Expression of an antisense Datura stramonium S-adenosylmethionine decarboxylase cDNA in tobacco: changes in enzyme activity, putrescine-spermidine ratio, rhizogenic potential, and response to methyl jasmonate.

Abstract

S-adenosylmethionine decarboxylase activity (SAMDC; EC 4.1.1.21) leads to spermidine and spermine synthesis through specific synthases which use putrescine, spermidine and decarboxylated S-adenosylmethionine as substrates. In order to better understand the regulation of polyamine (PA), namely spermidine and spermine, biosynthesis, a SAMDC cDNA of Datura stramonium was introduced in tobacco (Nicotiana tabacum L. cv. Xanthi) in antisense orientation under the CaMV 35S promoter, by means of Agrobacterium tumefaciens and leaf disc transformation. The effect of the genetic manipulation on PA metabolism, ethylene production and plant morphology was analysed in primary transformants (R0), and in the transgenic progeny (second generation, R1) of self-fertilised primary transformants, relative to empty vector-transformed (pBin19) and wild-type (WT) controls. All were maintained in vitro by micropropagation. Primary transformants, which were confirmed by Southern and northern analyses, efficiently transcribed the antisense SAMDC gene, but SAMDC activity and PA titres did not change. By contrast, in most transgenic R1 shoots, SAMDC activity was remarkably lower than in controls, and the putrescine-to-spermidine ratio was altered, mainly due to increased putrescine, even though putrescine oxidising activity (diamine oxidase, EC 1.4.3.6) did not change relative to controls. Despite the reduction in SAMDC activity, the production of ethylene, which shares with PAs the common precursor SAM, was not influenced by the foreign gene. Some plants were transferred to pots and acclimatised in a growth chamber. In these in vivo-grown second generation transgenic plants, at the vegetative stage, SAMDC activity was scarcely reduced, and PA titres did not change. Finally, the rhizogenic potential of in vitro-cultured leaf explants excised from antisense plants was significantly diminished as compared with WT ones, and the response to methyl jasmonate, a stress-mimicking compound, in terms of PA conjugation, was higher and differentially affected in transgenic leaf discs relative to WT ones. The effects of SAMDC manipulation are discussed in relation to plant generation, culture conditions and response to stress.

Full text

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Journal of Plant Physiology 162 (2005) 559—571
Expression of an antisense Datura stramonium
S-adenosylmethionine decarboxylase cDNA
in tobacco: changes in enzyme activity,
putrescine-spermidine ratio, rhizogenic potential,
and response to methyl jasmonate
Patrizia Torrigiania,?, Sonia Scaramaglia, Vanina Ziosia,
Melinda Mayerb, Stefania Biondia
aDipartimento di Biologia e.s., University of Bologna, Via Irnerio 42, 40126 Bologna, Italy
bDepartment of Genetics and Microbiology, Institute of Food Research, Colney Park, Norwich, UK
Received 21 April 2004; accepted 25 October 2004
Summary
S-adenosylmethionine decarboxylase activity (SAMDC; EC 4.1.1.21) leads to spermi-
dine and spermine synthesis through specific synthases which use putrescine,
spermidine and decarboxylated S-adenosylmethionine as substrates. In order to
better understand the regulation of polyamine (PA), namely spermidine and spermine,
biosynthesis, a SAMDC cDNA of Datura stramonium was introduced in tobacco
(Nicotiana tabacum L. cv. Xanthi) in antisense orientation under the CaMV 35S
promoter, by means of Agrobacterium tumefaciens and leaf disc transformation. The
effect of the genetic manipulation on PA metabolism, ethylene production and plant
morphology was analysed in primary transformants (R0), and in the transgenic progeny
(second generation, R1) of self-fertilised primary transformants, relative to empty
vector-transformed (pBin19) and wild-type (WT) controls. All were maintained in vitro
by micropropagation. Primary transformants, which were confirmed by Southern and
northern analyses, efficiently transcribed the antisense SAMDC gene, but SAMDC
activity and PA titres did not change. By contrast, in most transgenic R1 shoots, SAMDC
activity was remarkably lower than in controls, and the putrescine-to-spermidine
ratio was altered, mainly due to increased putrescine, even though putrescine
ARTICLE IN PRESS
www.elsevier.de/jplph
KEYWORDS
Antisense
transformation;
SAMDC;
Polyamines;
Methyl jasmonate;
Rhizogenesis;
Tobacco
0176-1617/$-see front matter & 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2004.10.008
Abbreviations: ADC, arginine decarboxylase; DAO, diamine oxidase; dcSAM, decarboxylated S-adenosylmethionine; ODC,
ornithine decarboxylase; MJ, methyl jasmonate, PA; polyamines; PCA, perchloric acid; PCR, polymerase chain reaction; SAM,
S-adenosylmethionine; SAMDC, S-adenosylmethionine decarboxylase, WT, wild-type
?Corresponding author. Tel.: +390512091291; fax: +39051242576.
E-mail address: patrizia.torrigiani@unibo.it (P. Torrigiani).

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oxidising activity (diamine oxidase, EC 1.4.3.6) did not change relative to controls.
Despite the reduction in SAMDC activity, the production of ethylene, which shares with
PAs the common precursor SAM, was not influenced by the foreign gene. Some plants
were transferred to pots and acclimatised in a growth chamber. In these in vivo-grown
second generation transgenic plants, at the vegetative stage, SAMDC activity was
scarcely reduced, and PA titres did not change. Finally, the rhizogenic potential of in
vitro-cultured leaf explants excised from antisense plants was significantly diminished
as compared with WTones, and the response to methyl jasmonate, a stress-mimicking
compound, in terms of PA conjugation, was higher and differentially affected in
transgenic leaf discs relative to WT ones. The effects of SAMDC manipulation are
discussed in relation to plant generation, culture conditions and response to stress.
& 2005 Elsevier GmbH. All rights reserved.
Introduction
Polyamines (PAs) are multivalent cations present
in all living organisms, which regulate chromatin
organisation, transcription and translation (Cohen
1998); therefore, they are required in cell division
and differentiation (Bagni and Torrigiani 1992),
including programmed cell death (Serafini-Fracas-
sini et al. 2002), and stress responses (Bouchereau
et al. 1999). Their synthesis starts from the amino
acids arginine and ornithine, which lead to putres-
cine, either indirectly via arginine decarboxylase
(ADC), or directly via ornithine decarboxylase
(ODC). Methionine is converted to S-adenosyl
methionine (SAM), which is primarily a methyl
donor and also an ethylene precursor (Ravanel et
al. 1998), and then to decarboxylated SAM (dcSAM)
via the key enzyme S-adenosylmethionine decar-
boxylase (SAMDC, Bennett et al. 2002). DcSAM is
the substrate, with putrescine and spermidine, for
synthases responsible for spermidine and spermine
synthesis, respectively (Hanzawa et al. 2000; Zhang
et al. 2003).
PA titres are regulated at the transcriptional,
post-transcriptional, translational and post-transla-
tional levels (Cohen 1998), and by conjugation to
hydroxycinnamic acids (Martin-Tanguy 1985). This
suggests that, although a rapid PA response is
required under some physiological or pathological
conditions, their cellular homeostasis is important.
Attempts have been made to alter endogenous PA
levels in various ways. Mutants were the first source
of plants with ‘‘non-physiological’’ PA levels (Malm-
berg and McIndoo 1983; Watson et al. 1998;
Hanzawa et al. 2000), but their scarcity has
hindered progress in understanding the regulation
of their metabolism. Attempts to genetically
modulate PA levels in plants have involved the
manipulation of putrescine biosynthesis with the
introduction in tobacco, carrot and rice of hetero-
logous ADC or ODC cDNA in sense orientation
resulting in variable increases in the target enzyme
activity and/or product (Kumar and Minocha 1998;
Mayer and Michael 2003; Trung-Nghia et al. 2003).
With respect to spermidine and spermine, plants
and cultured cells overexpressing the SAMDC gene
also display changes in enzyme activity and PA
levels compared with controls (Noh and Minocha
1994; Kumar et al. 1996; Rafart Pedros et al. 1999;
Quan et al. 2002; Thu-Hang et al. 2002; Waie and
Rajam 2003).
Less is known about antisense engineering of PA
metabolism. Rice callus lines containing an oat ADC
cDNA in antisense orientation showed decreased
ADC activity, which was reflected in depleted
putrescine and spermidine levels, although these
changes were reversed in regenerated plants
(Capell et al. 2000). Under the CaMV 35S promoter
or the tuber-specific patatin promoter, SAMDC
antisense potato plants exhibited reduced SAMDC
mRNA and activity, and spermidine levels (Kumar et
al. 1996; Rafart Pedros et al. 1999).
It is well known that alteration in PA titres is one
of the ways in which plants respond to biotic and
abiotic stress (Bouchereau et al. 1999; Walters
2003). In fact, rice plants overexpressing ADC or
SAMDC and tobacco overexpressing ODC have
exhibited enhanced osmotic stress resistance (Roy
and Wu 2001, 2002; Kumria and Rajam 2002; Waie
and Rajam 2003). Jasmonates are well-known
signalling molecules in stress responses and elicitors
of defense-related compounds (Creelman and Mullet
1997). Among jasmonate-induced genes, there are
those responsible for PA biosynthesis, with the
consequent accumulation of PAs, especially the
conjugated ones (Biondi et al. 2001, 2003).
The aim of the present work was to determine
whether downregulation of SAMDC can deplete
steady-state levels of higher PAs. To this end, an
antisense SAMDC cDNA of Datura stramonium, a
solanaceous species close to tobacco, was intro-
duced in tobacco under the CaMV 35S promoter.
Primary transformants (R0) and the segregating
progeny in the second generation (R1) were
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analysed, relative to wild type (WT) and empty
vector-containing (pBin19) controls, for (i) SAMDC
transcript levels, (ii) SAMDC activity, and (iii) free,
and soluble and insoluble conjugated PA titres. The
progeny were also analysed for diamine oxidase
(DAO) activity, ethylene production and morpholo-
gical changes. In addition, the rhizogenic potential
of these plants, which is very sensitive to changes
in the endogenous PA pool (Altamura et al. 1991;
Coue ´e et al. 2004), was evaluated using leaf
explants induced to form roots in the presence of
auxin. Finally, to examine whether antisense plants
exhibited an altered PA metabolism in response to a
stress-mimicking compound relative to WT ones,
leaf discs were incubated in a methyl jasmonate
(MJ)-containing solution, and PA accumulation was
investigated.
Results showed that, in most of the transgenic
progeny, SAMDC activity and PA levels were strongly
altered relative to controls, but only in micro-
propagated shoots, and that explants from in vivo-
grown plants displayed a depressed rhizogenic
potential, and a different response to MJ in terms
of PA conjugation.
Materials and methods
Construction of the antisense vector, plant
transformation and regeneration
A 1839-bp D. stramonium SAMDC cDNA (accession
number Y07768), sharing a 92% homology with the
tobacco SAMDC gene sequence (accession number
AF033100; Franceschetti et al. 2001), was sub-
cloned in an antisense orientation between a
cauliflower mosaic virus (CaMV) 35S RNA promoter
with duplicated enhancer sequences, and the CaMV
termination sequence in the vector pJIT60 (Guer-
ineau et al. 1992). The promoter, antisense cDNA
and terminator were then excised and ligated into
the KpnI and SalI sites of pBin19 (Bevan 1984). This
construct and an empty pBin19 vector were
transferred into Agrobacterium tumefaciens strain
LBA4404 by triparental mating as described by
Bevan (1984). Conventional leaf disc transforma-
tion of Nicotiana tabacum L. cv. Xanthi XHFD8 and
selection of plantlets was performed as described
previously (Mayer et al. 2001). Rooted primary
transformants (R0) were transferred to soil in a
greenhouse. Young leaves from three SAMDC anti-
sense (IV, V and VI) and three pBin19 control plants
were harvested one month later, at the vegetative
stage, and stored at ?801C until use for molecular
and biochemical analyses.
Seed germination in vitro and plant
cultivation
Seed obtained by self-pollination of WT plants,
empty vector-transformed plants (pBin19) and the
primary transformants IV, V and VI were surface-
sterilised with 5% commercial solution of sodium
hypochloride (6% active chloride) for 10min, rinsed
five times with sterile distilled water and germi-
nated in glass jars on MS medium (Murashige and
Skoog 1962). The latter was supplemented with
kanamycin in order to select for transgenic progeny
(R1). Selected seedlings of lines IV, V and VI were
subsequently micropropagated in vitro. Single
shoots were grown in test tubes on hormone-free
MS medium and subcultured every three weeks;
they constituted the second generation of in vitro
micropropagated shoots.
A number of these micropropagated shoots were
allowed to root; when they had 3–4 leaves and
were about 5-cm tall they were transferred to pots,
acclimatised and grown up to flowering in a growth
chamber (in vivo plants) under controlled light
(Philips SON-Tand HPI-T, 400W, 320mEm?2s?1) and
temperature (day/night temperature of 25/191C)
regimes, and with a 12-h photoperiod. They
constitutedinvivo-grown
plants, and these were used for morphometric
analyses. Internode number, plant height, and leaf
size were measured on three WTand pBin19 plants,
four plants of line IV and five of line V at
the vegetative and flowering stages. In vitro
micropropagated shoots, as well as leaves from
secondgeneration
ARTICLE IN PRESS
Table 1.
Experimental design
Plant generationType of analysis
Primary transformants
Plants IV, V and VISouthern blot
SAMDC northern blot
SAMDC activity
Polyamine titres
Second generation
transformants
In vitro micropropagated
shoots
PCR
SAMDC activity
Polyamine titres
DAO activity
Ethylene production
Morphology
SAMDC activity
Polyamine titres
Polyamine response to MJ
In vivo-grown plants
Explants from in vivo
plants
Rhizogenic potential
Expression of an antisense SAMDC in tobacco561

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in vivo-grown plants at the vegetative stage were
collected and stored at ?801C until use. Primary
(R0) and second generation (R1) transformants
were used for biochemical, molecular and morpho-
metric analyses, and leaves from in vivo-grown R1
plants were used as source of leaf discs and leaf
explants to study the response to MJ and the
rhizogenic potential according to the following
scheme (Table 1).
Molecular characterisation of transgenic
plants
Genomic DNA was extracted and restricted with
EcoRI for Southern blot analysis as described
previously (Mayer et al. 2001). The presence of
the pBIN19 vector was confirmed by hybridisation
to a 1955-bp PstI fragment encompassing the T-DNA
region (not shown). The transgene in SAMDC
antisense R0 plants was identified by hybridisation
to a 730-bp probe for the 35S promoter excised
from pJIT60 using KpnI and SmaI. Expression of the
transgene was confirmed by northern analysis using
the D. stramonium SAMDC cDNA as a probe. Three
primary transformants (plants IV, V, and VI) ex-
hibiting the highest level of SAMDC antisense mRNA
were selected for further experiments.
In the transgenic progeny (R1), the presence of
the D. stramonium sequence was confirmed by
genomic polymerase chain reaction (PCR) amplifi-
cation. The latter was carried out utilising the
Ready-To-Go PCR beads kit (Amersham Pharmacia
Biotech Italia, Milano, Italy) containing 200mM each
dNTPs and 1.5U of Taq DNA polymerase, with 1mg
DNA and 1mM of each primer, in a total volume
of 25ml. The forward primer was complementary
totheCaMV35Spromoter
GGTGGCTCCTACAAATGCCA-30); the reverse primer
was complementary to the D. stramonium SAMDC
transgene (50-TAGAGATGTGTATGACTG-30). The PCR
programme included 1 cycle at 941C for 3min,
1 cycle at 751C for 3min, 25 cycles at 941C for 15s,
25 cycles at 551C for 45s, 25 cycles at 721C for
1.5min and a final cycle at 721C for 10min. The
1-kb product was visualised by agarose gel electro-
phoresis.
sequence(50-
RNA extraction and northern blots
The presence of the transgene in primary
transformants (plants IV, V and VI) was also
confirmed by northern analysis. Total RNA was
extracted from ca. 200 to 300mg fresh weight leaf
samples using an RNeasy Plant Mini Kit (Qiagen,
Hilden, Germany) according to the manufacturer’s
instructions. RNA (15mg per track) was size-
fractionated on a 1.2% agarose formaldehyde gel
and transferred in 10?SSC (20?SSC: 0.3M sodium
citrate, 3.0M NaCl, pH 7) onto nylon membranes
(Hybond-N, Amersham Pharmacia Biotech Italia)
overnightaccording to
(Sambrook et al. 1989). RNA was cross-linked to
the membrane by exposure to UV at 312nm (Vilber
Lourmat, Marne ´ La Valle ´e, France) for 4min.
RNA blots were pre-hybridised at 421C for 2h and
hybridised at 421C for 18–20h with a (32P)dCTP-
labelled PCR fragment (random priming with a
Rediprime DNA labelling Kit, Amersham Pharmacia
Biotech Italia) of D. stramonium SAMDC as de-
scribed in Scaramagli et al. (1999). Following
hybridisation, membranes were washed as de-
scribed previously, and then exposed to X-ray film
at ?801C for 24h with intensifying screen (DuPont,
Wilmington, DE, USA). Equal loading of RNA on gels
was verified by reprobing stripped filters with an
Antirrhinum majus ubiquitin gene probe.
standard methods
Enzyme activity assays
The SAMDC activity (EC 4.1.1.21) assay was
performed by a radiometric method, as previously
described by Biondi et al. (2001). Enzyme extrac-
tion procedures were carried out in an ice bath.
Micropropagated shoots and leaves were homoge-
nised with 5 volumes of 0.1M Tris–HCl buffer, pH
7.6, containing 50mM EDTA. The homogenate was
centrifuged at 20000g for 30min at 41C; 0.2-ml
aliquots of the supernatant were incubated with
3.7kBqS-adenosyl-L-[carboxyl-14C]methionine
(2.07TBqmol?1, Amersham Pharmacia Biotech Ita-
lia) and 100mM unlabelled SAM in a final assay
volume of 0.5ml, and the rate of14CO2evolution
from SAM decarboxylation evaluated. At the end of
a 2-h incubation at 371C, the14CO2trapped in 2M
KOH (150mM) was counted in a Beckman LS 7800
beta counter.
For DAO activity assays (DAO, EC 1.4.3.6), shoots
were homogenised on ice with a mortar and pestle
in 3 volumes of 100mM potassium phosphate buffer,
pH 8, containing 2mM dithiothreitol. After centri-
fugation at 20000g for 30min at 41C, supernatants
were used to measure DAO activity by a radiometric
method based on the production of D1[14C]pyrroline
from [1,4-14C]putrescine as previously described
(Biondi et al. 2001). The [14C]pyrroline formed
during a 30-min incubation at 371C was immedi-
ately extracted in 0.5ml toluene; aliquots (100ml)
of the lipophilic phase were withdrawn and added
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to 2ml scintillation liquid (Ultima Gold), and the
radioactivity counted in a Beckman scintillation
counter. In both cases, protein content was
measuredusing Bradford’s
1976), with bovine serum albumin as the standard.
Experiments were repeated at least once (3–4
replicates each). Mean values were compared using
a one-way ANOVA to check for significant differ-
ences in enzyme activity levels between transgenic
individuals and WT controls.
method(Bradford
HPLC polyamine analysis
Leaves from primary transformants, in vitro
micropropagated R1 shoots, and leaves and leaf
explants from in vivo-grown R1 plants were
extracted in 10 volumes of 4% perchloric acid
(PCA), and centrifuged at 20000g for 30min at 41C.
Aliquots (0.3ml) of the supernatant (PCA-soluble
fraction), and of the resuspended pellet (PCA-
insoluble fraction) were subjected to acid hydro-
lysis (6N HCl at 1101C overnight) in order to release
PAs from their PCA-soluble and -insoluble conju-
gates, respectively. Aliquots (0.2ml) of the super-
natant, containing free PAs, and of the hydrolysed
supernatant and resuspended pellet were dansy-
lated, extracted in toluene and analysed by HPLC
(PU-980 Jasco, Tokyo, Japan) on a reverse phase
C18column (Spherisorb ODS2, 5-mm particle dia-
meter, 4.6?250mm, Waters, Wexford, Ireland)
using a programmed acetonitrile:water step gradi-
ent (Scaramagli et al. 1995). Eluted peaks were
detected by a spectrofluorometer (821-FP Jasco,
excitation 365nm, emission 510nm), and their
areas were recorded and integrated relative to
those of standard PAs using the JCL6000 software
(Jasco). Experiments were repeated at least once
(3 replicates each) and, where appropriate, mean
values were statistically analysed using a one-way
ANOVA to check for significant differences between
transgenic individuals and WT controls.
Ethylene measurement
Test tubes in which one week-old micropropa-
gated shoots were growing were sealed with air-
tight serum caps. After 18h, 10-ml gas samples
were withdrawn from the headspace and injected
into a Dani DS 86.01 (Dani, Milano, Italy) packed-
gas chromatograph equipped with a Poropak Q
column and a flame ionisation detector, as pre-
viously described (Biondi et al. 1998). The carrier
gas was nitrogen, at a flow rate of 16mlmin?1.
Rhizogenesis from leaf explants
Mature leaves from three WT and four antisense
(line V, plants C, E, F and G) in vivo-grown plants
were used as source of explants, which were
sterilised with a diluted (10%) commercial solution
of sodium hypochloride, rinsed three times, and
then cultured on a root-inducing MS medium
(Bellincampi et al. 1996) containing 2% sucrose,
0.8% agar and 0.6mM indoleacetic acid (IAA, Sigma-
Aldrich, Milano, Italy). Leaf explants (2?1cm),
bearing the mid-vein, were cultured (6 per Petri
dish) in the dark at 241C for 15d. The time course
of rhizogenesis was evaluated macroscopically at 9,
12 and 15d in culture on 120–180 explants. At
culture end, the mean number of roots per explant
and root length (total number of roots 500–800)
were measured, and data statistically treated with
the one-way ANOVA test to separate WT from
transgenic samples.
MJ treatment
Leaf discs (1-cm diameter) were excised, using a
cork borer, from fully expanded leaves of WT and
line V transgenic in vivo-grown plants, and floated
in Petri dishes on 20ml of 10mM potassium
phosphate buffer (pH 6) containing or not (controls)
10mMMJ(ServaElectrophoresis,
Germany), as previously described (Biondi et al.
2003). Since MJ was dissolved in ethanol, controls
contained the same concentration of ethanol
present in MJ treatments. After 48h of incubation
at 22711C in the light (50mmolm?2s?1) with a 16/
8h light/dark photoperiod, leaf discs were col-
lected, weighed, frozen in liquid nitrogen, and
stored at ?801C until further use.
Heidelberg,
Results
Primary transformants
The presence and expression of the D. stramo-
nium SAMDC sequence in primary transformants
was demonstrated by Southern and northern
analyses. The 35S sequence was present in genomic
DNA restricted with EcoRI from SAMDC antisense
plants (IV, V and VI), but absent from pBin19
controls (Fig. 1A). The SAMDC probe cross-hybri-
dised to tobacco SAMDC mRNA (Fig. 1B,C), but
SAMDC antisense plants IV, Vand VI also contained a
larger message corresponding to the D. stramonium
sequence which was absent in RNA from pBin19
controls. SAMDC activity, as well as free, and
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Expression of an antisense SAMDC in tobacco 563

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soluble- and insoluble conjugated PAs exhibited no
significant differences in SAMDC antisense primary
transformants relative to pBin19 plants (data not
shown).
Second generation transformants
Enzyme activities, polyamine titres and ethylene
production in micropropagated shoots
The presence of the foreign gene in all the
individuals selected was confirmed by PCR analysis
using specific primers for the D. stramonium
sequence (Fig. 2). In the antisense micropropa-
gated shoots, SAMDC activity was significantly
(po0.01 or 0.05) lower in all line IV (Fig. 3A), in
50% of line V (Fig. 3B), and in 86% of line VI
individuals (Fig. 3C) compared with that of WT and
pBin19 controls (both the average of three plants),
which were not significantly different from each
other.
Four antisense genotypes per line exhibiting
decreased SAMDC activity were analysed for PA
levels. They showed an alteration in the free
putrescine-to-spermidine ratio relative to WT and
pBin19 controls (which did not differ significantly
from each other), mainly due to elevation of
putrescineconcentration.
titres were enhanced in all transgenics up to
6-fold relative to controls,
did not increase, or increased to a lesser extent
Infact,putrescine
whilespermidine
(Fig. 4A,B,C). In contrast to the controls, the ratio
between the two amines in the individuals of the
three different lines ranged from 1 to as much as
10. Spermine was below the level of detection, and
no soluble or insoluble conjugates were detectable
in this juvenile physiological stage.
DAO activity, which could be responsible for
changes in putrescine titres, remained essentially
the same in WT (0.2270.05nmolmg?1protein
30min?1) and pBin19 (0.2370.08nmolmg?1pro-
tein 30min?1) controls, and in antisense genotypes
of the three lines (mean value 0.26nmolmg?1
protein 30min?1).
In addition, mean ethylene production from
shoots of the three SAMDC antisense lines was not
significantly different (17.073.8plg?1h?1) from
that of WT and pBin19 controls (18.673.3 and
16.273.25plg?1h?1, respectively).
SAMDC activity, polyamine titres, and
morphology of in vivo-grown plants
In mature leaves of in vivo-grown plants,
although SAMDC activity was modestly but signifi-
cantly (po0.05) downregulated in the 2 line IV
individuals, and in 4 out of 6 line V individuals
(Fig. 5), free, and soluble and insoluble-conjugated
PA titres (average values for each line) displayed no
significant differences compared with controls
(Table 2).
Morphometric analyses were performed on three
untransformed (WT) and three empty vector-
transformed (pBin19) controls, and on the above-
cited antisense tobacco plants of lines IV and V at
ARTICLE IN PRESS
Figure 1. Southern analysis of the D. stramonium SAMDC
gene (A) and SAMDC northern analysis (B, B0) in leaves
from antisense SAMDC primary transformants (IV, V and
VI) and empty vector-transformed (pBin19) tobacco
plants. Fifteen mg DNA and RNA were loaded per well.
The Southern blot was hybridised to the 35S fragment (A)
as described in Methods, and the northern to the D.
stramonium SAMDC cDNA (B) or to the Antirrhinum majus
ubiquitin probe as a loading control (C).
Figure 2. PCR analysis for the presence of the D.
stramonium SAMDC gene in antisense micropropagated
tobacco shoots of line IV, line V and line VI. Letters
representdifferentindividuals
* ¼ molecular-weight
? ¼ negative control.
ofeach line.
marker,+ ¼ positivecontrol,
P. Torrigiani et al.564

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both the vegetative and flowering stages. In
both vegetative (average number of internodes
12.772.1) and flowering plants (average number of
internodes 31.074.6), there were no significant
differences between WT or pBin19 controls and
antisense individuals in terms of average inter-
node number, or mature leaf length and width
(Table 3).
Rhizogenic response from leaf explants
The rhizogenic response of cultured leaf explants
from line V in vivo-grown plants C, E, F and G,
exhibiting depressed SAMDC activity, was evaluated
and compared with that of WT controls; empty
ARTICLE IN PRESS
Figure 3. SAMDC activity in 2 week-old in vitro micro-
propagated antisense SAMDC tobacco shoots of line IV
(A), line V (B) and line VI (C) compared with wild-type
(WT) and pBin19 (p19) controls. Data are the mean7SD
(n ¼ 6). Letters on the X-axis represent different
individuals. Asterisks indicate significant differences
from controls at po0.05 (*) or po0.01 (**).
Figure 4. Free polyamine levels in leaves from antisense
SAMDC in vitro micropropagated tobacco shoots of line IV
(A), line V (B) and line VI (C) compared with WT and
pBin19 (p19) controls. Pu, putrescine; Sd, spermidine.
Data are the mean7SD (n ¼ 6). Letters on the X-axis
represent different individuals. Asterisks indicate sig-
nificant differences from controls at po0.05 (*), po0.01
(**) and po0.001 (***).
Figure 5. SAMDC activity in leaves from in vivo-grown
WT tobacco plants and SAMDC antisense transgenic lines
IV and V. Data are the mean7SD (n ¼ 6). Asterisks
represent significant differences at po0.05. Letters on
the X-axis represent different individuals.
Expression of an antisense SAMDC in tobacco565

Page 8

vector-transformed controls were not checked
because their PA content was the same as in WT.
The time course of root formation showed that the
percentage of explants with macroscopic roots
increased with time (5–10% on day 9, 60% on day
12 and 100% on day 15), but without significant
differences between transgenic and WT genotypes
(Fig. 6A,B). At culture end, however, a difference
in rhizogenic response between control and anti-
sense plants occurred. In fact, the mean number of
roots per explant was 20% lower (po0.05) in
transgenic plants relative to controls (Fig. 6C) and
the mean root length (Fig. 6D) was 26% lower
(po0.001) in line V than in WT explants.
Response of leaf discs to MJ
Free, and soluble and insoluble-conjugated PA
titres were measured in leaf discs from WTand line
V plants treated or not with 10mM MJ. Again,
pBin19 controls were excluded from these experi-
ments because they did not display significant
differences in PA titres compared with WT. Free
PA composition showed a prevalence of putrescine
in WTand even more so in line Vuntreated explants
(putrescine-to-spermidine ratio 3.8 and 7.1, re-
spectively). Upon MJ treatment, free PA titres
decreased by about 50% in WT, but remained
unchanged in line V discs (Fig. 7A).
Upon treatment with MJ, soluble and insoluble
conjugated putrescine and spermidine accumula-
tion in transgenic explants was differentially
affected relative to WT ones (Fig. 7B,C). Thus,
soluble conjugated putrescine increased approxi-
mately three times in WTand 6 times in line V discs
(Fig. 7B). On the contrary, soluble conjugated
spermidine accumulated more in WT (5-fold) than
in transgenic (3.4-fold) explants. Equally, insoluble
ARTICLE IN PRESS
Table 2.
grown tobacco plants of WT, pBin19 and transgenic lines IV and V
Free, PCA-soluble and PCA-insoluble conjugated polyamine levels (nmolg?1FW) in leaves from in vivo-
FreeSoluble conjugatedInsoluble conjugated
Pu SdSm PuSd Sm PuSdSm
WT
pBin 19
Line IV
Line V
130721
172772
199792
120725
200743
210735
254735
207752
26712
19711
26711
1773
3717125
251794
350770
4127118
4267150
3517106
3617120
375764
2875
38715
22714
3178
104723
86716
110725
95712
63712
5578
5079
70713
3.170.8
4.070.5
5.371.2
3.570.7
Data are the mean7SD (n ¼ 6–10) for each line. Pu, putrescine; Sd, spermidine; Sm, spermine.
Table 3.
pBin19 and SAMDC antisense tobacco plants of lines IV and V. Data are the mean7SD (n ¼ 30–100) for each line
Flowering
Morphometric analyses (length and width of mature leaves) of in vivo-grown vegetative and flowering WT,
Vegetative
Leaf lengthLeaf widthLeaf lengthLeaf width
WT
pBin19
Line IV
Line V
17.3671.76
16.3371.73
19.5871.72
17.7972.40
11.6671.21
9.7370.77
12.1971.40
9.9271.61
26.3371.76
29.1773.22
32.2373.02
26.5673.04
14.7771.12
17.7572.38
17.5871.27
14.4171.78
Figure 6. Rhizogenesis from leaf explants of WT (A) and
line V (B) in vivo-grown vegetative plants cultured for
15d on a root-inducing medium containing 0.6mM IAA.
Mean number of roots per explant (C) and mean root
length (D) at culture end. Asterisks represent differences
at po0.001 (***) and po0.05 (*). Bar ¼ 1cm:
P. Torrigiani et al.566

Page 9

conjugated
spermidine 17.5-fold in MJ-treated WT discs, while
in line V ones putrescine increased 6.4-fold and
spermidine only 6.8-fold (Fig. 7C).
putrescineincreased4.4-foldand
Discussion
In the present paper, an antisense SAMDC cDNA
from D. stramonium was expressed in tobacco;
primary transformants (R0) and their transgenic
progeny (R1), belonging to three different lines,
were compared with WT and empty vector-trans-
formed (pBin19) controls in terms of PA metabo-
lism, morphogenic potential and response to the
stress-mimicking compound MJ.
In leaves of primary transformants, transcription
of the foreign gene had occurred but SAMDC
activity was unchanged relative to controls. Since
SAMDC is regulated at multiple levels, including
post-translational cleavage (Xiong et al. 1997;
Bennett et al. 2002; Hanfrey et al. 2002), it is
plausible that enzyme activity does not parallel
transcript accumulation; alternatively, the anti-
sense message may have not been correctly
translated (Lewin 2000). By contrast, in potato
primary transformants, constitutive overexpression
of an antisense SAMDC construct led to a 70–90%
reduction in SAMDC activity, and to a variable
decline in PA levels with a range of altered
phenotypes (Kumar et al. 1996). Similarly, under
the tuber-specific patatin promoter, some down-
regulation of SAMDC activity, and free and con-
jugated PA levels occurred but without phenotypic
changes in tubers (Rafart Pedros et al. 1999). In a
few cases, other PA biosynthetic genes, such as
ODC and ADC, were engineered in antisense
orientation. In tobacco transformed with an anti-
sense D. stramonium ODC cDNA, no changes in ODC
activity or PA levels were observed in R0 or R1
generations (Mayer and Michael 2003). In contrast,
previous studies with rice have shown that though
antisense ADC transgenic callus lines exhibited a
significant reduction of ADC (and ODC) activity, and
a decrease in putrescine and spermidine titres,
regenerated plants (R0) displayed no variations in
PA levels while the progeny (R1) did (Capell et al.
2000; Trung-Nghia et al. 2003). Also based on
results obtained with sense transformation (Bassie
et al. 2000), the authors underline the fact that,
besides promoter strength, the effects of transfor-
mation may depend upon developmental stage,
i.e., callus, regenerating tissue or shoots.
In accordance with this hypothesis, present data
show that, differently from R0, in second genera-
tion micropropagated transformants strong down-
regulation of SAMDC activity occurred in the
majority of individuals of each line. This, however,
did not correlate with depletion of free spermidine
levels (which in fact sometimes increased), and
led, instead, to the build-up of probably unutilised
putrescine, resulting in a strongly increased (up to
10) putrescine-to-spermidine ratio. Lack of corre-
lation between SAMDC activity and spermidine
levels is presumably due to the fact that spermidine
synthase, and not SAMDC, which only furnishes the
dcSAM substrate, is directly involved in spermidine
synthesis. In tobacco transformed with a truncated
SAMDC cDNA of Arabidopsis, exhibiting 7-fold up-
regulated SAMDC activity, spermidine levels were
unchanged despite dcSAM accumulation up to 400-
fold relative to controls (Hanfrey et al. 2002).
Although little is known about its regulation in
plants, spermidine synthase activity seems to
depend, at least in physiological conditions, upon
dcSAM availability, for which it has a higher affinity
than for putrescine (Yoon et al. 2000). However, in
tobacco overexpressing spermidine synthase, no
relationship was detected between SAMDC and
ARTICLE IN PRESS
Figure 7. Free (A), PCA-soluble conjugated (B) and PCA-
insoluble conjugated (C) polyamine content in leaf discs
of WTand line V tobacco plants incubated with 10mM MJ
for 48h. Pu, putrescine; Sd, spermidine, Sm, spermine.
Expression of an antisense SAMDC in tobacco 567

Page 10

spermidine synthase transcript levels or enzyme
activities suggesting independent regulatory me-
chanisms for the two genes (Franceschetti et al.
2004). On the other hand, in plants, SAMDC activity
is not putrescine-stimulated as in animal cells
(Xiong et al. 1997; Bennett et al. 2002); therefore,
putrescine accumulation may be compatible with
or may be the consequence of the observed
repression of SAMDC activity in micropropagated
shoots. Finally, low levels of SAMDC activity are
normally required in physiological conditions for
spermidine synthesis.
Lack of correlation between the extent of
enzyme activity and product accumulation has also
been reported for another PA biosynthetic enzyme;
in fact, overexpression of a D. stramonium sense
ODC cDNA in tobacco led to 25-fold increased
enzymeactivity butonly 2.5-fold putrescine
accumulation (Mayer and Michael 2003). The
authors suggest that PA homeostatic mechanisms
efficiently accommodate changes in enzyme activ-
ity since PA biosynthetic control is invested at
multiple independent steps. The fact that enzyme
activity represents single time-point measurements
of a rate of synthesis, while PA levels are the result
of several independent metabolic pathways should
also not be overlooked.
Although information is available on plants
expressing sense constructs (Noury et al. 2000;
Thu-Hang et al. 2002; Mayer and Michael 2003), as
regards antisense transformation information con-
cerning the R1 generation is limited, and practi-
cally absent with respect to micropropagated
plants. In vitro-cultured plants are exposed to a
variety of stresses, including osmotic, chemical and
wound stress. In the present work, substantial
SAMDC downregulation was observed only in micro-
propagated R1 shoots. This suggests that in vitro
culture-induced stress possibly induced the expres-
sion of the foreign gene, at least in terms of SAMDC
activity, which otherwise (R0 and in vivo-grown R1
plants) was partially or totally counteracted by
homeostatic adjustments. Paradoxically, this led to
spermidine accumulation rather than depletion.
One possible explanation for this may be that since
repression of SAMDC activity in such a juvenile
stage caused abundant putrescine accumulation,
the latter was metabolised to the higher PA. The
possible regulation of spermidine levels through an
altered biosynthesis or utilisation of spermine is
possible, but cannot be evaluated in micropropa-
gated tobacco shoots, because spermine is absent
or below detection levels.
In general, little is known about the metabolic
pathways upstream or downstream of the engi-
neered genes (Trung-Nghia et al. 2003; Mayer and
Michael 2003). The increase in putrescine titres
presently observed in transgenic micropropagated
shoots suggests that an upstream metabolic path-
way, i.e. that of DAO, may have been altered.
However, the differences observed in putrescine
oxidising activity remained within the range of
genotypic variability. Equally, despite the observed
reduction in SAMDC activity, ethylene production
was unaffected in micropropagated shoots, sug-
gesting that the two pathways are either not
competitive and/or that SAM was not limiting
under these conditions (Ravanel et al. 1998; Quan
et al. 2002).
Once the micropropagated shoots were allowed
to grow in vivo, the resulting transgenic plants
exhibited a strongly attenuated change in pattern;
in fact, although SAMDC activity was somewhat
decreased, no changes in free or conjugated PA
levels were observed. As far as the phenotype of
these plants is concerned, no changes were
observed. Genetic transformation, either sense or
antisense, involving PA biosynthetic genes, in most
cases, leads to no morphological changes, with a
few exceptions (Kumar et al. 1996; Rafart Pedros
et al. 1999; Masgrau et al. 1997), or unless a
truncated SAMDC construct is used (Hanfrey et al.
2002). By contrast, Arabidopsis mutants deficient
for ADC activity displayed an anomalous root
apparatus (Watson et al. 1998), while a mutant in
spermine synthase activity was found to be severely
affected in growth and cell elongation (Hanzawa
et al. 2000). These data suggest that altered plant
phenotypes may be better achieved by PA biosynth-
esis mutations.
Rhizogenesis is particularly sensitive to changes
in PA levels (Coue ´e et al. 2004). In root-forming
tobacco explants both free, and soluble and
insoluble conjugated PAs accumulate dramatically
(Torrigiani et al. 1989), and the perturbation of PA
biosynthesis interferes dramatically with organo-
genesis (Altamura et al. 1991). In a comparative
study on Solanum melongena explants induced to
form either adventitious shoots or roots, a differ-
ential response in terms of PA metabolism has been
reported (Scoccianti et al. 2000). In particular, root
formation, but not shoot formation, was associated
with a dramatic increase in soluble conjugated
spermidine, prior to and during meristemoid and
primordium formation. Furthermore, the conju-
gated-to-free spermidine, but not putrescine, ratio
was consistently higher in the organogenic borders
vs. the non-organogenic central zones of the
explants. Therefore, the diminished rhizogenic
response in transgenic line V explants, while not
correlating with the initial PA titre of leaf explants,
may be associated with their reduced capacity to
ARTICLE IN PRESS
P. Torrigiani et al.568

Page 11

accumulate conjugated spermidine under non-
physiological conditions, as documented here for
MJ-treated leaf discs. In fact, in root-forming
tobacco thin layers inhibition of SAMDC activity
led to a strong reduction of the rhizogenic
response, and of soluble and insoluble conjugated
spermidine levels (Altamura et al. 1991).
An emerging aspect of the manipulation of PA
biosynthesis is that increased putrescine or spermi-
dine levels are associated with increased stress
resistance (Roy and Wu 2001, 2002; Kumria and
Rajan 2002; Waie and Rajam 2003). It has been
previously shown that exposure to MJ of tobacco
leaf discs leads to a dramatic accumulation of
conjugated PAs (spermidine more than putrescine)
both in the PCA-soluble and -insoluble fraction, and
to increased SAMDC mRNA levels and activity
(Biondi et al. 2003). As observed in micropropa-
gated shoots, the free putrescine-to-spermidine
ratio was higher in SAMDC antisense leaf discs than
in WT ones, mainly due to enhanced putrescine
levels, this being possibly indicative of depressed
SAMDC activity. Moreover, under stress-mimicking
conditions (MJ treatment), the relative accumula-
tion of conjugated putrescine and spermidine in the
transgenic leaf discs was also different from WT
controls. Although in absolute terms antisense
explants exposed to MJ were able to accumulate
more conjugated PAs than WTones, the response to
the elicitor, quantified on the basis of fold increases
in soluble and insoluble conjugates in treated vs.
untreated explants, was differentially expressed in
the two genotypes, with putrescine more strongly
induced than spermidine in the transgenic discs.
This again led to an altered putrescine-to spermi-
dine ratio. Diversion of dcSAM from spermine to
spermidine biosynthesis in order to justify the
spermidine increase does not seem plausible in
leaf discs since spermine levels are one order of
magnitude lower than those of spermidine. Since
conjugates have a role, both physiological and
structural, in defence (Martin-Tanguy 1985; Keller
et al. 1996), their enhanced and differential
accumulation in transgenic leaf discs could be
relevant in stress responses. Possibly, an increase
in the putrescine-to-spermidine ratio renders the
plants more resistant to stress, though less prone to
growth and differentiation (depressed rhizogen-
esis). Besides the fact that the antisense mechan-
ism needs to be clarified, these results open new
perspectives for studies on response(s) to various
types of stress in transgenic plants with altered PA
metabolism.
In conclusion, the expression of an antisense
SAMDC in tobacco seems to be conditioned not only
by plant developmental stage, but also by imposed
culture conditions. In particular, a stress-inducing
event, such as in vitro culture or MJ treatment, is
needed to obtain a response in terms of changes in
PA levels.
Acknowledgements
This research was supported by funds from MIUR
(ex-60%) to PTand SB. The authors wish to thank Dr.
Anthony J. Michael (Institute of Food Research,
Norwich, UK) for stimulating discussions and critical
reading of the manuscript, Tullia Costa, Francesca
Paglierani and Elena Galassi for their excellent
contribution to the experimental work, and Gio-
vanni Bugamelli for technical assistance in growing
the plants.
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