Alkaloid Biosynthesis: Metabolism and Trafficking J¨ org Ziegler and Peter J. Facchini Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada; email: [email protected]; [email protected]Annu. Rev. Plant Biol. 2008. 59:735–69 The Annual Review of Plant Biology is online at plant.annualreviews.org This article’s doi: 10.1146/annurev.arplant.59.032607.092730 Copyright c 2008 by Annual Reviews. All rights reserved 1543-5008/08/0602-0735$20.00 Key Words benzylisoquinoline alkaloids, cellular compartmentalization, secondary metabolism, monoterpenoid indole alkaloids, tropane alkaloids, metabolic engineering Abstract Alkaloids represent a highly diverse group of compounds that are related only by the occurrence of a nitrogen atom in a heterocyclic ring. Plants are estimated to produce approximately 12,000 different alkaloids, which can be organized into groups according to their car- bon skeletal structures. Alkaloid biosynthesis in plants involves many catalytic steps, catalyzed by enzymes that belong to a wide range of protein families. The characterization of novel alkaloid biosyn- thetic enzymes in terms of structural biochemistry, molecular and cell biology, and biotechnological applications has been the focus of research over the past several years. The application of genomics to the alkaloid field has accelerated the discovery of cDNAs encoding previously elusive biosynthetic enzymes. Other technologies, such as large-scale gene expression analyses and metabolic engineering approaches with transgenic plants, have provided new insights into the regulatory architecture of alkaloid metabolism. 735 Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org by Washington State University on 11/01/11. For personal use only.
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ANRV342-PP59-29 ARI 29 March 2008 2:13
Alkaloid Biosynthesis:Metabolism and TraffickingJorg Ziegler and Peter J. FacchiniDepartment of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4,Canada; email: [email protected]; [email protected]
Annu. Rev. Plant Biol. 2008. 59:735–69
The Annual Review of Plant Biology is online atplant.annualreviews.org
This article’s doi:10.1146/annurev.arplant.59.032607.092730
AbstractAlkaloids represent a highly diverse group of compounds that arerelated only by the occurrence of a nitrogen atom in a heterocyclicring. Plants are estimated to produce approximately 12,000 differentalkaloids, which can be organized into groups according to their car-bon skeletal structures. Alkaloid biosynthesis in plants involves manycatalytic steps, catalyzed by enzymes that belong to a wide rangeof protein families. The characterization of novel alkaloid biosyn-thetic enzymes in terms of structural biochemistry, molecular andcell biology, and biotechnological applications has been the focus ofresearch over the past several years. The application of genomics tothe alkaloid field has accelerated the discovery of cDNAs encodingpreviously elusive biosynthetic enzymes. Other technologies, suchas large-scale gene expression analyses and metabolic engineeringapproaches with transgenic plants, have provided new insights intothe regulatory architecture of alkaloid metabolism.
Alkaloids are a diverse group of low-molecular-weight, nitrogen-containing com-pounds derived mostly from amino acids. Assecondary metabolites found in approximately20% of plant species, alkaloids are purportedto play a defensive role against herbivores andpathogens. Owing to their potent biologicalactivity, many of the approximately 12,000known alkaloids have been exploited as phar-maceuticals, stimulants, narcotics, and poi-
sons. Unlike most other types of secondarymetabolites, the many classes of alkaloids haveunique biosynthetic origins. Despite the di-versity of metabolic pathways, several techni-cal breakthroughs have recently contributedto impressive advancements in our under-standing of alkaloid biosynthesis. Recentapplications of genomics-based technolo-gies, such as expressed sequence tag (EST)databases, DNA microarrays, and proteomeanalysis, have accelerated the discovery of newcomponents and mechanisms involved in theassembly of alkaloids in plants. Our presentability to investigate secondary metabolismfrom a combined biochemical, molecular, cel-lular, and physiological perspective has im-proved our appreciation for the complex bi-ology of alkaloid pathways. In this review, wediscuss recent advances in our understandingof the metabolism and trafficking of alkaloids.
Alkaloid Biosynthetic Pathways
Monoterpenoid indole alkaloids. Themonoterpenoid indole alkaloids (MIA)comprise a family of structurally and phar-maceutically diverse alkaloids. Some of theapproximately 2,000 known compoundswidely used in medicine include vinblastinefor the treatment of cancer and ajmaline forantiarrythmic heart disorders. The majorsources for these compounds are Catharanthusroseus and Rauvolfia serpentina. MIAs are con-densation products of a nitrogen-containingindole moiety derived from tryptamineand a monoterpenoid component derivedfrom the iridoid glucoside secologanin(Figure 1). In the MIA biosynthetic pathway,
tryptophan decarboxylase (TDC) convertstryptophan to tryptamine; the gene encodingthis enzyme has been cloned from differentMIA-accumulating plants (25, 89, 178).Incorporation experiments with isotopicallylabeled glucose in C. roseus cell culturesshowed that the initial steps for the biosyn-thesis of the monoterpenoid skeleton proceedvia the triose phosphate/pyruvate pathway(22). Cognate cDNAs for 1-deoxy-d-xylulose5-phosphate reductoisomerase and 2C-methyl-d-erythritol 2,4-cyclodiphosphatesynthase were isolated from C. roseus andthe corresponding gene transcripts wereupregulated in MIA-producing cell cul-tures (163). The first committed step insecologanin biosynthesis is catalyzed bythe cytochrome P450 monooxygenasegeraniol-10-hydroxylase (Cyp76B6), whichwas purified and cloned from C. roseus (20).Several steps in the secologanin pathwayhave not been characterized, although theavailability of C. roseus EST databases (108)has led to the recent discovery of loganicacid methyltransferase (LAMT) (V. De Luca,personal communication). The final reactionof this early branch of the pathway involves anoxidative C-C cleavage catalyzed by a secondP450 monooxgenase (Cyp72A1), resulting inthe conversion of loganin to secologanin (70).
Strictosidine, the common precursor toall MIAs, is formed by a Pictet-Spenglercondensation of tryptamine and secologanin(Figure 1). Strictosidine synthase (STR)from R. serpentina was the first cDNA in-volved in alkaloid biosynthesis to be clonedand functionally expressed in microorgan-isms. STR orthologs from C. roseus and Ophi-orrhiza pumila have also been isolated (79,99, 178). The strictosidine glucose moietyis subsequently removed by a family 1 glu-cosyl hydrolase enzyme, strictosidine β-d-glucosidase (SGD); a cDNA for SGD wasfunctionally characterized from C. roseus andR. serpentina. SGD from R. serpentina showsexclusive substrate specificity toward stricto-sidine and closely related analogs (46, 50).The strictosidine-derived aglycone is subse-
quently converted via several unstable in-termediates to dehydrogeissoschizine, whichrepresents a key branchpoint intermediatethat leads to several diverse MIA pathways.The branch pathways proceeding throughtabersonine and polyneuridine aldehyde havebeen characterized most thoroughly. Otherbranch pathways, such as that leading tocatharanthine, remain poorly understood.
Hydroxylation of tabersonine at position16 represents the first reaction leadingto vindoline (Figure 1). The responsi-ble enzyme, tabersonine 16-hydroxylase(T16H, Cyp71D12), belongs to the P450monooxygenase family, and the correspond-ing cDNA was identified from a subsetencoding P450-dependent enzymes inducedby the treatment of C. roseus cultures withlight (140). O-Methylation at position 16,reduction of the 2,3-double bond, andN-methylation result in the conversion of 16-hydroxytabersonine to desacetoxyvindoline.Although cDNAs have not been isolated forthe enzymes that participate in these steps,the N- and O-methylating activities havebeen characterized in plant extracts (27, 37).The 2-oxoglutarate–dependent dioxygenasedesacetoxyvindoline 4-hydroxylase (D4H)produces desacetylvindoline, which is furtherconverted by the acetyl CoA–dependent de-sacetylvindoline 4-O-acetyltransferase (DAT)to vindoline (150, 162). Both enzymes havebeen cloned and characterized from C. roseus.DAT belongs to the BAHD (benzylalcohol O-acetyl-, anthocyanin-O-hydroxycinnamoyl-,anthranilate-N-hydroxycinnamoyl/benzoyl-,and deacetylvindoline 4-O-acetyltransferase)family of acyl-CoA dependent acyltrans-ferases; these proteins participate in a varietyof plant secondary metabolic pathways.Ultimately, vindoline is coupled to catha-ranthine by a nonspecific peroxidase toform vinblastine (145). A cDNA with 78%amino acid identity to DAT was obtainedfrom C. roseus and encodes minovincinine19-O-acetyltransferase, yielding echitove-nine, which is a second pathway originatingfrom the tabersonine branch (80). Whereas
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minovincinine acetyltransferase (MAT) showsresidual activity toward the DAT substrate,deacetoxyvindoline, DAT accepts only itsnatural substrate.
The polyneuridine aldehyde branch of theMIA pathway is initiated by the formation ofa bond, known as the sarpagan bridge, be-tween C5 and C16 of strictosidine-derived in-termediate compounds (Figure 1). The en-zyme characterized from crude extracts of R.serpentina shows NADPH and oxygen depen-dence, and is sensitive to P450 inhibitors, sug-gesting it is a P450 monooxygenase (139). Theresulting polyneuridine aldehyde undergoesdesterification to yield epi-vellosimine, whichis acetylated to form vinorine. Recombi-nant polyneuridine aldehyde esterase (PNAE)from R. serpentina shows esterase activity onlyto polyneuridine aldehyde and not to struc-turally related esters, and is a member of theα/β hydrolase superfamily (29). With approx-imately 30% identity on the amino acid levelto DAT and MAT, vinorine synthase (VS)from R. serpentina also belongs to the BAHDfamily of acyltransferases (11). Acetylajmalineis subsequently formed via a series of reac-tions that includes hydroxylation, two dou-ble bond reductions, and an N-methylation.The enzymes involved in these steps havebeen biochemically characterized as the P450-dependent monooxygenase vinorine hydrox-ylase (VH), which yields vomilenine, and theNADPH-dependent reductases vomileninereductase and 1,2-dehydrovomilenine reduc-tase (39, 44, 166). Acetylajamaline esterase(AAE) ultimately hydrolyzes the 17-O-acetylgroup to produce ajmaline. Active recombi-nant AAE, which could be produced only viaa virus expression system in Nicotiana ben-thamiana, shows slight substrate preferencefor acetylajmaline compared with norajma-line. The latter compound might representan intermediate in a parallel ajamaline biosyn-thetic pathway in which N-methylation occursafter deesterification (134). Unlike PNAE,AAE belongs to the GDSL lipase family. Rau-caffricine, a glucosylated derivative of vom-ilenine, is found in R. serpentina cultures. Al-
BIA:benzylisoquinolinealkaloid
Sanguinarine: anantimicrobialbenzylisoquinolinealkaloid sometimesused in oral hygieneproducts
though the glucosylating activity has not beenreported, researchers found a cDNA encod-ing the enzyme raucaffricine O-β-glucosidase(RG), responsible for deglycosylation of rau-caffricine (168). RG shares 60% amino acididentity with SGD and accepts strictosidine asa substrate, whereas SGD is not active againstraucaffricine.
Benzylisoquinoline alkaloids. Benzyliso-quinoline alkaloids (BIA) represent approxi-mately 2,500 elucidated structures and possesspotent pharmacological properties. The mostprominent compounds are the narcoticanalgesic morphine, the cough suppressantcodeine, the muscle relaxant papaverine, andthe antimicrobial agents sanguinarine andberberine. Collectively, these alkaloids occurmainly in the Papaveraceae, Ranunculaceae,Berberidaceae, and Menispermaceae; Papaversomniferum (opium poppy), Eschscholzia cali-forncia, Thalictrum species, and Coptis japonicaare the most extensively investigated species.BIA biosynthesis, which fundamentallyinvolves the condensation of two tyrosinederivatives, begins with the decarboxylationof tyrosine to tyramine or of dihydroxypheny-lalanine to dopamine by tyrosine decarboxy-lase (TYDC). TYDC constitutes a large genefamily; approximately 15 members are foundin opium poppy (33). Dopamine is the pre-cursor for the isoquinoline moiety, whereas4-hydroxyphenylacetaldehyde, resulting fromthe deamination of tyramine, is incorporatedas the benzyl component (Figure 2). As isthe case in MIA biosynthesis, BIA conden-sation is a Pictet-Spengler–type reaction andis catalyzed by the first committed step ofthe pathway, norcoclaurine synthase (NCS).The enzyme has been purified from Thalic-trum flavum and corresponding cDNAs havebeen isolated and functionally characterizedfrom opium poppy and T. flavum (87, 136,137). NCS is related to the pathogenesisrelated protein (PR) 10 and Bet v 1 allergenprotein families. However, homologous PR10proteins from opium poppy are not catalyt-ically active. Biochemical characterization of
recombinant T. flavum NCS has been ac-complished using a continuous enzyme assaybased on circular dichroism spectroscopythat follows the generation of the enzyme’schiral product. These studies revealed areaction mechanism that involves a two-step cyclization with a direct electrophilicaromatic substitution (91). Recently, asecond enzyme capable of producing onlythe (S )-norcoclaurine enantiomer was iso-lated from C. japonica; this enzyme displayssequence similarity to 2-oxoglutarate de-pendent dioxygenases (102). However, thisenzyme does not possess a 2-oxoglutarate-binding domain and requires ferrous ions,rather than 2-oxoglutarate or oxygen, foractivity. Remarkably, two proteins in twodifferent protein families can catalyze thesame reaction in vitro. Nevertheless, therelative participation of each enzyme in BIAbiosynthesis in vivo must still be determined.
The conversion of (S )-norcoclaurine to(S )-reticuline involves O-methylation at posi-tion 6, N-methylation, 3′-hydroxylation, anda second 4′-O-methylation (Figure 2). Nor-coclaurine 6-O-methyltransferase (6OMT)and 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) are both class IIO-methyltransferases that display strict re-giospecificity. Cognate cDNAs have been ob-tained for each enzyme from opium poppyand C. japonica (107, 121, 182). Additional6OMT homologs from Thalictrum tubero-sum were functionally characterized and ex-hibit a broader substrate specificity that in-
cludes catechols and phenylpropanoids inaddition to various BIA derivatives (42).Coclaurine N-methyltransferases (CNMT)have been cloned from opium poppy andC. japonica and are more closely related toS-adenosyl-L-methionine (SAM)-dependentcyclopropane fatty acid synthases than otherN-methyltransferases (19, 35). The hydroxy-lation of N-methylcoclaurine is catalyzed bya P450 monooxygenase classified in the Cypsubfamily 80B (65, 68, 125, 138).
(S )-Reticuline is the central pathway in-termediate from which most BIA structuraltypes are derived. Only the dimeric bisben-zylisoquinoline alkaloids are not producedvia (S )-reticuline. In this pathway, the cy-tochrome P450 enzyme Cyp80A1 catalyzesthe regio- and stereoselective oxidative C-O phenol coupling of the reticuline pre-cursor N-methylcoclaurine to produce thebisbenzylisoquinoline alkaloid berbamunine(78). Another exception where reticulinedoes not represent an intermediate com-pound might be the biosynthesis of the sim-ple N-demethylated BIA papaverine. The re-cent discovery of a 7-O-methyltransferasespecific for norreticuline (N7OMT, nor-reticuline 7-O-methyltransferase), but notN-methylated analogs, suggests that (S )-reticuline precursors might enter the path-way to papaverine (S. Pienkny, J. Ziegler& W. Brandt, manuscript in prepara-tion). A second O-methyltransferase thatacts on reticuline and catalyzes the forma-tion of laudanine was previously isolated
from opium poppy (121). (R,S )-Reticuline7-O-methyltransferase (7OMT) does not ac-cept N-demethylated BIA substrates, but is ac-tive toward phenolic compounds.
A major branch pathway that gives riseto many BIA classes begins with the forma-tion of (S )-scoulerine by the berberine bridgeenzyme (BBE) (Figure 2). This enzyme hasbeen cloned from several sources (28, 36, 138)and was recently characterized thoroughly(174, 175). BBE belongs to a novel familyof flavoproteins that possess two covalent at-tachment sites for flavin adenine dinucleotide(FAD); one is histidine and the other is cys-teine. The cysteinylation of the cofactor in-creases the midpoint redox potential to a valuehigher than that observed for other flavo-proteins, thereby facilitating hydride abstrac-tion of (S )-reticuline. This step represents thefirst half reaction toward the conversion to(S )-scoulerine. The biosynthesis of ben-zophenanthridine alkaloids is initiated by theformation of two methylenedioxy bridgesresulting in (S )-cheilanthifoline and (S )-stylopine (Figure 2). Both reactions are cat-alyzed by P450-dependent monooxygenasesand two cDNAs coding for stylopine syn-thase have been cloned from E. californica andclassified as Cyp719A2 and Cyp719A3 (67).Both recombinant proteins show the same re-giospecificity for methylendioxy bridge for-mation, but Cyp719A2 only converts (S )-cheilanthifoline to (S )-stylopine. In contrast,Cyp719A3 also accepts compounds withouta pre-existing methylenedioxy bridge. (S )-Stylopine is subsequently N-methylated to(S )-cis-N-methylstylopine by tetrahydropro-toberberine N-methyltransferase (TNMT).On the basis of homology to CNMT, acDNA encoding TNMT has been isolatedand functionally characterized from opiumpoppy (86). The enzyme shows a narrow sub-strate range in that it converts only tetrahy-droprotoberberine alkaloids with dimethoxyor methylenedioxy functional groups at C2/3and C9/10, respectively. TNMT is also oneof only a few plant enzymes able to cat-alyze the formation of quaternary ammo-
nium compounds. Subsequent hydroxylationby (S )-cis-N-methylstylopine 14-hydroxylaseyields protopine, which is further hydrox-ylated by protopine 6-hydroxylase to dihy-drosanguinarine. Both enzymes have been de-tected in protopine alkaloid-containing cellcultures, and their characterization suggeststhat they are P450 monooxygenases (132,156). Dihydrobenzophenanthridine oxidase,which converts dihydrosanguinarine to san-guinarine, has been purified from Sanguia-naria canadensis (7).
An alternative branch for the metabolismof (S )-scoulerine in some species involves theformation of (S )-tetrahydrocolumbamine byscoulerine 9-O-methyltransferase (SOMT)(Figure 2) (155). The cDNA encodingSOMT from C. japonica was the first re-ported clone for an O-methyltransferasespecifically implicated in BIA metabolism.All O-methyltransferases in the BIA pathwayexhibit considerable homology. Tetrahydro-columbamine is converted to columbamine,which is methylated by columbamineO-methyltransferase (CoOMT) to yieldpalmatine (106). Recombinant CoOMT isactive only against protoberberine alkaloidsubstrates, such as scoulerine or tetrahy-drocolumbamine, but not against otherBIA derivatives. The subsequent formationof a methylenedioxy bridge is catalyzedby canadine synthase, a P450 enzymethat belongs to the Cyp719A family (68).Cyp719A1 displays high substrate specificityfor tetrahydrocolumbamine and does notaccept columbamine. As such, a parallel pathto berberine via this columbamine is unlikely.(S )-Canadine, or (S )-tetrahydroberberineas it is also called, is oxidized by either (S )-canadine oxidase or (S )-tetrahydroberberineoxidase (STOX), which catalyze the samereaction but show substantially differentbiochemical properties.
Whereas all pathways downstream of reti-culine begin with the (S )-epimer, conversionto the (R )-epimer of reticuline is a requiredentry step into the morphinan alkaloid biosyn-thetic pathway (Figure 2). The epimerization
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of reticuline is a two-step process that in-volves the oxidation of (S )-reticuline by 1,2-dehydroreticuline synthase and the reductionof 1,2-dehydroreticuline to (R )-reticuline.Both steps have been characterized biochem-ically and the enzymes have been partially pu-rified (23, 63). Intramolecular carbon-carbonphenol coupling between C2 of the benzyland C4a of the isochinoline moiety leadsto the formation of salutaridine. This en-zyme belongs to the P450 monooxygenasefamily (48). The gene encoding salutaridinesynthase (SalSyn) was recently cloned fromopium poppy on the basis of its higher expres-sion in morphine-containing Papaver speciesand the enzyme was then functionally char-acterized (A. Gesell, F.C. Huang, J. Ziegler& T.M. Kutchan, manuscript in preparation).The SalSyn protein shows high homologyto the methylenedioxybridge-forming P450-dependent enzymes, and was classified asCyp719B1. The next step in the pathway iscatalyzed by salutaridine reductase (SalR); acognate cDNA was obtained via the sameapproach used for SalSyn (183). Functionalcharacterization of the recombinant enzymeshowed the stereospecific reduction of theketo group to 7(S )-salutaridinol, which is alsoreported for the purified enzyme from opiumpoppy (49). The enzyme belongs to the fam-ily of short chain dehydrogenases/reductases(SDR), but unlike many other enzymes inthis family it exhibits a higher molecularweight and is monomeric. The stereospecificreduction of salutaridine is required for thenext step, which is catalyzed by salutaridi-nol 7-O-acetyltransferase (SalAT). This en-zyme specifically acetylates the 7(S )-epimerof salutaridinol to salutaridinol-7-O-acetate(82). With considerable sequence homologyto the acetylating enzymes from the MIApathway, SalAT also belongs to the BAHDfamily of acetyltransferases (52). The in-troduced acetyl group is eliminated sponta-neously, leading to the formation of an oxidebridge between C-4 and C-5 to yield thebaine,the first pentacyclic alkaloid of the pathway(82). The final steps toward the biosynthesis of
SDR: short chaindehydrogenases/reductases
Calystegines:nortropane alkaloidsthought to serve asnutritional sourcesfor soilmicroorganisms
morphine consist of two demethylations andone reduction. Both the demethylation fromthebaine to neopinone, which isomerises tocodeinone, and from codeine to morphine arenot yet understood and no enzymes capable ofcatalyzing either reaction have been detected.Codeinone reductase has been purified andcloned from opium poppy, and, in contrastto SalR, belongs to the aldo-keto reductase(AKR) family (83, 160).
Tropane alkaloids and nicotine. Tropanealkaloids are an important class of plant-derived anticholinergic compounds, such ashyoscyamine and scopolamine, that occurin several Hyoscyamus, Atropa, and Daturaspecies. Calystegines, which function as se-lective glucosidase inhibitors, are more widelyspread than hyoscyamine and scopolamine inthe plant kingdom and occur mainly in theSolanaceae and Convolvulaceae (30). Nico-tine, the active principle in Nicotiana species,acts on nicotinic acetylcholine receptors tocause a variety of physiological effects, in-cluding addiction. Tropane alkaloid and nico-tine biosynthesis begin with the methyla-tion of putrescine to N-methylputrescineby putrescine N-methyltransferase (PMT)(Figure 3). The isolation of the cDNA en-coding PMT from tobacco was one of thefirst examples of the successful integrationof metabolite and gene expression profilesas a strategy to isolate cDNAs implicatedin plant secondary metabolite biosynthe-sis (61). Homologous cDNAs from otherplants that produce tropane alkaloids, suchas Hyoscyamus niger, Atropa belladonna, andSolanum tuberosum, have also been isolated(147, 153, 157). The second step in thepathway is the oxidative deamination of N-methylputrescine to 4-methylaminobutanalby N-methylputrescine oxidase (MPO). Thisenzyme belongs to a class of amine ox-idases that require copper as a cofactor.This property was exploited in a homology-based cloning strategy to isolate the MPOcDNA from tobacco (59). The central inter-mediate N-methyl-�1-pyrrolium cation for
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Ornithine
ODC
MPO
PMT
Spontaneous
TR-II
TR-I
Cyp80F1
H6H
Cyp82E4
Arginine
Putrescine
N-Methylputrescine
NADbiosynthesisintermediate 3,6-Dihydronicotinone
Nicotine
NornicotinePseudotropineTropinoneHygrine
LittorineTropine
Hyoscyamine aldehyde
Calystegine A3
Hyoscyamine
Calystegine B2Calystegine B1
Scopolamine
4-Methylaminobutanal N-Methyl-∆1-pyrroliumcation
Figure 3Biosynthesis of thetropane alkaloidshyoscyamine andscopolamine, thecalystegines, andnicotine.Molecular cloneshave been isolatedfor all enzymesshown.Abbreviations:Cyp80F1, littorinemutase/monooxygenase;Cyp82E4, nicotineN-demethylase;H6H, hyoscyamine6β-hydroxylase;MPO,methylputrescineoxidase; ODC,ornithinedecarboxylase;PMT, putrescineN-methyltransferase;TR-I, tropinonereductase I; TR-II,tropinonereductase II.
tropane alkaloid and nicotine biosynthesisresults from the spontaneous cyclization of4-methylaminobutanal. For nicotine, thecation is condensed with nicotinic acid to form3,6-dihydronicotine, which subsequently un-dergoes dehydrogenation to nicotine by en-zymes that remain poorly characterized.
Nicotine can be N-demethylated to form nor-nicotine, which is an undesirable derivativeowing to its role as the precursor of the car-cinogen N ′-nitrosonornicotine. On the basisof the differential abundance of transcriptscorresponding to several P450-encodingcDNAs in tobacco varieties acccumulating
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nicotine and nornicotine, a clone for nico-tine N-demethylase (NND) was identifiedand classified as Cyp82E4 (144, 176). Re-cently, Gavilano & Siminszky (45) describedan additional cDNA with NND activ-ity (Cyp82E5v2) that shows distinct tissue-specific expression in tobacco.
Although enzymatic activities have notbeen demonstrated, the condensation ofN-methyl-�1-pyrrolium cation with ace-toacetic acid is purported to yield hygrine,the precursor of tropane alkaloids (Figure 3).Hygrine is converted to tropinone, which issubsequently reduced to intermediates thatlead to hyoscyamine or calystegines depend-ing on the stereochemistry of the reduction.Tropinone reductase I (TR-I) catalyzes thereduction of tropinone to tropine, whichpossesses a 3α configuration. In contrast,tropinone reductase II (TR-II) catalyzes theformation of ψ-tropine, which has a 3β con-figuration. Cognate cDNAs show that bothNADPH-dependent enzymes belong to theSDR family and exhibit considerable aminoacid sequence similarity (76, 110, 111, 113).Domain-swapping experiments and site-directed mutagenesis led to the identificationof the substrate-binding domain responsiblefor the opposite stereospecificity of eachreductase. Tropine then condenses with the
phenylalanine-derived (R )-phenyllactate toyield littorine, which undergoes rearrange-ment to form hyoscyamine. Recently, Li andcoworkers (84) used functional genomicsbased on virus-induced gene silencing to iso-late a cytochrome P450 involved in littorinerearrangement from H. niger. This enzymewas classified as Cyp80F1 and it catalyzes theoxidation of (R )-littorine with rearrangementto hyoscyamine aldehyde. Finally, the epox-idation of hyoscyamine yields scopolaminevia a two-step reaction: 6β-hydroxylation ofthe tropane ring followed by intramolecularepoxide formation. The reaction is catalyzedby a 2-oxoglutarate–dependent dioxygenase,hyoscyamine 6β-hydroxylase (H6H), thecDNA for which has been cloned fromseveral tropane alkaloid-containing plants(88, 95, 154).
Purine alkaloids. Purine alkaloids arederived from purine nucleotides. Themost prominent members of this class ofcompounds are theobromine and caffeine.Purine alkaloid biosynthesis begins withthe N-methylation of xanthosine at position7 by 7-methylxanthosine synthase (XRS),which is also known as xanthosine 7-N-methyltanferase (XMT) (Figure 4). CognatecDNAs for XMT have been isolated from
Xanthosine
XMT
DXMT
CS
MXMT CS/TS7-Methylxanthosine
H2O Ribose
7-Methylxanthine
TheobromineCaffeine
Figure 4Biosynthesis of thepurine alkaloidscaffeine andtheobromine.Molecular cloneshave been isolatedfor all enzymesshown.Abbreviations: CS,caffeine synthase;DXMT, 1,7-dimethylxanthosinemethyltransferase;MXMT,7-methylxanthinemethyltransferase;TS, theobrominesynthase, XMT,xanthosine 7-N-methyltanferase.
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Coffea arabica (103, 158). The second step inthe purine alkaloid pathway is the hydrolysisof xanthosine to 7-methylxanthine, althoughthe responsible enzyme has not yet beenpurified or characterized. However, detailedstructural investigations of XMT suggest acoupled reaction for 7-N-methylation andnucleoside cleavage catalyzed by a singleenzyme (97). Several cDNAs with differentsubstrate specificities have been obtainedfrom tea, coffee, or cacao that catalyze the nexttwo N-methylations. Caffeine synthase (CS)performs dual methylations, first at position3 to form theobromine, and then at position1 to yield caffeine (74, 104). Several cDNAsencoding related enzymes that catalyze onlysingle N-methylations have been isolated, in-cluding 7-methylxanthine methyltransferase(MXMT1 and MXMT2) and theobrominesynthase (TS), which methylate position N-3,and 3,7-dimethylxanthine methyltransferase(DXMT), which catalyzes the final methy-
lation at position N-1 (117, 158). TheseN-methyltransferases possess more then80% amino acid similarity and phylogeneticanalysis suggests that they are more closelyrelated to carboxyl-methyltransferases thanto other N-methyltransferases.
Pyrrolizidine alkaloids. Pyrrolizidine alka-loids are produced constitutively in variousplants as a defense against hebivores and asa component of the complex chemical ecol-ogy between plants and animals (55). Thesealkaloids are composed of a necine base andone or more necic acids; the latter are re-sponsible for their structural diversity. Necinebiosynthesis begins with the condensation ofspermidine and putrescine to form homosper-midine by homospermidine synthase (HSS)(116) (Figure 5). The reaction mechanismis identical to that of deoxyhyposine syn-thase (DHS), which transfers the aminobutylmoiety to a lysine residue of the eukaryotic
Spermidine
Putrescine
Homospermidine
Necine base
HSS
HeliotrineSenecionine
Clivorine
Figure 5Biosynthesis ofpyrrolizidinealkaloids. Amolecular clonehas been isolatedfor only onebiosyntheticenzyme.Abbreviation: HSS,homospermidinesynthase.
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initiation factor 5A precursor protein. Ad-ditionally, both enzymes share extensive se-quence homology, suggesting the recruitmentof HSS from DHS, with subsequent opti-mization to facilitate the role of HSS in sec-ondary metabolism. Phylogenetic analysis ofHSS and DHS homologs from angiospermsthat represent several unrelated plant lineagessuggests at least four independent recruitmentevents for HSS (128). The remaining compo-nents of pyrrolizidine alkaloid metabolism arenot well understood.
The biomimetic exploitation of enzymes, es-pecially those that display strict stereospeci-ficity, is an appealing strategy for the commer-cial production of pharmaceutical alkaloids.Equally intriguing is the synthesis of novel al-kaloids via protein engineering aimed at al-tering the substrate specificity of biosyntheticenzymes. Recent attention has focused on elu-cidating the reaction and substrate bindingmechanisms of key alkaloid biosynthetic en-zymes. The structures of several enzymes havebeen determined and substrate-binding pock-ets have been characterized.
The first detailed structures of enzymes in-volved in plant alkaloid metabolism were ob-tained by X-ray crystallography for TR-I andTR-II, which show strict product specifici-ties and catalyze either tropine or ψ-tropineformation. Site-directed mutagenesis of se-lected amino acids in the substrate-bindingdomains resulted in a mutual conversion ofthe product specificities for TR-I and TR-II(114). A homology-based model of the three-dimensional structure of PMT was generatedon the basis of the crystal structure of the pu-tative ancestral enzyme spermidine synthase,which suggested the identity of amino acidsresponsible for the distinction between thesetwo enzymes (157).
Most X-ray crystallographic data havebeen obtained for enzymes implicated inMIA metabolism, especially those involved in
Homology-basedmodel: anatomic-resolutionprotein structurebased on amino acidsequence
Substrate docking:a mathematicalprocedure used toidentify ligand-binding sites inproteins
ajmaline biosynthesis. The structure of STRfrom R. serpentina revealed a novel six-bladedβ-propeller protein and site-directed muta-genesis showed the importance of a key gluta-mate residue in catalysis (93). The structure ofthe enzyme complexed with the natural sub-strates tryptamine and secologanine providedinsight into the architecture of the substratebinding sites, which could then be engineeredto accommodate several different tryptamineand secologanin analogs (18, 90). The three-dimensional structure of SGD revealed theexpected (β/α)8 barrel fold typical for family1 glycosidases (9). Site-directed mutagenesisidentified amino acid residues required forthe catalytic mechanism and the binding sitearchitecture. Ruppert and coworkers (133)reported preliminary X-ray crystallographicdata for the homologous enzyme RG. A com-parision of the structures of SGD and RG willbe interesting because both enzymes occur inthe same organism and catalyze the same typeof reaction, but differ in substrate specificity.VS was the first BAHD acyltransferase familyenzyme for which a three-dimensional struc-ture was obtained (92). The protein consistsof two major domains of similar size con-nected by a large loop. Whereas site-directedmutagenesis and structural analysis confirmthe importance of a HXXXD motif forcatalysis, a second highly conserved motif inmembers of the BAHD family is distant fromthe active site and is considered essential forproper folding rather than catalysis. Molec-ular modeling and site-directed mutagenesisestablished the classification of PNAE as amember of the α/β hydrolase superfamilyand identified the amino acid residues thatparticipate in the catalytic triad (96).
The structure of SalR (involved in BIAbiosynthesis) was analyzed by homology mod-elling (47). An additional helix near the cat-alytic site not found in multimeric SDR-typeenzymes appears to contribute to substratespecificity. Substrate docking studies and site-directed mutagenesis revealed several aminoacids implicated in the binding of the morphi-nan alkaloid precursor salutaridine. It will be
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Laticifer: a plantcell or vessel thatcontains latex
interesting to compare the substrate-bindingsite of SalR with that of COR, which specif-ically reduces codeinone, an alkaloid with asimilar structure, to salutaridine. The po-tential of homology modelling and substratedocking is well demonstrated in studies in-volving the O-methyltransferases implicatedin BIA metabolism (S. Pienkny, J. Ziegler &W. Brandt, manuscript in preparation). Al-though highly similar to 6OMT, the mod-elling and substrate docking results for a novelO-methyltransferase precluded norcoclaurineas a likely substrate, and rather suggestedthe O-methylation of norreticuline at posi-tion 7. Experimental evidence has confirmedthis prediction and led to the identification ofN7OMT. Furthermore, comparisons of the6OMT and N7OMT models identified theamino acids responsible for the distinct sub-strate specificity.
Recently, McCarthy & McCarthy (97) elu-cidated the structures of the two highly ho-mologous N-methyltransferases involved incaffeine biosynthesis, XMT and DXMT. Co-crystallization with the cofactor and eithersubstrate, coupled with structural compar-isons, revealed critical elements for substrateselectivity and catalysis.
REGULATION OF ALKALOIDBIOSYNTHETIC PATHWAYS
Transcriptome andMetabolome Analyses
Large-scale expression analyses have recentlybegun to provide a broad picture of the geneexpression profiles associated with alkaloidbiosynthesis. As a prerequisite for these stud-ies, several EST sequencing projects havebeen reported and the number of sequencesassociated with alkaloid-producing plantscontinues to increase. The first database con-sisted of 4,500 ESTs from the laticifers ofopium poppy (127). Further EST projectsinvolving this plant have yielded a total of25,000 ESTs from various tissues includingseedlings, stems, roots, and elicited cell cul-
tures (182, 183, 184). A recent floral genomeproject contributed 11,000 ESTs from theBIA-accumulating plant E. californica (17), andthe release of a C. japonica database has alsobeen announced (69, 75). NapGen, a consor-tium of Canadian investigators, has sequencedmore than 400,000 ESTs from a wide vari-ety of plants that produce health-related sec-ondary compounds. In this project, 46,000ESTs were sequenced from cell culturesof eight BIA-accumulating species includingE. californica, T. flavum, Nandina domestica,and Papaver bracteatum (D.K. Liscombe, J.Ziegler & P.J. Facchini, manuscript in prepa-ration). More than 56,000 ESTs have been ob-tained from leaf, leaf epidermis, and roots ofthe MIA-accumulating plant C. roseus (108).Large-scale sequencing projects for tropanealkaloid-containing plants, such as S. tubero-sum, are also well established. Although re-search in potato is primarily aimed at the dis-covery of genes involved in fungal resistance,the 62,000 ESTs from aerial and undergroundorgans are a good source to identify cDNAsimplicated in calystegine biosynthesis (131). Asequencing project targeting tropane alkaloidmetabolism yielded 2,300 ESTs from roots ofH. niger (84). In a similar effort to investigatethe transcriptional regulation associated withthe chemical composition of coffee, Lin andcoworkers (85) reported 47,000 ESTs fromCoffea canephora at different stages of seeddevelopment.
Large-scale transcriptome and metaboliteprofiling has been performed in opium poppycell cultures treated with a fungal elicitor(184), and in C. roseus and tobacco cell culturestreated with methyl jasmonate (51, 129). In allcases, researchers observed a coordinated in-crease in the expression of genes implicatedin alkaloid metabolism. Moreover, profoundchanges in the level of gene transcripts en-coding primary metabolic enzymes also oc-curred. Interestingly, transcripts involved inSAM recycling increased in all three sys-tems, indicating a high demand for this cofac-tor in the modification of alkaloid backbonestructures. As expected, genes implicated in
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aromatic amino acid metabolism were also in-duced in opium poppy and C. roseus in re-sponse to the increased demand for the pre-cursors of BIA and MIA biosynthesis (129,184). Surprisingly, the expression of genes as-sociated with nitrogen metabolism was not af-fected in opium poppy cultures, suggestingthat the cells have a constitutive and suffi-cient capacity for nitrogen assimilation evenduring periods of increased demand (184).However, substantial changes occurred in thelevels of primary metabolites involved in theproduction of energy molecules potentiallyrequired by enzymes and other componentsrequired by the induction of alkaloid biosyn-thesis (184). In C. roseus, the constructionof correlation networks that integrate tran-scriptomic and metabolomics data revealeda correlation between the expression of al-kaloid biosynthetic genes and correspondingmetabolic products (129).
Gene Regulationand Signal Transduction
Progress has continued on the identificationof promoter elements and transcription fac-tors involved in the regulation of several MIAbiosynthetic genes (165). The recent applica-tion of a genomics approach based on DNAmacroarrays constructed from ESTs has led tothe isolation of the first transcription factorputatively involved in the regulation of BIAmetabolism. Transcripts that encode a subsetof transcriptional regulators showed coordi-nate expression with respect to BIA biosyn-thetic genes in berberine-producing C. japon-ica cell cultures. One of these regulators, aWRKY transcription factor, was shown byRNAi and overexpression analysis to specif-ically regulate the expression of BIA biosyn-thetic genes (75). With respect to early signaltransduction events, considerable effort hasbeen focused on events associated with the in-duction of alkaloid metabolism in E. californicacell cultures, which accumulate benzophenan-thridine alkaloids such as sanguinarine in re-sponse to treatment with a fungal elicitor.
Metabolomics: thesystematic study ofthe chemicalfingerprints leftbehind by specificcellular processes
RNAi: RNAinterference
Two different signal transduction pathwayswere identified. One cascade is jasmonate-dependent and responds to high elicitorconcentrations. The other is jasmonate-independent and is triggered by low elic-itor concentrations (38). The jasmonate-independent pathway involves Gα proteinsthat interact and activate phospholipase A2,which leads to a transient proton efflux fromthe vacuole and a subsequent activation ofother cytoplasmic components (60, 164).
Transgenic Approaches
The application of transgenic technologiesto alkaloid-producing plants is primarily in-tended to increase the synthetic capacity ofdesired product via the overexpression of cer-tain genes, or to divert pathways to previ-ously under-represented or novel compoundsthrough posttranscriptional gene silencing.However, such experiments also provide in-sights into the regulatory architecture of alka-loid pathways, especially if the predicted out-come is not observed. Unexpected metabolicconsequences resulting from single-enzymeperturbations of alkaloid pathways suggest theexistence of key rate-limiting steps, potentialmultienzyme complexes, or unsuspected com-partmentalization.
Metabolic engineering of low-scopola-mine A. belladonna plants via the intro-duction of a constitutively expressed H6Htransgene led to an increase in scopolamineaccumulation (179). Similarly, a shift in theaccumulation of hyoscyamine in favor ofscopolamine occurred when the H6H trans-gene was introduced into A. beatica, suggest-ing that the enzyme is a rate-limiting stepin scopolamine biosynthesis (180). However,the unpredictability of metabolic engineeringin tropane alkaloid biosynthesis was demon-strated by the constitutive coexpression ofPMT and H6H. The transgenes caused onlymodest increases in alkaloid accumulationwhen expressed alone, but exhibited a syner-gistic effect on alkaloid levels when expressedtogether (181).
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Latex: the milkycytoplasm ofspecialized cellscalled laticifers
In MIA biosynthesis, the first attemptsat metabolic engineering focused on theexpression of constitutive TDC and STRtransgenes in C. roseus cell cultures (31).However, only unsustained increases in al-kaloid accumulation were reported, whichraises concerns about the actual relation-ship between the transgenes and the initialphenotype (16). Overexpression of enzymesresponsible for production of the indole moi-ety, such as anthranilate synthase (AS), re-sulted in higher levels of tryptophan, butnot alkaloids (64, 66). Monoterpenoid path-way reactions have long been consideredthe rate-limiting steps in MIA production.In this context, overexpression of a modi-fied 3-hydroxy-3-methylglutaryl-CoA reduc-tase leads to a predictable increase in al-kaloid content (8). The role of secologaninbiosynthesis in the control of flux into MIAmetabolism is underscored by the ectopicoverexpression of transcriptional regulators ofthe pathway. T-DNA activation tagging inC. roseus cell cultures resulted in the isola-tion of an octadecanoid-derivative responsiveCatharanthus AP2-domain protein (ORCA3),which activates the expression of genes en-coding AS, TDC, STR, D4H, cytochromeP450 reductase, and D-1-deoxyxylulose 5-phosphate synthase, but not those encod-ing geraniol 10-hydroxylase, SGD, and DAT(161). However, alkaloid accumulation in cellstransformed with a constitutively expressedORCA3 transgene occurred only in the pres-ence of exogenous loganin. The combinedoverexpression of transcriptional activators,such as ORCA2 and ORCA3, and the silenc-ing of repressors, such as the G-box bindingfactors 1 and 2 and the zinc finger proteinfamily, might be required to activate the en-tire pathway (100, 126, 143, 161).
Most of the transgenic work involving BIAmetabolism has focused on the modulationof BBE activity. Overexpression of this en-zyme in E. californica root cultures results inan increased accumulation of downstream al-kaloids and decreased levels of certain aminoacids (124). Conversely, antisense suppression
of BBE expression leads to undetectable lev-els of downstream metabolites and increasedcellular pools of amino acids, revealing the im-pact of perturbations in alkaloid metabolismon primary metabolism (123). Interestingly,antisense suppression of BBE does not re-sult in the accumulation of its substrate (S )-reticuline or any other upstream alkaloid, sug-gesting the occurrence of metabolic channelsthat are abolished in the absence of BBE.In contrast, RNAi lines targeting BBE in E.californica cell cultures result in high (S )-reticuline content (43). Furthermore, lauda-nine is detected, implying that the pathway isredirected toward the O-methylation of reti-culine by 7OMT. Transgenic opium poppyplants expressing an antisense-BBE constructshow increased flux into the morphinan andtetrahydrobenzylisoquinoline branch path-ways (40). Surprisingly, the BBE reactionproduct (S )-scoulerine also accumulates inthe latex of these transgenic plants, althoughthe roots show no alterations in metaboliteprofile. Whether these results are due to anadditional role of BBE in BIA metabolismremains unproven. Opium poppy plantstransformed with constitutively expressedor antisense-suppressed Cyp80B3, which en-codes N-methylcoclaurine 3′-hydroxylase,also show substantial modulations in alkaloidcontent (41). However, as expected the alka-loid profile is not altered because Cyp80B3acts early in the pathway. Overexpressionof COR1, the penultimate step in morphinebiosynthesis, also results in opium poppyplants with increased alkaloid content (81).This increase is attributable to higher levelsof morphine, codeine, and—for unexplainedreasons—thebaine, which is upstream ofCOR1 in the pathway. The overexpression ofone alkaloid biosynthetic gene might possiblycause the coordinate transcriptional inductionof other pathway genes. It would be interest-ing to subject these transgenic plants to mi-croarray analysis to potentially correlate alter-ations in transcript abundance with changesin alkaloid profile. In RNAi-silenced COR1plants, the expression of other known genes
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from the pathway is unaffected (4). However,the plants accumulate the far upstream inter-mediate (S )-reticuline rather than codeinone,the substrate of COR. Whether this is basedon a feedback mechanism that inhibits the en-tire morphinan branch pathway or the im-pairment of a required metabolic channel dueto the absence of COR is not known. Thepresence of multienzyme complexes in mor-phine biosynthesis is supported by experi-ments that involve the suppression of SalAT.RNAi-silenced SalAT plants show an accumu-lation of salutaridine, which is normally notabundant in opium poppy plants (3). This issurprising because salutaridinol, which doesnot accumulate, is the substrate for SalAT(Figure 2). Salutaridine might be channelledto thebaine through an enzyme complex thatincludes SalR and SalAT.
Experiments involving the overexpressionof two O-methyltransferases from the earlyBIA pathway in E. californica cells suggesteda rate-limiting role for 6OMT (69). Consti-tutive overexpression of 6OMT led to in-creased alkaloid content. In contrast, over-expression of 4′OMT had little effect. TheE. californica cell culture used in this studyappeared to lack a functional 6OMT, whichmight explain the strong effect of overexpress-ing a 6OMT transgene. Subsequent biochem-ical characterization of 4′OMT from E. califor-nica revealed low 6OMT activity, suggestinga role for 4′OMT as a surrogate for 6OMT tofacilitate BIA biosynthesis.
Transgenic approaches to alter caffeinebiosynthesis have recently focused on the gen-eration of C. arabica plants with reduced caf-feine content for the production of decaf-feinated coffee. Suppression of MXMT, thesecond N-methyltransferase in the pathway,results in the reduction of theobromine andcaffeine by 50% to 70% in C. arabica andC. canephora (118, 119). However, MXMT,XMT, and DXMT transcripts are all re-duced owing to the high homology betweenthe three genes. An alternative biotechno-logical strategy has targeted the productionof caffeine in non-caffeine-producing plants
to increase pest resistance. Overexpressionof all three N-methyltransferases in trans-genic Nicotiana tabacum leads to a substantial(5 ug g−1 fresh weight) accumulation of caf-feine in leaves (77, 159). This is sufficient toreduce by 50% pest damage caused by feedingof the tobacco cutworm Spodoptera litura.
ALKALOID TRAFFICKING
The cell biology of alkaloid metabolism inplants has recently emerged as an excitingfield of research. Although the biosyntheticpathways leading to various alkaloid typesare of polyphyletic origin, some intriguingparadigms are apparent, including the in-volvement of multiple cell types for alkaloidbiosynthesis and/or accumulation, the target-ing of different pathway enzymes to multiplesubcellular compartments, and the possibilitythat multienzyme complexes are ubiquitous.Such complex organization implicates exten-sive intra- and intercellular transport of path-way intermediates, products, and biosyntheticenzymes.
Alkaloids generally accumulate in specificcell types owing to their cytotoxicity andprobable role in plant defense responses. Forexample, alkaloids are sequestered to isolatedidioblasts and laticifers in C. roseus (151), rootendodermis and stem cortex/pith in T. flavum(138), and laticifers in opium poppy (13).The cellular localization of alkaloid pathwaysis remarkably diverse and complex. Work onthe cellular and developmental complexitiesand organization of alkaloid biosynthesis inC. roseus and opium poppy, in particular, haveestablished new paradigms in the cell biologyof secondary metabolism.
PMT and H6H, which catalyze the firstand last steps, respectively, in the biosyn-thesis of the tropane alkaloid scopolamine,localize to the pericycle in the roots of A.belladonna and Hyoscyamus muticus (56, 153,154) (Figure 6a). PMT also catalyzes the
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a b
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EpidermisCortex
Endodermis
Xylem
Xylem
Internal phloemparenchyma
Phloem
Epidermis
LaticiferIdioblast
Idioblast Epidermis
Parenchyma
Laticifer
Sieveelement
Companioncell
Vascularcambium
Xylem
Cortex
Endodermis
Xylem
Phloem
Phloem
Pericycle
Cortex
Endodermis
Xylem
Cortex
Protoderm
Stele
Phloem
Pericycle
Immatureendodermis
Palisademesophyll
Spongymesophyll
Figure 6Alkaloid biosynthetic pathways are associated with a diversity of cell types. The tissue-specificlocalization (red ) of enzymes and/or gene transcripts are shown for the biosynthesis of tropane alkaloidsin (a) Atropa belladonna and Hyoscyamus niger roots, (b) terpenoid indole alkaloids in Catharanthus roseusleaves, (c) pyrrolizidine alkaloids in Senecio vernalis roots, (d ) pyrrolizidine alkaloids in Eupatoriumcannabinum roots, (e) benzylisoquinoline alkaloids in Papaver somniferum vascular bundles, and( f ) benzylisoquinoline alkaloids in Thalictrum flavum roots.
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first step in nicotine biosynthesis and local-izes to the endodermis, outer cortex, andxylem in Nicotiana sylvestris (141, 142). In con-trast, TR-I, an intermediate enzyme in theproduction of hyoscyamine and scopolamine,resides in the endodermis and nearby corti-cal cells (112); thus, tropane alkaloid inter-mediates must also traffic between cell types(Figure 7). The specific pathway intermedi-ates that undergo intercellular translocationare not known. Interestingly, TR-II, whichprovides pseudotropine for the formation ofcalystegines, localizes to companion cells ofsieve elements in the phloem of potato (73).
Enzymes involved in MIA biosynthesis lo-calize to several different C. roseus organs andcell types. TDC and STR are abundant inroots (151), but also occur in photosyntheticorgans, whereas T16H (149), D4H (162), andDAT (150) are restricted to young leaves andother shoot organs where vindoline biosyn-thesis occurs. In situ hybridization and im-munocytochemical localization studies haveshown that Cyp72A1, TDC, and STR lo-calize to the epidermis of stems, leaves, andflower buds (70, 151) (Figure 6b). In roots,these enzymes occur in cells near the api-cal meristem. In contrast, D4H and DATare associated with laticifers and idioblastsof shoots, but are absent from roots. Lati-cifers and idioblasts are distributed through-out the mesophyll in leaves, often several celllayers away from the epidermis; thus, vin-doline biosynthesis involves at least two dis-tinct cell types and requires the intercellu-lar translocation of pathway intermediates.Moreover, gene transcripts that encode en-zymes involved in the MEP pathway, alongwith geraniol 10-hydroxylase, colocalize tothe internal phloem parenchyma of young C.roseus aerial organs (15, 120). These resultssuggest the translocation of vindoline biosyn-thetic intermediates from the internal phloemto the epidermis and from the epidermis tolaticifers and idioblasts (Figure 8). As is thecase in tropane alkaloid metabolism, the spe-cific pathway intermediates that undergo in-tercellular translocation in MIA biosynthe-
Cortex
Endodermis
Tropinone
Tropine
N-Methylputrescine
PutrescineHyoscyamine
ScopolamineOrnithine
OrnithineArginine
ArgininePericycle
Phloem Xylem
Figure 7Putative trafficking of pathway intermediates and products in tropanealkaloid metabolism. Red arrows show specific enzyme-catalyzed reactions.Blue arrows represent the purported intercellular translocation ofunspecified compounds.
sis in C. roseus are not known. Interestingly,the identification of gene transcripts encod-ing vindoline biosynthetic enzymes in specificcell types isolated by laser-capture microdis-section and enriched via carborundum abra-sion was interpreted to suggest that the leafepidermis is independently capable of produc-ing 16-methoxytabersonine (109). Upper leafepidermis has been implicated in several sec-ondary metabolic pathways (94), complicatingthe assignment of common precursors to spe-cific cell types. The role of internal phloem
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Cuticle
Epidermis
Laticifer
IPAP
IPAP
IPAP
IPAP
IPAP
IPAP
Palisademesophyll
Palisadeidioblast
Spongymesophyllidioblast
Vasculartissues
Tryptophan Loganic acid
Tryptamine + Secologanin
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16-Methoxytabersonine
MIAintermediates
?
MEP pathway
Geraniol10-Hydroxy-
geraniol
?
Deacetyl-vindoline
Vindoline
Deacetyl-vindoline
Vindoline
Deacetyl-vindoline
Vindoline
Figure 8Putative trafficking of pathway intermediates and products inmonoterpenoid indole alkaloid metabolism. Red arrows show specificenzyme-catalyzed reactions. Blue arrows represent the purportedintercellular translocation of unspecified compounds. IPAP, internalphloem-associated parenchyma.
and epidermis in the supply of terpenoid pre-cursors to alkaloid biosynthesis requires fur-ther evaluation.
Pyrrolizidine alkaloids also share a com-mon metabolic pathway in restricted yet di-verse taxa (54). In Senecio species, pyrrolizidinealkaloids are produced in actively growingroots as senecionine N-oxides, which aretransported via the phloem to above-groundorgans (54). Senecionine N-oxides are sub-sequently modified by one or two species-specific reactions (i.e., hydroxylation, dehy-
drogenation, epoxidation, or O-acetylation)that result in the unique pyrrolizidine alka-loid profile of different plants. Inflorescencesare the major sites of pyrrolizidine alkaloid ac-cumulation in Senecio jacobaea and Senecio ver-nalis; jacobine occurs in flowers. HSS is thefirst committed enzyme in pyrrolizidine alka-loid biosynthesis and localizes to the root en-dodermis and cortex adjacent to the phloemin S. vernalis (Figure 6c), which might re-flect a functional accommodation for sys-temic transport of pyrrolizidine alkaloids tothe stem (105). However, HSS is foundthroughout the root cortex in Eupatoriumcannabinum (Figure 6d ), which like S. vernalisis a member of the Asteraceae (6). In con-trast to the general monophyletic origin ofBIA biosynthesis (87), pyrrolizidine alkaloidpathways have evolved in at least four differ-ent angiosperm lineages (128). The differen-tial localization of a key enzyme was inter-preted to support the polyphyletic origin forpyrrolizidine alkaloid biosynthesis (6). In con-trast, the monophyletic origin of BIA biosyn-thesis (87) and the differential localization ofgene transcripts in T. flavum and opium poppysuggest the migration of pathway intermedi-ates between cell types.
In opium poppy, BIA accumulationoccurs in the articulated laticifers foundadjacent or proximal to sieve elements of thephloem (32). The cytoplasm of laticifers—orlatex—contains a full complement of cellularorganelles and many large vesicles to whichalkaloids are sequestered. Although laticiferswere long considered to be the site of BIAbiosynthesis and accumulation, the cellularlocalization of BIA biosynthetic enzymesand gene transcripts has shown that alkaloidsynthesis involves other cell types (13, 34,170). Initial in situ hybridization experimentsdemonstrated the localization of TYDC genetranscripts to the phloem, but not to laticifers(34). The morphinan pathway enzymessalutaridine synthase (Cyp719B1) and salu-taridine:NADPH 7-oxidoreductase (SalR)are also not detected in isolated latex (48, 49).Seven other biosynthetic enzymes—6OMT,
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CNMT, (S )-N-methylcoclaurine 3′-hydroxylase (Cyp80B3), 4′OMT, BBE,SalAT, and COR—localize to sieve elementsin opium poppy and their corresponding genetranscripts localize to associated companioncells (13, 135) (Figure 6e). The localizationof BIA metabolism components to phloemcells distinct from laticifers predicts the in-tercellular transport of alkaloid biosyntheticenzymes and pathway intermediates and/orproducts. The implication of sieve elementsalso breaks a long-standing paradigm inplant biology (Figure 9). Previously, sieveelements were not known to support complexmetabolism, and were assumed to possessonly a limited number of proteins requiredfor cell maintenance and solute transport.Recently, the physiological roles for sieve ele-ments have expanded to include the transportof information macromolecules such as RNA(72) and the biosynthesis of jasmonic acid(57), ascorbic acid (53), and defense-relatedcompounds (167). Sieve elements clearlypossess a previously unrealized biochemicalpotential.
T. flavum accumulates BIAs such as theantimicrobial agent berberine. In situ RNAhybridization analysis revealed the cell type–specific expression of BIA biosynthetic genesin roots and rhizomes of T. flavum (138). Inroots, gene transcripts for all nine enzymeslocalize to the pericycle—the innermost layerof the cortex—and adjacent cortical cells(Figure 6f ). In rhizomes, all biosyntheticgene transcripts are restricted to the proto-derm of leaf primordia. The protoderm local-ization of biosynthetic gene transcripts con-trasts with the tissue-specific accumulation ofBIAs. In roots, protoberberine alkaloids arerestricted to endodermal cells upon the initi-ation of secondary growth, and are distributedthroughout the pith and cortex in rhizomes.Thus, the cell type–specific localization ofBIA biosynthesis and accumulation are tem-porally and spatially separated in T. flavumroots and rhizomes, respectively. Despite theclose phylogenetic relationships between cor-responding biosynthetic enzymes (87), differ-
MIA biosynthetic enzymes are associated withat least three different cell types in C. roseus—geraniol 10-hydroxylase (G10H) is found ininternal phloem parenchyma of aerial organs(15), TDC, secologanin synthase, and STRare localized to the epidermis of aerial or-gans and the apical meristem of roots (70,151), and D4H and DAT are restricted tothe laticifers and idioblasts of leaves and stems(151); thus, vindoline pathway intermediatesmust be translocated between cell types. En-zymes involved in monoterpenoid indole alka-loid biosynthesis in C. roseus also localize to atleast five subcellular compartments—TDC,D4H, and DAT are in the cytosol (24), STRand the peroxidase involved in the couplingof MIA monomers localize to the vacuole(98, 99, 145), SGD is a soluble enzyme pur-ported to associate with the cytoplasmic faceof the endoplasmic reticulum (148), the P450-dependent monooxygenases G10H, secolo-ganin synthase (SLS), and T16H are integralendomembrane proteins (20, 149, 177), and
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Multienzymecomplex: a clusterof distinct enzymesthat catalyzeconsecutivemetabolic reactions
the N-methyltransferase involved in vindolinebiosynthesis localizes to thylakoid membranes(27). It is unclear whether some enzymes, suchas T16H, occur in the epidermis, or in lati-cifers and idioblasts. The complex compart-mentation of the monoterpenoid indole al-kaloid pathway suggests extensive subcellulartrafficking of pathway intermediates.
Several enzymes involved in BIA biosyn-thesis are associated with a subcellular com-partment other than the cytosol. In the san-guinarine branch pathway, density gradientfractionation suggested the localization ofBBE and P450 monooxygenases to micro-somes with a density slightly higher than thatof typical endoplasmic reticulum (ER) (5, 10,132, 156). Although BBE is not an integralmembrane protein, it is initially targeted tothe ER and subsequently sorted to a vacuolarcompartment (12). NCS is also predicted topossess a signal peptide for targeting to theER (137), and the final oxidation of alkaloids,such as dihydrosanguinarine, likely occurs inan ER-derived compartment (5). The associ-ation of BIA biosynthetic enzymes with en-domembranes led to speculation that special-ized alkaloid-synthesizing vesicles are presentin alkaloid-producing cells (5). Recently, how-ever, Cyp80B3, BBE, and sanguinarine werefound to colocalize with the ER in opiumpoppy cell cultures (2). Moreover, the induc-tion of sanguinarine biosynthesis correlateswith extensive dilations of the ER and theaccumulation of an electron-dense flocculentmaterial within the ER. Because the pH op-timum of BBE is ∼8.8 (146), sanguinarinebiosynthesis is unlikely to involve the vac-uole; thus, alkaloid metabolism could be en-tirely associated with the ER. Indeed, thevacuole might not even be the site of BIAaccumulation in cultured cells, as previouslythought. Instead, sanguinarine and related al-kaloids could be secreted and bound to cellwall components and reabsorbed, reduced tothe less-toxic compound dihydrosanguinar-ine, and further metabolized (171).
A unique subcellular compartmentaliza-tion of enzymes is also present in quinolizidine
alkaloid biosynthesis, which occurs in themesophyll of some legumes. Lysine decar-boxylase and the quinolizidine skeleton–forming enzyme have been detected inchloroplasts of Lupinus polyphyllus (172). 13α-Hydroxymultifluorine/13α-hydroxylupanineO-tigloylase is localized to the mitochondrialmatrix rather than to chloroplasts where denovo quinolizidine alkaloid biosynthesis isthought to occur (152). In contrast, epilupi-nine O-p-coumaryoltransferase is presentin a distinct organelle, but has not beenunambiguously localized.
Enzyme Complexesand Metabolic Channels
Many metabolic enzymes are generally as-sumed to interact physically or be in closeproximity with other enzymes that participatein common pathways (173). Theoretically,the existence of multienzyme complexes—also known as metabolic complexes/channelsor metabolons—promote the efficiency ofcellular metabolism. The direct transfer ofa pathway intermediate from one enzymeto another maintains a high local substrateconcentration, which avoids the dilution ofintermediates released into the cytoplasm.Enzyme complexes also eliminate competi-tion from other enzymes for the same sub-strate, increase the stability of intermediates,and minimize the deleterious effects of cyto-toxic compounds. Recent investigations of theinteractions between pathway enzymes havehinted at the importance of metabolic chan-nels in primary and secondary metabolism.The detection of multienzyme complexes hasbeen purported for flavonoid (1, 14, 58) andpolyamine (122) metabolism.
The results from the RNAi-mediatedsilencing of COR genes suggested the possibleexistence of a multienzyme complex in BIA(4). Although seven enzymatic steps occurbetween (S )-reticuline and codeinone—thesubstrate for COR—only (S )-reticuline accu-mulates at elevated levels. No intermediatesbetween (S )-reticuline and morphine are
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detected. Removal of COR was suggested todisrupt a metabolic channel composed of mor-phinan branch pathway enzymes, resulting inthe accumulation of an alkaloid intermediateproduced by enzymes that are not part ofthe same complex. Interestingly, thebaineand oripavine, intermediates upstream ofsubstrates acted upon by COR, accumulateto high levels in some opium poppy cul-tivars (101); thus, researchers expected anaccumulation of morphinan branch pathwayintermediates. An alternative interpretationnot considered by Allen and coworkers (4)is the suppression of 1,2-dehydroreticulinereductase as a possible side effect of silencingCOR. 1,2-Dehydroreticuline reductase is oneof two enzymes involved in the epimerizationof (S )-reticuline to (R )-reticuline. The po-tential homology between the two reductasescould lead to cosilencing.
PERSPECTIVE
Major progress in the elucidation of alka-loid biosynthetic pathways and their regula-tion has been obtained by application of largescale genomic tools. However, there are sev-eral techniques, whose applications are stillmore or less exclusively confined to model or-ganisms, that might further our understand-ing of plant alkaloid biosynthesis. Proteomeanalysis in alkaloid-accumulating plants hasbeen undertaken (26, 71, 115), but is still lim-ited by the lack of peptide mass databases forthe investigated species, which does not allowprotein identification by simple peptide massfingerprinting. Targeted induced local lesionsin genomes (TILLING) as a tool to discoverpoint mutations is also dependent on avail-
able sequence information (21). However, thegrowing sequence database makes the appli-cation of this technique feasible in studyingmutations in alkaloid-containing plants. Thedevelopment of sequencing techniques thatcan generate large datasets within short timeswill probably be applied to alkaloid produc-ing plants and will provide the basis for manysequence-based approaches that are now lim-ited to plants with sequenced genomes suchas Arabidopsis and rice. Whether genomesequencing projects in alkaloid-containingplants will be feasible remains to be seen, be-cause the genomes of the investigated plantsare very large (4.7 Gbp for C. roseus or 7.4 Gbpfor opium poppy compared with, for example,1 Gbp for rice). The predicted function of al-kaloid biosynthetic enzymes and other com-ponents based on the in vitro characterizationof gene products must be confirmed in vivo.However, functional genomics remains prob-lematic for most alkaloid-producing plantsowing to general limitations in establishedgenetic transformation technologies. Virus-induced gene silencing is a fast method for thegeneration of transiently transformed plantsand works well in a model organism suchas N. benthamiana (130), and protocols foropium poppy and E. californica have recentlybeen developed (62, 169). Thus, the impactof suppression of candidate genes on alkaloidbiosynthesis can be readily examined beforestable transformation is applied. Consideringthe progress in the investigation of alkaloidmetabolism achieved in the last 5 years, a lookto the future promises further strong devel-opments in the discovery of regulatory net-works that lead to alkaloid accumulation inplants.
SUMMARY POINTS
1. The application of genomics technologies has expedited the discovery of new alkaloidbiosynthetic genes that encode enzymes and regulatory proteins with novel functions.
2. Large-scale, integrated transcriptomics and metabolomics analyses are providing ini-tial hints about the regulation of alkaloid pathways.
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3. Structural analysis of alkaloid biosynthetic enzymes has enabled targeted mod-ifications of substrate-binding sites for the development of biomimetic alkaloidproduction.
4. Improved transformation protocols have facilitated the establishment of transgenicplants with tailored alkaloid profiles, providing additional insights into the regulationof pathways.
5. Alkaloid biosynthesis and accumulation are associated with a variety of cell types indifferent plants, including epidermis, endodermis, pericycle, phloem parenchyma,phloem sieve elements and companion cells, specialized mesophyll, and laticifers. Acommon paradigm is the involvement of multiple cell types and the implied transportof pathway intermediates and/or products.
6. The subcellular compartmentalization of alkaloid biosynthetic enzymes is as complexas the cell type–specific localization of gene transcripts, enzymes, and metabolites. Al-though the endoplasmic reticulum is a favored site for alkaloid formation, biosyntheticenzymes have been associated with chloroplast thylakoid membranes, mitochondria,vacuoles, and the cytosol.
FUTURE ISSUES
1. Despite impressive advances facilitated by the application of genomics technologies tothe discovery of novel genes that encode alkaloid biosynthetic enzymes, a substantialunderstanding of the regulatory components of alkaloid pathways has been achievedonly for terpenoid indole alkaloid metabolism. Genomics approaches should lead tothe identification of regulatory genes involved in benzylisoquinoline alkaloid synthesisand other alkaloid pathways.
2. The impact of posttranslational modification of alkaloid biosynthetic enzymes onproduct accumulation is a black box. Proteomic approaches might explain why theexpression levels of some biosynthetic genes do not correlate with alkaloid profiles.
3. Although the involvement of multiple cell types in the biosynthesis and accumula-tion of many alkaloids has been established, the identity of the pathway intermediatesand/or products that undergo intercellular transport is not known. Moreover, themechanisms of transport—symplastic involving plasmodesmata, or apoplastic involv-ing specific transporters or channels—are poorly understood.
4. The existence of metabolic channels as a common feature of alkaloid biosyntheticpathways has been widely purported, but only scant evidence is available to supporttheir existence. Establishing the interactions among biosynthetic enzymes and otherpathway components will facilitate the rational engineering of alkaloid metabolism inplants and microorganisms.
DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the objectivity ofthis review.
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ACKNOWLEDGMENTS
J.Z. was funded through a Canada Research Chair in Plant Metabolic Processes Biotechnologyheld by P.J.F. Research support from the Natural Sciences and Engineering Research Councilof Canada to P.J.F. is also gratefully acknowledged.
LITERATURE CITED
1. Achnine L, Blancaflor EB, Rasmussen S, Dixon RA. 2004. Colocalization of l-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling inphenylpropanoid biosynthesis. Plant Cell 16:3098–109
2. Alcantara J, Bird DA, Franceschi VR, Facchini PJ. 2005. Sanguinarine biosynthesis isassociated with the endoplasmic reticulum in cultured opium poppy cells after elicitortreatment. Plant Physiol. 138:173–83
3. Allen RS, Miller JA, Chitty JA, Fist AJ, Gerlach WL, Larkin PJ. 2007. Metabolic en-gineering of morphinan alkaloids by overexpression and RNAi suppression of salu-taridinol 7-O-acetyltransferase in opium poppy. Plant Biotechnol. J. doi:10.1111/j.1467-7652.2007.00293.x
4. Allen RS, Millgate AG, Chitty JA, Thisleton J, Miller JA, et al. 2004. RNAi-mediatedreplacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat.Biotechnol. 22:1559–66
5. Amann M, Wanner G, Zenk MH. 1986. Intracellular compartmentation of two enzymesof berberine biosynthesis in plant cell cultures. Planta 167:310–20
6. Anke S, Niemuller D, Moll S, Hansch R, Ober D. 2004. Polyphyletic origin ofpyrrolizidine alkaloids within the Asteraceae. Evidence from differential tissue expres-sion of homospermidine synthase. Plant Physiol. 136:4037–47
7. Arakawa H, Clark WG, Psenak M, Coscia CJ. 1992. Purification and characterization ofdihydrobenzophenanthridine oxidase from elicited Sanguinaria canadensis cell cultures.Arch. Biochem. Biophys. 299:1–7
8. Ayora-Talavera T, Chappell J, Lozoya-Gloria E, Loyola-Vargas VM. 2002. Overexpres-sion in Catharanthus roseus hairy roots of a truncated hamster 3-hydroxy-3-methylglutaryl-CoA reductase gene. Appl. Biochem. Biotechnol. 97:135–45
9. Barleben L, Panjikar S, Ruppert M, Koepke J, Stockigt J. 2007. Molecular architectureof strictosidine glucosidase: the gateway to the biosynthesis of the monoterpenoid indolealkaloid family. Plant Cell 19:2886–97
10. Bauer W, Zenk MH. 1991. Two methylenedioxy bridge forming cytochrome P-450 de-pendent enzymes are involved in (S )-stylopine biosynthesis. Phytochemistry 30:2953–62
11. Bayer A, Ma X, Stockigt J. 2004. Acetyltransfer in natural product biosynthesis–functionalcloning and molecular analysis of vinorine synthase. Bioorg. Med. Chem. 12:2787–95
12. Bird DA, Facchini PJ. 2001. Berberine bridge enzyme, a key branch-point enzyme inbenzylisoquinoline alkaloid biosynthesis, contains a vacuolar-sorting determinant. Planta213:888–97
13. Bird DA, Franceschi VR, Facchini PJ. 2003. A tale of three cell types: alkaloid biosynthesisis localized to sieve elements in opium poppy. Plant Cell 15:2626–35
14. Burbulis IE, Winkel-Shirley B. 1999. Interactions among enzymes of the Arabidopsisflavonoid biosynthetic pathway. Proc. Natl. Acad. Sci. USA 96:12929–34
15. Burlat V, Oudin A, Courtois M, Rideau M, St-Pierre B. 2004. Co-expression of threeMEP pathway genes and geraniol 10-hydroxylase in internal phloem parenchyma ofCatharanthus roseus implicates multicellular translocation of intermediates during the
www.annualreviews.org • Alkaloid Biosynthesis 759
Ann
u. R
ev. P
lant
Bio
l. 20
08.5
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5-76
9. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by W
ashi
ngto
n St
ate
Uni
vers
ity o
n 11
/01/
11. F
or p
erso
nal u
se o
nly.
ANRV342-PP59-29 ARI 29 March 2008 2:13
biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabo-lites. Plant J. 38:131–41
16. Canel C, Lopes-Cardoso MI, Whitmer S, van der Fits L, Pasquali G, et al. 1998. Ef-fects of overexpression of strictosidine synthase and tryptophan decarboxylase on alkaloidproduction by cell cultures of Catharanthus roseus. Planta 205:414–19
17. Carlson JE, Leebens-Mack JH, Wall PK, Zahn LM, Mueller LA, et al. 2006. EST databasefor early flower development in California poppy (Eschscholzia californica Cham., Papaver-aceae) tags over 6,000 genes from a basal eudicot. Plant Mol. Biol. 62:351–69
18. Chen S, Galan MC, Coltharp C, O’Connor SE. 2006. Redesign of a central enzyme inalkaloid biosynthesis. Chem. Biol. 13:1137–41
19. Choi KB, Morishige T, Shitan N, Yazaki K, Sato F. 2002. Molecular cloning and charac-terization of coclaurine N-methyltransferase from cultured cells of Coptis japonica. J. Biol.Chem. 277:830–35
20. Collu G, Unver N, Peltenburg-Looman AM, van der Heijden R, Verpoorte R, MemelinkJ. 2001. Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoidindole alkaloid biosynthesis. FEBS Lett. 508:215–20
21. Comai L, Henikoff S. 2006. TILLING: practical single-nucleotide mutation discovery.Plant J. 45:684–94
22. Contin A, van der Heijden R, Lefeber AW, Verpoorte R. 1998. The iridoid glucoside se-cologanin is derived from the novel triose phosphate/pyruvate pathway in a Catharanthusroseus cell culture. FEBS Lett. 434:413–16
23. De-Eknamkul W, Zenk MH. 1992. Purification and properties of 1,2-dehydroreticulinereductase from Papaver somniferum seedlings. Phytochemistry 31:813–21
24. De Luca V, Cutler AJ. 1987. Subcellular localization of enzymes involved in indolealkaloid biosynthesis in Catharanthus roseus. Plant Physiol. 85:1099–102
25. De Luca V, Marineau C, Brisson N. 1989. Molecular cloning and analysis of cDNAencoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases.Proc. Natl. Acad. Sci. USA 86:2582–86
26. Decker G, Wanner G, Zenk MH, Lottspeich F. 2000. Characterization of proteins inlatex of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresisand microsequencing. Electrophoresis 21:3500–16
27. Dethier M, De Luca V. 1993. Partial purification of an N-methyltransferase involved invindoline biosynthesis in Catharanthus roseus. Phytochemistry 32:673–78
28. Dittrich H, Kutchan TM. 1991. Molecular cloning, expression, and induction of berber-ine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloidsin the response of plants to pathogenic attack. Proc. Natl. Acad. Sci. USA 88:9969–73
29. Dogru E, Warzecha H, Seibel F, Haebel S, Lottspeich F, Stockigt J. 2000. The geneencoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesisin plants is an ortholog of the α/β-hydrolase super family. Eur. J. Biochem. 267:1397–406
30. Drager B. 2004. Chemistry and biology of calystegines. Nat. Prod. Rep. 21:211–2331. Facchini PJ. 2001. Alkaloid biosynthesis in plants: biochemistry, cell biology, molecular
32. Facchini PJ, Bird DA. 1998. Developmental regulation of benzylisoquinoline alkaloidbiosynthesis in opium poppy plants and tissue cultures. In Vitro Cell. Dev. Biol. Plant34:69–79
33. Facchini PJ, De Luca V. 1994. Differential and tissue-specific expression of a gene familyfor tyrosine/dopa decarboxylase in opium poppy. J. Biol. Chem. 269:26684–90
760 Ziegler · Facchini
Ann
u. R
ev. P
lant
Bio
l. 20
08.5
9:73
5-76
9. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by W
ashi
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n St
ate
Uni
vers
ity o
n 11
/01/
11. F
or p
erso
nal u
se o
nly.
ANRV342-PP59-29 ARI 29 March 2008 2:13
34. Facchini PJ, De Luca V. 1995. Phloem-specific expression of tyrosine/dopa decarboxylasegenes and the biosynthesis of isoquinoline alkaloids in opium poppy. Plant Cell 7:1811–21
35. Facchini PJ, Park SU. 2003. Developmental and inducible accumulation of gene tran-scripts involved in alkaloid biosynthesis in opium poppy. Phytochemistry 64:177–86
36. Facchini PJ, Penzes C, Johnson AG, Bull D. 1996. Molecular characterization of berber-ine bridge enzyme genes from opium poppy. Plant Physiol. 112:1669–77
37. Fahn W, Laussermair E, Deus-Neumann B, Stockigt J. 1985. Late enzymes of vindo-line biosynthesis S-adenosyl-L-methionine:11-O-demethyl-17-O-deacetylvindoline 11-O-methylase and unspecific acetylesterase. Plant Cell Rep. 4:337–40
38. Farber K, Schumann B, Miersch O, Roos W. 2003. Selective desensitization of jasmonate-and pH-dependent signaling in the induction of benzophenanthridine biosynthesis in cellsof Eschscholzia californica. Phytochemistry 62:491–500
39. Falkenhagen H, Stockigt J. 1995. Enzymatic biosynthesis of vomilenine, a key interme-diate of the ajmaline pathway, catalyzed by a novel cytochrome P450 dependent enzymefrom plant cell cultures of Rauvolfia serpentina. Z. Naturforsch. 1995:45–53
40. Frick S, Chitty JA, Kramell R, Schmidt J, Allen RS, et al. 2004. Transformation of opiumpoppy (Papaver somniferum L.) with antisense berberine bridge enzyme gene (anti-bbe)via somatic embryogenesis results in an altered ratio of alkaloids in latex but not in roots.Transgenic Res. 13:607–13
41. Frick S, Kramell R, Kutchan TM. 2007. Metabolic engineering with a morphine biosyn-thetic P450 in opium poppy surpasses breeding. Metab. Eng. 9:169–76
42. Frick S, Kutchan TM. 1999. Molecular cloning and functional expression of O-methyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis.Plant J. 17:329–39
43. Fujii N, Inui T, Iwasa K, Morishige T, Sato F. 2007. Knockdown of berberine bridgeenzyme by RNAi accumulates (S )-reticuline and activates a silent pathway in culturedCalifornia poppy cells. Transgenic Res. 16:363–75
44. Gao S, von Schumann G, Stockigt J. 2002. A newly-detected reductase from Rauvolfiacloses a gap in the biosynthesis of the antiarrhythmic alkaloid ajmaline. Planta Med.68:906–11
45. Gavilano LB, Siminszky B. 2007. Isolation and characterization of the cytochrome P450gene Cyp82E5v2 that mediates nicotine to nornicotine conversion in the green leaves oftobacco. Plant Cell. Physiol. 48:1567–74
46. Geerlings A, Ibanez MM, Memelink J, van Der Heijden R, Verpoorte R. 2000. Molecularcloning and analysis of strictosidine β-d-glucosidase, an enzyme in terpenoid indolealkaloid biosynthesis in Catharanthus roseus. J. Biol. Chem. 275:3051–56
47. Geissler R, Brandt W, Ziegler J. 2007. Molecular modeling and site-directed mutagenesisreveal the benzylisoquinoline binding site of the short-chain dehydrogenase/reductasesalutaridine reductase. Plant Physiol. 143:1493–503
48. Gerardy R, Zenk MH. 1993. Formation of salutaridine from (R )-reticuline by amembrane-bound cytochrome P-450 enzyme from Papaver somniferum. Phytochemistry32:79–86
49. Gerardy R, Zenk MH. 1993. Purification and characterization of salutaridine:NADPH7-oxidoreductase from Papaver somniferum. Phytochemistry 34:125–32
50. Gerasimenko I, Sheludko Y, Ma X, Stockigt J. 2002. Heterologous expression of a Rau-volfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoter-penoid indole alkaloids. Eur. J. Biochem. 269:2204–13
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ashi
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n St
ate
Uni
vers
ity o
n 11
/01/
11. F
or p
erso
nal u
se o
nly.
ANRV342-PP59-29 ARI 29 March 2008 2:13
51. Goossens A, Hakkinen ST, Laakso I, Seppanen-Laakso T, Biondi S, et al. 2003. A func-tional genomics approach toward the understanding of secondary metabolism in plantcells. Proc. Natl. Acad. Sci. USA 100:8595–600
52. Grothe T, Lenz R, Kutchan TM. 2001. Molecular characterization of the salutaridinol7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver som-niferum. J. Biol. Chem. 276:30717–23
53. Hancock RD, McRae D, Haupt S, Viola R. 2003. Synthesis of l-ascorbic acid in thephloem. BMC Plant Biol. 3:7
54. Hartmann T, Dierich B. 1998. Chemical diversity and variation of pyrrolizidine alkaloidsof the senecionine type: biological need or coincidence? Planta 206:443–51
55. Hartmann T. 2004. Plant-derived secondary metabolites as defensive chemicals in her-bivorous insects: a case study in chemical ecology. Planta 219:1–4
56. Hashimoto T, Hayashi A, Amano Y, Kohno J, Iwanari H, et al. 1991. Hyoscyamine6β-hydroxylase, an enzyme involved in tropane alkaloid biosynthesis, is localized at thepericycle of the root. J. Biol. Chem. 266:4648–53
57. Hause B, Hause G, Kutter C, Miersch O, Wasternack C. 2003. Enzymes of jasmonatebiosynthesis occur in tomato sieve elements. Plant Cell Physiol. 44:643–48
58. He XZ, Dixon RA. 2000. Genetic manipulation of isoflavone 7-O-methyltransferaseenhances biosynthesis of 4′-O-methylated isoflavonoid phytoalexins and disease resistancein alfalfa. Plant Cell 12:1689–702
59. Heim WG, Sykes KA, Hildreth SB, Sun J, Lu RH, Jelesko JG. 2007. Cloning and char-acterization of a Nicotiana tabacum methylputrescine oxidase transcript. Phytochemistry68:454–63
60. Heinze M, Steighardt J, Gesell A, Schwartze W, Roos W. 2007. Regulatory interaction ofthe Gα protein with phospholipase A2 in the plasma membrane of Eschscholzia californica.Plant J. doi:10.1111/j.1365–313X.2007.03300.x
61. Hibi N, Higashiguchi S, Hashimoto T, Yamada Y. 1994. Gene expression in tobaccolow-nicotine mutants. Plant Cell 6:723–35
62. Hileman LC, Drea S, Martino G, Litt A, Irish VF. 2005. Virus-induced gene silencing isan effective tool for assaying gene function in the basal eudicot species Papaver somniferum(opium poppy). Plant J. 44:334–41
64. Hong SB, Peebles CA, Shanks JV, San KY, Gibson SI. 2006. Expression of the Arabidop-sis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylasegenes in Catharanthus roseus hairy roots. J. Biotechnol. 122:28–38
65. Huang FC, Kutchan TM. 2000. Distribution of morphinan and benzo[c]phenanthridinealkaloid gene transcript accumulation in Papaver somninferum. Phytochemistry 53:555–64
66. Hughes EH, Hong SB, Gibson SI, Shanks JV, San KY. 2004. Metabolic engineeringof the indole pathway in Catharanthus roseus hairy roots and increased accumulation oftryptamine and serpentine. Metab. Eng. 6:268–76
67. Ikezawa N, Iwasa K, Sato F. 2007. Molecular cloning and characterization of methylene-dioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica.FEBS J. 274:1019–35
68. Ikezawa N, Tanaka M, Nagayoshi M, Shinkyo R, Sakaki T, et al. 2003. Molecular cloningand characterization of Cyp719, a methylenedioxy bridge-forming enzyme that belongsto a novel P450 family, from cultured Coptis japonica cells. J. Biol. Chem. 278:38557–65
762 Ziegler · Facchini
Ann
u. R
ev. P
lant
Bio
l. 20
08.5
9:73
5-76
9. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by W
ashi
ngto
n St
ate
Uni
vers
ity o
n 11
/01/
11. F
or p
erso
nal u
se o
nly.
ANRV342-PP59-29 ARI 29 March 2008 2:13
69. Inui T, Tamura K, Fujii N, Morishige T, Sato F. 2007. Overexpression of Coptis japonicanorcoclaurine 6-O-methyltransferase overcomes the rate-limiting step in benzylisoquino-line alkaloid biosynthesis in cultured Eschscholzia californica. Plant Cell Physiol. 48:252–62
70. Irmler S, Schroder G, St-Pierre B, Crouch NP, Hotze M, et al. 2000. Indole alka-loid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cy-tochrome P450 Cyp72A1 as secologanin synthase. Plant J. 24:797–804
71. Jacobs DI, Gaspari M, van der Greef J, van der Heijden R, Verpoorte R. 2005. Proteomeanalysis of the medicinal plant Catharanthus roseus. Planta 221:690–704
72. Jorgensen RA, Atkinson RG, Forster RL, Lucas WJ. 1998. An RNA-based informationsuperhighway in plants. Science 279:1486–87
73. Kaiser H, Richter U, Keiner R, Brabant A, Hause B, Drager B. 2006. Immunolocalisationof two tropinone reductases in potato (Solanum tuberosum L.) root, stolon, and tubersprouts. Planta 225:127–37
74. Kato M, Mizuno K, Crozier A, Fujimura T, Ashihara H. 2000. Caffeine synthase genefrom tea leaves. Nature 406:956–57
75. Kato N, Dubouzet E, Kokabu Y, Yoshida S, Taniguchi Y, et al. 2007. Identification of aWRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesisin Coptis japonica. Plant Cell Physiol. 48:8–18
76. Keiner R, Kaiser H, Nakajima K, Hashimoto T, Drager B. 2002. Molecular cloning,expression and characterization of tropinone reductase II, an enzyme of the SDR familyin Solanum tuberosum (L.). Plant Mol. Biol. 48:299–308
77. Kim YS, Uefuji H, Ogita S, Sano H. 2006. Transgenic tobacco plants producing caffeine:a potential new strategy for insect pest control. Transgenic Res. 15:667–72
78. Kraus PF, Kutchan TM. 1995. Molecular cloning and heterologous expression of a cDNAencoding berbamunine synthase, a C-O phenol-coupling cytochrome P450 from thehigher plant Berberis stolonifera. Proc. Natl. Acad. Sci. USA 92:2071–75
79. Kutchan TM. 1989. Expression of enzymatically active cloned strictosidine synthase fromthe higher plant Rauvolfia serpentina in Escherichia coli. FEBS Lett. 257:127–30
80. Laflamme P, St-Pierre B, De Luca V. 2001. Molecular and biochemical analysis ofa Madagascar periwinkle root-specific minovincinine-19-hydroxy-O-acetyltransferase.Plant Physiol. 125:189–98
81. Larkin PJ, Miller JA, Allen RS, Chitty JA, Gerlach WL, et al. 2007. Increasing mor-phinan alkaloid production by overexpressing codeinone reductase in transgenic Papaversomniferum. Plant Biotechnol. J. 5:26–37
82. Lenz R, Zenk MH. 1995. Acetyl coenzyme A:salutaridinol-7-O-acetyltransferase fromPapaver somniferum plant cell cultures. The enzyme catalyzing the formation of thebainein morphine biosynthesis. J. Biol. Chem. 270:31091–96
83. Lenz R, Zenk MH. 1995. Purification and properties of codeinone reductase (NADPH)from Papaver somniferum cell cultures and differentiated plants. Eur. J. Biochem. 233:132–39
84. Li R, Reed DW, Liu E, Nowak J, Pelcher LE, et al. 2006. Functional genomic analysis ofalkaloid biosynthesis in Hyoscyamus niger reveals a cytochrome P450 involved in littorinerearrangement. Chem. Biol. 13:513–20
85. Lin C, Mueller LA, McCarthy J, Crouzillat D, Petiard V, Tanksley SD. 2005. Coffeeand tomato share common gene repertoires as revealed by deep sequencing of seed andcherry transcripts. Theor. Appl. Genet. 112:114–30
86. Liscombe DK, Facchini PJ. 2007. Molecular cloning and characterization of tetrahy-droprotoberberine cis-N-methyltransferase, an enzyme involved in alkaloid biosynthesisin opium poppy. J. Biol. Chem. 282:14741–51
www.annualreviews.org • Alkaloid Biosynthesis 763
Ann
u. R
ev. P
lant
Bio
l. 20
08.5
9:73
5-76
9. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by W
ashi
ngto
n St
ate
Uni
vers
ity o
n 11
/01/
11. F
or p
erso
nal u
se o
nly.
ANRV342-PP59-29 ARI 29 March 2008 2:13
87. Liscombe DK, MacLeod BP, Loukanina N, Nandi OI, Facchini PJ. 2005. Evidence forthe monophyletic evolution of benzylisoquinoline alkaloid biosynthesis in angiosperms.Phytochemistry 66:2501–20
88. Liu T, Zhu P, Cheng KD, Meng C, He HX. 2005. Molecular cloning, expression andcharacterization of hyoscyamine 6β-hydroxylase from hairy roots of Anisodus tanguticus.Planta Med. 71:249–53
89. Lopez-Meyer M, Nessler CL. 1997. Tryptophan decarboxylase is encoded by two au-tonomously regulated genes in Camptotheca acuminata which are differentially expressedduring development and stress. Plant J. 11:1167–75
90. Loris EA, Panjikar S, Ruppert M, Barleben L, Unger M, et al. 2007. Structure-basedengineering of strictosidine synthase: auxiliary for alkaloid libraries. Chem. Biol. 14:979–85
92. Ma X, Koepke J, Bayer A, Fritzsch G, Michel H, Stockigt J. 2005. Crystallization andpreliminary X-ray analysis of native and selenomethionyl vinorine synthase from Rauvolfiaserpentina. Acta Crystallogr. D Biol. Crystallogr. 61:694–96
93. Ma X, Panjikar S, Koepke J, Loris E, Stockigt J. 2006. The structure of Rauvolfia serpentinastrictosidine synthase is a novel six-bladed β-propeller fold in plant proteins. Plant Cell18:907–20
94. Mahroug S, Courdavault V, Thiersault M, St-Pierre B, Burlat V. 2006. Epidermis is apivotal site of at least four secondary metabolic pathways in Catharanthus roseus aerialorgans. Planta 223:1191–200
95. Matsuda J, Okabe S, Hashimoto T, Yamada Y. 1991. Molecular cloning of hyoscyamine6β-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscya-mus niger. J. Biol. Chem. 266:9460–64
96. Mattern-Dogru E, Ma X, Hartmann J, Decker H, Stockigt J. 2002. Potential active-siteresidues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosyn-thesis, by modelling and site-directed mutagenesis. Eur. J. Biochem. 269:2889–96
97. McCarthy AA, McCarthy JG. 2007. The structure of two N-methyltransferases from thecaffeine biosynthetic pathway. Plant Physiol. 144:879–89
98. McKnight TD, Bergey DR, Burnett RJ, Nessler CL. 1991. Expression of enzymaticallyactive and correctly targeted strictosidine synthase in transgenic tobacco plants. Planta185:148–52
99. McKnight TD, Roessner CA, Devagupta R, Scott AI, Nessler CL. 1990. Nucleotide se-quence of a cDNA encoding the vacuolar protein strictosidine synthase from Catharanthusroseus. Nucleic Acids Res. 18:4939
100. Menke FL, Champion A, Kijne JW, Memelink J. 1999. A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str inter-acts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2.EMBO J. 18:4455–63
101. Millgate AG, Pogson BJ, Wilson IW, Kutchan TM, Zenk MH, et al. 2004. Analgesia:morphine-pathway block in top1 poppies. Nature 431:413–14
102. Minami H, Dubouzet E, Iwasa K, Sato F. 2007. Functional analysis of norcoclaurinesynthase in Coptis japonica. J. Biol. Chem. 282:6274–82
103. Mizuno K, Kato M, Irino F, Yoneyama N, Fujimura T, Ashihara H. 2003. The firstcommitted step reaction of caffeine biosynthesis: 7-methylxanthosine synthase is closelyhomologous to caffeine synthases in coffee (Coffea arabica L.). FEBS Lett. 547:56–60
764 Ziegler · Facchini
Ann
u. R
ev. P
lant
Bio
l. 20
08.5
9:73
5-76
9. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by W
ashi
ngto
n St
ate
Uni
vers
ity o
n 11
/01/
11. F
or p
erso
nal u
se o
nly.
ANRV342-PP59-29 ARI 29 March 2008 2:13
104. Mizuno K, Okuda A, Kato M, Yoneyama N, Tanaka H, et al. 2003. Isolation of a newdual-functional caffeine synthase gene encoding an enzyme for the conversion of 7-methylxanthine to caffeine from coffee (Coffea arabica L.). FEBS Lett. 534:75–81
105. Moll S, Anke S, Kahmann U, Hansch R, Hartmann T, Ober D. 2002. Cell-specificexpression of homospermidine synthase, the entry enzyme of the pyrrolizidine alkaloidpathway in Senecio vernalis, in comparison with its ancestor, deoxyhypusine synthase. PlantPhysiol. 130:47–57
106. Morishige T, Dubouzet E, Choi KB, Yazaki K, Sato F. 2002. Molecular cloning of colum-bamine O-methyltransferase from cultured Coptis japonica cells. Eur. J. Biochem. 269:5659–67
107. Morishige T, Tsujita T, Yamada Y, Sato F. 2000. Molecular characterization ofthe S-adenosyl-L-methionine:3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferaseinvolved in isoquinoline alkaloid biosynthesis in Coptis japonica. J. Biol. Chem. 275:23398–405
108. Murata J, Bienzle D, Brandle JE, Sensen CW, De Luca V. 2006. Expressed sequence tagsfrom Madagascar periwinkle (Catharanthus roseus). FEBS Lett. 580:4501–7
109. Murata J, De Luca V. 2005. Localization of tabersonine 16-hydroxylase and 16-OHtabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major siteof precursor biosynthesis in the vindoline pathway in Catharanthus roseus. Plant J. 44:581–94
111. Nakajima K, Hashimoto T, Yamada Y. 1993. Two tropinone reductases with differentstereospecificities are short-chain dehydrogenases evolved from a common ancestor. Proc.Natl. Acad. Sci. USA 90:9591–95
112. Nakajima K, Hashimoto T. 1999. Two tropinone reductases, that catalyze opposite stere-ospecific reductions in tropane alkaloid biosynthesis, are localized in plant root withdifferent cell-specific patterns. Plant Cell Physiol. 40:1099–107
113. Nakajima K, Oshita Y, Kaya M, Yamada Y, Hashimoto T. 1999. Structures and expres-sion patterns of two tropinone reductase genes from Hyoscyamus niger. Biosci. Biotechnol.Biochem. 63:1756–64
114. Nakajima K, Yamashita A, Akama H, Nakatsu T, Kato H, et al. 1998. Crystal structures oftwo tropinone reductases: different reaction stereospecificities in the same protein fold.Proc. Natl. Acad. Sci. USA 95:4876–81
115. Nawrot R, Kalinowski A, Gozdzicka-Jozefiak A. 2007. Proteomic analysis of Chelidoniummajus milky sap using two-dimensional gel electrophoresis and tandem mass spectrome-try. Phytochemistry 68:1612–22
116. Ober D, Hartmann T. 1999. Homospermidine synthase, the first pathway-specific en-zyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase. Proc.Natl. Acad. Sci. USA 96:14777–82
117. Ogawa M, Herai Y, Koizumi N, Kusano T, Sano H. 2001. 7-Methylxanthine methyltrans-ferase of coffee plants. Gene isolation and enzymatic properties. J. Biol. Chem. 276:8213–18
118. Ogita S, Uefuji H, Morimoto M, Sano H. 2004. Application of RNAi to confirm theo-bromine as the major intermediate for caffeine biosynthesis in coffee plants with potentialfor construction of decaffeinated varieties. Plant Mol. Biol. 54:931–41
119. Ogita S, Uefuji H, Yamaguchi Y, Koizumi N, Sano H. 2003. Producing decaffeinatedcoffee plants. Nature 423:823
www.annualreviews.org • Alkaloid Biosynthesis 765
Ann
u. R
ev. P
lant
Bio
l. 20
08.5
9:73
5-76
9. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by W
ashi
ngto
n St
ate
Uni
vers
ity o
n 11
/01/
11. F
or p
erso
nal u
se o
nly.
ANRV342-PP59-29 ARI 29 March 2008 2:13
120. Oudin A, Mahroug S, Courdavault V, Hervouet N, Zelwer C, et al. 2007. Spatial distri-bution and hormonal regulation of gene products from methyl erythritol phosphate andmonoterpene-secoiridoid pathways in Catharanthus roseus. Plant Mol. Biol. 65:13–30
121. Ounaroon A, Decker G, Schmidt J, Lottspeich F, Kutchan TM. 2003. (R,S )-Reticuline 7-O-methyltransferase and (R,S )-norcoclaurine 6-O-methyltransferase of Pa-paver somniferum—cDNA cloning and characterization of methyl transfer enzymes ofalkaloid biosynthesis in opium poppy. Plant J. 36:808–19
122. Panicot M, Minguet EG, Ferrando A, Alcazar R, Blazquez MA, et al. 2002. Apolyamine metabolon involving aminopropyl transferase complexes in Arabidopsis. PlantCell 14:2539–51
123. Park SU, Yu M, Facchini PJ. 2002. Antisense RNA-mediated suppression of ben-zophenanthridine alkaloid biosynthesis in transgenic cell cultures of California poppy.Plant Physiol. 128:696–706
124. Park SU, Yu M, Facchini PJ. 2003. Modulation of berberine bridge enzyme levels in trans-genic root cultures of California poppy alters the accumulation of benzophenanthridinealkaloids. Plant Mol. Biol. 51:153–64
125. Pauli HH, Kutchan TM. 1998. Molecular cloning and functional heterologous expres-sion of two alleles encoding (S )-N-methylcoclaurine 3′-hydroxylase (Cyp80B1), a newmethyl jasmonate-inducible cytochrome P-450-dependent monooxygenase of benzyliso-quinoline alkaloid biosynthesis. Plant J. 13:793–801
126. Pauw B, Hilliou FA, Martin VS, Chatel G, de Wolf CJ, et al. 2004. Zinc finger proteinsact as transcriptional repressors of alkaloid biosynthesis genes in Catharanthus roseus. J.Biol. Chem. 279:52940–48
127. Pilatzke-Wunderlich I, Nessler CL. 2001. Expression and activity of cell-wall-degradingenzymes in the latex of opium poppy, Papaver somniferum L. Plant Mol. Biol. 45:567–76
128. Reimann A, Nurhayati N, Backenkohler A, Ober D. 2004. Repeated evolution of thepyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages. PlantCell. 16:2772–84
129. Rischer H, Oresic M, Seppanen-Laakso T, Katajamaa M, Lammertyn F, et al. 2006.Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthusroseus cells. Proc. Natl. Acad. Sci. USA 103:5614–19
130. Robertson D. 2004. VIGS vectors for gene silencing: many targets, many tools. Annu.Rev. Plant Biol. 55:495–519
131. Ronning CM, Stegalkina SS, Ascenzi RA, Bougri O, Hart AL, et al. 2003. Comparativeanalyses of potato expressed sequence tag libraries. Plant Physiol. 131:419–29
132. Rueffer M, Zenk MH. 1987. Enzymatic formation of protopines by a microsomal cy-tochrome P-450 system of Corydalis vaginans. Tetrahedron Lett. 28:5307–10
133. Ruppert M, Panjikar S, Barleben L, Stockigt J. 2006. Heterologous expression, purifica-tion, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plantenzyme specifically involved in Rauwolfia alkaloid biosynthesis. Acta Crystallogr. Sect. FStruct. Biol. Cryst. Commun. 62:257–60
134. Ruppert M, Woll J, Giritch A, Genady E, Ma X, Stockigt J. 2005. Functional expressionof an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expressionsystem. Planta 222:888–98
135. Samanani N, Alcantara J, Bourgault R, Zulak KG, Facchini PJ. 2006. The role of phloemsieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opiumpoppy. Plant J. 47:547–63
766 Ziegler · Facchini
Ann
u. R
ev. P
lant
Bio
l. 20
08.5
9:73
5-76
9. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by W
ashi
ngto
n St
ate
Uni
vers
ity o
n 11
/01/
11. F
or p
erso
nal u
se o
nly.
ANRV342-PP59-29 ARI 29 March 2008 2:13
136. Samanani N, Facchini PJ. 2002. Purification and characterization of norcoclaurine syn-thase. The first committed enzyme in benzylisoquinoline alkaloid biosynthesis in plants.J. Biol. Chem. 277:33878–83
137. Samanani N, Liscombe DK, Facchini PJ. 2004. Molecular cloning and characterizationof norcoclaurine synthase, an enzyme catalyzing the first committed step in benzyliso-quinoline alkaloid biosynthesis. Plant J. 40:302–13
138. Samanani N, Park SU, Facchini PJ. 2005. Cell type-specific localization of transcriptsencoding nine consecutive enzymes involved in protoberberine alkaloid biosynthesis.Plant Cell 17:915–26
139. Schmidt D, Stockigt J. 1995. Enzymatic formation of the sarpagan-bridge: a key step inthe biosynthesis of sarpagine- and ajmaline-type alkaloids. Planta Med. 61:254–58
140. Schroder G, Unterbusch E, Kaltenbach M, Schmidt J, Strack D, et al. 1999. Light-induced cytochrome P450-dependent enzyme in indole alkaloid biosynthesis: tabersonine16-hydroxylase. FEBS Lett. 458:97–102
141. Shoji T, Nakajima K, Hashimoto T. 2000. Ethylene suppresses jasmonate-induced geneexpression in nicotine biosynthesis. Plant Cell Physiol. 41:1072–76
142. Shoji T, Winz R, Iwase T, Nakajima K, Yamada Y, Hashimoto T. 2002. Expression pat-terns of two tobacco isoflavone reductase-like genes and their possible roles in secondarymetabolism in tobacco. Plant Mol. Biol. 50:427–40
143. Siberil Y, Benhamron S, Memelink J, Giglioli-Guivarc’h N, Thiersault M, et al. 2001.Catharanthus roseus G-box binding factors 1 and 2 act as repressors of strictosidine synthasegene expression in cell cultures. Plant Mol. Biol. 45:477–88
144. Siminszky B, Gavilano L, Bowen SW, Dewey RE. 2005. Conversion of nicotine to norni-cotine in Nicotiana tabacum is mediated by Cyp82E4, a cytochrome P450 monooxygenase.Proc. Natl. Acad. Sci. USA 102:14919–24
145. Sottomayor M, Lopez-Serrano M, DiCosmo F, Ros Barcelo A. 1998. Purification andcharacterization of α-3′,4′-anhydrovinblastine synthase (peroxidase-like) from Catharan-thus roseus (L.) G. Don. FEBS Lett. 428:299–303
146. Steffens P, Nagakura N, Zenk MH. 1985. Purification and characterization of the berber-ine bridge enzyme from Berberis beaniana cell cultures. Phytochemistry 24:2577–83
147. Stenzel O, Teuber M, Drager B. 2006. Putrescine N-methyltransferase in Solanum tubero-sum L., a calystegine-forming plant. Planta 223:200–12
148. Stevens LH, Blom TJM, Verpoorte R. 1993. Subcellular localization of tryptophan de-carboxylase, strictosidine synthase and strictosidine glucosidase in suspension-culturedcells of Catharanthus roseus and Tabernaemontana divaricata. Plant Cell Rep. 12:573–76
149. St-Pierre B, De Luca V. 1995. A cytochrome P-450 monooxygenase catalyzes the firststep in the conversion of tabersonine to vindoline in Catharanthus roseus. Plant Physiol.109:131–139
150. St-Pierre B, Laflamme P, Alarco AM, De Luca V. 1998. The terminal O-acetyltransferaseinvolved in vindoline biosynthesis defines a new class of proteins responsible for coenzymeA-dependent acyl transfer. Plant J. 14:703–13
151. St-Pierre B, Vazquez-Flota FA, De Luca V. 1999. Multicellular compartmentation ofCatharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathwayintermediate. Plant Cell 11:887–900
152. Suzuki H, Koike Y, Murakoshi I, Saito K. 1996. Subcellular localization of acyltransferasesfor quinolizidine alkaloid biosynthesis in Lupinus. Phytochemistry 42:1557–62
153. Suzuki K, Yamada Y, Hashimoto T. 1999. Expression of Atropa belladonna putrescineN-methyltransferase gene in root pericycle. Plant Cell Physiol. 40:289–97
www.annualreviews.org • Alkaloid Biosynthesis 767
Ann
u. R
ev. P
lant
Bio
l. 20
08.5
9:73
5-76
9. D
ownl
oade
d fr
om w
ww
.ann
ualr
evie
ws.
org
by W
ashi
ngto
n St
ate
Uni
vers
ity o
n 11
/01/
11. F
or p
erso
nal u
se o
nly.
ANRV342-PP59-29 ARI 29 March 2008 2:13
154. Suzuki K, Yun DJ, Chen XY, Yamada Y, Hashimoto T. 1999. An Atropa belladonnahyoscyamine 6β-hydroxylase gene is differentially expressed in the root pericycle andanthers. Plant Mol. Biol. 40:141–52
155. Takeshita N, Fujiwara H, Mimura H, Fitchen JH, Yamada Y, Sato F. 1995.Molecular cloning and characterization of S-adenosyl-L-methionine:scoulerine-9-O-methyltransferase from cultured cells of Coptis japonica. Plant Cell Physiol. 36:29–36
156. Tanahashi T, Zenk MH. 1990. Elicitor induction and characterization of microsomalprotopine-6-hydroxylase, the central enzyme in benzophenanthridine alkaloid biosyn-thesis. Phytochemistry 29:1113–22
157. Teuber M, Azemi ME, Namjoyan F, Meier AC, Wodak A, et al. 2007. Putrescine N-methyltransferases-a structure-function analysis. Plant Mol. Biol. 63:787–801
158. Uefuji H, Ogita S, Yamaguchi Y, Koizumi N, Sano H. 2003. Molecular cloning andfunctional characterization of three distinct N-methyltransferases involved in the caffeinebiosynthetic pathway in coffee plants. Plant Physiol. 132:372–80
159. Uefuji H, Tatsumi Y, Morimoto M, Kaothien-Nakayama P, Ogita S, Sano H. 2005.Caffeine production in tobacco plants by simultaneous expression of three coffee N-methyltrasferases and its potential as a pest repellant. Plant Mol. Biol. 59:221–27
160. Unterlinner B, Lenz R, Kutchan TM. 1999. Molecular cloning and functional expressionof codeinone reductase: the penultimate enzyme in morphine biosynthesis in the opiumpoppy Papaver somniferum. Plant J. 18:465–75
161. van der Fits L, Memelink J. 2000. ORCA3, a jasmonate-responsive transcriptional regu-lator of plant primary and secondary metabolism. Science 289:295–97
162. Vazquez-Flota F, De Carolis E, Alarco AM, De Luca V. 1997. Molecular cloningand characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don.Plant Mol. Biol. 34:935–48
163. Veau B, Courtois M, Oudin A, Chenieux JC, Rideau M, Clastre M. 2000. Cloning andexpression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus.Biochim. Biophys. Acta 1517:159–63
164. Viehweger K, Schwartze W, Schumann B, Lein W, Roos W. 2006. The Gα proteincontrols a pH-dependent signal path to the induction of phytoalexin biosynthesis inEschscholzia californica. Plant Cell 18:1510–23
165. Vom Endt D, Soares e Silva M, Kijne JW, Pasquali G, Memelink J. 2007. Identificationof a bipartite jasmonate-responsive promoter element in the Catharanthus roseus ORCA3transcription factor gene that interacts specifically with AT-hook DNA-binding proteins.Plant Physiol. 144:1680–89
166. von Schumann G, Gao S, Stockigt J. 2002. Vomilenine reductase–a novel enzyme cat-alyzing a crucial step in the biosynthesis of the therapeutically applied antiarrhythmicalkaloid ajmaline. Bioorg. Med. Chem. 10:1913–18
167. Walz C, Giavalisco P, Schad M, Juenger M, Klose J, Kehr J. 2004. Proteomics of cur-curbit phloem exudate reveals a network of defence proteins. Phytochemistry 65:1795–804
168. Warzecha H, Gerasimenko I, Kutchan TM, Stockigt J. 2000. Molecular cloning andfunctional bacterial expression of a plant glucosidase specifically involved in alkaloidbiosynthesis. Phytochemistry 54:657–66
169. Wege S, Scholz A, Gleissberg S, Becker A. 2007. Highly efficient virus-induced genesilencing (VIGS) in California poppy (Eschscholzia californica): an evaluation of VIGS as astrategy to obtain functional data from nonmodel plants. Ann. Bot. 100:641–49
768 Ziegler · Facchini
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ANRV342-PP59-29 ARI 29 March 2008 2:13
170. Weid M, Ziegler J, Kutchan TM. 2004. The roles of latex and the vascular bundle inmorphine biosynthesis in the opium poppy, Papaver somniferum. Proc. Natl. Acad. Sci.USA. 101:13957–62
171. Weiss D, Baumert A, Vogel M, Roos W. 2006. Sanguinarine reductase, a key enzyme ofbenzophenanthridine detoxification. Plant Cell Environ. 29:291–302
172. Wink M, Hartmann T. 1982. Localization of the enzymes of quinolizidine alkaloidbiosynthesis in leaf chloroplasts of Lupinus polyphyllus. Plant Physiol. 70:74–77
173. Winkel BS. 2004. Metabolic channeling in plants. Annu. Rev. Plant Biol. 55:85–107174. Winkler A, Hartner F, Kutchan TM, Glieder A, Macheroux P. 2006. Biochemical evi-
dence that berberine bridge enzyme belongs to a novel family of flavoproteins containinga bi-covalently attached FAD cofactor. J. Biol. Chem. 281:21276–85
175. Winkler A, Kutchan TM, Macheroux P. 2007. 6-S-Cysteinylation of bi-covalently at-tached FAD in berberine bridge enzyme tunes the redox potential for optimal activity.J. Biol. Chem. 282:24437–43
176. Xu D, Shen Y, Chappell J, Cui M, Nielsen M. 2007. Biochemical and molecular charac-terization of nicotine demethylase in tobacco. Physiol. Plant. 129:307–19
177. Yamamoto H, Katano N, Ooi A, Inoue K. 2000. Secologanin synthase which catalyzesthe oxidative cleavage of loganin into secologanin is a cytochrome P450. Phytochemistry53:7–12
178. Yamazaki Y, Sudo H, Yamazaki M, Aimi N, Saito K. 2003. Camptothecin biosyntheticgenes in hairy roots of Ophiorrhiza pumila: cloning, characterization and differential ex-pression in tissues and by stress compounds. Plant Cell Physiol. 44:395–403
179. Yun DJ, Hashimoto T, Yamada Y. 1992. Metabolic engineering of medicinal plants:transgenic Atropa belladonna with an improved alkaloid composition. Proc. Natl. Acad. Sci.USA. 89:11799–803
180. Zarate R, el Jaber-Vazdekis N, Medina B, Ravelo AG. 2006. Tailoring tropane alkaloidaccumulation in transgenic hairy roots of Atropa baetica by overexpressing the gene en-coding hyoscyamine 6β-hydroxylase. Biotechnol. Lett. 28:1271–77
181. Zhang L, Ding R, Chai Y, Bonfill M, Moyano E, et al. 2004. Engineering tropane biosyn-thetic pathway in Hyoscyamus niger hairy root cultures. Proc. Natl. Acad. Sci. USA 101:6786–91
182. Ziegler J, Diaz-Chavez ML, Kramell R, Ammer C, Kutchan TM. 2005. Comparativemacroarray analysis of morphine containing Papaver somniferum and eight morphinefree Papaver species identifies an O-methyltransferase involved in benzylisoquinolinebiosynthesis. Planta 222:458–71
183. Ziegler J, Voigtlander S, Schmidt J, Kramell R, Miersch O, et al. 2006. Comparativetranscript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis. Plant J. 48:177–92
184. Zulak KG, Cornish A, Daskalchuk TE, Deyholos MK, Goodenowe DB, et al. 2007. Genetranscript and metabolite profiling of elicitor-induced opium poppy cell cultures revealsthe coordinate regulation of primary and secondary metabolism. Planta 225:1085–106
The Role of Glutathione in Photosynthetic Organisms:Emerging Functions for Glutaredoxins and GlutathionylationNicolas Rouhier, Stephane D. Lemaire, and Jean-Pierre Jacquot � � � � � � � � � � � � � � � � � � � � �143
