Transcriptional Regulation and Transport of Terpenoid Indole Alkaloid in Catharanthus roseus

20 Pages·2016·8.72 MB·English

International Journal o f
Molecular Sciences
Review
Transcriptional Regulation and Transport of
Terpenoid Indole Alkaloid in Catharanthus roseus:
Exploration of New Research Directions
JiaqiLiu1,2,JunjunCai3,RuiWang2,*andShihaiYang1,*
1 CollegeofChineseHerbalMedicine,JilinAgriculturalUniversity,Changchun130047,China;
[email protected]
2 CropResearchInstitute,SichuanAcademyofAgriculturalSciences,Chengdu610066,China
3 WestChinaHospital,SichuanUniversity,Chengdu610066,China;[email protected]
* Correspondence:[email protected](R.W.);[email protected](S.Y.);
Tel.:[phone](R.W.);[phone](S.Y.);
Fax:[phone](R.W.);[phone](S.Y.)
AcademicEditor:MarcelloIriti
Received:4November2016;Accepted:22December2016;Published:28December2016
Abstract: As one of the model medicinal plants for exploration of biochemical pathways and
molecular biological questions on complex metabolic pathways, Catharanthus roseus synthesizes
morethan100terpenoidindolealkaloids(TIAs)usedforclinicaltreatmentofvariousdiseasesand
fornewdrugdiscovery. Giventhatextensivestudieshaverevealedthemajormetabolicpathways
andthespatial-temporalbiosynthesisofTIAinC.roseusplant,littleisknownaboutsubcellularand
inter-cellulartraffickingorlong-distancetransportofTIAendproductsorintermediates,aswellas
theirregulation. Whilethesetransportprocessesareindispensableformulti-organelle,-tissueand
-cellbiosynthesis,storageandtheirfunctions,greateffortshavebeenmadetoexplorethesedynamic
cellular processes. Progress has been made in past decades on transcriptional regulation of TIA
biosynthesisbytranscriptionfactorsaseitheractivatorsorrepressors;recentstudiesalsorevealed
severaltransportersinvolvedinsubcellularandinter-cellularTIAtrafficking. However,manydetails
and the regulatory network for controlling the tissue-or cell-specific biosynthesis, transport and
storageofserpentineandajmalicineinroot,catharanthineinleafandroot,vindolinespecificallyin
leafandvinblastineandvincristineonlyingreenleafandtheirbiosyntheticintermediatesremaintobe
determined.Thisreviewistosummarizetheprogressmadeinbiosynthesis,transcriptionalregulation
andtransportofTIAs. Basedonanalysisoforganelle,tissueandcell-typespecificbiosynthesisand
progresses in transport and trafficking of similar natural products, the transporters that might
be involved in transport of TIAs and their synthetic intermediates are discussed; according to
transcriptomeanalysisandbioinformaticapproaches,thetranscriptionfactorsthatmightbeinvolved
inTIAbiosynthesisareanalyzed. Furtherdiscussionismadeonabroadcontextoftranscriptional
andtransportregulationinordertoguideourfutureresearch.
Keywords: terpenoid indole alkaloids; biosynthesis; transcription factor; transporter; regulatory
network;compartmentation
1. Introduction
Plant secondary metabolites are often produced either in certain tissues or cells under stress
conditionsorinducedbyvariousdevelopmental,hormonalandenvironmentalcues. Thebiosynthesis
of secondary metabolites is often tightly regulated at transcriptional levels. Even in a plant cell
where secondary metabolites are synthesized, the multiple enzymes and their reactions are often
compartmentalizedintovarioussubcellularorganelles,suchaschloroplast,endoplasmicreticulum
Int.J.Mol.Sci.2017,18,53;doi:10.3390/ijms18010053 www.mdpi.com/journal/ijms
Int.J.Mol.Sci.2017,18,53 2of20
(ER), vacuoles, as well as apoplastic spaces. It is also often observed that the synthetic site of
secondarymetabolitesisusuallydifferentfromthesitewherethesechemicalsfunction,asdefensive
compounds against insects, bacteria or fungal pathogens. Thus, efficient transport of precursors,
metabolic intermediates and the end products for biosynthesis, storage or function, are of critical
in whole plant secondary metabolism and their biological significance. Many studies support
the idea that transporters, either across membranes or intra-cellular trafficking, or inter-cellular
long distance transport, provide another layer of regulation for metabolic flux [1,2]. Furthermore,
thesetransmembranetransporters,vesicletraffickingcomponents,aswellasproteincarriersarealso
regulatedattranscriptionallevels[3]. Therefore,transcriptionalandtransportregulationofsecondary
metabolitebiosynthesisarecriticalresearchtopics.
Catharanthus roseus (Madagascar periwinkle) is a perennial herb belonging to the family
Apocynaceae. Itproducesover100differentterpenoidindolealkaloids(TIAs),someofwhichexhibit
strongpharmacologicalactivitiesandareessentiallyusedinclinicaltreatmentofvariousdiseases[4].
Vinblastine and vincristine, which have been used clinically to treat cancers since 1950s, are the
mostvaluabledimericTIAsinC.roseus[5]. ThesetwodimericTIAsareproducedintraceamount
in C. roseus by couplingvindoline and catharanthine, both of whichhave also been reported with
anti-bacterialactivities,anti-diabeticpropertiesanddiureticactions[6]. OtherTIAsfromC.roseussuch
asajmalicineandserpentineareusedinanti-hypertensiveandanti-neuro-inflammatoryagents[6].
Duetoextremelylowyieldofthehighlyvaluablevinblastineandvincristine,substantialeffortsin
pastdecadeshaveputonlarge-scalecellculture,bioreactorprocessingbiotechnologyandmetabolic
engineeringtoimprovetheirproductioninordertomeettheincreasingdemandsfromthemarket.
However,thesuccessisverylimited. IthasbeenrealizedthatunderstandingoftheTIAbiosynthesis,
transportandtheirregulationmayempowerourabilitytoapplynewandrobustmolecularandgenetic
toolsinmetabolicengineeringoftheproductionofthesevaluablemetabolites[7–10]. Severalreviews
were published about the organ-, tissue- and cell-specific as well as transcriptional regulation of
TIAbiosynthesis[8,11]. However,thebreakthroughsmaderecentlyonTIAbiosynthesisregulation
and particularly, the intra- and inter-cellular TIA transport, from molecular biology, genomic and
transcriptomicspointsofviewhaveprovidedsignificantinsightsintotheseimportantdynamiccellular
processes[12]. ThisreviewistosummarizethesemostrecentprogressesmadeonTIAbiosynthesis,
transcriptionalregulationandtheirtransportandtoemploytranscriptomeanalysisforfurtherlooking
forTIAtranscriptionalregulatorsthatarepotentiallyinvolvedintheTIAbiosynthesis.
2. TheBiosyntheticPathwayoftheTIAsinCatharanthusroseus
Inrecentdecades,C.roseushasbeenextensivelystudiedforelucidatingthecomplexbiosynthetic
pathways of TIAs and now it has become an ideal medicinal plant for in-depth investigating the
complexmolecularmechanismsforTIAbiosynthesisandtransportaswellastheirtranscriptional
regulation. All the TIAs in C. roseus are derived from the central precursor strictosidine,
whichisacondensedproductofthetryptophanpathway-derivedtryptamineandtheseco-iridoid
pathway-derivedsecologaninbystrictosidinesynthase(STR)(Figure1). Studiesonthebiosynthetic
pathways of tryptamine and secologain have been carried out for years. Two enzymes have been
revealedfortheircriticalrolesintryptaminebiosynthesis,anthranilatesynthase(AS)andtryptophan
decarboxylase (TDC) [7,13], however, specific process remains to be determined. In seco-iridoid
pathway, secologanin is finally generated through eight steps after the hydrolysis of geranyl
diphosphate (GPP) to geraniol by geraniol synthase (GES) [14], in such pathway, all the enzymes
involvedhavebeenidentifiedandthereversiblereactionfrom10-oxogeranialto7-deoxyloganetic
acid has also been illuminated, while the recent study did not found the intermediate product
iridotrialinthisreaction[15–17]. Subsequently,thecentralprecursorstrictosidineisconvertedinto
strictosidine aglycoside by strictosidine β-D-glucosidase (SGD) [18], and strictosidine aglycoside
can be used to synthesize various kinds of TIAs. Crystal structure studies detected two new
cathenamine reductases (CRs), namely, heteroyohimbine synthase (HYS) and tetrahydroalstonine
Int.J.Mol.Sci.2017,18,53 3of20
synthase(THAS).HYSreducescathenamineintoajmalicineand19-epi-ajmalicine, whileTHASis
predomIinnt. aJ.n Mtolly. Srcie. 2s0p17o, n18s, i5b3 lefortheconversionoftetrahydroalstoninefromstrictosidinea3g olf y19c on[19].
Alstonineandserpentinearetheoxidationproductsoftetrahydroalstonineandajmalinerespectively.
HYS reduces cathenamine into ajmalicine and 19-epi-ajmalicine, while THAS is predominantly
Although the specialized oxidation enzyme has not been isolated, the oxidation conversion from
responsible for the conversion of tetrahydroalstonine from strictosidine aglycon [19]. Alstonine and
ajmalinseertpoensteinrpe eanret itnhee opxirdeasteionnt phroadsubctese onf toetbrasheyrdvreodalsitnonpinlea nantdv aajmcuaolinlees reasnpedctiivselmy. aAinlthlyouagchc tuhme ulated
in rootssp[e2c0ia]l.izedV ionxdidoaltiinone eisnzaymnei mhaps onrotat nbteebni oissoylanttehde, ttihce porxeidcuatriosno rcoonfvevrisniocnr ifsrtoimne aajmnadlinve intob lastine.
Since thseerpceynttoinceh proremseent Ph4a5s 0beeenn zoybmseerveCdY iPn 7p1lDan1t Vv2acutaolbees rasnodn iins em3a-oinxlyy gaeccnuamseula(tTed3 Oin) raonotds [a2n0]. alcohol
Vindoline is an important biosynthetic precursor of vincristine and vinblastine. Since the cytochrome
dehydrogenase (ADHL), tabersonine3-reductase (T3R) have been identified, the biosynthesis
P450 enzyme CYP71D1V2 tabersonine3-oxygenase (T3O) and an alcohol dehydrogenase (ADHL),
process from tabersonine to vindoline is well understood. However, the enzymes converting the
tabersonine3-reductase (T3R) have been identified, the biosynthesis process from tabersonine to
strictosidine-aglycone into tabersonine remain to be identified. On the other hand, the whole
vindoline is well understood. However, the enzymes converting the strictosidine-aglycone into
biosyntthaebseirssonoifnea rneomtahienr top bree ciduernstoifriedc.a Othna trhaen otthhienr eharnedm, tahien wshtoole bbeiosryenvtheeaslies do,f aanloththoeur gprheciutrssopr utative
intermecdatihaatera,nsthteinme mremadaienns intoe b,eh raesvebaeleedn, aildtheonutgifih eitds pfourtataivleo inngtertmimedeia[t2e,1 s]t.emAmftaedrentihnee, bhiaoss byenetnh esis of
vindolinideenatnifdiedc afothr aar alonntgh itnime,e t[h2e1]y. Aarfteerc othuep bleiodsyinntthoesdisi mof evriincdaollkinael oaindds ,caat-h3a(cid:48)r,a4n(cid:48)-thainnhe,y tdhreoyv ainreb lastine
(AVLB),cowuiptlhedt heinptoe rodximidearisce aa-l3k(cid:48)a,l4o(cid:48)i-dasn, hay-d3′r,4o′v-ainnhbyldarsotvinineblsaysntinthe as(eA(VPLRBX), 1)w[i2th2 ].tThhe evpearlouxaidbalseeT IAsin
a-3′,4′-anhydrovinblastine synthase (PRX1) [22]. The valuable TIAs in C. roseus, vinblastine and
C. roseus, vinblastine and vincristine, were eventually derived from AVLB through multiple steps,
vincristine, were eventually derived from AVLB through multiple steps, which remain to be
whichreumndaeitnermtoinbeedu. ndetermined.

Figure 1. Schematic biosynthetic pathways for terpenoid indole alkaloids (TIAs) in Catharanthus
Figure 1. Schematic biosynthetic pathways for terpenoid indole alkaloids (TIAs) in
roseus. The updated TIA biosynthetic pathways are presented by incorporating the most recently
Catharanthus roseus. The updated TIA biosynthetic pathways are presented by incorporating the
published results. Question marks in red indicate unknown process or enzymes for the reactions.
most reAcebbnrtelvyiaptiuonbsl isahree:d DrXesSu, lt1s-d.eQoxuye-Dst-xioynlulomsea rk5-sphinosprheadte insdynicthaatese;u nDkXnRo, w1n-deporxoyc-De-sxsylourloseen zymes
for the5-rpehaocstpihoantse . Arbedburectvoiiasotimoenrsasea; re:MDCTX, S, M1-EdPe oxyc-yDti-dxyyltlrualnossfeeras5e-;p hoCsMphKa, te 4s-y(cnytthidaisnee; DXR,
1-deoxy5-′D-d-ixpyhlousplohsoe)-25--Cp-hmoesthpyhl-aDt-eerryetdhruitcotlo iksionmasee;r aMseE;CMS,C T2-,CM-mEePthcyyletriydthyrlittroal n2s,f4e-rcaycsleo;dCipMhoKsp,h4a-t(ec ytidine
5(cid:48)-diphosysnpthhaos)e-;2 -CH-DmSe, thhyyld-rDox-eyrmyetthhyrilbtoultenkyiln a4s-ed;ipMhoEspChaSt,e 2-sCyn-mthaesteh; ylHeDryRt,h rhityodlro2xy,4m-ceythcyllobdutiepnhylo sphate
4-diphosphate reductase; IDI, isopentenyl diphosphate isomerases; IPP isomerase; GPPS, geranyl
synthase; HDS, hydroxymethylbutenyl 4-diphosphate synthase; HDR, hydroxymethylbutenyl
diphosphate synthase; GES, geraniol synthase; CPR, cytochrome P450 reductase; G10H, geraniol
4-diphosphatereductase; IDI,isopentenyldiphosphateisomerases; IPPisomerase; GPPS,geranyl
10-hydroxylase; 10HGO, 10-hydroxygeraniol dehydrogenase; IS, iridoid synthase; IO, iridoid
diphospohxiadtaeses; y7n-DthLaGsTe,; 7G-dEeoSx,ygloegraanneitoicl ascyidn tghluacsoes;ylCtraPnRsf,ercayset;o DchLr7oHm, 7e-dPeo4x5y0logreandiuc catcaids eh;ydGro1x0yHla,seg; eraniol
10-hydrLoAxMylTa,s elo;ga1n0icH aGciOd ,m1et0h-yhltyradnrsofexryasgee; rSaLnSi,o slecdoleohgyandinro sgyennthaassee;; AISS,, ainrtihdroanidilatse ysnytnhthaassee;; TIDOC,, iridoid
oxidaset;r7y-pDtoLpGhaTn, d7e-dcaeroboxxyylloagsea; nSeTtRic, astcriicdtogsilduicnoe ssyylntrthaanssef;e SraGsDe,; DstrLic7tHos,id7i-ndee oβ-xDy-glolugcaonsiidcaascei; dTh16yHd2r,o xylase;
tabersonine 16-hydroxylase 2; 16OMT, 16-hydroxytabersonine-16-O-methyltransferase; T3O,
LAMT, loganic acid methyltransferase; SLS, secologanin synthase; AS, anthranilate synthase;
tabersonine 3-oxygenase; T3R, tabersonine 3-reductase; NMT, N-methyltransferase; D4H,
TDC, tryptophan decarboxylase; STR, strictosidine synthase; SGD, strictosidine β-D-glucosidase;
desacetoxyvindoline 4-hydroxylase; DAT, acetyl CoA: deacetylvindoline 4-O-acetyltransferase; CR,
T16H2, tabersonine 16-hydroxylase 2; 16OMT, 16-hydroxytabersonine-16-O-methyltransferase;
cathenamine reductases; THAS, tetrahydroalstonine synthase; HYS, heteroyohimbine synthase;
T3O, taPbReXrs1o, an-i3n′,4e′-a3n-hoyxdyrgoevninabslaes;tinTe3 sRy,ntthaabsee;r PsoEnRi, npeuta3t-irveed puercotxaisdea;seN. MT, N-methyltransferase; D4H,
des acetoxyvindoline 4-hydroxylase; DAT, acetyl CoA: deacetylvindoline 4-O-acetyltransferase;
CR,cathenaminereductases;THAS,tetrahydroalstoninesynthase;HYS,heteroyohimbinesynthase;
PRX1,a-3(cid:48),4(cid:48)-anhydrovinblastinesynthase;PER,putativeperoxidase.
Int.J.Mol.Sci.2017,18,53 4of20
3. TranscriptionalRegulationofTIABiosynthesis
Plenty of evidence indicates that the synthesis of TIAs is strictly regulated by transcription
factors that target on the key structural genes. Elicitors such as yeast, jasmonate (JA) and related
oxylipins, hormonessuchasauxinsaswellasenvironmentalcuescanstimulateTIAbiosynthesis
in C. roseus [23,24]. It has been revealed that these stimuli-promoted TIA biosyntheses happen
via transcriptional regulation of TIA synthetic genes, such as STR, TDC, G10H. The mechanisms
for regulating the biosynthesis of different TIA end products are complicated and diversified;
manytranscriptionfactorshavebeencharacterizedfortheirregulatoryfunctionsandsomeofthem
havebeensuccessfullyappliedforTIAproduction[24].
Therearemanyexamplesthatapplieddifferenttranscriptionfactorstoimprovethecontentof
plantsecondarymetabolites.OverexpressionofTSAR1orTSAR2inbasichelix-loop-helix(bHLH)gene
familyinMedicagotruncatulahairyrootscausedtheup-regulationoftriterpenesaponinbiosynthetic
genes and increased accumulation of triterpene saponins [24]. A novel APETALA2/Ethylene
Response Factors (AP2/ERF) family transcription factor PsAP2 from Papaver somniferum was
overexpressedintobacco,whichshowedimprovedresistancetobothabioticandbioticstresses[25].
AfterexpressingsevenMYBtranscriptionfactors(Dof1.1,IQD1-1,MYB28,MYB29,MYB34,MYB51
andMYB122)involvedinaliphaticandindolicglucosinolate(GSL)biosynthesisinChinesecabbage,
thealiphaticandindolicGSLcontentsarechanged[26]. OverexpressionofAaERF1andAaERF2from
Artemisia annua could increase the accumulation of artemisinin and artemisinic acid in transgenic
A. annua plants [27]. Thus, it is an effective way to apply effective transcription factors for plant
secondarymetabolicengineering.
Two AP2/ERF family transcription factors ORCA2 and ORCA3 were characterized as the
critical regulators for TIA biosynthesis. They could specifically bind to the JERE (jasmonate and
elicitor-responsiveelement)inSTRpromoterandcanbeup-regulatedbyJA[28]. InC.roseushairy
roots,theup-regulatedexpressionofORCA2significantlychangedthetranscriptsofmanystructural
genesinTIAbiosynthesis,suchasAS,TDC,G10H,LAMT,STR,T16H,PRX1,D4H,SGDandDAT;
moreover, the induced ORCA2 also caused the changes of the expressions of several TF encoding
genes,suchasORCA3,ZCT1,ZCT2,ZCT3andCrMYC2(Figure2). Accordingly,theaccumulationof
catharanthine,ajmalicine,serpentineandtabersoninewerealsochangeddramaticallyafterORCA2
induction[29]. AnotherstudyfoundthatvindolineintheORCA2transgenichairyrootscouldbe
significantlyincreasedcomparedtothecontrollines[30]. Therefore,ORCA2playsanimportantrolein
theregulationofTIAmetabolism. AnotherAP2/ERFfactorORCA3wasisolatedbyT-DNAactivation
tagging approach, which could also regulate the expressions of many TIA synthetic genes [31].
InC.roseuscellsuspensioncultures,overexpressingORCA3causedtheup-regulationofTDC,STR,SLS,
CPR,D4H,ASandDXS,variableexpressionofSGD,aswellasunaffectedexpressionofG10HandDAT.
Therefore,overexpressionofORCA3significantlyinducedtryptamineinshikimatepathway,whileit
didnotcausesecologanininseco-iridoidpathway. Also,feedingloganintoORCA3-overexpressed
lines significantly increase TIA production [31]. Partly different from the results of C. roseus cell
suspensioncultures,overexpressionofORCA3and,alongwithJAelicitationinhairyroot,induced
theexpressionlevelsofAS,DXS,SLSandSTR,decreasedtheexpressionofSGD,butnotaffected
TDC,G10H,CPR,GBFsandORCA2. Thedifferencesmayresultfromdifferentregulatorymechanisms
betweentwotypesoftissueculturesystems[32]. Therefore,theoverexpressionofORCA3alonein
two types of tissue cultures could not cover all the pathway genes, such as G10H, SGD and DAT.
Anotherstudyshowsthatco-overexpressionofORCA3andSGDinC.roseushairyrootsresultedina
significantincreaseofmanyTIAs[33]. Co-overexpressionofG10HandORCA3inC.roseusplantand
hairyrootsbothincreasedtheaccumulationofTIAs,indicatingthatco-overexpressionofregulator
andcriticalsyntheticgeneisanefficientwaytoincreasemetaboliteproduction[34]. BesidesJA,other
ORCA3inducersarefoundrecently. FeedingartemisinicacidtoC.roseusmeristematiccellsresultedin
theincreaseofthetranscriptlevelofORCA3,whichindicatedthatartemisinicacidmaybeanother
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waytoinduceORCA3[35]. AnotherstudyshowsthatinoculationoffungalendophytesinC.roseus
couldup-regulatetheexpressionofORCA3andenhancetheaccumulationofvindoline[36].
ThefactthatORCAscanbeinducedbynotonlyJAbutalsootherelicitorimpliestheexpressions
oftwoORCAsareregulatedbymoretranscriptionfactors,suchasCrMYC2. Asapositiveregulator
fortheexpressionsofORCAs,CrMYC2wasinitiallyisolatedthroughayeastone-hybridscreening
systemusingatetramerofG-boxfromtheSTRpromoterasbait,whileitcannotcontroltheactive
oftheSTRpromoter[37]. CrMYC2canregulatetheexpressionORCA3geneviabindingtoaspecific
sequenceinthejasmonate-responsiveelement(JRE)fromORCA3promoter[38]. Overexpressionor
knockdownofCrMYC2significantlychangedtheexpressionofORCAsandalsohadastrongeffect
ontheaccumulationofcatharanthineandtabersonine, butshowednoinfluenceonSTRandTDC
expression,whichindicatedthatCrMYC2iscriticalfortheexpressionofMeJA-responsiveORCAs
andtheaccumulationofalkaloidbutitcouldnotregulatesuchstructuralgenesasSTRandTDC[39].
AbHLHtranscriptionfactorBIS1fromcladeIVaisolatedfromC.roseuscouldregulatetheexpression
ofstructuralgenesthatORCA3cannotcover[40]. OverexpressionofBIS1inC.roseuscellsandhairy
rootscausedasignificantincreaseintheexpressionoftheseco-iridoidpathwaygenesupstreamof
LAMTandthe2-C-methyl-D-erythritol-4-phosphate(MEP)pathwaygenes. Meanwhile,loganicacid,
secologaninandstrictosidinewerehighlyaccumulatedintheBIS1-overexpressingcells. However,
theaccumulationofTIAsintransgenichairyrootswasnotincreased,whichmayberesultedfrom
thedown-regulationofORCA3targetgenesLAMT,SLS,TDC,andSGD.Decreasedexpressionofthe
ORCA3targetgeneswasnotcausedbydecreasedexpressionofORCA2orORCA3,norofanyother
knownC.roseusTFencodinggenepreviouslylinkedwithregulationoftheMIApathway,andthus
involvesother,yetunknown,regulatorymechanisms. AnotherbHLHtranscriptionfactorBIS2was
identifiedrecently,whichisthehomologofBIS1thatcanformhomo-orheterodimerswithBIS1. Same
withBIS1,overexpressingBIS2inC.roseussuspensioncellscouldalsoup-regulateMEPaswellas
seco-iridoidpathwaygenes,knockdownofBIS2completelyabolishedtheJA-inducedup-regulation
oftheseco-iridoidpathwaygenesandtheaccumulationofTIAs. TheexpressionofBIS2couldbe
regulatedbyBISs,whiletheBISs-bindingsitesofBIS2promoterremaintobedetermined. BIS1and
BIS2couldbothregulatethestructuralgenesthatORCA2andORCA3cannotaffect,suchasG10H[41].
Therefore,BISstogetherwithORCAsmaycontrolthewholeupstreampathwayofTIAbiosynthesis
andsupportsufficientsyntheticprecursorofTIAs.
TwoAP2/ERFproteinsformaclusterwithORCA3,ORCA4andORCA5werecloned,whichare
homologoustoORCA3. TheexpressionofORCA4andORCA5canbealsoinducedbyJAlikeORCA3.
However,unlikeORCA3,ORCA4andORCA5cannotbedirectlyregulatedbyCrMYC2,theymaybe
regulatedbyothertranscriptionfactors. Moreover,overexpressionofORCA4inC.roseushairyroots
significantlyincreasedthetranscriptslevelsofgenesinbothtryptophanpathwayandseco-iridoid
pathway,andalsoincreasedseveralTIAs,especiallytabersonine. Therefore,ORCA4isfunctionally
overlappingbutdivergentwithORCA3[42].
CrWRKY1,belongingtothegroupIIIWRKYsuperfamily,wasidentifiedinC.roseus,whichcanbe
inducedbyseveralphytohormonesandpreferentiallyexpressesinroots[43]. StudiesfoundCrWRKY1
regulatesTDCbybindingWboxelementinTDCpromoter. OverexpressionofCrWRKY1inC.roseus
hairyrootsincreasedseveralkeypathwaygenes,suchasAS,DXS,SLS,SGD,especiallyTDC;aswellas
TFencodinggenes,suchasZCTs,whileitrepressesthetranscriptionalactivatorsORCA2,ORCA3,and
CrMYC2. Moreover,theaccumulationofserpentinewassignificantlyincreased,whilecatharanthine
wasdecreased. Therefore,CrWRKY1isanidealcandidatetoregulateserpentinebranchtoproduce
moreajmalicineandserpentine.
CrBPF1 is a kind of MYB transcription factor that binds to BA region in STR promoter.
OverexpressingCrBPF1inC.roseushairyrootschangedtheexpressionofmanypathwaygenes. Asfor
regulatorygenes,overexpressingCrBPF1increasedthetranscriptsofORCA3,CrMYC1,CrMYC2,BIS1,
GBF2andZCTs,butlittleaffectedtheexpressionsofORCA2andCrWRKY1. Althoughoverexpression
of CrBPF1 could not obviously increase the accumulation of TIAs and even caused the decrease
Int.J.Mol.Sci.2017,18,53 6of20
ofserpentine,itcouldextensivelyregulateTIApathwaygenesandincreasetheexpressionofTIA
transcriptionalrepressors,whichmaybeagooddirectiontotheresearchofTIAbiosynthesis[44].
IthasbeenreportedthatusingG-boxelementintheSTRpromotersasbaittoscreenC.roseus
yeastexpressionlibraryresultedinidentificationofG-boxbindingfactorsCrGBF1andCrGBF2[37,45].
BothCrGBF1andCrGBF2actastranscriptionalrepressorsoftheSTRviabindingtotheNRelement
Int. J. Mol. Sci. 2017, 18, 53 6 of 19
in STR promoter, which indicated that GBFs may play an important role in the regulation of the
expressionofSItT hRasa bneden trheepoarctecdu tmhaut luastiinogn Go-bfoTx IeAlesm.ent in the STR promoters as bait to screen C. roseus
yeast expression library resulted in identification of G-box binding factors CrGBF1 and CrGBF2
ThreeCys2/His2-typezincfingertranscriptionfactorsfromC.roseus,ZCT1,ZCT2andZCT3,
[37,45]. Both CrGBF1 and CrGBF2 act as transcriptional repressors of the STR via binding to the NR
wereisolatedthroughayeastone-hybridscreeningsystemusinganelicitor-responsiveDBelement
element in STR promoter, which indicated that GBFs may play an important role in the regulation of
in TDC prthoem exoptreerssaiosn bofa SitTR[ 4a6n]d. thAe allccoumf uthlaetimon orfe TpIrAess. s the activities of STR and TDC promoters in
trans-activatioTnhraees sCayyss2,/Hwish2-itcyhpem zianyc fpinrgoebr atrbalnyscbripetiroens ufalctteodrs ffrroomm Ct. hroeseeuxs,i sZtCeTn1c, eZCoTf2a apndo tZeCnTt3r, epression
were isolated through a yeast one-hybrid screening system using an elicitor-responsive DB element
domain,LxLxLmotifintheC-terminalregionofZCTs. Inaddition,theZCTproteinscanalsorepress
in TDC promoter as bait [46]. All of them repress the activities of STR and TDC promoters in
thetranscriptionalactivatingactivationoftheORCAs,andarefoundtobeinducedbyyeastextract
trans-activation assays, which may probably be resulted from the existence of a potent repression
(YE)andJdAo.mHaion,w LexvLxeLr, mthoetirfe ina rtehes eCv-teerrmalindail frfeegrieonn coefs ZbCeTtsw. Iene nadZdiCtioTn1, ,tZheC ZTC2Ta pnrdoteZinCs Tca3n. Talhsoe STRand
TDCpromroepterersss tbhien tdrainnsgcrispittieosnaol facZtiCvaTti1ng, ZacCtivTa2tioanr eofd thifef OerReCnAtsf, raonmd aroen feouonfd ZtoC bTe 3in,dauncedd tbhye yesatsrtu cturesof
extract (YE) and JA. However, there are several differences between ZCT1, ZCT2 and ZCT3. The
ZCT1,ZCT2andZCT3arealsodifferent[46]. Furthermore,thefunctionsofthemarepartiallydifferent.
STR and TDC promoters binding sites of ZCT1, ZCT2 are different from one of ZCT3, and the
ZCT1andZCT2actasrepressorsofhydroxymethylbutenyl4-diphosphatesynthase(HDS)whileZCT3
structures of ZCT1, ZCT2 and ZCT3 are also different [46]. Furthermore, the functions of them are
hasnoeffepcatrtoianllyH dDiffSer[e4n7t.] .ZACTr1e canedn tZsCtuT2d yacst haos wresprtehsasotrss iloefn hcyindrgoxZyCmTet1hywlbautsennyol t4s-duifpfihcoisepnhattteo increase
TIA produsycntitohanseo (rHtDhSe) wexhpiler eZsCsTio3 nhaos fntoh eeffeTcIt Aonb HioDsSy [n47th]. eAt ircecgeennt setsu,dyw shhiocwhs mthaaty sibleencrinegs uZlCteTd1 from the
was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes, which
remainedelevatedexpressionlevelofZCT3. TheseresultsrevealthattheZCTsmayplayoverlapping
may be resulted from the remained elevated expression level of ZCT3. These results reveal that the
butdistinctfunctionsinTIAbiosynthesis[48].
ZCTs may play overlapping but distinct functions in TIA biosynthesis [48].

Figure2. FRigeugrue l2a.t iRoenguolaftiTonI Aof bTiIoAs ybinostyhnetthiecticp aptahthwwaayyss bbyy trtarnascnrsipctrioipn tfiaocntorfsa cint oCrasthianranCthautsh arorsaenust.h usroseus.
Various structural genes (marked in blue) in TIA biosynthetic pathway are regulated by different
Variousstructuralgenes(markedinblue)inTIAbiosyntheticpathwayareregulatedbydifferent
transcription factors (TFs). ORCAs are believed to be key regulators that directly bind to the
transcription factors (TFs). ORCAs are believed to be key regulators that directly bind to the
promoters of structural genes involved in TIA biosynthesis. Several TFs such as WRKYs not only
promoterscoonftrsotlr uthcet uexrparlesgseionne osf isntrvuoctluvreald gienneTs IbAut balisoos yrengtuhlaetes itsh.e SeexpvreerssailonT Fofs ostuhecrh TaFss iWnclRudKinYgs notonly
control theMYexCp2,r OesRsCioAns, oZfCTsst,r uetcct. uSroamle gTeFns essucbhu ats aMlsYoC2r ecgouullda nteot tdhierecetxlyp rreegsusliaoten thoef eoxtphreesrsioTnF sof including
structural genes in biosynthetic pathways. Therefore, these TFs form a transcriptional regulatory
MYC2, ORCAs, ZCTs, etc. Some TFs such as MYC2 could not directly regulate the expression of
network for TIA biosynthetic pathways. Among them, some TFs, such as BIS2, ZCT2 and WRKY2
structural genes in biosynthetic pathways. Therefore, these TFs form a transcriptional regulatory
(marked in green), are negative regulators, whereas most TFs (marked in red) are positively regulate
networkfoTrIAT bIAiosybniothseysins.t hDeXtSi,c 1p-daetohxwy-Da-yxsy.luAlosme o5-npghotshpehmate, ssyonmtheasTe; FGs1,0sHu, cgheraasnioBlI S102-,hyZdCroTx2ylaasne;d WRKY2
(markedinCPgRre, ceynto),charroemnee Pg4a50ti rveedurcetgasuel; aLtAoMrsT,,w lohgaenrieca ascimd mosetthTylFtrsan(msfearraksee; dSLiSn, sreecdol)ogaarneinp soysnitthivaseel;y regulate
TIAbiosynSTthRe, sstirsic.toDsiXdiSn,e 1sy-dntehoasxey; S-DG-Dx, ystlruicltoossiedi5n-ep βh-Do-sgpluhcoastiedassye;n CthR,a csaeth;eGna1m0Hine, rgeedruactnaisoesl; 1T016-hHy2,d roxylase;
tabersonine 16-hydroxylase 2; 16OMT, 16-hydroxytabersonine-16-O-methyltransferase; T3O,
CPR,cytochromeP450reductase;LAMT,loganicacidmethyltransferase;SLS,secologaninsynthase;
tabersonine 3-oxygenase; T3R, tabersonine 3-reductase; NMT, N-methyltransferase; D4H,
STR, stricdtoessaicdeitnoxeyvsinydnotlhinaes 4e-h;ydSrGoxDyl,asse;t rDiActTo, saicdetiynl eCoβA-: Dde-gacleutyclovsiniddoalsinee; 4-CO-Rac,etcyalttrhanesnfearmasein. e reductases;
T16H2, tabersonine 16-hydroxylase 2; 16OMT, 16-hydroxytabersonine-16-O-methyltransferase;

T3O, tabersonine 3-oxygenase; T3R, tabersonine 3-reductase; NMT, N-methyltransferase; D4H,
desacetoxyvindoline4-hydroxylase;DAT,acetylCoA:deacetylvindoline4-O-acetyltransferase.
Int.J.Mol.Sci.2017,18,53 7of20
4. TransportofTIAsinandbetweenOrgans,Tissues,CellsandSubcellularCompartments
Tissue, cell-specific and multi-organelle-participated synthesis of specific TIAs have been
extensively studied with various techniques, including in situ hybridization, immunoblot,
transcriptomicanalysisandGFP-fusionofmetabolicenzymesortransporters. Highlycomplicated
regulationofvariousregulatoryandstructuralgenes,translocationofmetabolicenzymesandtransport
ofmetabolicintermediatesthroughdifferenttissues,cellsandsubcellularcompartmentsarerequired
forefficientbiosynthesisofTIAsuponvarioushormonalandenvironmentalcues[49]. Threemodesof
transportofalkaloidsinplantshavebeenreported: inter-organ,inter-cellularandintra-cellular[50].
Thetypicalinter-organalkaloidstransportsareforberberineandnicotine:berberineissynthesized
in the root of Coptis japonicais and then transported to the rhizome through a long distance [51];
nicotineisproducedonlyintherootofNicotianatabacumandtransportedtotheleavesoftheplantvia
thexylem[52]. InC.roseus,synthesisofsomeTIAssuchasvindolineandcatharanthine,mainlytakes
placeinyoungleavesandstems,whereassynthesisofotherssuchasajmalicineandserpentinemainly
occursinroots. Theyalsodisplaycomplexinter-cellularandintra-cellulartransport.
5. Inter-CellularTransportIsRequiredforBiosynthesisofTIAs
Studies have revealed that TIA biosynthetic pathway in aerial organs of C. roseus occurs in
four cell types: internal phloem-associated parenchyma (IPAP), epidermal cells, laticifers and
idioblasts [53,54]. The IPAP cells exist in the periphery of stem pith or in traxylary on the upper
part of the vascular bundles in leaves and are full of chloroplast with the plastid-located MEP
pathway enzymes [53,55]. MEP pathway, which is required for the development and function of
chloroplast, happens mostly in young green tissues [56]. The early step of seco-iridoid pathway,
geraniolconversioninto10-oxogeranialviageraniol10-hydroxylase(G10H)areco-localizedinthe
IPAPcellsofyoungtissuessuchasleavesandroots[11,57]. Iridoidoxidase(IO),7-deoxyloganetic
acidglucosyltransferase(7-DLGT)and7-deoxyloganicacidhydroxylase(DL7H)arealsolocalized
totheIPAPcellsandtheirtranscripts, whicharefourtimesmoreabundantinthewholeleafthan
in the epidermis, while loganic acid methyl-transferase (LAMT) and secologanin synthase (SLS)
are preferentially expressed in the leaf epidermis. Loganic acid, as the intermediate, is assumed
to move from the IPAP cells to the epidermis to convert into secologain under the catalyzing of
LAMTandSLS[58–62]. Tryptamineandsecologaninarecondensedtoformstrictosidineinepidermal
cell of young developing shoots and leaves, meanwhile, STR- and SGD- catalyzed central steps
also happen in the epidermis (Figure 3) [63]. Strictosidine is the central precursor of TIAs, some
ofwhicharedirectlyformedinepidermis,suchasajmalineandcatharanthine. Ajmalineismainly
accumulated in root epidermis, while catharanthine is mainly secreted to the surface of leaves by
the ATP Binding cassette (ABC) transporter CrTPT2 to transport it from the epidermis to the leaf
surfaceinthewaxexudates(Figure3)[64]. Asforvindoline,thelateprecursordesacetoxyvindoline
forvindolinebiosynthesisisformedinepidermis,theenzymestabersonine16-hydroxylase2(T16H2),
16-hydroxytabersonine-16-O-methyltransferase(16OMT),T3O/T3RandN-methyltransferase(NMT)
arepreferentiallylocalizedinepidermis[31,32]. However,Desacetoxyvindoline4-hydroxylase(D4H)
and acetyl-CoA:4-O-deacetylvindoline 4-O-acetyltransferase (DAT), catalyzing the last two steps
are confirmed to localize idioblasts and laticifers of leaves, stems and flowers [65]. Therefore, the
intermediatesdesacetoxyvindolineorotheraccessoryproductsneedtobetransportedfromepidermis
toidioblastsorlaticifersforatwo-stepcollaborationtoformvindoline[66],butwhetheritisnecessary
or how vindoline is transported out of idioblasts and laticifers remains unknown. Whether some
stagesofthetranslocationareaccomplishedpassivelythroughthesymplasmorarecontrolledby
plasmodesmata-localizedproteinsisstillanopenquestion.
Int.J.Mol.Sci.2017,18,53 8of20
Int. J. Mol. Sci. 2017, 18, 53 8 of 19

Figure 3. Tissue-specific biosynthesis and inter-cellular transport of TIAs through known and
Figure3.Tissue-specificbiosynthesisandinter-cellulartransportofTIAsthroughknownandunknown
unknown transporters in Catharanthus roseus. The main TIA-biosynthesizing cells in the leaf of C.
transportersinCatharanthusroseus.ThemainTIA-biosynthesizingcellsintheleafofC.roseusinclude
roseus include palisade and spongy mesophyll cells, internal phloem-associated parenchyma (IPAP),
palisade and spongy mesophyll cells, internal phloem-associated parenchyma (IPAP), epidermal
epidermal cells, laticifers and idioblasts. So far, most transporters possibly involved in transport of
cells, laticifers and idioblasts. So far, most transporters possibly involved in transport of TIA
TIA intermediates or end products between these cells are unknown. The symbol “?” indicates the
intermediates or end products between these cells are unknown. The symbol “?” indicates the
unknown transportation system of inter-cellular transport. The only identified transporter
unknowrensptornansisbpleo rftoart iroenlesaysisntgem ofo fcainthtaerra-cnethlliunlea roturta nosf puoprpt.eTr heepiodnelrymiadl ecnetlilfis eodnttora cnustpicolret eisr raens pAoBnCsi ble
forreletarasninspgoortfecr aTthPaTr2a. nWthhienteheoru ptloafsmuopdpeesrmeaptaid, esyrmmbaollcizeellds oinn tboluceu tbicallles,i saraen aAlsBoC intvraonlvsepdo rinte rTITAP T2.
Whethienrteprlmasemdiaotde etsramnasptao,rts ybemtwbeoelniz deidffeinrebnltu tyepbeas lolsf ,caelrles raelmsoaiinn vtoo blvee ddetienrmTiInAedin. termediatetransport
betweendifferenttypesofcellsremaintobedetermined.
6. Intra-Cellular Transport of TIA Intermediates and End Products
6. Intra-CellularTransportofTIAIntermediatesandEndProducts
The MEP pathway primarily takes place in the stroma of plastids or stromules in IPAP cells
[11,67]. However, isopentenyl diphosphate isomerase (IDI), which catalyzes the interconversion of
The MEP pathway primarily takes place in the stroma of plastids or stromules in IPAP
isopentenyl diphosphate (IPP) and dimethylally diphosphate (DMAPP) to produce plenty of
cells[11,67]. However,isopentenyldiphosphateisomerase(IDI),whichcatalyzestheinterconversion
isoprenoids, was targeted to plastids, mitochondria and peroxisome [68]. Therefore, geraniol needs
of isopentenyl diphosphate (IPP) and dimethylally diphosphate (DMAPP) to produce plenty of
to be exported from the stroma by uncharacterized plastid inner or outer envelope transporter or
isoprenoids,wastargetedtoplastids,mitochondriaandperoxisome[68]. Therefore,geraniolneeds
some efficient metabolic flux into the cytosol of IPAP cells [69]. Geraniol is the substrate of the
to be evxapcuoorltaerd mfreommbrtahnee- storro menadbopylausnmcihc arreatcictuerluizme d(EpRla)-satsisdociinanteedr oGr10oHu teinr etnhev eplorpodeutcrtaionns poofr ter
or som1e0-heyffidcroiexnytgemraentiaobl o[7li0c], flwuhxichin itso futrhtehecr yctoonsvoelrtoefd IiPnAtoP thcee llolsga[n6i9c ]a.ciGd ebrya annio ElRi-sastshoeciastuebds Ptr4a5t0e of
the vaDcuLo7lHa.r 1m0-heymdbrorxayngee-raonrioel nddeohypdlarosmgeincasree t(i1c0uHluGmO) (aEnRd )7--aDssLoGcTia atered foGu1n0dH in itnhet hcyetopsorol dinu IcPtAioPn of
10-hydcreollxsy. gLeorgaanniico la[c7id0 ]a,nwdh sieccholiosgfaunritnh aerrec soynnvtheerstiezdedin itno tthhee clyotogsaonl iocfa ecpididebrymaanl cEeRlls-,a ssisnoccei aLtAedMPT 450
DL7H.a1n0d- hEyRd-arnocxhyogreedr aPn4i5o0l SdLeSh yhdavroe gbeenena seen(s1u0reHdG toO l)oacantde i7n- DthLeG cTytoasroel foofu tnhde einpidtheermciyst o[7s1o]l. TinDICP AP
involved in the shikimate pathway is localized to the cytosol of the epidermis. Tryptamine and
cells. Loganicacidandsecologaninaresynthesizedinthecytosolofepidermalcells,sinceLAMTand
secologanin are transported by unidentified transporters from cytosol into the vacuole, in which STR
ER-anchoredP450SLShavebeenensuredtolocateinthecytosoloftheepidermis[71]. TDCinvolved
condenses them into strictosidine [63]. The vacuole-accumulated strictosidine or its hydrolyzed
intheshikimatepathwayislocalizedtothecytosoloftheepidermis. Tryptamineandsecologaninare
product aglycon needs to be transported out of the vacuole into the cytosol. SGD was a highly stable
transportedbyunidentifiedtransportersfromcytosolintothevacuole,inwhichSTRcondensesthem
supramolecular complex within the nucleus, indicating that transportation of strictosidine across the
intostrtiocntoospildasint eb[y6 3u]n.iTdhenetvifaiecdu otrlea-nascpcourmteru lpaltaeyds satr iccrtiotisciadl inroeleo rinit schonytdrorollliynzge dTIpAr obdiouscytnathgelysicso n[63n]e. eds
tobetrSatnriscptoosritdeidneo augtlyocfotnhee ivs acocunvoelertiendt ointtho eTcIAytso, ssuocl.hS aGs Dcatwhaarsaanthhiignhe laynsdt atabbleerssuonpirnaem ino tlheceu clyatroscoolm ofp lex
withinetphiedenrumcilse.u Tsh,ei ncodnicvaetrisniognt ohfa ttatbrearnsospnionret atoti ovinndooflsintrei cotcocsuirdsi nine vaacrrioousss cthometpoanrtompelanstst. bTyheu fnirisdte tnwtoifi ed
transpostretpers pcaltaaylyszaedcr bityi cTa1l6rHol aenidn 1c6oOnMtroTl lwinegreT loIAcalbizioesdy innt thhees cisyt[o6s3o]l. [S7t2r]i.c Tto16siHd iins eana gElRy-caonncheoirsecdo Pn4v5e0r ted
into TIAs, such as catharanthine and tabersonine in the cytosol of epidermis. The conversion of

tabersonine to vindoline occurs in various compartments. The first two steps catalyzed by T16H
and 16OMT were localized in the cytosol [72]. T16H is an ER-anchored P450 and can release
Int.J.Mol.Sci.2017,18,53 9of20
Int. J. Mol. Sci. 2017, 18, 53 9 of 19
16-ahnydd rcoaxny traebleearsseo n1i6n-ehytodrtohxeyctyabtoesrosolnviinaei ttsoe xthpeo sciyntgoscoalt avlyiat icitss iteextpoowsianrgd cthatealcyyttioc ssoilte[7 t3o,7w4a]r.dN MtheE is
likceylytotsoobl e[7l3o,c7a4l]i.z NedMinE tihse lilkeealfye ptoid beer mloicsailnizaesds oinci athtieo nlewafi tehpcidhelormroips lians tatshsyolcaiaktoioidn mweimthb crhalnoersop[7la5s–t7 7]
(Fitghuyrleak4o).id membranes [75–77] (Figure 4).

FigFuigruer4e. 4S. uSubbcceelllulullaarr ccoommppaarrttmmeennttaattioionn oof fTTIAIA biboisoysnythnethsiess aisnda nindteinr-t eorr- inotrrain-cterlal-uclealrl utrlaanrstproarnts opfo rt
ofTTIAIAs sini nCaCthaatrhaanrtahnutsh ruosseuross. eTuIsA. bTioIsAynbthioetsiyc nptahtehtwicayp ainth aweraiayl oinrgaanesr ioafl Co.r rgoasneuss oofccCur.sr ions efuousro cceclul rs
types: internal phloem-associated parenchyma (IPAP), epidermal cells, laticifers and idioblasts. An
in four cell types: internal phloem-associated parenchyma (IPAP), epidermal cells, laticifers and
ATP-binding cassette transporters TPT2 can transport catharanthine from epidermal cells to deposit
idioblasts. AnATP-bindingcassettetransportersTPT2cantransportcatharanthinefromepidermal
on cuticle. The transporters transport TIA intermediates between these cells, such as
cellstodepositoncuticle.ThetransporterstransportTIAintermediatesbetweenthesecells,suchas
desacetoxyvindoline from epidermal cells to laticifers and idioblasts for vindoline biosynthesis and
desacetoxyvindolinefromepidermalcellstolaticifersandidioblastsforvindolinebiosynthesisand
loganic acid communicates between the epidermal cells and IPAP cells, remain unknown, as
loganicacidcommunicatesbetweentheepidermalcellsandIPAPcells,remainunknown,asindicated
indicated by question marks. In each type of cells, primarily, the epidermal cells, the vacuole, the
byquestionmarks. Ineachtypeofcells,primarily,theepidermalcells,thevacuole,thechloroplast
chloroplast and possibly, the nucleus, are involved in the TIA biosynthesis. However, transporters or
andpossibly,thenucleus,areinvolvedintheTIAbiosynthesis. However,transportersortransport
transport mechanisms for cross-membrane communication of these intermediates are largely
mechanismsforcross-membranecommunicationoftheseintermediatesarelargelyunknown.Howdo
unknown. How do two important precursors secolognain and tryptamine are transported into the
two important precursors secolognain and tryptamine are transported into the vacuole for their
vacuole for their condensation into strictosidine by vacuole-localized STR? Are they transported by
condensationintostrictosidinebyvacuole-localizedSTR?AretheytransportedbyMATEtransporters?
MATE transporters? What is it necessary for SGD localization in the nucleus, as reported, since
WhatisitnecessaryforSGDlocalizationinthenucleus,asreported,sinceaglyconisstillneedto
aglycon is still need to be transported out of the nucleus? How the product of T3O/T3R is transported
betransportedoutofthenucleus?HowtheproductofT3O/T3Ristransportedintothechloroplast
into the chloroplast for methylation by the granule-localized NMT? All these questions need to be
formethylationbythegranule-localizedNMT?Allthesequestionsneedtobeansweredtogivea
answered to give a clear scenario of intermediate transport or trafficking during the TIA
clearscenarioofintermediatetransportortraffickingduringtheTIAbiosynthesis.Abbreviationsare:
biosynthesis. Abbreviations are: SGD, strictosidine β-D-glucosidase; STR, strictosidine synthase; TDC,
SGtDry,psttorpichtaonsi ddeincearβbo-Dx-yglalusec;o DsiXdSa,s e1;-dSeToRx,ys-tDr-ixcytolusildosine e5-spyhnothspahsea;teT DsyCn,thtrayspe;t oDpXhRa,n 1-ddeecoaxryb-oDx-yxlyalsuel;oDseX S,
1-d5e-pohxyos-Dph-xaytelu losreed5u-pchtooissopmhaetreassey; nthaMseC;TD, XR,M1-EdPe oxyc-yDt-ixdyyllutrlaonssefe5r-apshe;o sphCaMteKre, duc4t-o(icsyotmidienrea se;
MC5’T-d,MiphEoPspcyhtoi)d-y2-lCtr-amnesftehryals-De;-eCryMthKr,it4o-l( cyktinidaisnee; 5M’-dEiCpSh, os2p-Ch-om)-e2t-hCy-lmereytthhyrli-toDl- e2ry,4t-hcryictololdkiipnhaossep;hMaEteC S,
2-Csy-mntehtahsye;l erHyDthSr,i tohly2d,r4o-xcyymcleotdhiyplhbuotsepnhyal te4-sdyinpthhoasspeh;aHte DsSy,nhthyadsreo; xyHmDeRt,h yhlybdurtoexnyyml e4t-hdyiplbhuotesnpyhla te
syn4-tdhiapsheo;spHhDatRe ,rehdyudcrtaosxey; mIDeIt,h yIPlbPu itseonmyelra4s-ed; ipGhPoPsSp, hgaetreanryeldduipchtaossep;haItDe Is,ynIPthPaseis; oGmEeSr,a sgee;ranGioPlP S,
gesryanntyhladsiep;h Go1s0pHh,a tgeersaynniothl 1a0se-h;yGdrEoSx,ylgaesrea; n1i0oHlGsyOn, t1h0a-hsey;drGo1x0yHge,ragneiroaln dieohly1d0r-ohgyednraosxe;y IlDasSe,; ir1id0oHidG O,
10-shyyndthraosxey; g7eDrLanS,i o7l-ddeeohxyydlorgoagneentaics ea;ciIdD sSy,nitrhidaoseid; DsLyGntTh, a7s-ed;e7oDxyLloSg,a7n-deteico xaycildog galnuecotiscyaltcriadnssfyernatshea; se;
DLDGLT7,H7,- d7-edoexoyxlyolgoagnaentiicc aacciidd 7g-hluycdorsoyxlytrlaasnes; fLerAaMseT;,D loLg7aHn,ic7 -adceido xmyelothgyalntriacnascfiedra7s-eh; ySdLrSo, xsyeclaosloeg;aLnAinM T,
logsaynnitchaascei; d methyTlt1r6aHns2f,e rase; SLtSab,esrescoonliongea nin syn1th6-ahsyed;rTox1y6lHas2e, taberson2;i ne 16-hy1d6rOoMxyTla, se
2;1166-hOyMdrTo,xy1t6a-bheyrdsornoixnyet-a1b6e-Ors-omneitnhey-l1tr6a-nOs-fmereatshey; lTtr3aOn,s ftearbaesres;onTi3nOe ,3t-aobxyegrseonnaisne;e T33-oRx, ytgaebnerassoen;inTe3 R,
tab3e-rresdonuicntaes3e;- rNedMuTc,t Nas-em;eNthMyTlt,raNn-smfeerathsey;l tDra4nHs,f dereasasece;tDox4yHv,inddeosalicneet o4x-hyyvdinrodxoylliansee;4 D-hAyTd,r aocxeytylals Ceo;DA:A T,
acedteyalcCetoyAlv:idndeaoclientey l4v-iOn-daocelitnyeltr4a-nOs-faecreatsye.l transferase.
Int.J.Mol.Sci.2017,18,53 10of20
Laticifersandidioblastsarethecellslocatedinstemsandleaveswheretabersoninederivative
converts into vindoline. The last two vindoline synthetic enzymes DAT and D4H are localized in
cytoplasmandnucleusofthelaticifersandidioblastscells[74]. Vindolineisformedinthecytosol
oflaticifersandidioblastsandtransportedintothevacuolebyaspecificprotonantiporter,whichis
energized by the V-H+-ATPase. Catharanthine and AVLB are also taken up into the vacuole of
epidermalcellsbyanuncharacterizedantiporterthroughanH+-dependentmechanism(Figure4)[78].
SincecatharanthineandvindolinearenotinthevacuoleofthesamecellsinC.roseusleaves,theyare
coupledtoformbisindolealkaloidsvinblastineandvincristine,onlybecausestimulationfromexternal
environmentcouldbreakdownthespatialseparation. However,aslongasbisindolealkaloidforms,
theyaretransportedandaccumulatedinthevacuoles.
7. TheBiochemical,MolecularBiologicalAspectsofTIATransporters
TIAs present in C. roseus tissues for many physiological functions. Catharanthine can inhibit
the growth of fungal zoospores and shows insect toxicity at physiological concentrations on the
surface of C. roseus leaves. The complex developmental, environmental, hormonal, organ- and
cell-specificcuesregulatethegenesinvolvedinthebiosynthesisofTIAs[77].Proteinkinasecascade-or
calcium-mediatedsignalingisimportantfortheregulationofmethyljasmonate-dependentandyeast
elicitor-inducedTIAbiosynthesisinC.roseuscells[79–81]. Thesefactors-induceddenovobiosynthesis
andactivesecretionofTIAscostlotsofenergy.Thelong-distancetransportofendproductsorsecretion
ofTIAstothetargetsitesalsorequiresenergy[64]. ThevacuolarsequestrationoftheseTIAsmore
likelydependsonsecondarytransporters,suchasMATEormultidrugresistancetransporter[78].
Thebiosynthesisofintermediatesaswellasend-productsofTIAshappensindifferenttissues
andorgans,andtheyaresubsequentlytransportedtothesitesforthenextreactions,storage,orfor
theirphysiologicalfunctions. Therefore,theefficientlong-distancetransportisessentiallyrequired.
Usually, three basic transport or trafficking processes exist for organic compounds in plant cells,
membrane vesicle trafficking of substances, protein-aided transport/subcellular trafficking and
membranetransporters-mediatedacross-membranetransport[3,82].
AlthoughgreatattentionwaspaidtothetransportofTIAslongtimeago,nowitisrecognized
thatapassivediffusionisusuallyimpossiblefortransportofhighlychargedTIAs,likewiseforan
unspecific ion-trap mechanism [20,83]. Plenty of evidence indicates that many alkaloids such as
berberine in Coptis japonica, nicotine in tobacco, or terpenoids are transported across the plasma
membranebyABCtransporters. Forexample,CjMDR1expressedinthexylemoftherhizome[51]
andCjABCB2expressedincellsaroundthexylemoftherhizome[84]areinvolvedinthetransportof
berberinetotheplacewhereberberineissynthesized;NtNUP—aplasmamembranenicotine—actsas
aprotonsymporterofnicotine[85];multidrugandtoxiccompoundextrusion(MATE)transporters
act as vacuolar sequestration of nicotine [86,87]; NpPDR1—a plasma membrane pleiotropic drug
resistance-typeABCtransporterinNicotianaplumbaginifolia—transportsditerpenesclareolantifungal
compound[88]. OverexpressionofCjMDR1inC.roseuscellculturespromotedasignificantuptakeand
accumulationofajmalicineandtetrahydroalstoninecomparedwithcontrollinesafterfeedingthese
alkaloids[89]. TheseresultssuggestthatTIAscanbetransportedbysuchtypeofABCtransporter.
Proteomic study on two independent cell lines with different TIAs metabolism reveal that some
differentiallyexpressedtransporterspossiblyinvolvedinthetransportofTIAs,includingsevenABCG
proteins,threemultidrugresistancepumps,twomultidrugresistance-associatedproteins,threesoluble
ABCtransporters(ABCEandABCFsubfamilies)andoneforeachMATEandpeptidetransporter[90].
InC.roseus,TIAsareprovedtobeaccumulatedinthevacuolesbyvarioustechniques[78,91,92].
OnestudyindicatesthataspecificprotonantiportsystemcouldtrapTIAsinthevacuolesofC.roseus
mesophyllcells. TheuptakeofvindolineintotheisolatedtonoplastvesicleswasdependentonATP
andH+gradientacrossvacuolarmembrane,sinceitwassharplysuppressedbyH+gradientdissipators
butunaffectedbyABCtransporterinhibitor[78]. Interestingly,catharanthineandanhydrovinblastine
are also incorporated into the vacuole through an H+-dependent mechanism. These indicate that