Nitrogen Assimilation, Abiotic Stress and Glucose 6-Phosphate

14 Pages·2016·2.51 MB·English

by  Sergio Esposito

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.:[phone];Fax:[phone]
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. Kruger,N.J.; vonSchaewen,A.Theoxidativepentosephosphatepathway: Structureandorganisation.
Curr.Opin.PlantBiol.2003,6,236–246.[CrossRef]
2. Soderberg,T.Biosynthesisofribose-5-phosphateanderythrose-4-phosphateinarchaea: Aphylogenetic
analysisofarchaealgenomes.Archaea2005,1,347–352.[CrossRef][PubMed]
3. Stincone,A.; Prigione,A.; Cramer,T.; Wamelink,M.M.C.; Campbell,K.; Cheung,E.; Olin-Sandoval,V.;
Grüning,N.-M.;Krüger,A.;Alam,M.T.;etal.Thereturnofmetabolism:Biochemistryandphysiologyofthe
pentosephosphatepathway.Biol.Rev.Camb.Philos.Soc.2014,90,927–963.[CrossRef][PubMed]
4. Esposito,S.;Guerriero,G.;Vona,V.;Rigano,V.D.;Carfagna,S.;Rigano,C.Glutamatesynthaseactivitiesand
proteinchangesinrelationtonitrogennutritioninbarley:Thedependenceondifferentplastidicglucose-6P
dehydrogenaseisoforms.J.Exp.Bot.2005,56,55–64.[CrossRef][PubMed]
5. Michels,A.K.;Wedel,N.;Kroth,P.G.;Germany,A.K.M.Diatomplastidspossessaphosphoribulokinase
withanalteredregulationandnooxidativepentosephosphatepathway1.PlantPhysiol.2005,137,911–920.
[CrossRef][PubMed]
6. Debnam,P.M.;Emes,M.J.Subcellulardistributionofenzymesoftheoxidativepentosephosphatepathway
inrootandleaftissues.J.Exp.Bot.1999,50,1653–1661.[CrossRef]
7. Au,S.W.;Naylor,C.E.;Gover,S.;Vandeputte-Rutten,L.;Scopes,D.A.;Mason,P.J.;Luzzatto,L.;Lam,V.M.;
Adams,M.J.Solutionofthestructureoftetramerichumanglucose6-phosphatedehydrogenasebymolecular
replacement.ActaCrystallogr.D.Biol.Crystallogr.1999,55,826–834.[CrossRef][PubMed]
8. Oesterhelt,C.;Klocke,S.;Holtgrefe,S.;Linke,V.;Weber,A.P.M.;Scheibe,R.Redoxregulationofchloroplast
enzymesinGaldieriasulphurariainviewofeukaryoticevolution.PlantCellPhysiol. 2007,48,1359–1373.
[CrossRef][PubMed]