ANRV342-PP59-29 ARI 29 March 2008 2:13

Alkaloid Biosynthesis:
Metabolism and Trafcking
Ziegler and Peter J. Facchini
Jorg
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4,

Canada; email: pfacchin@ucalgary.ca; joerg.ziegler@ucalgary.ca
by Washington State University on 11/01/11. For personal use only.

Annu. Rev. Plant Biol. 2008. 59:73569 Key Words

The Annual Review of Plant Biology is online at
plant.annualreviews.org
This articles doi:
10.1146/annurev.arplant.59.032607.092730
benzylisoquinoline alkaloids, cellular compartmentalization,
secondary metabolism, monoterpenoid indole alkaloids, tropane
alkaloids, metabolic engineering

Copyright  c 2008 by Annual Reviews. Abstract

All rights reserved
Alkaloids represent a highly diverse group of compounds that are
1543-5008/08/0602-0735$20.00 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 eld 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
 ANRV342-PP59-29 ARI 29 March 2008 2:13

sons. Unlike most other types of secondary

Contents
ALKALOID METABOLISM . . . . . . .
Alkaloid Biosynthetic Pathways . . .
Structure-Function Relationships
in Alkaloid Biosynthetic
Enzymes . . . . . . . . . . . . . . . . . . . . . .
REGULATION OF ALKALOID
BIOSYNTHETIC PATHWAYS . .
Transcriptome and Metabolome
Analyses . . . . . . . . . . . . . . . . . . . . . .
Gene Regulation and Signal
Transduction . . . . . . . . . . . . . . . . . .
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
metabolites, the many classes of alkaloids have
unique biosynthetic origins. Despite the di-
736
versity of metabolic pathways, several techni-
736
cal breakthroughs have recently contributed
to impressive advancements in our under-
standing of alkaloid biosynthesis. Recent
747
applications of genomics-based technolo-
gies, such as expressed sequence tag (EST)
748
databases, DNA microarrays, and proteome
analysis, have accelerated the discovery of new
748
components and mechanisms involved in the
assembly of alkaloids in plants. Our present
749

ability to investigate secondary metabolism

Transgenic Approaches . . . . . . . . . . . 749
from a combined biochemical, molecular, cel-
ALKALOID TRAFFICKING . . . . . . . 751
lular, and physiological perspective has im-
by Washington State University on 11/01/11. For personal use only.

Cell TypeSpecic Localization

of Alkaloid Biosynthetic
Enzymes . . . . . . . . . . . . . . . . . . . . . .
Subcellular Compartmentalization
of Alkaloid Biosynthetic
Enzymes . . . . . . . . . . . . . . . . . . . . . .
Enzyme Complexes and Metabolic
Channels. . . . . . . . . . . . . . . . . . . . . .
PERSPECTIVE . . . . . . . . . . . . . . . . . . . .
ALKALOID METABOLISM
Alkaloids are a diverse group of low-
molecular-weight, nitrogen-containing com-
EST: expressed pounds derived mostly from amino acids. As
sequence tag
secondary metabolites found in approximately
MIA: 20% of plant species, alkaloids are purported
monoterpenoid
to play a defensive role against herbivores and
indole alkaloid
pathogens. Owing to their potent biological
activity, many of the approximately 12,000
known alkaloids have been exploited as phar-
maceuticals, stimulants, narcotics, and poi-
proved our appreciation for the complex bi-
ology of alkaloid pathways. In this review, we
751
discuss recent advances in our understanding
of the metabolism and trafcking of alkaloids.
755
Alkaloid Biosynthetic Pathways
756 Monoterpenoid indole alkaloids. The
757 monoterpenoid indole alkaloids (MIA)
comprise a family of structurally and phar-
maceutically diverse alkaloids. Some of the
approximately 2,000 known compounds
widely used in medicine include vinblastine
for the treatment of cancer and ajmaline for
antiarrythmic heart disorders. The major
sources for these compounds are Catharanthus
roseus and Rauvola serpentina. MIAs are con-
densation products of a nitrogen-containing
indole moiety derived from tryptamine
and a monoterpenoid component derived
from the iridoid glucoside secologanin
(Figure 1). In the MIA biosynthetic pathway,

Figure 1
Biosynthesis of the monoterpenoid indole alkaloids vinblastine and ajmaline. Enzymes for which
corresponding molecular clones have been isolated are shown in bold. Abbreviations: AAE, acetylajmalan
esterase; ANAMT, acetylnorajmalan methyltransferase; Cyp72A1, secologanin synthase; Cyp76B6,
geraniol 10-hydroxylase; D4H, desacetoxyvindoline 4-hydroxylase; DAT, deacetylvindoline
4-O-acetyltransferase; DHVR, dihydrovomilenine reductase; LAMT, loganic acid methyltransferase;
MAT, minovincinine acetyltransferase; NAMT, norajmalan methyltransferase; NMT,
N-methyltransferase; PER, peroxidase; PNAE, polyneuridine aldehyde esterase; RG, raucaffricine
O--glucosidase; SBE, sarpagan bridge enzyme; SGD, strictosidine -d-glucosidase; STR, strictosidine
synthase; T16H, tabersonine 16-hydroxylase; TDC, tryptophan decarboxylase; VH, vinorine
hydroxylase; VR, vomilenine reductase; VS, vinorine synthase.
736 Ziegler Facchini
 ANRV342-PP59-29 ARI 29 March 2008 2:13

Geraniol

Cyp76B6 LAMT

7-Deoxyloganic acid Loganic acid Loganin L-Tryptophan

Cyp72A1 TDC
10-Hydroxygeraniol LAMT

7-Deoxyloganin Secologanin Tryptamine

10-Oxogeraniol 7-Deoxyloganetic acid STR

Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

SGD

16-Methoxytabersonine 16-Hydroxytabersonine Strictosidine aglycoside Strictosidine

by Washington State University on 11/01/11. For personal use only.

T16H

SBE PNAE

Polyneuridine aldehyde epi-Vellosimine

16-Methoxy-2,3-dihydro- Tabersonine Dehydrogeissoschizine
3-hydroxytabersonine VS

NMT
VH

Vomilenine Vinorine
Lochnericine 6,7-Dehydrominovincinine
VR
MAT
Desacetoxyvindoline

DHVR
D4H

1,2-Dihydrovomilenine Acetylnorajmaline
Hrhammericine 6,7-Dehydroechitovenine
ANAMT
AAE
MAT
Deacetylvindoline

DAT
Norajmaline Acetylajmaline
19-O-Acetylhrhammericine
NAMT AAE

Vindoline Cantharanthine
RG Ajmaline
PER

Raucaffricine
Vinblastine

www.annualreviews.org Alkaloid Biosynthesis 737

 ANRV342-PP59-29
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.
ARI 29 March 2008 2:13
tryptophan decarboxylase (TDC) converts
tryptophan to tryptamine; the gene encoding
this enzyme has been cloned from different
MIA-accumulating plants (25, 89, 178).
Incorporation experiments with isotopically
labeled glucose in C. roseus cell cultures
showed that the initial steps for the biosyn-
thesis of the monoterpenoid skeleton proceed
via the triose phosphate/pyruvate pathway
(22). Cognate cDNAs for 1-deoxy-d-xylulose
5-phosphate reductoisomerase and 2C-
methyl-d-erythritol 2,4-cyclodiphosphate
synthase were isolated from C. roseus and
the corresponding gene transcripts were
upregulated in MIA-producing cell cul-
tures (163). The rst committed step in
secologanin biosynthesis is catalyzed by
the cytochrome P450 monooxygenase
geraniol-10-hydroxylase (Cyp76B6), which
was puried and cloned from C. roseus (20).
Several steps in the secologanin pathway
have not been characterized, although the
availability of C. roseus EST databases (108)
has led to the recent discovery of loganic
acid methyltransferase (LAMT) (V. De Luca,
personal communication). The nal reaction
of this early branch of the pathway involves an
oxidative C-C cleavage catalyzed by a second
P450 monooxgenase (Cyp72A1), resulting in
the conversion of loganin to secologanin (70).
Strictosidine, the common precursor to
all MIAs, is formed by a Pictet-Spengler
condensation of tryptamine and secologanin
(Figure 1). Strictosidine synthase (STR)
from R. serpentina was the rst cDNA in-
volved in alkaloid biosynthesis to be cloned
and functionally expressed in microorgan-
isms. STR orthologs from C. roseus and Ophi-
orrhiza pumila have also been isolated (79,
99, 178). The strictosidine glucose moiety
is subsequently removed by a family 1 glu-
cosyl hydrolase enzyme, strictosidine -d-
glucosidase (SGD); a cDNA for SGD was
functionally characterized from C. roseus and
R. serpentina. SGD from R. serpentina shows
exclusive substrate specicity toward stricto-
sidine and closely related analogs (46, 50).
The strictosidine-derived aglycone is subse-
738 Ziegler Facchini
quently converted via several unstable in-
termediates to dehydrogeissoschizine, which
represents a key branchpoint intermediate
that leads to several diverse MIA pathways.
The branch pathways proceeding through
tabersonine and polyneuridine aldehyde have
been characterized most thoroughly. Other
branch pathways, such as that leading to
catharanthine, remain poorly understood.
Hydroxylation of tabersonine at position
16 represents the rst reaction leading
to vindoline (Figure 1). The responsi-
ble enzyme, tabersonine 16-hydroxylase
(T16H, Cyp71D12), belongs to the P450
monooxygenase family, and the correspond-
ing cDNA was identied from a subset
encoding P450-dependent enzymes induced
by the treatment of C. roseus cultures with
light (140). O-Methylation at position 16,
reduction of the 2,3-double bond, and
N-methylation result in the conversion of 16-
hydroxytabersonine to desacetoxyvindoline.
Although cDNAs have not been isolated for
the enzymes that participate in these steps,
the N- and O-methylating activities have
been characterized in plant extracts (27, 37).
The 2-oxoglutaratedependent dioxygenase
desacetoxyvindoline 4-hydroxylase (D4H)
produces desacetylvindoline, which is further
converted by the acetyl CoAdependent de-
sacetylvindoline 4-O-acetyltransferase (DAT)
to vindoline (150, 162). Both enzymes have
been 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 variety
of plant secondary metabolic pathways.
Ultimately, vindoline is coupled to catha-
ranthine by a nonspecic peroxidase to
form vinblastine (145). A cDNA with 78%
amino acid identity to DAT was obtained
from C. roseus and encodes minovincinine
19-O-acetyltransferase, yielding echitove-
nine, which is a second pathway originating
from the tabersonine branch (80). Whereas
 ANRV342-PP59-29 ARI 29 March 2008 2:13
minovincinine acetyltransferase (MAT) shows
residual activity toward the DAT substrate,
deacetoxyvindoline, DAT accepts only its
natural substrate.
The polyneuridine aldehyde branch of the
MIA pathway is initiated by the formation of
a 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). The
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
resulting polyneuridine aldehyde undergoes
desterication to yield epi-vellosimine, which
is acetylated to form vinorine. Recombi-
by Washington State University on 11/01/11. For personal use only.
nant polyneuridine aldehyde esterase (PNAE)
from R. serpentina shows esterase activity only
to polyneuridine aldehyde and not to struc-
turally related esters, and is a member of the
/ hydrolase superfamily (29). With approx-
imately 30% identity on the amino acid level
to DAT and MAT, vinorine synthase (VS)
from R. serpentina also belongs to the BAHD
family of acyltransferases (11). Acetylajmaline
is 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 have
been biochemically characterized as the P450-
dependent monooxygenase vinorine hydrox-
ylase (VH), which yields vomilenine, and the
NADPH-dependent reductases vomilenine
reductase and 1,2-dehydrovomilenine reduc-
tase (39, 44, 166). Acetylajamaline esterase
(AAE) ultimately hydrolyzes the 17-O-acetyl
group to produce ajmaline. Active recombi-
nant AAE, which could be produced only via
a virus expression system in Nicotiana ben-
thamiana, shows slight substrate preference
for acetylajmaline compared with norajma-
line. The latter compound might represent
an intermediate in a parallel ajamaline biosyn-
thetic pathway in which N-methylation occurs
after deesterication (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-
though the glucosylating activity has not been
reported, researchers found a cDNA encod-
ing the enzyme raucaffricine O--glucosidase
BIA:
(RG), responsible for deglycosylation of rau- benzylisoquinoline
caffricine (168). RG shares 60% amino acid alkaloid
identity with SGD and accepts strictosidine as Sanguinarine: an
a substrate, whereas SGD is not active against antimicrobial
raucaffricine. benzylisoquinoline
alkaloid sometimes
used in oral hygiene
Benzylisoquinoline alkaloids. Benzyliso-
products
quinoline alkaloids (BIA) represent approxi-
mately 2,500 elucidated structures and possess
potent pharmacological properties. The most
prominent compounds are the narcotic
analgesic morphine, the cough suppressant
codeine, the muscle relaxant papaverine, and
the antimicrobial agents sanguinarine and
berberine. Collectively, these alkaloids occur
mainly in the Papaveraceae, Ranunculaceae,
Berberidaceae, and Menispermaceae; Papaver
somniferum (opium poppy), Eschscholzia cali-
forncia, Thalictrum species, and Coptis japonica
are the most extensively investigated species.
BIA biosynthesis, which fundamentally
involves the condensation of two tyrosine
derivatives, begins with the decarboxylation
of tyrosine to tyramine or of dihydroxypheny-
lalanine to dopamine by tyrosine decarboxy-
lase (TYDC). TYDC constitutes a large gene
family; approximately 15 members are found
in opium poppy (33). Dopamine is the pre-
cursor for the isoquinoline moiety, whereas
4-hydroxyphenylacetaldehyde, resulting from
the deamination of tyramine, is incorporated
as the benzyl component (Figure 2). As is
the case in MIA biosynthesis, BIA conden-
sation is a Pictet-Spenglertype reaction and
is catalyzed by the rst committed step of
the pathway, norcoclaurine synthase (NCS).
The enzyme has been puried from Thalic-
trum avum and corresponding cDNAs have
been isolated and functionally characterized
from opium poppy and T. avum (87, 136,
137). NCS is related to the pathogenesis
related protein (PR) 10 and Bet v 1 allergen
protein families. However, homologous PR10
proteins from opium poppy are not catalyt-
ically active. Biochemical characterization of
www.annualreviews.org Alkaloid Biosynthesis 739
 ANRV342-PP59-29 ARI 29 March 2008 2:13

TYDC NCS
L-Dopa Dopamine

(S)-Norcoclaurine

4-Hydroxyphenylacetaldehyde 6OMT
Papaverine
CoOMT

Palmatine Columbamine (S)-Coclaurine

CNMT

Cyp719A1
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

Cyp80A1 Bisbenzylisoquinoline
alkaloids
Cyp80B3
by Washington State University on 11/01/11. For personal use only.

(S)-Canadine (S)-Tetrahydrocolumbamine (S)-3'-Hydroxy- (S)-N-Methylcoclaurine

N-methylcoclaurine

STOX SOMT 4'OMT

DRS DRR
BBE

Berberine (S)-Scoulerine (S)-Reticuline 1,2-Dehydroreticuline (R)-Reticuline

CFS 7OMT Cyp719B1

SalR

Cyp719A2
(S)-Stylopine (S)-Cheilanthifoline Laudanine
Salutaridinol Salutaridine

TNMT SalAT

MSH
(S)-cis-N-Methylstylopine Protopine Thebaine
Salutaridinol-7-O-acetate

P6H

Spontaneous
Dihydrosanguinarine 6-Hydroxyprotopine Neopinone Oripavine Morphinone

DBOX Spontaneous COR

COR

Sanguinarine Codeinone Codeine Morphine

740 Ziegler Facchini

 ANRV342-PP59-29 ARI 29 March 2008 2:13

recombinant T. avum NCS has been ac-
complished using a continuous enzyme assay
based on circular dichroism spectroscopy
that follows the generation of the enzymes
chiral product. These studies revealed a
reaction mechanism that involves a two-
step cyclization with a direct electrophilic
aromatic substitution (91). Recently, a
second enzyme capable of producing only
the (S )-norcoclaurine enantiomer was iso-
lated from C. japonica; this enzyme displays
sequence similarity to 2-oxoglutarate de-
pendent dioxygenases (102). However, this
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
cludes catechols and phenylpropanoids in
addition to various BIA derivatives (42).
Coclaurine N-methyltransferases (CNMT)
have been cloned from opium poppy and
C. japonica and are more closely related to
S-adenosyl-L-methionine (SAM)-dependent
cyclopropane fatty acid synthases than other
N-methyltransferases (19, 35). The hydroxy-
lation of N-methylcoclaurine is catalyzed by
a P450 monooxygenase classied in the Cyp
subfamily 80B (65, 68, 125, 138).
(S )-Reticuline is the central pathway in-
termediate from which most BIA structural

enzyme does not possess a 2-oxoglutarate- types are derived. Only the dimeric bisben-
binding domain and requires ferrous ions, zylisoquinoline alkaloids are not produced
rather than 2-oxoglutarate or oxygen, for via (S )-reticuline. In this pathway, the cy-
by Washington State University on 11/01/11. For personal use only.

activity. Remarkably, two proteins in two tochrome P450 enzyme Cyp80A1 catalyzes
different protein families can catalyze the the regio- and stereoselective oxidative C-
same reaction in vitro. Nevertheless, the O phenol coupling of the reticuline pre-
relative participation of each enzyme in BIA cursor N-methylcoclaurine to produce the
biosynthesis in vivo must still be determined. bisbenzylisoquinoline alkaloid berbamunine
The conversion of (S )-norcoclaurine to (78). Another exception where reticuline
(S )-reticuline involves O-methylation at posi- does not represent an intermediate com-
tion 6, N-methylation, 3 -hydroxylation, and pound might be the biosynthesis of the sim-
a second 4 -O-methylation (Figure 2). Nor- ple N-demethylated BIA papaverine. The re-
coclaurine 6-O-methyltransferase (6OMT) cent discovery of a 7-O-methyltransferase
and 3 -hydroxy-N-methylcoclaurine 4 -O- specic for norreticuline (N7OMT, nor-
methyltransferase (4 OMT) are both class II reticuline 7-O-methyltransferase), but not
O-methyltransferases that display strict re- N-methylated analogs, suggests that (S )-
giospecicity. Cognate cDNAs have been ob- reticuline precursors might enter the path-
tained for each enzyme from opium poppy way to papaverine (S. Pienkny, J. Ziegler
and C. japonica (107, 121, 182). Additional & W. Brandt, manuscript in prepara-
6OMT homologs from Thalictrum tubero- tion). A second O-methyltransferase that
sum were functionally characterized and ex- acts on reticuline and catalyzes the forma-
hibit a broader substrate specicity that in- tion of laudanine was previously isolated

Figure 2
Biosynthesis of the benzylisoquinoline alkaloids berberine, morphine, and sanguinarine. Enzymes for
which corresponding molecular clones have been isolated are shown in bold. Abbreviations: 4 OMT,
3 -hydroxy-N-methylcoclaurine 4 -O-methyltransferase; 6OMT, norcoclaurine 6-O-methyltransferase;
7OMT, reticuline 7-O-methyltransferase; BBE, berberine bridge enzyme; CFS, cheilanthifoline
synthase; CNMT, coclaurine N-methyltransferase; CoOMT, columbamine O-methyltransferase; COR,
codeinone reductase; Cyp719A1, canadine synthase; Cyp719A2, stylopine synthase; Cyp719B1,
salutaridine synthase; Cyp80A1, berbamunine synthase; Cyp80B3, N-methylcoclaurine 3 -hydroxylase;
DBOX, dihydrobenzophenanthridine oxidase; DRR, 1,2-dehydroreticuline reductase; DRS,
1,2-dehydroreticuline synthase; MSH, N-methylstylopine 14-hydroxylase; NCS, norcoclaurine synthase;
P6H, protopine 6-hydroxylase; SalAT, salutaridinol 7-O-acetyltransferase; SalR, salutaridine:NADPH
7-oxidoreductase; SOMT, scoulerine 9-O-methyltransferase; STOX, (S )-tetrahydroxyprotoberberine
oxidase; TNMT, tetrahydroprotoberberine cis-N-methyltransferase; TYDC, tyrosine decarboxylase.

www.annualreviews.org Alkaloid Biosynthesis 741

 ANRV342-PP59-29
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.
ARI 29 March 2008 2:13
from opium poppy (121). (R,S )-Reticuline
7-O-methyltransferase (7OMT) does not ac-
cept N-demethylated BIA substrates, but is ac-
tive toward phenolic compounds.
A major branch pathway that gives rise
to many BIA classes begins with the forma-
tion of (S )-scoulerine by the berberine bridge
enzyme (BBE) (Figure 2). This enzyme has
been cloned from several sources (28, 36, 138)
and was recently characterized thoroughly
(174, 175). BBE belongs to a novel family
of avoproteins that possess two covalent at-
tachment sites for avin 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 value
higher than that observed for other avo-
proteins, thereby facilitating hydride abstrac-
tion of (S )-reticuline. This step represents the
rst half reaction toward the conversion to
(S )-scoulerine. The biosynthesis of ben-
zophenanthridine alkaloids is initiated by the
formation of two methylenedioxy bridges
resulting in (S )-cheilanthifoline and (S )-
stylopine (Figure 2). Both reactions are cat-
alyzed by P450-dependent monooxygenases
and two cDNAs coding for stylopine syn-
thase have been cloned from E. californica and
classied as Cyp719A2 and Cyp719A3 (67).
Both recombinant proteins show the same re-
giospecicity for methylendioxy bridge for-
mation, but Cyp719A2 only converts (S )-
cheilanthifoline to (S )-stylopine. In contrast,
Cyp719A3 also accepts compounds without
a 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, a
cDNA encoding TNMT has been isolated
and functionally characterized from opium
poppy (86). The enzyme shows a narrow sub-
strate range in that it converts only tetrahy-
droprotoberberine alkaloids with dimethoxy
or methylenedioxy functional groups at C2/3
and C9/10, respectively. TNMT is also one
of only a few plant enzymes able to cat-
alyze the formation of quaternary ammo-
742 Ziegler Facchini
nium compounds. Subsequent hydroxylation
by (S )-cis-N-methylstylopine 14-hydroxylase
yields protopine, which is further hydrox-
ylated by protopine 6-hydroxylase to dihy-
drosanguinarine. Both enzymes have been de-
tected in protopine alkaloid-containing cell
cultures, and their characterization suggests
that they are P450 monooxygenases (132,
156). Dihydrobenzophenanthridine oxidase,
which converts dihydrosanguinarine to san-
guinarine, has been puried from Sanguia-
naria canadensis (7).
An alternative branch for the metabolism
of (S )-scoulerine in some species involves the
formation of (S )-tetrahydrocolumbamine by
scoulerine 9-O-methyltransferase (SOMT)
(Figure 2) (155). The cDNA encoding
SOMT from C. japonica was the rst re-
ported clone for an O-methyltransferase
specically implicated in BIA metabolism.
All O-methyltransferases in the BIA pathway
exhibit considerable homology. Tetrahydro-
columbamine is converted to columbamine,
which is methylated by columbamine
O-methyltransferase (CoOMT) to yield
palmatine (106). Recombinant CoOMT is
active only against protoberberine alkaloid
substrates, such as scoulerine or tetrahy-
drocolumbamine, but not against other
BIA derivatives. The subsequent formation
of a methylenedioxy bridge is catalyzed
by canadine synthase, a P450 enzyme
that belongs to the Cyp719A family (68).
Cyp719A1 displays high substrate specicity
for tetrahydrocolumbamine and does not
accept columbamine. As such, a parallel path
to berberine via this columbamine is unlikely.
(S )-Canadine, or (S )-tetrahydroberberine
as it is also called, is oxidized by either (S )-
canadine oxidase or (S )-tetrahydroberberine
oxidase (STOX), which catalyze the same
reaction but show substantially different
biochemical properties.
Whereas all pathways downstream of reti-
culine begin with the (S )-epimer, conversion
to the (R )-epimer of reticuline is a required
entry step into the morphinan alkaloid biosyn-
thetic pathway (Figure 2). The epimerization
 ANRV342-PP59-29 ARI 29 March 2008 2:13
of reticuline is a two-step process that in-
volves the oxidation of (S )-reticuline by 1,2-
dehydroreticuline synthase and the reduction
of 1,2-dehydroreticuline to (R )-reticuline.
Both steps have been characterized biochem-
ically and the enzymes have been partially pu-
ried (23, 63). Intramolecular carbon-carbon
phenol coupling between C2 of the benzyl
and C4a of the isochinoline moiety leads
to the formation of salutaridine. This en-
zyme belongs to the P450 monooxygenase
family (48). The gene encoding salutaridine
synthase (SalSyn) was recently cloned from
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
opium poppy on the basis of its higher expres-
sion in morphine-containing Papaver species
and the enzyme was then functionally char-
by Washington State University on 11/01/11. For personal use only.
acterized (A. Gesell, F.C. Huang, J. Ziegler
& T.M. Kutchan, manuscript in preparation).
The SalSyn protein shows high homology
to the methylenedioxybridge-forming P450-
dependent enzymes, and was classied as
Cyp719B1. The next step in the pathway is
catalyzed by salutaridine reductase (SalR); a
cognate cDNA was obtained via the same
approach used for SalSyn (183). Functional
characterization of the recombinant enzyme
showed the stereospecic reduction of the
keto group to 7(S )-salutaridinol, which is also
reported for the puried enzyme from opium
poppy (49). The enzyme belongs to the fam-
ily of short chain dehydrogenases/reductases
(SDR), but unlike many other enzymes in
this family it exhibits a higher molecular
weight and is monomeric. The stereospecic
reduction of salutaridine is required for the
next step, which is catalyzed by salutaridi-
nol 7-O-acetyltransferase (SalAT). This en-
zyme specically acetylates the 7(S )-epimer
of salutaridinol to salutaridinol-7-O-acetate
(82). With considerable sequence homology
to the acetylating enzymes from the MIA
pathway, SalAT also belongs to the BAHD
family of acetyltransferases (52). The in-
troduced acetyl group is eliminated sponta-
neously, leading to the formation of an oxide
bridge between C-4 and C-5 to yield thebaine,
the rst pentacyclic alkaloid of the pathway
(82). The nal steps toward the biosynthesis of
morphine consist of two demethylations and
one reduction. Both the demethylation from
thebaine to neopinone, which isomerises to
SDR: short chain
codeinone, and from codeine to morphine are dehydrogenases/
not yet understood and no enzymes capable of reductases
catalyzing either reaction have been detected. Calystegines:
Codeinone reductase has been puried and nortropane alkaloids
cloned from opium poppy, and, in contrast thought to serve as
to SalR, belongs to the aldo-keto reductase nutritional sources
for soil
(AKR) family (83, 160).
microorganisms
Tropane alkaloids and nicotine. Tropane
alkaloids are an important class of plant-
derived anticholinergic compounds, such as
hyoscyamine and scopolamine, that occur
in several Hyoscyamus, Atropa, and Datura
species. Calystegines, which function as se-
lective glucosidase inhibitors, are more widely
spread than hyoscyamine and scopolamine in
the plant kingdom and occur mainly in the
Solanaceae and Convolvulaceae (30). Nico-
tine, the active principle in Nicotiana species,
acts on nicotinic acetylcholine receptors to
cause a variety of physiological effects, in-
cluding addiction. Tropane alkaloid and nico-
tine biosynthesis begin with the methyla-
tion of putrescine to N-methylputrescine
by putrescine N-methyltransferase (PMT)
(Figure 3). The isolation of the cDNA en-
coding PMT from tobacco was one of the
rst examples of the successful integration
of metabolite and gene expression proles
as a strategy to isolate cDNAs implicated
in plant secondary metabolite biosynthe-
sis (61). Homologous cDNAs from other
plants that produce tropane alkaloids, such
as Hyoscyamus niger, Atropa belladonna, and
Solanum tuberosum, have also been isolated
(147, 153, 157). The second step in the
pathway is the oxidative deamination of N-
methylputrescine to 4-methylaminobutanal
by N-methylputrescine oxidase (MPO). This
enzyme 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 MPO
cDNA from tobacco (59). The central inter-
mediate N-methyl-1 -pyrrolium cation for
www.annualreviews.org Alkaloid Biosynthesis 743
 ANRV342-PP59-29 ARI 29 March 2008 2:13

ODC

Putrescine

Ornithine PMT Arginine

NAD
biosynthesis
intermediate 3,6-Dihydronicotinone
N-Methylputrescine
MPO

Spontaneous

4-Methylaminobutanal N-Methyl-1-pyrrolium
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

cation Nicotine

Figure 3
Cyp82E4
by Washington State University on 11/01/11. For personal use only.

Biosynthesis of the
tropane alkaloids
hyoscyamine and TR-II
scopolamine, the
calystegines, and
nicotine. Hygrine Tropinone Pseudotropine Nornicotine
Molecular clones TR-I
have been isolated
for all enzymes
shown.
Abbreviations:
Cyp80F1, littorine
mutase/
monooxygenase; Tropine Littorine
Cyp82E4, nicotine
N-demethylase; Cyp80F1
H6H, hyoscyamine
6-hydroxylase;
MPO,
Calystegine A3
methylputrescine
oxidase; ODC,
ornithine Hyoscyamine Hyoscyamine aldehyde
decarboxylase;
PMT, putrescine H6H
N-
methyltransferase;
TR-I, tropinone Calystegine B1 Calystegine B2
reductase I; TR-II,
tropinone
reductase II. Scopolamine

tropane alkaloid and nicotine biosynthesis
results from the spontaneous cyclization of
4-methylaminobutanal. For nicotine, the
cation is condensed with nicotinic acid to form
3,6-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 derivative
owing to its role as the precursor of the car-
cinogen N  -nitrosonornicotine. On the basis
of the differential abundance of transcripts
corresponding to several P450-encoding
cDNAs in tobacco varieties acccumulating

744 Ziegler Facchini

 ANRV342-PP59-29 ARI 29 March 2008 2:13

nicotine and nornicotine, a clone for nico- phenylalanine-derived (R )-phenyllactate to

tine N-demethylase (NND) was identied
and classied as Cyp82E4 (144, 176). Re-
cently, Gavilano & Siminszky (45) described
an additional cDNA with NND activ-
ity (Cyp82E5v2) that shows distinct tissue-
specic expression in tobacco.
Although enzymatic activities have not
been demonstrated, the condensation of
N-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 is
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
yield littorine, which undergoes rearrange-
ment to form hyoscyamine. Recently, Li and
coworkers (84) used functional genomics
based on virus-induced gene silencing to iso-
late a cytochrome P450 involved in littorine
rearrangement from H. niger. This enzyme
was classied as Cyp80F1 and it catalyzes the
oxidation of (R )-littorine with rearrangement
to hyoscyamine aldehyde. Finally, the epox-
idation of hyoscyamine yields scopolamine
via a two-step reaction: 6-hydroxylation of
the tropane ring followed by intramolecular

subsequently reduced to intermediates that epoxide formation. The reaction is catalyzed

lead to hyoscyamine or calystegines depend- by a 2-oxoglutaratedependent dioxygenase,
ing on the stereochemistry of the reduction. hyoscyamine 6-hydroxylase (H6H), the
by Washington State University on 11/01/11. For personal use only.

Tropinone reductase I (TR-I) catalyzes the cDNA for which has been cloned from
reduction of tropinone to tropine, which several tropane alkaloid-containing plants
possesses a 3 conguration. In contrast, (88, 95, 154).
tropinone reductase II (TR-II) catalyzes the
formation of -tropine, which has a 3 con- Purine alkaloids. Purine alkaloids are
guration. Cognate cDNAs show that both derived from purine nucleotides. The
NADPH-dependent enzymes belong to the most prominent members of this class of
SDR family and exhibit considerable amino compounds are theobromine and caffeine.
acid sequence similarity (76, 110, 111, 113). Purine alkaloid biosynthesis begins with
Domain-swapping experiments and site- the N-methylation of xanthosine at position
directed mutagenesis led to the identication 7 by 7-methylxanthosine synthase (XRS),
of the substrate-binding domain responsible which is also known as xanthosine 7-N-
for the opposite stereospecicity of each methyltanferase (XMT) (Figure 4). Cognate
reductase. Tropine then condenses with the cDNAs for XMT have been isolated from
Figure 4
Biosynthesis of the
purine alkaloids
caffeine and
XMT theobromine.
Molecular clones
H2O Ribose have been isolated
7-Methylxanthine for all enzymes
shown.
Abbreviations: CS,
caffeine synthase;
Xanthosine 7-Methylxanthosine MXMT CS/TS
DXMT, 1,7-
dimethylxanthosine
methyltransferase;
MXMT,
DXMT 7-methylxanthine
CS methyltransferase;
TS, theobromine
synthase, XMT,
Caffeine Theobromine xanthosine 7-N-
methyltanferase.

www.annualreviews.org Alkaloid Biosynthesis 745

 ANRV342-PP59-29 ARI 29 March 2008 2:13

Coffea arabica (103, 158). The second step in
the purine alkaloid pathway is the hydrolysis
of xanthosine to 7-methylxanthine, although
the responsible enzyme has not yet been
puried or characterized. However, detailed
structural investigations of XMT suggest a
coupled reaction for 7-N-methylation and
nucleoside cleavage catalyzed by a single
enzyme (97). Several cDNAs with different
substrate specicities have been obtained
from tea, coffee, or cacao that catalyze the next
two N-methylations. Caffeine synthase (CS)
performs dual methylations, rst at position
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
lation at position N-1 (117, 158). These
N-methyltransferases possess more then
80% amino acid similarity and phylogenetic
analysis suggests that they are more closely
related to carboxyl-methyltransferases than
to other N-methyltransferases.
Pyrrolizidine alkaloids. Pyrrolizidine alka-
loids are produced constitutively in various
plants as a defense against hebivores and as
a component of the complex chemical ecol-
ogy between plants and animals (55). These
alkaloids are composed of a necine base and

3 to form theobromine, and then at position one or more necic acids; the latter are re-
1 to yield caffeine (74, 104). Several cDNAs sponsible for their structural diversity. Necine
encoding related enzymes that catalyze only biosynthesis begins with the condensation of
by Washington State University on 11/01/11. For personal use only.

single N-methylations have been isolated, in-
cluding 7-methylxanthine methyltransferase
(MXMT1 and MXMT2) and theobromine
synthase (TS), which methylate position N-3,
and 3,7-dimethylxanthine methyltransferase
(DXMT), which catalyzes the nal methy-
spermidine and putrescine to form homosper-
midine by homospermidine synthase (HSS)
(116) (Figure 5). The reaction mechanism
is identical to that of deoxyhyposine syn-
thase (DHS), which transfers the aminobutyl
moiety to a lysine residue of the eukaryotic

Spermidine

HSS
Homospermidine

Putrescine

Necine base

Figure 5
Biosynthesis of
pyrrolizidine
alkaloids. A
molecular clone
has been isolated
for only one
biosynthetic
enzyme.
Abbreviation: HSS,
homospermidine Senecionine Heliotrine
synthase. Clivorine

746 Ziegler Facchini

 ANRV342-PP59-29 ARI 29 March 2008 2:13
initiation factor 5A precursor protein. Ad-
ditionally, both enzymes share extensive se-
quence homology, suggesting the recruitment
of HSS from DHS, with subsequent opti-
mization to facilitate the role of HSS in sec-
ondary metabolism. Phylogenetic analysis of
HSS and DHS homologs from angiosperms
that represent several unrelated plant lineages
suggests at least four independent recruitment
events for HSS (128). The remaining compo-
nents of pyrrolizidine alkaloid metabolism are
not well understood.
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
Structure-Function Relationships
in Alkaloid Biosynthetic Enzymes
by Washington State University on 11/01/11. For personal use only.
The biomimetic exploitation of enzymes, es-
pecially those that display strict stereospeci-
city, 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 specicity of biosynthetic
enzymes. Recent attention has focused on elu-
cidating the reaction and substrate binding
mechanisms of key alkaloid biosynthetic en-
zymes. The structures of several enzymes have
been determined and substrate-binding pock-
ets have been characterized.
The rst detailed structures of enzymes in-
volved in plant alkaloid metabolism were ob-
tained by X-ray crystallography for TR-I and
TR-II, which show strict product specici-
ties and catalyze either tropine or -tropine
formation. Site-directed mutagenesis of se-
lected amino acids in the substrate-binding
domains resulted in a mutual conversion of
the product specicities for TR-I and TR-II
(114). A homology-based model of the three-
dimensional structure of PMT was generated
on the basis of the crystal structure of the pu-
tative ancestral enzyme spermidine synthase,
which suggested the identity of amino acids
responsible for the distinction between these
two enzymes (157).
Most X-ray crystallographic data have
been obtained for enzymes implicated in
MIA metabolism, especially those involved in
ajmaline biosynthesis. The structure of STR
from R. serpentina revealed a novel six-bladed
-propeller protein and site-directed muta-
Homology-based
genesis showed the importance of a key gluta- model: an
mate residue in catalysis (93). The structure of atomic-resolution
the enzyme complexed with the natural sub- protein structure
strates tryptamine and secologanine provided based on amino acid
sequence
insight into the architecture of the substrate
binding sites, which could then be engineered Substrate docking:
a mathematical
to accommodate several different tryptamine
procedure used to
and secologanin analogs (18, 90). The three- identify ligand-
dimensional structure of SGD revealed the binding sites in
expected (/)8 barrel fold typical for family proteins
1 glycosidases (9). Site-directed mutagenesis
identied amino acid residues required for
the catalytic mechanism and the binding site
architecture. Ruppert and coworkers (133)
reported preliminary X-ray crystallographic
data for the homologous enzyme RG. A com-
parision of the structures of SGD and RG will
be interesting because both enzymes occur in
the same organism and catalyze the same type
of reaction, but differ in substrate specicity.
VS was the rst BAHD acyltransferase family
enzyme for which a three-dimensional struc-
ture was obtained (92). The protein consists
of two major domains of similar size con-
nected by a large loop. Whereas site-directed
mutagenesis and structural analysis conrm
the importance of a HXXXD motif for
catalysis, a second highly conserved motif in
members of the BAHD family is distant from
the active site and is considered essential for
proper folding rather than catalysis. Molec-
ular modeling and site-directed mutagenesis
established the classication of PNAE as a
member of the / hydrolase superfamily
and identied the amino acid residues that
participate in the catalytic triad (96).
The structure of SalR (involved in BIA
biosynthesis) was analyzed by homology mod-
elling (47). An additional helix near the cat-
alytic site not found in multimeric SDR-type
enzymes appears to contribute to substrate
specicity. Substrate docking studies and site-
directed mutagenesis revealed several amino
acids implicated in the binding of the morphi-
nan alkaloid precursor salutaridine. It will be
www.annualreviews.org Alkaloid Biosynthesis 747
 ANRV342-PP59-29
Laticifer: a plant
cell or vessel that
contains latex
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.
ARI 29 March 2008 2:13
interesting to compare the substrate-binding
site of SalR with that of COR, which specif-
ically reduces codeinone, an alkaloid with a
similar structure, to salutaridine. The po-
tential of homology modelling and substrate
docking is well demonstrated in studies in-
volving the O-methyltransferases implicated
in 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 novel
O-methyltransferase precluded norcoclaurine
as a likely substrate, and rather suggested
the O-methylation of norreticuline at posi-
tion 7. Experimental evidence has conrmed
this prediction and led to the identication of
N7OMT. Furthermore, comparisons of the
6OMT and N7OMT models identied the
amino acids responsible for the distinct sub-
strate specicity.
Recently, McCarthy & McCarthy (97) elu-
cidated the structures of the two highly ho-
mologous N-methyltransferases involved in
caffeine biosynthesis, XMT and DXMT. Co-
crystallization with the cofactor and either
substrate, coupled with structural compar-
isons, revealed critical elements for substrate
selectivity and catalysis.
REGULATION OF ALKALOID
BIOSYNTHETIC PATHWAYS
Transcriptome and
Metabolome Analyses
Large-scale expression analyses have recently
begun to provide a broad picture of the gene
expression proles associated with alkaloid
biosynthesis. As a prerequisite for these stud-
ies, several EST sequencing projects have
been reported and the number of sequences
associated with alkaloid-producing plants
continues to increase. The rst database con-
sisted of 4,500 ESTs from the laticifers of
opium poppy (127). Further EST projects
involving this plant have yielded a total of
25,000 ESTs from various tissues including
seedlings, stems, roots, and elicited cell cul-
748 Ziegler Facchini
tures (182, 183, 184). A recent oral genome
project contributed 11,000 ESTs from the
BIA-accumulating plant E. californica (17), and
the release of a C. japonica database has also
been announced (69, 75). NapGen, a consor-
tium of Canadian investigators, has sequenced
more than 400,000 ESTs from a wide vari-
ety of plants that produce health-related sec-
ondary compounds. In this project, 46,000
ESTs were sequenced from cell cultures
of eight BIA-accumulating species including
E. californica, T. avum, 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 of
the MIA-accumulating plant C. roseus (108).
Large-scale sequencing projects for tropane
alkaloid-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 underground
organs are a good source to identify cDNAs
implicated in calystegine biosynthesis (131). A
sequencing project targeting tropane alkaloid
metabolism yielded 2,300 ESTs from roots of
H. niger (84). In a similar effort to investigate
the transcriptional regulation associated with
the chemical composition of coffee, Lin and
coworkers (85) reported 47,000 ESTs from
Coffea canephora at different stages of seed
development.
Large-scale transcriptome and metabolite
proling has been performed in opium poppy
cell cultures treated with a fungal elicitor
(184), and in C. roseus and tobacco cell cultures
treated with methyl jasmonate (51, 129). In all
cases, researchers observed a coordinated in-
crease in the expression of genes implicated
in alkaloid metabolism. Moreover, profound
changes in the level of gene transcripts en-
coding primary metabolic enzymes also oc-
curred. Interestingly, transcripts involved in
SAM recycling increased in all three sys-
tems, indicating a high demand for this cofac-
tor in the modication of alkaloid backbone
structures. As expected, genes implicated in
 ANRV342-PP59-29 ARI 29 March 2008 2:13
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, suggesting
that the cells have a constitutive and suf-
cient capacity for nitrogen assimilation even
during periods of increased demand (184).
However, substantial changes occurred in the
levels of primary metabolites involved in the
production of energy molecules potentially
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
required by enzymes and other components
required by the induction of alkaloid biosyn-
thesis (184). In C. roseus, the construction
by Washington State University on 11/01/11. For personal use only.
of correlation networks that integrate tran-
scriptomic and metabolomics data revealed
a correlation between the expression of al-
kaloid biosynthetic genes and corresponding
metabolic products (129).
Gene Regulation
and Signal Transduction
Progress has continued on the identication
of promoter elements and transcription fac-
tors involved in the regulation of several MIA
biosynthetic genes (165). The recent applica-
tion of a genomics approach based on DNA
macroarrays constructed from ESTs has led to
the isolation of the rst transcription factor
putatively involved in the regulation of BIA
metabolism. Transcripts that encode a subset
of 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, a
WRKY transcription factor, was shown by
RNAi and overexpression analysis to specif-
ically regulate the expression of BIA biosyn-
thetic genes (75). With respect to early signal
transduction events, considerable effort has
been focused on events associated with the in-
duction of alkaloid metabolism in E. californica
cell cultures, which accumulate benzophenan-
thridine alkaloids such as sanguinarine in re-
sponse to treatment with a fungal elicitor.
Two different signal transduction pathways
were identied. One cascade is jasmonate-
dependent and responds to high elicitor
Metabolomics: the
concentrations. The other is jasmonate- systematic study of
independent and is triggered by low elic- the chemical
itor concentrations (38). The jasmonate- ngerprints left
independent pathway involves G proteins behind by specic
cellular processes
that interact and activate phospholipase A2,
which leads to a transient proton efux from RNAi: RNA
interference
the vacuole and a subsequent activation of
other cytoplasmic components (60, 164).
Transgenic Approaches
The application of transgenic technologies
to alkaloid-producing plants is primarily in-
tended to increase the synthetic capacity of
desired product via the overexpression of cer-
tain genes, or to divert pathways to previ-
ously under-represented or novel compounds
through 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 metabolic
consequences resulting from single-enzyme
perturbations of alkaloid pathways suggest the
existence of key rate-limiting steps, potential
multienzyme complexes, or unsuspected com-
partmentalization.
Metabolic engineering of low-scopola-
mine A. belladonna plants via the intro-
duction of a constitutively expressed H6H
transgene led to an increase in scopolamine
accumulation (179). Similarly, a shift in the
accumulation of hyoscyamine in favor of
scopolamine occurred when the H6H trans-
gene was introduced into A. beatica, suggest-
ing that the enzyme is a rate-limiting step
in scopolamine biosynthesis (180). However,
the unpredictability of metabolic engineering
in tropane alkaloid biosynthesis was demon-
strated by the constitutive coexpression of
PMT and H6H. The transgenes caused only
modest increases in alkaloid accumulation
when expressed alone, but exhibited a syner-
gistic effect on alkaloid levels when expressed
together (181).
www.annualreviews.org Alkaloid Biosynthesis 749
 ANRV342-PP59-29 ARI 29 March 2008 2:13
In MIA biosynthesis, the rst attempts
at metabolic engineering focused on the
expression of constitutive TDC and STR
Latex: the milky
cytoplasm of transgenes in C. roseus cell cultures (31).
specialized cells However, only unsustained increases in al-
called laticifers kaloid accumulation were reported, which
raises concerns about the actual relation-
ship between the transgenes and the initial
phenotype (16). Overexpression of enzymes
responsible for production of the indole moi-
ety, such as anthranilate synthase (AS), re-
sulted in higher levels of tryptophan, but
not alkaloids (64, 66). Monoterpenoid path-
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
way reactions have long been considered
the rate-limiting steps in MIA production.
In this context, overexpression of a modi-
by Washington State University on 11/01/11. For personal use only.
ed 3-hydroxy-3-methylglutaryl-CoA reduc-
tase leads to a predictable increase in al-
kaloid content (8). The role of secologanin
biosynthesis in the control of ux into MIA
metabolism is underscored by the ectopic
overexpression of transcriptional regulators of
the pathway. T-DNA activation tagging in
C. roseus cell cultures resulted in the isola-
tion of an octadecanoid-derivative responsive
Catharanthus AP2-domain protein (ORCA3),
which activates the expression of genes en-
coding AS, TDC, STR, D4H, cytochrome
P450 reductase, and D-1-deoxyxylulose 5-
phosphate synthase, but not those encod-
ing geraniol 10-hydroxylase, SGD, and DAT
(161). However, alkaloid accumulation in cells
transformed with a constitutively expressed
ORCA3 transgene occurred only in the pres-
ence of exogenous loganin. The combined
overexpression of transcriptional activators,
such as ORCA2 and ORCA3, and the silenc-
ing of repressors, such as the G-box binding
factors 1 and 2 and the zinc nger protein
family, might be required to activate the en-
tire pathway (100, 126, 143, 161).
Most of the transgenic work involving BIA
metabolism has focused on the modulation
of BBE activity. Overexpression of this en-
zyme in E. californica root cultures results in
an increased accumulation of downstream al-
kaloids and decreased levels of certain amino
acids (124). Conversely, antisense suppression
750 Ziegler Facchini
of BBE expression leads to undetectable lev-
els of downstream metabolites and increased
cellular pools of amino acids, revealing the im-
pact of perturbations in alkaloid metabolism
on 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 channels
that 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 is
redirected toward the O-methylation of reti-
culine by 7OMT. Transgenic opium poppy
plants expressing an antisense-BBE construct
show increased ux into the morphinan and
tetrahydrobenzylisoquinoline branch path-
ways (40). Surprisingly, the BBE reaction
product (S )-scoulerine also accumulates in
the latex of these transgenic plants, although
the roots show no alterations in metabolite
prole. Whether these results are due to an
additional role of BBE in BIA metabolism
remains unproven. Opium poppy plants
transformed with constitutively expressed
or antisense-suppressed Cyp80B3, which en-
codes N-methylcoclaurine 3 -hydroxylase,
also show substantial modulations in alkaloid
content (41). However, as expected the alka-
loid prole is not altered because Cyp80B3
acts early in the pathway. Overexpression
of COR1, the penultimate step in morphine
biosynthesis, also results in opium poppy
plants with increased alkaloid content (81).
This increase is attributable to higher levels
of morphine, codeine, andfor unexplained
reasonsthebaine, which is upstream of
COR1 in the pathway. The overexpression of
one alkaloid biosynthetic gene might possibly
cause the coordinate transcriptional induction
of 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 changes
in alkaloid prole. In RNAi-silenced COR1
plants, the expression of other known genes
 ANRV342-PP59-29 ARI 29 March 2008 2:13
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 based
on a feedback mechanism that inhibits the en-
tire morphinan branch pathway or the im-
pairment of a required metabolic channel due
to the absence of COR is not known. The
presence 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 not
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
abundant in opium poppy plants (3). This is
surprising because salutaridinol, which does
not accumulate, is the substrate for SalAT
by Washington State University on 11/01/11. For personal use only.
(Figure 2). Salutaridine might be channelled
to thebaine through an enzyme complex that
includes SalR and SalAT.
Experiments involving the overexpression
of two O-methyltransferases from the early
BIA pathway in E. californica cells suggested
a 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. The
E. californica cell culture used in this study
appeared to lack a functional 6OMT, which
might 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, suggesting
a role for 4 OMT as a surrogate for 6OMT to
facilitate BIA biosynthesis.
Transgenic approaches to alter caffeine
biosynthesis 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, the
second N-methyltransferase in the pathway,
results in the reduction of theobromine and
caffeine by 50% to 70% in C. arabica and
C. canephora (118, 119). However, MXMT,
XMT, and DXMT transcripts are all re-
duced owing to the high homology between
the three genes. An alternative biotechno-
logical strategy has targeted the production
of caffeine in non-caffeine-producing plants
to increase pest resistance. Overexpression
of all three N-methyltransferases in trans-
genic Nicotiana tabacum leads to a substantial
Idioblast: an
(5 ug g1 fresh weight) accumulation of caf- individual cell that
feine in leaves (77, 159). This is sufcient to differs greatly from
reduce by 50% pest damage caused by feeding its neighbors
of the tobacco cutworm Spodoptera litura.
ALKALOID TRAFFICKING
The cell biology of alkaloid metabolism in
plants has recently emerged as an exciting
eld of research. Although the biosynthetic
pathways leading to various alkaloid types
are of polyphyletic origin, some intriguing
paradigms are apparent, including the in-
volvement of multiple cell types for alkaloid
biosynthesis and/or accumulation, the target-
ing of different pathway enzymes to multiple
subcellular compartments, and the possibility
that multienzyme complexes are ubiquitous.
Such complex organization implicates exten-
sive intra- and intercellular transport of path-
way intermediates, products, and biosynthetic
enzymes.
Cell TypeSpecific Localization
of Alkaloid Biosynthetic Enzymes
Alkaloids generally accumulate in specic
cell types owing to their cytotoxicity and
probable role in plant defense responses. For
example, alkaloids are sequestered to isolated
idioblasts and laticifers in C. roseus (151), root
endodermis and stem cortex/pith in T. avum
(138), and laticifers in opium poppy (13).
The cellular localization of alkaloid pathways
is remarkably diverse and complex. Work on
the cellular and developmental complexities
and organization of alkaloid biosynthesis in
C. roseus and opium poppy, in particular, have
established new paradigms in the cell biology
of secondary metabolism.
PMT and H6H, which catalyze the rst
and 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
www.annualreviews.org Alkaloid Biosynthesis 751
 ANRV342-PP59-29 ARI 29 March 2008 2:13

a Pericycle b
Phloem Idioblast Epidermis

Internal phloem Palisade

parenchyma mesophyll
Xylem
Spongy
Phloem mesophyll

Xylem
Endodermis Idioblast Laticifer
Cortex Epidermis
Epidermis
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

c d
by Washington State University on 11/01/11. For personal use only.

Phloem
Phloem

Xylem Xylem

Endodermis Endodermis
Cortex Cortex

Immature
e f endodermis

Pericycle
Xylem

Vascular
cambium
Companion Sieve
cell element
Laticifer Stele

Cortex
Protoderm
Parenchyma

Figure 6
Alkaloid biosynthetic pathways are associated with a diversity of cell types. The tissue-specic
localization (red ) of enzymes and/or gene transcripts are shown for the biosynthesis of tropane alkaloids
in (a) Atropa belladonna and Hyoscyamus niger roots, (b) terpenoid indole alkaloids in Catharanthus roseus
leaves, (c) pyrrolizidine alkaloids in Senecio vernalis roots, (d ) pyrrolizidine alkaloids in Eupatorium
cannabinum roots, (e) benzylisoquinoline alkaloids in Papaver somniferum vascular bundles, and
( f ) benzylisoquinoline alkaloids in Thalictrum avum roots.

752 Ziegler Facchini

 ANRV342-PP59-29 ARI 29 March 2008 2:13

rst step in nicotine biosynthesis and local-

izes to the endodermis, outer cortex, and
xylem in Nicotiana sylvestris (141, 142). In con-
trast, TR-I, an intermediate enzyme in the Cortex
production of hyoscyamine and scopolamine,
resides in the endodermis and nearby corti-
cal cells (112); thus, tropane alkaloid inter-
mediates must also trafc between cell types
(Figure 7). The specic pathway intermedi- Tropinone
ates that undergo intercellular translocation Tropine
are not known. Interestingly, TR-II, which Endodermis
provides pseudotropine for the formation of
calystegines, localizes to companion cells of
N-Methylputrescine
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

sieve elements in the phloem of potato (73).

Hyoscyamine
Enzymes involved in MIA biosynthesis lo- Putrescine
calize to several different C. roseus organs and
by Washington State University on 11/01/11. For personal use only.

Scopolamine
Pericycle Ornithine Arginine
cell types. TDC and STR are abundant in
roots (151), but also occur in photosynthetic
organs, whereas T16H (149), D4H (162), and
DAT (150) are restricted to young leaves and Ornithine
Arginine
other shoot organs where vindoline biosyn-
thesis occurs. In situ hybridization and im-
Phloem Xylem
munocytochemical localization studies have
shown that Cyp72A1, TDC, and STR lo-
calize to the epidermis of stems, leaves, and
ower buds (70, 151) (Figure 6b). In roots,
these enzymes occur in cells near the api-
cal meristem. In contrast, D4H and DAT
are associated with laticifers and idioblasts
of shoots, but are absent from roots. Lati-
cifers and idioblasts are distributed through- Figure 7
out the mesophyll in leaves, often several cell Putative trafcking of pathway intermediates and products in tropane
layers away from the epidermis; thus, vin- alkaloid metabolism. Red arrows show specic enzyme-catalyzed reactions.
doline biosynthesis involves at least two dis- Blue arrows represent the purported intercellular translocation of
unspecied compounds.
tinct cell types and requires the intercellu-
lar translocation of pathway intermediates.
Moreover, gene transcripts that encode en- sis in C. roseus are not known. Interestingly,
zymes involved in the MEP pathway, along the identication of gene transcripts encod-
with geraniol 10-hydroxylase, colocalize to ing vindoline biosynthetic enzymes in specic
the internal phloem parenchyma of young C. cell types isolated by laser-capture microdis-
roseus aerial organs (15, 120). These results section and enriched via carborundum abra-
suggest the translocation of vindoline biosyn- sion was interpreted to suggest that the leaf
thetic intermediates from the internal phloem epidermis is independently capable of produc-
to the epidermis and from the epidermis to ing 16-methoxytabersonine (109). Upper leaf
laticifers and idioblasts (Figure 8). As is the epidermis has been implicated in several sec-
case in tropane alkaloid metabolism, the spe- ondary metabolic pathways (94), complicating
cic pathway intermediates that undergo in- the assignment of common precursors to spe-
tercellular translocation in MIA biosynthe- cic cell types. The role of internal phloem

www.annualreviews.org Alkaloid Biosynthesis 753

 ANRV342-PP59-29 ARI 29 March 2008 2:13

drogenation, epoxidation, or O-acetylation)

Cuticle that result in the unique pyrrolizidine alka-
loid prole of different plants. Inorescences
Tryptophan Loganic acid
are the major sites of pyrrolizidine alkaloid ac-
Tryptamine + Secologanin cumulation in Senecio jacobaea and Senecio ver-
Epidermis nalis; jacobine occurs in owers. HSS is the
Strictosidine rst committed enzyme in pyrrolizidine alka-
16-Methoxytabersonine loid biosynthesis and localizes to the root en-
dodermis and cortex adjacent to the phloem
in S. vernalis (Figure 6c), which might re-
ect a functional accommodation for sys-
Palisade
mesophyll temic transport of pyrrolizidine alkaloids to
MIA the stem (105). However, HSS is found
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

intermediates Palisade throughout the root cortex in Eupatorium

? idioblast
cannabinum (Figure 6d ), which like S. vernalis
Deacetyl-
is a member of the Asteraceae (6). In con-
by Washington State University on 11/01/11. For personal use only.

Deacetyl- vindoline
vindoline trast to the general monophyletic origin of
Vindoline
Vindoline BIA biosynthesis (87), pyrrolizidine alkaloid
Laticifer
pathways have evolved in at least four differ-
ent angiosperm lineages (128). The differen-
Deacetyl-
vindoline tial localization of a key enzyme was inter-
MEP pathway
? preted to support the polyphyletic origin for
Vindoline
Geraniol pyrrolizidine alkaloid biosynthesis (6). In con-
10-Hydroxy- Spongy
IPAP mesophyll trast, the monophyletic origin of BIA biosyn-
geraniol
idioblast
IPAP IPAP thesis (87) and the differential localization of
IPAP gene transcripts in T. avum and opium poppy
suggest the migration of pathway intermedi-
Vascular
tissues ates between cell types.
IPAP
IPAP In opium poppy, BIA accumulation
occurs in the articulated laticifers found
Figure 8 adjacent or proximal to sieve elements of the
Putative trafcking of pathway intermediates and products in phloem (32). The cytoplasm of laticifersor
monoterpenoid indole alkaloid metabolism. Red arrows show specic latexcontains a full complement of cellular
enzyme-catalyzed reactions. Blue arrows represent the purported
organelles and many large vesicles to which
intercellular translocation of unspecied compounds. IPAP, internal
phloem-associated parenchyma. alkaloids are sequestered. Although laticifers
were long considered to be the site of BIA
and epidermis in the supply of terpenoid pre- biosynthesis and accumulation, the cellular
cursors to alkaloid biosynthesis requires fur- localization of BIA biosynthetic enzymes
ther evaluation. and gene transcripts has shown that alkaloid
Pyrrolizidine alkaloids also share a com- synthesis involves other cell types (13, 34,
mon metabolic pathway in restricted yet di- 170). Initial in situ hybridization experiments
verse taxa (54). In Senecio species, pyrrolizidine demonstrated the localization of TYDC gene
alkaloids are produced in actively growing transcripts to the phloem, but not to laticifers
roots as senecionine N-oxides, which are (34). The morphinan pathway enzymes
transported via the phloem to above-ground salutaridine synthase (Cyp719B1) and salu-
organs (54). Senecionine N-oxides are sub- taridine:NADPH 7-oxidoreductase (SalR)
sequently modied by one or two species- are also not detected in isolated latex (48, 49).
specic reactions (i.e., hydroxylation, dehy- Seven other biosynthetic enzymes6OMT,

754 Ziegler Facchini

 ANRV342-PP59-29 ARI 29 March 2008 2:13
CNMT, (S )-N-methylcoclaurine 3 -
 Parenchyma
hydroxylase (Cyp80B3), 4 OMT, BBE,
SalAT, and CORlocalize to sieve elements
in opium poppy and their corresponding gene
transcripts localize to associated companion
cells (13, 135) (Figure 6e). The localization
of BIA metabolism components to phloem
cells distinct from laticifers predicts the in-
tercellular transport of alkaloid biosynthetic
enzymes and pathway intermediates and/or
products. The implication of sieve elements
also breaks a long-standing paradigm in
plant biology (Figure 9). Previously, sieve
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
elements were not known to support complex
metabolism, and were assumed to possess
only a limited number of proteins required
by Washington State University on 11/01/11. For personal use only.
for cell maintenance and solute transport.
Recently, the physiological roles for sieve ele-
ments have expanded to include the transport
of information macromolecules such as RNA
(72) and the biosynthesis of jasmonic acid
(57), ascorbic acid (53), and defense-related
compounds (167). Sieve elements clearly
possess a previously unrealized biochemical
potential.
T. avum accumulates BIAs such as the
antimicrobial agent berberine. In situ RNA
hybridization analysis revealed the cell type
specic expression of BIA biosynthetic genes
in roots and rhizomes of T. avum (138). In
roots, gene transcripts for all nine enzymes
localize to the pericyclethe innermost layer
of the cortexand adjacent cortical cells
(Figure 6f ). In rhizomes, all biosynthetic
gene transcripts are restricted to the proto-
derm of leaf primordia. The protoderm local-
ization of biosynthetic gene transcripts con-
trasts with the tissue-specic accumulation of
BIAs. In roots, protoberberine alkaloids are
restricted to endodermal cells upon the initi-
ation of secondary growth, and are distributed
throughout the pith and cortex in rhizomes.
Thus, the cell typespecic localization of
BIA biosynthesis and accumulation are tem-
porally and spatially separated in T. avum
roots and rhizomes, respectively. Despite the
close phylogenetic relationships between cor-
responding biosynthetic enzymes (87), differ-
Companion
cell
Laticifer
Alkaloid
biosynthetic Alkaloids
enzymes
Sieve
element
Figure 9
Putative trafcking of benzylisoquinoline alkaloid biosynthetic enzymes
and pathway intermediates and/or products.
ent cell types are involved in the biosynthesis
and accumulation of BIAs in opium poppy, T.
avum, and perhaps C. japonica.
Subcellular Compartmentalization
of Alkaloid Biosynthetic Enzymes
MIA biosynthetic enzymes are associated with
at least three different cell types in C. roseus
geraniol 10-hydroxylase (G10H) is found in
internal phloem parenchyma of aerial organs
(15), TDC, secologanin synthase, and STR
are localized to the epidermis of aerial or-
gans and the apical meristem of roots (70,
151), and D4H and DAT are restricted to
the laticifers and idioblasts of leaves and stems
(151); thus, vindoline pathway intermediates
must be translocated between cell types. En-
zymes involved in monoterpenoid indole alka-
loid biosynthesis in C. roseus also localize to at
least ve subcellular compartmentsTDC,
D4H, and DAT are in the cytosol (24), STR
and the peroxidase involved in the coupling
of MIA monomers localize to the vacuole
(98, 99, 145), SGD is a soluble enzyme pur-
ported to associate with the cytoplasmic face
of the endoplasmic reticulum (148), the P450-
dependent monooxygenases G10H, secolo-
ganin synthase (SLS), and T16H are integral
endomembrane proteins (20, 149, 177), and
www.annualreviews.org Alkaloid Biosynthesis 755
 ANRV342-PP59-29 ARI 29 March 2008 2:13
the N-methyltransferase involved in vindoline
biosynthesis localizes to thylakoid membranes
(27). It is unclear whether some enzymes, such
Multienzyme
complex: a cluster as T16H, occur in the epidermis, or in lati-
of distinct enzymes cifers and idioblasts. The complex compart-
that catalyze mentation of the monoterpenoid indole al-
consecutive kaloid pathway suggests extensive subcellular
metabolic reactions
trafcking 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 gradient
fractionation suggested the localization of
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
BBE and P450 monooxygenases to micro-
somes with a density slightly higher than that
of typical endoplasmic reticulum (ER) (5, 10,
by Washington State University on 11/01/11. For personal use only.
132, 156). Although BBE is not an integral
membrane protein, it is initially targeted to
the ER and subsequently sorted to a vacuolar
compartment (12). NCS is also predicted to
possess a signal peptide for targeting to the
ER (137), and the nal oxidation of alkaloids,
such as dihydrosanguinarine, likely occurs in
an ER-derived compartment (5). The associ-
ation of BIA biosynthetic enzymes with en-
domembranes led to speculation that special-
ized alkaloid-synthesizing vesicles are present
in alkaloid-producing cells (5). Recently, how-
ever, Cyp80B3, BBE, and sanguinarine were
found to colocalize with the ER in opium
poppy cell cultures (2). Moreover, the induc-
tion of sanguinarine biosynthesis correlates
with extensive dilations of the ER and the
accumulation of an electron-dense occulent
material within the ER. Because the pH op-
timum of BBE is 8.8 (146), sanguinarine
biosynthesis is unlikely to involve the vac-
uole; thus, alkaloid metabolism could be en-
tirely associated with the ER. Indeed, the
vacuole might not even be the site of BIA
accumulation in cultured cells, as previously
thought. Instead, sanguinarine and related al-
kaloids could be secreted and bound to cell
wall components and reabsorbed, reduced to
the less-toxic compound dihydrosanguinar-
ine, and further metabolized (171).
A unique subcellular compartmentaliza-
tion of enzymes is also present in quinolizidine
756 Ziegler Facchini
alkaloid biosynthesis, which occurs in the
mesophyll of some legumes. Lysine decar-
boxylase and the quinolizidine skeleton
forming enzyme have been detected in
chloroplasts of Lupinus polyphyllus (172). 13-
Hydroxymultiuorine/13-hydroxylupanine
O-tigloylase is localized to the mitochondrial
matrix rather than to chloroplasts where de
novo quinolizidine alkaloid biosynthesis is
thought to occur (152). In contrast, epilupi-
nine O-p-coumaryoltransferase is present
in a distinct organelle, but has not been
unambiguously localized.
Enzyme Complexes
and Metabolic Channels
Many metabolic enzymes are generally as-
sumed to interact physically or be in close
proximity with other enzymes that participate
in common pathways (173). Theoretically,
the existence of multienzyme complexes
also known as metabolic complexes/channels
or metabolonspromote the efciency of
cellular metabolism. The direct transfer of
a pathway intermediate from one enzyme
to another maintains a high local substrate
concentration, which avoids the dilution of
intermediates 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 the
interactions between pathway enzymes have
hinted at the importance of metabolic chan-
nels in primary and secondary metabolism.
The detection of multienzyme complexes has
been purported for avonoid (1, 14, 58) and
polyamine (122) metabolism.
The results from the RNAi-mediated
silencing of COR genes suggested the possible
existence of a multienzyme complex in BIA
(4). Although seven enzymatic steps occur
between (S )-reticuline and codeinonethe
substrate for CORonly (S )-reticuline accu-
mulates at elevated levels. No intermediates
between (S )-reticuline and morphine are
 ANRV342-PP59-29 ARI 29 March 2008 2:13
detected. Removal of COR was suggested to
disrupt a metabolic channel composed of mor-
phinan branch pathway enzymes, resulting in
the accumulation of an alkaloid intermediate
produced by enzymes that are not part of
the same complex. Interestingly, thebaine
and oripavine, intermediates upstream of
substrates acted upon by COR, accumulate
to high levels in some opium poppy cul-
tivars (101); thus, researchers expected an
accumulation of morphinan branch pathway
intermediates. An alternative interpretation
not considered by Allen and coworkers (4)
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
is the suppression of 1,2-dehydroreticuline
reductase as a possible side effect of silencing
COR. 1,2-Dehydroreticuline reductase is one
by Washington State University on 11/01/11. For personal use only.
of two enzymes involved in the epimerization
of (S )-reticuline to (R )-reticuline. The po-
tential homology between the two reductases
could lead to cosilencing.
PERSPECTIVE
Major progress in the elucidation of alka-
loid biosynthetic pathways and their regula-
tion has been obtained by application of large
scale genomic tools. However, there are sev-
eral techniques, whose applications are still
more or less exclusively conned to model or-
ganisms, that might further our understand-
ing of plant alkaloid biosynthesis. Proteome
analysis in alkaloid-accumulating plants has
been undertaken (26, 71, 115), but is still lim-
ited by the lack of peptide mass databases for
the investigated species, which does not allow
protein identication by simple peptide mass
ngerprinting. Targeted induced local lesions
in genomes (TILLING) as a tool to discover
point mutations is also dependent on avail-
SUMMARY POINTS
1. The application of genomics technologies has expedited the discovery of new alkaloid
biosynthetic 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.
able sequence information (21). However, the
growing sequence database makes the appli-
cation of this technique feasible in studying
mutations in alkaloid-containing plants. The
development of sequencing techniques that
can generate large datasets within short times
will probably be applied to alkaloid produc-
ing plants and will provide the basis for many
sequence-based approaches that are now lim-
ited to plants with sequenced genomes such
as Arabidopsis and rice. Whether genome
sequencing projects in alkaloid-containing
plants will be feasible remains to be seen, be-
cause the genomes of the investigated plants
are very large (4.7 Gbp for C. roseus or 7.4 Gbp
for 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 characterization
of gene products must be conrmed in vivo.
However, functional genomics remains prob-
lematic for most alkaloid-producing plants
owing to general limitations in established
genetic transformation technologies. Virus-
induced gene silencing is a fast method for the
generation of transiently transformed plants
and works well in a model organism such
as N. benthamiana (130), and protocols for
opium poppy and E. californica have recently
been developed (62, 169). Thus, the impact
of suppression of candidate genes on alkaloid
biosynthesis can be readily examined before
stable transformation is applied. Considering
the progress in the investigation of alkaloid
metabolism achieved in the last 5 years, a look
to the future promises further strong devel-
opments in the discovery of regulatory net-
works that lead to alkaloid accumulation in
plants.
www.annualreviews.org Alkaloid Biosynthesis 757
 ANRV342-PP59-29 ARI 29 March 2008 2:13

3. Structural analysis of alkaloid biosynthetic enzymes has enabled targeted mod-

ications of substrate-binding sites for the development of biomimetic alkaloid
production.
4. Improved transformation protocols have facilitated the establishment of transgenic
plants with tailored alkaloid proles, providing additional insights into the regulation
of pathways.
5. Alkaloid biosynthesis and accumulation are associated with a variety of cell types in
different plants, including epidermis, endodermis, pericycle, phloem parenchyma,
phloem sieve elements and companion cells, specialized mesophyll, and laticifers. A
common paradigm is the involvement of multiple cell types and the implied transport
of pathway intermediates and/or products.
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

6. The subcellular compartmentalization of alkaloid biosynthetic enzymes is as complex

as the cell typespecic localization of gene transcripts, enzymes, and metabolites. Al-
by Washington State University on 11/01/11. For personal use only.

though the endoplasmic reticulum is a favored site for alkaloid formation, biosynthetic
enzymes 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 to
the discovery of novel genes that encode alkaloid biosynthetic enzymes, a substantial
understanding of the regulatory components of alkaloid pathways has been achieved
only for terpenoid indole alkaloid metabolism. Genomics approaches should lead to
the identication of regulatory genes involved in benzylisoquinoline alkaloid synthesis
and other alkaloid pathways.
2. The impact of posttranslational modication of alkaloid biosynthetic enzymes on
product accumulation is a black box. Proteomic approaches might explain why the
expression levels of some biosynthetic genes do not correlate with alkaloid proles.
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 intermediates
and/or products that undergo intercellular transport is not known. Moreover, the
mechanisms of transportsymplastic involving plasmodesmata, or apoplastic involv-
ing specic transporters or channelsare poorly understood.
4. The existence of metabolic channels as a common feature of alkaloid biosynthetic
pathways has been widely purported, but only scant evidence is available to support
their existence. Establishing the interactions among biosynthetic enzymes and other
pathway components will facilitate the rational engineering of alkaloid metabolism in
plants and microorganisms.

DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the objectivity of
this review.

758 Ziegler Facchini

 ANRV342-PP59-29 ARI 29 March 2008 2:13

ACKNOWLEDGMENTS
J.Z. was funded through a Canada Research Chair in Plant Metabolic Processes Biotechnology
held by P.J.F. Research support from the Natural Sciences and Engineering Research Council
of Canada to P.J.F. is also gratefully acknowledged.

LITERATURE CITED
1. Achnine L, Blancaor EB, Rasmussen S, Dixon RA. 2004. Colocalization of l-
phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in
phenylpropanoid biosynthesis. Plant Cell 16:3098109
2. Alcantara J, Bird DA, Franceschi VR, Facchini PJ. 2005. Sanguinarine biosynthesis is
associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor
treatment. Plant Physiol. 138:17383
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

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-
by Washington State University on 11/01/11. For personal use only.

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-mediated
replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat.
Biotechnol. 22:155966
5. Amann M, Wanner G, Zenk MH. 1986. Intracellular compartmentation of two enzymes
of berberine biosynthesis in plant cell cultures. Planta 167:31020
6. Anke S, Niemuller D, Moll S, Hansch R, Ober D. 2004. Polyphyletic origin of
pyrrolizidine alkaloids within the Asteraceae. Evidence from differential tissue expres-
sion of homospermidine synthase. Plant Physiol. 136:403747
7. Arakawa H, Clark WG, Psenak M, Coscia CJ. 1992. Purication and characterization of
dihydrobenzophenanthridine oxidase from elicited Sanguinaria canadensis cell cultures.
Arch. Biochem. Biophys. 299:17
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. 97:13545
9. Barleben L, Panjikar S, Ruppert M, Koepke J, Stockigt J. 2007. Molecular architecture
of strictosidine glucosidase: the gateway to the biosynthesis of the monoterpenoid indole
alkaloid family. Plant Cell 19:288697
10. Bauer W, Zenk MH. 1991. Two methylenedioxy bridge forming cytochrome P-450 de-
pendent enzymes are involved in (S )-stylopine biosynthesis. Phytochemistry 30:295362

11. Bayer A, Ma X, Stockigt J. 2004. Acetyltransfer in natural product biosynthesisfunctional
cloning and molecular analysis of vinorine synthase. Bioorg. Med. Chem. 12:278795
12. Bird DA, Facchini PJ. 2001. Berberine bridge enzyme, a key branch-point enzyme in
benzylisoquinoline alkaloid biosynthesis, contains a vacuolar-sorting determinant. Planta
213:88897
13. Bird DA, Franceschi VR, Facchini PJ. 2003. A tale of three cell types: alkaloid biosynthesis
is localized to sieve elements in opium poppy. Plant Cell 15:262635
14. Burbulis IE, Winkel-Shirley B. 1999. Interactions among enzymes of the Arabidopsis
avonoid biosynthetic pathway. Proc. Natl. Acad. Sci. USA 96:1292934
15. Burlat V, Oudin A, Courtois M, Rideau M, St-Pierre B. 2004. Co-expression of three
MEP pathway genes and geraniol 10-hydroxylase in internal phloem parenchyma of
Catharanthus roseus implicates multicellular translocation of intermediates during the

www.annualreviews.org Alkaloid Biosynthesis 759

 ANRV342-PP59-29 ARI 29 March 2008 2:13

biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabo-

lites. Plant J. 38:13141
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 alkaloid
production by cell cultures of Catharanthus roseus. Planta 205:41419
17. Carlson JE, Leebens-Mack JH, Wall PK, Zahn LM, Mueller LA, et al. 2006. EST database
for early ower development in California poppy (Eschscholzia californica Cham., Papaver-
aceae) tags over 6,000 genes from a basal eudicot. Plant Mol. Biol. 62:35169
18. Chen S, Galan MC, Coltharp C, OConnor SE. 2006. Redesign of a central enzyme in
alkaloid biosynthesis. Chem. Biol. 13:113741
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. 277:83035
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

20. Collu G, Unver N, Peltenburg-Looman AM, van der Heijden R, Verpoorte R, Memelink
J. 2001. Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid
indole alkaloid biosynthesis. FEBS Lett. 508:21520
by Washington State University on 11/01/11. For personal use only.

21. Comai L, Henikoff S. 2006. TILLING: practical single-nucleotide mutation discovery.

Plant J. 45:68494
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 Catharanthus
roseus cell culture. FEBS Lett. 434:41316
23. De-Eknamkul W, Zenk MH. 1992. Purication and properties of 1,2-dehydroreticuline
reductase from Papaver somniferum seedlings. Phytochemistry 31:81321
24. De Luca V, Cutler AJ. 1987. Subcellular localization of enzymes involved in indole
alkaloid biosynthesis in Catharanthus roseus. Plant Physiol. 85:1099102
25. De Luca V, Marineau C, Brisson N. 1989. Molecular cloning and analysis of cDNA
encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases.
Proc. Natl. Acad. Sci. USA 86:258286
26. Decker G, Wanner G, Zenk MH, Lottspeich F. 2000. Characterization of proteins in
latex of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresis
and microsequencing. Electrophoresis 21:350016
27. Dethier M, De Luca V. 1993. Partial purication of an N-methyltransferase involved in
vindoline biosynthesis in Catharanthus roseus. Phytochemistry 32:67378
28. Dittrich H, Kutchan TM. 1991. Molecular cloning, expression, and induction of berber-
ine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloids
in the response of plants to pathogenic attack. Proc. Natl. Acad. Sci. USA 88:996973
29. Dogru E, Warzecha H, Seibel F, Haebel S, Lottspeich F, Stockigt J. 2000. The gene
encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis
in plants is an ortholog of the /-hydrolase super family. Eur. J. Biochem. 267:1397406
30. Drager B. 2004. Chemistry and biology of calystegines. Nat. Prod. Rep. 21:21123
31. Facchini PJ. 2001. Alkaloid biosynthesis in plants: biochemistry, cell biology, molecular
regulation, and metabolic engineering applications. Annu. Rev. Plant Physiol. Plant Mol.
Biol. 52:2966
32. Facchini PJ, Bird DA. 1998. Developmental regulation of benzylisoquinoline alkaloid
biosynthesis in opium poppy plants and tissue cultures. In Vitro Cell. Dev. Biol. Plant
34:6979
33. Facchini PJ, De Luca V. 1994. Differential and tissue-specic expression of a gene family
for tyrosine/dopa decarboxylase in opium poppy. J. Biol. Chem. 269:2668490

760 Ziegler Facchini

 ANRV342-PP59-29 ARI 29 March 2008 2:13

34. Facchini PJ, De Luca V. 1995. Phloem-specic expression of tyrosine/dopa decarboxylase

genes and the biosynthesis of isoquinoline alkaloids in opium poppy. Plant Cell 7:181121
35. Facchini PJ, Park SU. 2003. Developmental and inducible accumulation of gene tran-
scripts involved in alkaloid biosynthesis in opium poppy. Phytochemistry 64:17786
36. Facchini PJ, Penzes C, Johnson AG, Bull D. 1996. Molecular characterization of berber-
ine bridge enzyme genes from opium poppy. Plant Physiol. 112:166977
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 unspecic acetylesterase. Plant Cell Rep. 4:33740
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 cells
of Eschscholzia californica. Phytochemistry 62:491500

39. Falkenhagen H, Stockigt J. 1995. Enzymatic biosynthesis of vomilenine, a key interme-
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

diate of the ajmaline pathway, catalyzed by a novel cytochrome P450 dependent enzyme
from plant cell cultures of Rauvola serpentina. Z. Naturforsch. 1995:4553
by Washington State University on 11/01/11. For personal use only.

40. Frick S, Chitty JA, Kramell R, Schmidt J, Allen RS, et al. 2004. Transformation of opium
poppy (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. 13:60713
41. Frick S, Kramell R, Kutchan TM. 2007. Metabolic engineering with a morphine biosyn-
thetic P450 in opium poppy surpasses breeding. Metab. Eng. 9:16976
42. Frick S, Kutchan TM. 1999. Molecular cloning and functional expression of O-
methyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis.
Plant J. 17:32939
43. Fujii N, Inui T, Iwasa K, Morishige T, Sato F. 2007. Knockdown of berberine bridge
enzyme by RNAi accumulates (S )-reticuline and activates a silent pathway in cultured
California poppy cells. Transgenic Res. 16:36375
44. Gao S, von Schumann G, Stockigt J. 2002. A newly-detected reductase from Rauvola
closes a gap in the biosynthesis of the antiarrhythmic alkaloid ajmaline. Planta Med.
68:90611
45. Gavilano LB, Siminszky B. 2007. Isolation and characterization of the cytochrome P450
gene Cyp82E5v2 that mediates nicotine to nornicotine conversion in the green leaves of
tobacco. Plant Cell. Physiol. 48:156774
46. Geerlings A, Ibanez MM, Memelink J, van Der Heijden R, Verpoorte R. 2000. Molecular
cloning and analysis of strictosidine -d-glucosidase, an enzyme in terpenoid indole
alkaloid biosynthesis in Catharanthus roseus. J. Biol. Chem. 275:305156
47. Geissler R, Brandt W, Ziegler J. 2007. Molecular modeling and site-directed mutagenesis
reveal the benzylisoquinoline binding site of the short-chain dehydrogenase/reductase
salutaridine reductase. Plant Physiol. 143:1493503
48. Gerardy R, Zenk MH. 1993. Formation of salutaridine from (R )-reticuline by a
membrane-bound cytochrome P-450 enzyme from Papaver somniferum. Phytochemistry
32:7986
49. Gerardy R, Zenk MH. 1993. Purication and characterization of salutaridine:NADPH
7-oxidoreductase from Papaver somniferum. Phytochemistry 34:12532
50. Gerasimenko I, Sheludko Y, Ma X, Stockigt J. 2002. Heterologous expression of a Rau-
vola cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoter-
penoid indole alkaloids. Eur. J. Biochem. 269:220413

www.annualreviews.org Alkaloid Biosynthesis 761

 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 plant
cells. Proc. Natl. Acad. Sci. USA 100:8595600
52. Grothe T, Lenz R, Kutchan TM. 2001. Molecular characterization of the salutaridinol
7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver som-
niferum. J. Biol. Chem. 276:3071723
53. Hancock RD, McRae D, Haupt S, Viola R. 2003. Synthesis of l-ascorbic acid in the
phloem. BMC Plant Biol. 3:7
54. Hartmann T, Dierich B. 1998. Chemical diversity and variation of pyrrolizidine alkaloids
of the senecionine type: biological need or coincidence? Planta 206:44351
55. Hartmann T. 2004. Plant-derived secondary metabolites as defensive chemicals in her-
bivorous insects: a case study in chemical ecology. Planta 219:14
56. Hashimoto T, Hayashi A, Amano Y, Kohno J, Iwanari H, et al. 1991. Hyoscyamine
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

6-hydroxylase, an enzyme involved in tropane alkaloid biosynthesis, is localized at the

pericycle of the root. J. Biol. Chem. 266:464853
57. Hause B, Hause G, Kutter C, Miersch O, Wasternack C. 2003. Enzymes of jasmonate
by Washington State University on 11/01/11. For personal use only.

biosynthesis occur in tomato sieve elements. Plant Cell Physiol. 44:64348

58. He XZ, Dixon RA. 2000. Genetic manipulation of isoavone 7-O-methyltransferase
enhances biosynthesis of 4 -O-methylated isoavonoid phytoalexins and disease resistance
in alfalfa. Plant Cell 12:1689702
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. Phytochemistry
68:45463
60. Heinze M, Steighardt J, Gesell A, Schwartze W, Roos W. 2007. Regulatory interaction of
the G protein with phospholipase A2 in the plasma membrane of Eschscholzia californica.
Plant J. doi:10.1111/j.1365313X.2007.03300.x
61. Hibi N, Higashiguchi S, Hashimoto T, Yamada Y. 1994. Gene expression in tobacco
low-nicotine mutants. Plant Cell 6:72335
62. Hileman LC, Drea S, Martino G, Litt A, Irish VF. 2005. Virus-induced gene silencing is
an effective tool for assaying gene function in the basal eudicot species Papaver somniferum
(opium poppy). Plant J. 44:33441
63. Hirata K, Poeaknapo C, Schmidt J, Zenk MH. 2004. 1,2-Dehydroreticuline synthase,
the branch point enzyme opening the morphinan biosynthetic pathway. Phytochemistry
65:103946
64. Hong SB, Peebles CA, Shanks JV, San KY, Gibson SI. 2006. Expression of the Arabidop-
sis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase
genes in Catharanthus roseus hairy roots. J. Biotechnol. 122:2838
65. Huang FC, Kutchan TM. 2000. Distribution of morphinan and benzo[c]phenanthridine
alkaloid gene transcript accumulation in Papaver somninferum. Phytochemistry 53:55564
66. Hughes EH, Hong SB, Gibson SI, Shanks JV, San KY. 2004. Metabolic engineering
of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of
tryptamine and serpentine. Metab. Eng. 6:26876
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. 274:101935
68. Ikezawa N, Tanaka M, Nagayoshi M, Shinkyo R, Sakaki T, et al. 2003. Molecular cloning
and characterization of Cyp719, a methylenedioxy bridge-forming enzyme that belongs
to a novel P450 family, from cultured Coptis japonica cells. J. Biol. Chem. 278:3855765

762 Ziegler Facchini

 ANRV342-PP59-29 ARI 29 March 2008 2:13

69. Inui T, Tamura K, Fujii N, Morishige T, Sato F. 2007. Overexpression of Coptis japonica
norcoclaurine 6-O-methyltransferase overcomes the rate-limiting step in benzylisoquino-
line alkaloid biosynthesis in cultured Eschscholzia californica. Plant Cell Physiol. 48:25262

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 identication of cy-
tochrome P450 Cyp72A1 as secologanin synthase. Plant J. 24:797804
71. Jacobs DI, Gaspari M, van der Greef J, van der Heijden R, Verpoorte R. 2005. Proteome
analysis of the medicinal plant Catharanthus roseus. Planta 221:690704
72. Jorgensen RA, Atkinson RG, Forster RL, Lucas WJ. 1998. An RNA-based information
superhighway in plants. Science 279:148687
73. Kaiser H, Richter U, Keiner R, Brabant A, Hause B, Drager B. 2006. Immunolocalisation
of two tropinone reductases in potato (Solanum tuberosum L.) root, stolon, and tuber
sprouts. Planta 225:12737
74. Kato M, Mizuno K, Crozier A, Fujimura T, Ashihara H. 2000. Caffeine synthase gene
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

from tea leaves. Nature 406:95657

75. Kato N, Dubouzet E, Kokabu Y, Yoshida S, Taniguchi Y, et al. 2007. Identication of a
by Washington State University on 11/01/11. For personal use only.

WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis

in Coptis japonica. Plant Cell Physiol. 48:818
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 family
in Solanum tuberosum (L.). Plant Mol. Biol. 48:299308
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. 15:66772
78. Kraus PF, Kutchan TM. 1995. Molecular cloning and heterologous expression of a cDNA
encoding berbamunine synthase, a C-O phenol-coupling cytochrome P450 from the
higher plant Berberis stolonifera. Proc. Natl. Acad. Sci. USA 92:207175
79. Kutchan TM. 1989. Expression of enzymatically active cloned strictosidine synthase from
the higher plant Rauvola serpentina in Escherichia coli. FEBS Lett. 257:12730
80. Laamme P, St-Pierre B, De Luca V. 2001. Molecular and biochemical analysis of
a Madagascar periwinkle root-specic minovincinine-19-hydroxy-O-acetyltransferase.
Plant Physiol. 125:18998
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 Papaver
somniferum. Plant Biotechnol. J. 5:2637
82. Lenz R, Zenk MH. 1995. Acetyl coenzyme A:salutaridinol-7-O-acetyltransferase from
Papaver somniferum plant cell cultures. The enzyme catalyzing the formation of thebaine
in morphine biosynthesis. J. Biol. Chem. 270:3109196
83. Lenz R, Zenk MH. 1995. Purication 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 of
alkaloid biosynthesis in Hyoscyamus niger reveals a cytochrome P450 involved in littorine
rearrangement. Chem. Biol. 13:51320
85. Lin C, Mueller LA, McCarthy J, Crouzillat D, Petiard V, Tanksley SD. 2005. Coffee
and tomato share common gene repertoires as revealed by deep sequencing of seed and
cherry transcripts. Theor. Appl. Genet. 112:11430
86. Liscombe DK, Facchini PJ. 2007. Molecular cloning and characterization of tetrahy-
droprotoberberine cis-N-methyltransferase, an enzyme involved in alkaloid biosynthesis
in opium poppy. J. Biol. Chem. 282:1474151

www.annualreviews.org Alkaloid Biosynthesis 763

 ANRV342-PP59-29 ARI 29 March 2008 2:13

87. Liscombe DK, MacLeod BP, Loukanina N, Nandi OI, Facchini PJ. 2005. Evidence for
the monophyletic evolution of benzylisoquinoline alkaloid biosynthesis in angiosperms.
Phytochemistry 66:250120
88. Liu T, Zhu P, Cheng KD, Meng C, He HX. 2005. Molecular cloning, expression and
characterization of hyoscyamine 6-hydroxylase from hairy roots of Anisodus tanguticus.
Planta Med. 71:24953
89. Lopez-Meyer M, Nessler CL. 1997. Tryptophan decarboxylase is encoded by two au-
tonomously regulated genes in Camptotheca acuminata which are differentially expressed
during development and stress. Plant J. 11:116775
90. Loris EA, Panjikar S, Ruppert M, Barleben L, Unger M, et al. 2007. Structure-based
engineering of strictosidine synthase: auxiliary for alkaloid libraries. Chem. Biol. 14:979
85
91. Luk LY, Bunn S, Liscombe DK, Facchini PJ, Tanner ME. 2007. Mechanistic studies on
norcoclaurine synthase of benzylisoquinoline alkaloid biosynthesis: an enzymatic Pictet-
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

Spengler reaction. Biochemistry 46:1015361

92. Ma X, Koepke J, Bayer A, Fritzsch G, Michel H, Stockigt J. 2005. Crystallization and
by Washington State University on 11/01/11. For personal use only.

preliminary X-ray analysis of native and selenomethionyl vinorine synthase from Rauvola
serpentina. Acta Crystallogr. D Biol. Crystallogr. 61:69496

93. Ma X, Panjikar S, Koepke J, Loris E, Stockigt J. 2006. The structure of Rauvola serpentina
strictosidine synthase is a novel six-bladed -propeller fold in plant proteins. Plant Cell
18:90720
94. Mahroug S, Courdavault V, Thiersault M, St-Pierre B, Burlat V. 2006. Epidermis is a
pivotal site of at least four secondary metabolic pathways in Catharanthus roseus aerial
organs. Planta 223:1191200
95. Matsuda J, Okabe S, Hashimoto T, Yamada Y. 1991. Molecular cloning of hyoscyamine
6-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscya-
mus niger. J. Biol. Chem. 266:946064
96. Mattern-Dogru E, Ma X, Hartmann J, Decker H, Stockigt J. 2002. Potential active-site
residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosyn-
thesis, by modelling and site-directed mutagenesis. Eur. J. Biochem. 269:288996
97. McCarthy AA, McCarthy JG. 2007. The structure of two N-methyltransferases from the
caffeine biosynthetic pathway. Plant Physiol. 144:87989
98. McKnight TD, Bergey DR, Burnett RJ, Nessler CL. 1991. Expression of enzymatically
active and correctly targeted strictosidine synthase in transgenic tobacco plants. Planta
185:14852
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 Catharanthus
roseus. 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. 18:445563
101. Millgate AG, Pogson BJ, Wilson IW, Kutchan TM, Zenk MH, et al. 2004. Analgesia:
morphine-pathway block in top1 poppies. Nature 431:41314
102. Minami H, Dubouzet E, Iwasa K, Sato F. 2007. Functional analysis of norcoclaurine
synthase in Coptis japonica. J. Biol. Chem. 282:627482
103. Mizuno K, Kato M, Irino F, Yoneyama N, Fujimura T, Ashihara H. 2003. The rst
committed step reaction of caffeine biosynthesis: 7-methylxanthosine synthase is closely
homologous to caffeine synthases in coffee (Coffea arabica L.). FEBS Lett. 547:5660

764 Ziegler Facchini

 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 new
dual-functional caffeine synthase gene encoding an enzyme for the conversion of 7-
methylxanthine to caffeine from coffee (Coffea arabica L.). FEBS Lett. 534:7581
105. Moll S, Anke S, Kahmann U, Hansch R, Hartmann T, Ober D. 2002. Cell-specic
expression of homospermidine synthase, the entry enzyme of the pyrrolizidine alkaloid
pathway in Senecio vernalis, in comparison with its ancestor, deoxyhypusine synthase. Plant
Physiol. 130:4757
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 of
the S-adenosyl-L-methionine:3 -hydroxy-N-methylcoclaurine 4 -O-methyltransferase
involved in isoquinoline alkaloid biosynthesis in Coptis japonica. J. Biol. Chem. 275:23398
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

405
108. Murata J, Bienzle D, Brandle JE, Sensen CW, De Luca V. 2006. Expressed sequence tags
from Madagascar periwinkle (Catharanthus roseus). FEBS Lett. 580:45017
by Washington State University on 11/01/11. For personal use only.

109. Murata J, De Luca V. 2005. Localization of tabersonine 16-hydroxylase and 16-OH

tabersonine-16-O-methyltransferase to leaf epidermal cells denes them as a major site
of precursor biosynthesis in the vindoline pathway in Catharanthus roseus. Plant J. 44:581
94
110. Nakajima K, Hashimoto T, Yamada Y. 1993. cDNA encoding tropinone reductase-II
from Hyoscyamus niger. Plant Physiol. 103:146566
111. Nakajima K, Hashimoto T, Yamada Y. 1993. Two tropinone reductases with different
stereospecicities are short-chain dehydrogenases evolved from a common ancestor. Proc.
Natl. Acad. Sci. USA 90:959195
112. Nakajima K, Hashimoto T. 1999. Two tropinone reductases, that catalyze opposite stere-
ospecic reductions in tropane alkaloid biosynthesis, are localized in plant root with
different cell-specic patterns. Plant Cell Physiol. 40:1099107
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. 63:175664
114. Nakajima K, Yamashita A, Akama H, Nakatsu T, Kato H, et al. 1998. Crystal structures of
two tropinone reductases: different reaction stereospecicities in the same protein fold.
Proc. Natl. Acad. Sci. USA 95:487681
115. Nawrot R, Kalinowski A, Gozdzicka-Jozeak A. 2007. Proteomic analysis of Chelidonium
majus milky sap using two-dimensional gel electrophoresis and tandem mass spectrome-
try. Phytochemistry 68:161222
116. Ober D, Hartmann T. 1999. Homospermidine synthase, the rst pathway-specic en-
zyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase. Proc.
Natl. Acad. Sci. USA 96:1477782
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 conrm theo-
bromine as the major intermediate for caffeine biosynthesis in coffee plants with potential
for construction of decaffeinated varieties. Plant Mol. Biol. 54:93141
119. Ogita S, Uefuji H, Yamaguchi Y, Koizumi N, Sano H. 2003. Producing decaffeinated
coffee plants. Nature 423:823

www.annualreviews.org Alkaloid Biosynthesis 765

 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 and
monoterpene-secoiridoid pathways in Catharanthus roseus. Plant Mol. Biol. 65:1330
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 somniferumcDNA cloning and characterization of methyl transfer enzymes of
alkaloid biosynthesis in opium poppy. Plant J. 36:80819
122. Panicot M, Minguet EG, Ferrando A, Alcazar R, Blazquez MA, et al. 2002. A
polyamine metabolon involving aminopropyl transferase complexes in Arabidopsis. Plant
Cell 14:253951
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. 128:696706
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

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 benzophenanthridine
by Washington State University on 11/01/11. For personal use only.

alkaloids. Plant Mol. Biol. 51:15364

125. Pauli HH, Kutchan TM. 1998. Molecular cloning and functional heterologous expres-
sion of two alleles encoding (S )-N-methylcoclaurine 3 -hydroxylase (Cyp80B1), a new
methyl jasmonate-inducible cytochrome P-450-dependent monooxygenase of benzyliso-
quinoline alkaloid biosynthesis. Plant J. 13:793801
126. Pauw B, Hilliou FA, Martin VS, Chatel G, de Wolf CJ, et al. 2004. Zinc nger proteins
act as transcriptional repressors of alkaloid biosynthesis genes in Catharanthus roseus. J.
Biol. Chem. 279:5294048
127. Pilatzke-Wunderlich I, Nessler CL. 2001. Expression and activity of cell-wall-degrading
enzymes in the latex of opium poppy, Papaver somniferum L. Plant Mol. Biol. 45:56776
128. Reimann A, Nurhayati N, Backenkohler A, Ober D. 2004. Repeated evolution of the
pyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages. Plant
Cell. 16:277284
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 Catharanthus
roseus cells. Proc. Natl. Acad. Sci. USA 103:561419
130. Robertson D. 2004. VIGS vectors for gene silencing: many targets, many tools. Annu.
Rev. Plant Biol. 55:495519
131. Ronning CM, Stegalkina SS, Ascenzi RA, Bougri O, Hart AL, et al. 2003. Comparative
analyses of potato expressed sequence tag libraries. Plant Physiol. 131:41929
132. Rueffer M, Zenk MH. 1987. Enzymatic formation of protopines by a microsomal cy-
tochrome P-450 system of Corydalis vaginans. Tetrahedron Lett. 28:530710

133. Ruppert M, Panjikar S, Barleben L, Stockigt J. 2006. Heterologous expression, purica-
tion, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant
enzyme specically involved in Rauwola alkaloid biosynthesis. Acta Crystallogr. Sect. F
Struct. Biol. Cryst. Commun. 62:25760
134. Ruppert M, Woll J, Giritch A, Genady E, Ma X, Stockigt J. 2005. Functional expression
of an ajmaline pathway-specic esterase from Rauvola in a novel plant-virus expression
system. Planta 222:88898
135. Samanani N, Alcantara J, Bourgault R, Zulak KG, Facchini PJ. 2006. The role of phloem
sieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opium
poppy. Plant J. 47:54763

766 Ziegler Facchini

 ANRV342-PP59-29 ARI 29 March 2008 2:13

136. Samanani N, Facchini PJ. 2002. Purication and characterization of norcoclaurine syn-
thase. The rst committed enzyme in benzylisoquinoline alkaloid biosynthesis in plants.
J. Biol. Chem. 277:3387883
137. Samanani N, Liscombe DK, Facchini PJ. 2004. Molecular cloning and characterization
of norcoclaurine synthase, an enzyme catalyzing the rst committed step in benzyliso-
quinoline alkaloid biosynthesis. Plant J. 40:30213
138. Samanani N, Park SU, Facchini PJ. 2005. Cell type-specic localization of transcripts
encoding nine consecutive enzymes involved in protoberberine alkaloid biosynthesis.
Plant Cell 17:91526

139. Schmidt D, Stockigt J. 1995. Enzymatic formation of the sarpagan-bridge: a key step in
the biosynthesis of sarpagine- and ajmaline-type alkaloids. Planta Med. 61:25458

140. Schroder G, Unterbusch E, Kaltenbach M, Schmidt J, Strack D, et al. 1999. Light-
induced cytochrome P450-dependent enzyme in indole alkaloid biosynthesis: tabersonine
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

16-hydroxylase. FEBS Lett. 458:97102

141. Shoji T, Nakajima K, Hashimoto T. 2000. Ethylene suppresses jasmonate-induced gene
expression in nicotine biosynthesis. Plant Cell Physiol. 41:107276
by Washington State University on 11/01/11. For personal use only.

142. Shoji T, Winz R, Iwase T, Nakajima K, Yamada Y, Hashimoto T. 2002. Expression pat-
terns of two tobacco isoavone reductase-like genes and their possible roles in secondary
metabolism in tobacco. Plant Mol. Biol. 50:42740
143. Siberil Y, Benhamron S, Memelink J, Giglioli-Guivarch N, Thiersault M, et al. 2001.
Catharanthus roseus G-box binding factors 1 and 2 act as repressors of strictosidine synthase
gene expression in cell cultures. Plant Mol. Biol. 45:47788
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 102:1491924
145. Sottomayor M, Lopez-Serrano M, DiCosmo F, Ros Barcelo A. 1998. Purication and
characterization of -3 ,4 -anhydrovinblastine synthase (peroxidase-like) from Catharan-
thus roseus (L.) G. Don. FEBS Lett. 428:299303
146. Steffens P, Nagakura N, Zenk MH. 1985. Purication and characterization of the berber-
ine bridge enzyme from Berberis beaniana cell cultures. Phytochemistry 24:257783
147. Stenzel O, Teuber M, Drager B. 2006. Putrescine N-methyltransferase in Solanum tubero-
sum L., a calystegine-forming plant. Planta 223:20012
148. Stevens LH, Blom TJM, Verpoorte R. 1993. Subcellular localization of tryptophan de-
carboxylase, strictosidine synthase and strictosidine glucosidase in suspension-cultured
cells of Catharanthus roseus and Tabernaemontana divaricata. Plant Cell Rep. 12:57376
149. St-Pierre B, De Luca V. 1995. A cytochrome P-450 monooxygenase catalyzes the rst
step in the conversion of tabersonine to vindoline in Catharanthus roseus. Plant Physiol.
109:131139
150. St-Pierre B, Laamme P, Alarco AM, De Luca V. 1998. The terminal O-acetyltransferase
involved in vindoline biosynthesis denes a new class of proteins responsible for coenzyme
A-dependent acyl transfer. Plant J. 14:70313
151. St-Pierre B, Vazquez-Flota FA, De Luca V. 1999. Multicellular compartmentation of
Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway
intermediate. Plant Cell 11:887900
152. Suzuki H, Koike Y, Murakoshi I, Saito K. 1996. Subcellular localization of acyltransferases
for quinolizidine alkaloid biosynthesis in Lupinus. Phytochemistry 42:155762
153. Suzuki K, Yamada Y, Hashimoto T. 1999. Expression of Atropa belladonna putrescine
N-methyltransferase gene in root pericycle. Plant Cell Physiol. 40:28997

www.annualreviews.org Alkaloid Biosynthesis 767

 ANRV342-PP59-29 ARI 29 March 2008 2:13

154. Suzuki K, Yun DJ, Chen XY, Yamada Y, Hashimoto T. 1999. An Atropa belladonna
hyoscyamine 6-hydroxylase gene is differentially expressed in the root pericycle and
anthers. Plant Mol. Biol. 40:14152
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. 36:2936
156. Tanahashi T, Zenk MH. 1990. Elicitor induction and characterization of microsomal
protopine-6-hydroxylase, the central enzyme in benzophenanthridine alkaloid biosyn-
thesis. Phytochemistry 29:111322
157. Teuber M, Azemi ME, Namjoyan F, Meier AC, Wodak A, et al. 2007. Putrescine N-
methyltransferases-a structure-function analysis. Plant Mol. Biol. 63:787801
158. Uefuji H, Ogita S, Yamaguchi Y, Koizumi N, Sano H. 2003. Molecular cloning and
functional characterization of three distinct N-methyltransferases involved in the caffeine
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

biosynthetic pathway in coffee plants. Plant Physiol. 132:37280

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-
by Washington State University on 11/01/11. For personal use only.

methyltrasferases and its potential as a pest repellant. Plant Mol. Biol. 59:22127
160. Unterlinner B, Lenz R, Kutchan TM. 1999. Molecular cloning and functional expression
of codeinone reductase: the penultimate enzyme in morphine biosynthesis in the opium
poppy Papaver somniferum. Plant J. 18:46575
161. van der Fits L, Memelink J. 2000. ORCA3, a jasmonate-responsive transcriptional regu-
lator of plant primary and secondary metabolism. Science 289:29597
162. Vazquez-Flota F, De Carolis E, Alarco AM, De Luca V. 1997. Molecular cloning
and 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. 34:93548
163. Veau B, Courtois M, Oudin A, Chenieux JC, Rideau M, Clastre M. 2000. Cloning and
expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus.
Biochim. Biophys. Acta 1517:15963
164. Viehweger K, Schwartze W, Schumann B, Lein W, Roos W. 2006. The G protein
controls a pH-dependent signal path to the induction of phytoalexin biosynthesis in
Eschscholzia californica. Plant Cell 18:151023
165. Vom Endt D, Soares e Silva M, Kijne JW, Pasquali G, Memelink J. 2007. Identication
of a bipartite jasmonate-responsive promoter element in the Catharanthus roseus ORCA3
transcription factor gene that interacts specically with AT-hook DNA-binding proteins.
Plant Physiol. 144:168089
166. von Schumann G, Gao S, Stockigt J. 2002. Vomilenine reductasea novel enzyme cat-
alyzing a crucial step in the biosynthesis of the therapeutically applied antiarrhythmic
alkaloid ajmaline. Bioorg. Med. Chem. 10:191318
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 and
functional bacterial expression of a plant glucosidase specically involved in alkaloid
biosynthesis. Phytochemistry 54:65766
169. Wege S, Scholz A, Gleissberg S, Becker A. 2007. Highly efcient virus-induced gene
silencing (VIGS) in California poppy (Eschscholzia californica): an evaluation of VIGS as a
strategy to obtain functional data from nonmodel plants. Ann. Bot. 100:64149

768 Ziegler Facchini

 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 in
morphine biosynthesis in the opium poppy, Papaver somniferum. Proc. Natl. Acad. Sci.
USA. 101:1395762
171. Weiss D, Baumert A, Vogel M, Roos W. 2006. Sanguinarine reductase, a key enzyme of
benzophenanthridine detoxication. Plant Cell Environ. 29:291302
172. Wink M, Hartmann T. 1982. Localization of the enzymes of quinolizidine alkaloid
biosynthesis in leaf chloroplasts of Lupinus polyphyllus. Plant Physiol. 70:7477
173. Winkel BS. 2004. Metabolic channeling in plants. Annu. Rev. Plant Biol. 55:85107
174. Winkler A, Hartner F, Kutchan TM, Glieder A, Macheroux P. 2006. Biochemical evi-
dence that berberine bridge enzyme belongs to a novel family of avoproteins containing
a bi-covalently attached FAD cofactor. J. Biol. Chem. 281:2127685
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.
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

J. Biol. Chem. 282:2443743

176. Xu D, Shen Y, Chappell J, Cui M, Nielsen M. 2007. Biochemical and molecular charac-
terization of nicotine demethylase in tobacco. Physiol. Plant. 129:30719
by Washington State University on 11/01/11. For personal use only.

177. Yamamoto H, Katano N, Ooi A, Inoue K. 2000. Secologanin synthase which catalyzes
the oxidative cleavage of loganin into secologanin is a cytochrome P450. Phytochemistry
53:712
178. Yamazaki Y, Sudo H, Yamazaki M, Aimi N, Saito K. 2003. Camptothecin biosynthetic
genes in hairy roots of Ophiorrhiza pumila: cloning, characterization and differential ex-
pression in tissues and by stress compounds. Plant Cell Physiol. 44:395403
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. 89:11799803
180. Zarate R, el Jaber-Vazdekis N, Medina B, Ravelo AG. 2006. Tailoring tropane alkaloid
accumulation in transgenic hairy roots of Atropa baetica by overexpressing the gene en-
coding hyoscyamine 6-hydroxylase. Biotechnol. Lett. 28:127177
181. Zhang L, Ding R, Chai Y, Bonll 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. Comparative
macroarray analysis of morphine containing Papaver somniferum and eight morphine
free Papaver species identies an O-methyltransferase involved in benzylisoquinoline
biosynthesis. Planta 222:45871
183. Ziegler J, Voigtlander S, Schmidt J, Kramell R, Miersch O, et al. 2006. Comparative
transcript and alkaloid proling in Papaver species identies a short chain dehydrogenase/
reductase involved in morphine biosynthesis. Plant J. 48:17792
184. Zulak KG, Cornish A, Daskalchuk TE, Deyholos MK, Goodenowe DB, et al. 2007. Gene
transcript and metabolite proling of elicitor-induced opium poppy cell cultures reveals
the coordinate regulation of primary and secondary metabolism. Planta 225:1085106

www.annualreviews.org Alkaloid Biosynthesis 769

 AR342-FM ARI 26 March 2008 19:43

Annual Review of
Plant Biology

Contents Volume 59, 2008

Our Work with Cyanogenic Plants

Eric E. Conn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

New Insights into Nitric Oxide Signaling in Plants

Angelique Besson-Bard, Alain Pugin, and David Wendehenne p p p p p p p p p p p p p p p p p p p p p p p p p 21
by Washington State University on 11/01/11. For personal use only.

Plant Immunity to Insect Herbivores

Gregg A. Howe and Georg Jander p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 41
Patterning and Polarity in Seed Plant Shoots
John L. Bowman and Sandra K. Floyd p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 67
Chlorophyll Fluorescence: A Probe of Photosynthesis In Vivo
Neil R. Baker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 89
Seed Storage Oil Mobilization
Ian A. Graham p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p115
The Role of Glutathione in Photosynthetic Organisms:
Emerging Functions for Glutaredoxins and Glutathionylation
Nicolas Rouhier, Stephane D. Lemaire, and Jean-Pierre Jacquot p p p p p p p p p p p p p p p p p p p p p143
Algal Sensory Photoreceptors
Peter Hegemann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p167
Plant Proteases: From Phenotypes to Molecular Mechanisms
Renier A.L. van der Hoorn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p191
Gibberellin Metabolism and its Regulation
Shinjiro Yamaguchi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p225
Molecular Basis of Plant Architecture
Yonghong Wang and Jiayang Li p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p253
Decoding of Light Signals by Plant Phytochromes
and Their Interacting Proteins
Gabyong Bae and Giltsu Choi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p281
Flooding Stress: Acclimations and Genetic Diversity
J. Bailey-Serres and L.A.C.J. Voesenek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p313

v
 AR342-FM ARI 26 March 2008 19:43

Roots, Nitrogen Transformations, and Ecosystem Services

Louise E. Jackson, Martin Burger, and Timothy R. Cavagnaro p p p p p p p p p p p p p p p p p p p p p p p341
A Genetic Regulatory Network in the Development of Trichomes
and Root Hairs
Tetsuya Ishida, Tetsuya Kurata, Kiyotaka Okada, and Takuji Wada p p p p p p p p p p p p p p p p p p365
Molecular Aspects of Seed Dormancy
Ruth Finkelstein, Wendy Reeves, Tohru Ariizumi, and Camille Steber p p p p p p p p p p p p p p p387
Trehalose Metabolism and Signaling
Matthew J. Paul, Lucia F. Primavesi, Deveraj Jhurreea, and Yuhua Zhang p p p p p p p p417
Auxin: The Looping Star in Plant Development
Rene Benjamins and Ben Scheres p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

Regulation of Cullin RING Ligases

by Washington State University on 11/01/11. For personal use only.

Sara K. Hotton and Judy Callis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p467

Plastid Evolution
Sven B. Gould, Ross F. Waller, and Geoffrey I. McFadden p p p p p p p p p p p p p p p p p p p p p p p p p p p p p491
Coordinating Nodule Morphogenesis with Rhizobial Infection
in Legumes
Giles E.D. Oldroyd and J. Allan Downie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p519
Structural and Signaling Networks for the Polar Cell Growth
Machinery in Pollen Tubes
Alice Y. Cheung and Hen-ming Wu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p547
Regulation and Identity of Florigen: FLOWERING LOCUS T Moves
Center Stage
Franziska Turck, Fabio Fornara, and George Coupland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p573
Plant Aquaporins: Membrane Channels with Multiple Integrated
Functions
Christophe Maurel, Lionel Verdoucq, Doan-Trung Luu, and Veronique Santoni p p p p595
Metabolic Flux Analysis in Plants: From Intelligent Design
to Rational Engineering
Igor G.L. Libourel and Yair Shachar-Hill p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p625
Mechanisms of Salinity Tolerance
Rana Munns and Mark Tester p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p651
Sealing Plant Surfaces: Cuticular Wax Formation by Epidermal Cells
Lacey Samuels, Ljerka Kunst, and Reinhard Jetter p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p683
Ionomics and the Study of the Plant Ionome
David E. Salt, Ivan Baxter, and Brett Lahner p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p709

vi Contents
 AR342-FM ARI 26 March 2008 19:43

Alkaloid Biosynthesis: Metabolism and Trafcking

Jorg Ziegler and Peter J. Facchini p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p735
Genetically Engineered Plants and Foods: A Scientists Analysis
of the Issues (Part I)
Peggy G. Lemaux p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p771

Indexes

Cumulative Index of Contributing Authors, Volumes 4959 p p p p p p p p p p p p p p p p p p p p p p p p813

Cumulative Index of Chapter Titles, Volumes 4959 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p818
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org

Errata
by Washington State University on 11/01/11. For personal use only.

An online log of corrections to Annual Review of Plant Biology articles may be found
at http://plant.annualreviews.org/

Contents vii