Available online at www.sciencedirect.
com
ScienceDirect
Making iridoids/secoiridoids and monoterpenoid indole alkaloids:
progress on pathway elucidation
Vincenzo De Luca1, Vonny Salim1,2, Antje Thamm 1,
Sayaka Atsumi Masada1,3 and Fang Yu1,4
Members of the Acanthaceae, Apocynaceae, Bignoniaceae,
Caprifoliaceae, Gentianaceae, Labiatae, Lamiaceae, Loasaceae,
Loganiaceae, Oleaceae, Plantaginaceae, Rubiaceae,
Saxifragaceae, Scrophulariaceae, Valerianaceae, and
Verbenaceae plant families are well known to accumulate
thousands of bioactive iridoids/secoiridoids while the
Apocynaceae, Loganiaceae and Rubiaceae families also
accumulate thousands of bioactive monoterpenoid indole
alkaloids (MIAs), mostly derived from the secologanin and
tryptamine precursors. Several large-scale RNA-sequencing
projects have greatly advanced the tools available for identifying
candidate genes whose gene products are involved in the
biosynthesis of iridoids/MIAs. This has led to the rapid
comparative bioinformatics guided elucidation of several key
remaining steps in secologanin biosynthesis as well as other steps
in MIA biosynthesis. The availability of these tools will permit broad
scale biochemical and molecular description of the reactions
required for making thousands of iridoid/MIAs. This information
will advance our understanding of the evolutionary and ecological
roles played by these metabolites in Nature and the genes will be
used for biotechnological production of useful iridoids/MIAs.
Addresses
1
Department of Biological Sciences, Brock University, 500 Glenridge
Avenue, St. Catharines, Ontario L2S 3A1, Canada
2 pathways.
Biochemistry and Molecular Biology, Michigan State University, East
Lansing, MI 48824-1319, United States
3
Division of Pharmacognosy, Phytochemistry and Narcotics, National
Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-
8501, Japan
4
School of Biological Engineering, Dalian Polytechnic University, #1
Qinggongyuan, Dalian, Liaoning 116034, China
Corresponding authors: De Luca, Vincenzo (vdeluca@brocku.ca), Salim,
Vonny (vsalim@msu.edu), Thamm, Antje (smasadaatsumi@nihs.go.jp),
Masada, Sayaka Atsumi (at07ty@badger.ac.brocku.ca) and Yu, Fang
(fyu0506@gmail.com)
Current Opinion in Plant Biology 2014, 19:35–42
This review comes from a themed issue on Physiology and
metabolism
Edited by Sarah E O’Connor and Thomas P Brutnell
For a complete overview see the Issue and the Editorial
Available online 5th April 2014
1369-5266/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.pbi.2014.03.006
Introduction
Plants are the source of many thousands of complex
chemicals that include specialized metabolites such as
www.sciencedirect.com
phenols, terpenes and alkaloids. The alkaloids alone
contain nitrogen in their basic structures that are derived
mainly from the a few amino acid precursors and that
confer unique biological activities to this class of mol-
ecules. In spite of their important pharmacological activi-
ties and medicinal uses, the pathways of alkaloid
biosynthesis remain poorly understood. This lack of
progress has been attributed to the unavailability of
the substrate intermediates required for detecting the
biochemical reactions in multistep pathways and to
inherent difficulties associated with the slow traditional
forward genetic approaches for identifying the genes
involved.
The present review will focus on the recent use of
large-scale transcriptome projects [Phytometasyn
(http://www.phytometasyn.com/; [1,2]); Medicinal
Plant Genomics Consortium (http://www.medicinal
plantgenomics.msu.edu/; [2,3,4]); Medicinal Plant
Transcriptome Project (http://www.uic.edu/pharmacy/
MedPlTranscriptome/index.html)] and CATHACyc
(http://www.cathacyc.org/; [5]) that are speeding up the
discovery of monoterpenoid indole alkaloid biosynthesis
Monoterpenoid indole alkaloids
The monoterpenoid indole alkaloids (MIAs) represent
one of the largest and most biologically active classes of
special metabolites found in thousands of species of
plants of the Apocynaceae, Loganiaceae and Rubiaceae
families. These MIAs include the Catharanthus roseus
vinblastine/vincristine and Camptotheca acuminata/Ophior-
hiza pumila camptothecin anticancer alkaloids, the anti-
malarial quinine from Cinchona ledgeriana/C. succirubra and
the antihypertensive drug ajmalicine from Catharanthus
rosues/Rauvolfia serpentina. The pathways involved
require the condensation of secologanin with tryptamine
to yield strictosidine that is then converted to several
thousand MIAs found in Nature. While almost 200 MIAs
have been described in C. roseus, catharanthine and vindo-
line are present in greatest abundance and have great
commercial value since dimers of these two compounds
are drugs used in cancer chemotherapy. The Cathar-
anthus literature related to MIA biosynthesis, its organ-
ization in different leaf cell types [6–8] and its
biotechnological applications [9] has recently (been)
reviewed and has been extensively reviewed over the
past 10 years.
Current Opinion in Plant Biology 2014, 19:35–42
36 Physiology and metabolism
Missing genes for the supply of geraniol in
iridoid biosynthesis
Higher plants have cytosolic mevalonic acid and plastidic
methyl erythritol phosphate (MEP) pathways that pro-
vide isoprenoid precursors for a large range of monoter-
pene, sesquiterpene, diterpene, triterpene, tetraterpene
and polyterpene. In ‘‘range of monoterpene, sesquiterpene,
diterpene, triterpene, tetraterpene and polyterpene products’’
roseus, most of MEP pathway genes have been charac-
terized and were shown to be preferentially expressed in
specialized Internal Phloem Associated Parenchyma
(IPAP) cells (Figure 1; [6,7]) in order to supply precursors
for the assembly of iridoid precursors required for the
biosynthesis of secologanin in C. roseus. These IPAP cells
also preferentially express geraniol 10-hydroxylase
(G10H) (Figure 1, reaction 2) and 10-hydroxygeraniol
oxidoreductase (10HGO) (Figure 1, reaction 3), while
the two terminal reactions in secologanin biosynthesis are
known to be preferentially expressed in the leaf
epidermis (Figure 1, reactions 8 and 9 [6]). However
several genes including isopenenyl diphosphate synthase,
geraniol synthase (Figure 1, reaction 1) and other gene
products responsible the formation of loganic acid
remained to be described (Figure 1, reactions 4–7; iridoid
synthase/monoterpene cyclase, 7-deoxyloganetic acid
synthase, 7-deoxyloganetic acid glucosyltransferase, and
7-deoxyloganic acid hydroxylase).
Isopentenyl diphosphate isomerase (IDI) mediates the
equilibrium and supply of isopentenyl diphosphate (IPP)
and dimethylallyl diphosphate (DMAPP) required for the
assembly of geranyl pyrophosphate (GPP). Recent stu-
dies have described a single gene in Catharanthus with
alternative transcription sites to generate different iso-
forms of CrIDI that appear to be targeted to peroxisomes,
mitochondria and plastids [10]. Presumably the plastid
targeted isoform would also be expressed in IPAP cells
where it would participate in providing IPP and DMAPP
for biosynthesis of GPP.
While the MEP pathway is responsible for the formation
of IPP and DMAPP, geranyl pyrophosphate synthase
(GPPS) is required for the formation of GPP. Recently
several CrGPPS genes identified from C. roseus, through
database searches (www.ncbi.nlm.nih.gov/and http://
medicinalplantgenomics.msu.edu) [11] were functionally
characterized. Three separate types of CrGPPS were
functionally characterized with two of them being
expressed in chloroplasts and one of them more likely
to be targeted to mitochondria. While the results com-
plemented previous studies on the functional character-
ization and plastid localization of one of these genes [12]
several other CrGPPS genes were also described to be
present in Catharanthus [11] that make it difficult to
establish how many of these genes provide precursor
GPP for the biosynthesis of iridoids. Therefore, it is
interesting and perhaps understandable that neither
Current Opinion in Plant Biology 2014, 19:35–42
study reported if any of these CrGPPS genes might be
preferentially expressed in IPAP cells.
The molecular cloning of C. roseus geraniol synthase
(CrGS) was also recently described and recombinant
protein was shown to convert geraniol pyrophosphate
(GPP) to geraniol when expressed in Escherichia coli
[13]. A significant finding of this study was that CrGS
appears to be preferentially expressed in IPAP cells
where it converts MEP-derived GPP to produce the
geraniol required for the G10H that produces 10-hydro-
xygeraniol. IPAP cells are known to convert geraniol to
10-oxogeranial via G10H (Figure 1, reaction 2) and
10HGO (Figure 1, reaction 3). Together these character-
izations of CrGPPS and CrGS complete the metabolic
connection of the MEP pathway to G10H.
Assembly of secologanin
Over 2500–3000 iridoids/secoiridoids with a wide variety of
biological properties [14,15] have been characterized after
isolation from members of the Acanthaceae, Apocynaceae,
Bignoniaceae, Caprifoliaceae, Gentianaceae, Labiatae,
Lamiaceae, Loasaceae, Loganiaceae, Oleaceae, Plantagi-
naceae, Rubiaceae, Saxifragaceae, Scrophulariaceae,
Valerianaceae, and Verbenaceae plant families. These
unusual monoterpenoids contain a methylcyclopentan-
[c]-pyran skeleton typically fused to a six-membered ox-
ygen containing heterocycle. While iridoids participate in
MIA biosynthesis within the Apocynaceae, Loganiaceae
and Rubiaceae families this mostly involves a single iri-
doid, secologanin that provides a C10 or C9 or ocassonally a
C8 fragment to the formation of MIAs. However the levels
of MIAs produced may be small compared to the levels of
iridoids that accumulate within MIA producing plant
species. Recent studies in C. roseus have shown that the
levels of secologanin in Catharanthus leaves were 10–15
times higher than those of the major MIAs, catharanthine
and vindoline [16–18]. This raises important questions
about the biological importance of maintaining high levels
of secologanin in Catharanthus and perhaps in other MIA
accumulating plant species where little documentation
exists about the levels of these iridoids. The mobility of
iridoids was recently documented when photosynthesis
was measured in Snapdragon (Antirrhinum majus) leaves by
feeding them with 14CO2 [19]. Measurements showed that
47% of the phloem mobile 14C-photoassimilate was the
iridoid antirrhinoside with sucrose making up the rest. The
study suggested that rapid conversion of the products of
photosynthesis to toxic antirrhinoside could provide a se-
lective advantage to iridoid producing species by deterring
herbivory by phloem feeding insects and this was sup-
ported in other studies with Alonsoa meridionalis and Asar-
ina barclaiana describing the phloem mobility of
antirrhinoside [20]. This raises interesting questions about
the mechanisms of iridoid biosynthesis that might involve
biochemical cellular specialization events that may be
common to iridoid and/or MIA producing plant species
www.sciencedirect.com
 Iridoid/secoiridoid and monoterpenoid indole alkaloid pathway elucidation De Luca et al. 37

Figure 1

upper & lower epidermis

N
CrTPT2
catharanthine
N
transporter
H
CO2CH3
strictosidine
(12)
upper cuticle catharanthine H3CO N
H CO2CH3
upper epidermis secologanin tryptamine
(9) (11) 16-methoxytabersonine
idioblast loganin tryptophan
(8)
loganic acid ?

?
IPAP
laticifer loganic acid
(7) Laticifers &
7-deoxyloganic acid
ldioblasts
(6)
EPAP 7-deoxyloganetic acid
(5)
Iridodial
idioblast H3CO N OAc
(4) CH3
CO2CH3
10-oxogeranial
lower epidermis (3) Last 4 steps in
lower cuticle 10-hydroxygeraniol vindoline
(2) biosynthesis
geraniol IPAP
(1) cells
MEP pathway

Current Opinion in Plant Biology

Biosynthesis of secologanin/MIAs and transporters required for their assembly. The methylerythritol phosphate pathway supplies precursors through
geraniol synthase (1) to form loganic acid via geraniol hydroxylase (2), 10-hydroxygeraniol oxidoreductase (3), iridodial cyclase (4), 7-deoxyloganetic
acid synthase (5), 7-deoxyloganetic acid glucosyltransferase (6) and deoxyloganic acid 7-hydroxylase (7) in internal phloem associated (IPAP) cells but
not in external phloem associated (EPAP) cells. Loganic acid is transported by unknown mechanisms to the leaf epidermis to be converted to
secologanin by loganic acid methyltransferase (8) and secologanin synthase (9). The leaf epidermis tryptophan decarboxylase (11) converts
tryptophan into tryptamine that is used with secologanin by strictosidine synthase to generate strictosidine. Strictosidine b glucosidase activity
generates the labile strictosidine aglycone that serves to produce different ring re-arrangements leading to the formation of corynanthe (example
ajmalicine), iboga (example catharanthine) and aspidosperma (example tabersonine) alkaloids through a series of reactions that have not been
characterized at the molecular level. The catharanthine is exported through the CrTPT2 transporter to accumulate in the upper and lower cuticles of
Catharanthus leaves. Tabersonine is converted by tabersonine-16-hydroxylase and 16-hydroxytabersonine-16-O-methyltransferase to generate 16-
methoxytabersonine. This MIA is then mobilized by an unknown transport mechanism to laticifers and idioblasts where the remaining four steps in
vindoline biosynthesis will take place and vindoline may be transferred to the tonoplast by a vacuolar membrane-associated proton antiport system
that remains to be characterized at the molecular level.

such as specialized iridoid biosynthesis IPAP cells
(Figure 1).
The key cyclization step for conversion of 10-oxogeranial
to cis–trans-nepetalactol was identified by using a tran-
scriptomic approach (http://www.medicinalplantgen-
omics.msu.edu/) to identify NADPH-using enzymes
that appeared to be co-regulated with other MIA pathway
genes (Figure 1; reaction 4) [21]. While this iridoid
cylcase showed 67% identity to progesterone-5b-
reductase that reduces a double bond in the biosynthesis
of cardenolides in Digitalis purpurea its expression was
restricted to the same IPAP cells where G10H is prefer-
entially expressed. While the recombinant iridoid cyclase
converted 10-oxogeranial to cis–trans-nepetalactol, it was
unable to catalyze the reduction of progesterone. In
contrast to all known monoterpene cyclases that involve
a cation indermediate produced from geranyl dipho-
sphate, this unique cyclase used 10-oxogeranial in an
NAD(P)H requiring reaction. The role of iridoid cyclase
was corroborated by virus induced gene silencing (VIGS)
since silenced plants expressed lower cyclase levels and
accumulated lower levels of the major MIAs, cathar-
anthine and vindoline.

www.sciencedirect.com Current Opinion in Plant Biology 2014, 19:35–42

38 Physiology and metabolism

The successful identification, cloning and functional
characterization of 7-deoxyloganetic acid synthase
(7DLS) [16] and 7-deoxyloganic acid 7-hydroxylase
(DL7H) [17] involved a bioinformatic search [6,22,23]
for homologous cytochrome P 450 (CYP) candidate genes
from annotated express sequence tag databases of seven
secologanin/MIA producing plant species from the Apoc-
ynaceae family, one secologanin/quinoline alkaloid produ-
cing species from the Rubiaceae family and one
secologanin producing species from the Caprifoliaceae
family (Figure 2, databases found at www.phytometa-
syn.ca). This screen identified three C. roseus candidate
genes that were expressed only in secologanin accumulat-
ing plant species, but not in dozens of other transcriptome
databases from other species of plants that do not accumu-
late this metabolite. Selected genes were then silenced by
VIGS in C. roseus and silenced plants were monitored for a
decline in secologanin/MIA levels (Figure 2; [16,17]).
The silencing of 7DLS and DL7H genes triggered signifi-
cant declines of secologanin/MIAs in suppressed plants
and in the case of DL7H suppressed lines they accumulated
significant amounts of the DL7H substrate, 7-deoxylo-
ganic acid. Functional expression of the 7DLS and
DL7H genes in yeast identified them as authentic CYPs
involved in the biosynthesis of 7-deoxyloganetic acid and
loganic acid, respectively (Figure 1, reactions 5 and 7)).
Gardenia jasminoides that accumulates the iridoids genipo-
side and gardenoside was used to identify the first known
iridoid glucosyltransferase gene [24] from cell cultures
that were shown to glucosylate genipin to form genipo-
side and gardenoside, respectively. The molecular clon-
ing approach involved the design of primers based on a
conserved plant secondary product glycosyltransferase

Figure 2

16OMT
100%

T16H2
MECS

LAMT
50%
35%

UGT7
UGT8
G10H

DL7H
7DLS

TPT2
SGD

NMT
DXR

GES
DXS

TDC

DAT
STR

D4H
SLS
IDI1

IS

Catharanthus roseus
Catharanthus ovalis
Catharanthus longifolius
Amsonia hubrichtii
Vinca minor
Rauvolfia serpentina
Tabernaemontata elegans
Lonicera japonica
Cinchona ledgeriana
Camptotheca acuminata
Selection of candidate genes

Toolkit:
– gene expression profiling
– virus induced gene silencing
– targeted metabolite profiling
– functional identification of recombinant proteins

rapid pathway discovery

Current Opinion in Plant Biology

Candidate gene discovery of biosynthetic pathways through comparative transcriptomics. Comparative heat map of some known MEP, iridoid and
MIA pathway enzymes in C. roseus to candidate gene products found in Catharanthus ovalis, Catharanthus longifolius, Amsonia hubrichtii, Vinca minor,
Rauvolfia serpentina, Tabernaemontana elegans, Lonicera japonica, Cinchona ledgeriana and Camptotheca acuminata. protein amino. The amino acid
sequence similarities range from 33 to100% (Supplementary Table 1). The amino acid identities of genes from different species were imported into
Cluster 3.0 (Michiel de Hoon, University of Tokyo; http://bonsai.hgc.jp/mdehoon/software/cluster/) and hierarchical clustering of species was done
with uncentered correlation and centroid linkage. The heat map was visualized in Tree View [34]. The selected candidate gene is then tested with a
toolkit that might be used in a series of steps available for a plant species, leading to functional gene discovery. Abbreviations of genes in the figure
are: [DXS (1-deoxy-D-xylulose-5-phosphate-synthase); DXR (1-deoxy-D-xylulose-5-phosphate reductase); IDII; MECS; (4-cytidyl-diphospho-2-C-
methyl-D-erythritol synthase); GES (geraniol synthase); G10H (geraniol 10-hydroxylase); IS (iridodial cyclase); 7DLS (7-deoxyloganetic acid synthase);
UGT7, (7-deoxyloganetic acid glucosyltransferase) UGT8, (weak 7-deoxyloganetic acid glucosyltransferase); DL7H (deoxyloganic acid 7-hydroxylase);
LAMT (loganic acid methyltransferase); SLS (secologanin synthase); TDC (tryptophan decarboxylase); STR (strictosidine synthase); SGD (strictosidine
b glucosidase); T16H (tabersonine-16-hydroxylase); 16OMT (16-hydroxytabersonine-16-O-methyltransferase); NMT (16-methoxytabersonine-N-
methyltransferase); D4H (deacetoxyvindoline 4-hydroxylase); DAT (deacetylvindoline 4-O-acetyltransferase); TPT2 (catharanthine transporter)].

Current Opinion in Plant Biology 2014, 19:35–42 www.sciencedirect.com

 Iridoid/secoiridoid and monoterpenoid indole alkaloid pathway elucidation De Luca et al. 39
box that ultimately led to the cloning, functional expres-
sion of 13 candidate GTs and the identification of a single
candidate that preferentially glucosylated 7-deoxyloga-
netin and genipin, showed weak activity towards loga-
netin and was inactive with 7-deoxyloganetic acid. These
results strongly suggested that methylation of the
carboxyl group occurred before glucosylation in contrast
to the results obtained for the loganic acid O-methyl-
transferase reaction from C. roseus that takes place after
glucosyltation [25]. The same molecular approach to
clone iridoid GTs in Catharanthus cell cultures com-
bined with mining of a Catharanthus database found in
the PlantGDB server (http://plantgdb.org/cgi-bin/blast/
PlantGDBblast) with the Gardenia clone led to the
identification of three candidate iridoid GTs [18]. One
was a higly specific 7-deoxyloganetic acid GT (UGT8)
whose expression was restricted to Catharanthus leaves
and was found in a Catharanthus leaf EST database
(http://www.phytometasyn.ca/). The other two were 7-
deoxyloganetin GTs (UGT6 and UGT7) but UGT7 had
weak iridoid GT activity and did also glucosylate sub-
strates like curcumin, genistein, luteolin, and kaemp-
ferol. In additional studies, tissues were abraded with
carborundum particles to preferentially extract RNA
from Catharanthus leaf tissues to show that both
UGT6 and UGT8 were preferentially expressed within
the leaf rather than in the epidermis where the last two
steps in secologanin biosynthesis were preferentially
expressed. In situ hybridization studies localized expres-
sion UGT8 to IPAP cells.
The presence of separate gene products (UDP6 and
UGT8) that can glucosylate 7-deoxyloganetic acid in
leaves and 7-deoxyloganetin in cell suspension cultures,
respectively, raise the possibility the separate pathways
may be involved in secologanin biosynthesis in leaves and
roots. These data as well as other research (reviewed in
[26]) indicates that the organization of MIA biosynthesis
in roots is quite different from that of leaves. The charac-
terization of root MIA pathways remains an important and
key objective that requires urgent attention. These
recent discoveries complete the characterization of the
eight genes required to form secologanin from geraniol
and should facilitate metabolic engineering efforts to
produce iridoids or MIAs in plants or in heterologous
systems such as yeast [6–8,9]. However it remains to be
determined what transport mechanisms might be
involved in the export of loganic acid from IPAP cells,
its transport to leaf epidermis and its import into these
cells for subsequent O-methylation and ring opening to
produce secologanin (Figure 1).
Discovery of new MIA pathway genes
Several genes involved in MIA biosynthesis have been
identified and functionally characterized. These include
tryptophan decarboxylase, strictosidine synthase, stricto-
sidine b glucosidase, tabersonine 16-hydroxylase, taber-
www.sciencedirect.com
sonine 19-hydroxylase, 16-hydroxytabersonine-16-O-
methyltransferase, 2,3-dihydro-3hydroxytabersonine-N-
methyltransferase, deacetoxyvindoline-4-hydroxylase
deacetylvindoline-O-acetyltransferase, vacuolar class III
peroxidase and NADPH cytochrome C reductase [6–
8,9]. However the genes responsible for numerous reac-
tions that convert strictosidine aglycone and intermediates
to form the three key classes of MIAs represent a major
challenge to the understanding of their biosynthesis.
A recent study showed that T16H is actually encoded by
two distinct cytochrome P 450 genes (CYP71D12,
CYP71D351) with the newly reported gene
(CYP71D351) being the main isoform expressed in Cath-
aranthus leaf epidermis while the second isoform was
mainly expressed in floral tissues [27]. The two T16H
genes share 82% amino acid sequence identity suggesting
a common ancestral origin. While the high substrate
specificity of both gene products for tabersonine makes
it difficult to understand why independent genes should
be expressed in separate Catharanthus organs, this study
did help to clarify a number of unexplained results about
the complexity of T16H expression in Catharanthus,
including the failure to detect the CYP71D12 form of
T16H in leaves by in situ hybridization when CYP71D351
form of T16H was successfully detected in leaf epidermis
[27]. This raises an important question concerning the
cell-type specific expression of the CYP71D12 form of
T16H in developing flowers. This information might help
to reveal if this form of the enzyme might be involved in
other biochemical processes, in addition to this reaction in
developing flowers.
The organization of MIA biosynthesis in different cell
types in C. roseus leaves (Figure 1) show that the last two
steps in secologanin biosynthesis, the decarboxylation of
tryptamine, the assembly of strictosidine and its conver-
sion to catharanthine and 16-methoxytabersonine occurs
in the leaf epidermis while the remaining four steps in
vindoline biosynthesis takes place inside the leaf within
mesophyll cells and/or in specialized idioblasts/laticifers.
The biosynthesis of catharanthine has been suggested to
occur in leaf epidermal cells by the almost exclusive
presence of this MIA in chloroform extracts of leaf
surfaces [28]. In addition, the lack of vindoline in
chloroform extracts and its presence within leaves sup-
ported the localization of the late stages of vindoline
biosynthesis to the mesophyll and provided a plausible
explanation for the low levels of vindoline/catharanthine
dimers found in Catharanthus leaf tissues.
The presence of catharanthine on leaf surfaces and of
vindoline within mesophyll was used to suggest that
transport mechanisms must be present in order to allow
movement of catharanthine and 16-methoxytabersonine
from the leaf epidermis to each cellular compartment
(Figure 1; [28]). The leaf surface localization of
Current Opinion in Plant Biology 2014, 19:35–42
40 Physiology and metabolism
catharanthine stimulated a search for possible alkaloid
transporters that might be involved in the secretion
process. This led to the discovery [29] of an ATP-
binding cassette transporter that is expressed predomi-
nantly in the epidermal cells of young leaves and that
appears to be responsible for the secretion of cathar-
anthine to the leaf surface [28]. Phylogenetic analysis
(http://www.phytometasyn.ca/) suggested that the cath-
aranthine transporter appears to be restricted to plant
species active in MIA biosynthesis and is closely related
to a common plasma membrane ABC transporter found in
Arabidopsis, barley and rice involved in cuticle assembly.
These specialized plasma membrane ABC-PDR-cathar-
anthine-like transporters were also identified in plants
from distinct geographical origins (Eurasian Vinca minor,
African African Tabernamontana elegans, Indian R. serpen-
tina, South American C. ledgeriana, and North American
Amsonia hubrichtii). The presence of such a transporter in
plants from diverse locations was used to suggest that the
appearance of MIA biosynthesis in plants may have
occurred coincidentally with the evolution of such
secretory mechanisms and that many more plants from
this family secrete their MIAs to their respective surfaces
[28,29].
A separate recent biochemical study documented the
presence in Catharanthus leaves of a vacuolar mem-
brane-associated proton antiport system that requires a
pH gradient generated by an ATPase and/or pryropho-
sphatase tonoplast pump to transport vindoline [30].
This detailed study confirmed earlier research that
described such a transporter found in vacuoles isolated
from Catharanthus cell cultures [31,32]. Mobilization of
vindoline occurred through an active ATP-mediated or
PPi-mediated trans-tonoplast transporter that followed
Michaelis-Menton saturation kinetics. The study also
suggested with less convincing evidence that cathar-
anthine and AVLB might also be transported across
tonoplast membranes by the same system. In this context
isolated vacuoles were shown to contain similar levels of
vindoline and catharanthine, but no AVLB could be
detected. It is of great importance that this vindoline
transporter be cloned and characterized in order to further
elucidate the intercellular organization of MIA biosyn-
thesis (Figure 1).
Future directions
This short review highlights the most recent develop-
ments in our knowledge of MIA biosynthesis and illus-
trates how large-scale sequencing projects of non-model
medicinal plants is providing novel tools and approaches
for identifying target genes by performing mass transcrip-
tome analyses combined with phylogenetic studies in
relation to the known phytochemical composition of
target plant species. This information combined with
tools such as VIGS, carborundum abrasion, in situ hybrid-
ization and metabolite profiling are being used to rapidly
Current Opinion in Plant Biology 2014, 19:35–42
narrow down candidate genes for functional expression
and discovery of their biochemical roles. One of the most
important recent developments has been the elucidation
of the steps leading to the formation of secologanin. This
will have important future impacts for production of
iridoids/MIAs in various heterologous microorganisms/
yeast/plants beginning with systems that produce stricto-
sidine, the central precursor of most MIAs, as attested by
several personal communications and studies making
their way towards publication [33]. These production
systems will have a huge impact on the production of
any number of iridoids or MIAs that have particular uses
in various human activities. In addition the elucidation of
the secologanin pathway is likely to have important con-
sequences for the enhanced general discovery of iridoid
pathways in any number of desired plant species. Progress
will be rapid for elucidating the remaining steps in
biosynthesis and transport of vindoline, catharanthine,
camptothecin and many other biologically active mol-
ecules from plants. The possibility that most MIAs occur
on the above ground surfaces of plants needs to be studied
and elaborated upon with fundamental studies on the
evolutionary processes involved as well as the chemical
and environmental ecology that may be responsible
selecting this secretory process.
Acknowledgements
We wish to express our apologies to colleagues whose work has not been
highlighted due to space constraints. We gratefully acknowledge funding
from a Discovery Grant provided by National Sciences and Engineering
Research Council of Canada and from a Canada Research Chair in Plant
Biotechnology to Vincenzo De Luca.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.pbi.2014.03.006.
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