Metabolite Diversity in Alkaloid Biosynthesis

20 Pages·2016·1.31 MB·English

ThePlantCell,Vol.28:1926–1944,August2016,www.plantcell.orgã2016AmericanSocietyofPlantBiologists.Allrightsreserved.
Metabolite Diversity in Alkaloid Biosynthesis: A Multilane
(Diastereomer) Highway for Camptothecin Synthesis in
Camptotheca acuminata
RadinSadre,aMariaMagallanes-Lundback,aSujanaPradhan,b,1VonnySalim,a,1AlexMesberg,aA.DanielJones,a,b
andDeanDellaPennaa,2
aDepartmentofBiochemistryandMolecularBiology,MichiganStateUniversity,EastLansing,Michigan48824-1319
bDepartmentofChemistry,MichiganStateUniversity,EastLansing,Michigan48824-1319
ORCIDID:0000-0002-8412-2579(R.S.)
Camptothecin is a monoterpene indole alkaloid (MIA) used to produce semisynthetic antitumor drugs. We investigated
camptothecinsynthesisinCamptothecaacuminatabycombiningtranscriptomeandexpressiondatawithreversegenetics,
biochemistry, and metabolite profiling. RNAi silencing of enzymes required for the indole and seco-iridoid (monoterpene)
componentsidentifiedtranscriptionalcrosstalkcoordinatingtheirsynthesisinroots.Metaboliteprofilingandlabelingstudies
of wild-type and RNAi lines identified plausible intermediates for missing pathway steps and demonstrated nearly all
camptothecinpathwayintermediatesarepresentasmultipleisomers.UnlikepreviouslycharacterizedMIA-producingplants,
C.acuminatadoesnotsynthesize3-a(S)-strictosidineasitscentralMIAintermediateandinsteadusesanalternativeseco-
iridoid pathway that produces multiple isomers of strictosidinic acid. NMR analysis demonstrated that the two major
strictosidinic acid isomers are (R) and (S) diastereomers at their glucosylated C21 positions. The presence of multiple
diastereomers throughout the pathway is consistent with their use in synthesis before finally being resolved to a single
camptothecin isomer after deglucosylation, much as a multilane highway allows parallel tracks to converge at a common
destination.Amodel“diastereomer”pathwayforcamptothecinbiosynthesisinC.acuminataisproposedthatfundamentally
differsfrompreviouslystudiedMIApathways.
INTRODUCTION is impractical and the production of semisynthetic derivatives
reliesentirelyoncamptothecinisolatedfromthebarkandseeds
Monoterpene indole alkaloids (MIAs) account for thousands of
of C. acuminata and Nothapodytes nimmoniana (Lorence and
specialized metabolites produced by plant species from the
Nessler,2004).Theincreasingworldwidedemandforcamptothecin
ordersCornalesandGentianales.Muchofthischemicaldiversity
requiresexplorationofmoreeconomicalandsustainablealternatives
originatesfromthecommonprecursor3-a(S)-strictosidine,which
foritsproduction.
isformedbystereospecificcondensationoftheindolemetabolite
Despite the medicinal importance of camptothecin, its bio-
tryptamine and the monoterpene secologanin (De Luca et al.,
synthesis by plants remains undeciphered. Since its original dis-
2014).MIAmetabolicdiversitylikelyevolvedasacomponentof
coveryinC.acuminata,camptothecinwasfoundtoaccumulatein
plant adaptation to changing environments and as a defensive
severalAsianandAfricantropicalandsubtropicalplantspeciesfrom
agent against various biotic stresses. Many MIAs, including
unrelated orders and families, including species of Ophiorrhiza
camptothecin,targetspecificcellularprocessesinmammalsand
(Wink,2003;GopalakrishnanandShankar,2014).Earlyradiolabeling
have important pharmacological activities and medicinal uses.
experimentsinC.acuminatademonstratedthattryptophanandits
Camptothecin was first identified during the 1960s as a novel
decarboxylationproducttryptamineweremetabolicprecursors,as
antitumoralkaloidofCamptothecaacuminata(Cornales),atree
was a mixture of the monoterpene alcohols geraniol and its cis-
nativetoSouthernChina(Walletal.,1966).Itsmodeofactionisthe isomer nerol, leading to classification of camptothecin as a MIA
specificinactivationoftopoisomeraseIresultingincelldeathby
(SherihaandRapoport,1976)despiteithavingaquinoline,andnot
apoptosis (Wright et al., 2015). Semisynthetic analogs derived
indole,ringsystem.SubsequentstableisotopelabelingandNMR
fromcamptothecinexhibitimprovedpharmacologicalproperties
experimentsdemonstratedthatstrictosamideservedasaprecursor
andclinicalefficacyrelativetocamptothecinandarewidelyused
for camptothecin in C. acuminata (Hutchinson et al., 1979). The
totreatlung,colorectal,cervical,andovariancancers(Liuetal.,
co-occurrence of camptothecin in Ophiorrhiza pumila with the
2015).Asformanyalkaloids,chemicalsynthesisofcamptothecin
structurallyrelatedalkaloidspumilosideanddeoxypumilosidesug-
gested they are intermediates in the camptothecin biosynthetic
1Theseauthorscontributedequallytothiswork. pathway(Aimietal.,1989).Notably,pumilosidewasalsodetectedin
[email protected]. C.acuminata(Carteetal.,1990;Montoroetal.,2010).Recentstudies
Theauthorresponsiblefordistributionofmaterialsintegraltothefindings ofcamptothecinbiosynthesisinO.pumilahairyrootsdemonstrated
presentedinthisarticleinaccordancewiththepolicydescribedinthe
thatstrictosidine,derivedfromthecondensationoftryptamineand
Instructions for Authors (www.plantcell.org) is: Dean DellaPenna
([email protected]). theseco-iridoidsecologanin,isrequiredforcamptothecinsynthesis
www.plantcell.org/cgi/doi/10.1105/tpc.16.00193 inthisspecies(Asanoetal.,2013;Yamazakietal.,2013).
CamptothecinBiosynthesisinCamptotheca 1927
Theseco-iridoidpathwayleadingtostrictosidinewasonlyre- collected from wild-type plants grown under greenhouse con-
centlyelucidatedintheMIA-producingplantCatharanthusroseus. ditions,and70%aqueousacetonitrileextractsweresubjectedto
Geraniol8-oxidase(CYP76B6)catalyzeshydroxylationoftheMEP nontargetedUHPLC/MSanalysisusinga52-minreverse-phase
pathway-derivedisoprenoidgeraniolto8-hydroxygeraniol,which chromatographic separation. Nineteen metabolites were anno-
undergoes further oxidation to the dialdehyde 8-oxogeranial (1) tatedbasedonaccuratemassmeasurementsofpositiveionsand
by8-hydroxygeranioloxidoreductase(Höferetal.,2013).Theen- fragments observed in UHPLC/MS and UHPLC/MS/MS mass
zyme iridoid synthase catalyzes the subsequent conversion of spectra(Figure1,Table1;SupplementalTable1).Wild-typeC.
8-oxogeranial (1) to iridodial (Geu-Flores et al., 2012), which is acuminataaccumulatedtryptamine(4),theiridoidsloganicacid
oxidizedfurtherto7-deoxyloganeticacidby7-deoxyloganeticacid (2),andsecologanicacid(3)aswellastheMIAstrictosidinicacid
synthase(CYP76A26)(Salimetal.,2014).7-Deoxyloganeticacidis (5), but lacked detectable levels of their methyl-esterified de-
glycosylated to 7-deoxyloganic acid (Asada et al., 2013), which rivativesloganin,secologanin,andstrictosidine(numbersinbold
undergoessubsequenthydroxylationbyCYP72A224toyieldlo- numberscorrespondtothechemicalstructuresshowninTable1
ganic acid, whose carboxyl group isthen O-methylatedto form andFigure1).Thissuggeststhatincontrasttothebeststudied
loganin(Murataetal.,2008;Salimetal.,2013).Secologaninsyn- MIA-producingplantsRauvolfiaserpentinaandC.roseusthatuse
thase(CYP72A1)thencatalyzestheoxidativeringopeningoflo- methyl-esterifiedintermediates(DeLucaetal.,2014),C.acumi-
ganintosecologanin,whichcontainsanaldehydegroupsuitablefor natausesanalternativeseco-iridoidpathwaywithcarboxylicacid
condensation with the amino group of tryptamine (Irmler et al., intermediatesuptostrictosidinicacid.Severalpoststrictosidinic
2000).Strictosidinesynthase,aPictet-Spenglerase,catalyzesthe acid metabolites that we propose as intermediates in the
stereospecific condensation of tryptamine and secologanin to camptothecinpathwaywerealsodetected(Table1).Inaddition
3-a(S)-strictosidine(BracherandKutchan,1992).Itwasproposed to the previously reported metabolites strictosamide (6) and
thatstrictosidineissubsequentlychanneledintothebiosynthesis pumiloside (10) (Hutchinson et al., 1979; Carte et al., 1990;
of the downstream MIAs strictosamide, pumiloside, and deoxy- Montoroetal.,2010),weidentifiedmetaboliteswithexactmo-
pumilosidetoyieldcamptothecininO.pumila(Asanoetal.,2013). lecularmassesandfragmentionsconsistentwithstrictosamide
However, poststrictosidine reaction steps, enzymes, and their epoxide(7),strictosamidediol(8),strictosamideketolactam(9),
sequence(s)remainunknownincamptothecin-producingplants. anddeoxypumiloside(11)(Table1).Surprisingly,mostofthese
TheMedicinalPlantConsortiumrecentlymadetheassembled MIAswerepresentinmultipleisomericformswithidenticalexact
transcriptome, gene expression, and metabolite profiles for C. molecularandfragmentionmassesthatweresolvedandnum-
acuminata publicly available (http://medicinalplantgenomics. beredaccordingtotheirrelativeelutionorder.Notethatallisomers
msu.edu/,http://metnetdb.org/mpmr_public/).Here,wecom- ofacompoundhavethesameboldednumberandthatadditional
bined analysesoftranscriptomeandexpressiondatasetswith descriptorsareusedtodifferentiateisomers(e.g.,isomer1and
reverse genetics, biochemistry, and metabolite profiling to in- isomer 2). Strictosidinic acid (5) and strictosamide epoxide (7)
vestigatecamptothecinbiosynthesisinsoil-grownC.acuminata each exhibited three isomers, while two isomers each were
plants.Twoessentialgenesforcamptothecinbiosynthesis,those detected for strictosamide (6), strictosamide ketolactam (9),
encoding tryptophandecarboxylaseandiridoidsynthase,were pumiloside(10),anddeoxypumiloside(11).
identifiedanddownregulatedbyRNAi,andtheconsequencesfor Wenextquantifiedtherelativelevelsofthesemetabolitesand
metabolite and gene expression profiles determined. Our key theirisomersthatcouldbedetectedinextractsoffivedifferent
findingsarethatincontrasttootherwell-studiedMIAproducing tissuesforthreewild-typeplants(Figure2).Asexpected,shoot
plants,includingO.pumila(Yamazakietal.,2003b;Asanoetal., apex and young leaf shared similar metabolite profiles and ac-
2013),C.acuminatadoesnotsynthesizestrictosidineandinstead cumulatedmostmetabolitesatdistinctlyhigherlevelscompared
usesanalternativeseco-iridoidpathwaytoproducestrictosidinic withrootand/ormatureleaf.Tryptamine(4)wasonlydetectedin
acidcomposedofamixtureofglucosidediastereomersvaryingin stem,shootapex,andyoungleaf.Loganicacid(2)contentwas
stereochemistryatC21.Thesestrictosidinicaciddiastereomers highestinstem,lowerinotherphotosynthetictissues,andabsent
arechanneledintothecamptothecinpathway,andmultipledia- fromroot.Secologanicacid(3)wasdetectedinallfivetissueswith
stereomers of other intermediates are present throughout the levelsinphotosynthetictissues2-to3-foldhigherthaninroots.
pathway, comparable to a multilane highway in which parallel Themajoraccumulationsitesforstrictosidinicacid(5)wereshoot
lanesleadtoasingledestination,inthiscaseasingleisomerof apexandyoungleafwithprogressivelylowerlevelsinstem,root,
camptothecin.Finally,weidentifynovelalkaloidsthatareplau- and mature leaf. Surprisingly, the levels of strictosamide (6)
sibleintermediatesformissingstepsinthepathwayusingstable werenearlyidenticalinall tissues.Strictosamideepoxide(7),
isotopelabelingandtandemmassspectrometry. strictosamide diol (8), and strictosamide ketolactam (9) could
not be reliably quantified due to their low abundances in all
RESULTS C.acuminatatissues.Thetissuedistributionprofileofpumiloside
(10)wassimilartothatofstrictosidinicacid(5),withthehighest
IdentificationandCharacterizationofCamptothecin levels being in shoot apex and young leaf and lowest levels in
PathwayIntermediatesinC.acuminata matureleaf.Deoxypumiloside(11)wasonlydetectedinstem,root,
and atextremelylow levelsinshoot apex. All tissues contained
To assess the abundance of putative intermediates of the camptothecin(12)withyoungleafandshootapexaccumulatingthe
camptothecinpathwayinC.acuminata,fivedifferenttissues(root, highestlevels.Itisnoteworthythattherelativeamountsofisomers
green stems, shoot apex, and young and mature leaves) were ofstrictosidinicacid(5)andpoststrictosidinicacidintermediates
1928 ThePlantCell
Figure1. ProposedPathwayforCamptothecinBiosynthesisinC.acuminata.
Camptothecin(12)issynthesizedfromthecentralprecursorstrictosidinicacid(5)derivedfromcondensationoftryptamine(4)andtheiridoidsecologanic
acid(3),amonoterpenoidglycoside.Invivolabelingexperimentswithdeuterated[a,a,b,b-d]-tryptamineresultedinlabelingofstrictosidinicacidand
4
downstreammetaboliteswiththenumberandthelikelypositionsofdeuterium(D)indicatedinred.Enzymeactivitiesareindicated.TDC1andCYC1were
identified and characterized in this study. 7-DLS, 7-deoxyloganetic acid synthase; 7-DLGT, 7-deoxyloganetic acid glucosyltransferase; 7-DLH,
7-deoxyloganicacidhydroxylase;SLAS,secologanicacidsynthase;STRAS,strictosidinicacidsynthase;DH,dehydration;RD,reduction.
variedamongtissues(Figure2).Inmosttissues,oneofthetwo 1H-13C coupling constants, which are sensitive to orientation
majorisomerswasmoreabundant;forexample,strictosamide(6) relativetootherbondsandhavebeenusedtoestablishrelative
isomer1predominatesinrootsandstems,whileisomer2pre- bondorientationsinavarietyofmetabolites(Marquezetal.,2001).
dominatesinshootapicesandleaves.Ingeneral,rootsandstems Thesecouplingconstants(1J )were170Hzforisomer2and
C-H
havesimilarisomerprofilesasdoyoungandmatureleaves. 178Hzforisomer3,thelargestisomer-relateddifferenceamong
The presence of multiple isomeric MIA metabolites in all1J valuesandconsistentwithtwoepimersdifferinginthe
C-H
UHPLC/MSmetaboliteprofilesofC.acuminatatissueswithin- stereochemistryofC-glycosylationatposition21(Figure3,Table
distinguishableMS/MSspectra(Table1;SupplementalTable1) 2). Based on similarities to NMR spectra reported for 21(S)-
suggestedtheydifferinstereochemicalconfigurations,buttheir strictosidine,weassignstrictosidinicacidisomer3tohavethe
chromatographicresolutionindicatestheyarediastereomers.To 21(R)configurationandstrictosidinicacidisomer2tohave21(S)
assessthenatureoftheseisomeric compounds,thetwomost configuration (Figure 3), the latter being in common with the
abundantisomersofstrictosidinicacid(5),whichwerepresentin publishedstructureandcouplingconstantsof21(S)-strictosidine
sufficientquantitytobepurified,weresubjectedtoanassortment (Patthy-Lukátsetal.,1997).NMRdataforstereocentersatpo-
ofone-andtwo-dimensionalNMRspectroscopicanalyses.The sitions3,15,and20andthecarbohydrateresonancesforisomers
mostsubstantialdifferenceswereobservedin the1Hchemical 2and3didnotdiffersubstantially,consistentwiththedifference
shiftsatposition21(Table2;SupplementalDataSetS1),whichis beingtheconfigurationatposition21.Thesetwodiastereomer
thepositionofglycosylation.Subsequentcoupledheteronuclear configurationsaremostlikelyformedearlieriniridoidbiosynthesis
singlequantumcoherence(cHSQC)NMRspectraweregenerated bythespontaneousringopeningandclosingof7-deoxyloganetic
to assess whether relative stereochemical configuration of hy- acidthatformsC2-hydroxyepimers,whicharethenglycosylated
drogensatchiralpositionscouldbeassessedbymeasuringthe (Figure 4). The presence of multiple isomers for most of the
CamptothecinBiosynthesisinCamptotheca 1929
Table1.RelevantCompoundsDetectedinWild-TypeC.acuminata
Experimental
Retention Calculatedm/z Precursorm/z FragmentIon(s)Observedin TDC1-RNAiNo.of
AnnotatedMetabolite Formula Time(min) for[M+H]+ for[M+H]+ MS/MSSpectra(m/z) DeuteriumAtoms
Tryptamine(4) C H N 8.0 161.1079 161.1065 144 4
10 12 2
Loganicacid(2) C H O 13.6(2isomers)a 377.1448 377.1450 359,215,197,179,161,151,137, 0
16 24 10
133,123,109,81
Secologanicacid(4) C H O 15.1(2isomers)a 375.1291 375.1290 213,195,177,151,125,109,107, 0
16 22 10
95,79,77
Strictosidinicacid(5) C H NO 23.9(isomer1) 517.2186 517.2198 500,355,338,320,269/268,251, n.d.(isomer1)
26 32 2 9
24.8(isomer2) 194,180/181,168/170,151, 4(isomer2)
26.5(isomer3) 156,144,130,125 4(isomer3)
Strictosamide(6) C H NO 37.6(isomer1) 499.2080 499.2081 337,319,267,171,144 4(isomer1)
26 30 2 8
41.5(isomer2) 4(isomer2)
Strictosamide C H NO 22.8(isomer1) 515.2030 515.2021 353,335,309,291,283,265,263, n.d.(isomer1)
26 30 2 9
epoxide(7) 24.8(isomer2) 237,209,183,184,155,144 n.d.(isomer2)
26.5(isomer3) n.d.(isomer3)
Strictosamidediol(8) C H NO 20.1 533.2135 533.2170 371,353,283,265,185,160,142, n.d.
26 32 2 10
132
Strictosamide C H NO 22.5(isomer1) 531.1979 531.1953 369,351,341,299,281,271,253, n.d.(isomer1)
26 30 2 10
ketolactam(9) 22.7(isomer2) 194,176,158,148,130,124, n.d.(isomer2)
106
Pumiloside(10) C H NO 30.2(isomer1) 513.1873 513.1890 351,333,315,305,281,235,140 2(isomer1)
26 28 2 9
32.9(isomer2) n.d.(isomer2)
Deoxypumiloside(11) C H NO 36.1(isomer1) 497.1924 497.1930 335,265,247,219,183,169,142, 2(isomer1)
26 28 2 8
38.2(isomer2) 97 2(isomer2)
Camptothecin(12) C H NO 34.3 349.1188 349.1198 305,277,249,219/220,168 2
20 16 2 4
Metabolitesinroot,stem,shootapex,andleafextractswereseparatedbya52-minUHPLC/MS/MSmethodandarelistedwiththeirprecursorand
fragmentions.Fragmentionsobtainedafterlossofaglucoseunit(162D)arehighlightedinbold.Onlystemtissuecontaineddetectablelevelsofall
metaboliteisomerslistedhere.Thelastcolumninthetablesummarizestheresultsofinvivolabelingexperimentswith[a,a,b,b-d]-tryptamineinTDC1-
4
RNAiplants,whichdonotaccumulatetryptamine-derivedMIAswithouttryptaminesupplementation.AfterincubationofTDC1-RNAiapicalcuttings
with[a,a,b,b-d]-tryptaminefor6weeks,severaldeuteratedMIAsweredetectableinstemextracts.Thenumberofdeuteriumatomsincorporatedinto
4
detectablemetabolitesisindicatedinthelastcolumn,withn.d.indicatingcompoundsthatwerebelowthelimitofdetectionindeuteriumlabeling
experiments.
aLoganic acid and secologanic acid exist also as multiple isomers that were not resolved with this UHPLC/MS method but were resolved using
adifferentchromatographysystem(SupplementalFigures1and2).
later biosynthetic intermediates suggests that C. acuminata yield the quinolinone ring of pumiloside (10). The subsequent
7-deoxyloganeticacidglucosyltransferaseiseitherapromiscuous reduction to deoxypumiloside (11), deglycosylation, and other
enzyme that can accommodate both R- and S-isomers of metabolicconversionsleadtotheendproductcamptothecin(12).
7-deoxyloganetic acid or that multiple stereospecific C. acu- This model pathway takes into account the formation and re-
minata7-deoxyloganeticacidglycosyltransferasesexist.Under tentionofmultipleglycosidicisomers(diastereomers)forpathway
our52-minchromatographicconditions,thefirstiridoidswecan intermediatesfrom7-deoxyloganeticacidtodeoxypumiloside(11).
detect, loganic acid (2) and secologanic acid (3), appeared as
single chromatographic peaks; however, two isomers were re- IdentificationofCandidateGenesInvolvedin
solved for each using a different chromatography column and
CamptothecinBiosynthesis
gradient(SupplementalFigures1and2).Thus,theisomersob-
servedthroughoutthepathwayarelikelyglucosidicisomersthat Genes responsible for the production of camptothecin in C.
havetheiroriginearlyiniridoidsynthesis. acuminataarelargelyunknown.Apreviousstudydemonstrated
On the basis of these observations, a model pathway for that C. acuminata possesses two differentially expressed tryp-
camptothecinsynthesisinC.acuminataisproposed(Figure1), tophandecarboxylasegenes,TDC1andTDC2,forthesynthesis
where tryptamine (4) and the aldehyde-containing iridoid of tryptamine (López-Meyer and Nessler, 1997). Transcriptome
secologanicacid(3)(ratherthansecologanin)arecoupledvia datafromtheMedicinalPlantGenomicsResource(MPGR)da-
aPictet-Spenglerreaction,leadingtostrictosidinicacid(5).Intra- tabase (Góngora-Castillo et al., 2012) indicated that TDC1 ex-
moleculardehydrativecyclizationofstrictosidinicacidyieldsstric- pressionismoderatetohighinleaves,roots,andyoungbarkofC.
tosamide(6).Conversionoftheindoleringsystemtothequinoline acuminata, while TDC2 transcript is barely detectable in most
ringispostulatedtooccurviamultistepoxidationtostrictosamide planttissues,withtheexceptionofimmatureandmaturefruit.In
ketolactam(9)followedbycondensationandeliminationofwaterto matureleaves,roots,andyoungbark,TDC1transcriptlevelsare
1930 ThePlantCell
Figure2. TissueDistributionProfilesofProposedCamptothecinPathwayMetabolitesinWild-TypeC.acuminata.
Tissueswerecollectedfromwild-typeplantsthathadbeenundergreenhousecultivationinsoilfor8monthsand70%acetonitrileextractswereanalyzed
usinga15-mingradientelutionmethodforUHPLC/MS.Multipleisomersweredetectedforstrictosidinicacid(Figure1,compound5)andpoststrictosidinic
acidmetabolites(Figure1,compounds6,10,11,and12).AveragevaluesareshownwithSD(n=3)forthemostabundantandquantifiableisomers.SA,shoot
apex;YL,youngleaf;ML,matureleaf.
100-, 200-, and 600-fold, respectively, higher than TDC2 tran- tissue-specificexpressionprofilesfor53,154uniqueC.acuminata
scriptlevels.SinceTDC1isexpressedintissuesthataccumulate transcriptsin18tissuesfromdifferentdevelopmentalstagessuch
camptothecin,weconsidereditthemostpromisingcandidatefor as 10-d-old seedlings to fruiting trees (Góngora-Castillo et al.,
synthesizingtheindoleprecursorofcamptothecin.Thepredicted 2012). Prior to coexpression analysis, genes exhibiting low
TDC1 protein shares 68 and 74% sequence identities with the transcriptabundances(log fragmentsperkilobaseoftranscript
2
tryptophandecarboxylasesofC.roseus(DeLucaetal.,1989)and permillionmappedreads[FPKM]lowerthan2)inrootorimmature
O.pumila(Yamazakietal.,2003a),respectively. bark were removed from the data set, leaving 26,874 unique
To select additional candidate camptothecin biosynthesis transcriptsforfurtheranalyses.Additionally,asiridoidsynthesisis
genes,theMPGRtranscriptomedatawereusedforcoexpression known to involve cyclization, oxidation, and glycosylation re-
analyses.Wepostulatedthatseco-iridoidpathwaygeneswould actions(Miettinenetal.,2014),1146transcriptsencodingputative
becoexpressedwithTDC1tocoordinatesynthesisoftheseco- proteases, histone and histone-modifying proteins, ribosomal
iridoid and indole precursors needed for camptothecin pro- proteins, tRNA synthetases, and proteins related to ubiq-
duction.TheoriginalMPGRdatasetincludeddevelopmentaland uitinylationwereconsideredunlikelytohavedirectinvolvementin
CamptothecinBiosynthesisinCamptotheca 1931
Table2.NMRChemicalShiftsandCouplingConstantsforStrictosidinicAcidIsomersIsolatedfromC.acuminataLeafTissueasMeasuredfrom
J-Resolved1HSpectraand1H-13CcHSQCSpectra
Carbonnumber 1Hshift(ppm)and3J 13Cshift(ppm)and1J 1Hshift(ppm)and3J 13Cshift(ppm)and1J
H-H C-H H-H C-H
andgroup couplingconstants(Hz) couplingconstants(Hz) couplingconstants(Hz) couplingconstants(Hz)
Isomer2 Isomer3
3(CH) 4.45(dd),3J=3.4,7.7Hz 50.8,1J=144Hz 4.56(dd),3J=3.6,7.9Hz 53.1,1J=143Hz
5(CH) 3.29(m) 41.3,1J=143,145Hz; 3.45(m) 39.6,1J=139,141Hz
2
3.75(m) 3.50(m)
6(CH) 3.01(m) 18.1,1J=131,133Hz 2.99(m) 18.2,1J=129,131Hz
2
3.29(m) 3.04(m)
9(CH) 7.56(d),3J=7.9Hz 117.7,1J=159Hz 7.55(d),3J=7.9Hz 117.6,1J=158Hz
10(CH) 7.15,3J=7.4Hz 119.1,1J=159Hz 7.13,3J=7.3Hz 118.9,1J=158Hz
11(CH) 7.23,3J=7.6Hz 121.9,1J=159Hz 7.23,3J=7.6Hz 121.8,1J=158Hz
12(CH) 7.42,3J=8.2Hz 110.7,1J=159Hz 7.45,3J=8.2Hz 110.8,1J=158Hz
14(CH) 2.15(dd),3J=4.6,11.1Hz; 33.7,1J=128,129Hz 1.98(m) 34.1,1J=130,131Hz
2
2.40(dd),3J=5.9,11.6Hz 2.46(m)
15(CH) 3.00(m) 32.6,1J=137Hz 2.96(m) 32.9,1J=135Hz
17(CH) 7.67(s) 151.7,1J=191Hz 7.35(s) 148.4,1J=192Hz
18(CH) 5.32(d),3J=10.7Hz; 117.5,1J=155,158Hz 5.39(d),3J=11.0Hz; 118.4,1J=154,161Hz
2
5.43(d),3J=17.3Hz 5.47(d),3J=17.3Hz
19(CH) 5.96(dd),3J=10.3,17.5Hz 134.8,1J=156Hz 6.19(dd),3J=10.2,17.2Hz 134.8,1J=152Hz
20(CH) 2.70(m) 44.2,1J=134Hz 2.74(m) 44.9,1J=132Hz
21(CH) 5.93(d),3J=9.5Hz 95.1,1J=170Hz 5.64(d),3J=8.2Hz 95.5,1J=178Hz
19(CH) 4.92(d),3J=8.0Hz 98.9,1J=163Hz 4.85(d),3J=7.9Hz 98.8,1J=162Hz
29(CH) 3.44(m) 76.5,1J=145Hz 3.41(m) 76.5,1J=145Hz
39(CH) 3.41(m) 77.3,1J=145Hz 3.35(m) 77.3,1J=145Hz
49(CH) 3.26(m) 70.2,1J=145Hz 3.28(m) 70.3,1J=145Hz
59(CH) 3.24(m) 73.3,1J=145Hz 3.24(m) 73.3,1J=145Hz
69(CH) 3.68(dd),3J=4.95,12.2Hz; 61.6,1J=141,142Hz 3.76(dd),3J=6.61,12.3Hz; 61.5,1J=131,132Hz
2
4.12(dd),3J=2.17,12.1Hz 4.05(dd),3J=2.40,12.6Hz
Notabledifferencesin1Hchemicalshiftsand1J couplingconstantswereobservedforposition21(highlightedinboldinthetable).
C-H
specializedmetabolismandwerealsoremoved,leavingadataset C.roseusiridoidtranscriptionfactorbHLHIRIDOIDSYNTHESIS1
of25,725transcriptsforhierarchicalclusteranalyses. (BIS1)(VanMoerkerckeetal.,2015),alongwithaputativeortholog
Hierarchicalclusteranalysisidentifiedasubclusterof23tran- toproteinS,ana/b-hydrolasesuperfamilyproteinassociatedwith
scriptsthatencodesevenproteinswithhighestidentitytoknown seco-iridoid/MIAsynthesizingcellsinC.roseus(Leménageretal.,
steps in the seco-iridoid pathway. These include geraniol-8- 2005) (Figure 5). Like TDC1, most of the seco-iridoid pathway
oxidase(G8O),CYCLASE1(CYC1),andCYC2,whicharemem- candidate genes in this cluster showed moderate to high ex-
bersoftheprogesterone5-b-reductasefamily,7-deoxyloganetic pressioninrootandimmaturebark,buttheyalsohadlowerex-
acidsynthase,andglucosyltransferase,aputativeorthologofthe pression than TDC1 in other tissues (e.g., mature leaf), which
Figure3. StructuresoftheTwoMajorStrictosidinicAcidIsomersIsolatedfromC.acuminataLeafTissue.
NMRanalyses(Table2)showthatthetwomajorstrictosidinicacidisomers(isomers2and3)differinstereochemicalconfigurationatpositionC21,thesiteof
glucosylation.
1932 ThePlantCell
Figure4. FormationofIridoidDiastereomers.
Equilibriumbetweentheopenandclosedringconformationsof7-deoxyloganeticacidyieldsdiastereomersattheC2hydroxylgroup.
resultedintheirclusteringapartfromTDC1(Figure4).Thetryp- transcriptsencodingstepsofthetryptophanandMEPpathways
tophanandMEPpathwaysprovidesubstratesfromintermediary were identified by homology searches (Supplemental Data Set
metabolismfortryptamineandiridoidsynthesis,respectively.To S2),andtheirexpressionprofilessubjectedtohierarchicalcluster
assess whether members of these pathways have expression analysis together with transcripts of the iridoid subcluster
patterns similar to that of the iridoid cluster, C. acuminata (Supplemental Figure 3 and Supplemental Data Set S3). This
Figure5. HeatMapofExpressionDataofCandidateGenesfortheSeco-IridoidBranchofCamptothecinBiosynthesis.
Hierarchicalclusteringof25,725transcriptswasgeneratedbasedonaveragelinkageofPearsoncorrelationcoefficientsoflog-transformedFPKM
2
expressionvaluesfromtheMPGRwebsite(http://medicinalplantgenomics.msu.edu/)withMeVv4.9(Saeedetal.,2006).Asubclusterencompassing
23genesisshownwithGenBankaccessionnumberslistedinparentheses.Thecolorscaledepictstranscriptabundance(expressedaslog-transformed
2
FPKM).Reddotsindicatethepositionofcandidatesfortheseco-iridoidpathway,andforcomparison,theexpressionprofile(expressedaslog-transformed
2
FPKM)ofTDC1isshown(yellowdot).CYC1andTDC1,bothhighlightedinbold,havebeencharacterizedinthisstudy.Notethatlistsofcandidategenes,
GenBankaccessionnumbers,MPGRtranscriptidentifiers,andFPKMvaluesaregiveninSupplementalDataSets2and3.HMTD,heavymetaltrans-
port/detoxification;GUF,geneofunknownfunction.
CamptothecinBiosynthesisinCamptotheca 1933
analysisshowedthattheexpressionpatternofmosttryptophan
andMEPpathwaygenesisquitedifferentfromthatoftheiridoid
subcluster, with a few notable exceptions: one gene encoding
a tryptophan synthase subunit and one gene encoding an an-
thranilatesynthasesubunit.
CYC1ShowsIridoidSynthaseActivityinVitro
InC.roseus,iridoidsynthaseisamemberoftheprogesterone
5-b-reductase family that catalyzes the NAD(P)H-dependent
conversion of 8-oxogeranial (1) to iridodial/nepetalactol in MIA
biosynthesis(Geu-Floresetal.,2012;Munkertetal.,2015).This
suggestedthatoneorbothoftheC.acuminataprogesterone5-b-
reductasefamilymembersinourgenecluster(Figure5,CYC1and
CYC2) might encode orthologous iridoid synthase activities
in camptothecin biosynthesis. The C. acuminata transcriptome
encodes at least seven progesterone 5-b-reductase family
members,ofwhichCYC1andCYC2,with65and59%aminoacid
sequence identity, respectively, have the highest identities to
C. roseus iridoid synthase. In comparative analysis of the phy-
logeneticrelationshipsbetweenpredictedmembersofthepro-
gesterone-5-b-reductasefamilyinC.roseus,C.acuminata,and
R.serpentina,CYC1andCYC2formedseparate,well-resolved
clades(SupplementalFigure4andSupplementalDataSetS4).
To examine the catalytic activities of CYC1 and CYC2 in
comparison to the previously characterized C. roseus iridoid
synthase (Geu-Flores et al., 2012), the respective coding
sequenceswereexpressedinEscherichiacoliandrecombinant
His-tagged proteins purified by affinity chromatography. The
purified enzymes were subsequently tested in colorimetric de-
hydrogenaseassaysforproteinfunctionalitythatarebasedonthe
NAD(P)H-dependentreductionofnitrobluetetrazoliumchlorideto
the dark-blue formazan product. These experiments indicated
Figure 6. Iridoid Synthase Assay with Recombinant CYC1 and CYC2
thatallthreeenzymeswerefunctionalandrequiredNAD(P)Has
Enzymes.
cofactorforactivity(SupplementalFigure5).Thepurifiedproteins
werethenassayedforiridoidsynthaseactivitywith8-oxogeranial Purifiedrecombinantproteinswereassayedforiridoidsynthaseactivityin
(1)assubstrateusingachemicallysynthesizedsubstrate(Geu- reactionmixtureswith8-oxogeranial(Figure1,compound1)intheabsence
orpresenceofNADPH.Assayswereextractedwithdichloromethaneand
Floresetal.,2012)composedofamixtureofthecis-transisomers
analyzedbyGC/MS.Therespectivetotalionchromatogramsareshownin
8-oxogeranial (1)/8-oxoneral in an approximate 2:1 ratio. In-
comparisonto that obtained fora control assay with C.roseusiridoid
cubationofCYC1withsubstrateandNADPHresultedinnearly
synthase(atog,reactionproducts;handi,8-oxoneraland8-oxogeranial
completeconversionofthesubstratetosevencis-transisomersof
substrates,respectively).InthepresenceofNADPH,CYC1andC.roseus
iridodial/nepetalactol that were indistinguishable in gas chro- iridoid synthase catalyzed conversion of the substrate to reduction
matographicretentiontimesandmassspectratothoseobtained products while no products were detectable in assays with CYC2. In-
with the C. roseus iridoid synthase control (Figure 6). These data cubationoftheproteinsinreactionmixtureslackingNADPHdidnotresult
clearly demonstrated that like C. roseus iridoid synthase, indetectableproducts;CYC1minusNADPHisshownasarepresentative
C. acuminata CYC1 catalyzes the reductive cyclization of trace.
8-oxogeranial(1).AssayswithCYC2didnotresultinanydetectable
products,indicatingthatCYC2maynotuse8-oxogeranial(1)as
forTDC1orCYC1andtransgenicplantswereregeneratedinvitro.
substrateinplanta(Figure6).
Rootedantibiotic-resistantplantletsweretransferredfrominvitro
SilencingofCYC1andTDC1ExpressioninC. culturetosoil cultivationundergreenhouseconditions. Withthe
acuminataPlants exceptionofdecreasedresistancetoredspidermiteinfestation,
RNAi plants did not show any obvious phenotypic differences
CYC1 and TDC1 were selected as targets for reverse genetics comparedwithwild-typeplants(SupplementalFigure6).
analysesbygenesilencing.Wedevelopedastabletransformation Afteragrowthandacclimationperiodof3to4monthsunder
methodtointegrateRNAi-mediatingexpressioncassettesintothe greenhousecultivation,RNAi-mediatedsuppressionofthetarget
C.acuminatagenomeusingthepHellsgate12system(Helliwelland genes was evaluated in independent transformation events by
Waterhouse,2005).C.acuminatacotyledonexplantsweretrans- quantitativereal-timePCR.SinceTDC1andCYC1expressionin
formedbyAgrobacteriumtumefaciensharboringRNAiconstructs wild-typeplantsishighestinimmaturebark,stemtissuewasused
1934 ThePlantCell
todeterminemRNAabundancesfortargetgenesinRNAiplants cultivationforabouteightmonths.RNAwasextractedfromroots,
relativetountransformedwild-typecontrolplants.InthesixTDC1- greenstems,andyoungdevelopingleaves,andtheaverageTDC1
RNAi plants analyzed, TDC1 stem transcript levels were de- andCYC1mRNAlevelswerecompared(Figure8).Inwild-type
creasedtoaslowas3%ofwild-typelevels(Figure7A).Consistent plants,TDC1transcriptlevelswere;2-foldhigherinstemsthan
with this observation, camptothecin levels in mature leaves of rootsandyoungleavesandbarelydetectableinthesetissuesin
TDC1-RNAi plants were reduced by as much as 1000-fold TDC1-RNAi lines (Figure 8A). CYC1 expression in wild-type
comparedwithwild-typeplants(Figure7A).Similaranalyseswere plants was highest in stems and ;40-fold that of roots and
performedonnineteenCYC1-RNAiplantswhereamuchwider young leaves (Figure 8B). The average CYC1 mRNA level in
range of silencing and impact on leaf camptothecin levels was greenstemsofCYC1-RNAilineswasstronglyreducedto4%of
observed(Figure7B).ApproximatelyhalfoftheCYC1-RNAilines thewildtype,whileinrootsandyoungleaves,itwasonlyre-
hadstemCYC1transcriptlevelsbelow15%ofthewildtype.This duced to 49 and 27% of wild-type levels, respectively, sug-
group of CYC1-RNAi lines also had 15- to 400-fold lower gesting that target-gene silencing was less effective in these
camptothecinlevelsinmatureleavesthandidwild-typeplants. twotissues(Figure8B).
Taken together, these data indicate that stable RNAi trans-
formationwassuccessfulinC.acuminataandthattheexpres- TDC1-RNAiPlantsAreDeficientinIndoleAlkaloidsand
sion of both TDC1 and CYC1 are necessary for camptothecin
AccumulateLoganicAcidinRoots
biosynthesis.
Targetgeneimpactwasfurtherexaminedinfiveindependent To further evaluate the impact of TDC1 gene silencing on
transformed lines of TDC1-RNAi and CYC1-RNAi that had se- camptothecin biosynthesis, extracts from roots, stems, shoot
verelyreducedmatureleafcamptothecinlevelsandthreewild- apices, and young and mature leaves were subjected to
type lines independently regenerated through tissue culture. UHPLC/MSanalyses.Incontrasttothewild-typecontrols,TDC1-
Plants used for these studies had been under greenhouse RNAilinesdidnotaccumulatedetectablelevelsoftryptamine(4),
strictosidinic acid (5), or any of the poststrictosidinic acid me-
tabolitesinFigure1,includingthenewlyproposedpathwayin-
termediatesstrictosamideepoxide(7),strictosamidediol(8),and
strictosamideketolactam(9).Bycontrast,thelevelsoftheiridoids
loganicacid(2)andsecologanicacid(3)instems,shootapices,
andleavesofTDC1-RNAiplantswerenotsignificantlydifferent
from wild-type levels (Figure 9). However, all TDC1-RNAi lines
accumulated loganic acid (2) in roots, a metabolite below de-
tectioninwild-typeroots(Figure9).
To assess whether the absence of strictosidinic acid (5) and
poststrictosidinic acid metabolites in theTDC1-RNAi lines was
directlyattributabletotryptamine(4)deficiency,feedingexperi-
ments with deuterated tryptamine were performed. Apical cut-
tings from TDC1-RNAi and wild-type plants were incubated in
aqueous solutions with or without tryptamine-a,a,b,b-d . After
4
6weeks,stemsandmatureleaveswerecollectedandextracts
preparedandanalyzedviaUHPLC/MS.Asexpected,incubation
ofwild-typeapicalcuttingswithdeuteratedtryptamineresultedin
little incorporation of label into camptothecin pathway inter-
mediates due to the substantial intrinsic pool of unlabeled
tryptamineinstemandshootapex(;100nmolpergramfresh
weight;Figure2).However,whenTDC1-RNAicuttingswerein-
cubated with deuterated tryptamine, deuterium-labeled camp-
tothecin pathway intermediates were detected in stemsand to
alesserextentinleaves.Fourdeuteriumatomsfromtryptamine-d
4
wereincorporatedintostrictosidinicacid(5)isomers2and3and
Figure7. CorrelationAnalysesbetweenStemTranscriptAbundanceof
strictosamide(6)isomers1and2,whiletwodeuteriumatomswere
theRNAi-TargetedGenewiththeMatureLeafCamptothecinContentof
incorporatedintopumiloside(10)isomer1,deoxypumiloside(11)
TransgenicRNAiPlantsandWild-TypePlants.
isomers 1 and 2, and camptothecin (12), providing further evi-
DataareshownforTDC1-RNAiplants(A)andforCYC1-RNAiplants(B)in dence for the role of these metabolites as intermediates in
comparisontowild-typeplants(filledtriangles).Eachcirclerepresentsone
camptothecinsynthesis(Table1).Becausetheabsolutelevelsof
RNAiplantfromanindependenttransformationevent,withfilledcircles
all pathway compounds in deuterated tryptamine TDC1-RNAi
indicatinglinesthatwereselectedforfurtherin-depthanalyses.Forthewild
cuttings were still much lower than in wild-type cuttings, in-
type,threeindependentlinesweretakenthroughtissuecultureandre-
corporationoflabelintolessabundantisomerscouldnotbere-
generation.Stemsandmatureleaveswerecollectedfromplantsthathad
beenundergreenhousecultivationinsoilforapproximately4months.Note liablydetermined.Thelikelypositionsofdeuteriumlabelswithin
thatthevaluesareplottedondoublelog scale. thedifferentpathwayintermediatesareindicatedinFigure1.
10
CamptothecinBiosynthesisinCamptotheca 1935
weredecreasedinCYC1-RNAilinesrelativetothoseinthewild
type,insometissuestobelowthedetectionlimit(Table3).Isomer
compositionswerealsoradicallyalteredincertaintissuesofthe
CYC1-RNAi lines compared with average wild-type levels. For
example, strictosidinic acid (5) was composed of 39% isomer
2[21(S)isomer]and61%isomer3[21(R)isomer]inmatureleaves
ofthewildtype,whereasinCYC1-RNAilines,isomer3wasmore
dominant(94%).Strictosamide(6)inrootsandstemsofthewild
type consisted mainly of isomer 1 (74 and 87%, respectively),
whileisomer2wasexclusivelyaccumulatedinCYC1-RNAilines.
Pumiloside(10)instemsofthewildtypewascomposedof41%
isomer2,whereasinCYC1-RNAilines,pumilosideisomer1was
the only detectable isomer. Surprisingly, deoxypumiloside (11)
isomercompositionsdidnotsignificantlydifferbetweenwild-type
andCYC1-RNAiplants.Thesecombinedresultsfortheimpactof
CYC1-RNAiareconsistentwiththemetaboliteprofilesofTDC1-
RNAi lines and the proposed pathway for camptothecin bio-
synthesis(Figures1and9).
TranscriptionalCrosstalkbetweentheIndoleandIridoid
Figure8. ExpressionAnalysesoftheRNAi-TargetedGeneinRoot,Young
BiosyntheticPathways
Stems,andYoungLeavesofTDC1-RNAiPlantsandCYC1-RNAiPlants
RelativetotheWildType.
Totestwhetherindoleandiridoidprecursorsynthesismightbe
Tissues were collected from plants that had been under greenhouse coordinated,weanalyzedtheexpressionofadditionalcandidate
cultivationfor8months.Averagetranscriptcopynumberswerenormalized genesinRNAiplantscomparedwiththewildtype.CYC1,CYC2,
toACTIN6mRNA(n=5and3forRNAiandwild-typeplants,respectively) and G8O (geraniol-8-oxidase; Figure 5) mRNA levels were de-
andareshownforTDC1-RNAiplants(A)andCYC1-RNAiplants(B)relative terminedandcomparedintherootsandgreenstemsofTDC1-
tothewildtype.Notethattheyaxisisdiscontinuoustoallowdepictionof
RNAi and wild-type plants (Figure 10). All three genes were
thesignificantdifferencesinRNAitargetgenecopynumbersbetweenwild-
expressedmosthighlyinstems(Figures10Band10D),withCYC2
type and RNAi lines. Asterisks indicate significant differences in RNAi
mRNAlevelsnotbeingsignificantlydifferentinanygenotypeor
plants(unpairedttest;*P<0.05;**P<0.001)relativetothewildtype.
tissue. However, in TDC1-RNAi roots, CYC1 and G8O mRNA
levelswereincreased9-and40-fold,respectively,relativetothe
wildtype(Figure10A),suggestingthatinresponsetothesevere
ImpairedIndoleAlkaloidBiosynthesisinCYC1-RNAiPlants reductionintryptamineandcamptothecinbiosynthesis(Figure9)
IsAccompaniedbyIncreasedTryptamineLevels themonoterpenebranchofthepathwayisspecificallyupregu-
latedinrootsofTDC1-RNAiplants.CYC1-RNAilineswithstrong
We similarly assessed the impact of CYC1-RNAi by analyzing
impactonCYC1expressionandmatureleafcamptothecinlevels
pathway metabolite levels in roots, stems, shoot apices, and
were similarly assessed for TDC1, CYC2, and G8O expression
youngandmatureleavesoffiveindependentCYC1-RNAilinesin
(Figures10Cand10D).WhileG8OandCYC2transcriptlevelswere
comparisontothewildtype.Inadditiontoasharpreductionin
notsignificantlydifferentbetweenCYC1-RNAilinesandwild-type
camptothecinlevelsinalltissuestoaslowas2%ofwild-type
plants, TDC1 expression was significantly increased 3-fold in
levels,alltissuesofCYC1-RNAilinesaccumulatedsubstantially
roots (Figure 10C), suggesting that in response to the severe
higherlevelsoftryptamine(4)comparedwiththewildtype(Figure
reductioninlevelsofmonoterpeneintermediates(Figure9,Table
9).Incontrasttowild-typeandTDC1-RNAiplants,whichlacked
3)andcamptothecinbiosynthesisinCYC1-RNAilines,theindole
detectablelevelsoftryptamineinrootsandmatureleaves,CYC1-
branch is upregulated. These data indicate the existence of
RNAirootsandmatureleavescontained;250nmoltryptamine
atranscriptionalcrosstalkmechanismbetweentheindoleandthe
pergramfreshweight(Figure9).Instems,shootapices,andyoung
iridoidbranchofthecamptothecinpathwayinC.acuminataroots.
leavesoftheCYC1-RNAilines,tryptaminelevelsapproachedor
exceeded 1000 nmol per gram fresh weight (15- to 23-fold in-
creasesrelativetothewildtype). DISCUSSION
NoneoftheCYC1-RNAi linesaccumulated detectablelevels
ofloganicacid(2)inanytissue,andinmosttissues,thelevels TheC.acuminataSeco-IridoidPathwayGenerates
of secologanic acid (3), strictosidinic acid (5), and the post-
Isomeric,AcidicPrecursorsforCamptothecinBiosynthesis
strictosidinicacidintermediateswerealsoreducedatleast6-fold
relative to the wild type (Figure 9, Table 3), with strictosamide Our results indicate that C. acuminata (order Cornales) uses
epoxide(7),strictosamidediol(8),andstrictosamideketolactam a seco-iridoid pathway that fundamentally differs from that in
(9)levelsbeingreducedtobelowthedetectionlimit.Withregardto C. roseus and R. serpentina, two plant species in the order
the isomer composition of strictosidinic acid (5) and post- Gentianales in which seco-iridoid and MIA synthesis has been
strictosidinicacidmetabolites,thelevelsofallidentifiedisomers most extensively studied (De Luca et al., 2014). C. roseus and