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The cell and developmental biology
of alkaloid biosynthesis
Vincenzo De Luca and Benoit St Pierre
Plants produce unique natural products as a result of gene mutation and subsequent adaptation of metabolic pathways to create new secondary metabolites. However, their biosynthesis
and accumulation remains remarkably under the control of the biotic and abiotic environments.
Alkaloid biosynthesis, which requires the adaptation of cellular activities to perform specialized
metabolism without compromising general homeostasis, is accomplished by restricting
product biosynthesis and accumulation to particular cells and to defined times of plant development. The cell and developmental biology of alkaloid biosynthesis, which is remarkably
complex, evolved in part by recruiting pre-existing enzymes to perform new functions.
A
lkaloids are low molecular weight nitrogen-containing
substances with characteristic toxicity and pharmacological activity. These properties, which have traditionally
been exploited by humans for hunting, execution and warfare,
have also been used for the treatment of disease1. About 20% of
plant species accumulate alkaloids, which are mostly derived
from the amino acids, Phe, Tyr, Trp, Lys and Orn. In addition, the
monoterpenoid indole alkaloids, which form a large class of complex compounds, are derived from Trp and terpenoid precursors.
Over 12 000 different alkaloids have been described, indicating
their structural and biosynthetic diversity compared to that of
other secondary metabolites2 (Table 1).
Our extensive knowledge about the chemistry and pharmacology of alkaloids has led to their use in a range of medical applications. However, little is known about their biosynthesis or about
the factors that regulate alkaloid production2–5. Recently, intensive
efforts have elucidated the basic biochemistry and molecular biology of some alkaloid pathways and, more generally, of secondary
metabolism6. These discoveries are now enabling more rapid consensus cloning and identification of highly specific biochemical
reactions involved in the production of interesting alkaloids4,7,8.
These results support chemical reasoning that the diversification
of secondary metabolism originated from the elaboration of a few
central intermediates that could be modified through oxidation,
followed by the protection of hydroxyl groups by acylation, glucosylation, methylation, or among others, prenylation (Fig. 1). In
this context, the Arabidopsis genome, which has yet to be completely sequenced, contains large gene families for each of these
classes of biochemical reactions, including cytochrome P450
mono-oxygenases, dioxygenases, acyltransferases, methyltransferases and glucosyltransferases6.
Additional studies have begun to illustrate that alkaloid biosynthesis is not a random process, but is highly ordered with respect
Table 1. Plant secondary metabolites
with known structures
Secondary metabolites
Alkaloids
Terpenoids
Phenylpropanoids
Others
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April 2000, Vol. 5, No. 4
Number of structures
12 000
30 000
2500
2500
to plant development, controlling the expression of pathways within
organs, within specific cells, or within organelles inside those
cells. Here we review recent developments in our understanding
of the cell and developmental biology of alkaloid biosynthesis and
their biological origins.
Alkaloids of the Solanaceae
The Solanaceae produce a variety of interesting biologically active
products including the steroid alkaloids solanidine, nicotine (Fig. 1)
and tropane alkaloids (Fig. 2). For example, it is well known that
domestication of potatoes involved the successful elimination of
toxic steroid alkaloids in tubers while retaining steroid alkaloids
in other plant parts for biotic defense. The tropane alkaloids,
which have a colorful history of human use, have inspired the
creation of several synthetic analogs. These are used to control
motion sickness, as powerful bronchodilators to treat chronic
bronchitis, as antimuscarinic drugs for the control of Parkinson’s
disease, or as midriatics, which are used to dilate the pupil of the
eye to facilitate surgery. In the case of nicotine, breeding efforts
have focused on increasing the levels of this alkaloid in tobacco
for smoking purposes. The continuing appeal of cigarettes to
humans can be explained by the rapid absorption, transport and
binding of nicotine to acetylcholine receptors in the brain and the
ensuing pleasure obtained.
Root-specific scopolamine and nicotine biosynthesis
Roots have been shown to elaborate a remarkable variety of
secondary metabolites9, including alkaloids, flavonoids and
terpenoids. Nicotine and tropane alkaloids, which are mainly produced in roots as suggested by reciprocal grafting experiments10,
accumulate mostly within vacuoles2 of plant roots and leaves. The
early pathway leading to biosynthesis of both nicotine (Fig. 1) and
tropane (Fig. 2) alkaloids involves the formation of a pyrrolidine
ring that is derived from putrescine via the sequential action of
an S-adenosyl-L-methionine-dependent putrescine-N-methyltransferase (PMT), a diamine oxidase and a spontaneous chemical rearrangement. The presence of PMT is unique to alkaloid-producing
plants and it appears to have evolved from spermidine synthase11
(SPDS), which catalyzes the transfer of the aminopropyl moiety of
decarboxylated S-adenosyl-L-methionine to form spermine.
PMT enzyme activity has primarily been detected within roots
of tropane alkaloid-producing Atropa belladonna, Hyoscyamus
niger and Datura stramonium12, and this has been confirmed by
RNA gel blot analyses for A. belladonna 13. Histochemical analysis
of transgenic A. belladonna expressing b-glucuronidase behind
1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01575-2
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the PMT promotor shows that GUS is
H3C
expressed specifically in root pericycle
cells13 (Fig. 2). The pericycle-specific
CH3
expression of hyoscyamine-6b-hydroxyN
CH3
lase, which catalyzes the last step in
CH3
N
scopolamine biosynthesis, has also been
H
demonstrated by in situ hybridization studH3CO2C
Solanidine
14
ies (Fig. 2). Localization of the first and
N
O
Anhydrolast steps in scopolamine biosynthesis in
+
vinblastine
Gal ORha
N
the pericycle, suggested that the rest of the
H
OGlc
16
2
pathway could also be localized there.
CH3
3 4
H3CO
N
OAc
In addition this suggests that if the root
Nicotine
H
CO2CH3
CH3 OH
pericycle is the manufacturing centre for
tropane alkaloids, its proximity to the adjaO
O
CH3
cent xylem cells probably facilitates the
N
N
transport of alkaloids to leaves, where they
O
O
are known to accumulate.
OCH3
O
H
N-methylputrescine is converted to
H
H3CO
O
N
tropinone, through an incompletely underOCH3
H3CO
O
CH3
O
N
stood pathway (Fig. 2) that involves the
H
Berberine
CH3
O
participation of phenylalanine-derived
Macarpine
phenyllactic acid15. Tropinone is then con(+)-13 -Tigloyloxylupanine
verted to tropine or to pseudotropine,
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respectively, by tropinone reductase (TR) I
Fig. 1. Typical substitution reactions involved in the diversification of alkaloid structures.
or II, which catalyze opposite stereospeAnhydro-vinblastine is composed of catharanthine (upper molecule) and vindoline (lower
16
cific reductions . These enzymes are part
molecule). Vindoline is derived from tabersonine by sequential aromatic hydroxylation at posiof a short chain dehydrogenase or reduction 16 (red), 16-O-methylation (blue), hydration at position 2,3 (gray), N-methylation (pink),
tase gene family, with numerous homolohydroxylation at position 4 (orange) and 4-O-acetylation (green). Steroid alkaloids, such as
gous genes occurring in several plant
solanidine are glycosides produced by the action of specific O-glycosyltransferases, which
species that do not make tropane alkaloids.
transfer sugars such as galactose, glucose and rhamnose (red) to the free or glycosylated
aglycone. Nicotine is derived from N-methylputrescine and nicotinic acid. PutrescineThis suggests that some plant homologs
N-methyltransferase transfers the methyl group (red) from S-adenosylmethionine. The
might function in other primary metabolic
quinolizidine alkaloid (1)-13a-tigloyloxylupanine is produced by acylation (green) of 13pathways, from which the TR enzymes
hydroxylupanine. Berberine and macarpine are isoquinoline alkaloids that accumulate in plants
were possibly recruited.
such as Eschscholtia californica and Papaver somniferum. Biosynthesis of berberine4 requires
In contrast with the pericycle-specific
the participation of two flavinylated oxidases and a cytochrome P450, whereas biosynthesis of
expression observed for the first and last
the more highly oxidized alkaloid, macarpine, involves six cytochromes P450 in addition to
steps in scopolamine biosynthesis, TR-1
two other oxidases. Some of the reactions involving cytochromes P450 are highlighted in red
protein is expressed strongly only in the
in the berberine and macarpine molecules. Several intermediate methyl groups are highlighted
17
endodermis and outer cortex (Fig. 2). The
in blue on the berberine and macarpine molecules. These reactions are catalyzed by substratedifferent cellular compartmentation of PMT,
specific O-methyltransferases that produce desired methylation patterns.
TR-1 and H6H indicates that biosynthetic
intermediates must be shuttling between the
pericycle and the endodermis to complete
the biosynthetic cycle leading to the production of scopolamine.
biosynthesis of tropane alkaloids, such as those that convert
Further studies to determine the sights of PMT, TR I and H6H phenylalanine into phenyl-lactic acid, and those involved in the
expression have shown that only roots express these enzyme formation of the tropate ester precursor of hyoscyamine15. This
activities and contain the proteins that react to antibodies raised information could be useful to complete our understanding of the
against each enzyme17. Dissection of young roots into 5-mm seg- cell-specific compartmentation of tropane alkaloid biosynthesis.
ments has revealed that the expression of each protein is restricted
to a region 1–3 cm from the root apex, whereas they are absent in Indole alkaloid biosynthesis in the Catharanthus roseus
more differentiated root sections. In more mature roots, reactive model system
PMT, TR I and H6H proteins are only detected in the region Catharanthus roseus (Madagascar periwinkle) produces monoter1–3 cm from the root apex of the developmentally younger lateral penoid indole alkaloids that are derived from the shikimate and
roots. These results confirm that tropane alkaloid biosynthesis is the deoxyxylulose phosphate pathways. The biosynthesis of the
developmentally regulated, in addition to being cell-type specific. indole moiety requires tryptamine, which is derived from tryptoThe localization of PMT to the pericycle17, where amino phan by the action of tryptophan decarboxylase (TDC). The
acids transported through the vascular tissue are unloaded, would biosynthesis of the terpenoid moiety requires secologanin, which
allow ready access to ornithine or arginine, the precursors of is derived from geraniol via a series of enzymatic conversions19,20.
putrescine18. N-methylputrescine or some biosynthetic intermedi- The committed step in monoterpenoid indole alkaloid biosyntheate could then be transported to the endodermis for further elabo- sis involves a vacuole-specific21 strictosidine synthase (Str1),
ration into tropine. It is unclear if tropine is then further elaborated which catalyzes the stereospecific condensation of tryptamine and
into hyoscyamine before being transported back to the pericycle secologanin to yield 3-a(S)-strictosidine (Fig. 3). Several thoufor conversion into scopolamine. In this context, it would be rele- sand strictosidine-derived indole alkaloids have been charactervant to identify the location of other enzymes involved in the ized, a few of which are used for the treatment of hypertension and
April 2000, Vol. 5, No. 4
169
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(Pericycle-specific)
PMT
NH 2 CH 3
H 2N
N -methylputrescine
H 3 CN
NH 2
H2 N
Putrescine
(a)
Tropinone
O
TR-1
(Endodermis and outer cortex-specific)
H 3 CN
Phe
Tropine
HO
H 3 CN
CH 2 OH
O
Hyoscyamine
H6H
OC
IC
H
(Pericycle-specific)
(c)
H 3 CN
O
Scopolamine
(b)
Ep
CH 2 OH
En
P
O
H
Fig. 2. Histochemical localization of tropane alkaloid biosynthesis in root cross-sections. (a)
Putrescine-N-methyltransferase (PMT) promotor GUS fusion expression has been detected
by GUS staining in roots of Atropa belladonna. Reproduced, with permission, from Ref. 13.
Immunological localization of (b) tropinone reductase (TR-1) and (c) hyoscyamine 6bhydroxylase (H6H) in roots of Hyoscyamus niger. (b) and (c) reproduced, with permission,
from Ref. 17. The three biochemical steps leading to scopolamine biosynthesis and their cellular location are depicted, respectively, in red (PMT), green (TR-1) and blue (H6H). The
Phenyllactic acid component of hyoscyamine is derived from Phe. Abbreviations:
Ep, epidermis; OC, outer cortex; IC, inner cortex; En, endodermis; P, pericycle.
for several antineoplastic ailments22. In particular, vinblastine
and vincristine from C. roseus are valuable chemotherapeutic agents
currently used in the treatment of several cancerous diseases (Fig. 1).
Indole alkaloid biosynthesis is under cell-, tissue-, developmentand environment-specific control
The commercial importance of vinblastine and vincristine have
led to considerable efforts to produce these chemicals in high
yielding callus, cell suspension, and hairy root culture systems,
but without apparent success23. The ability of these culture systems to accumulate catharanthine and tabersonine, but not vindoline, suggested that the biosynthesis of catharanthine and
vindoline is differentially regulated and that vindoline biosynthesis
is under a more rigid cell-, tissue-, development- and environment-specific control than that of catharanthine19,20. This hypothesis was further corroborated by studies that suggested that the
ability to synthesize and accumulate vindoline reappeared with
the regeneration of shoots and in shoot tissue cultures23.
The use of germinating seedlings of C. roseus growing in the
light or in its absence have been of great importance in elucidating
how vindoline is made in the intact plant and in dissecting the
unique morphogenetic- and environment-specific events involved
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April 2000, Vol. 5, No. 4
in activating the vindoline pathway19. If
Catharanthus seeds are germinated and
grown in the absence of light, they accumulate high levels of tabersonine, as well as
small amounts of four other intermediates
between tabersonine and vindoline. The
growth of etiolated seedlings in the presence
of light, stimulated the quantitative, largescale turnover of tabersonine and the intermediates of vindoline by sequential aromatic
hydroxylation, O-methylation, hydration, Nmethylation, hydroxylation and O-acetylation
(Fig. 1). These results suggest that light treatment activates the late stages of vindoline
biosynthesis, and that three hydroxylases, Omethyltransferase, N-methyltransferase and
O-acetyltransferase are involved in the conversion of tabersonine into vindoline19,20.
Further biochemical studies have resolved
that:
• Tabersonine-16-hydroxylase belongs to
the class of cytochrome P450-dependent
mono-oxygenases, which are usually
associated with the external face of the
endoplasmic reticulum24.
• N-methyltransferase is associated with
chloroplast thylakoids19.
• Terminal desacetoxyvindoline-4-hydroxylase (D4H)25 and deacetylvindoline4-O-acetyltransferase (DAT)8 are cytosolic enzymes (Fig. 1).
These results indicate that the cytosolic
face of the endoplasmic reticulum, the
vacuole, the chloroplast thylakoid and the
cytoplasmic compartment of the cell are all
involved in vindoline biosynthesis.
Cell-specific distribution of TDC, STR1,
D4H and DAT
The genes for cytochrome P450 tabersonine16-hydroxylase (Ref. 26), D4H (Ref. 25) and
DAT (Ref. 8) have been cloned recently.
Detailed immunological and molecular studies strongly suggest that
D4H and DAT are only expressed in the above-ground plant parts,
whereas TDC and STR1 occur throughout the plant.
C. roseus has simple, elliptical mesomorphic leaves (Box 1)
composed of several cell types27. The upper and lower epidermis
are composed of thin-walled cells arranged in a single layer, whereas
the mesophyll is arranged into a single layer of elongated palisade
parenchyma on the adaxial side and a thicker multicellular spongy
parenchyma on the abaxial side of the leaf (Fig. 2). In addition,
unbranched, nonarticulated laticifers (Box 1) are associated with
the veins, which are curved and diverge from the midrib at a
35–458. Branching from these are smaller veins usually composed
of a tracheary element and a laticifer. The palisade and spongy
mesophyll also contain idioblasts (Box 1), which are identified by
their distinctive yellow autoflorescence when the leaves are illuminated by blue light, and by their distinctively larger size than
the surrounding mesophyll cells.
In situ RNA hybridization and immunocytochemistry27 have
established that TDC and STR1 are localized to the epidermis of
stems, leaves (Fig. 3) and flower buds, whereas they appear in
most protoderm and cortical cells around the apical meristem of
root tips (Fig. 3). D4H and DAT are associated with laticifers and
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idioblast cells of leaves, stems and flower buds (Fig. 3). These
results provide significant new evidence that, in addition to intracellular specialization involving multiple cellular compartments,
at least two cell types requiring intercellular translocation of a
pathway intermediate are needed to allow the biosynthesis of
vindoline in C. roseus.
Additional studies27 have shown that alkaloid biosynthesis only
occurs transiently during early leaf, stem and root development. In
situ hybridization and immunocytochemical studies have revealed
that alkaloid biosynthesis follows a basipetal distribution in young
expanding leaves. This expression pattern, together with the
results obtained in young roots (Fig. 3), suggests that alkaloid
biosynthesis is activated in young rapidly dividing cells, whereas
it is down regulated in more mature tissues.
The results raise interesting questions about:
(1) The number of pathway steps occurring in the epidermis or in
the root apex.
(2) Whether alkaloid biosynthesis in roots and in epidermal tissues
serves different biological and ecological functions by producing different indole alkaloid end-products.
(3) Which of these tissues produces specific intermediates for
vindoline biosynthesis.
Box 1. Glossary
Idioblast – specialized plant cell that contains a distinctive chemical
composition compared with surrounding cells.
Laticifer – there are two main types, both are long and narrow and
can be branched or unbranched.
Nonarticulated (non-branched) laticifer – specialized cell that contains a milky, chemically complex fluid called latex. Non-articulated
laticifers initiate as single cells and grow to a large size by intrusive
growth (pushing their way between other cells). They continue to
grow as the plant grows and become giant coenocytic cells.
Mesomorphic leaf – leaves adapted to environments where there is
adequate soil moisture.
(4) How intermediates are mobilized into laticifers and idioblasts.
(5) Why this elaborate cell-specific expression is required.
(6) Which alkaloids accumulate in laticifers, idioblasts, epidermis
and roots.
Although it is not known which alkaloids are made in the
epidermis apart from strictosidine, it is well known that roots
UE
Leaf
(a) Epidermal cell
COOH
N
H
STR1
NH2TDC
N
H
?
NH2
CHO
H
H3COOC
N
H H
H
OGlc
O
NH
H
H
H3COOC
OGlc
O
LE
PI
(b) Idioblast or laticifer cell
N
CL
N
D4H
DAT
H
H
N
H3CO
H
CH3 HO CO2CH3
N
H3CO
OAC
H
CO
CH3
2
CH3 HO
SI
T
Root
(c) Cortical cell
COOH
N
H
NH2TDC
STR1
N
H
NH2
CHO
H
H3COOC
H
O
?
N
H H
OGlc
H
H3COOC
NH
H
C
OGlc
O
Fig. 3. Cell-specific localization of tdc, str1, d4h and dat mRNAs in developing Catharanthus leaves and roots27. Leaf or root cross-sections
were reacted with antisense digoxigenin-labeled transcripts. Hybridized transcripts were localized with antidigoxigenin-alkaline phosphatase
congugate followed by BCIP/nitro tetrazolium color development. (a) Both tdc and str1 (data not shown) are expressed in the upper and lower
leaf epidermis. (b) Both d4h (data not shown) and dat are expressed in leaf idioblasts and laticifers. (c) Both tdc and str1 (data not shown) are
expressed in root protoderm and cortical cells around the apical meristem of root tips. Abbreviations: C, root cortex; CL, cross-connecting
laticifer cells; LE, lower epidermis; PI, palisade mesophyll-associated idioblast cells; SI, spongy mesophyll-associated idioblast cell; T, tracheid; UE, upper epidermis. Reproduced, with permission, from Ref. 27.
April 2000, Vol. 5, No. 4
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Table 2. Xylem and phloem transport of alkaloidsa
Compounds
Xylem
Phloem
2
2
2
2
3
3
3
3
3
3
2
2
Quinolizidine alkaloids
Pyrrolizidine alkaloids
Aconitine
Polyhydroxy alkaloids
Nicotine alkaloids
Tropane alkaloids
a
Reproduced, with permission, from Ref. 5.
Abbreviations: 3, transported; 2, not transported.
accumulate many alkaloids, including catharanthine, tabersonine
and derivatives of tabersonine. Therefore it is possible that rootsynthesized catharanthine and tabersonine are transported via
xylem and laticifer-associated tracheids to supply these precursors
to laticifers. This is plausible because there are several examples of
phloem and xylem-based transport of alkaloids2,5 (Table 2). The
laticifer could convert tabersonine into vindoline, which would then
be coupled by a non-specific peroxidase-mediated process28 with
catharanthine to yield the antineoplastic agents vinblastine and vincristine. Therefore the localization of the late stages of vindoline
biosynthesis to the laticifer might ensure sequestration of the toxic
antineoplastic agents that would be produced with the presence of
catharanthine and vindoline in the same cell. However, the ability
of shoot cultures lacking roots, to accumulate vindoline23, suggests
roots might not be the only source of catharanthine and tabersonine.
The chloroplast as the site of quinolizidine alkaloid
biosynthesis
Biosynthesis of lysine-derived quinolizidine alkaloids, which are
found in many legumes, such as lupins (Lupinus), appears to occur
within the mesophyll chloroplasts of green leaves5. A biochemicallocalization study has been performed with two acyltransferases
that catalyze terminal acylations leading to the production, respectively, of (1) p-coumaroylepilupinine and (2) 13a tigloyloxymultiflorine29 (Fig. 1). This study in Lupinus albus demonstrated
that one acylation occurred within the cytoplasm and the other in
mitochondria, but not within chloroplasts. These findings suggest
that although chloroplasts might be responsible for the formation
of the quinolizidine skeleton5, further modifications must be
occurring after intracellular transport to the cytosol and to the
mitochondria. The need for tigloylation to occur within the mitochondria can be explained by the fact that the acyl donor, tigloylCoA, which is derived via a three-step process from isoleucine, is
produced within mitochondria in animal systems. Therefore an
analogous pathway might be present in plants. The endproducts of
biosynthesis are thought to accumulate particularly within the vacuoles of the epidermis of lupin plants, where their defensive roles
can be most effectively exploited by the plant2,5. This adds the
mitochondria to the growing list of cellular organelles that might
participate in alkaloid biosynthesis. These studies also illustrate
how plants have been opportunistic to adapt primary metabolic
processes, as well as the organelles in which they evolved, to
permit the diversification of secondary metabolism.
Isoquinoline alkaloid biosynthesis
The biosynthesis of isoquinoline alkaloids3,4,30 proceeds via the
decarboxylation of tyrosine or DOPA to yield the respective
amine precursors. Dopamine and p-hydroxyphenylacetaldehyde
are then combined by the action of (S)-norcoclaurine synthase to
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April 2000, Vol. 5, No. 4
yield (S)-norcoclaurine, which is a central precursor to several
thousand isoquinoline alkaloids. In situ-hybridization studies in
opium poppy (Papaver) with tyrosine/DOPA decarboxylase
(TYDC) have revealed that the expression of two subgroups of
differentially expressed genes are restricted to the metaphloem
and to the protoxylem of vascular tissues in mature stems and
roots31. Opium poppy alkaloids accumulate within laticifers,
which are derived from the metaphloem. The restriction of TYDC
expression to these particular cells illustrates the strict control to
which this gene family is subject.
Further biochemical localization studies30 have been performed
using Berberis wilsoniae cell cultures. Berberine bridge enzyme
(BBE) and (S)-tetrahydroberberine oxidase, which catalyze the
third to last and final steps of berberine (Fig. 1) biosynthesis, are
localized to a membrane fraction with a different specific gravity
from those of the endoplasmic reticulum and plant vacuoles. The
biochemical localization results were in part corroborated when
the berberine bridge enzyme was cloned32 and it was shown to
contain a 22 amino acid signal peptide directing it to the endoplasmic reticulum. Additional studies in the Catharanthus model
system for indole alkaloid biosynthesis have also shown that Str1
contains a putative signal peptide that is cleaved from the mature
protein33, which appears to be located within plant vacuoles21.
These results suggest that unique specialized cellular compartments
in plants serve as scaffolds for alkaloid biosynthesis.
Several vacuolar homologs of BBE (Accession no. Al080254)
and Str1 (Accession no. P92976) have been identified in Arabidopsis recently, which is not known to make alkaloids. The presence of
these homologs could indicate that the BBE and Str1 reactions occur
in the vacuole because these genes evolved from common ancestors
that had other vacuolar functions. Therefore, the complex intracellular compartmentation of alkaloid biosynthesis might have occurred
as a consequence of adapting compartmented reactions of primary
metabolism to participate in alkaloid biosynthesis. The recent
cloning of homospermidine synthase34, the first pathway-specific
enzyme of pyrrolizidine alkaloid biosynthesis, clearly showed that it
had evolved from deoxyhypusine synthase (DHS). DHS is highly
conserved among eukaryotes and archaebacteria where it catalyzes
the first step in the activation of translation initiation factor 5A,
which is essential for eukaryotic cell proliferation. This is a further
example of recruiting a gene involved in a primary process to
perform a pathway-specific function in alkaloid biosynthesis
Conclusions
Recently, rapid progress has been made in our understanding of
the biochemistry, molecular biology and cell biology of alkaloid
biosynthesis in plants. This fundamental understanding is
expected to lead to rapid progress in the isolation of almost any
desired gene. The availability of these tools have been used to
study the cell and developmental processes leading to the synthesis and accumulation of alkaloids that are often toxic to the host as
well as to potential predators. The data from several different
alkaloid-producing plants suggests that their biosynthesis and
accumulation involve a highly regulated process that includes
cell-, tissue-, development- and environment-specific controls.
The evolution of alkaloid pathways together with their cellular
compartmentation appears to be closely associated with the primary reactions from which they have evolved. With the availability of an increasing number of genes involved in alkaloid
biosynthesis, increasing efforts will be made to identify the regulators35 that are associated with the development of specialized
cell-types that accommodate alkaloid biosynthesis and accumulation. Continuing studies promise to yield exciting new discoveries
about the biological origins of alkaloid biosynthesis in plants.
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Acknowledgements
We thank Takashi Hashimoto for the photographs used in
Figure 2. We also thank Michael Wink for sharing unpublished
reviews. This work was supported by a grant to V.D.L. from the
Natural Sciences and Engineering Council of Canada.
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gene is differentially expressed in the root pericycle and anthers. Plant Mol.
Biol. 40, 141–152
15 Zabetakis, I. et al. (1998) The biosynthetic relationship between littorine and
hyoscyamine in transformed roots of Datura stramonium. Plant Cell Rep. 18,
341–345
16 Nakajima, K. et al. (1993) Two tropinone reductases with different
stereospecificities are short chain dehydrogenases evolved from a common
ancestor. Proc. Natl. Acad. Sci. U. S. A. 90, 9591–9595
17 Nakajima, K. and Hashimoto, T. (1999) Two tropinone reductases, that
catalyse opposite stereospecific reductions in tropane alkaloid biosynthesis,
are localized in plant root with different cell-specific patterns. Plant Cell
Physiol. 40, 1099–1107
18 Hashimoto, T. and Yamada, Y. (1994) Alkaloid biogenesis: molecular aspects.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 257–285
19 De Luca, V. (1993) Indole alkaloid biosynthesis. In Methods in Plant
Biochemistry. Enzymes of Secondary Metabolism (Vol. 9) (Lea, P., ed.), pp.
345–368, Academic Press
20 Meijer, A.M. et al. (1993) Regulation of enzymes and genes involved in
terpenoid indole alkaloid biosyntheis in Catharanthus roseus. J. Plant Res.
(Special Issue) 3, 145–164
21 McKnight, T.D. et al. (1991) Expression of enzymatically active and
correctly targeted strictosidine synthase in transgenic tobacco plants. Planta
185, 148–152
22 Van Tellingen, O. et al. (1992) Pharmacology, bio-analysis and
pharmacokinetics of the vinca alkaloids and semi-synthetic derivatives.
Anticancer Res. 12, 1699–1715
23 Van der Heijden, R. et al. (1989) Cell and tissue cultures of Catharanthus
roseus (L.) G. Don: a literature survey. Plant Cell, Tissue Organ Cult.
18, 231–280
24 St-Pierre, B. and De Luca, V. (1995) A cytochrome P450 monooxygenase
catalyses the first step in the conversion of tabersonine to vindoline in
Catharanthus roseus. Plant Physiol. 109, 131–139
25 Vazquez-Flota, F. et al. (1997) Molecular cloning and characterization of
desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate-dependent dioxygenase
involved in the biosynthesis of vindoline in Catharanthus roseus. Plant Mol.
Biol. 34, 935–948
26 Schroder, G. et al. (1999) Light-induced cytochrome P450-dependent enzyme
in indole alkaloid biosynthesis: tabersonine 16-hydroxylase. FEBS Lett.
458, 97–102
27 St-Pierre, B. et al. (1999) Multicellular compartmentation of Catharanthus
roseus alkaloid biosynthesis predicts intercellular translocation of a pathway
intermediate. Plant Cell 11, 887–900
28 Sottomayor, M. et al. (1998) Purification and characterization of a-39,49anhydrovinblastine synthase (peroxidase-like) from Catharanthus roseus (L.)
G. Don. FEBS Lett. 428, 299–303
29 Suzuki, H. et al. (1996) Subcellular localization of acyltransferases for
quinolizidine alkaloid biosynthesis in Lupinus. Phytochemistry 42, 1557–1562
30 Kutchan, T.M. and Zenk, M. (1993) Enzymology and molecular biology of
benzophenanthridine alkaloid biosynthesis. J. Plant Res. (Special issue) 3, 165–173
31 Facchini, P.J. and De Luca, V. (1995) Phloem-specific expression of
tyrosine/dopa decarboxylase genes and the biosynthesis of isoquinoline
alkaloids in opium poppy. Plant Cell 7, 1811–1821
32 Dittrich, H. and Kutchan, T.M. (1991) Molecular cloning, expression and
induction of berberine bridge enzyme, an enzyme essential to the formation of
benzophenanthridine alkaloids in the response to pathogen attack. Proc. Natl.
Acad. Sci. U. S. A. 88, 9969–9973
33 McKnight, T.D. et al. (1990) Nucleotide sequence of a cDNA encoding the
vacuolar protein strictosidine synthase from Catharanthus roseus. Nucleic
Acids Res. 18, 4939
34 Ober, D. and Hartmann, T. (1999) Homospermidine synthase, the first
pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from
deoxyhypusine synthase. Proc. Natl. Acad. Sci. U. S. A. 96, 14777–14782
35 Menke, F.L. et al. (1999) A novel jasmonate- and elicitor-responsive element in
the periwinkle secondary metabolite biosynthetic gene Str interacts with a
jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2.
EMBO J. 18, 4455–4463
Vincenzo De Luca* is at Novartis Agribusiness Biotechnology
Research Inc., 3054 Cornwallis Road, Research Triangle Park,
NC 27709-2257, USA; Benoit St Pierre is at Laboratoire de
Physiologie Végétale, EA 2106, UFR des Sciences et Techniques,
Université de Tours, Parc de Grandmont, 37200 TOURS, France
(tel 133 247 367101; fax 133 247 367042;
e-mail saint.pierre@univ-tours.fr).
*Author for correspondence (tel 11 919 541 8575;
fax 11 919 541 8585; e-mail vince.deluca@nabri.novartis.com).
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173
Reviews
The cell and developmental biology
of alkaloid biosynthesis
Vincenzo De Luca and Benoit St Pierre
Plants produce unique natural products as a result of gene mutation and subsequent adaptation of metabolic pathways to create new secondary metabolites. However, their biosynthesis
and accumulation remains remarkably under the control of the biotic and abiotic environments.
Alkaloid biosynthesis, which requires the adaptation of cellular activities to perform specialized
metabolism without compromising general homeostasis, is accomplished by restricting
product biosynthesis and accumulation to particular cells and to defined times of plant development. The cell and developmental biology of alkaloid biosynthesis, which is remarkably
complex, evolved in part by recruiting pre-existing enzymes to perform new functions.
A
lkaloids are low molecular weight nitrogen-containing
substances with characteristic toxicity and pharmacological activity. These properties, which have traditionally
been exploited by humans for hunting, execution and warfare,
have also been used for the treatment of disease1. About 20% of
plant species accumulate alkaloids, which are mostly derived
from the amino acids, Phe, Tyr, Trp, Lys and Orn. In addition, the
monoterpenoid indole alkaloids, which form a large class of complex compounds, are derived from Trp and terpenoid precursors.
Over 12 000 different alkaloids have been described, indicating
their structural and biosynthetic diversity compared to that of
other secondary metabolites2 (Table 1).
Our extensive knowledge about the chemistry and pharmacology of alkaloids has led to their use in a range of medical applications. However, little is known about their biosynthesis or about
the factors that regulate alkaloid production2–5. Recently, intensive
efforts have elucidated the basic biochemistry and molecular biology of some alkaloid pathways and, more generally, of secondary
metabolism6. These discoveries are now enabling more rapid consensus cloning and identification of highly specific biochemical
reactions involved in the production of interesting alkaloids4,7,8.
These results support chemical reasoning that the diversification
of secondary metabolism originated from the elaboration of a few
central intermediates that could be modified through oxidation,
followed by the protection of hydroxyl groups by acylation, glucosylation, methylation, or among others, prenylation (Fig. 1). In
this context, the Arabidopsis genome, which has yet to be completely sequenced, contains large gene families for each of these
classes of biochemical reactions, including cytochrome P450
mono-oxygenases, dioxygenases, acyltransferases, methyltransferases and glucosyltransferases6.
Additional studies have begun to illustrate that alkaloid biosynthesis is not a random process, but is highly ordered with respect
Table 1. Plant secondary metabolites
with known structures
Secondary metabolites
Alkaloids
Terpenoids
Phenylpropanoids
Others
168
April 2000, Vol. 5, No. 4
Number of structures
12 000
30 000
2500
2500
to plant development, controlling the expression of pathways within
organs, within specific cells, or within organelles inside those
cells. Here we review recent developments in our understanding
of the cell and developmental biology of alkaloid biosynthesis and
their biological origins.
Alkaloids of the Solanaceae
The Solanaceae produce a variety of interesting biologically active
products including the steroid alkaloids solanidine, nicotine (Fig. 1)
and tropane alkaloids (Fig. 2). For example, it is well known that
domestication of potatoes involved the successful elimination of
toxic steroid alkaloids in tubers while retaining steroid alkaloids
in other plant parts for biotic defense. The tropane alkaloids,
which have a colorful history of human use, have inspired the
creation of several synthetic analogs. These are used to control
motion sickness, as powerful bronchodilators to treat chronic
bronchitis, as antimuscarinic drugs for the control of Parkinson’s
disease, or as midriatics, which are used to dilate the pupil of the
eye to facilitate surgery. In the case of nicotine, breeding efforts
have focused on increasing the levels of this alkaloid in tobacco
for smoking purposes. The continuing appeal of cigarettes to
humans can be explained by the rapid absorption, transport and
binding of nicotine to acetylcholine receptors in the brain and the
ensuing pleasure obtained.
Root-specific scopolamine and nicotine biosynthesis
Roots have been shown to elaborate a remarkable variety of
secondary metabolites9, including alkaloids, flavonoids and
terpenoids. Nicotine and tropane alkaloids, which are mainly produced in roots as suggested by reciprocal grafting experiments10,
accumulate mostly within vacuoles2 of plant roots and leaves. The
early pathway leading to biosynthesis of both nicotine (Fig. 1) and
tropane (Fig. 2) alkaloids involves the formation of a pyrrolidine
ring that is derived from putrescine via the sequential action of
an S-adenosyl-L-methionine-dependent putrescine-N-methyltransferase (PMT), a diamine oxidase and a spontaneous chemical rearrangement. The presence of PMT is unique to alkaloid-producing
plants and it appears to have evolved from spermidine synthase11
(SPDS), which catalyzes the transfer of the aminopropyl moiety of
decarboxylated S-adenosyl-L-methionine to form spermine.
PMT enzyme activity has primarily been detected within roots
of tropane alkaloid-producing Atropa belladonna, Hyoscyamus
niger and Datura stramonium12, and this has been confirmed by
RNA gel blot analyses for A. belladonna 13. Histochemical analysis
of transgenic A. belladonna expressing b-glucuronidase behind
1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01575-2
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the PMT promotor shows that GUS is
H3C
expressed specifically in root pericycle
cells13 (Fig. 2). The pericycle-specific
CH3
expression of hyoscyamine-6b-hydroxyN
CH3
lase, which catalyzes the last step in
CH3
N
scopolamine biosynthesis, has also been
H
demonstrated by in situ hybridization studH3CO2C
Solanidine
14
ies (Fig. 2). Localization of the first and
N
O
Anhydrolast steps in scopolamine biosynthesis in
+
vinblastine
Gal ORha
N
the pericycle, suggested that the rest of the
H
OGlc
16
2
pathway could also be localized there.
CH3
3 4
H3CO
N
OAc
In addition this suggests that if the root
Nicotine
H
CO2CH3
CH3 OH
pericycle is the manufacturing centre for
tropane alkaloids, its proximity to the adjaO
O
CH3
cent xylem cells probably facilitates the
N
N
transport of alkaloids to leaves, where they
O
O
are known to accumulate.
OCH3
O
H
N-methylputrescine is converted to
H
H3CO
O
N
tropinone, through an incompletely underOCH3
H3CO
O
CH3
O
N
stood pathway (Fig. 2) that involves the
H
Berberine
CH3
O
participation of phenylalanine-derived
Macarpine
phenyllactic acid15. Tropinone is then con(+)-13 -Tigloyloxylupanine
verted to tropine or to pseudotropine,
Trends in Plant Science
respectively, by tropinone reductase (TR) I
Fig. 1. Typical substitution reactions involved in the diversification of alkaloid structures.
or II, which catalyze opposite stereospeAnhydro-vinblastine is composed of catharanthine (upper molecule) and vindoline (lower
16
cific reductions . These enzymes are part
molecule). Vindoline is derived from tabersonine by sequential aromatic hydroxylation at posiof a short chain dehydrogenase or reduction 16 (red), 16-O-methylation (blue), hydration at position 2,3 (gray), N-methylation (pink),
tase gene family, with numerous homolohydroxylation at position 4 (orange) and 4-O-acetylation (green). Steroid alkaloids, such as
gous genes occurring in several plant
solanidine are glycosides produced by the action of specific O-glycosyltransferases, which
species that do not make tropane alkaloids.
transfer sugars such as galactose, glucose and rhamnose (red) to the free or glycosylated
aglycone. Nicotine is derived from N-methylputrescine and nicotinic acid. PutrescineThis suggests that some plant homologs
N-methyltransferase transfers the methyl group (red) from S-adenosylmethionine. The
might function in other primary metabolic
quinolizidine alkaloid (1)-13a-tigloyloxylupanine is produced by acylation (green) of 13pathways, from which the TR enzymes
hydroxylupanine. Berberine and macarpine are isoquinoline alkaloids that accumulate in plants
were possibly recruited.
such as Eschscholtia californica and Papaver somniferum. Biosynthesis of berberine4 requires
In contrast with the pericycle-specific
the participation of two flavinylated oxidases and a cytochrome P450, whereas biosynthesis of
expression observed for the first and last
the more highly oxidized alkaloid, macarpine, involves six cytochromes P450 in addition to
steps in scopolamine biosynthesis, TR-1
two other oxidases. Some of the reactions involving cytochromes P450 are highlighted in red
protein is expressed strongly only in the
in the berberine and macarpine molecules. Several intermediate methyl groups are highlighted
17
endodermis and outer cortex (Fig. 2). The
in blue on the berberine and macarpine molecules. These reactions are catalyzed by substratedifferent cellular compartmentation of PMT,
specific O-methyltransferases that produce desired methylation patterns.
TR-1 and H6H indicates that biosynthetic
intermediates must be shuttling between the
pericycle and the endodermis to complete
the biosynthetic cycle leading to the production of scopolamine.
biosynthesis of tropane alkaloids, such as those that convert
Further studies to determine the sights of PMT, TR I and H6H phenylalanine into phenyl-lactic acid, and those involved in the
expression have shown that only roots express these enzyme formation of the tropate ester precursor of hyoscyamine15. This
activities and contain the proteins that react to antibodies raised information could be useful to complete our understanding of the
against each enzyme17. Dissection of young roots into 5-mm seg- cell-specific compartmentation of tropane alkaloid biosynthesis.
ments has revealed that the expression of each protein is restricted
to a region 1–3 cm from the root apex, whereas they are absent in Indole alkaloid biosynthesis in the Catharanthus roseus
more differentiated root sections. In more mature roots, reactive model system
PMT, TR I and H6H proteins are only detected in the region Catharanthus roseus (Madagascar periwinkle) produces monoter1–3 cm from the root apex of the developmentally younger lateral penoid indole alkaloids that are derived from the shikimate and
roots. These results confirm that tropane alkaloid biosynthesis is the deoxyxylulose phosphate pathways. The biosynthesis of the
developmentally regulated, in addition to being cell-type specific. indole moiety requires tryptamine, which is derived from tryptoThe localization of PMT to the pericycle17, where amino phan by the action of tryptophan decarboxylase (TDC). The
acids transported through the vascular tissue are unloaded, would biosynthesis of the terpenoid moiety requires secologanin, which
allow ready access to ornithine or arginine, the precursors of is derived from geraniol via a series of enzymatic conversions19,20.
putrescine18. N-methylputrescine or some biosynthetic intermedi- The committed step in monoterpenoid indole alkaloid biosyntheate could then be transported to the endodermis for further elabo- sis involves a vacuole-specific21 strictosidine synthase (Str1),
ration into tropine. It is unclear if tropine is then further elaborated which catalyzes the stereospecific condensation of tryptamine and
into hyoscyamine before being transported back to the pericycle secologanin to yield 3-a(S)-strictosidine (Fig. 3). Several thoufor conversion into scopolamine. In this context, it would be rele- sand strictosidine-derived indole alkaloids have been charactervant to identify the location of other enzymes involved in the ized, a few of which are used for the treatment of hypertension and
April 2000, Vol. 5, No. 4
169
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(Pericycle-specific)
PMT
NH 2 CH 3
H 2N
N -methylputrescine
H 3 CN
NH 2
H2 N
Putrescine
(a)
Tropinone
O
TR-1
(Endodermis and outer cortex-specific)
H 3 CN
Phe
Tropine
HO
H 3 CN
CH 2 OH
O
Hyoscyamine
H6H
OC
IC
H
(Pericycle-specific)
(c)
H 3 CN
O
Scopolamine
(b)
Ep
CH 2 OH
En
P
O
H
Fig. 2. Histochemical localization of tropane alkaloid biosynthesis in root cross-sections. (a)
Putrescine-N-methyltransferase (PMT) promotor GUS fusion expression has been detected
by GUS staining in roots of Atropa belladonna. Reproduced, with permission, from Ref. 13.
Immunological localization of (b) tropinone reductase (TR-1) and (c) hyoscyamine 6bhydroxylase (H6H) in roots of Hyoscyamus niger. (b) and (c) reproduced, with permission,
from Ref. 17. The three biochemical steps leading to scopolamine biosynthesis and their cellular location are depicted, respectively, in red (PMT), green (TR-1) and blue (H6H). The
Phenyllactic acid component of hyoscyamine is derived from Phe. Abbreviations:
Ep, epidermis; OC, outer cortex; IC, inner cortex; En, endodermis; P, pericycle.
for several antineoplastic ailments22. In particular, vinblastine
and vincristine from C. roseus are valuable chemotherapeutic agents
currently used in the treatment of several cancerous diseases (Fig. 1).
Indole alkaloid biosynthesis is under cell-, tissue-, developmentand environment-specific control
The commercial importance of vinblastine and vincristine have
led to considerable efforts to produce these chemicals in high
yielding callus, cell suspension, and hairy root culture systems,
but without apparent success23. The ability of these culture systems to accumulate catharanthine and tabersonine, but not vindoline, suggested that the biosynthesis of catharanthine and
vindoline is differentially regulated and that vindoline biosynthesis
is under a more rigid cell-, tissue-, development- and environment-specific control than that of catharanthine19,20. This hypothesis was further corroborated by studies that suggested that the
ability to synthesize and accumulate vindoline reappeared with
the regeneration of shoots and in shoot tissue cultures23.
The use of germinating seedlings of C. roseus growing in the
light or in its absence have been of great importance in elucidating
how vindoline is made in the intact plant and in dissecting the
unique morphogenetic- and environment-specific events involved
170
April 2000, Vol. 5, No. 4
in activating the vindoline pathway19. If
Catharanthus seeds are germinated and
grown in the absence of light, they accumulate high levels of tabersonine, as well as
small amounts of four other intermediates
between tabersonine and vindoline. The
growth of etiolated seedlings in the presence
of light, stimulated the quantitative, largescale turnover of tabersonine and the intermediates of vindoline by sequential aromatic
hydroxylation, O-methylation, hydration, Nmethylation, hydroxylation and O-acetylation
(Fig. 1). These results suggest that light treatment activates the late stages of vindoline
biosynthesis, and that three hydroxylases, Omethyltransferase, N-methyltransferase and
O-acetyltransferase are involved in the conversion of tabersonine into vindoline19,20.
Further biochemical studies have resolved
that:
• Tabersonine-16-hydroxylase belongs to
the class of cytochrome P450-dependent
mono-oxygenases, which are usually
associated with the external face of the
endoplasmic reticulum24.
• N-methyltransferase is associated with
chloroplast thylakoids19.
• Terminal desacetoxyvindoline-4-hydroxylase (D4H)25 and deacetylvindoline4-O-acetyltransferase (DAT)8 are cytosolic enzymes (Fig. 1).
These results indicate that the cytosolic
face of the endoplasmic reticulum, the
vacuole, the chloroplast thylakoid and the
cytoplasmic compartment of the cell are all
involved in vindoline biosynthesis.
Cell-specific distribution of TDC, STR1,
D4H and DAT
The genes for cytochrome P450 tabersonine16-hydroxylase (Ref. 26), D4H (Ref. 25) and
DAT (Ref. 8) have been cloned recently.
Detailed immunological and molecular studies strongly suggest that
D4H and DAT are only expressed in the above-ground plant parts,
whereas TDC and STR1 occur throughout the plant.
C. roseus has simple, elliptical mesomorphic leaves (Box 1)
composed of several cell types27. The upper and lower epidermis
are composed of thin-walled cells arranged in a single layer, whereas
the mesophyll is arranged into a single layer of elongated palisade
parenchyma on the adaxial side and a thicker multicellular spongy
parenchyma on the abaxial side of the leaf (Fig. 2). In addition,
unbranched, nonarticulated laticifers (Box 1) are associated with
the veins, which are curved and diverge from the midrib at a
35–458. Branching from these are smaller veins usually composed
of a tracheary element and a laticifer. The palisade and spongy
mesophyll also contain idioblasts (Box 1), which are identified by
their distinctive yellow autoflorescence when the leaves are illuminated by blue light, and by their distinctively larger size than
the surrounding mesophyll cells.
In situ RNA hybridization and immunocytochemistry27 have
established that TDC and STR1 are localized to the epidermis of
stems, leaves (Fig. 3) and flower buds, whereas they appear in
most protoderm and cortical cells around the apical meristem of
root tips (Fig. 3). D4H and DAT are associated with laticifers and
trends in plant science
Reviews
idioblast cells of leaves, stems and flower buds (Fig. 3). These
results provide significant new evidence that, in addition to intracellular specialization involving multiple cellular compartments,
at least two cell types requiring intercellular translocation of a
pathway intermediate are needed to allow the biosynthesis of
vindoline in C. roseus.
Additional studies27 have shown that alkaloid biosynthesis only
occurs transiently during early leaf, stem and root development. In
situ hybridization and immunocytochemical studies have revealed
that alkaloid biosynthesis follows a basipetal distribution in young
expanding leaves. This expression pattern, together with the
results obtained in young roots (Fig. 3), suggests that alkaloid
biosynthesis is activated in young rapidly dividing cells, whereas
it is down regulated in more mature tissues.
The results raise interesting questions about:
(1) The number of pathway steps occurring in the epidermis or in
the root apex.
(2) Whether alkaloid biosynthesis in roots and in epidermal tissues
serves different biological and ecological functions by producing different indole alkaloid end-products.
(3) Which of these tissues produces specific intermediates for
vindoline biosynthesis.
Box 1. Glossary
Idioblast – specialized plant cell that contains a distinctive chemical
composition compared with surrounding cells.
Laticifer – there are two main types, both are long and narrow and
can be branched or unbranched.
Nonarticulated (non-branched) laticifer – specialized cell that contains a milky, chemically complex fluid called latex. Non-articulated
laticifers initiate as single cells and grow to a large size by intrusive
growth (pushing their way between other cells). They continue to
grow as the plant grows and become giant coenocytic cells.
Mesomorphic leaf – leaves adapted to environments where there is
adequate soil moisture.
(4) How intermediates are mobilized into laticifers and idioblasts.
(5) Why this elaborate cell-specific expression is required.
(6) Which alkaloids accumulate in laticifers, idioblasts, epidermis
and roots.
Although it is not known which alkaloids are made in the
epidermis apart from strictosidine, it is well known that roots
UE
Leaf
(a) Epidermal cell
COOH
N
H
STR1
NH2TDC
N
H
?
NH2
CHO
H
H3COOC
N
H H
H
OGlc
O
NH
H
H
H3COOC
OGlc
O
LE
PI
(b) Idioblast or laticifer cell
N
CL
N
D4H
DAT
H
H
N
H3CO
H
CH3 HO CO2CH3
N
H3CO
OAC
H
CO
CH3
2
CH3 HO
SI
T
Root
(c) Cortical cell
COOH
N
H
NH2TDC
STR1
N
H
NH2
CHO
H
H3COOC
H
O
?
N
H H
OGlc
H
H3COOC
NH
H
C
OGlc
O
Fig. 3. Cell-specific localization of tdc, str1, d4h and dat mRNAs in developing Catharanthus leaves and roots27. Leaf or root cross-sections
were reacted with antisense digoxigenin-labeled transcripts. Hybridized transcripts were localized with antidigoxigenin-alkaline phosphatase
congugate followed by BCIP/nitro tetrazolium color development. (a) Both tdc and str1 (data not shown) are expressed in the upper and lower
leaf epidermis. (b) Both d4h (data not shown) and dat are expressed in leaf idioblasts and laticifers. (c) Both tdc and str1 (data not shown) are
expressed in root protoderm and cortical cells around the apical meristem of root tips. Abbreviations: C, root cortex; CL, cross-connecting
laticifer cells; LE, lower epidermis; PI, palisade mesophyll-associated idioblast cells; SI, spongy mesophyll-associated idioblast cell; T, tracheid; UE, upper epidermis. Reproduced, with permission, from Ref. 27.
April 2000, Vol. 5, No. 4
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Table 2. Xylem and phloem transport of alkaloidsa
Compounds
Xylem
Phloem
2
2
2
2
3
3
3
3
3
3
2
2
Quinolizidine alkaloids
Pyrrolizidine alkaloids
Aconitine
Polyhydroxy alkaloids
Nicotine alkaloids
Tropane alkaloids
a
Reproduced, with permission, from Ref. 5.
Abbreviations: 3, transported; 2, not transported.
accumulate many alkaloids, including catharanthine, tabersonine
and derivatives of tabersonine. Therefore it is possible that rootsynthesized catharanthine and tabersonine are transported via
xylem and laticifer-associated tracheids to supply these precursors
to laticifers. This is plausible because there are several examples of
phloem and xylem-based transport of alkaloids2,5 (Table 2). The
laticifer could convert tabersonine into vindoline, which would then
be coupled by a non-specific peroxidase-mediated process28 with
catharanthine to yield the antineoplastic agents vinblastine and vincristine. Therefore the localization of the late stages of vindoline
biosynthesis to the laticifer might ensure sequestration of the toxic
antineoplastic agents that would be produced with the presence of
catharanthine and vindoline in the same cell. However, the ability
of shoot cultures lacking roots, to accumulate vindoline23, suggests
roots might not be the only source of catharanthine and tabersonine.
The chloroplast as the site of quinolizidine alkaloid
biosynthesis
Biosynthesis of lysine-derived quinolizidine alkaloids, which are
found in many legumes, such as lupins (Lupinus), appears to occur
within the mesophyll chloroplasts of green leaves5. A biochemicallocalization study has been performed with two acyltransferases
that catalyze terminal acylations leading to the production, respectively, of (1) p-coumaroylepilupinine and (2) 13a tigloyloxymultiflorine29 (Fig. 1). This study in Lupinus albus demonstrated
that one acylation occurred within the cytoplasm and the other in
mitochondria, but not within chloroplasts. These findings suggest
that although chloroplasts might be responsible for the formation
of the quinolizidine skeleton5, further modifications must be
occurring after intracellular transport to the cytosol and to the
mitochondria. The need for tigloylation to occur within the mitochondria can be explained by the fact that the acyl donor, tigloylCoA, which is derived via a three-step process from isoleucine, is
produced within mitochondria in animal systems. Therefore an
analogous pathway might be present in plants. The endproducts of
biosynthesis are thought to accumulate particularly within the vacuoles of the epidermis of lupin plants, where their defensive roles
can be most effectively exploited by the plant2,5. This adds the
mitochondria to the growing list of cellular organelles that might
participate in alkaloid biosynthesis. These studies also illustrate
how plants have been opportunistic to adapt primary metabolic
processes, as well as the organelles in which they evolved, to
permit the diversification of secondary metabolism.
Isoquinoline alkaloid biosynthesis
The biosynthesis of isoquinoline alkaloids3,4,30 proceeds via the
decarboxylation of tyrosine or DOPA to yield the respective
amine precursors. Dopamine and p-hydroxyphenylacetaldehyde
are then combined by the action of (S)-norcoclaurine synthase to
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April 2000, Vol. 5, No. 4
yield (S)-norcoclaurine, which is a central precursor to several
thousand isoquinoline alkaloids. In situ-hybridization studies in
opium poppy (Papaver) with tyrosine/DOPA decarboxylase
(TYDC) have revealed that the expression of two subgroups of
differentially expressed genes are restricted to the metaphloem
and to the protoxylem of vascular tissues in mature stems and
roots31. Opium poppy alkaloids accumulate within laticifers,
which are derived from the metaphloem. The restriction of TYDC
expression to these particular cells illustrates the strict control to
which this gene family is subject.
Further biochemical localization studies30 have been performed
using Berberis wilsoniae cell cultures. Berberine bridge enzyme
(BBE) and (S)-tetrahydroberberine oxidase, which catalyze the
third to last and final steps of berberine (Fig. 1) biosynthesis, are
localized to a membrane fraction with a different specific gravity
from those of the endoplasmic reticulum and plant vacuoles. The
biochemical localization results were in part corroborated when
the berberine bridge enzyme was cloned32 and it was shown to
contain a 22 amino acid signal peptide directing it to the endoplasmic reticulum. Additional studies in the Catharanthus model
system for indole alkaloid biosynthesis have also shown that Str1
contains a putative signal peptide that is cleaved from the mature
protein33, which appears to be located within plant vacuoles21.
These results suggest that unique specialized cellular compartments
in plants serve as scaffolds for alkaloid biosynthesis.
Several vacuolar homologs of BBE (Accession no. Al080254)
and Str1 (Accession no. P92976) have been identified in Arabidopsis recently, which is not known to make alkaloids. The presence of
these homologs could indicate that the BBE and Str1 reactions occur
in the vacuole because these genes evolved from common ancestors
that had other vacuolar functions. Therefore, the complex intracellular compartmentation of alkaloid biosynthesis might have occurred
as a consequence of adapting compartmented reactions of primary
metabolism to participate in alkaloid biosynthesis. The recent
cloning of homospermidine synthase34, the first pathway-specific
enzyme of pyrrolizidine alkaloid biosynthesis, clearly showed that it
had evolved from deoxyhypusine synthase (DHS). DHS is highly
conserved among eukaryotes and archaebacteria where it catalyzes
the first step in the activation of translation initiation factor 5A,
which is essential for eukaryotic cell proliferation. This is a further
example of recruiting a gene involved in a primary process to
perform a pathway-specific function in alkaloid biosynthesis
Conclusions
Recently, rapid progress has been made in our understanding of
the biochemistry, molecular biology and cell biology of alkaloid
biosynthesis in plants. This fundamental understanding is
expected to lead to rapid progress in the isolation of almost any
desired gene. The availability of these tools have been used to
study the cell and developmental processes leading to the synthesis and accumulation of alkaloids that are often toxic to the host as
well as to potential predators. The data from several different
alkaloid-producing plants suggests that their biosynthesis and
accumulation involve a highly regulated process that includes
cell-, tissue-, development- and environment-specific controls.
The evolution of alkaloid pathways together with their cellular
compartmentation appears to be closely associated with the primary reactions from which they have evolved. With the availability of an increasing number of genes involved in alkaloid
biosynthesis, increasing efforts will be made to identify the regulators35 that are associated with the development of specialized
cell-types that accommodate alkaloid biosynthesis and accumulation. Continuing studies promise to yield exciting new discoveries
about the biological origins of alkaloid biosynthesis in plants.
trends in plant science
Reviews
Acknowledgements
We thank Takashi Hashimoto for the photographs used in
Figure 2. We also thank Michael Wink for sharing unpublished
reviews. This work was supported by a grant to V.D.L. from the
Natural Sciences and Engineering Council of Canada.
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Vincenzo De Luca* is at Novartis Agribusiness Biotechnology
Research Inc., 3054 Cornwallis Road, Research Triangle Park,
NC 27709-2257, USA; Benoit St Pierre is at Laboratoire de
Physiologie Végétale, EA 2106, UFR des Sciences et Techniques,
Université de Tours, Parc de Grandmont, 37200 TOURS, France
(tel 133 247 367101; fax 133 247 367042;
e-mail saint.pierre@univ-tours.fr).
*Author for correspondence (tel 11 919 541 8575;
fax 11 919 541 8585; e-mail vince.deluca@nabri.novartis.com).
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