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Biosynthesis of anthraquinones in cell cultures of the Rubiaceae

Article in Plant Cell Tissue and Organ Culture · December 2001

DOI: 10.1023/A:1012758922713

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 Plant Cell, Tissue and Organ Culture 67: 201–220, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
201

Review

Biosynthesis of anthraquinones in cell cultures of the Rubiaceae

Ying-Shan Han, Robert Van der Heijden & Robert Verpoorte∗

Division of Pharmacognosy, Leiden / Amsterdam Center for Drug Research, Gorlaeus Laboratories, P.O.
Box 9502, 2300 RA Leiden, The Netherlands (∗ requests for offprints; Fax: +31-(0)71-527-4511; E-mail:
verpoort@lacdr.leidenuniv.nl)

Received 29 January 2001; accepted in revised form 9 July 2001

Key words: anthraquinones, biosynthesis, metabolic engineering, methylerythritol 4-phosphate, mevalonic acid,
o-succinylbenzoic acid, Rubiaceae

Abstract
Plants and their derived cell and tissue cultures in the family Rubiaceae accumulate a number of anthraquinones.
There are two main biosynthetic pathways leading to anthraquinones in higher plants: the polyketide pathway
and the chorismate/o-succinylbenzoic acid pathway. The latter occurs in the Rubiaceae for the biosynthesis of
Rubia type anthraquinones. In this pathway, ring A and B of the Rubia type anthraquinones are derived from
shikimic acid, α-ketoglutarate via o-succinylbenzoate, whereas ring C is derived from isopentenyl diphosphate,
a universal building block for all isoprenoids. At present, it is known that isopentenyl diphosphate is formed via
the mevalonic acid pathway or the 2-C-methyl-D-erythritol 4-phosphate pathway. Recent findings demonstrate that
the 2-C-methyl-D-erythritol 4-phosphate pathway, not the mevalonic acid pathway, is involved in the formation of
isopentenyl diphosphate, which constitutes ring C of anthraquinones in the Rubiaceae. This review summarizes the
latest results of studies on the biosynthetic pathways, the enzymology and regulation of anthraquinone biosynthesis,
as well as aspects of the metabolic engineering. Furthermore, biochemical and molecular approaches in functional
genomics, which facilitate elucidation of anthraquinone biosynthetic pathways, are briefly described.

Abbreviations: AQ – anthraquinone; 2,4-D – 2,4-dichlorophenoxyacetic acid; CDP-ME – 4-cytidyl diphospho-2

C-methyl-D-erythritol; CHAPS – 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; DHNA – 1,4-
dihydroxynaphthoic acid; DMAPP – 3,3-dimethylallyl diphosphate; DOXP – 1-deoxy-D-xylulose 5-phosphate;
DXR – 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS – 1-deoxy-D-xylulose 5-phosphate synthase;
E-4-P – erythrose 4-phosphate; ER – endoplasmic reticulum; ESTs – expressed sequence tags; GAP – glycer-
aldehyde 3-phosphate; HMG-CoA – 3-hydroxy-3-methylglutaryl Coenzyme A; HMGR – HMG-CoA reductase;
IPK – isopentenyl monophosphate kinase; IAA – 3-indoleacetic acid; ICS – isochorismate synthase; IPP – iso-
pentenyl diphosphate; MEC – 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; MeJA – methyl jasmonate; MEP
– 2-C-methyl-D-erythritol 4-phosphate; MVA – mevalonic acid; NAA – 1-naphthaleneacetic acid; OSB – o-
succinylbenzoic acid; PDHC – pyruvate dehydrogenase complex; TCA – trichloroacetic acid; TIA – terpenoid
indole alkaloids; TPP – thiamine diphosphate

Introduction cluding Rubiaceae, Rhamnaceae, Polygonaceae and

Anthraquinones (AQs) (Figure 1) are an important
group of natural products occurring in bacteria, fungi,
lichens and higher plants (Thomson, 1971, 1987,
1996; Muzychkina, 1998). In higher plants, they
are found in a large number of plant families, in-
Leguminosae.
The family Rubiaceae comprises about 450 gen-
era and 6500 species and includes trees, shrubs and
infrequently herbs. Plants belonging to this family
are known to contain substantial amounts of AQs,
especially in the roots. Cell and tissue cultures of Ru-
202
Table 1. Anthraquinones (AQs) from cell cultures and plants of the Rubiaceae in literature since 1985 (species; source:
reference)
Cinchona ledgeriana; cell culture: AQs; Van der
Leer et al., 1991.
C. robusta; cell culture: robustaquinone A∗ , B∗ ,
C∗ , D∗ , E∗ , F∗ , G∗ , H∗ , copareolatin 6-methyl
ether, 1,3,8-trihydroxy-2-methoxy-AQ ; Schripsema
et al., 1999
Damnacanthus indicus; rhizomes: 5-hydroxy-1,2-
methylenedioxy-AQ∗ , damnacanthal, juzunol,
damnacanthol; Koyama et al., 1992
Faramea cyanea; heartwood: rubiadin, 3-hydroxy-
2-methyl-AQ, 3-hydroxy-1-methoxy-2-
methoxy methyl-AQ (damnacanthol-ω-methyl
ether), 1,3-dihydroxy-2-methoxyl-AQ (lucidin-ω-
methyl ether); Ferrari et al., 1985
Galium sinaicum; roots: 6-hydroxyanthragallol 1,3-
dimethyl ether∗ , 6 (or 7) hydroxymethyl-
anthragallol 1,3-dimethyl ether∗ , alizarin 1-methyl
ether, anthragallol 1,3-dimethyl ether; Halim et al.,
1992
G. sinaicum; roots: 7-methylanthragallol 1,3-
dimethyl ether∗ , 7-methyl-anthragallol 2-methyl
ether∗ , 6-methylanthragallol 3-methyl ether∗ , 8-
hydroxyanthragallol 2,3-dimethyl ether∗ , 7-
formylanthragallol 1,3-dimethyl ether∗ ,
copareolatin 5,7-dimethyl ether∗ , copareolatin 6,7-
dimethyl ether∗ , 6-methoxylucidin ω-ethyl ether∗ ,
6-hydroxyxanthopurpurin∗ , bisinaiquinone [10,2 -
bi(9-hydroxy-3-methyl- 1,4-anthraquinonyl)] ∗ ; El-
Gamal et al., 1995
G. sinaicum; roots: 6,7-dimethoxy
xanthopurpurin∗ , 6-hydroxy-7-methoxy rubiadin∗ ,
5-hydroxy-6-hydroxymethyl anthragallol 1,3-
dimethyl ether∗ , 7-carboxy anthragallol 1,3-
dimethyl ether∗ , anthragallol 1-methyl ether 3-O-β-
D -glucopyranoside∗, anthragallol 1-methyl ether 3-
O-rutinoside∗ , anthragallol 3-O-rutinoside∗ ,
alizarin 1-methyl ether 2-O-primeveroside∗ ; El-
Gamal et al., 1996
G. spurium; whole plants: 8-hydroxy-3-methoxy-7-
methyl-1,2-methylenedioxy-9,10-AQ∗ , 2,8-
dihydroxy-1,3-dimethoxy-7 -methyl-9,10-AQ∗ , 1-
hydroxy-2-methyl-AQ, lucidin-ω-methyl ether, 2-
methoxy-6-methyl-1,3,5-trih ydroxy-AQ, rubiadin,
purpurin-1-methyl ether, alizarin-1-methyl ether, 6-
methylalizarin, xanthopurpurin; Koyama et al.,
1993
G. verum; cell cultures: 1,3-dihydroxy-2-
methoxymethyl -AQ, 1,3-dimethoxy-2-hydroxy-AQ,
1,3-dihydroxy-2-acetoxy-AQ, 1-hydroxy-2-
hydroxymethyl-AQ, 1,3-dihydroxy-2-methyl-AQ,
1-methoxy-2-hydroxy-AQ, 1,3-dihydroxy-2-
hydroxymethyl -6-methoxy-AQ, 1,6-dihydroxy-2-
methyl-AQ; Banthorpe and White, 1995
G. verum; cell cultures in bioreactor: AQs;
Christiane et al., 1995
G. verum; immobilized cells: AQs; Heike et al.,
1996
Hedyotis dichotoma; roots: 1,4-dihydroxy-2,3-
dimethoxy-AQ, 2,3-dimethoxy-9-hydroxy-1,4-AQ;
Hamzah et al., 1997
H. diffusa; whole plants: 2,3-dimethoxy-6-methyl-
AQ; Ho et al., 1986
H. herbacea; whole plants: 2-hydroxymethyl-10-
hydroxy-1, 4-AQ∗ , 1,4-dihydroxy-2-hydroxymethyl-
AQ, 2,3-dimethoxy-9-hydroxy-1,4-AQ, 1,4-
dihydroxy-2,3-dimethoxy-AQ; Permana et al., 1999
Knoxia valerianoides; whole plants: 2-
ethoxymethylknoxiavaledin∗ , 2-
formylknoxiavaledin∗ , 2-
hydroxymethylknoxiavaledin∗ , knoxiadin,
damnacanthal, nordamncanthal, ibericin, 3-
methylalizarin, damnacanthol; Zhou et al., 1994
Morinda citrifolia; heartwood: physcion,
morindone, physcion-8-O-[α-L-arabinopyranosyl
(1→3)β-D-galactopyranosyl (1→6)- β-D-
Singh, 1993
M. citrifolia; cell culture: AQs; Bassetti et al., 1996
M. citrifolia; cell culture: alizarin-primeveroside,
lucidin-primeveroside; Hagendoorn et al., 1997;
Van der Plas et al., 1998
M. citrifolia; stem: nordamnacanthal,
damnacanthal; Do et al., 1999
M. elliptica; roots: 2-formyl-1-hydroxy-AQ∗ , 1-
hydroxy-2-methyl-AQ, nordamnacanthal,
damnacanthal, lucidin-ω-methyl ether, rubiadin,
soranjidiol, morindone, rubiadin-1-methyl ether,
alizarin-1-methyl ether, morindone-5-methyl ether;
Ismail et al., 1997

Table 1. Continued
M. elliptica; cell culture: anthragallol-1,2-dimethyl
ether, purpurin-1-methyl ether, nordamnacanthal,
alizarin-1-Me ether, rubiadin, soranjidiol, lucidin-
ω- methyl ether, morindone; Abdullah et al., 1998;
Jasril et al., 2000
M. lucida; roots: 3-hydroxy-AQ-2-carbaldehyde,
nordamnacanthal, damnacanthal, 3-hydroxy-2-
hydroxymethyl-AQ, damnacanthol, tectoquinone,
1-hydroxy-2-methyl-AQ, rubiadin-1-methyl ether,
morindon, 1,5,6-trimethoxy-2-methyl-AQ, alizarin-
1-methyl ether; Rath et al., 1995
M. lucida; stem bark, roots: digitolutein, rubiadin
1-methyl ether, damnacanthal; Koumaglo et al.,
1992
M. officinalis; cell culture: AQs; Xiang and Guo,
1997
M. umbellate; plants: alizarin 2-methyl ether,
alizarin, munjistin methyl ester, 2-hydroxy-AQ-3-
aldehyde; Chang and Chen, 1995
Neonauclea calycina; plants: damnacanthal,
rubiadin 1-methyl ether, nordamnacanthal,
morindone, damnacanthal, lucidin 3-O-
primeveroside, morindone 6-O-primeveroside;
Tosa et al., 1998
Ophiorrhiza pumila; cell culture: 2-methyl-AQ, 1-
hydroxy-2-methyl-AQ, 3-hydroxy-2-methyl-AQ, 3-
hydroxy-AQ-2-carbaldehyde, 1-hydroxy-2-
hydroxymethyl-AQ, 3-hydroxy-2-hydroxymethyl-
AQ, 1-hydroxy-2-hydroxymethyl-3-methoxy-AQ
(lucidin 3-methylether)∗ , 1,3-dihydroxy-2-methyl-
AQ, 1,3-dihydroxy-2-methoxymethyl-AQ, 1,3-
dihydroxy-2-hydroxymethyl-AQ, 2-n-
butoxymethyl-1,3-dihydroxy -AQ (lucidin ω-butyl
ether)∗ ; Kitajima et al., 1998
Prismatomeris sessiliflora; roots: rubiadin,
rubiadin-1-methyl ether; Likhitwitayawuid et al.,
1999
Rubia akane; roots: 1-hydroxy-2-methyl-AQ, 2-
ethoxycarbonyl-1-hydroxy-AQ ∗ ; Okuyama et al.,
1990
R. akane; cell culture: 1,2-dihydroxy-AQ; Mizutani
et al., 1997
203
R. akane; cell culture: alizarin, purpurin, 2-methyl-
1,3,6-trihydroxy-AQ, purpuroxanthin; Endo et al.,
1997
R. akane; cell culture: alizarin; Shim et al., 1999
R. cordifolia; roots: 1,3-dihydroxy-2-
methoxymethyl -AQ, 1-methoxy-2-methoxymethyl-
3-hydroxy -AQ, 4-hydroxy-2-carboxy-AQ, 1,4-
dihydroxy-2-hydroxymethyl -AQ, 1-hydroxy-2-
hydroxymethyl-AQ; Vidal-Tessier et al., 1987
R. cordifolia; roots: dihydromollugin∗ , 2-
carbomethoxy-3-(3 -hydroxy) isopentyl-1,4-
naphthohydroqui none 4-O-β-glucoside∗ , 2-methyl-
1,3,6-trihydroxy-9-10-AQ 3-O-β-glucoside∗ , 2-
methyl-1,3,6-trihydroxy-9,1 0-AQ 3-O-(3 -O-
acetyl)-α-rhamnosyl (1→2)-β-glu coside∗ , 2-
methyl-1,3,6-trihydroxy-9,1 0-AQ 3-O-(3 ,6 -O-
diacetyl)-α-rhamnosyl (1→2)-β-glu coside∗ , 2-
methyl-1,3,6-trihydroxy-9,1 0-AQ 3-O-(4 ,6 -O-
diacetyl)-α-rhamnosyl (1→2)-β-glucoside∗ ;
Itokawa et al., 1989
R. cordifolia; roots: alizarin, 1-hydroxy-2-methyl-
9,10-AQ, 1,3,6-trihydroxy-2-methyl-9,1 0-AQ-3-O-
(6 -O-acetyl)-α-L-rhamnosyl (1→2)-β-D-glucoside,
1,3,6-trihydroxy-2-methyl-9,10-AQ-3-O-(6 -O-
acetyl)-β-D-glucoside∗ , 2-carbomethoxy-3-prenyl-
1,4-n aphthohydroquinonedi-β-D-glucoside,
rubimallin, β-sitosterol, daucosterol; Qiao et al.,
1990
R. cordifolia; roots: 2-methyl-1,3,6-trihydroxy-AQ,
1-hydroxy-AQ, 1,2,4-trihydroxy-9,10-AQ, 2-
methyl-1,3,6-trihydroxy-9,1 0-AQ 3-O-β-D-
glucoside, 1,2-dihydroxy-9,10-AQ 2-O-β-D-
xylosyl-(1→6)-β-D-glucoside, 1,3-dihydroxy-2-
hydroxymethyl-9,10-AQ 3-O-β-D-xylosyl-(1→6)-
β-D-glucoside, 2-methyl-1,3,6-trihydroxy-9,1 0-AQ
3-O-β-D-xylosyl-(1→2)-β-D-(6 -O-acetyl)-
glucoside∗ ; Wang et al., 1992
R. cordifolia; hairy root culture: purpurin; Shin et
al., 1996
R. cordifolia; plant: rubiadin; Tripathi et al., 1997
R. cordifolia; callus culture: munjistin, purpurin;
Mischenko et al., 1999
204
Table 1. Continued
R. lanceolata; roots: mollugin, 1-hydroxy-2-
methyl-AQ, 2-methylquinizarin, 3-carbomethoxy-1-
hydroxy-AQ, lucidin ethyl ether, rubiadin, alizarin,
ω-hydroxypachybasin∗ , digiferruginol∗ ; Kuo et al.,
1995
R. oncotricha; roots: 3-carbomethoxy-1-hydroxy-
9,10 -AQ∗ , 1-hydroxy-2-methyl-AQ,
xanthopurpurin, 2-methyl-1,3,6-trihydroxy-AQ,
tectoquinone, alizrain 2-methyl ether; Itokawa et
al., 1991
R. peregrina; hairy root culture: alizarin; Lodhi and
Charlwood, 1996; Lodhi et al., 1996
R. schumanniana; roots: 1,2-methyl-1,3,6-
trihydroxy-9,10-AQ-3-O-α-L-rhamnopyranosyl
(1→2)-β-D-(6 -acetyl)-glucopyranoside, 2-methyl-
1,3,6-trihydroxy-9,10-AQ-3-O-α-L-
rhamnopyranosyl (1→2)-β-D-glucopyranoside, 1-
hydroxy-2-hydroxymethylene-9,10-AQ-11-O-β-D-
glucopyranosyl-(1→6)-β-D-glucopyranoside∗ ; Liu
et al., 1991
R. sylvatica; roots: 1-hydroxy-2-methyl-AQ, 1,3,6-
trihydroxy-2-methyl-AQ-3-O-(6 -O-acetyl)-α-
rhamnosyl-(1→2)-β-D-glucoside, 1,3,6-trihydroxy-
2-methyl-AQ-3-O-α-rhamnosyl-(1→2)-β-D-
glucoside, 1,3,6-trihydroxy-2-methyl-AQ-3-O-
(3 ,6 -O-diacetyl)-α-rhamnosyl (1→2)-β-D-
glucoside, 1,3,6-trihydroxy-2-methyl-AQ-3-O-
(4 ,6 -O-diacetyl)-α-rhamnosyl-(1→2)-β-D-
glucoside, 1,3,6-trihydroxy-2-methyl-AQ-3-O-β-D-
glucoside; Hou and Wang, 2000
∗ Reported as novel compound.
Figure 1. Structure and carbon numbering of anthraquinones.
R. tinctorum; roots: 1-hydroxy-2-methyl-AQ,
tectoquinone, nordamnacanthal, 1-hydroxy-2-
methoxy-AQ, 1,3-dihydroxy-2-ethoxymethyl-AQ,
alizarin, lucidin, lucidin primeveroside, ruberythric
acid, rubiadin, xanthopurpurin, 1,3-dihydroxy-2-
methoxymethyl-AQ, munjistin methylester, 7-
hydroxy-2-methyl-AQ∗ ; Kawasaki et al., 1992
R. tinctorum; hairy root culture: alizarin, purpurin,
lucidin; Sato et al., 1991
R. tinctorum; hairy root culture: nordamnacanthal,
alizarin; van der Heijden et al., 1994
R. tinctorum; hairy root culture: AQs; Masahiro et
al., 1994
R. tinctorum; callus cultures: ruberythric acid,
rubiadine-3-glucoside; Stara et al., 1995
R. tinctorum; adventitous roots: alizarin,
ruberythric acid, lucidin, lucidin-3-primeveroside,
nordamnacanthal, munjistin, pseudopurpurin,
purpurin; Sato et al., 1991; 1997
R. tinctorum; roots: lucidin; Westendorf et al., 1998
R. tinctorum; roots: 1-hydroxy-2-hydroxymethyl-
AQ 3-glucoside, 2-hydroxymethyl-AQ 3-glucoside,
3,8-dihydroxy-2-hydroxymethyl-AQ 3-glucoside,
alizarin, lucidin-ω-ethyl ether, lucidin
primeveroside; El-Emary et al., 1998
R. tinctorum; hairy root culture: alizarin; Mantrova
et al., 1999
biaceous plants can also accumulate large amounts of
AQs and in some cases the content of AQs in cell cul-
tures even exceeds that of the parent plant. Wijnsma
and Verpoorte (1986) listed the AQs found in the Ru-
biaceae. This information has been updated to include
AQs isolated from the plants or cell cultures of mem-
bers of the Rubiaceae in the last decade (Table 1). The
presence of AQs gives many plants of the Rubiaceae
commercial importance. The root of Rubia tinctorum
(madder) is a source of its natural dyes (Angelini et
al., 1997). In addition, AQs from Rubiaceous plants
have been reported to exhibit some interesting in vitro
biological activities: antimicrobial (Sittie et al., 1999),
antifungal (Rath et al., 1995), hypotensive, analgesic
(Younos et al., 1990), antimalarial (Koumaglo et al.,

1992; Sittie et al., 1999), antioxidant (Tripathi et al.,
1997), antileukemic and mutagenic functions (Chang
and Chen, 1995; Ismail et al., 1997).
In the past years, our knowledge of the AQ bio-
synthesis has increased considerably, but still many
steps are not yet well known. The advent of new tech-
nologies in functional genomics, which accelerate the
discovery of new genes and enzymes, should help in
elucidating AQ biosynthetic pathways. With this per-
spective, we here review the present knowledge of
AQ biosynthetic pathways, as well as the knowledge
of regulation of AQ biosynthesis by external factors
and at the molecular level. Recent discoveries of the
terpenoid and isochorismate biosynthetic pathways,
both of importance for AQs, are also briefly dealt with.
Biosynthetic pathways leading to AQs
Major routes of biosynthesis of AQs have been re-
viewed previously (Leistner, 1985; Wijnsma and Ver-
poorte, 1986; Suzuki and Matsumoto, 1988; Inouye
and Leistner, 1988; Koblitz, 1988; Van Den Berg and
Labadie, 1989; Leistner, 1995). In general, quinones
from higher plants are derived from various precurs-
ors and are produced via different pathways (Leistner,
1981; Inouye and Leistner, 1988). The established
literature reports two main distinct biosynthetic path-
ways leading to AQs in higher plants: The polyketide
pathway and the chorismate/o-succinylbenzoic acid
pathway.
The polyketide pathway
The polyketide pathway occurs mainly in fungi and
some higher plant families as Leguminosae, Rham-
naceae and Polygonaceae (Simpson, 1987; Inouye and
Leistner, 1988). In this pathway, AQs are formed
from one acetyl-CoA unit extended by seven malonyl-
CoA units via an octaketide chain (Figure 2). The
polyketide pathway has been extensively studied in
Streptomyces spp. (Hutchinson and Colombo, 1999).
These types of AQs often exhibit a characteristic sub-
stitution pattern, i.e. they are substituted in both ring A
and C of AQ structure. Chrysophanol and emodin are
typically substituted with hydroxy groups in both ring
A and C and they occur in fungi and higher plants. In
Rumex (Polygonaceae) and Rhamnus (Rhamnaceae),
these two AQs were shown to be biosynthesized via
the polyketide pathway (Leistner, 1971). Enzyme sys-
tems, catalyzing the synthesis of polyketides, have
205
been well documented in bacteria, but detected only
rarely in higher plants (Bernard, 1999).
The chorismate/o-succinylbenzoic acid pathway
In the family Rubiaceae, the genera Galium, Morinda,
Rubia and Cinchona have been extensively used for
AQ biosynthetic studies. AQs in the Rubiaceae are
considered to be of the Rubia type, i.e. ring A and
B are biosynthetically derived from chorismic acid
and α-ketoglutarate via o-succinylbenzoic acid (OSB),
whereas ring C is formed from isopentenyl diphos-
phate (IPP) via the terpenoid pathway (Figure 3)
(Leistner, 1981, 1985).
Biosynthesis of ring A and B
Early work on feeding experiments established that
ring A and B of AQs in Rubia (Leistner, 1981),
Morinda (Leistner, 1975) and Galium (Bauch and
Leistner, 1978; Inoue et al., 1984) are derived
from shikimic acid (Leistner and Zenk, 1967), α-
ketoglutarate via o-succinylbenzoate (Figure 3-I, 3-II).
In the biosynthesis of ring A and B, chorismate de-
rived from the shikimate pathway is first converted
to isochorismate. This reaction is catalyzed by the
enzyme isochorismate hydroxymutase or isochoris-
mate synthase (ICS; EC 5.4.99.6) (Poulsen and Ver-
poorte, 1991). Isochorismate in its turn is converted
into o-succinyl benzoic acid (OSB) in the presence
of α-ketoglutarate and thiamine diphosphate (TPP),
catalyzed by OSB synthase. One of the intermediates
in the formation of OSB from isochorismic acid ap-
pears to be 2-succinyl-6-hydroxy-2,4-cyclohexadiene-
1-carboxylate (Simantiras and Leistner, 1991). OSB
is subsequently activated at the aliphatic carboxyl
group to produce an OSB-CoA ester. This reac-
tion is catalyzed by OSB:CoA ligase (Simantiras and
Leistner, 1992). Ring closure of OSB-CoA results
in the formation of 1,4-dihydroxy-2-naphthoic acid
(DHNA), which gives rise to ring A and B of AQs.
AQs in Cinchona are somewhat unique in that they
are substituted in both ring A and C (Schripsema et
al., 1999). To a lesser extent, this substitution pattern
has also been found in other genera of the Rubiaceae,
such as Galium (Koyama et al., 1993) and Morinda
(Leistner, 1973a). Normally, this pattern is typical of
AQs formed via the polyketide pathway. Schripsema
et al. (1999) proposed that AQs in Cinchona might be
derived from caffeic acid or another phenylpropanoid
and not from chorismate/o-succinylbenzoic acid. Ac-
cording to the labeling patterns of phenylalanine pro-
206
Figure 2. Polyketide pathway for the biosynthesis of anthraquinones.
duced in R. tinctorum after incorporation of [1-13C]
glucose (Eichinger et al., 1999), it is known that caf-
feic acid (Figure 4) is derived from phenylalanine via
the phenylpropanoid pathway (Strack, 1997). If ring
A and B of the AQs are derived from caffeic acid
or another phenylpropanoid, this should result in 13 C
enrichment in either C-8, C-9, C-11 in ring A and B
(Figure 4-I) or C-5, C-10, C-12 in ring A and B of
the AQs (Figure 4-II). However, the observed labeling
patterns (Figure 4-III) of lucidin primeveroside in R.
tinctorum (Eichinger et al., 1999) and robustaquinone
B in Cinchina ‘Robusta’ (Han et al., 2001) show en-
richments in C-8, C-10, C-11 of AQs. These results
are in contradiction with a phenylpropanoid precursor
such as caffeic acid, instead they are in agreement with
the chorismate/o-succinylbenzoate pathway.
The enzymology of AQ biosynthesis in the Rubi-
aceae is still not completely understood. At present,
relatively few steps in this pathway have been partially
characterized to the enzyme level. ICS has been isol-
ated and characterized from Rubiaceous cell cultures
(Ledüc et al., 1991, 1997; Van Tegelen et al., 1999a).
The ics gene was cloned from AQ-synthesizing cell
cultures of Morinda citrifolia (Van Tegelen et al.,
1999b). OSB synthase was detected in AQ-producing
heterotrophic Galium and photoautotrophic M. lucida
cell cultures (Simantiras and Leistner, 1989, 1991).
OSB:CoA ligase has also been purified and charac-
terized from AQ-producing cell cultures of Galium
mollugo (Heide et al., 1982; Sieweke and Leistner,
1992). The Galium OSB:CoA ligase has a molecular
mass of 66 kDa and two isoelectric points at pH 7.5
and 8.7. Characterization of the enzymes catalyzing
the remaining steps and the intermediates after OSB
still remains to be investigated.
Biosynthesis of ring C
The subsequent prenylation of DHNA at C-3 in Ru-
biaceous plants (Inoue et al., 1979, 1984) results in
a naphthoquinol (or naphthoquinone). The formation
of ring C of AQs results from the cyclization via C-
C bond formation between the aromatic ring of the
naphthoquinone and an isoprene unit IPP or DMAPP
(Figure 3).
Since the 1950s, it was accepted that IPP is formed
from the acetyl-CoA via mevalonic acid (MVA) path-
way (Bach et al., 1999; Bochar et al., 1999) (Figures
3-III and 5-I), which was considered as ubiquitous in
all living organisms. Recently, a non-MVA pathway,
termed as the 2-C-methyl-D-erythritol 4-phosphate
(MEP) pathway (Rohmer, 1999; Lichtenthaler, 1999;
Eisenreich et al., 2001) (Figures 3-IV and 5-II), was
discovered. In this pathway, IPP is formed from 1-
deoxy-D-xylulose 5-phosphate (DOXP) by condens-
ation of glyceraldehyde 3-phosphate (GAP) and pyr-
uvate. This pathway was initially identified in bacteria
(Rohmer et al., 1993) and was subsequently found in
green algae and plastids of higher plants. This raises
the question about the origin of IPP, incorporated to
ring C of the AQs in the Rubiaceae, whether it is either
derived from the MVA or the MEP pathway.
The mevalonic acid pathway. The MVA pathway
(Figure 5-I) in relation to AQ biosynthesis has been
well studied. Early work has shown that feeding of
[2-14C] mevalonic acid to root system (Leistner and
Zenk, 1968) and plants (Burnett and Thomson, 1968)
of R. tinctorum resulted in incorporation into AQs.
These results established that ring C of Rubia type
AQs are derived from mevalonic acid, possibly via
DMAPP (Figure 3). However, the incorporation rates
were low, from 0.02 to 0.06%. In cell cultures of M.
citrifolia, no incorporation of [2-14C] mevalonic acid
into morindone or alizarin was observed (Leistner,
1973a). When [5-14C] mevalonic acid was fed to cell
cultures of R. tinctorum, the incorporation rate into al-
izarin was only 0.003% (Leistner, 1973b). Recently, it
was shown that the MEP pathway, instead of the MVA
pathway, is involved in the formation of IPP, incorpor-
ated into ring C of AQs (Eichinger et al., 1999; Han
 207

Figure 3. Biosynthetic pathways leading to anthraquinones in the Rubiaceae. DHNA – 1,4-dihydroxy-2-naphthoic acid; DMAPP – 3,3-di-
methylallyl diphosphate; E-4-P – erythrose 4-phosphate; GAP – glyceraldehyde 3-phosphate; HMG-CoA – 3-hydroxy-3-methylglutaryl
coenzyme A; IPP – isopentenyl diphosphate; MVA – mevalonic acid; MEP – 2-C-methyl-D-erythritol 4-phosphate; OSB – o-succinylbenzoic
acid; PEP – phosphoenol pyruvate; TCA – tricarboxylic acid; TPP – thiamine diphosphate. Enzymes: (1) isochorismate synthase; (2)
o-succinylbenzoate synthase; (3) OSB:CoA ligase; (4) IPP isomerase.

et al., 2001). It was also shown that all higher plants
appear to utilize both the MVA and the MEP pathways
with apparent cross-talk between the MVA and the
MEP pathways across compartments (Ramos-Valdivia
et al., 1997a; Goese et al., 1999). This possibly ex-
plains why labeled mevalonic acid was incorporated
into AQs in low levels.
The MEP pathway. On the basis of retrobiosynthetic
nuclear magnetic resonance analysis of the incorpor-
ation of [1-13C]glucose into AQs in cell cultures of
208
Figure 4. The predicted labeling patterns of ring A and B in AQs via the phenylpropanoid pathway (I, II) and the observed labeling pattern of
ring A and B (III) after incorporation of [1-13 C] glucose (From Eichinger et al., 1999; Han et al., 2001).
R. tinctorum (Eichinger et al., 1999) and C. ‘Robusta’
(Han et al., 2001), it was shown that the MEP pathway,
not the MVA pathway, is involved in the formation of
IPP, which constitutes ring C of AQs in the above cul-
tures. Feeding 13 C- and 14 C-labeled pyruvate to cell
cultures of M. citrifolia (Stalman et al., 2001) resulted
in incorporation into the AQs. Addition of lovastatin
(or mevinolin), a highly specific inhibitor of the en-
zyme HMG-CoA reductase in the MVA pathway, did
not inhibit the biosynthesis of AQs in cell cultures of
C. ‘Robusta’ (Han et al., unpublished data). These
results also support the view that the MEP pathway
is involved in the formation of ring C of AQs in the
Rubiaceae.
In the MEP pathway (Figure 5-II), the initial re-
action is the formation of DOXP by the condensation
of GAP with pyruvate. This reaction is catalyzed by
1-deoxy-D-xylulose 5-phosphate synthase (DXS), a
novel type of transketolase (Lois et al., 1998). The
dxs gene was cloned and characterized from E. coli
(Sprenger et al., 1997; Lois et al., 1998), other bac-
teria (Altincicek et al., 2000; Kuzuyama et al., 2000a;
Hahn et al., 2001) and higher plants (Mandel et al.,
1996; Lange et al., 1998b; Bouvier et al., 1998;
Miller et al., 1999; Chahed et al., 2000; Lois et
al., 2000). In the second step, DOXP is converted
into 2-C-methyl-D-erythritol 4-phosphate (MEP) by
the enzyme DOXP reductoisomerase (DXR). The dxr
genes have been cloned and characterized in plants
(Lange and Croteau, 1999b; Schwender et al., 1999;
Veau et al., 2000), certain bacteria (Takahashi et al.,
1998; Kuzuyama et al., 2000b; Altincicek et al., 2000;
Grolle et al., 2000). It was recently shown that MEP
was converted to 4-cytidyl diphospho-2C-methyl-D-
erythritol (CDP-ME). This conversion is catalyzed by
CDP-ME synthase (Rohdich et al., 1999; Kuzuyama
et al., 2000c), encoded by the ispD gene (formerly
designated ygbP). CDP-ME synthase (IspD protein)
was cloned from Arabidopsis thaliana and the over-
expressed recombinant protein in E. coli was purified
to homogeneity with a mass of 29 kDa (Rohdich et al.,
2000b). CDP-ME is further converted to 2-C-methyl-
D -erythritol 2,4- cyclodiphosphate (MEC), catalyzed
by the consecutive action of two proteins: CDP-ME
kinase (Lüttgen et al., 2000; Rohdich et al., 2000a) and
MEC synthase (Herz et al., 2000; Veau et al., 2000;
Hahn et al., 2001), which are encoded by the genes
ispE (formerly designated ychB) and ispF (formerly
designated ygbB), respectively. On the other hand, the
YchB protein, designated as isopentenyl monophos-
phate kinase (IPK), was isolated from E. coli and
peppermint (Mentha piperita) (Lange and Croteau,
1999a). IPK catalyzes the formation of IPP from iso-
pentenyl monophosphate at extremely low rates, 178
 209

Figure 5. Pathways for IPP biosynthesis. ADP – adenosine diphosphate; ATP – adenosine 5 -triphosphate; CMP – cytidine monophosphate;
CTP – cytosine 5 -triphosphate; DMAPP = 3,3-dimethylallyl diphosphate; IPP = isopentenyl diphosphate; NADP – nicotinamide adenine
dinucleotide phosphate; NADPH – nicotinamide adenine dinucleotide phosphate, reduced. I: the MVA pathway Enzymes: (1) acetoacetyl-CoA
thiolase; (2) HMG-CoA synthase; (3) HMG-CoA reductase; (4) MVA kinase; (5) MVAP kinase; (6) MVAPP decarboxylase. II: The MEP
Pathway (From Rohdich et al., 2000a) Genes: dxs – 1-deoxy-D-xylulose 5-phosphate synthase; dxr – 1-deoxy-D-xylulose 5-phosphate re-
ductase; ispD – 4-cytidyl diphosphate-2-C-methyl-D-erythritol synthase; ispE – 4-cytidyl-diphosphate-2-C-methyl-D-erythritol kinase; ispF –
2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase.
210
± 81 and 1.43 pmol·g−1 ·s−1 in E. coli and pepper-
mint, respectively. This reaction was suggested as the
last step in the MEP pathway. However, Rohdich et al.
(2000a) showed that the over-expressed IspE (YchB)
protein from tomato (Lycopersicon esculentum) did
not show any IPK activity, even when very high con-
centrations of the recombinant enzymes were used.
Also, the IspE protein of E. coli accepts CDP-ME,
but not isopentenyl monophosphate, as a substrate
(Kuzuyama et al., 2000c). So, it was argued that the
detected low IPK activity could not be metabolically
relevant (Rohdich et al., 2000a). The remaining re-
action steps leading to IPP still remain to be fully
elucidated. With all the available information on the
MEP pathway from bacteria and other plants, further
studies of the involvement of this pathway in AQ bio-
synthesis can be done directly at the enzyme and gene
levels.
After formation of IPP, it is converted to DMAPP,
the activated monomer building block for all isopren-
oids. This reversible conversion is catalyzed by the
enzyme IPP isomerase (reviewed by Ramos-Valdivia
et al., 1997a). IPP isomerase activity was found in
induced AQ-accumulating cell cultures of C. robusta
(Ramos-Valdivia et al., 1997b). The purified two iso-
forms of IPP isomerase have K m values for IPP of 5.1
and 0.95 µM, respectively. The activity of IPP iso-
merase was also found in AQ-producing cell cultures
of M. citrifolia and R. tinctorum (Ramos-Valdivia et
al., 1998).
Late steps in AQ biosynthesis
Most AQs in the Rubiaceae have substitutions, e.g.
hydroxy, methoxy and methyl groups in the ring C. To
a lesser extent, some AQs in this family have substi-
tutions in both ring A and C (Wijnsma and Verpoorte,
1986). These substitution groups might be introduced
by hydroxylation or methylation (Poulton, 1981) in the
late stage of AQ biosynthesis. In cell cultures of M.
citrifolia (Leistner, 1973a, 1975), it was shown that
the hydroxy groups attached to ring A of morindone
were not derived from the hydroxy groups of shikimic
acid. Instead, the hydroxy groups are introduced at a
later stage of the biosynthetic pathway. Many AQs are
stored in the form of glycosides, due to glycosylation
(Van der Plas et al., 1998). Five AQ-specific glucosyl-
transferases were partially purified from C. succirubra
(Khouri et al., 1987).
Regulation of AQ biosynthesis
AQ biosynthesis in cell cultures is regulated in re-
sponse to both environmental (light, nutritional com-
ponents, elicitors, etc.) and endogenous (metabolic
and developmental) stimuli. Compartmentation is also
an important component of regulation (Hrazdina,
1994).
Environmental factors
A number of environmental factors influence the bio-
synthesis of AQs in cell cultures, including light, plant
growth regulators, nutritional factors, biosynthetic
precursors and elicitors.
Light often exerts an influence on the formation of
secondary metabolites in plant cell cultures. Generally
illumination of the cell cultures reduces AQ yield. In
cell cultures of C. ledgeriana, the highest AQ yield
was obtained in the dark-grown cultures (Harkes et
al., 1985). In cell cultures of R. cordifolia, AQ form-
ation was repressed by irradiation of blue or red light
(Suzuki et al., 1985). In 18-month old cell cultures of
M. elliptica, absence of light increased AQ content in
growth medium and production medium by 70% and
100%, respectively (Abdullah et al., 1998).
Plant growth regulators play an important role
in the induction and repression of biosynthetic path-
ways leading to secondary metabolites. In cell
cultures of Morinda, Galium and Cinchona, 2,4-
dichlorophenoxyacetic acid (2,4-D) was shown to sup-
press AQ production, while 1-naphthaleneacetic acid
(NAA) promoted AQ production (Zenk et al., 1984;
Leistner, 1995). However, 2,4- D induces, while NAA
almost completely suppresses, AQ formation in G.
spurium (Schulte et al., 1984). In cell cultures of C.
succirubra, increasing concentrations of NAA (1.25
– 12.5 ppm) or 3-indoleacetic acid (IAA) (2.5 – 25
ppm), in the absence of cytokinin, enhanced both cell
growth and AQ production, whereas, increasing con-
centration of 2,4-D (0.25 – 2.5 ppm) inhibited both
growth and AQ production (Khouri et al., 1986). How-
ever, addition of cytokinin did not affect either cell
growth or AQ production. In hairy root cultures of
R. tinctorum, IAA resulted in the maximal growth
rate and the highest AQ production, while kinetin
had no effect (Sato et al., 1991). In cell cultures
of M. citrifolia (Van der Plas et al., 1995), it was
shown that there are two developmental states: an AQ
non-producing state with rapid cell division and an
AQ-producing state with inhibited cell division. Ad-

dition of 2,4-D resulted in a rapid switch from an AQ
producing to a non-AQ-producing state (Hagendoorn
et al., 1997). It was recently shown that the inhibition
of AQ biosynthesis in M. citrifolia occurs because of
the repression of ics gene expression by 2,4-D (Van
Tegelen et al., 1999b). All these findings indicate that
AQ synthesis is regulated in a different way by the
type and concentration of auxin, rather than by the
presence of cytokinin. The type of carbon source and
its concentration influence both growth and AQ pro-
duction. Higher concentrations of sucrose (6–18%)
inhibited growth in the presence of phytohormones
(IAA or NAA). However, in phytohormone-free me-
dium, 12% sucrose resulted in maximal growth and
AQ production (Sato et al., 1991). Photoautotrophic
cell cultures of M. lucida, lipoquinones such as phylo-
quinone and plastoquinone were formed in medium
without sucrose under relatively high light intensit-
ies (5000 – 6000 lux), but no AQs were formed.
When such cultures were transferred to fresh me-
dium containing sucrose and cultured in the dark, AQs
were produced, but lipoquinones disappeared. Man-
nitol could not replace sucrose, indicating that AQ
formation is dependent on the presence of sucrose (Ig-
bavboa et al., 1985). This suggests that sucrose not
only plays an important nutritive role, but might also
a regulatory role in secondary product formation in
the cell cultures. It was also shown that sugars have
important signaling functions throughout the plant life
cycle (Smeekens, 2000). In cell cultures of M. citrifo-
lia, sucrose was better than glucose for the formation
of AQs (Zenk et al., 1975). In contrast, sucrose was
inferior to glucose in cell cultures of R. cordifolia (Su-
zuki et al., 1984). Similarly, glucose resulted in higher
yield of AQs in elicited cell cultures of C. ‘Robusta’
(Han et al., 2001). These results clearly show that each
culture has its own specific requirements for growth
regulators and carbohydrates for secondary metabolite
production (Schulte et al., 1984; Harkes et al., 1985;
Khouri et al., 1986; Robins et al., 1986).
Addition of known or putative precursors can en-
hance AQ production. In Morinda cell cultures, ad-
dition of OSB resulted in a remarkable increase of
AQ production (Zenk et al., 1975; Bauch and Leist-
ner, 1978). Addition of pyruvate, a precursor of the
MEP pathway, at a level of 20 mM in the growth
medium resulted in a 4-fold increase in AQ produc-
tion in cell cultures of M. citrifolia (Stalman et al.,
2001), even in the presence of inhibitory levels of
2,4-D. It was also found that L-tryptophan and its
precursors such as anthranilate were potent inhibitors
211
of AQ formation in cell cultures of M. citrifolia (El-
Shagi et al., 1984) and C. ledgeriana (Robins et al.,
1986, Van der Leer et al., 1991). Some pathway spe-
cific inhibitors have a marked effect on growth and
AQ production. AQ synthesis in cell cultures of G.
mollugo was blocked by glyphosate, an inhibitor of
the shikimic acid pathway. This inhibition can be alle-
viated by the addition of chorismate and OSB. This
demonstrates that glyphosate inhibits a step in the
biosynthetic sequence from shikimate to chorismate
(Amrhein et al., 1980), a precursor for AQ synthesis.
Similarly, glyphosate decreased AQ production in cell
cultures of C. ledgeriana, and this inhibition was alle-
viated by the addition of L-tyrosine, L-phenylalanine
and L-tryptophan (Robins et al., 1986). It was found
that mevinolin, a highly specific inhibitor of the en-
zyme HMG-CoA reductase in the MVA pathway, did
not inhibit the AQ biosynthesis in cell cultures of C.
‘Robusta’ (Han et al., unpublished data).
The biosynthesis of phytoalexin metabolites in
plant cell cultures is often induced or enhanced by
biotic elicitors such as pathogenic microorganisms,
cellulase, pectinase and yeast preparations and abi-
otic stresses such as wounding, treatment of heavy
metals, and jasmonates (Boller, 1995). AQ production
in Cinchona cell cultures can be induced by the addi-
tion of elicitors, such as homogenates of Phytophthora
cinnamomi (Wijnsma et al., 1985; Ramos-Valdivia et
al., 1997c). Jasmonates are key signals of defense
gene expression. Exogenously applied jasmonates in-
duce transcriptional activities of genes involved in the
biosynthesis of secondary metabolites in plant cell cul-
tures (Gundlach et al., 1992; Mueller et al., 1993;
Reymond and Farmer, 1998). In cell cultures of R.
tinctorum, rubiadin was increased 11-fold by the ad-
dition of methyl jasmonate (MeJA) (Gundlach et al.,
1992). In transformed hairy roots of R. tinctorum, ad-
dition of MeJA resulted in a five to eight-fold increase
of AQ contents compared to the control (Mantrova
et al., 1999). In adventitious root cultures of R. tinc-
torum, addition of CuCl2 induced phytochelatins and
affected the production of AQ pigments, but addition
of glutathione alone resulted in a marked increase in
AQ production in adventitious root cultures of R. tinc-
torum, particularly lucidin-3-O-primeveroside (Sato
et al., 1997). Chitosan not only acts as an elicitor,
but permeabilizes plant membranes. In immobilized
cells of M. citrifolia, chitin-50 stimulated AQ form-
ation (Heike et al., 1996). It was found that silicone
A stimulated AQ production and release in a two-
liquiD-phase culture of M. citrifolia (Bassetti et al.,
212
1996). The silicon treatment in the two-liquid-phase
system (5 ml n-hexadecane/50 ml medium) resulted in
a 150% increase of the overall secondary metabolite
productivity.
Regulation of AQ biosynthesis at the enzyme and gene
levels
In the Rubiaceae, chorismate is not only a precursor
for AQs, but also for a large variety of metabol-
ites, such as aromatic amino acids. Thus, chorismate
represents a major branch-point intermediate in plant
metabolism (Poulsen and Verpoorte, 1991; Knaggs,
1999; Herrmann et al., 1999). ICS could be a regu-
latory enzyme for the channeling of chorismate into
AQ biosynthesis in the Rubiaceae. In AQ-producing
cell cultures of R. tinctorum, it was shown that the
increase in AQ biosynthesis is preceded by a propor-
tional increase in ICS activity and its transcript level
(Van Tegelen et al., 1999c). In elicited cell cultures of
C. robusta, the accumulation of AQs was preceded by
a 2.5-fold transient induction of IPP isomerase activ-
ity. In cell cultures of M. citrifolia and R. tinctorum,
the accumulation of AQs was also accompanied by
a change in the activity of IPP isomerase (Ramos-
Valdivia et al., 1997c). This suggests that IPP iso-
merase channels isoprene units into AQ biosynthesis
(Ramos-Valdivia et al., 1998).
In the MVA pathway, HMGR is considered to be
a rate-limiting enzyme in mammalian metabolism, es-
pecially for cholesterol. It also has been demonstrated
to be a regulatory point for the synthesis of sterols and
phytoalexins in plants (Stermer et al., 1994; Schalleret
al., 1995; Weissenborn et al., 1995). In cell cultures
of L. erythrorhizon (Lange et al., 1998a), a naph-
thoquinone pigment shikonin is produced. The carbon
skeleton of shikonin is partly derived from the MVA
pathway. It was found that HMGR enzyme activity
and mRNA levels closely correlated with the accu-
mulation of shikonin derivatives. White light, which
inhibits shikonin formation, also strongly suppressed
HMGR gene expression. This suggests that HMGR
plays a significant role in the regulation of shikonin
biosynthesis at the transcriptional level.
In the biosynthesis of secondary metabolites in
which the MEP pathway is involved, many examples
show that DXS and/or DXR are likely to be regulat-
ory enzymes involved in channeling intermediates into
different pathways. At present, it has been shown that
the biosynthesis of ubiquinone (Harker and Bramley,
1999) and the carotenoids lycopene or zeaxanthin
(Matthews and Wurtzel, 2000) in E. coli was enhanced
by overexpression of a heterologous dxs gene. In E.
coli overexpressing a dxs gene from Synechococcus
leopoliensis, the content of DMAPP was significantly
increased, whereas overexpression of the dxr gene
did not lead to an increased level of DMAPP. So it
was suggested that dxs, but not dxr, catalyzes a rate-
limiting step of the MEP pathway (Miller et al., 2000).
It was also found that overexpression of the gene dxs,
dxr and idi, encoding an IPP isomerase, stimulated
carotenogenesis up to 3.5-fold in the transformed E.
coli (Albrecht et al., 1999). In peppermint, the rate
of monoterpene biosynthesis was preceded by an in-
crease in DXS mRNA levels in oil gland secretory
cells from leaves at different developmental stages
(Lange et al., 1998b). It was also shown that colon-
isation of roots from wheat, maize, rice and barley by
an arbuscular mycorrhizal fungi involves strong induc-
tion of transcript levels of two key enzymes DXS and
DXR of the MEP pathway. This induction is temporar-
ily and spatially correlated with specific and concom-
itant accumulation of two classes of apocarotenoids
(Walter et al., 2000). In the terpenoid indole alkal-
oids (TIA) producing cells of C. roseus, it was found
that the accumulation of DXS transcripts is associated
with TIA production (Chahed et al., 2000). It was also
shown that DXS mRNA accumulation strongly cor-
related with carotenoid synthesis during tomato fruit
development (Lois et al., 2000). DXS /or DXR might
be regulatory enzymes in AQ biosynthesis in the Ru-
biaceae. Studies on correlation of dxs transcript levels
with AQ accumulation and cloning of the gene dxs are
under way in our laboratory.
Compartmentation
The subcellular compartmentation of a pathway plays
a central role in regulating the biosynthesis of sec-
ondary metabolites. The MEP pathway is primar-
ily operative in plastids of higher plants, whereas
the MVA pathway is likely located in the cytoplasm
(Lichtenthaler et al., 1999). In photoautotrophic cell
cultures of M. citrifolia, irregular or distorted plast-
ids containing starch grains were observed in AQ-
producing cells (Yamamoto et al., 1987). This sug-
gests that plastids play an important role in AQ pro-
duction. In addition, highly elongated rough endoplas-
mic reticulum (ER) observed in AQ-producing cells
suggests that rough ER may also participate in AQ
synthesis. At present, the exact process of AQ syn-
thesis within the cell is not yet known. However, it

is likely that the biosynthesis of AQs in the Rubiaceae
requires at least four compartments, the plastids for the
formation of isoprene moiety, the ER and the cytosol
for further steps in the pathway and finally the vacuole
for the storage of AQs. Apparently, the sites of AQ
synthesis and for storage are separated by eliminating
product feedback inhibition. However, studies on the
enzymes and intermediates of AQ biosynthesis and
their localization are needed to elucidate the sites as
well as the control mechanism of AQ biosynthesis.
Metabolic engineering of AQ biosynthesis
Since the 1980s, plant genetic engineering has made
great progress and led to the commercial introduction
of transgenic crops in the 1990s. Recently, a golden
rice was developed through metabolic engineering (Ye
et al., 2000), where a complete β-carotene (provitamin
A) biosynthetic pathway has been successfully intro-
duced into rice endosperm and the resulting transgenic
rice exhibited a clearly visible yellow color, due to
the carotenoid accumulation. In the past years, there
has also been much progress in metabolic engineering
of plant secondary metabolite biosynthetic pathways
(Verpoorte and Alfermann, 2000).
There have been a few reports on metabolic engin-
eering of AQ pathway in plant cell cultures. In trans-
formed hairy root cultures of R. peregrina, the accu-
mulation of total AQs and the major AQ alizarin were
found to be approximately 2-fold and 3-fold higher,
respectively, compared to field grown roots (Lodhi and
Charlwood, 1996). Moreover, the gene coding for ICS
from E. coli was also introduced (Lodhi et al., 1996).
The ICS activity in the transgenic roots containing the
ics gene was found to be twice to that in control roots.
The accumulation of total AQs was 20% higher than
that in control. In our laboratory, an ics gene from
C. roseus was introduced into M. citrifolia. Only the
cell line containing ics in sense orientation showed
ICS activity. However, the AQ contents in sense and
antisense cell lines were not different (Talou et al., un-
published data). A genetically transformed hairy root
cultures derived from AQ-producing madder plants
were developed (Kuzovkina et al., 1999).
Novel approaches to elucidate biosynthetic
pathways
The advent of genome sequence information has
greatly accelerated the identification of genes (Canel,
213
1999). In the initial step of the MEP pathway, syn-
thesis of DOXP requires an acyloin condensation re-
action of GAP with pyruvate, whereby pyruvate is
decarboxylated. This type of reaction is also cata-
lyzed by the well-documented pyruvate decarboxylat-
ing enzyme such as the E1 component (EC 1.2.4.1)
of the pyruvate dehydrogenase complex (PDHC) or
of pyruvate decarboxylase (EC 4.1.1.1). Likewise,
thiamin-dependent transketolases could also catalyze
the transfer of an activated acetaldehyde group from
pyruvate to GAP. Thus it was hypothesized that a DXS
enzyme might share sequence motifs with the E1 sub-
unit of PDHC and with transketolases. After screening
E. coli genome for such genes encoding products sim-
ilar to transketolase and E1, dxs gene was successfully
identified in E. coli (Sprenger et al., 1997). Functional
genomics approaches, which use combined computa-
tional and expression-based analysis of large amounts
of sequence information, are emerging as powerful
tools for gene discovery and characterization. From
the secretory cells of the oil glands responsible for
essential oil biosynthesis in peppermint, mRNA was
obtained and subsequently used to generate a cDNA
library. Led by bioinformatic processing of the data
to candidate genes putatively involved in essential oil
biosynthesis and secretion, the expressed sequence
tags (ESTs) from the cDNA library were heterolog-
ously expressed in E. coli. Functional characterization
of the corresponding recombinant proteins revealed
the ESTs involved in essential oil biosynthesis, secre-
tion and transportation (Lange et al., 2000). Recently,
DNA microarrays, consisting of thousands of indi-
vidual gene sequences printed in a high-density array
on a glass microscope slide, provide an exciting new
tool for studying gene expression on a very large scale
(Graves, 1999; Sundberg, 2000). Using microarrays,
gene expression profiles were attained for Arabidop-
sis thaliana (Schena et al., 1995), Saccharomyces
cerevisiae (DeRisi et al., 1997). A recent study by use
of DNA microarrays allowed the discovery of a novel
alcohol acyltransferase gene involved in flavour bio-
genesis in strawberry (Aharoni et al., 2000). The next
step, protein microarrays (MacBeath and Schreiber,
2000; Emili and Cagney, 2000) will be used for pro-
filing of proteins encoded by differentially expressed
cDNA clones. Proteomics is the systematic analysis of
the proteins expressed by the genome. It is a promising
approach for the identification of enzymes involved
in secondary metabolism (Jacobs et al., 2000). In
alkaloid-producing cells of C. roseus with treatment
of 2,4-D and/or zeatin, the proteomic investigations
214

Figure 6. A silver stained 2D-gel of a protein extract of seven-day-old suspension cultured cells of Morinda citrifolia. The proteins were
precipitated with 10% TCA in acetone and dissolved in 9.5 M urea, 2% CHAPS, 0.8% carrier ampholytes, 1% dithiotreitol. One hundred
microgram of protein was loaded on a 18 cm IPG-strip pH 4–7 (Jacobs et al, unpublished).

showed that there were changes of 24 polypeptides,
of which a 28 kDa polypeptide showed a close correl-
ation with alkaloid production (Carpin et al., 1997).
With the above-mentioned novel approaches, proteo-
mics seems to be a suitable approach for elucidation of
the yet unknown steps in AQ-biosynthesis. A first step
for the proteomics of Morinda has been made in our
laboratory. A 2D-gel of a protein extract of cultured
cells of M. citrifolia is presented in Figure 6 (Jac-
obs et al., unpublished data). A proteome analysis of
M. citrifolia cells under AQ-producing and non-AQ-
producing states may identify the proteins involved in
AQ-production.
Conclusion and perspective
In the past decade, there has been much progress in
understanding the AQ biosynthesis in the Rubiaceae.
Some genes encoding enzymes of AQ biosynthesis
have been identified following the purification of the
corresponding enzymes. The MEP pathway has been
shown to be involved in the AQ biosynthesis in the
Rubiaceae. Although much progress was made, our
knowledge of the AQ biosynthesis still needs to be
highly enhanced, particularly in the areas such as
the site of AQ biosynthesis, compartmentation, the
elements and mechanisms responsible for AQ biosyn-
thesis and regulation at the enzyme and gene levels. To
answer these questions, it is necessary to integrate cell
physiological, biochemical, molecular biological and
genetic approaches.
In the next few years, the new approaches such as
microarrays, proteomics will certainly become power-
ful tools in biology. These technologies should bene-
fit studies on analyzing gene expression, identifying
genes/enzymes and regulatory mechanisms respons-
ible for AQ biosynthesis. The more profound know-
ledge of AQ biosynthesis will advances the devel-
opment of metabolic engineering of AQ biosynthetic
pathways. Eventually, we shall be able to manipulate
AQ biosynthesis in the Rubiaceae through metabolic
engineering in the near future.

Acknowledgements
Financial support of the ‘Van Leersum Fonds’ is grate-
fully acknowledged. We would like to thank Ir. D.I.
Jacobs, Dr. D. Hallard and Drs. M. van Rijssen
for their contributions to the Morinda 2D-gel exper-
iments.
References
Abdullah MA, Ali AM, Marziah M, Lajis NH & Ariff AB (1998)
Establishment of cell suspension cultures of Morinda elliptica
for the production of anthraquinones. Plant Cell Tiss. Org. Cult.
54: 173–182
Aharoni A, Keizer LC, Bouwmeester HJ, Sun Z, Alvarez-Huerta M,
Verhoeven HA, Blaas J, Houwelingen MML, Vos RCHD, Van
der Voet H, Jansen RC, Guis M, Mol J, Davis RW, Schena M, Van
Tunen AJ & O’Connell AP (2000) Identification of the SAAT
gene involved in strawberry flavor biogenesis by use of DNA
microarrays. Plant Cell 12: 647–662
Albrecht M, Misawa N & Sandmann G (1999) Metabolic engin-
eering of the terpenoid biosynthetic pathway of Escherichia coli
for production of the carotenoids β-carotene and zeaxanthin.
Biotech. Lett. 21: 791–795
Altincicek B, Hintz M, Sanderbrand S, Wiesner J, Beck E & Jomaa
H (2000) Tools for discovery of inhibitors of the 1-deoxy–
xylulose 5-phosphate (DOXP) synthase and DOXP reductoi-
somerase: An approach with enzymes from the pathogenic bac-
terium Pseudomonas aeruginosa. FEMS Microbiol. Lett. 190:
329–333
Amrhein N, Deus B, Gehrke P & Steinrücken HC (1980) The site
of the inhibition of the shikimate pathway by glyphosphate. Plant
Physiol. 66: 830–834
Angelini LG, Pistelli L, Belloni P, Bertoli A & Panconesi S (1997)
Rubia tinctorum a source of natural dyes: Agronomic evaluation,
quantitative analysis of alizarin and industrial assays. Ind. Crops
Prod. 6: 303–311
Bach TJ, Boronat A, Campos N, Ferrer A & Vollack KU (1999)
Mevalonate biosynthesis in plants. Crit. Rev. Biochem. Mol.
Biol. 34: 107–122
Bassetti L, Pijnenburg J & Tramper J (1996) Silicone-stimulated
anthraquinone production and release by Morinda citrifolia in
a two-liquid-phase system. Biotechnol. Lett. 18: 377–382
Banthorpe DV & White JJ (1995) Novel anthraquinones from un-
differentiated cell cultures of Galium verum. Phytochemistry 38:
107–111
Bauch HJ & Leistner E (1978) Aromatic metabolites in cell suspen-
sion cultures of Galium mollugo. Planta Med. 33: 105–123
Bernard JR (1999) Biosynthesis of polyketides (other than actino-
mycete macrolides). Nat. Prod. Rep. 16: 425–484
Bochar DA, Friesen JA, Stauffacher CV & Rodwell VW (1999)
Biosynthesis of mevalonic acid from acetyl-CoA. In: Barton D &
Nakanishi K (eds) Comprehensive Natural Products Chemistry,
Vol 2 (pp 15–44). Pergamon, Oxford
Boller T (1995) Chemoperception of microbial signals in plant cells.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 189–214
Bouvier F, d’Harlingue A, Suire C, Backhaus RA & Camara B
(1998) Dedicated roles of plastid transketolases during the early
onset of isoprenoid biogenesis in pepper fruits. Plant Physiol.
117: 1423–1431
215
Burnett AR & Thomson RH (1968) Naturally occurring quinones.
Part XV. Biogenesis of the anthraquinones in Rubia tinctorum L.
(Madder). J. Chem. Soc. C. 2437–2441
Canel C (1999) From genes to phytochemicals: the genomics ap-
proach to the characterization and utilization of plant secondary
metabolism. Acta Hortic. 51–57
Carpin S, Quelhazi L, Filali M, Chénieux JC, Rideau M & Hamdi
S (1997) The relationship between the accumulation of a 28 kD
polypeptide and that of indole alkaloids in Catharanthus roseus
cell suspension cultures. J. Plant Physiol. 150: 452–457
Chahed L, Oudin A, Guivarc’h N, Hamdi S, Chénieux J, Rideau M
& Clastre M (2000) 1-deoxy-D-xylulose 5-phosphate synthase
from periwinke: cDNA identification and induced gene expres-
sion in terpenoid indole alkaloid-producing cells. Plant Physiol.
Biochem. 38: 559–566
Chang P & Chen C (1995) Isolation and characterization of anti-
tumor anthraquinones from Morinda umbellata. Chin. Pharm. J.
(Taipei) 47: 347–353
Christiane LP, Heike D & Dietrich K (1995) Bioreactors for plant
cell cultures. BioTec 7: 28–32
DeRisi JL, Iyer VR & Brown PO (1997) Exploring the metabolic
and genetic control of gene expression on a genomic scale.
Science 278: 680–686
Do QV, Pharm GD, Mai NT, Phan TPP, Nguyen HN, Jea YY & Ahn
BZ (1999) Cytotoxicity of some anthraquinones from the stem
of Morinda citrifolia growing in Vietnam. Tap Chi Hoa Hoc 37:
94–97
Eichinger D, Bacher A, Zenk MH & Eisenreich W (1999) Quantit-
ative assesment of metabolic flux by 13 C NMR analysis. Biosyn-
thesis of anthraquinones in Rubia tinctorum. J. Am. Chem. Soc.
121: 7469–7475
Eisenreich W, Rohdich F & Bacher A (2001) Deoxyxylulose phos-
phate pathway to terpenoids. Trends Plant Sci. 6: 78–84
El-Emary NA & Backheet EY (1998) Three hydroxymethylanthra-
quinone glycosides from Rubia tinctorum. Phytochemistry 49:
277–279
El-Gamal AA, Takeya K, Itokawa H, Halim AF, Amer MM, Saad
HA & Awad SA (1995) Anthraquinones from Galium sinaicum.
Phytochemistry 40: 245–251
El-Gamal AA, Takeya K, Itokawa H, Halim AF, Amer MM, Saad
HA & Awad SA (1996) Anthraquinones from the polar fractions
of Galium sinaicum. Phytochemistry 42: 1149–1155
El-Shagi H, Schulte U & Zenk MH (1984) Specific inhibition of
anthraquinone formation by amino compounds in Morinda cell
cultures. Naturwissenschaften 71: 267
Emili AQ & Cagney G (2000) Large-scale functional analysis using
peptide or protein arrays. Nat. Biotechnol. 18: 393–397
Endo M, Sakata K & Katayama A (1997) The pigments in the callus
of Rubia akane and their dyeing properties. Nippon Sanshigaku
Zasshi 66: 107–112
Ferrari F, Monache GD & Lima RA (1985) Two naphthopyran de-
rivatives from Faramea cyanea. Phytochemistry 24: 2753–2755
Goese M, Kammhuber K, Bacher A, Zenk MH & Eisenreich W
(1999) Biosynthesis of bitter acids in hops. A 13 C-NMR and 2 H-
NMR study on the building blocks of humulone. Eur. J. Biochem.
263: 447–454
Graves DJ (1999) Powerful tools for genetic analysis come of age.
Trends Biotechnol. 17: 127–134
Grolle S, Bringer-Meyer S & Sahm H (2000) Isolation of the
dxr gene of Zymomonas mobilis and characterization of the
1-deoxy-D-xylulose 5-phosphate reductoisomerase. FEMS Mi-
crobiol. Lett. 191: 131–137
216
Gundlach H, Müller MJ, Kutchan TM & Zenk MH (1992) Jasmonic
acid is a signal transducer in elicitor-induced plant cell cultures.
Proc. Natl. Acad. Sci. USA 89: 2389–2393
Hagendoorn MJM, Jamar DCL, Meykamp B & Van der Plas LHW
(1997) Cell division versus secondary metabolite production in
Morinda citrifolia cell suspensions. J. Plant Physiol. 150: 325–
330
Hahn FM, Eubanks LM, Testa CA, Blagg BSJ, Baker JA & Poulter
CD (2001) 1-deoxy-D-xylulose 5-phosphate synthase, the gene
product of open reading frame (ORF) 2816 and ORF 2895 in
Rhodobacter capsulatus. J. Bacteriol. 183: 1–11
Halim AF, El-Fattah HA, El-Gamal AA & Thomson RH (1992) An-
thraquinones from Galium sinaicum. Phytochemistry 31: 355–
356
Hamzah AS, Jasmani H, Ahmad R & Baba AR (1997) New anthra-
quinones from the roots of Hedyotis dichotoma. J. Nat. Prod. 60:
36–37
Han YS, Van der Heijden R, Lefeber AWM, Erkelens C & Verpoorte
R (2001) Biosynthesis of anthraquinones in cell cultures of Cin-
chona ‘Robusta’ proceeds via the methylerythritol 4-phosphate
pathway. Phytochemistry (accepted).
Harkes PAA, Krijbolder L, Libbenga KR, Wijnsma R, Aremge TN
& Verpoorte R (1985) Influence of various media constituents
on growth of Cinchona ledgeriana tissue cultures and the pro-
duction of alkaloids and anthraquinones therein. Plant Cell Tiss.
Org. Cult. 4: 199–214
Harker M & Bramley PM (1999) Expression of prokaryotic 1-
deoxy-D-xylulose 5-phosphatases in E. coli increases carotenoid
and ubiquinone biosynthesis. FEBS Lett. 448: 115–119
Heide L, Kolkmann R, Arendt S & Leistner E (1982) Enzymatic
synthesis of o-succinylbenzoyl-CoA in cell-free extracts of AQ
producing Galium mollugo L. cell suspension cultures. Plant Cell
Rep. 1: 180–182
Heike H, Heike D & Dietrich K (1996) Biosynthesis and accumu-
lation of anthraquinones in Galium verum. Immobilized cells.
Immobilization and two-phase culturing of plant cell cultures.
BioTec. 8: 42, 47–49
Herrmann, Klaus M, Weaver & Lisa M (1999) The shikimate
pathway. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 473–503
Herz S, Wungsintaweekul J, Schuhr CA, Hecht S, Lüttgen H,
Sagner S, Fellermeier M, Eisenreich W, Zenk MH & Bacher
A (2000) Biosynthesis of terpenoids: YgbB protein converts
4-diphosphocytidyl-2C-methyl-D-erythri tol 2-phosphate to 2C-
methyl-D-erythritol 2,4-cyclodiphosphate. Proc. Natl. Acad. Sci.
USA 97: 2486–2490
Ho TI, Chen GP, Lin YC, Lin YM & Chen FC (1986) An anthra-
quinone from Hedyotis diffusa. Phytochemistry 25: 1988–1989
Hrazdina G (1994) Compartmentation in phenolic metabolism. Acta
Hort. 381: 86–96
Hutchinson CR & Colombo AL (1999) Genetic engineering of dox-
orubicin production in Streptomyces peucetius: A review. J. Ind.
Microbiol. Biotechnol. 23: 647–652
Igbavboa U, Sieweke HJ, Leistner E, Röwer I, Hüsemann W &
Barz W (1985) Alternative formation of anthraquinones and
lipoquinones in heterotrophic and photoautotrophic cell suspen-
sion cultures of Morinda lucida Benth. Planta 166: 537–544
Inoue K, Shiobara Y, Nayeshiro H, Inouye H, Wilson G & Zenk
MH (1979) Site of prenylation in anthraquinone biosynthesis in
cell cultures of Galium mollugo. J. Chem. Soc., Chem. Comm.
957–959
Inoue K, Shiobara Y, Nayeshiro H, Inouye H, Wilson G & Zenk MH
(1984) Biosynthesis of anthraquinones and related compounds
in Galium mollugo cell suspension cultures. Phytochemistry 23:
307–311
Inouye H & Leistner E (1988) Biosynthesis of quinones. In: Patai S
& Rappoport Z (eds) The Chemistry Of Quinonoid Compounds,
Vol II (pp 1293–1349). John Wiley & Sons Ltd., New York
Ismail NH, Ali AM, Aimi N, Kitajima M, Takayama H & Lajia NH
(1997) Anthraquinone from Morinda elliptica. Phytochemistry
45: 1723–1725
Itokawa H, Qiao Y & Takeya K (1989) Anthraquinones and naph-
thohydroquinones from Rubia cordifolia. Phytochemistry 28:
3465–3468
Itokawa H, Qiao Y & Takeya K (1991) Anthraquinones, naph-
thoquinones and naphthohydroquinones from Rubia oncotricha.
Phytochemistry 30: 637–640
Jacobs DI, Van der Heijden R & Verpoorte R (2000) Proteom-
ics in plant biotechnology and secondary metabolism research.
Phytochem. Anal. 11: 277–287
Jasril, Lajis NH, Abdullah MA, Ismail NH, Ali AM, Marziah M,
Ariff AB, Kitajima M, Takayama H & Aimi N (2000) Anthra-
quinones from cell suspension culture of Morinda elliptica. Nat.
Prod. Sci. 6: 40–43
Kawasaki Y, Goda Y & Yoshihira K (1992) The mutagenic constitu-
ents of Rubia tinctorum. Chem. Pharm. Bull. 40: 1504–1509
Khouri H, Ibrahim RK & Rideau M (1986) Effects of nutritional
and hormonal factors on growth and production of anthraquinone
glucosides in cell suspension cultures of Cinchona succirubra.
Plant Cell Rep. 5: 423–426
Khouri H, Ibrahim RK & Rideau M (1987) Purification and some
properties of five anthraquinone-specific glucosyltransferases
from Cinchona succirubra cell suspension culture. Phytochem-
istry 26: 2531–2535
Kitajima M, Fischer U, Nakamura M, Ohsawa M, Ueno M,
Takayama H, Unger M, Stöckigt J & Aimi N (1998) An-
thraquinones from Ophiorrhiza pumila tissue and cell cultures.
Phytochemistry 48:107–111
Knaggs AR (1999) The biosynthesis of shikimate metabolites. Nat.
Prod. Rep. 16: 525–560
Koblitz H (1988) Anthraquinones. In: Vasil I (ed) Cell Culture And
Somatic Cell Genetics Of Plants, Vol 1 (pp 113–139). Academic
Press, Orlando
Koumaglo K, Gbeassor M, Nikabu O, Souza CD & Werner W
(1992) Effects of three compounds extracted from Morinda
lucida on Plasmodium falciparum. Planta Med. 58: 533–534
Koyama J, Okatani T, Tagahara K, Kouno I & Irie H (1992)
Anthraquinones of Damnacanthus indicus. Phytochemistry 31:
709–710
Koyama J, Ogura T & Tagahara K (1993) Anthraquinones of
Galium spurium. Phytochemistry 33: 1540–1542
Kuo SC, Chen PR, Lee SW & Chen ZT (1995) Constituents of Ru-
biaceous plants in Taiwan. Part 2. Constituents of roots of Rubia
lanceolata hayata. J. Chin. Chem. Soc. (Taipei) 42: 869–871
Kuzovkina IN, Mantrova OV, Al’terman IE & Yakimov SA (1999)
Culture of genetically transformed hairy roots derived from
anthraquinone-producing European madder plants. Russ. J. Plant
Physiol. 46: 248–251
Kuzuyama T, Takagi M, Takahashi S & Seto H (2000a) Cloning
and characterization of 1-deoxy-D-xylulose 5-phosphate syn-
thase from Streptomyces sp. strain CL190, which uses both
the mevalonate and nonmevalonate pathways for isopentenyl
diphosphate biosynthesis. J. Bacteriol. 182: 891–897
Kuzuyama T, Takahashi S, Takagi M, Kaneda K & Seto H (2000b)
Characterization of 1-deoxy-D-xylulose 5-phosphate reductoi-
somerase, an enzyme involved in isopentenyl diphosphate bio-
synthesis, and identification of its catalytic amino acid resides. J.
Biol. Chem. 275: 19928–19932

Kuzuyama T, Takagi M, Kaneda K, Dairi T & Seto H (2000c) Form-
ation of 4-(cytidine 5 -diphospho)-2-C-methyl-erythritol from
2-C-methyl-erythritol 4-phosphate by 2-C-methyl-erythritol 4-
phosphate cytidylyltransferase, a new enzyme in the non-
mevalonate pathway. Tetrahedron Lett. 41: 703–706
Lange BM, Severin K, Bechthold A & Heide L (1998a) Regulatory
role of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A re-
ductase for shikonin biosynthesis in Lithospermum erythrorhizon
cell suspension cultures. Planta 204: 234–241
Lange BM, Wildung MR, McCaskill D & Croteau R (1998b) A
family of transketolases that directs isoprenoid biosynthesis via
a mevalonate-independent pathway. Proc. Natl. Acad. Sci. USA
95: 2100–2104
Lange BM & Croteau R (1999a) Isopentenyl diphosphate bio-
synthesis via a mevalonate-independent pathway: isopentenyl
monophosphate kinase catalyzes the terminal enzymatic step.
Proc. Natl. Acad. Sci. USA 96: 13714–13719
Lange BM & Croteau R (1999b) Isoprenoid biosynthesis via a
mevalonate-independent pathway in plants: cloning and hetero-
logous expression of 1-deoxy-D-xylulose-5-phosphate reductoi-
somerase from peppermint. Arch. Biochem. Biophys. 365: 170–
174
Lange BM, Wildung MR, Stauber EJ, Sanchez C, Pouchnik D &
Croteau R (2000) Probing essential oil biosynthesis and secretion
by functional evaluation of expressed sequence tags from mint
glandular trichomes. Proc. Natl. Acad. Sci. USA 97: 2934–2939
Ledüc C, Ruhnau P & Leistner E (1991) Isochorismate hy-
droxymutase from Rubiaceae cell suspension cultures. Plant Cell
Rep. 10: 334–337
Ledüc C, Birgel I, Müler R & Leistner E (1997) Isochorismate hy-
droxymutase from cell-suspension culture of Galium mollugo L.
Planta 202: 206–210
Leistner E (1971) A second pathway leading to anthraquinones in
higher plants. Phytochemistry 10: 3015—3020
Leistner E (1973a) Biosynthesis of morindone and alizarin in in-
tact plants and cell suspension cultures of Morinda citrifolia.
Phytochemistry 12: 1669—1674
Leistner E (1973b) Mode of incorporation of precursors into al-
izarin (1,2-dihydroxy-9,10-anthraquinone). Phytochemistry 12:
337–345
Leistner E (1975) Isolation, identification and biosynthesis of an-
thraquinones in cell suspension cultures of Morinda citrifolia.
Planta Med. Suppl. 214–224
Leistner E (1981) Biosynthesis of Plant Quinones. In: Conn EE
(ed) The Biochemistry Of Plants, Vol 7 (pp 403–423). Academic
Press, London
Leistner E (1985) Biosynthesis of chorismate-derived quinones in
plant cell cultures. In: Neumann KH, Barz W & Reinhard E (eds)
Primary and Secondary Metabolism of Plant Cell Cultures (pp
215–224). Springer-Verlag, Berlin, New York
Leistner E (1995) XVI Morinda species: Biosynthesis of quinones
in cell cultures. In: YPS Bajaj (ed) Biotechnology In Agriculture
And Forestry, Medicinal And Aromatic Plants VIII Vol. 33 (pp
296–307). Springer-Verlag, Berlin Heidelberg
Leistner E & Zenk MH (1967) Incorporation of shikimic acid into
1,2-dihydroxy-anthraquinone (alizarin) by Rubia tinctorum L.
Tetrahedron Lett. 475–476
Leistner E & Zenk MH (1968) Mevalonic acid a precursor of
the substituted benzenoid ring of Rubiaceae anthraquinones.
Tetrahedron Lett. 1395–1396
Lichtenthaler HK (1999) The 1-deoxy-D-xylulose-5-phosphate
pathway of isoprenoids biosynthesis in plants. Ann. Rev. Plant
Physiol. Plant Mol. Biol. 50: 47–65
217
Likhitwitayawuid K, Dej-adisai S, Jongbunprasert V & Krungkrai
J (1999) Antimalarials from Stephania venosa, Prismatomeris
sessiliflora, Diospyros montana and Murraya siamensis. Planta
Med. 65: 754–756
Liu YL, Chen BZ, Bai YL, Duddeck H & Hiegemann M (1991)
Digiferruginol glycoside from the roots of Rubia schumanniana.
Phytochemistry 30: 947–949
Lodhi AH & Charlwood BV (1996) Agrobacterium rhizogenes-
mediated transformation of Rubia peregrina L: In vitro ac-
cumulation of anthraquinones. Plant Cell Tiss. Org. Cult. 46:
103–108
Lodhi AH, Bongaerts RJM, Verpoorte R, Coomber SA & Charl-
wood BV (1996) Expression of bacterial isochorismate synthase
(EC 5.4.99.6) in transgenic root cultures of Rubia peregrina.
Plant Cell Rep. 16: 54–57
Lois LM, Campos N, Putra SR, Danielsen K, Rohmer M & Boronat
A (1998) Cloning and characterization of a gene from Escheri-
chia coli encoding a transketolase-like enzyme that catalyzes the
synthesis of 1-deoxy-D-xylulose 5-phosphate, a common pre-
cursor for isoprenoid, thiamin, and pyridoxol biosynthesis. Proc.
Natl. Acad. Sci. USA 95: 2105–2110
Lois LM, Rodriguez-Concepcion M, Gallego F, Campos N &
Boronat A (2000) Carotenoid biosynthesis during tomato fruit
development: Regulatory role of 1-D-deoxyxylulose 5-phosphate
synthase. Plant J 22: 503–513
Lüttgen H, Rohdich F, Herz S, Wungsintaweekul J, Hecht S, Fischer
M, Schuhr CA, Fellermeier M, Sagner S, Zenk MH, Bacher A
& Eisenreich W (2000) Biosynthesis of terpenoids: YchB pro-
tein of Escherichia coli phosphorylates the 2-hydroxy group of
4-diphosphocytidyl-2-C-methyl-D-erythritol. Proc. Natl. Acad.
Sci. USA 97: 1062–1067
MacBeath G & Schreiber SL (2000) Printing proteins as microar-
rays for high-throughput function determination. Science 289:
1760–1763
Mandel MA, Feldmann KA, Herrera-Estrella L, Rocha-Sosa M
& León P (1996) CLA1, a novel gene required for chloro-
plast development, is highly conserved in evolution. Plant J. 9:
649–658
Mantrova OV, Dunaeva MV, Kuzovkina IN, Schneider B & Müller-
Uri F (1999) Effect of methyl jasmonate on anthraquinone
biosynthesis in transformed madder roots. Russ. J. Plant Physiol.
46: 276–279
Masahiro K, Koji M, Masahito T, Setsuji T & Takahito I (1994) Pro-
duction and release of anthraquinones pigments by hairy roots of
madder. J. Ferment. Bioeng. 77: 103–106
Matthews PD & Wurtzel ET (2000) Metabolic engineering of
carotenoid accumulation in E. coli by modulation of the isopren-
oid precursor pool with expression of deoxyxylulose phosphate
synthase. Appl. Microbiol. Biotechnol. 53: 396–400
Miller B, Heuser T & Zimmer W (1999) A Synechococcus leo-
poliensis SAUG 1402-1 operon harboring the 1-deoxyxylulose
5-phosphate synthase gene and two additional open reading
frames is functionally involved in the dimethylallyl diphosphate
synthesis. FEBS Lett. 460: 485–490
Miller B, Heuser T & Zimmer W (2000) Functional involve-
ment of a deoxy-D-xylulose 5-phosphate reductoisomerase gene
harboring locus of Synechococcus leopoliensis in isoprenoid
biosynthesis. FEBS Lett. 481: 221–226
Mischenko NP, Fedoreyev SA, Glazunov VP, Chernoded GK, Bul-
gakov VP & Zhuravlev YN (1999) Anthraquinone production by
callus cultures of Rubia cordifolia. Fitoterapia 70: 552–557
Mizutani H, Hashimoto O, Nakashima R & Nagai J (1997) Anthra-
quinone production by cell suspension cultures of Rubia akane
NAKAI. Biosci. Biotech. Biochem. 61: 1743–1744
218
Mueller MJ, Brodschelm W, Spannagl E & Zenk MH (1993)
Signaling in the elicitation process is mediated through the oct-
adecanoid pathway leading to jasmonic acid. Proc. Natl. Acad.
Sci. USA 90: 7490–7494
Muzychkina RA (1998) Natural Anthraquinones: Biological and
Physicochemical Properties. PHASIS, Moscow
Okuyama E, Sato K & Yoshihira K (1990) 2-ethoxycarbonyl-1-
hydroxyanthraquinone from Rubia akane. Phytochemistry 29:
3973–3974
Permana D, Lajis NH, Othman AG, Ali AM, Aimi N, Kitajima M
& Takayama H (1999) Anthraquinones from Hedyotis herbacea.
J. Nat. Prod. 62: 1430–1431
Poulsen C & Verpoorte R (1991) Roles of chorismate mutase, iso-
chorismate synthase and anthranilate synthase in plants. Phyto-
chemistry 30: 377–386
Poulton JE (1981) Transmethylation and demethylation reactions
in the metabolism of secondary plant products. In: Conn EE
(ed) The Biochemistry of Plants, Vol 7 (pp 667–723). Academic
Press, London
Ramos-Valdivia AC, Van der Heijden R & Verpoorte R (1997a)
Isopentenyl diphosphate isomerase: A core enzyme in isopren-
oid biosynthesis. A review of its biochemistry and function. Nat.
Prod. Rep. 591–603
Ramos-Valdivia AC, Van der Heijden R & Verpoorte R (1997b)
Purification and characterization of two isoforms of isopentenyl-
diphosphate isomerase from elicitor-treated Cinchona robusta
cells. Eur. J. Biochem. 249: 161–170
Ramos-Valdivia AC, Van der Heijden R & Verpoorte R (1997c)
Elicitor-mediated induction of anthraquinone biosynthesis and
regulation of isopentenyl diphosphate isomerase and farnesyl
diphosphate synthase activities in cell suspension cultures of
Cinchona robusta. Planta 203: 155–161
Ramos-Valdivia AC, Van der Heijden R & Verpoorte R (1998) Iso-
pentenyl diphosphate isomerase and prenyltransferase activities
in Rubiaceous and Apocynaceous cultures. Phytochemistry 48:
961–969
Rath G, Ndonzao M & Hostettmann K (1995) Antifungal anthra-
quinones from Morinda lucida. Int. J. Pharmacogn. 33: 107–114
Reymond P & Farmer EE (1998) Jasmonate and salicylate as global
signals for defense gene expression. Curr. Opin. Plant Biol. 1:
404–411
Robins RJ, Payne J & Rhodes MJC (1986) The production of anthra-
quinones by cell suspension cultures of Cinchona ledgeriana.
Phytochemistry 25: 2327–2334
Rohdich F, Wungsintaweekul J, Fellermeier M, Sagner S, Herz
S, Kis K, Eisenreich W, Bacher A & Zenk MH (1999)
Cytidine 5 -triphosphate-dependent biosynthesis of isoprenoids:
YgbP protein of Escherichia coli catalyzes the formation of 4-
diphosphocytidyl-2-C-methylerythrito l. Proc. Natl. Acad. Sci.
USA 96: 11758–11763
Rohdich F, Wungsintaweekul J, Lüttgen H, Fischer M, Eisenreich
W, Schuhr CA, Fellermeier M, Schramek N, Zenk MH & Bacher
A (2000a) Biosynthesis of terpenoids: 4-diphosphocytidyl-2-C-
methyl-D-erythritol kinase from tomato. Proc. Natl. Acad. Sci.
USA 97: 8251–8256
Rohdich F, Wungsintaweekul J, Eisenreich W, Richter G, Schuhr
CA, Hecht S, Zenk MH & Bacher A (2000b) Biosynthesis
of terpenoids: 4-diphosphocytidyl-2-C-methyl- D-erythritol syn-
thase of Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 97:
6451–6456
Rohmer M, Knani M, Simonin P, Sutter B & Sahm H (1993) Iso-
prenoid biosynthesis in bacteria: A novel pathway for early steps
leading to isopentenyl diphosphate. Biochem. J 295: 517–524
Rohmer M (1999) The discovery of a mevalonate-independent path-
way for isoprenoid biosynthesis in bacteria, algae and higher
plants. Nat. Prod. Rep. 16: 565–574
Sato K, Yamazaki T, Okuyama E, Yoshihira K & Shimomura K
(1991) Anthraquinones production by transformed root cultures
of Rubia tinctorum: Influence of phytohormones and sucrose
concentration. Phytochemistry 30: 1507–1509
Sato K, Kubota H, Goda Y, Yamada T & Maitani T (1997) Gluta-
thione enhanced anthraquinone production in adventitious root
cultures of Rubia tinctorum. Plant Biotechnol. (Tokyo) 14: 63–66
Schaller H, Grausem B, Benveniste P, Chye ML, Tan CT, Song
YH & Chua NH (1995) Expression of the Hevea brasilien-
sis (H.B.K.) Müll. Arg. 3-hydroxy-3-methylglutaryl-coenzyme
A reductase 1 in tobacco results in sterol overproduction. Plant
Physiol. 109: 761–770
Schena M, Shalon D, Davis RW & Brown PO (1995) Quantitative
monitoring of gene expression patterns with a complementary
DNA microarray. Science 270: 467–470
Schripsema J, Ramos-Valdivia AC & Verpoorte R (1999) Ro-
bustaquinones, novel anthraquinones from an elicited Cinchona
robusta suspension culture. Phytochemistry 51: 55–60
Schulte U, El-Shagi H & Zenk MH (1984) Optimization of 19 Rubi-
aceae species in cell suspension cultures of Cinchona ledgeriana.
Plant Cell Rep. 3: 51–54
Schwender J, Müller C, Zeidler J & Lichtenthaler HK (1999)
Cloning and heterologous expression of a cDNA encoding 1-
deoxy-D-xylulose-5-phosphate reductoisomerase of Arabidopsis
thaliana. FEBS Lett. 455: 140–144
Shim JJ, Shin JH, Pai T, Chung IS & Lee HJ (1999) Permeabil-
ization of elicited suspension culture of madder (Rubia akane
Nakai) cells for release of anthraquinones. Biotechnol. Tech. 13:
249–252
Shin S & Kim Y (1996) Production of anthraquinone derivat-
ives by hairy roots of Rubia cordifolia var. pratensis. Saengyak
Hakhoechi 27: 301–308
Sieweke H & Leistner E (1992) O-succinylbenzoate: Coenzyme A
ligase from anthraquinone producing cell suspension cultures of
Galium mollugo. Phytochemistry 31: 2329–2335
Simantiras M & Leistner E (1989) Formation of o-succinylbenzoic
acid from iso-chorismic acid in protein extracts from
anthraquinone-producing plant cell suspension cultures.
Phytochemistry 28: 1381–1382
Simantiras M & Leistner E (1991) Cell free synthesis of o-
succinylbenzoic acid in protein extracts from anthraquinone and
phylloquinone (vitamin K1) producing plant cell suspension cul-
tures. Occurrence of intermediates between isochorismic acid
and o-succinylbenzoic acid. Z. Naturforsch. 46c 364–370
Simantiras M & Leistner E (1992) O-succinylbenzoate:coenzyme A
ligase from anthraquinone producing cell suspension cultures of
Galium mollugo. Phytochemistry 31: 2329–2335
Simpson TJ (1987) The biosynthesis of polyketides. Nat. Prod. Rep.
4: 339–376
Sittie AA, Lemmich E, Olsen CE, Hviid L, Kharazmi A, Nkrumah
FK & Christensen SB (1999) Structure-activity studies: In vitro
antileishmanial and antimalarial activities of anthraquinones
from Morinda lucida. Planta Med. 65: 259–261
Smeekens S (2000) Sugar-induced signal transduction in plants.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 49–81
Sprenger GA, Schörken U, Wiegert T, Grolle S, DeGraaf AA, Taylor
SV, Begley TP, Bringer-Meyer S & Sahm H (1997) Identification
of a thiamin-dependent synthase in Escherichia coli required for
the formation of the 1-deoxy-D-xylulose 5-phosphate precursor
to isoprenoids, thiamin and pyridoxol. Proc. Natl. Acad. Sci.
USA 94: 12859–12862

Srivasta M & Singh J (1993) A new anthraquinone glycoside from
Morinda citrifolia. Int. J. Pharmacog. 31: 182–184
Stalman M (2001) Metabolic regulation of anthraquinone biosyn-
thesis in cell cultures of Morinda citrifolia. Ph.D Thesis (pp
53–72). Univ. of Nijmegen, The Netherlands
Stara D, Suchy V & Blanarik P (1995) Tissue culture of Rubia tinc-
torum and production of anthraquinones. Cesk. Slov. Farm. 44:
167–1695
Stermer BA, Bianchini GM & Korth KL (1994) Regulation of
HMG-CoA reductase activity in plants: Review. J. Lip. Res. 35:
1133–1140
Strack D (1997) Phenolic metabolism. In: Dey PM & Harborne JB
(eds) Plant Biochemistry (pp 387–416). Academic Press, Inc.,
San Diego
Sundberg SA (2000) High-throughput and ultra-high-throughput
screening: Solution- and cell-based approaches. Curr. Opin.
Biotechnol. 11: 47–53
Suzuki H, Matsumoto T & Mikami Y (1984) Effects of nutritional
factors on the formation of anthraquinone by Rubia cordifolia
plant cells in suspension culture. Agric. Biol. Chem. 48: 603–610
Suzuki H, Matsumoto T & Mikami Y (1985) Effects of physical
factors and surface active agents on the formation of anthra-
quinone by Rubia cordifolia cells in suspension culture. Agric.
Biol. Chem. 48: 519–520
Suzuki H & Matsumoto T (1988) Anthraquinone: production by
plant cell culture. In: Bajaj YPS (ed) Biotechnology In Agricul-
ture and Forestry, Medicinal and Aromatic Plants I, Vol. 4 (pp
237–250). Springer-Verlag, Berlin Heidelberg
Takahashi S, Kuzuyama T, Watanabe H & Seto H (1998) A 1-
deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the
formation of 2-C-methyl-D-erythritol 4-phosphate in an altern-
ative nonmevalonate pathway for terpenoid biosynthesis. Proc.
Natl. Acad. Sci. USA 95: 9879–9884
Thomson RH (1971) Naturally Occurring Quinones (2nd edn).
Academic Press, London
Thomson RH (1987) Naturally Occurring Quinones. III. Recent
Advances. Chapman and Hall, London
Thomson RH (1996) Naturally Occuring Quinones. IV. Chapman
and Hall, London
Tosa H, Linuma M, Asai F, Tanaka T, Nozaki H, Ikeda S, Tsutsui
K, Tsutsui K, Yamada M & Fujimori S (1998) Anthraquinones
from Neonauclea calycina and their inhibitory activity against
DNA topoisomerase II. Biol. Pharm. Bull. 21: 641–642
Tripathi YB, Sharma M & Manickam M (1997) Rubiadin, a new
antioxidant from Rubia cordifolia. Ind. J. Biochem. Biophys. 34:
302–306
Van den Berg AJJ & Labadie RP (1989) Quinones. In: Harborne
JB (ed) Methods In Plant Biochemistry, Vol 1 (pp 451–491).
Academic Press Limited, London
Van der Heijden R, Verpoorte R, Hoekstra SS & Hoge JHC (1994)
Nordamnacanthal, a major anthraquinone from an Agrobac-
terium rhizogenes induced root culture of Rubia tinctorum. Plant
Physiol. Biochem. 32: 399–404
Van der Leer T, Wijnsma R, Van der Heijden R, Verpoorte R &
Svendsen AB (1991) A comparative study of the effects of L-
tryptophan and tryptamine on a nonalkaloid producing cell sus-
pension culture of Cinchona ledgeriana. Plant Physiol. Biochem.
29: 91–98
Van der Plas LHW, Eijkelboom C & Hagendoorn MJM (1995) Re-
lation between primary and secondary metabolism in plant cell
suspensions. Plant Cell Tiss. Org. Cult. 43: 111–116
Van der Plas LHW, Hagendoorn MJM & Jamar DCL (1998) An-
thraquinones glycosylation and hydrolysis in Morinda citrifolia
219
cell suspensions: regulation and function. J. Plant Physiol. 152:
235–241
Van Tegelen LJP, Bongaerts RJM, Croes AF, Verpoorte R &
Wullems GJ (1999a) Isochorismate synthase isoforms from
elicited cell cultures of Rubia tinctorum. Phytochemistry 51:
263–269
Van Tegelen LJP, Stalman M, Wind J, Vernooij J, Croes AF &
Wullems GJ (1999b) Role of isochorismate synthase in choris-
mate partitioning in anthraquinone-synthesizing cell cultures of
Morinda citrifolia. Ph.D Thesis (pp 77–89). Univ. of Nijmegen,
The Netherlands
Van Tegelen LJP, Toebes A, Stalman M, Croes AF & Wullems
GJ (1999c) Role of isochorismate synthase in the regulation
of anthraquinone biosynthesis in elicited cell cultures of Rubia
tinctorum. Ph.D Thesis (pp 63–73). Univ. of Nijmegen, The
Netherlands
Veau B, Courtois M, Oudin A, Chénieux JC, Rideau M & Clastre M
(2000) Cloning and expression of cDNAs encoding two enymes
of the MEP pathway in Catharanthus roseus. Biochim. Biophys.
Acta 1517: 159–163
Verpoorte R & Alfermann AW (2000) Metabolic Engineering
Of Plant Secondary Metabolism. Kluwer Academic Publishers,
Dordrecht, The Netherlands
Vidal-Tessier AM, Delaveau P & Champion B (1987) New an-
thraquinones of Rubia cordifolia L. roots. Ann. Pharm. Fr. 45:
261–167
Walter MH, Fester T & Strack D (2000) Arbuscular mycorrhizal
fungi induce the non-mevalonate methylerythritol 4-phosphate
pathway of isoprenoid biosynthesis correlated with accumulation
of the ‘yellow pigment’ and other apocarotenoids. Plant J. 21:
571–578
Weissenborn DL, Denbow CJ, Laine M, Lång S, Yang Z, Yu
X & Cramer CL (1995) HMG-CoA reductase and terpenoid
phytoalexins: molecular specialization within a complex path-
way. Physiol. Plant. 93: 393–400
Westendorf J, Pfau W & Schulte A (1998) Carcinogenicity and
DNA adduct formation observed in ACI rats after long-term
treatment with madder root, Rubia tinctorum L. Carcinogenesis
19: 2163–2168
Wijnsma R, Go JTKA, Van Weerden IN, Harkes PAA, Verpoorte R
& Baerheim-Svendsen A (1985) Anthraquinones as phytoalexins
in cell and tissue cultures of Cinchona sp. Plant Cell Rep. 4: 241–
244
Wijnsma R & Verpoorte R (1986) Anthraquinones in the Rubiaceae.
In: Herz W, Grisebach GW & Kirby Ch Tamm (eds) Prog. Chem.
Org. Nat. Prod., Vol. 49 (pp 79–149). Springer-Verlag, Vienna,
New York
Xiang H & Guo Y (1997) Studies on the production of anthra-
quinone by plant cell suspension culture. Huanan Ligong Daxue
Xuebao, Ziran Kexueban 25: 62–67
Yamamoto H, Tabata M & Leistner E (1987) Cytological changes
associated with induction of anthraquinone synthesis in pho-
toautotrophic cell suspension cultures of Morinda lucida. Plant
Cell Rep. 6: 187–190
Ye X, Al-Babili S, Klöti A, Zhang J, Lucca P, Beyer P & Potrykus
I (2000) Engineering the provitamin A (-carotene) biosynthetic
pathway into (carotenoid-free) rice endosperm. Science 287:
303–305
Younos C, Rolland A, Fleurentin J, Lanhers M, Misslin R & Mor-
tier F (1990) Analgesic and behavioural effects of Morinda
citrofolia. Planta Med. 56: 430–434
Zenk MH, El-Shagi H & Schulte U (1975) Anthraquinone pro-
duction by cell suspension cultures of Morinda citrifolia. Planta
Med. (Suppl) 79–101
 220

Zenk MH, Schulte U & El-Shagi H (1984) Regulation of an- Zhou Z, Jiang SH, Zhu DY, Lin LZ & Cordell GA (1994) An-
thraquinone formation by phenoxyacetic acids in Morinda cell thraquinones from Knoxia valerianoides. Phytochemistry 36:
cultures. Naturwissenschaften 71: 266 765–768

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