Research

Differential gene expression of ARGININE

Blackwell Publishing, Ltd.

DECARBOXYLASE ADC1 and ADC2 in Arabidopsis

thaliana: characterization of transcriptional regulation
during seed germination and seedling development
Irène Hummel1, Gildas Bourdais1, Gwenola Gouesbet1, Ivan Couée1, Russell L. Malmberg2 and
Abdelhak El Amrani1
1
Centre National de la Recherche Scientifique, Université de Rennes 1, UMR 6553 ECOBIO, Campus de Beaulieu, bâtiment 14A, F-35042 Rennes Cedex,
France; 2Plant Biology Department, University of Georgia, Athens, GA 30602-7271, USA

Summary

Author for correspondence: • In plants, polyamines can generally be synthesized by the ornithine decarboxylase
Abdelhak El Amrani and arginine decarboxylase pathways. However, the model plant Arabidopsis
Tel: +33 223235124 thaliana appears to possess only the arginine decarboxylase pathway. As two
Fax: +33 223235026
Email: abdelhak.el-
paralogous ARGININE DECARBOXYLASE (ADC) genes are present in Arabidopsis,
amrani@Univ-rennes1.fr we investigated differential expression and potential differences of promoter activity
during seedling development and under specific stress conditions.
Received: 19 February 2004
Accepted: 16 April 2004 • Promoter activities were studied in stable homozygotic transformants harbouring
promoter::reporter gene fusions.
• Under temperate conditions, ADC2 promoter activity was strongly associated
with seed germination, root and leaf development, whereas ADC1 promoter activity
was low during vegetative development. Light, sucrose and ethylene were shown
to be important regulators of ADC2 promoter activity. By contrast, in roots
and leaves of plantlets subjected to chilling treatment the ADC1 paralogue
showed high promoter activity whereas ADC2 promoter activity was considerably
decreased.
• In situations of seed germination, root development and response to chilling, the
modifications of promoter activities were associated with changes in mRNA levels,
emphasizing the involvement of transcriptional regulation in ARGININE DECAR-
BOXYLASE gene expression.
Key words: Arabidopsis, arginine decarboxylase, chilling, development, transcrip-
tional regulation, stress.

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© New Phytologist (2004) doi: 10.1111/j.1469-8137.2004.01128.x

has been found to use both arginine and ornithine as substrate

Introduction
Polyamines, which are present in prokaryotic and eukaryotic
organisms (Chang et al., 2000), appear to be important for
growth and development of higher plants ( Watson et al.,
1998; Locke et al., 2000; Hanfrey et al., 2001). In animals and
fungi the main synthesis pathway of the diamine putrescine
occurs through an ornithine decarboxylase (ODC)-catalysed
reaction. However, a membrane-associated decarboxylase
in rat brain (Regunathan & Reis, 2000). In plants and some
bacteria, distinct ODC-catalysed and arginine decarboxylase
(ADC)-catalysed pathways have been thoroughly characterized.
After putrescine synthesis, aminopropyl groups are added
sequentially to form the triamine spermidine and the tetra-
amine spermine.
It is generally considered that, in higher plants, the activi-
ties of ODC and ADC undergo distinct regulation depending

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on developmental and physiological conditions. The ODC
activity is generally increased in actively dividing cells (Heimer
et al., 1979), and is localized primarily in the meristematic
zones (Schwartz et al., 1986). By contrast, ADC is active in
elongating cells, in embryonic cells, and in cells under various
stress conditions (Flores, 1991). Although nonenzymatic
decarboxylation of ornithine can occasionally be detected in
Arabidopsis thaliana, neither ODC enzymatic activity nor spe-
cific inhibition of the ODC assay has been clearly character-
ized (Hanfrey et al., 2001). Moreover, genome-wide analysis
in Arabidopsis did not identify any intact or degraded ODC
sequence, or any ODC expressed sequence tag. Arabidopsis is
therefore presently considered to be the only plant, and one of
the two eukaryotic organisms (the other being the protozoan
Trypanosoma cruzi), lacking ODC activity (Hanfrey et al.,
2001).
In this species the formation of putrescine would thus
result from ADC-catalysed pathways. Moreover, the corre-
sponding ADC gene appears to have been duplicated at the
origin of the Brassicaceae family, thus yielding two paralogues,
generally called ADC1 and ADC2 (Galloway et al., 1998). In
Arabidopsis, ADC1 and ADC2 are located on chromosomes II
and IV, respectively. The protein sequences derived from these
two genes show 80% homology. However, the enzyme activ-
ities of these proteins may differ somewhat, and analysis of
N-terminal sequences indicates that subcellular location
could be different (Hanfrey et al., 2001). Characterization of
specific roles for ADC and ODC in plants that possess ODC,
the absence of ODC in Arabidopsis, and the presence of two
distinct ADC in this species emphasize the potential speciali-
zation of ADC1 and ADC2 in Arabidopsis.
In order to study these potentially different roles, the pro-
moter activities of ADC1 and ADC2 were studied in stable
homozygotic transformants of Arabidopsis harbouring the
promoter of either ADC1 ( pADC1) or ADC2 ( pADC2 ) fused
to the GUS reporter gene. The present work reports the
importance of pADC2 activity during seed germination and
seedling development, and the importance of pADC1 activity
specifically in the response of chilling. Situations of contrasted
pADC activity were compared with changes in mRNA levels.
Seed germination, root development and response to chilling
are thus shown to be characterized by transcriptional activa-
tion of ADC genes.
Materials and Methods
Plant material and growth conditions
Arabidopsis seeds of Wassilewskija (WS) ecotype were grown
axenically on 1 × Murashige and Skoog medium (M5519,
Sigma), 0.8% (w/v) agar, in the absence or presence of sucrose
(3%, w/v). Growth was performed under temperate (17/
22°C night/day, 16 h light period) or chilling (5/10°C night/
day, 14 h light period) conditions. Growth of mature plants
New Phytologist (2004) 163: 519–531
was carried out in a growth chamber at 25°C under fluorescent
light (16 h light). In the case of salt treatment, plantlets were
grown on 1 × MS agar under temperate conditions, then
transferred for 4 or 24 h onto 1 × MS agar medium supple-
mented with 300 m NaCl. The first generation of stable
transformants carrying pADC1::GUS or pADC2::GUS con-
structs, as previously published (El Amrani et al., 2002), were
used to generate the homozygotic transgenic lines used in
the present study.
Selection of homozygotic transgenic lines
Transformation of Arabidopsis with pADC1::GUS or
pADC2::GUS constructs has been described previously
(El Amrani et al., 2002). The ADC1/ADC2 identity of
the promoter::GUS fusions was directly confirmed by
sequencing, as reported previously (El Amrani et al., 2002).
T1 seeds were harvested in bulk and sown on a kanamycin-
containing medium (100 µg l−1). Kanamycin-resistant
plants were planted on soil, and the T2 seeds were harvested
after 2 months from individual T1 plants. The number of
loci of integrated T-DNA copies was indicated by segregation
of the kanamycin-resistance phenotype in T2 progeny.
Transgenic lines showing a kanr : kans ratio of 3 : 1 were
considered to be single-locus for the T-DNA insertion.
Homozygotic lines of the T3 progeny were used for analysis of
promoter activity.
Genetic crosses
Flowers of eto3 and etr1 ethylene mutants were emasculated
with a fine forceps and immediately pollinated. For all the
crosses, pADC1::GUS or pADC2::GUS homozygotic lines
were used as male parents. Resulting F1 seeds were sown on
kanamycin (100 µg l−1)-containing medium. These F1 hetero-
zygous lines expressed both the pADC::GUS transformation
and the dominant etr1 and eto3 mutations.
Northern blot analysis
PCR was performed with the primers 5′-AATCGTGGAG-
AGTTTCGGGT-3′ and 5′-ACCACTCGGATCTGTAACTT-
3′ (ADC1 ) or 5′-CGGTGATGTTTTTATCCCGG-3′ and
5′-TTGCT TGATGAACCAT TGGA-3′ (ADC2) which
amplify, respectively, a 508 bp fragment from ADC1
(Genbank) U52851 (238–270 bp upstream and downstream
of the translation initiation codon, respectively) or a 1171 bp
fragment from ADC2 (Genbank) BT000682 (66–1237 bp
downstream of the initiation start codon). The resulting
fragments were isolated from an agarose gel and cloned into
the pGEMT vector (Promega). Distinct digoxigenin-labelled
probes were prepared by amplification of DNA fragments in
the presence of digoxigenin-11-dUTP (Roche Diagnostics,
Germany) as suggested by the manufacturer.
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Northern analysis was performed with Dig High Prime
DNA labelling and Detection Starter KitII (Roche Diag-
nostics). Total RNA from Arabidopsis plantlets was extracted
with a Total RNA Isolation System kit (Promega). RNA
(10 µg) was mixed with sample buffer containing ethidium
bromide, separated by electrophoresis through 1% (w/v)
agarose gel containing 2% formaldehyde and MOPS 1× and
capillary-blotted with 20 × saline sodium citrate (SSC) onto
Zeta-Probe GT genomic Tested Blotting membranes (Bio-
Rad). RNA was fixed to the membrane by UV.
Prehybridization (2 h) and hybridization (overnight) were
performed in hybridization buffer (DIG Easy Hyb, Boe-
hringer Mannheim) at 50°C. After hybridization, membranes
were washed for 5 min twice with 2 × SSC, 1% SDS at ambi-
ent temperature, and for 15 min twice with 0.1 × SSC, 1%
SDS at 50°C. The hybridized probes were immunodetected
with an alkaline phosphatase-conjugated antidigoxigenin
antibody and visualized with chemiluminescence substrate
(CSPD, Roche) following the supplier’s instructions (Roche
Diagnostics).
Bio-informatics and sequence information
Multiple-sequence alignments were constructed using
CW ver. 1.7 (Thompson et al., 1994). Analysis of
binding sites for transcription factors was carried out with
the PlantCARE database on plant cis-acting regulatory ele-
ments (Lescot et al., 2002) at http://intra.psb.ugent.be:8080/
PlantCARE/.
Histochemical analysis
Histochemical GUS staining was performed as described
previously by Jefferson et al. (1987). Plant tissues containing
pADC1::GUS or pADC2::GUS fusions were stained for GUS
activity at 37°C overnight, unless otherwise indicated in figure
legends. Tissues were washed with sodium phosphate buffer
(50 m, pH 7) then left overnight in 70% ethanol. No back-
ground staining was observed in any kanamycin-sensitive plants.
Results
Characterization of homozygotic promoter::GUS
transgenic lines
Stable transformants of Arabidopsis heterozygotic lines carrying
pADC1::GUS or pADC2::GUS constructs (El Amrani et al.,
2002) were used to generate homozygotic transgenic plants.
In the present work, only homozygotic lines from the T3
progeny were used for analysis of reporter gene expression as
described in Materials and Methods. At least 10 independent
transgenic lines were analysed for each construct. Despite
the expected variability caused by the position effect, all
pADC2::GUS transgenic homozygotic lines displayed
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Research 521
similar patterns of reporter GUS expression. By contrast,
pADC1::GUS transformation resulted in a significant (> 40%)
proportion of homozygotic kanamycin-resistant lines showing
no GUS staining in a wide range of growth conditions. The
pADC1::GUS lines showing maximum GUS staining were
chosen for subsequent experiments.
The pADC1::GUS and pADC2::GUS homozygotic trans-
genic lines showed contrasting patterns of reporter gene
expression in flowering plants which had grown under tem-
perate conditions. In pADC1::GUS lines, high levels of GUS
staining were found in stigmata and staminae and particularly
in pollen grains, whereas pADC2::GUS lines showed no sig-
nificant promoter activity in pollen grains. These pADC2::GUS
lines showed more generalized expression in floral organs and
strong GUS staining in the stigmata. Thus reporter gene
expression in floral organs from homozygotic lines (results not
shown) was similar to previous results on heterozygotic lines
(El Amrani et al., 2002).
Previous analysis of the 1500 bp proximal regions of ADC1
and ADC2 promoters had shown that they had a low level of
homology and possessed a complex array of putative cis-acting
regulatory elements (El Amrani et al., 2002). Moreover, one
specific feature of pADC1 was shown to be the presence of a
copy of a transposable element (El Amrani et al., 2002). Fur-
ther analysis was carried out using the PlantCARE database
(Lescot et al., 2002) to identify putative plant regulatory ele-
ments, especially those showing a contrasting pattern of pres-
ence/absence between pADC1 and pADC2. The transcription
initiation sites of ADC1 (Accession No. U52851) and ADC2
(Accession No. BT000682) were deduced from the latest
issues of cDNA sequences (October 2002, National Center
for Biotechnological Information), and are indicated in the
nucleotide sequences of the promoter regions (Fig. 1) by
position +1. The ATG start codons of ADC1 and ADC2 are,
respectively, 382 and 519 bp downstream from the transcrip-
tion initiation sites. The pollen-specific regulatory element
AAATGA (Weterings et al., 1995) was present six times in
both orientations in pADC1, and only once in pADC2
(Fig. 1). Multiple copies of cis-acting regulatory elements
(Argüello-Astorga & Herrera-Estrella, 1996) involved in light
responsiveness were present in both promoters (data not
shown). Whereas pADC2 presented five sucrose-responsive
elements (SURE), pADC1 contained only one SURE element
(Fig. 1). The ADC1 promoter, but not the ADC2 promoter,
contained two copies of cis-acting regulatory elements (LTR)
involved in the response to low temperature (Fig. 1). The
dehydration-responsive element (DRE) was found only in the
ADC1 promoter, and STRE, a stress-responsive element, was
found in both promoters (Fig. 1). Both ADC1 and ADC2
promoters contained ERE, an ethylene-responsive element
(Fig. 1). Thus a number of relevant cis-acting regulatory ele-
ments such as SURE, LTR and pollen-specific regulatory ele-
ments showed a contrasting pattern of distribution between
pADC1 and pADC2.
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Fig. 1 continued. Sequence comparison of ADC1 and ADC2 promoters. Alignment of ADC1 and ADC2 promoters is shown; gaps are inserted
into the sequences for optimal alignment. Transcription sites are indicated by +1; 5′ UTR regions are in italic. Initiation ATG codons indicated in
bold. Putative TATA boxes are boxed. The transposable element present in the ADC1 promoter (El Amrani et al., 2002) is highlighted against
a grey background, flanked by imperfect terminal inverted repeats (white characters against a grey background). The pollen-specific regulatory
element (Weterings et al., 1995) is highlighted against a black background. LTR, low-temperature responsive element (Dunn et al., 1998);
SURE, sucrose-responsive element (Ishiguro & Nakamura, 1994); STRE, stress-responsive element (Siderius & Mager, 1997); DRE, dehydration-
responsive element (Yamaguchi-Shinozaki & Shinozaki, 1994); ERE, ethylene-responsive element (Itzhaki et al., 1994).

of pADC2 promoter was therefore characteristic of the

Activities of pADC1 and pADC2 during seed
germination and early vegetative growth
Whereas pADC2, but not pADC1, activity was high in mature
siliques, mature and dry seeds showed no activity of pADC1
and pADC2 (Fig. 2a). However, during sensu stricto seed
germination from imbibition up to emergence of the rad-
icle (Bewley, 1997), GUS histochemical analysis showed a
contrasting pattern of activity between pADC1 and pADC2.
No GUS staining was observed in pADC1::GUS transgenic
lines (Fig. 2b). By contrast, pADC2::GUS transgenic lines
showed strong activity which started at an early stage of
imbibition with staining of testa, cotyledons and embryonic
axes (Fig. 2b). High activity of pADC2 was then detected in
the emerging radicle (Fig. 2b). The transcriptional activation
germination process.
Analysis of reporter GUS expression during seedling growth
and leaf emergence was carried out under conditions of optimal
growth under temperate conditions. To obtain optimal root
development (Jones & Grierson, 2003), plantlets were grown
in the presence of 3% sucrose (Fig. 3). Whereas in pADC1::GUS
transgenic lines light GUS staining was detected in cotyledons
and limbs of first leaves (Fig. 3a), pADC2::GUS transgenic
lines showed strong GUS staining in cotyledons and in both
petioles and limbs of first leaves (Fig. 3d).
No pADC1-driven GUS staining was detectable in any part
of the root system, in either the primary root or lateral roots
(Fig. 3b). By contrast, the ADC2 promoter was highly active
in roots (Fig. 3d), especially in the root apical meristem, with

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decreasing activity in subapical and differentiating regions of
the root, except in vascular tissues where GUS staining was
maintained at high level. Closer histochemical analysis during
root development showed that pADC2 promoter activity was
closely related not only to the apical meristem, but also to
lateral root primordium (LRP) formation ( Fig. 3e–j). Activity
of pADC2 was high in the pericycle cells of xylem poles during
the first anticlinal divisions that initiate LRP formation
(Fig. 3e). By contrast, pericycle cells at the opposite side of the
stele showed significantly lower pADC2 activity ( Fig. 3e). The
pADC2 activity remained high after the start of periclinal
divisions in the LRP, and during the eight different stages of
lateral root development described by Malamy & Benfey
(1997), until emergence from the parent root (Fig. 3e–j). After
emergence, reporter gene expression in pADC2::GUS trans-
genic lines was maintained in the axis of the secondary root,
especially in the apical zone, as occurred in the primary root.
Effects of light and sucrose on the activities of pADC1
and pADC2
Dark growth of seedlings in the absence of sucrose under
temperate conditions resulted in the absence of GUS staining
in roots and shoots of pADC1::GUS (Fig. 4e,f ) and
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Fig. 2 Histochemical localization of GUS
activity in transgenic lines harbouring ADC1
and ADC2 promoter::GUS fusions during
seed maturation and germination of
Arabidopsis. (a) GUS activity during seed
maturation, when seeds are still attached to
parent tissues. (b) GUS activity during sensu
stricto seed germination from imbibition up
to emergence of the radicle. Seeds were sown
on 1 × MS agar, incubated 48 h at 4°C to
break dormancy, then placed in a growth
chamber as described in Materials and
Methods, and stained after 0, 10, 24, 36 and
48 h germination.
pADC2::GUS (Fig. 4a,b) transgenic seedlings. These results
strongly contrasted with pADC2 activity in light-grown
seedlings in the absence (Fig. 4i) and presence (Fig. 3c) of
sucrose. However, sucrose treatment of dark-grown seed-
lings restored GUS staining in both roots and shoots of
pADC2::GUS transgenic lines (Fig. 4c,d), showing that
pADC2 was highly responsive to sucrose and indicating that
sucrose may also be a component of ADC2 induction by light.
By contrast, sucrose treatment did not modify GUS staining
in roots of dark-grown pADC1::GUS transgenic seedlings, but
gave a slight activation of pADC1 in cotyledons (Fig. 4g,h).
Effects of chilling on activities of pADC1 and pADC2
When germination and seedling growth were carried out at
low temperature (5°C night, 10°C day) the relative pattern
of pADC1 and pADC2 activities was greatly affected, with
pADC2 activity significantly decreasing in roots and leaves
(Fig. 4i–l), whereas pADC1 activity increased in leaves and
was induced in roots (Fig. 4m–p). The latter effect was
particularly striking. When chilling treatment was applied
to plantlets that had previously grown under temperate
conditions, strong GUS staining was observed in roots of
pADC1::GUS transgenic lines after 24 h treatment (data not
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Fig. 3 Histochemical localization of GUS activity in transgenic lines harbouring ADC1 and ADC2 promoter::GUS fusions during early stages of
seedling development and during lateral root development of Arabidopsis. Seedlings were grown in the presence of 3% sucrose. (a) 15-d-old
seedling; (b) close-up of the root apex of pADC1::GUS lines. (c) 15-d-old seedling; (d) close-up of the root apex of pADC2::GUS lines. (e–j)
GUS activity localization at different stages of lateral root primordium development as described by Malamy & Benfey (1997); (e) stage I, arrows
point to new cell walls indicating anticlinal division in the pericycle; (f) stage II, arrows point to new cell walls indicating a periclinal division which
divided the lateral root primordium (LRP) into two layers; (g) stage IV, initiation of formation of fourth layer of LRP; (h) stage V, cells of the LRP
undergo expansion; (i) stage VI, formation of elongated cells reminiscent of vascular elements; (j) emerging LRP and beginning of first divisions
of meristematic initials.

shown), whereas under temperate conditions these lines
presented no detectable GUS staining in roots. By contrast,
saline stress (300 m NaCl) for 4 h under conditions that
have been shown to induce gene responses (Knight et al., 1997)
and ADC expression (Gong et al., 2001) in the Col0 genetic
background did not modify ADC promoter activities, either
in pADC1::GUS (Fig. 3s,t) or in pADC2::GUS transgenic
lines (Fig. 4q,r). Longer salt treatment (24 h) did not result in
any increase of promoter activity (data not shown).
Activities of pADC1 and pADC2 in ethylene-response
mutant backgrounds
Given the importance of ethylene in germination and
seedling growth (Leon & Sheen, 2003), and the link between
ethylene and polyamine synthetic pathways (Locke et al., 2000),
transcriptional activity of ADC promoters was investigated
in ethylene-response mutant backgrounds (Fig. 5a–e) under
conditions of maximal pADC2 activity (Figs 3, 4). The
transgenic pADC::GUS lines were crossed with etr1, an
ethylene-insensitive mutant impaired in ethylene perception
and signal input (Schaller & Bleecker, 1995), or with eto3, a
mutant that overproduces ethylene in etiolated seedlings
(Woeste et al., 1999). The latter mutant shows a characteristic
ethylene-induced ‘triple-response’ phenotype in a constitutive
manner (Guzman & Ecker, 1990). The progeny were
heterozygous for the T-DNA and for eto3 and etr1 dominant
mutations, which allowed us to use the F1 generation for
ADC transcriptional expression. The transgenic pADC::GUS ×
eto3 lines exhibited the triple response when growth was
carried out in the dark. Low or no GUS staining was observed
during dark growth in the presence of sucrose in the
pADC1::GUS lines, and in the progeny obtained from
crosses between pADC1::GUS and both etr1 and eto3 mutants
(data not shown). By contrast, pADC2::GUS × etr1 lines
showed significantly decreased GUS activity under illuminated
(Fig. 5a,b) and dark (Fig. 5c,d) conditions, indicating that
perception and transduction of the ethylene signal may be
involved in transcriptional regulation of pADC2. No significant
variation of GUS expression was observed when pADC2::GUS
fusion was expressed in the eto3 mutant background (Fig. 5c,e).
Differential accumulation of ADC1 and ADC2 mRNA
ADC1 and ADC2 mRNA levels were investigated by
Northern blot analysis in situations of contrasted promoter
activities in the absence of sucrose. High ADC2 promoter
activity and undetectable ADC1 promoter activity during

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Fig. 4 Effects of environmental cues on histochemical localization of GUS activity in pADC1::GUS and pADC2::GUS transgenic lines of
Arabidopsis. (a–d) pADC2::GUS lines grown in dark condition without sucrose (a, cotyledons; b, root) or with sucrose (c, cotyledons; d, root).
(e–h) pADC1::GUS lines grown in dark conditions without sucrose (e, cotyledons; f, root) or with sucrose (g, cotyledons, h, root). (i–l)
pADC2::GUS lines grown in the absence of sucrose under temperate conditions (i, shoot; j, root apex) or at low temperature (k, shoot; l, root
apex). (m–p) pADC1::GUS lines grown in the absence of sucrose under temperate conditions (m, shoot; n, root apex) or at low temperature
(o, shoot; p, root apex). (q–t) pADC2::GUS lines (q, shoot; r, root apex) or pADC1::GUS lines (s, shoot; t, root apex) grown in the absence of
sucrose and in the presence of 300 m NaCl under temperate conditions.

germination (Fig. 2b) were associated with high levels of
ADC2 mRNA and very low levels of ADC1 mRNA in
imbibed seeds, as shown in Fig. 6(a). Both ADC1 and ADC2
mRNA were found in 15-d-old plantlets grown under
temperate conditions (Fig. 6b). This was consistent with the
detection of both ADC1 and ADC2 promoter activities in
leaves under temperate conditions (Fig. 4i,m). However, the
contrasting activities of ADC1 and ADC2 promoters (Fig. 4i,m)
did not result in highly contrasting levels of mRNA (Fig. 6b),
although repeated Northern analysis showed consistently
higher levels of ADC2 mRNA than of ADC1 mRNA (data
not shown). Northern blot analysis was also carried out
in plantlets grown at low temperature. By contrast with
temperate conditions, enhanced ADC1 promoter activity at
low temperature (Fig. 4o,p) was associated with a strong
increase of ADC1 mRNA levels. Conversely, ADC2 mRNA
levels decreased at low temperature (Fig. 6c) in accordance
with lower pADC2 activity (Fig. 4k,l).
Discussion
Differential activity of the promoters of ADC1
and ADC2
The existence of two ADC genes in Arabidopsis (Galloway
et al., 1998) is in agreement with the duplicated status of

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Fig. 5 ADC promoter activities in ethylene
mutant eto3 and etr1 genetic backgrounds.
The pADC2::GUS line, or progeny obtained
from crosses of pADC2::GUS line and eto3
mutant or etr1, are indicated. Growth was
carried out in the presence of 3% sucrose
under light or dark conditions as indicated. To
avoid excess coloration GUS staining was
arrested after 2 h of reaction.
Fig. 6 ADC1 and ADC2 transcript levels in Arabidopsis. Northern
blot analysis of ADC1 and ADC2 in (a) imbibed seeds (36–48 h);
(b) 15-d-old plantlets grown in the absence of sucrose under
temperate conditions; (c) at low temperature. 10 µg total RNA was
separated by agarose-gel electrophoresis and blotted onto a nylon
membrane. Hybridizations were done with specific DIG-labelled
probes against ADC1 or ADC2 genes. Ethidium bromide staining was
used to ensure equal loading in the gel.
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Research 527
its genome (Arabidopsis Genome Initiative, 2000). The
stabilization of genome organization has provided the
opportunity for functional divergence of the two paralogues.
Although the protein sequences of ADC1 and ADC2 show
a high degree of homology, the possibility of divergence in
terms of substrate specificity and enzymatic regulation cannot
be discounted. Biochemical analysis of mammalian ADC has
shown that it could use both ornithine and arginine, although
the specific ODC inhibitor, difluoromethylornithine, had no
effect on enzyme activity with either substrate ( Regunathan &
Reis, 2000). Previous studies have emphasized the hypothesis
that Arabidopsis ADC1 and ADC2 may be targeted to dif-
ferent subcellular localizations (Hanfrey et al., 2001). This
implies action on distinct metabolic pools and thus strongly
suggests important functional differences. In addition to
possible variation of protein and enzyme functions between
ADC1 and ADC2, important developmental and stress-
response modifications of mRNA expression between ADC1
and ADC2 in Arabidopsis have been reported recently (Soyka
& Heyer, 1999; Perez-Amador et al., 2002; Piotrowski et al.,
2003).
Many studies have insisted on the importance of post-
transcriptional and post-translational regulation of ADC
accumulation and activity (Malmberg & Cellino, 1994;
Borrell et al., 1996; Watson & Malmberg, 1996). Thus in
Arabidopsis plantlets stressed by potassium deficiency, ADC
mRNA did not correlate with increase of ADC enzyme activity
(Watson & Malmberg, 1996). However, in other situations,
such as response to acid and salt stresses, ADC mRNA levels
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were found to correlate with ADC activity (Perez-Amador
et al., 1995; Chattopadhyay et al., 1997). However, analysis
of ADC1 and ADC2 promoters showed striking differences,
such as various cis-acting regulatory elements, and the pres-
ence of a transposable element specifically in the promoter of
ADC1 (El Amrani et al., 2002). In the present study analysis
of homozygotic pADC::GUS transgenic lines confirmed that
in imbibed seeds, seedlings, roots, stems, leaves and flowers
under temperate conditions, pADC1 activity was significantly
more development- and tissue-limited than pADC2, which
had a more general pattern of activity. The more restricted
pattern of activity for pADC1 may be related to the presence
of the transposable element through disruption of cis-acting
regulatory elements, as has recently been shown for the Zea
mays a1 gene (Pooma et al., 2002), or through epigenetic regu-
lation. Introduction of the pADC1::GUS transgene resulted
in over 40% of silenced lines, whereas pADC2::GUS lines all
showed reporter gene activity. The ADC1 promoter is likely to
be more sensitive to epigenetic regulation as its sequence con-
tains a transposable element that is present in nearly 2000
copies in the Arabidopsis genome (El Amrani et al., 2002). In
plants and animals DNA methylation is involved in herit-
ability and flexibility of epigenetic states, and it has been shown
that transposable elements are the primary targets of genomic
DNA methylation (Okamoto & Hirochika, 2001). As inser-
tion of transposable elements may influence expression of
neighbouring genes via DNA methylation, epigenetic regu-
lation may be one of the mechanisms controlling ADC1
expression.
However, situations of highly contrasting promoter activity
were shown to correlate with corresponding variation of
mRNA levels. Thus Piotrowski et al. (2003) have reported
ADC2 mRNA to be at much higher levels than ADC1 mRNA
in roots. This is, at least partially, consistent with the differen-
tial activities of ADC1 and ADC2 promoters in roots of plants
grown under temperate conditions (Fig. 3b,d). By contrast, in
young leaves and rosette leaves both pADC1 and pADC2
showed significant activity (Fig. 3a,d), and both ADC1 and
ADC2 mRNA have been shown to be present (Perez-Amador
et al., 2002; Piotrowski et al., 2003). Detection of pADC1
and pADC2 activities in inflorescences was also associated
with the detection of ADC1 and ADC2 transcripts in flowers
and siliques (Soyka & Heyer, 1999). Thus these various
relationships between ADC1 and ADC2 promoter activities
and mRNA levels strongly suggested that both promoter
sequences were functional. Moreover, the reporter gene strategy
had previously shown that pADC1, but not pADC2, was
highly active in pollen grains (El Amrani et al., 2002). This
was partially correlated with the presence of the sequence
AAATGA, which has been described as capable of driving pollen-
specific expression independently of orientation ( Weterings
et al., 1995). This sequence was present six times in both ori-
entations in the ADC1 promoter, and only once in the ADC2
promoter. This provided strong evidence that evolution had
New Phytologist (2004) 163: 519–531
selected divergent cis-acting regulatory elements in the pro-
moter sequences of the two ADC paralogues, and that these
divergent elements were functional. Moreover, this strongly
indicated that the presence of the transposable element did
not interfere negatively with this pollen-specific expression,
and may even contribute to pollen-specific expression through
the presence of an additional AAATGA sequence (Fig. 1).
Finally, the striking differences of tissue-specific promoter
activity clearly showed that ADC genes were not transcribed
at a basic level in all tissues, with subsequent regulation at
post-transcriptional and post-translational levels, and correla-
tively suggested that transcriptional control may be important
for ADC gene expression.
High ADC2 gene expression during seed germination
and seedling growth under temperate conditions
Seed maturation is followed by embryo developmental arrest
during seed dehydration; on germination, embryo arrest
is lifted and cell division resumes (Raz et al., 2001). These
developmental stages are highly regulated and are of great
importance in the life cycle of monocarpic species such as
Arabidopsis. The ADC paralogues were found to show
contrasting promoter activities during these developmental
stages. None of these genes showed activity during seed
maturation and dehydration. During germination, which
starts with uptake of water by the quiescent dry seed and
terminates with emergence of the embryonic axis (Bewley,
1997), no ADC1 promoter activity was observed, whereas
ADC2 promoter activity was high as early as 10 h after seed
imbibition, and before primary root emergence (Fig. 2b).
This correlated with the detection of significant levels of
ADC2 mRNA in imbibed seeds (Fig. 6a). It has been shown
that germination of barley seed was promoted by addition of
exogenous polyamines (putrescine, spermidine and spermine),
and it is suggested that endogenous polyamines may play
a growth-promoting role complementary to ethylene in the
normal course of barley germination (Locke et al., 2000). At
the end of the germination process, ADC2 promoter activity
was high in the emerging radicle. Involvement of ADC in
germination and radicle extrusion is in agreement with the
general idea that ADC is active in elongating cells (Nam et al.,
1997). The specific reason why the ADC2 gene is upregulated
during this process remains to be elucidated.
Under temperate conditions, pADC2 was highly active in
seedling and plantlet roots, which is in accordance with the
higher level of ADC2 transcripts in roots (Piotrowski et al.,
2003). A high level of ADC transcripts in roots is also in gen-
eral agreement with high ADC activity in roots (Hanfrey
et al., 2001) and a high level of putrescine in roots (Watson
et al., 1998; Piotrowski et al., 2003). Moreover, the pattern of
pADC2::GUS activity reveals a tight relationship with rhizo-
genesis processes. Lateral root formation involves the stimula-
tion of pericycle cells to proliferate and create a new root
www.newphytologist.org © New Phytologist (2004)

meristem. Formation of the lateral root primordium may
be divided into different stages that can be characterized by
histology, cell division patterns, and gene expression (Malamy
& Benfey, 1997). Activation of pADC2 was found to be
induced at stage I of primordium development, before the
first periclinal division occurs. At later stages of development,
strong GUS staining was observed in the whole primordium.
Arabidopsis spe mutants, which are characterized by low
levels of ADC activity, are deeply affected in root develop-
ment, with enhanced lateral root formation in single mutants
and a compact root system in double mutants (Watson et al.,
1998). A number of previous studies have associated ADC
activity with root growth and root branching (Biondi et al.,
1993; Watson et al., 1998; Hummel et al., 2002). The present
study shows that this association is correlated with activation
of the ADC2 promoter in the early stages of lateral root
formation. However, to our knowledge no study has yet
addressed the mechanisms of polyamine action on root
development.
Activation of both ADC1 and ADC2 promoters was found
in the aerial parts of plantlets, with pADC2 showing much
higher activity than pADC1. ADC1 and ADC2 mRNA levels
have been reported to be equivalent in plantlet leaves
(Piotrowski et al., 2003). Similarly, ADC1 and ADC2 mRNA
levels in whole plantlets showed much less contrast (Fig. 6b)
than the differences in promoter activity (Fig. 4i–n). Thus
important post-transcriptional regulation probably acts on
ADC1 and ADC2 transcripts. Expression of ADC2 gene in
stems is in accordance with increased ADC mRNA levels and
increased ADC activity in hypocotyls of germinating soybean
(Nam et al., 1997). Expression of ADC1 and ADC2 genes in
leaves is in accordance with the physiological importance of
polyamines in leaves for photosynthetic activity (Chang et al.,
2000). At least one of the ADC proteins is likely to be targeted
to the chloroplast (Perez-Amador et al., 2002), and the oat
ADC has been shown to be localized in the chloroplast
(Borrell et al., 1995). Moreover, light was found to be a strong
inducer for ADC1 and ADC2 promoter activity, with no or
little promoter activity during dark growth (Fig. 4a–n). This
strong induction by light was consistent with the presence of
many putative light-responsive cis-acting regulatory elements
in the promoters of both ADC1 and ADC2. Similarly, the
promoter region of the carnation ADC gene is rich in light-
responsive, cis-acting regulatory elements, and ADC tran-
scripts increase tenfold after light exposure (Chang et al., 2000).
In the case of ADC2, promoter activity was also increased in
the dark in the presence of exogenous sucrose, especially in
roots (Fig. 4a–d). This was consistent with the presence of
numerous sucrose-responsive, cis-acting regulatory elements
in the promoter of ADC2. It is thus tempting to speculate that
this induction by sucrose, resulting in high pADC2 activity in
roots, may be part of shoot–root relationships during light
growth of plants through the transport of sucrose from shoot
to root.
© New Phytologist (2004) www.newphytologist.org
Research 529
The pattern of pADC2 activity during seedling develop-
ment was found to be affected in dominant ethylene-mutant
backgrounds, even in the presence of light and sucrose, which
normally resulted in high pADC2 activity (Fig. 3). Gallardo
et al. (2002) have recently highlighted the importance of
S-adenosylmethionine synthesis for seedling development of
Arabidopsis. This is consistent with an essential role of endo-
genous ethylene during seedling development, which has also
been described in other species (Petruzzelli et al., 2000). Thus
ADC2 would be part of an array of genes activated by ethylene
during seedling development.
High ADC1 gene expression in response to chilling
Chilling, which has been shown to increase ADC activity and
polyamine levels (Lee 1997; Shen et al., 2000; He et al., 2002)
had a strong effect on ADC1 and ADC2 promoter activity.
Strikingly, growth under chilling conditions thoroughly
modified the respective patterns of pADC1 and pADC2 act-
ivity, with pADC1 and pADC2 becoming, respectively, highly
active and poorly active in roots (Fig. 4i–p). This strong effect
appeared to be a specific response to chilling rather than a
general response to stress, as salt treatment did not change
the patterns of ADC1 and ADC2 promoter activity. The
chilling effect was correlated to changes in mRNA level, and
consistent with the specific presence of two copies of a low-
temperature response element in the promoter of ADC1 and
with the potential impact of the transposable element on
gene expression, as a copy of this low-temperature-response
element is part of the ADC1 transposable element (Fig. 1).
Moreover, changes in temperature may also affect epigenetic
control of ADC1 promoter activity. Further work should
therefore determine whether this ADC1/ADC2 functional
divergence derives directly from the presence of the trans-
posable element in the ADC1 promoter.
The functional role of ADC activity in response to chilling
is highlighted by previously reported correlations between
agmatine accumulation and frost resistance in wheat (Racz
et al., 1996), and by the involvement of polyamines and
increased ADC activity in chilling tolerance of cucumber
(Shen et al., 2000). In Arabidopsis, D-Arg inhibition of ADC
drastically reduced plantlet development at low temperature
and caused symptoms of chilling injury (data not shown).
Thus in Arabidopsis the polyamine response to chilling is
shown to correlate with transcriptional activation of the
ADC1 promoter. This switching effect of chilling on ADC1
and ADC2 relative expression therefore provides a good
experimental model for understanding the specific roles of
ADC1 and ADC2.
Acknowledgements
We thank the Nottingham Arabidopsis stock center for
providing the eto3 and etr1 ethylene mutants.
New Phytologist (2004) 163: 519–531
530 Research
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