,838 3081 10,493 2990 6692 281

Decarboxylation NADP-ME cp E At1g79750 Malate + NADP!pyruvate

+NADPH +CO2

1120.5a 1 4649 1334 87 12,192

DiT2/DCT cp T At5g64280 Malate (out) + X!malate (in) + X Not

needed

Not needed

212 149 7 545

pyruvate exporter

cp T Unknown Pyruvate (in) + X!pyruvate

(out) + X

Not needed

Not needed

? ? ? ?

NAD-ME mi E At2g13560 Malate + NAD!pyruvate +NADH +CO2 177.5a 2624 172a 14 7 29

DIC mi T At2g22500 Malate (out) + P or SO4

(in)!malate (in) + P or SO4

(out)

455 577 6 29 2 147

pyruvate exporter

mi T Unknown Pyruvate (in) + X!pyruvate

(out) + X

? ? Not

needed Not needed

Not needed

Not needed

PEP-CK cy E At4g37870 OAA + ATP!PEP + ADP + CO2 8819 106 42 1364 21 7801

AAC mi T At3g08580 ATP (in) + ADP (out)!ATP

(out) + ADP (in), stoichiometry not clear for P

461 578 487 83 35 115

Transfer acid generation

plAspAT cp E At4g31990 OAA + aminoacid!Asp + ketoacid 49 151 1476 436 1797 51

plMDH cp E At5g58330 OAA + NADPH!malate + NADP 632 478 6165 702 2621 95

DiT1/OMT cp T AT5G12860 OAA (out) + malate (in)!malate

(out) + OAA (in)

203 409 744 310 476 22

DiT2/DCT cp T AT5G64280 OAA (out) + Asp (in)!Asp

(out) + OAA (in)

33 153 212 149 7 545

mAspAT mi E At2g30970 OAA + aminoacid!Asp + ketoacid 16 3885 246 23 13 8

mMDH mi E AT1G53240 OAA + NADH!malate + NAD 164 721 185 77 28 104

cytAspAT cy E At1g62800 OAA + aminoacid!Asp + ketoacid 1273 5 44 24 19 91

cytMDH cy E AT1G04410 OAA + NADH!malate + NAD 735 4 5 311 463 1083

AlaAT ? E At1g17290 Pyruvate + amino acid!

alanine + ketoacid

3000 2538 2628 997 349 222

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ofmodules whichcanbelinkeddifferently indifferent plants.ThereactionsinvolvedintheC4cyclearecharac- terized by a high degree of compartmentation. In the majorityofplants,thecarboxylationanddecarboxylation modulesareseparatedintodifferentcelltypes,theme- sophyllandbundlesheath,butinavarietyofChenopdia- ceae and Hydrilla they also occur in the same cell[15].

Manyenzymesinvolvedin theC4cycleresidein orga- nelles rather than in the cytosol [4]. Their localization necessitates the presence of transport proteins which connect the reactions of the C4 cycle to the cytosol [16].Inturn,thecytosolsofthePEPCcontainingmeso- phylltissue andtheRubisCOcontainingbundlesheath tissueshare asymplasticconnection[17–20].Molecular identificationofthetransportproteinslagsbehindiden- tificationof theenzymes. Thenecessarytransport func- tionsforeachmodulewillbehighlightedinthisreview.

Modulesof theC4cycle

Regenerationmodule

The regeneration module produces the CO2 acceptor, phosphoenolpyruvate (PEP), from pyruvate and phos- phate. Itis invariablylocalized to thechloroplasts with thepyruvatephosphatedikinase(alsoknownaspyruvate orthophosphate dikinase) as the central enzyme [4,21–

23].ItproducesPEP,pyrophosphate(PP)andadenosine monophosphosphate (AMP) from pyruvate, phosphate and ATP.The module requires theaction of pyropho- sphorylase(PPase)andAMPkinase(AMK)todrivethe reactionandfunnelPPandAMPbackintometabolismas phosphate and ADP [23]. The module is only 80%

specificto themesophyllin Zeamays[24]and Sorghum bicolor[25]basedontranscriptabundances(Table1,data extracted from [24]), but also controlled at the post- transcriptionallevel[26].Twoprototypesofthemodule arecurrentlyknown(Figure1bandc).Bothusethesame enzymes but different transport proteins. All species whichimport pyruvatein asodium dependentmanner, thatisallC4speciestestedsofarwiththeexception of theAndropogonae [27]likely use theBASS2/NHD/PPT transportsystem(Figure1b).BASS2wasidentifiedasthe probablepyruvate transporter based on expression pat- ternssimilartootherC4genes,thatis,highlyexpressedin C4speciesanddifferentially expressedbetweenC3and C4sisterspecies[5,7]andsubsequently biochemically characterized [28]. It indeed transports pyruvate in exchangefor sodium,isspecific forthesodiumionover lithium, is a highly abundant protein in the sodium dependentpyruvatetransporting C4speciesand,ifmu- tatedinaC3plant,producesapyruvatetransportrelated phenotype[28].Itactsinconcertwithasodiumprotein exchanger(NHD) whichexchangesthesodiumforpro- tons.Theprotonistransportedbackoverthemembrane with the exportof PEP and import of phosphate. The transport across the chloroplast envelope thus involves three transport proteins which catalyze a net flux of pyruvate and phosphate in and PEP out with sodium

86 Physiologyandmetabolism

Table1(Continued) ModuleGene nameCompartmentActionArabidopsis IDbReactionP.maximumG.gynandraF.trinerviaZ.mays wholeleafZ.mays mesophyllZ.mays BS IntegrationTPTcpTAt5g46110TP(in)+3-PGA(out)!TP (out)+3-PGA(in)27576022355573020691551 PGKcpEAT3G127803-PGA+ATP!2,3 bisphosphoglycerate+ADP1568269821606442630774 GAP-DHcpEAT1G429702,3bisphosphoglycerate+ NADPH!TP+NADP

689645205930446303509 PDH-kinasemiEAT3G06483PDHprotein+ATP!PDH protein-phosphorylated+ATP920393118674314319 aMaybemismappedtowrongMEduringcross-speciesmapping. bRepresentativegene,notalwaysthebestblasthit.

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and protons cycling [28]. The transcripts of all three transportersarehighlyabundantinC4plantsanddiffer- entiallyexpressedcomparedtosisterC3species(Table1) [5,7,8].

TheC4Andropogonaesuchassorghumormaizeareknown toexchangepyruvateforprotonsdirectly[27]andconse- quently, neither BASS2 nor NHD can be detected as highlyabundantin maize(Table 1)[25].Insteadof the conjunct action of BASS and NHD, the Andropogonae haveanalternativetransportproteininmesophyllchlor- oplastswhich,todate,hasnotbeencharacterizedatthe molecularlevel(Figure1c,Table1).Candidatesforthe chloroplast pyruvate importer in maize were suggested basedonproteomiccomparisonofmaizeandpeachloro- plast envelopes: mesophyll envelope proteins (Mep) Mep1-4(Table2)[16].Apyruvatetransporterisexpected to behighlyabundantin maize mesophyllcells.Ofthe candidates originally proposed, Mep1 and to a lesser degree Mep3a, fit the above criterium. In A. thaliana, thetransporterorthologoustoMep1carriesglycolateand glycerate during photorespiration [29] and is named PLGG1.

Carboxylationmodule

Theproductoftheregenerationmodule,PEP,iscarbox- ylated by PEPC in the cytosol of the cell using HCO3

.PEPCisalwayshighlyupregulatedinC4species (Table 1)[5,7,8,9,30].The HCO3

for PEPCis pro- duced throughthe action of acytosolic carbonic anhy- drase (CA). Abundant cytosolic CA activity can be achieved by upregulating the cytosolic CA [5] or by retargetingthealreadyabundantplastidicCA[31,32].Its mesophyll specificity is regulated post-transcriptionally [36]. The importance of the enzyme apparently varies among species. In Flaveria bidentis, reduction of CA drastically reduced fitness under greenhouseconditions [33]butinmaize,reductionofCAonlyhasminoreffects [34].Themoduleisspecifictothemesophyll(Table1).

Notransport proteinsarerequiredfor thismodule.

Decarboxylationmodule

ThreealternativesarerealizedfordecarboxylatingtheC4

acid(Figure1d–f).Basedontheavailableglobaldatasets,

NAD-MEbasedand NADP-MEbaseddecarboxylation appeartoexcludeeachotherwhileeithercanbesupple- mented with PEP-CK based decarboxylation [5,6–

8,9,10]. NADP-ME based decarboxylation occurs in the chloroplasts and requires the import of malate and theexportofpyruvate(Figure1d).TranscriptsofNADP- ME are reliably of higher abundance in F. bidentis (Table 1) [6,7] aswellas in Z.mays,Setaria viridis,and Echinochloa glabrescens [8,9,10]. The transport protein catalyzing the import of malate and/or the export of pyruvate is unknown at the molecular level (Table 1).

The dicarboxylate transporter DiT2 (alternate name DCT)wasconsideredacandidatetransporterformalate in maize [35], however, the Flaveria trinervia DiT2 is unabletocatalyzethereactionbasedoninvitroanalysisof the transport substrates [36].DiT2 ismoderately upre- gulatedinconjunctionwithNADP-ME[6–8]andDiT2 is strongly expressed in maize bundle sheath cells (Table1)andS.bicolorbundlesheathcells[25].Mutant analysisofmaizeshowedsymptomsconsistentwitharole in malateimportintheC4cycle[37]raisingtheques- tionwhethertheoriginalassaywasunsuitablefordetect- ingtheexchange[36]orwhetherFlaveriaandmaizemay use different transporters (Figure 1d). The molecular identity of the pyruvate exporter is currently unknown but it cannot be excluded that pyruvate may rely on diffusiontopassthechloroplastmembraneinitselectro- neutral form, pyruvic acid [38,39], especially given the high concentrations of pyruvate generated by NADP- ME. The module is specific to the bundle sheath (Table 1).

The NAD-ME based decarboxylation module is built around the mitochondrial NAD-ME which is highly upregulated in theC4species usingthis type of decar- boxylation (Figure 1e) [5,8,40]. Its high expression is accompaniedbytheexpressionofadicarboxylatecarrier, DIC(Table1)[5,8].Amongthemitochondrialcarriers thatacceptmalateorOAA,DICisuniqueasitcatalyzes netcarbonimportasmalate/phosphateormalate/sulfate exchange[41].Incaseof phosphateexchange,theelec- tricalfieldiscapableofdrivingtheexchangeasmalateis negatively charged twice and phosphate is negatively charged three times. The resultingcytosolic phosphate

Table2

Candidatetransportersforreactionuncharacterizedatthemolecularlevel;alldatagivenasreadspermillion;P.maximumvaluestaken fromBra¨utigametal.(2014),Z.mayswholeleafvaluestakenfromSekhonetal.(2011),leafVT_13,G.gynandravaluesremappedfrom Kulahogluetal.(2014),leafstage4,F.trinerviavaluesaveragedfromMallmannetal.(2014),Z.maysmesopyllandbundlesheath(BS) datafromChangetal.(2012);action:E=enzyme;T=transporter

Originalname AGI MaizeID P.maximum G.gynandra F.trinervia Z.mays wholeleaf

Z.maysM Z.maysBS

Mep1 At1g32080 GRMZM5G873519 103 172 128 345 340 535

Mep2 At5g23890 GRMZM2G077222 38 200 169 32 0 0

Mep3 At5g12470 GRMZM2G138258 22

185 1072 498 20 2734

Mep4 At5g12470 GRMZM2G305851 3 33 113 9

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orsulfate needstobereimported, likelyas protonsym- port[8].IfinsteadtheC4acidisimportedintothebundle sheath mitochondria as aspartate, the import protein remains unknown as the DICs of Arabidopsis do not acceptaminoacids[41].

The decarboxylation yields mitochondrial pyruvate which hasto beexported to thecytosol. The pyruvate exporteriscurrentlynotknown;itisfeasiblethatpyru- vatecrossesthemitochondrialmembraneaspyruvicacid by diffusion because at least at high concentrations, pyruvatecanmovebydiffusion in vesicles[38].UCP1, upregulated in both NAD-ME and NADP-ME plants maintainsredoxpoiseinphotosynthetictissues[42],but nootherknownmitochondrialtransportproteinisconsis- tentlyupregulatedintheC4speciesassayedbyRNA-seq.

ThethirddecarboxylationmoduleisrunbyPEP-CKand occursinthecytosol(Figure1f).TheactionofPEP-CK requiresoneATPperreactionwhichislikelysuppliedby themitochondriaas theplastidic ATPexchangerisnot relevantduringday-timemetabolism[43].ExportofATP ismediatedbyAAC[44],agenetranscriptionally abun- dantin manyspecies(Table 1)but specificallyupregu- latedintheC4speciesMegathyrsusmaximuswhichreliesto alargedegreeonPEP-CK[8].Alternatively,PEPmaybe metabolized to pyruvate directly by cytosolic pyruvate kinasewhichwillsupplytheATPforthedecarboxylation reactionmaking theATPexportfrommitochondriaun- necessary.

Transferacidgenerationmodule

TheOAAgeneratedbyPEPCinthemesophyllisimme- diateconvertedtomalateoraspartatebeforetransferand the pyruvate generated by MEs in the bundle sheath (Figure 1d and e) can be converted to alanine before transfer. The localization and role of the transfer acid generationisnotwellcharacterized.

MostC4speciesanalyzedharboratranscriptionallyhighly abundant alanine aminotransferase (AlaAT) (Table 1) [5,7,8] of which the localization is unclear. In dicots, theAlaATappearstobeorganelle-localizedinwhichcase additional transport processes were required. In maize, AlaAT is evenly distributed between mesophyll and bundlesheath(Table1).

Plants containAspAT and MDH in chloroplasts, mito- chondria and the cytosol and each can be upregulated and/or abundantin C4species (Table 1).All C4 plants analyzedtodatewhichuseNADP-MEbaseddecarbox- ylationshowchloroplast-dependentconversionreactions (Figure 1g). OAA is reduced in the chloroplasts after counter-exchange withmalate bydicarboxylate translo- cator1(DiT1)acrosstheplastidenvelope[45].Alterna- tively, the OAA is converted to aspartate (Asp) in chloroplastsbyplastidicaspartatetransaminase(AspAT)

[12,46]afterimportbyDiT2[36]inexchangeforaspar- tate.Bothenzymesandtransportersaremoderatelyupre- gulated [7]; in maize, AspAT, MDH, and DiT1 are mesophyllspecificwhileDiT2isbundlesheath specific (Table1).Thetransaminationreactionrequiresanamino groupdonorinthechloroplastwhichmaycomedirectly fromtheNassimilationorfromaplastidicAlaATreaction realizingtheregenerationofpyruvate.InthemixedPEP- CK/NAD-ME M. maximus, the cytosolic AspAT and MDHareabundant(Table1)[8].TheNAD-MEspecies G. gynandra highly expresses a mitochondria localized AspAT [5,13]. It is unclear which transport protein catalyzestheexchangeof aspartateandOAAacrossthe mitochondrial envelope (Figure 1h); the DIC does not transportaminoacids[41],andforDTCthetransportof aminoacidswas nottested[47].

Metabolicintegration

TheC4cyclerunsatopC3metabolism,interactswithit, and creates different environments in mesophyll and bundle sheath cells all of which require fine tuning.

For example, abundant expression of TPT [5,16]

(Table1)couplesthepartsoftheCalvinBensonBassham cycle localized in the mesophyll and bundle sheath [24,48].TheshuttlemayalsotransferATPandreducing equivalents in the form of triosephosphates [16]. C4

metabolismcreateslargepoolsofthetransfermetabolites whichneedtobeprotectedfromtheunderlyingmetabo- lism,forexamplebyreducingentryintheTCAcyclevia pyruvatedehydrogenasecomplexkinase[8].Integration intotheunderlyingmetabolismmaybedifferentnotonly basedtheuseofdecarboxylationenzymeineachspecies but mayalso carry further speciesspecific solutions for metabolic adjustment. Different comparative RNA-seq papers have reported a range of possible adaptations including reduction of photorespiration in all systems studiedtodate[8,9],reductioninaminoacidandprotein synthesisinsomespecies[5,7,9]andchangesinamino acidmetabolismforG.gynandropsisandM.maximus [8].

The reductionin photorespiratorygene expression was suggestedasthemolecularchangewhichfixestheC4trait [49].The connectionofthetwo celltypesand efficient intercellulartransfer is critical [8], but so far understu- died.

Summary andoutlook

RNA-seqhascontributedtothemolecularidentification ofnovel[28]andknown[5,8]transportersof theC4

cycleand identified theenzymesof theC4cycleatthe molecularlevelinmanyspecies(Table1).Theblueprint oftheC4cycleiscomplete for NAD-MEandPEP-CK module (Figure 1e and f) [8], but less well defined for AndropogonaeNADP-MEspeciesasthemesophyllpy- ruvate transporter remains unknown (Figure 1b). Al- though the genes involved in transfer acid generation appear defined,their intra-cellularand inter-cellularlo- calizationand potentialneedfor additional transporters

88 Physiologyandmetabolism

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remain unclear. Additional RNA-seqexperiments may define the modules ever more clearly for new C4

species while combined RNA-seq and genome-seq experiments will move the analyses to the next step:

identifying the regulatorycircuits underlying the high abundance detected byRNA-seq. Expressionpatterns ofC4cyclegenesaresimilartothoseofphotosynthetic genes [30,50,51] alreadysuggesting a common regula- tory system.

References and recommendedreading

Papersofparticularinterest,publishedwithintheperiodofreview, havebeenhighlightedas:

ofspecialinterest ofoutstandinginterest

1. WeberAPM,vonCaemmererS:Plastidtransportand metabolismofC(3)andC(4)plantscomparativeanalysisand possiblebiotechnologicalexploitation.CurrOpinPlantBiol 2010,13:257-265.

2. Bra¨utigamA,WeberAPM: InTransportProcessesConnecting theReactionsofC4Photosynthesis,vol.32.Editedby

RaghavendraAS,SageRF.Springer;2011.

3. OsborneCP,SackL:EvolutionofC(4)plants:anewhypothesis foraninteractionofCO(2)andwaterrelationsmediatedby planthydraulics.PhilosTransRSocB-BiolSci2012, 367:583-600.

4. HatchMD:C-4photosynthesisauniqueblendofmodified biochemistry,anatomyandultrastructure.BiochimBiophys Acta1987,895:81-106.

5.

Bra¨utigamA,KajalaK,WullenweberJ,SommerM,GagneulD, WeberKL,CarrKM,GowikU,MassJ,LercherMJetal.:AnmRNA blueprintforC4photosynthesisderivedfromcomparative transcriptomicsofcloselyrelatedC3andC4species.Plant Physiol2011,155:142-156.

TheinitialtranscriptomicspaperdemonstratesthattheC4traitcanbe tracedatthetranscriptionallevel.

6. MallmannJ,HeckmannD,BrautigamA,LercherMJ,WeberAPM, WesthoffP,GowikU:Theroleofphotorespirationduringthe evolutionofC-4photosynthesisinthegenusFlaveria.Elife 2014:3.

7. GowikU,BrautigamA,WeberKL,WeberAPM,WesthoffP:

EvolutionofC4photosynthesisinthegenusFlaveria:how manyandwhichgenesdoesittaketomakeC4? PlantCell 2011,23:2087-2105.

8. Bra¨utigamA,SchlieskyS,Ku¨lahogluC,OsborneCP,WeberAPM:

TowardsanintegrativemodelofC4photosyntheticsubtypes:

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