Secondary metabolism in root and callus cultures of Hyoscyamus muticus L.: The relationship between morphological organisation and response to methyl jasmonate

Abstract

Production of acid-soluble conjugated di- and polyamines, like that of other secondary metabolites, is enhanced by exposure to methyl jasmonate (MJ). We investigated this metabolic response, and activities of enzymes involved in putrescine (Put) and tropane alkaloid biosynthesis, in root cultures of Hyoscyamus muticus and compared it with that of callus cultures. In root cultures, free Put and N-methylputrescine (mPut) increased upon treatment with MJ, whereas in callus cultures mPut levels were not affected. Differently from roots, conjugated amines were scarce or absent in callus cultures, and accumulated only transiently upon treatment with MJ. Arginine decarboxylase, ornithine decarboxylase and diamine oxidase activities in root cultures were strongly stimulated by treatment with MJ, but were inhibited in callus cultures. Exposure to MJ also enhanced putrescine N-methyltransferase activity in root cultures more than in callus cultures. These results are discussed in relation to the different capacity for tropane alkaloid production in the two culture systems.

Full text

Secondary metabolism in root and callus cultures of Hyoscyamus
muticus L.: the relationship between morphological organisation and
response to methyl jasmonate
Stefania Biondi
a,
*, Sonia Scaramagli
a
, Kirsi-Marja Oksman-Caldentey
b
,
Ferruccio Poli
a
a
Dipartimento di Biologia e.s., Universita` di Bologna, Via Irnerio 42, I-40126 Bologna, Italy
b
VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT Espoo, Finland
Received 20 March 2002; received in revised form 6 June 2002; accepted 6 June 2002
Abstract
Production of acid-soluble conjugated di- and polyamines, like that of other secondary metabolites, is enhanced by exposure to
methyl jasmonate (MJ). We investigated this metabolic response, and activities of enzymes involved in putrescine (Put) and tropane
alkaloid biosynthesis, in root cultures of Hyoscyamus muticus and compared it with that of callus cultures. In root cultures, free Put
and N-methylputrescine (mPut) increased upon treatment with MJ, whereas in callus cultures mPut levels were not affected.
Differently from roots, conjugated amines were scarce or absent in callus cultures, and accumulated only transiently upon treatment
with MJ. Arginine decarboxylase, ornithine decarboxylase and diamine oxidase activities in root cultures were strongly stimulated
by treatment with MJ, but were inhibited in callus cultures. Exposure to MJ also enhanced putrescine N-methyltransferase activity
in root cultures more than in callus cultures. These results are discussed in relation to the different capacity for tropane alkaloid
production in the two culture systems. #2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Hyoscyamus muticus ; Methyl jasmonate; Polyamine metabolism; Tissue culture; Tropane alkaloids
1. Introduction
The diamine putrescine (Put) and the higher poly-
amines derived from it, i.e. spermidine (Spd) and
spermine (Spm), are regarded as plant growth regulators
involved in many aspects of growth, morphogenesis and
response to stress [1], and as such belong to the primary
metabolism. In many plants, and particularly in Sola-
naceae, di- and polyamines covalently bound to pheno-
lic compounds (particularly hydroxycinnamic acids) are
also very abundant. These hydroxycinnamic acid amides
(HCAs), also known as acid-soluble conjugated di-/
poly-amines, accumulate during flowering and pathogen
attack in tobacco plants [2];ininvitro-cultured explants
([3] and references therein), including root cultures of
Hyoscyamus [4,5] and Datura [6], they often come to
represent a large proportion of the total di-/poly-amine
pool. Although the precise role of HCAs remains to be
elucidated, their endogenous concentrations, their phe-
nolic partners and the fact that conjugation appears to
be an irreversible process suggest that they are likely to
be secondary metabolites.
Hyoscyamus muticus L. (Egyptian henbane) is an
important medicinal plant belonging to the Solanaceae
which produces tropane alkaloids; of these, the main
constituent is hyoscyamine with small quantities of
scopolamine. Together with nicotine, tropane alkaloids
are derived from Put which is, in turn, synthesised from
ornithine or arginine via ornithine- (ODC) and arginine-
decarboxylase (ADC) activities, respectively. The first
specific precursor of the tropane alkaloid pathway is N-
methylputrescine (mPut), whose formation from Put is
catalysed by putrescine N-methyltransferase (PMT),
and hence represents the first committed step in tropane
alkaloid biosynthesis [7]. Thus, Put plays a pivotal role
between two biosynthetic pathways, i.e. tropane alka-
* Corresponding author. Tel.: /39-51-2091291; fax: /39-51-
242576
E-mail address: sbiondi@alma.unibo.it (S. Biondi).
Plant Science 163 (2002) 563 /569
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loids vs. polyamines, and occupies a crucial position at
the interface between primary and secondary metabo-
lism.
Jasmonates are lipid-derived compounds regarded as
‘non traditional’ plant hormones [8] which exert numer-
ous effects on plant growth and development. In recent
years, however, the most studied aspect of jasmonates is
their role in plant defense. The action of jasmonic acid
(JA) or methyl jasmonate (MJ) as elicitors of secondary
metabolites in plants is well known. Indeed upon
exposure of plant cells, tissues or organs to jasmonates,
the production of several classes of alkaloids [9 /11],
phenolics [12] and coumarins [13], as well as HCAs
[5,14,15], is strongly stimulated.
Differently from morphologically intact root cultures,
unorganised tissue or cell cultures often lose the capacity
to produce alkaloids [16]. In fact, hyoscyamine is present
only in trace amounts in callus [17] or cell [18] cultures
of Hyoscyamus . In order to shed some light on the
interplay between MJ-induced changes and develop-
mental control of the interface between primary and
secondary metabolism, two morphologically different in
vitro culture systems, callus vs. roots, the former
spontaneously derived from the latter and both growing
on hormone-free medium, were comparatively analysed.
In this comparative analysis between an unorganised
(callus) and an organised (roots) system, we tried to
answer the following questions: (1) Is callus unable to
produce secondary metabolites other than hyoscyamine/
scopolamine (e.g. HCAs) and, if so, can MJ overcome
this inhibition? (2) What are the responses to MJ in
terms of diamine (Put, mPut) and polyamine (Spd, Spm)
biosynthesis and accumulation? (3) How are the activ-
ities of two enzymes, PMT and diamine oxidase (DAO),
the latter also involved in diamine and tropane alkaloid
metabolism, affected by treatment with MJ?
Results show that root cultures exhibited MJ-induced
overproduction of free and/or conjugated di- and
polyamines, whereas callus cultures exhibited only a
transient accumulation of amine conjugates in response
to MJ. Elicitor-induced effects on enzyme activities were
also attenuated or opposite in callus as compared to
root cultures.
2. Materials and methods
2.1. Plant material and tissue culture
Root cultures of H. muticus were established and
maintained on agar-solidified hormone-free B5 [19]
medium as described by Biondi et al. [5]. They were
routinely subcultured every 10 /15 days by transferring
1-cm long apices to fresh medium. Root-derived callus
cultures were maintained on the same hormone-free B5
medium and were subcultured at monthly intervals.
Both types of cultures were grown in the dark at 239
/
18C.
2.2. Elicitor treatments
Roots and calli were transferred to liquid B5 medium
in Erlenmeyer flasks placed on a shaker 7 days before
initiating treatment with MJ as described previously [5].
Roots and calli were harvested at different times after
addition of 1 or 100 mM MJ, and stored at 
/80 8C until
use.
2.3. Di- and poly-amine analysis
For the TLC analysis of free and conjugated di- and
polyamines, aliquots of the supernatant and hydrolysed
supernatant, respectively were dansylated and extracted
in toluene following the method described in Biondi et
al. [15]. Chromatograms were run with cyclohexane /
ethylacetate (3:2) to separate the polyamines, and with
cyclohexane/ethylacetate/diethylamine (14:5:1) to se-
parate the diamines Put and mPut. Spots co-migrating
with standard Put, mPut, Spd and Spm were scraped off
and the powder eluted in 2 ml acetone to measure the
fluorescence using a Jasco FP-770 spectrofluorimeter.
2.4. ADC and ODC activity assays
Samples (:/1 g FW) were homogenised on ice in 5
volumes of 100 mM Tris /HCl, pH 8.5, containing 50
mM pyridoxalphosphate and 5 mM dithiothreitol, and
centrifuged at 18 000 /gfor 30 min at 4 8C. ODC and
ADC activity assays were performed essentially as
described in Biondi et al. [15]. They consisted in
measuring the
14
CO
2
evolution from 7.4 kBq of
L
-
[1-
14
C]ornithine (sp. act. 2.11 TBq mol
1
, Amersham,
Milano, Italy) or 22.2 kBq of
DL
-[U-
14
C]arginine (sp.
act. 11 TBq mol
1
, Amersham), respectively after a 2-h
incubation at 37 8C of aliquots of the supernatant or
resuspended pellet, and in the presence of unlabelled
substrate (1 mM arginine or ornithine).
2.5. Putrescine N-methyltransferase activity assay
PMT activity was evaluated using the method of Feth
et al. [20] with some modifications. Tissue ( :/0.5/1g
FW) was extracted on ice with 3 volumes of 100 mM
potassium phosphate buffer, pH 7.5, containing 5 mM
EDTA, 10 mM mercaptoethanol, 0.5% sodium ascor-
bate and 2% polyethyleneglycol 400 (buffer A), and the
homogenate centrifuged at 27 000 /gfor 30 min. The
supernatant was loaded onto a Sephadex G25 pre-
packed PD-10 column (Amersham Pharmacia Biotech
Italia) equilibrated and eluted with 50 mM potassium
phosphate buffer containing 1 mM EDTA and 5 mM
mercaptoethanol, pH 8 (buffer B). The reaction was
S. Biondi et al. / Plant Science 163 (2002) 563 /569564

performed by incubating 100 ml of the purified super-
natant with 20 ml 25 mM Put (final concentration 3.6
mM), 8 ml10mMS-adenosylmethionine (final concen-
tration 0.6 mM) and 12 ml buffer B at 37 8C for 30 min.
After stopping the reaction by heating in boiling water,
65 mM borate/KOH buffer and a solution of dan-
sylchloride (5.4 mg ml
1
acetonitrile) was added to the
incubation mixture. After heating at 60 8C for 15 min,
the dansylated amines were extracted by adding 0.5 ml
toluene and mixing on a Vortex for 30 s. An aliquot (400
ml) of the toluene was then removed, taken to dryness
and the dry residue resuspended in a fixed volume of
acetonitrile which was then injected into the HPLC. The
chromatographic conditions for the separation of dan-
sylated mPut were as described by Feth et al. [20]. The
retention time of mPut was 24 min.
2.6. Diamine oxidase activity assay
Oxidative activity was assayed by a radiometric
method [21] that measures [
14
C]D
1
-pyrroline formation
from [
14
C]Put. Roots were homogenised in 3 volumes of
100 mM potassium phosphate buffer, pH 8, containing
2 mM dithiothreitol, and centrifuged at 18 000 /gfor
30 min. Aliquots of the supernatant and resuspended
pellet were pre-incubated separately at room tempera-
ture for 45 min and then incubated at 37 8C for 30 min
with 7.4 kBq [1,4-
14
C]Put (sp. act. 4.37 TBq mol
1
,
Amersham) and 22.5 mg catalase. Unlabelled substrate
(Put or mPut) was also added at a final concentration of
100 mM. After stopping the reaction, the [
14
C]-labelled
products were extracted in 2 ml toluene, and radio-
activity was measured in a scintillation counter (LS
7800, Beckman).
2.7. Protein determination
Protein content of extracts used for the enzyme assays
was determined according to Bradford [22], using BSA
as standard.
3. Results
When left undisturbed for several months, roots
spontaneously formed callus at the distal (cut) end.
These calli could continue indefinite growth after being
separated from the roots and subcultured on the same
hormone-free medium. Thus, roots and their morpho-
logically disorganised counterparts (calli) were perfectly
comparable from the point of view of external growth
conditions.
3.1. Free and conjugated amines
The most represented free amine in root cultures was
Put, followed by mPut, Spd and Spm (Fig. 1A and B). A
short-term exposure (24 h) to 1 mM MJ did not affect
endogenous levels of the free polyamines Spd and Spm,
but strongly enhanced those of the diamines Put and
mPut (Fig. 1A). The conjugated di-/poly-amine pool in
roots was not much larger than that of free amines, and
conjugated mPut was not detectable at 24 h (Fig. 1C).
Soluble conjugates, especially of Spd, increased after a
24-h exposure to the elicitor (Fig. 1C). After 3 days in
the presence of MJ, free di-/poly-amines were unaffected
(Fig. 1B) but conjugated amines were still higher than in
controls (Fig. 1D).
In callus cultures grown in the absence of MJ, free
mPut was undetectable at 24 h (Fig. 2A), whereas a
rather high level of free mPut, comparable to that of
roots, appeared on day 3 (Fig. 2B). A short-term
treatment (24 h) of callus with the higher (100 mM)
concentration of MJ slightly enhanced free Put levels;
free Spd and Spm increased with both concentrations of
the elicitor, and by about 2-fold with the higher
concentration (Fig. 2A). After 3 days in the presence
of either concentration of MJ, free Put increased several-
fold, while mPut levels remained unchanged relativeto
controls (Fig. 2B); polyamines increased slightly (by
about 50/60%). A long-term exposure (7 days) to MJ
either did not significantly alter (1 mM) or caused a
marked decline (100 mM) in the cellular content of all
free di-/poly-amines, with Spm falling below detectable
levels (data not shown).
The main difference between root and callus cultures
was the scarcity of PCA-soluble conjugated amines in
the latter, with conjugated mPut and Spm below
detection levels. MJ, however, induced a marked but
Fig. 1. Free (A, B) and PCA-soluble conjugated (C, D) diamine and
polyamine content in roots of H. muticus treated (MJ) or not (control)
with 1 mM MJ for 24 h (A, C) or 3 days (B, D).
S. Biondi et al. / Plant Science 163 (2002) 563 /569 565

transient accumulation of conjugated Put and Spd after
a 24-h exposure (Fig. 2C), but after 3 and 7 days
conjugates were again undetectable both in controls and
MJ-treated callus cultures (data not shown).
3.2. ADC and ODC activities
In root extracts, Put biosynthetic enzyme activities
were detected both in the soluble and precipitable
fraction. In both cases, a 6-day treatment with 1 mM
MJ enhanced activity by about 2-fold for both enzymes
and in both fractions (Table 1). Under our assay
conditions, ADC activity in callus extracts was below
detection level, while ODC activity was present both in
the supernatant and pellet. After exposure to MJ,
soluble ODC activity in callus was lower than in
controls, and no longer detectable in the particulate
fraction (Table 1).
3.3. Putrescine N-methyltransferase activity
Root and callus cultures displayed comparable levels
of PMT activity in the absence of elicitor (Fig. 3). In
roots, exposure to either 1 or 100 mM MJ-induced an
approximately 5-fold increase in PMT activity. In callus
cultures, the lower concentration of MJ enhanced
enzyme activity about 3-fold, and the higher concentra-
tion caused only a 60% increase in activity. Conse-
quently, in the presence of 100 mM MJ, PMT activity in
roots was five times higher than in calli.
3.4. Diamine oxidase activity
In control roots, oxidative activity in the presence of
added unlabelled mPut was slightly higher (67 and 30%
in the supernatant and pellet, respectively) than with Put
(Fig. 4A and C). Upon treatment with 1 mM MJ, roots
displayed about 4- to over 10-fold increases in soluble
DAO activity (Fig. 4A). While in control roots activity
in the pellet was similar to or only slightly higher than in
the supernatant, in MJ-treated ones activity was mark-
edly higher in the former fraction. In fact, the most
dramatic enhancement (almost 25 times) of DAO
activity occurred in the pellet fraction and in the
presence of Put (Fig. 4C). In callus cultures, DAO
activity was higher when unlabelled Put was supplied
rather than mPut (Fig. 4B and D). In contrast with root
cultures, soluble activity in callus was higher (about
twice) than the pelletable one, and 1 mM MJ inhibited
activity in both fractions (Fig. 4B and D).
4. Discussion
In an earlier study using a hairy root clone of H.
muticus obtained by transformation with Agrobacterium
tumefaciens carrying the T-DNA from A. rhizogenes ,JA
was shown to cause only a small increase in hyoscya-
mine tissue content compared with untreated controls
[5]. In the same study, a marked accumulation of the
Fig. 2. Free (A, B) and PCA-soluble conjugated (C) diamine and
polyamine content in callus cultures of H. muticus treated or not
(control) with 1 or 100 mM MJ for 24 h (A, C) or 3 days (B).
Table 1
Soluble (SN) and precipitable (PT) ADC and ODC activities in roots
and calli of H. muticus exposed or not (control) for 6 days with 1 mM
MJ
ADC ODC
pmol (mg protein)
1
2h
1
nmol (mg protein)
1
2h
1
SN PT SN PT
Roots
Control 229914 7492 18.690.6 3.790.5
MJ 434913 179940 36.794.8 6.490.9
Callus
Control n.d. n.d. 3.391.8 7.593.1
MJ n.d. n.d. 1.890.5 n.d.
n.d., Not detectable.
Fig. 3. PMT activity in roots and calli of H. muticus treated or not
(control) for 7 days with 1 or 100 mM MJ.
S. Biondi et al. / Plant Science 163 (2002) 563 /569566

precursors Put and mPut, and of soluble di- and poly-
amine conjugates was observed in another hairy root
clone (A4) of H. muticus after a 7-day exposure to
exogenous jasmonates. Similar results were obtained
with a hairy root clone (KB7) transformed with the
hyoscyamine-6b-hydroxylase gene, and thus exhibiting
detectable levels of scoplamine as well as hyoscyamine.
Upon exposure to 100 mM MJ for variable periods of
time (2 to 7 days), free and conjugated amine titres
increased several-fold while only a slight increase in
tissue content of the two alkaloids was observed
(unpublished data). Previous results therefore indicate
that increased precursor availability does not lead to
enhanced end product formation while another second-
ary metabolic route is induced, namely the one leading
to HCA formation, suggesting that further research is
still needed to identify the critical points in the tropane
alkaloid pathway, and to understand what triggers one
secondary metabolic pathway rather than another.
In order to begin to address these questions, the
capacity of hyoscyamine-producing (roots) and -non
producing (calli) tissues to respond to elicitation by MJ
was compared in terms of free and conjugated di-/poly-
amine formation, and activities of enzymes involved in
Put and tropane alkaloid biosynthesis.
Present results show that the short-term response (24
h) of roots to 1 mM MJ resulted in several-fold increases
in free Put/mPut and in conjugated Spd, and a
consistent but less marked increase in conjugated Put
levels. The stimulatory effect of MJ on conjugate
formation was still activeonday3.
In callus cultures, the free Put pool size was markedly
enhanced by the same concentration of elicitor only
after 3 days. This may be because at 24 h much of the
available free Put was conjugated. Later on (day 7),
neither free nor conjugated Put accumulation, nor that
of other conjugates, was stimulated by MJ; this is in
marked contrast with the response observed in hairy
root clone KB7, and in other transformed or non-
transformed root clones [5]. Results also show that,
contrary to root cultures, mPut levels in callus were not
enhanced by MJ. This indirectly indicates that alkaloid
production is also not inducible by the elicitor in these
cultures.
The fact that treatment with MJ failed to induce Put/
mPut accumulation in callus is in accord with its lower
Put biosynthetic activities (ADC, ODC) compared with
root cultures, and with the fact that, in the former, these
activities were not enhanced by the elicitor. Robins et al.
[23] reported that ADC was the more important activity
for hyoscyamine biosynthesis in Datura root cultures,
but the relative contribution of ADC and ODC to HCA
formation is still uncertain [6,24]. Furthermore, Robins
et al. [6] used specific inhibitors to show that the
formation of polyamines was favoured over that of
tropane alkaloids. Our results indicating that in tissues
producing little or no alkaloids, ADC activity is
undetectable, while free Put, Spd and Spm are present
are in keeping with these notions.
MJ has been previously shown to upregulate gene
expression and/or activity of ADC and ODC in a hairy
root clone of H. muticus [5] and in shoot-forming
tobacco thin layer explants [15]. Imanishi et al. [25]
reported induction by MJ of ODC mRNA levels in
tobacco cell cultures, while Lee et al. [26] reported
increased activity of ADC in rice seedlings during MJ-
induced chilling tolerance. Thus, the inhibitory effect of
MJ on these activities in H. muticus callus cultures is
remarkable, and shows that morphologically disorga-
nised tissues have a distinctly different behaviour from
nicotine- or alkaloid-producing cultures.
An interesting feature reported here and previously
observed in tobacco thin layers [15] was the marked MJ-
induced increase in ADC and ODC activities associated
with the pellet fraction of root extracts. This suggests
that MJ acts preferentially on organelle-associated
activity whose occurrence has been documented (re-
viewed by Cohen [1]). Callus cultures differed from roots
also with respect to this particular response whose
significance remains to be established.
Another striking difference between root and callus
cultures was the fact that conjugated amines were scarce
(24 h) or absent (3 and 7 days) in the latter. Thus,
morphologically disorganised cultures of H. muticus
lose the capacity to synthesise hyoscyamine [17,18] as
well as HCAs. Put and Spd conjugates were nevertheless
transiently induced by MJ also in callus cultures. This
again is in contrast with root cultures where both a rapid
and long-term response to MJ is possible.
Conjugate over-accumulation means that MJ stimu-
lated the amounts of the free amine and/or of the
phenolic partners. Indeed, MJ has been shown to induce
phenylalanine ammonia lyase (PAL) activity in tobacco
Fig. 4. DAO activity in the supernatant (A, B) and pellet (C, D) of
root (A, C) and callus cultures (B, D) of H. muticus treated or not with
1mM MJ for 7 days. Activity was assayed in the presence of 100 mM
Put or mPut.
S. Biondi et al. / Plant Science 163 (2002) 563 /569 567

[13], and PAL poly(A)/RNA in soybean [9] cell
cultures. Thus, enhanced conjugate levels in MJ-treated
tissues may either serve to detoxify cells from excess
phenolics and/or to buffer major alterations in free
amines since these have a role as plant growth regula-
tors. Alternatively, they may have a function in protect-
ing plant cells exposed to stress, wounding or infection
[27], in this case mimicked by exogenous MJ. The
generally low-/short-lasting response of callus to MJ in
terms of HCA accumulation may reflect an inability to
up-regulate not only ADC/ODC but also PAL activity,
a typical secondary metabolic pathway which, together
with that of tropane alkaloids, callus cultures probably
lose.
From studies on in vitro cultures of various Solana-
ceae [4,7,23], a good correlation between PMT activity
and the capacity to produce nicotine and tropane
alkaloids has been demonstrated. In addition, exogen-
ous application of MJ-induced pmt gene expression in
N. sylvestris [28] and tobacco cell suspension cultures
[25]. Somewhat unexpectedly our results show that, in
the absence of elicitor, PMT activity levels were
comparable in tropane alkaloid-producing and non-
producing cultures (i.e. roots and calli, respectively). It
must be noted, however, that activity was assayed in the
presence of a saturating concentration of substrate.
Consequently, it does not reflect the physiological
situation, but does indicate that both culture types
probably have comparable levels of enzyme protein. It
is also noteworthy that the response to elicitor treatment
was quite different: in roots, MJ enhanced activity about
5-fold at both concentrations tested, whereas in callus
cultures the lower concentration of MJ was much more
effective than the higher one, and maximum stimulation
was only 3-fold. This differential dose /response effect of
the elicitor on the two culture types deserves further
investigation. Again, the fact that PMT activity was
measured in vitro with a high exogenous supply of
substrate may explain why MJ-stimulated activity in
callus was not reflected in increased endogenous mPut
levels.
DAO from cultured roots of H. niger displayed a
marked preference for mPut as substrate compared with
cadaverine or Put [29]. In our comparative analysis of
root and callus cultures, we noted that oxidative activity
in the former was higher when unlabelled mPut was
supplied compared with Put; the opposite was true for
callus. This seems to be in accord with the different
capacity for alkaloid biosynthesis. Another major dif-
ference between the two culture types was the fact that
in callus cultures soluble activity was higher than the
pelletable one, and that activity in both fractions was
inhibited, rather than enhanced, by MJ. In the cyto-
plasm, the DAO-catalysed oxidative deamination of Put
may serve to regulate cellular levels of this important
plant growth regulator. In addition, immunohistochem-
ical studies haveprovided evidence for a cell wall-
associated DAO where the H
2
O
2
generated by diamine
oxidation could be utilised by peroxidases in the
polymerisation and cross-linking of cell wall compo-
nents [30]. Indeed, reactive oxygen species (ROS), such
as H
2
O
2
, are a central component of the plant’s defense
machinery in that they also act as direct toxicants to
pathogens, and signal later defense reactions such as
phytoalexin synthesis ([31] and references therein). It is
therefore not unlikely that DAO activity, which is
stimulated by application of MJ also in tobacco explants
[15],isinvolved in defense reactions through its ROS
(H
2
O
2
)-producing function. In addition, a DAO (also
known as N-methylputrescine oxidase) is responsible for
the formation of N-methyl-D
1
-pyrrolinium cation from
mPut, the second step along the biosynthetic pathway
from Put towards tropane alkaloids. Thus, in our root
cultures, MJ-stimulated DAO activity in both soluble
and precipitable fractions can be interpreted as a means
to (a) control Put levels whose biosynthesis was en-
hanced by the elicitor; (b) favour cell wall stiffening; (c)
stimulate the production of defense compounds (i.e.
alkaloids). Callus cells are apparently unable to respond
to the elicitor also with respect to this defense process.
Plant tissue cultures have long been proposed as
useful systems for the large-scale production of phyto-
chemicals. In cultures which produce the desired com-
pound(s), a number of approaches have been adopted to
increase yield: selection of high-producing lines, culture
medium composition, treatment with elicitors. Genetic
transformation with genes encoding enzymes involved in
biosynthetic pathways remains a possibility [32] but is
presently limited by our lack of understanding of the
regulation of these pathways. Another important aspect
concerns the fact that secondary metabolism represents
one facet of plant differentiation; consequently, mor-
phologically undifferentiated cultures of many species
fail to produce secondary metabolites. Results of the
present work indicate that the combined use of elicita-
tion by MJ, and the comparison between root and callus
cultures provides a useful system to investigate elicitor
perception, intracellular signalling and the regulation of
secondary metabolism.
Acknowledgements
The experimental work performed by A. Golinelli, M.
Macrı` and L. Guerra is gratefully acknowledged. Many
thanks to P. Torrigiani and F. Antognoni for critical
revision of the manuscript. This research was supported
by funds from the Italian MURST (ex-60%) to S.B. and
F.P.
S. Biondi et al. / Plant Science 163 (2002) 563 /569568

References
[1] S.S. Cohen, A Guide to the Polyamines, Oxford University Press,
New York, 1998.
[2] H.E. Flores, J. Martin-Tanguy, Polyamines and plant secondary
metabolites, in: R.D. Slocum, H.E. Flores (Eds.), Biochemistry
and Physiology of Polyamines in Plants, CRC Press, Boca Raton,
Florida, 1991, pp. 57 /76.
[3] V. Scoccianti, E. Sgarbi, D. Fraternale, S. Biondi, Organogenesis
from Solanum melongena L. (eggplant) cotyledon explants is
associated with hormone-modulated enhancement of polyamine
biosynthesis and conjugation, Protoplasma 211 (2000) 51 /63.
[4] T. Hashimoto, Y. Yukimune, Y. Yamada, Putrescine and
putrescine N-methyltransferase in the biosynthesis of tropane
alkaloids in cultured roots of Hyoscyamus albus. I. Biochemical
studies, Planta 178 (1989) 123 /130.
[5] S. Biondi, S. Fornale´, K.-M. Oksman-Caldentey, M. Eeva, S.
Agostani, N. Bagni, Jasmonates induce over-accumulation of
methylputrescine and conjugated polyamines in Hyoscyamus
muticus L. root cultures, Plant Cell Rep. 19 (2000) 691/697.
[6] R.J. Robins, A.J. Parr, N.J. Walton, Studies on the biosynthesis
of tropane alkaloids in Datura stramonium L. transformed root
cultures. 2. On the relative contribution of
L
-arginine and
L
-
ornithine to the formation of the tropane ring, Planta 183 (1991)
196 /201.
[7] N. Hibi, T. Fujita, M. Hatano, T. Hashimoto, Y. Yamada,
Putrescine N-methyltransferase in cultured roots of Hyoscyamus
albus , Plant Physiol. 100 (1992) 826 /835.
[8] R.A. Creelman, J.E. Mullet, Oligosaccharins, brassinolides, and
jasmonates: non-traditional regulators of plant growth, develop-
ment, and gene expression, Plant Cell 9 (1997) 1211/1223.
[9] H. Gundlach, M.J. Mu
¨ller, T.M. Kutchan, M.H. Zenk, Jasmonic
acid is a signal transducer in elicitor-induced plant cell cultures,
Proc. Natl. Acad. Sci. USA 89 (1992) 2389 /2393.
[10] R.J. Aerts, D. Gisi, E. De Carolis, V. De Luca, T.W. Baumann,
Methyl jasmonate vapor increases the developmentally controlled
synthesis of alkaloids in Catharanthus and Cinchona seedlings,
Plant J. 5 (1994) 635 /643.
[11] I. Zabetakis, R. Edwards, D. O’Hagan, Elicitation of tropane
alkaloid biosynthesis in transformed root cultures of Datura
stramonium, Phytochemistry 50 (1999) 53 /56.
[12] J. Lee, T. Vogt, J. Schmidt, B. Parthier, M. Lo
¨bler, Methyljas-
monate-induced accumulation of coumaroyl conjugates in barley
leaf segments, Phytochemistry 44 (1997) 589 /592.
[13] M. Sharan, G. Taguchi, K. Gonda, T. Jouke, M. Shimosaka, N.
Hayashida, M. Okazaki, Effects of methyl jasmonate and elicitor
on the activation of phenylalanine ammonia-lyase and the
accumulation of scopoletin and scopolin in tobacco cell cultures,
Plant Sci. 132 (1998) 13 /19.
[14] J.C. Mader, Effects of jasmonic acid, silver nitrate and
L
-AOPP
on the distribution of free and conjugated polyamines in roots and
shoots of S. tuberosum in vitro, J. Plant Physiol. 154 (1999) 79 /
88.
[15] S. Biondi, S. Scaramagli, F. Capitani, M.M. Altamura, P.
Torrigiani, Methyl jasmonate upregulates biosynthetic gene
expression, oxidation and conjugation of polyamines, and inhibits
shoot formation in tobacco thin layers, J. Exp. Bot. 52 (2001)
231 /242.
[16] R.J. Robins, E.G. Bent, J.C.M. Rhodes, Studies on the biosynth-
esis of tropane alkaloids in Datura stramonium L. transformed
root cultures. 3. The relationship between morphological integrity
and alkaloid biosynthesis, Planta 185 (1991) 385/390.
[17] H.E. Flores, P. Filner, Metabolic relationships of putrescine,
GABA and alkaloids in cell and root cultures of Solanaceae, in:
K.-H. Neuman, W. Barz, E. Reinhard (Eds.), Primary and
Secondary Metabolism of Plant Cell Cultures, Springer, Berlin,
Heidelberg, 1985, pp. 174 /185.
[18] K.-M. Oksman-Caldentey, A. Strauss, Somaclonal variation of
scopolamine content in protoplast-derived cell culture clones of
Hyoscyamus muticus , Planta Medica 52 (1986) 6 /12.
[19] O.L. Gamborg, R.A. Miller, K. Ojima, Nutrient requirements of
suspension cultures of soybean root cells, Exp. Cell Res. 50 (1968)
151/158.
[20] F. Feth, H.A. Arfmann, V. Wray, K.G. Wagner, Determination
of putrescine N-methyltransferase by high performance liquid
chromatography, Phytochemistry 24 (1985) 921 /923.
[21] T. Okuyama, Y. Kobayashi, Determination of diamine oxidase
activity by scintillation counting, Arch. Biochem. Biophys. 95
(1961) 242 /250.
[22] M.M. Bradford, A rapid and sensitive method for the quantita-
tion of microgram quantities of protein utilizing the principle of
protein-dye binding, Anal. Biochem. 72 (1976) 248 /254.
[23] R.J. Robins, A.J. Parr, J. Payne, N.J. Walton, M.J.C. Rhodes,
Factors regulating tropane-alkaloid production in a transformed
root culture of a Datura candida /D. aurea hybrid, Planta 181
(1990) 414 /422.
[24] D. Burtin, J. Martin-Tanguy, M. Paynot, N. Rossin, Effects of
the suicide inhibitors of arginine and ornithine decarboxylase
activities on organogenesis, growth, free polyamine and hydro-
xycinnamoyl putrescine levels in leaf explants of Nicotiana Xanthi
n.c. cultivated in vitro on a medium producing callus formation,
Plant Physiol. 89 (1989) 104 /110.
[25] S. Imanishi, K. Hashizume, M. Nakakita, H. Kojima, Y.
Matsubayashi, T. Hashimoto, Y. Sakagami, Y. Yamada, K.
Nakamura, Differential induction by methyl jasmonate of genes
encoding ornithine decarboxylase and other enzymes involved in
nicotine biosynthesis in tobacco cell cultures, Plant Mol. Biol. 38
(1998) 1101 /1111.
[26] T.-M. Lee, H.-S. Lur, Y.-H. Lin, C. Chu, Physiological and
biochemical changes related to methyl jasmonate-induced chilling
tolerance of rice (Oryza sativaL.) seedlings, Plant Cell Environ.
19 (1996) 65 /74.
[27] A. Bouchereau, A. Aziz, F. Larher, J. Martin-Tanguy, Poly-
amines and environmental challenges: recent development, Plant
Sci. 140 (1999) 103 /125.
[28] T. Shoji, Y. Yamada, T. Hashimoto, Jasmonate induction of
putrescine N-methyltransferase gene in the root of Nicotiana
sylvestris , Plant Cell Physiol. 41 (2000) 831/839.
[29] T. Hashimoto, A. Mitani, Y. Yamada, Diamine oxidase from
cultured roots of Hyoscyamus niger , Plant Physiol. 93 (1990)
216/221.
[30] R. Angelini, G. Rea, R. Federico, R. D’Ovidio, Spatial distribu-
tion and temporal accumulation of mRNA encoding diamine
oxidase during lentil (Lens culinaris Medicus) seedling develop-
ment, Plant Sci. 119 (1996) 103 /113.
[31] D. Scheel, Resistance response physiology and signal transduc-
tion, Curr. Op. Plant Biol. 1 (1998) 305 /310.
[32] F. Sato, T. Hashimoto, A. Hachiya, K. Tamura, K. Choi, T.
Morishige, H. Fujimoto, Y. Yamada, Metabolic engineering of
plant alkaloid biosynthesis, Proc. Natl. Acad. Sci. USA 98 (2001)
367/372.
S. Biondi et al. / Plant Science 163 (2002) 563 /569 569