plants Review Nitrogen Assimilation, Abiotic Stress and Glucose 6-Phosphate Dehydrogenase: The Full Circle of Reductants SergioEsposito DipartimentodiBiologia-UniversitàdiNapoli“FedericoII”,ComplessoUniversitariodiMonteSant’Angelo, ViaCinthia4,Naples80126,Italy;[email protected];Tel.:+39-081-679124;Fax:+39-081-679233 AcademicEditor:MaurizioChiurazzi Received:3April2016;Accepted:3May2016;Published:11May2016 Abstract: Glucose 6 phosphate dehydrogenase (G6PDH; EC 1.1.1.49) is well-known as the main regulatory enzyme of the oxidative pentose phosphate pathway (OPPP) in living organisms. Namely, in Planta, different G6PDH isoforms may occur, generally localized in cytosol and plastids/chloroplasts. These enzymes are differently regulated by distinct mechanisms, still far from being defined in detail. In the last decades, a pivotal function for plant G6PDHs during theassimilationofnitrogen,providingreductantsforenzymesinvolvedinnitratereductionand ammoniumassimilation,hasbeendescribed. Morerecently,severalstudieshavesuggestedamain roleofG6PDHtocounteractdifferentstressconditions,amongthesesalinityanddrought,withthe involvement of an ABA depending signal. In the last few years, this recognized vision has been greatlywidened,duetostudiesclearlyshowingthenon-conventionalsubcellularlocalizationofthe differentG6PDHs,andthepeculiarregulationofthedifferentisoforms. Thewholebodyofthese considerationssuggestsacentralquestion: howdotheplantcellsdistributethereductantscoming fromG6PDHandbalancetheirequilibrium? Thisreviewexploresthepresentknowledgeaboutthese mechanisms,inordertoproposeaschemeofdistributionofreductantsproducedbyG6PDHduring nitrogenassimilationandstress. Keywords: oxidative pentose phosphate pathway; redox regulation; nitrogen assimilation; abioticstress 1. TheRolesofOPPPandItsRegulationbyG6PDHinPlanta Theoxidativepentosephosphatepathway(OPPP)isanalmostubiquitouspathwaypresentinall EukaryaandmostBacteria[1];onlyArcheaappeartobemissingacompleteOPPP[2]. Thewholeprocessconsistsoftheoxidationofglucose-6-phosphate(G6P)topentose-P,byevolving oneCO moleculeandreducingtwomoleculesofNADP+toNADPH(Figure1);ithasbeenestimated 2 that 15% to 30% of hexose phosphate oxidized to glyceraldehyde-3P in a plant cell is processed byOPPP. Thewholepathwayhasbeenconsideredforalongtimetoplaypivotalroleincellmetabolism, representingthecentralpointofmanycellularprocesses,suchasthesupplyofcarbonskeletonsfor nucleotidesynthesis;and,asinalleukaryoticcells,beingamajorsourceofNADPH;OPPPiscriticalto maintainredoxbalanceunderstresssituations,e.g.,duringcellproliferation,inageing,andincancer cells[3]. Plants2016,5,24;doi:10.3390/plants5020024 www.mdpi.com/journal/plants Plants2016,5, 24 2of14 Plants 2016, 5, 24 2 of 13 (A) (B) Figure 1. Design of conventional (A) and alternative (B) oxidative pentose phosphate pathway Figure1.Designofconventional(A)andalternative(B)oxidativepentosephosphatepathway(OPPP) (OPPP) according to [1]. Intermediates peculiar of the OPPP are highlighted in orange; intermediates accordingto[1].IntermediatespeculiaroftheOPPParehighlightedinorange;intermediatescommon common to glycolysis are highlighted in light blue; intermediates common to Calvin–Benson cycle toglycolysisarehighlightedinlightblue;intermediatescommontoCalvin–Bensoncyclearehighlighted ainreli ghhigthglriegehnt.eEdn izny mligehsta grereheignh. lEignhztyemdiens yaerlelo hwig.hClOighetveodl vined yiesllhoiwgh. lCigOht2e edvionlvreedda ins dhiNgAhlDigPhHtedre dinu rceedd 2 and NADPH reduced in blue. To avoid confusion, the inter-conversion of glyceraldehyde-3P and inblue. Toavoidconfusion, theinter-conversionofglyceraldehyde-3Panddi-hydroxy-acetone-P di-hydroxy-acetone-P by triose phosphate isomerase (TPI; EC 5.3.1.1) is omitted. List of enzymes by triose phosphate isomerase (TPI; EC 5.3.1.1) is omitted. List of enzymes abbreviations: abbreviations: G6PDH, Glucose-6-phosphate 1-dehydrogenase (EC 1.1.1.49); 6PGL, G6PDH, Glucose-6-phosphate 1-dehydrogenase (EC 1.1.1.49); 6PGL, 6-Phosphogluconolactonase 6-Phosphogluconolactonase (EC 3.1.1.31); 6PGDH, 6-Phosphogluconate dehydrogenase (EC3.1.1.31);6PGDH,6-Phosphogluconatedehydrogenase(decarboxylating)(EC1.1.1.44);R5P-ISO, (Rdiebcoasreb-o5-xpyhlaotsinpgh)a te(EiCso m1.e1r.a1s.4e4()E; CR55.P3-.1IS.6O);, RR5iPb-oEsPeI-,5R-pihbuoslopshea-t5e- pihsoosmpehraatsee3 -(eEpCim 5e.r3a.1se.6()E; CR55P.1-.E3.P1I),; TRAib,uTlorasne-s5a-lpdhoolasspeh(aEteC 32-e.2p.i1m.2e);raTsKe ,(ETCra 5n.s1k.3e.t1o)l;a TsAe,( ETCran2.s2a.l1d.1o)l;asHeP (EI,CH 2e.x2o.1s.e2-)6; -TpKh,o Tsrpahnastkeeitsoolamsee r(aEsCe (2E.2C.15.1.3);. 1H.9P).I,M Hoedxiofiseed-6f-rpohmos[p1]h.ate isomerase (EC 5.3.1.9). Modified from [1]. Plants2016,5, 24 3of14 PartoftheintermediatesofOPPP(glyceraldehyde-3Pandfructose-6P)aresharedwithglycolysis, and,inphotosyntheticorganisms,someothers(e.g.,erythrose-4Pandpentose-P)arecommontothe Calvin–Bensoncycleaswell(Figure1);moreover,inplantcells,anot-secondaryroleisgivenbythe furnishingofE4Pandtriose-Pforshikimatepathwayduringchlorophyllbiosynthesis. Thisgeneratesaverycomplexwebofmetabolicpathways,apparentlynotstrictlyrelated,andon theotherhand,stillfarfrombeingfullyelucidatedintheirregulation,relativeactivities,andfluxes. ThefirstpartoftheOPPPcanbedescribedasacharacteristicirreversibleoxidativephase,inwhich CO hasevolvedfromG6P,andNADP+reducedtoNADPH;afollowingreversiblepartre-generates 2 hexose-Pstartingfrompentose-P. The initial reactions are carried out by glucose-6P dehydrogenase (EC 1.1.1.49-G6PDH), producing6-phospho-gluconolactone,whichisthenhydrolyzedbya6-phosphogluconolactonase (6PGL-EC3.1.1.31)to6-phosphogluconicacid;successively,aphosphogluconicaciddehydrogenase (EC1.1.1.44-6PGDH)detachesaCO molecule,reducingaNADP+toNADPH,thusformingribose-5P. 2 Thesereactionsareregulatedandlimitedbythefirststep: G6PDHreactionisthecontrollingenzyme ofthewholepathway,giventhatitsactivityisabletopacethefullcyclerate(Figure1). Thefollowingstepsoftheregeneratingphaseareplayedbyribulose-5Pepimerase,ribose-5P isomerase,transketolaseandtransaldolase,reconvertingdifferentthree-toseven-(orpossiblyeight-) carbonphosphorylatedsugars;allofthesereactionsaregenerallyconsideredneartoequilibrium[1]. Inplants,theOPPPsubcellularlocalizationsuggestsacomplexnetworkofcoordinationofcarbon pathways in cells: although the cytosolic OPPP cycle represents the major part of the measured activity(about60%–85%ofthetotal,[4]),theexistenceofacompleteOPPPconfinedintheplastidial compartmenthasbeenwidelydemonstrated. Interestingly,thisschemeisnotalwaysconfirmedinotherorganismspossessingphotosynthesis, suchasDiatoms,whereonlythecytosolicOPPPispresent,andmostoftheenzymesofthepathway areabsentbythesepeculiarplastids[5]. Itshouldbeemphasizedthatmanystudieshavedemonstratedthat,inplantcells,thecytosolic OPPP is not always complete, and, apart from G6PDH and 6PGDH, some of the enzymes of the regenerativesegmentofthepathwaymightbemissinginthecytosol[6]. Ontheotherhand,relative tocompartmentedOPPP,atleasttwodistinctG6PDHsarepresentinhigherplants,withdifferent regulatoryproperties,thussuggestingthatchloroplasts,andheterotrophicplastidscouldbeequipped withdifferentlymodulatedOPPPs. 2. G6PDHIsoformsinPlants Plants’G6PDHisanactivehomotetramer(200–250kDa)orhomodimer(100–120kDa)formedby subunits(50–60kDa). Theassemblyofthesubunitsispossiblyplayedbysalinebridgesasdescribed forhumanG6PDH:thisstructureisstabilizedbyNADP+;whentheNADP+/NADPHratioislow,the enzymesplitsintoitsinactivemonomers[7]. Thecomparisonof44aminoacidsequencesencodingforG6PDHfromBacteria,algae,Fungi, Metazoa and Planta, generates a phylogenetic tree organized in different main branches: the first comprisesG6PDHsfromProcarya,diatoms,Fungiandanimals,localizedinthecytosol;anadjacent branchwithallthecytosolicisoformsfromplants. Asecondmainbranch, clusteringalltheplant plastidic G6PDHs, splits in four different arms: P1-G6PDH (chloroplastic isoform), green algae chloroplasticisoforms,P0isoforms(itsrolewillbediscussedlater)andfinallyP2-G6PDHs(plastidic isoform)(Figure2). ItisinterestingtounderlinethedistantpositionofDiatoms’G6PDHandRhodophyta’sG6PDH inthistree: itisworthrememberingthatHeterokontophytachloroplastsoriginatedbyasecondary endosymbiosiseventwitharedalga(orastrictlyrelatedunknownorganism). InDiatoms,theOPPP issolelycytosolic[5];theGaldieraG6PDHsequenceherereportedislocatedinthechloroplasts[8],but clusteredwithcy-G6PDHs(evenifveryclosetoPlantaeP1-G6PDH)(Figure2). Plants2016,5, 24 4of14 Plants 2016, 5, 24 4 of 13 CCoommmmoonnllyy,, ththeeG 6GPD6PHDsHeq useenqcueepnrcees enptrsesthenettsy ptihcael mtyoptiifcsaol fdmeohtyifdsr oogfe nadseehsy:dAroRgoesnsmasaens-: foAld Rmoosstimfaonn-fothlde mNo-ttiefr moni ntuhse, Na-ntdermNiAnuDsP, +a-nbdin NdiAnDgPs+i-tbeinwdeinreg sfoitue nwde.re Tfhouenpdu. tTahtiev epuatcattiivvee raecgtiivoen r(eYgRioIDn H(YYRLIDGHKEY)LGprKeEse)n ptrseasetnytrso as ytyl-rroessyidl-urees,idwuhei,c whhisicdh iisst idnicsttiivnectoivfec oyft ocsyotoliscoilsico fisoorfmorsm, isn, sitnesatdeaodf oaf pah pehneynlayllaanlainnein,ec,h cahraacratecrtiesrtiisctiocf ocfo cmopmaprtamrtemnetendteidso ifsoorfmorsm. s. FFiigguurree 22.. PPhhyylologgeenneetitci ctrtereee ooff GG66PPDDHH iissooffoorrmmss ffrroomm vvaarriioouuss lliivviinngg oorrggaanniissmmss.. BBluluee aanndd vviioolleett hhiigghhlilgighhtsts ddeessiiggnnaattee ccyyttoossoolilcic iissooffoorrmmss ((ssiimmppllyy iinnddiiccaatteedd aass GG66PPDDHH iinn BBaacctteerriiaa)).. GGrreeeenn hhiigghhlliigghhtt iinnddiiccaatteess tthheec hclholroorpolpaslatisctiPc1 Pis1o foisromfos.rmYesl.l oYwelinlodwic aitnesditchaetecas tatlhyeti ccaalltyaliyntaicctailvlye Pi0n-aiscotifvoerm Ps0.-Oisroafnogremfso.r theplastidialP2-G6PDHisoforms.RedhighlightisforRedAlgaeisoform.Otherdetailsinthetext. Orange for the plastidial P2-G6PDH isoforms. Red highlight is for Red Algae isoform. Other details in the text. 2.1. CytosolicG6PDH(Cy-G6PDH) 2.1. Cytosolic G6PDH (Cy-G6PDH) Generally,inhigherplantsatleasttwocytosolicisoformsarepresent,differentlyexpressedin varioGuesnteisrsaulleys, [in9, 1h0ig].her plants at least two cytosolic isoforms are present, differently expressed in variouTsh teisbsuioecsh [e9m,10ic]a. l properties of cy-G6PDH have been studied in potato [11] and in barley (HorTdehuem bviuolcghaerme),icbaolt hporonppeurtriiefise dofe nczyy-Gm6ePfDroHm hroaovtes [b1e2e]na nsdturdeiceodm ibni npaonttaetnoz y[1m1]e [a1n3d]. in barley (HordTeuhme vcuyltgoasroel)i,c bGot6hP oDnH pugreinfieerda lelnyzsyhmoew fsroaml orowotsse [n1s2i]t iavnitdy rteocormedbuincainngt epnozwymere; [t1h3e]. enzyme is reguTlahtee dcbyytoNsoAliDc PGH6P/DNHA DgPe+neraratilolya nshdoiwtiss cao mlopwe tsiteinvseiltyiviintyh ibtoit erdedbuycNinAg DpPoHwe[1r1; ,t1h3e]. enzyme is regulaAterde gbuyl aNtiAonDoPfHc/yNtoAsDolPic+ irsaotfioor amnda citti ivsi tcyombypseutigtiavre-slye ninsihnigbimteedc bhyan NisAmDhPaHs b[1e1e,n13s]u. ggested: this wouAld rceognutlraotliorno ootf nciytrtoosgoelnica insodfosurmlfu arcatisvsiitmy iblayt sioungaorn-sdenepsienngd meneccehoanficsamrb hoanss btaeteuns soufgtgheestpelda:n tth,iisn woroduelrd tcoomntarnoal groeotth eniatrmoigneona acnidd ssyunlftuhre saisss[i1m3]il.ation on dependence of carbon status of the plant, in orderR toec menatnsatgued itehse daemminoon satcriadte sythnethaecstiivs a[1ti3o]n. ofcy-G6PDHfromArabidopsis. thaliana(A.thaliana)is contRroelcleedntb sytuthdeieps hdoesmphoonrsytrlaatteio tnheo faTcthivr-a4t6io7nb yofG clyy-cGo6gPeDnHSy fnrtohmas AerKaibnidaospes3is(. AthSaKliaαn)au (pAo. nthsaallitanstar)e isss ctoonsturostllaeidn tbhye itnhcer epahseodsprheoqruyelsattioofnr eodfu Tctharn-4ts6[71 4b]y. Glycogen Synthase Kinase 3 (ASKα) upon salt stress to sustain the increased request of reductants [14]. 2.2. ChloroplasticG6PDH(P1-G6PDH) 2.2. Chloroplastic G6PDH (P1-G6PDH) ThepresenceofaG6PDHactivityconfinedinchloroplastshasbeenwidelydemonstrated[15,16]: thiscThhleo ropprelassetniccee nozfy ma eG,6gPenDeHra lalyctdiveifityn ecdoansfiPn1e-dG 6inP DcHhl,oirsorpelvaesrtssi bhlyasi nbheiebnit ewdibdyellyig hdtetmoognusatrraantetede [a1n5,1e6ffi]:c tihenist cphhloortoopsylanstthice seins,zyanmde,a gveonideraalfluyt dileefcinyecdle a;sin Pt1h-Ge6dPaDrkHth, iiss rienvheibrsiitbiolyn iinshriebmitoedve bdy, laingdhtt thoe gOuPaPraPnitseea catniv eaftfeicdietnotp prhoodtuocseyrnetdhuesciisn,g aneqdu aivvoailden at sfu[1ti6l]e. cycle; in the dark this inhibition is removed, and the OPPP is activated to produce reducing equivalents [16]. Plants2016,5, 24 5of14 LightinhibitionofthechloroplasticG6PDHisexplainedbytheredoxmodificationsthroughthe ferredoxin/thioredoxinsystem[1]: ithasbeendemonstratedthatthisregulationiscarriedoutbyat leasttwocysteineresiduesontheN-terminusoftheactiveproteinsequence[17]. ChloroplasticP1-G6PDHhasbeenshowntobehighlysensitivetoNADPH(Ki <8µM), NADPH actingasacompetitiveinhibitor[11,18];moreover,thechloroplasticisoformiscontrolledbyanumber ofotherfactors,suchasNADPH/NADP+ratio,Mg++andribulose-5P[4,16]. 2.3. PlastidialG6PDH(P2-G6PDH) AsecondG6PDHisoformisdetectableinplantrootsuponnitrogensupply;thisactivityis,at leastinpart,responsibleofthedoublingofOPPPrateinroottissues;molecularstudiesconfirmedthe existenceofthissecond,plastidialenzyme[12,19,20]. ThisplastidialG6PDH(referredasP2-G6PDH)isexpressedinnearlyallplantorgans[9,21],andit exhibitsdistinctkineticpropertieswithrespecttobothcytosolicandchloroplasticG6PDHs[12,20–22]. TheactivityofthisisoformistightlymodifieduponreductionpossiblyinasimilarwayasforP1-G6PDH, evenifthepossiblyofaspecificthioredoxinclassinteractingwithP2-G6PDHcouldbesuggested[23,24]. A detailed kinetics study indicated that plastidial P2-G6PDH reaction follows an ordered sequential mechanism [25], and that this activity is primarily regulated by the NADPH/NADP+ ratio, [12,26]; interestingly, the different sensitivity to NADPH represents the peculiar difference betweenchloroplasticandplastidialG6PDHs: P2-G6PDHisconsiderablerefractorytoinhibitionby NADPH,showingaKi >40µM,avalue5–10foldhigherwithrespecttothevaluesknownfor NADPH cytosolicandchloroplasticisoforms[4]. 2.4. TheEnigmaticP0-G6PDH AgenomewideanalysisinA.thalianadepictedacomplexpatternforG6PDHisoformsinhigher plants,withgenesencodingforonechloroplastic,twoplastidic,twocytosolicG6PDH;tothosefully functionalencodingsequences,asingulargene(namedG6PD4)encodingforanon-functionalenzyme was identified [9]. At the time, this was classified as a pseudo gene, but further research found animportantroleforthisprotein,nowdescribedasP0-G6PDH. Ontheotherhand,previousfindingsintriguinglysuggestedapossiblepresenceoftheOPPPinthe peroxisomes[27,28]. Recently,ithasbeenelegantlydemonstratedthat,uponoxidativestress,thereis anassemblyinthecytosolofP1-G6PDHsubunitswithP0-G6PDH[29]:Thesehetero-dimersareableto entertheperoxisomes,duetothepresence,intheC-terminalofP0-G6PDH,ofaperoxisometargeting sequence(PTS),exposedforpreferentialtargetingintheseorganelles. Similarly,specific6PGLand 6PGDHhavebeenrecentlylocalizedinArabidopsisperoxisomes:thiswouldcreateanefficientNADPH producingmachineryintheperoxisomesuponstressorforpossiblespecificNADPHrequirementin theseorganelles[28–31]. 2.5. TheSubcellularG6PDHLocalizationIsNotObviousinPlantCells Despiteofthecategorizationofthedifferentisoforms,manyevidenceswouldsuggestthatthe subcellularandtissuelocalizationofG6PDHsinplantcellsarenotconstant,orobvious. Asamajor example,inA.thaliana,thechloroplastic(P1-G6PDH,encodedbyG6PD1),andoneofthetwoplastidial P2-G6PDHs(encodedbyG6PD2)showkineticpropertiesthatareoppositeofthosedescribedforall theotherplantsstudiedsofar,G6PDH1exhibitingalowNADPHsensitivity,andG6PDH2ahigh sensitivitytoNADPH[9]. Further,theexclusiveoccurrenceofP1-G6PDHinchloroplasts,whichhasbeendescribedwithout uncertainness for decades, has been disputed in the last few years for the newly demonstrated interactionwithP0-G6PDH[29,31]. Recent studies still debate about the localization of plastidial isoform: transcript analysis demonstrated that P2-G6PDH is expressed in roots and non photosynthetic tissues (as expected) inpotatoandArabidopsis[21]. Ontheotherhand,bothtranscriptsandabundanceofP2-G6PDHcould Plants2016,5, 24 6of14 bedetected,evenifalowerextent,ingreentissues[4,21].Atmoment,itisfarfrombeingdemonstrated ifthisisoformpresentintheleavesreallyoccursinphotosyntheticcellsorindifferenttissuesofthe leaves(e.g.,epidermis,stomata,phloem,etc.). 3. G6PDHsandNitrogenAssimilation Themainrouteofnitrogenassimilationinplanttissuesinvolvestheglutaminesynthetase(GS; EC 6.1.1.3)/glutamate synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14) cycle[32]. Inhigherplants,GSispresentasaplastidialenzyme,andseveralcytosolicisoformsaredifferently distributedintissues. Ontheotherhand,Fd-GOGATandNADH-GOGATisoformsarebothlocated intheplastids. InleaftissuesthereisthebulkofFd-GOGATactivity,involvedinphotorespiratory ammoniaassimilation;NADH-GOGATisthemajorisoformintheroots,andisinvolvedintheprimary nitrogenassimilation[32]. Therelationshipsbetweencarbonmetabolismandnitrogenassimilationhavebeenwidelystudied, andmanystudiesconcernedtheeffectsofammoniumonrespirationandOPPP[4,19,20,26,33]. Thereductionofnitratetoammoniumandtheformationofglutamatebothrequirereductants, suppliedbyphotosyntheticprocessinthelight;inthedark,andinheterotrophictissues,theoxidative processesgeneratethereducingpowerandATP. Nitritereductaserequiresferredoxinaselectrondonor;inbarleyrootsferredoxin-ADP+reductase differsfromitsleafcounterpart[33]:Asconsequence,highlevelsofNADPHarenotessentialtoreduce ferredoxin,suggestingthatG6PDHplaysacentralroleduringnitrateassimilationinheterotrophic tissues[19]. Intriguingly, bothG6PDHand6PGDHactivitiesincreasedundernitrateassimilation, stronglysuggestingacoordinationbetweenthenitrogenmetabolismandOPPP[34]. Conclusively, nitritereductionisdirectlyconnectedtoCO evolutionfromC1ofG6P,clearly 2 indicatingthatOPPPisactivatedduringnitrogenassimilation[19]. Overwhelmingly, the promoter sequences of nitrite reductase, FNR, ferredoxin and G6PDH all present the same NIT-2 motif, a nitrogen metabolism regulating factor [35], confirming the molecularcoordinationoftheexpressionofbothG6PDHandenzymesandproteinsdirectlyinvolved innitrogenmetabolism. The tight relationship between OPPP and nitrogen metabolism is not confined within nitrate reductionprocess: GOGATactivityisclearlysupportedbyplastidicG6PDH,whichisabletosatisfy theincreasedrequestofreducingpoweruponammoniumassimilation. InN-starvedbarleyroots,onlythecytosolicisoformcanbedetectedbyWesternblotting,[4,26], andareducedexpressionoftheplastidicisoformwasobserved[34];supplyofnitrogenresultsinaprompt increaseofG6PDHactivity,partiallyascribingtotheanewlysynthetizedplastidialisoform[4,26]. FurtherevidenceofthestrictcorrelationbetweenplastidialOPPPandGOGATactivityhasbeen given by experiments on isolated organelles: root plastids are able to synthesize glutamate using GOGATonlyifsubstratesoftheOPPP(e.g.,G6Porpentose-P)arepresent[19,20];thekineticofthe G6P-dependentglutamatesynthesisinrootplastidssuggestedthatG6PDHisabletosupportmaximal levelsofglutamatesynthesisevenundersub-saturatingconditions[19,20]. Basedontheevidencesgivenbyanumberofpapers,ageneralschemeforthestrictrelationship between G6PDHs and GOGAT isoforms can be hypothesized: nitrogen caused an increase of NADH-GOGAT activity in the roots [4]; the reducing power in these tissues for NADH-GOGAT islikelyfurnishedbyP2-G6PDH,whichisinturninducedbynitrogen(possiblyunderthestimuliof glutaminelevels,[26]). The low sensitivity to NADPH inhibition would help to sustain P2-G6PDH for the supply of reductantsduringnitrogenassimilationintheroots;andinthelight/darktransitionintheleaves[4,34–36]. In the leaves, P1- and P2-G6PDHs would supply reducing power for Fd-GOGAT enzyme, in thefirstdarkphase,whenthehaltinphotosynthesisproduceshighammoniumlevelsduebothto photorespirationanddeaminationofglutamineandasparagine[34,37]. Plants2016,5, 24 7of14 Intriguingly,itcouldbearguedthat-uponhighnutrientavailability-theabilityofP2-G6PDHto maintainpartofitsactivityunderhighNADPH/NADP+ratioswouldallowthesupportthenitrogen assimilationinthelight[3,4,36]. ThestrictrelationshipbetweenOPPPandnitrogenmetabolismhasbeenrecentlyinvestigated by a genetic approach, demonstrating that N assimilation genes are expressed by a sugar sensing mechanism,initsturnrequiringOPPPactivity. Therefore,theexpressionofnitrateassimilationgenes inthenucleusofrootcellsispromotedbyasignalemanatingfromOPPPactivityintheplastid[38]. 4. G6PDHsuponAbioticStress It has been suggested that the early response to the abiotic stress would involve the OPPP[14,39–41], which represent a true metabolic sensor during response to oxidative stress [42]. ThepossiblephysiologicalandbiochemicalrolesplayedbyOPPP,andnamelyG6PDH,inplanttissues duringdifferentstresshavebeeninvestigated[29,43,44]: ChangesinOPPPhavebeendemonstrated uponcoldstress[45,46],heat[47],metalspollution[48–51],drought[52,53]andsalinity[18,54,55]. Furthermore, manyevidencessuggestthatthisroleintheresponsetooxidativestresscanbe widenedtopathogensattack[56–59],butthiswillnotbediscussedinthisreviewfurther. First evidences for a direct G6PDH involvement in stress response were given examining short-term genes involved in the response to salinity in wheat [54]; among these WESR (Wheat EarlySaltResponding)genes,WESR5hadbeenidentifiedasencodingforacytosolicG6PDH[41]. TheincreaseofbothexpressionandactivityofG6PDHhasbeenextensivelydescribedduring salt stress [18,43,44,58] which presumably activate the whole set of the enzymes of OPPP [54]. Salinitycaused an increase of G6PDH activity in barley roots, regardless of nitrogen supply; this increaseisdependentonthede-novosynthesisofG6PDH,possiblyascribedtothecytosolicisoform; thus,itisevidentthattheincreaseofG6PDHactivityinducedbysaltstressfallsoutsidethenormal patternofphysiologicalconditions[18]. ThiseffectcanbeascribedtoanABAsignalingpathway,activatingtheABAsignalingcascade, andinducingthosegeneswhichpresentABREelementsinthepromoterregion[60],suchasG6PDH isoforms in wheat [54], tomato [53], rice [60], barley [13,39] and other plants. Correspondingly, in barleyrootsexposedtoexogenousABAroot,P2-G6PDHoccurrenceandactivitywasenhanced[39], indicating an ABA-responsive pathway for G6PDH expression; this hypothesis has been recently confirmedintomato,whereanalogousincreasesinexpressionof9-cis-epoxycarotenoiddioxygenase (NCED—theABAsynthetizingenzyme),proteinphosphatase2C-type(PP2C—thetargetoftheABA receptorsPYR-PYL/RCAR)areassociatedtocy-G6PDHexpression,occurrenceandactivity,inboth hydroponic,greenhouseandfieldgrowingconditions[53]. Furthermore,cy-G6PDHwouldusuallyabletomaintainanadequateNADPH/NADP+ ratio in the cytosol but, under severe stress conditions, a diversion of NADPH produced for nitrogen metabolism by P2-G6PDH does occur, in order to counteract the dangerous effects of stress, thus haltingnutrientassimilation[18]. TransgenictobaccooverexpressingaplastidicG6PDHexhibitedno visiblephenotype,butshowedmetabolismalterations,suggestingthatP2-G6PDHmaybeessentialin balancingtheredoxchargeinorganelles[39]. DifferentmodificationsofG6PDHisoforms,affectingbothactivityandkineticpropertiesupon stresscouldhavebeenproposed: chaperoninsaction,sugarsignalingandphosphorylation[14,61,62]. OPPPhasbeensuggestedasamajorpathwayinvolvedinthesugarinductionofnitrogen(and sulfur)carriersinplantroots. Thissignalingpathwaywouldcoordinatenutrientuptake,andtheir assimilation,togetherwiththeprovidingofNADPHnecessaryforaminoacidsynthesis[61]. SaltstressactivatesthesynthesisofGlycogensynthasekinase3fromA.thaliana(ASKα);inits turn,ASKαisabletophosphorylatecy-G6PDHonThr-467,thusstimulatingthecatalyticactivity,and enhancingNADPHproductiontocounteractROSexcess[14];thereforetheantioxidantsystemisable toreduceH O towaterbytheglutathioneperoxidasecycleorbytheascorbate-glutathionecycle[53]. 2 2 Plants2016,5, 24 8of14 5. HowDoesthePlantCellCopewiththeDistributionofReductantsProducedbyG6PDH? Alltheserecentlydiscoveredpatternsofregulation—andsubcellularlocalization—ofdifferent G6PDHisoformsshedanewlightintheemergingcomplexroleofthispivotalenzymeinthefurnishing ofreductantsforbothnutrientassimilationandstressresponseinplantcells. Thus, a general picture of the involvement of OPPP in the response to abiotic stress in plant cells can be designed: the whole pathway would be able to provide reductants to counteract the effects of stress (e.g., ROS production); of course, due to its central role in nutrient assimilation, this action would be exerted by diverging the reductants physiologically utilized during nitrogen assimilation. Therefore,abioticstressconditionswouldcauseapartialbutconsistentdistractionof NADPH-producedbytheOPPP-frombasalmetabolismtoROSscavenging[22,46]. Anyway,howcanthereductantsproducedbyG6PDHbeaddressedtowardsnitrogenmetabolism, orstressresponse? Howcouldthismechanismbetriggered? The oxidative damage induced by stress increased NADPH synthesis by OPPP; it can be hypothesized that the need to maintain of steady-state level of H O in cells during stress would 2 2 allowthefunctionofhydrogenperoxideasasignalinducinganenhancedG6PDHexpressionand activity[63]. Furthermore,theG6PDHinvolvementinstressresponsecouldintersectnitrogenmetabolismin amorecomplexpattern. Nitricoxide(NO)hasbeendemonstratedasinvolvedinresponsestomost ofstressinplantcells[64,65];amongthedifferentpathwaysynthetizingNOinplants,apreferential roleisplayedbynitratereductase(NR),whoseactivityisdirectlyenhancedbyG6PDHuponsalinity in roots [66]. It has been proposed that G6PDH would increase NADPH levels stimulating the NR-dependent NO production, thus enhancing the activities of antioxidant pathways, in order to scavengetheROSinducedbysaltstress[64,66];asimilarmechanismhasbeensuggestedinsoybean rootsduringcadmiumstress[49]. Ofcourse,aquestionraisesabouthowtheplantcelldealswiththediversionofreductantsand the increase of G6PDH activity: which are the fast-responding factors connecting OPPP to abiotic stress and, at the same time, able to turn off (or at least lowering) the supply of reductants for nitrogenmetabolism? RecentstudiesstronglysuggestthatthioredoxinscouldplaytheroleofregulatorsofG6PDH activity. Evenifthishasbeenknownfordecades,andwidelyrecognized,recentstudiesindicatethat thedifferentG6PDHisoformsarenotallregulatedbythesamethioredoxins,andoftentheiractivity canmemodulatedbysinglespecificclass(orsubclasses)ofTRXsatadifferentextent[23,24,67,68]. Namely, P1-G6PDH appears to be modulated by TRX f [23,67]; it has been previously suggested Trx m as a modulator of P2-G6PDH activity, but this was obtained using crude bacterial extracts overexpressinghistaggedrecombinantprotein[17];recently,aspecificmodulationbyTRXm,andnot TRXf,ofhighlypurifiedrecombinantP2-G6PDHfromPopulustrichocarpahasbeensuggested[69]. Thus, TRXs could represent a highly flexible system to modulate the activity of the different G6PDH isoforms, (and possibly their subcellular localization as well), resulting in the optimal distributionofreductantsproducedbytheplantcell. Ageneralschemecouldbedefined,takinginaccountthedifferentregulatorymechanismsexisting onthisenzyme(Figure3). Under“normal”conditions(e.g.,light,irrigation,nutrientavailabilityoptimal)NADPHproduced byG6PDHcanbeutilizedfornitrogenassimilation,e.g.,byGOGATandNiR(Figure3A).Whenstress conditionsappear,differentsignalstriggerthediversionofNADPHfromnitrogenassimilationto abioticstressresponse(Figure3B).Firstly,anABAsignalingpathwayshouldbeabledodivertthe reductantsforROSdetoxification:thismayoccurbothbymovingheterodimersP1-P0intoperoxisomes, andincreasingtheactivityofplastidialP2-G6PDH. Further, a major mechanism would be represented by cy-G6PDH phosphorylation by Ask-α, specificallyconnectedtoplantstressresponse. Plants2016,5, 24 9of14 Plants 2016, 5, 24 9 of 13 In the dark, or in heterotrophic tissues, NADPH is usually provided by OPPP in the cytosol In the dark, or in heterotrophic tissues, NADPH is usually provided by OPPP in the cytosol (Figure (Figure3C),andanenhancedG6PDHactivitywouldsupportreductantsincreasedrequestuponstress 3C), and an enhanced G6PDH activity would support reductants increased request upon stress (Figure (Fi3gDu)r. eIn3 Dch)l.oIrnopchlalsotrso ipn ldasatrsk,i nandda rink ,naonnd-pihnontoosny-npthhoettioc spylnasthtiedtsi,c Pp1l-aGs6tiPdDs,HP, 1a-nGd6 PP2D-GH6,PaDnHd Pw2o-Gul6dP bDe H woaublled tboe caobulentteorcaoctu tnhtee reafcfetcthtse oeff fsetcrtessso, frsetsrpesesc,tirveeslpye. cAtitv tehlye. mAotmtheenmt, oitm ceonutl,di tocnoluyl dbeo nsplyecbuelastpeedc uthlaatte, d thasitm,siilamr iltaor Pto1/PP10/-GP60P-GDH6P DhyHbrhidy bernidzyemnezsy, ma erse,-adirree-cdtiiornec toifo nGo6PfDGH6P DcoHuldco ouclcduor cicnu rpeinropxeisroomxiesso mofe s ofhheetteerrootrtoropphhici ctistsisuseuse, bs,ubt uthtitsh sihsosuhlodu bled pbreopperrolyp einrvlyesitnigvaetsetdig iant tehde ifnuttuhree f(uFtiguurere( F3Dig)u. re3D). FigFuigruer3e. T3.h eTfhaet efaotfer eodfu rcetadnutcstadnetrsi vdederfivroemd Gfrloumco sGel-u6cPodsee-h6yPd rdoegheyndarsoeg(eGn6aPseD H(G)6,aPnDdHm), oarendg emneorrael ly Oxgiednaetriavlelyp Oenxtiodsaetivpeh opsepnhtoastee pphaothspwhaayte( OpaPthPwP)a,yi n(OpPlaPnPt),c einll sp.la(Ant) .ceInllsp. h(Aot)o. sIny npthhoettoicsycnetlhlse,tcicy tcoelslos,l ic cytosolic OPPP provides NADPH for basal metabolism in the light. Normally, photosynthesis is able OPPP provides NADPH for basal metabolism in the light. Normally, photosynthesis is able to to sustain the request of electrons for nitrogen metabolism. Chloroplastic P1-G6PDH results sustaintherequestofelectronsfornitrogenmetabolism. ChloroplasticP1-G6PDHresultsinhibited inhibited by NADPH and thus OPPP is inactive in the organelles. (B). In the leaves, under stress byNADPHandthusOPPPisinactiveintheorganelles. (B).Intheleaves,understressconditions, conditions, there is an increase of cytosolic OPPP in order to supply NADPH for stress response. An thereisanincreaseofcytosolicOPPPinordertosupplyNADPHforstressresponse. Anincreased increased expression of cy-G6PDH occurs, together with expression and synthesis of P1- and expressionofcy-G6PDHoccurs,togetherwithexpressionandsynthesisofP1-andP0-G6PDH:this P0-G6PDH: this causes the formation of heterodimers directed to peroxisomes; there is the activation causestheformationofheterodimersdirectedtoperoxisomes; thereistheactivationofaspecific of a specific machinery formed by oxidative section of OPPP to counteract the stress; or for different machineryformedbyoxidativesectionofOPPPtocounteractthestress;orfordifferentmetabolic metabolic functions in specific plant organs. (C). In the dark, or in heterotrophic tissues—under functionsinspecificplantorgans.(C).Inthedark,orinheterotrophictissues—underphysiological physiological conditions—chloroplastic (or plastidial) OPPP are activated by P1-G6PDH or conditions—chloroplastic(orplastidial)OPPPareactivatedbyP1-G6PDHorP2-G6PDH,respectively, P2-G6PDH, respectively, providing reductants for nitrogen assimilation in the dark/heterotrophic providingreductantsfornitrogenassimilationinthedark/heterotrophicconditions. Asinleaves, conditions. As in leaves, cytosolic OPPP provides NADPH for basal metabolism. (D). In the dark, or cytosolicOPPPprovidesNADPHforbasalmetabolism.(D).Inthedark,orinheterotrophictissues, in heterotrophic tissues, under stress there is an increase of both cytosolic OPPP (by cy-G6PDH), and understressthereisanincreaseofbothcytosolicOPPP(bycy-G6PDH),andchloroplastic/plastidial chloroplastic/plastidial OPPPs (by P1-G6PDH or P2-G6PDH, respectively), in order to counteract the OPPPs(byP1-G6PDHorP2-G6PDH,respectively),inordertocounteractthestress.Itispossibleto stress. It is possible to hypothesize (?) the formation of heterodimers directed to peroxisomes (dotted hypothesize(?)theformationofheterodimersdirectedtoperoxisomes(dottedgreyarrows),butthis grey arrows), but this has not been proven yet. It must be underlined that P2-G6PDH is able to hasnotbeenprovenyet. ItmustbeunderlinedthatP2-G6PDHisabletomaintainahighrateeven maintain a high rate even at NADPH/NADP+ ratios normally easily inhibiting P1-G6PDH, thus atNADPH/NADP+ratiosnormallyeasilyinhibitingP1-G6PDH,thussustainingthestressresponse sustaining the stress response in root/heterotrophic tissues (e.g. enduring drought/salt stress inroot/heterotrophictissues(e.g. enduringdrought/saltstressconditions). Abbreviations: NTR, conditions). Abbreviations: NTR, nitrate transporters; NR, Nitrate reductase; NiR, Nitrite reductase; nitratetransporters;NR,Nitratereductase;NiR,Nitritereductase;GS,glutaminesynthetase;GOGAT, GS, glutamine synthetase; GOGAT, glutamate synthase; Fd, Ferredoxin (red,reduced/ox, oxidated); glutamatesynthase; Fd, Ferredoxin(red,reduced/ox, oxidated); R5P,ribose-5P;E4P,erythrose-4P; R5P, ribose-5P; E4P, erythrose-4P; Glu, glutamate; Gln glutamine; 2-OG, 2 oxoglutarate; PTS, Glpue,rgolxuitsaommaet eta;rGgelntingglu steaqmuiennec;e2. -OG,2oxoglutarate;PTS,peroxisometargetingsequence. Plants2016,5, 24 10of14 6. Conclusions Inplantcells,G6PDHplaysacentralroleinbothprovidingreductantsandprecursorsforbasal metabolism,andtocounteracttheoxidativeburstuponstressconditions. Therefore,G6PDHcanbe consideredasamainfactorofcellredoxpoise,determiningsensitivitytostress. Thecompartmentedchloroplastic(P1-G6PDH)andplastidial(P2-G6PDH)isoformsareconnected withthefurnishingofreductantsfornitrogenassimilation,butuponstressconditions,bothactivities canberecruitedforresistanceand/ortolerancestrategies. The interconnections between OPPP, nitrogen metabolism, and stress response are multiple: the ABA signaling pathway plays a central role in the induction of G6PDH upon stress and the reductantsproducedbyOPPPcanbedivertedfromnitrogenassimilationtowardstheROSscavenging; moreoverthioredoxinsmodulatingactivityofthedifferentG6PDHisoformscouldselectivelytrigger thedifferentisoforms. Furthermore,itshouldberememberedthat,inhigherplants,nitricoxidecanbesynthetizedby nitritereductaseuponhighlevelsofNADPH(increaseduponstress)creatingaloopbetweenOPPP, nitrogenassimilationandNOsignaling. Last but not the least, the synthesis of P0-G6PDH upon stress would be able to form hybrid G6PDHsredirectedindifferentorganelles(e.g.,peroxisomes)tocounteractoxidativestress. ThedefinitionofacrystalstructureofplantG6PDHisoforms,andamorecleardefinitionofthe kinetic and inhibitory mechanisms are still required in order to correctly define their role in plant cell metabolism. Although in the last decade a huge leap has been done in the understanding of controllingmechanismsoverlyingG6PDHactivityinplants,thereisstillalongpathtoreachthegoal ofacomprehensivedefinitionoftheregulationofthispivotalenzymeinplants. Acknowledgments:TheAuthorwouldliketothankallthestudents,Ph.D.andscientistswho—throughoutthe years—hadworkedonG6PDHinhislaboratory.Particularly,thanksto(inalphabeticalorder):ManuelaCardi, DanielaCastiglia,AlessiaDeLillo,MyriamFerrara,andGeaGuerriero. Iamindebttomanycolleaguesforsuggestions,andhelpfuldiscussionsaboutG6PDH.Iwishtothankatleast afewofthem:MikeJ.Emes,Jean-PierreJacquot,AntjievonSchaewen,andMirkoZaffagnini. ConflictsofInterest:Theauthordeclaresnoconflictofinterest. References 1. 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