Journal of Experimental Botany, Vol. 57, No. 15, pp.

4155–4169, 2006
doi:10.1093/jxb/erl193 Advance Access publication 22 November, 2006

RESEARCH PAPER

Cloning and characterization of Arabidopsis and Brassica

juncea flavin-containing amine oxidases

Tze Soo Lim, Thiruvetipuram Rajam Chitra, Ping Han, Eng Chong Pua and Hao Yu*
Department of Biological Sciences and Temasek Life Sciences Laboratory, National University of Singapore,

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Singapore 117543

Received 18 July 2006; Accepted 7 September 2006

Abstract of FAO activity on shoot regeneration was exerted

Polyamines (PAs) are low molecular weight metabolites
involved in various physiological and developmental
processes in eukaryotic and prokaryotic cells. The
cellular PA level is regulated in part by the action of
amine oxidases (AOs) including copper diamine oxi-
dases (DAOs) and flavoprotein polyamine oxidases
(PAOs). In this study, the isolation and characterization
of flavin amine oxidases (FAOs) from Brassica juncea
(BJFAO) and Arabidopsis (ATFAO1) are reported that
were clustered in the same group as polyamine
oxidases from maize (MPAO) and barley (BPAO1) and
monoamine oxidases from mammalian species.
ATFAO1 was temporally and spatially regulated in
Arabidopsis and showed distinct expression patterns
in response to different stress treatments. To investi-
gate the in vivo function of FAO, transgenic Arabidopsis
plants expressing sense, antisense, and double-
stranded BJFAO RNAs were generated and those with
altered activity of FAOs were selected for further
characterization. It was found that the shoot regenera-
tion response in transgenic plants was significantly
affected by the modulated PA levels corresponding to
FAO activities. Tissues that originated from transgenic
plants with down-regulated FAO activity were highly
regenerative, while those from transgenic plants with
upregulated FAO activity were poorly regenerative. The
shoot regeneration capacity in these transgenic plants
was related to the levels of individual PAs, suggesting
that FAO affects shoot regeneration by regulating
cellular PAs. Furthermore, it was found that the effect
downstream of the Enhancer of Shoot Regeneration
(ESR1) gene, which may function in a branch of the
cytokinin signalling pathway.
Key words: Arabidopsis thaliana, cytokinin, flavin-containing
amine oxidase, polyamine, shoot regeneration.
Introduction
Polyamines (PAs) are polycations of low molecular mass
that are found ubiquitously in all living organisms. They
play an important role in growth and development of
prokaryotes and eukaryotes. In plants, the most common
aliphatic PAs are putrescine (Put), the tri-amine spermidine
(Spd), and the tetra-amine spermine (Spm) (Galston and
Sawhney, 1990). Many other di- and polyamines are present
in plants and other organisms, such as 1,3-diaminopropane
and cadaverine (1,5-diaminopentane). Unusual PAs have
also been identified in bacteria, algae, fungi, animals, and
higher plants (Niitsu and Samejima, 1993).
Plant PAs often occur in free form as soluble PAs.
However, they can be conjugated to low molecular mass
molecules by the formation of an amide linkage or bound
to macromolecules such as proteins. Because of their
polycationic nature, PAs can interact with anionic macro-
molecules such as DNA, RNA, phospholipids, and certain
proteins (Tiburcio et al., 1997). Therefore, they can play
important roles in stabilization and protection of these
molecules to maintain cell homeostasis, growth, and
tumorigenesis (Wallace et al., 2003).

* To whom correspondence should be addressed. E-mail: dbsyuhao@nus.edu.sg or yuhao@tll.org.sg

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; ABA, abscisic acid; ACC, 1-aminocyclopropane-1-carboxylate; AO, amine oxidase; AVG, 2-
aminoethoxyvinyl glycine; BA, benzyladenine; CaMV, cauliflower mosaic virus; CIM, callus-inducing medium; Cu-AO, copper amine oxidase; DAO, diamine
oxidase; FAD-AO, flavin adenine dinucleotide amine oxidase; FAO, flavin-amine oxidase; GA, gibberellic acid; GABA, c-aminobutyric acid; H2O2, hydrogen
peroxide; MAO, monoaminase oxidase; MeJA, methyl jasmonic acid; PA, polyamine; PAO, polyamine oxidase; PEG, polyethylene glycol; Put, putrescine; SA,
salicylic acid; SIM, shoot-inducing medium; Spd, spermidine; Spm, spermine; UTR, untranslated region.

ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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4156 Lim et al.
In plants, PAs have been shown to play important roles in
a myriad range of physiological processes during growth
and development, including embryogenesis, dormancy
breaking of tubers and seed germination, stimulation and
development of flower buds, flower and leaf senescence,
fruit set, development and ripening, and in vitro organo-
genesis (Evans and Malmberg, 1989). Apart from plant
growth and development, PAs are also involved in the
response to environmental stimuli such as osmotic stress,
mineral deficiency, salinity (Bouchereau et al., 1999), and
pathogen infection (Walters, 2003). Plants usually respond
to stress by increasing their endogenous PA content,
accompanied by an increase in the activity of enzymes
involved in PA biosynthesis. It was therefore suggested that
PAs might play a role in plant survival under stress
(Bouchereau et al., 1999).
PAs are oxidatively deaminated by the action of amine
oxidases (AOs), which can be divided into copper-containing
AOs (Cu-AOs; EC 1.4.3.6) and FAD-dependent AOs
(FAD-AOs; EC 1.4.3.4). In plants, Cu-AO, also known as
DAO, catabolizes Put to produce 4-aminobutyraldehyde
concomitantly with hydrogen peroxide (H2O2) and ammo-
nia, while Spd and Spm are degraded primarily by FAD-AO,
also known as PAO. Together with H2O2 and diaminopro-
pane that is converted to b-alanine, 4-aminobutyraldehyde
and aminopropylpyrroline are produced from the oxidation
of Spd and Spm, respectively. 4-Aminobutyraldehyde
cyclizes spontaneously to yield D1-pyrroline that can be
further oxidized to form c-aminobutyric acid (GABA) by
the action of pyrroline dehydrogenase (Flores and Filner,
1985). GABA can be transaminated and oxidized to form
succinic acid, which is subsequently incorporated into the
Krebs cycle. This degradation pathway can thus ensure the
recycling of carbon and nitrogen from PAs.
It is important to underline that the enzymes involved in
PA catabolism and the products derived from their oxi-
dation have been implicated in some essential physiological
processes (Martin-Tanguy, 1997; Šebela et al., 2001). For
example, several lines of evidence indicate that H2O2,
a product of PA oxidation, can drive the cross-linking of
cell wall proteins and serve as a secondary messenger to
trigger the plant hypersensitive response (Levine et al.,
1994) as well as increase disease resistance (Kachroo et al.,
2003) and somatic embryogenesis (Luo et al., 2001). PAOs
are also involved in the production of uncommon PAs, such
as norspermidine, norspermine, cardopentamine, caldohex-
amine, homocaldopentamine, and monocaldohecamine,
that may be involved in co-ordinating the growth response
of various organisms under extreme environmental con-
ditions (Philips and Kuehn, 1991).
In recent years, major efforts have been dedicated to
perturbation of PA production in transgenic plants through
genes that overexpress or down-regulate enzymes involved
in the PA metabolic pathways (Bastola and Minocha, 1995;
Bhatnagar et al., 2001; Lepri et al., 2001). The majority of
these studies have focused on the PA biosynthetic pathways
and only a few on the catabolic pathways (Wisniewski and
Brewin, 2000; Rea et al., 2004). In the latter scenario,
PAOs have been isolated mostly from monocotyledonous
species, especially cereals belonging to the Graminae (Šebela
et al., 2001), where maize PAO (MPAO) (Tavladoraki
et al., 1998) and barley PAOs (BPAO1 and BPAO2)
(Cervelli et al., 2001) are the most studied members of this
class. Hitherto, evidence for the occurrence of PAO in
dicotyledonous species has been reported only once from
Medicago sativa L. (alfalfa) (Bagga et al., 1991), where
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only the enzyme activity of PAO was examined.
In this study, the isolation and functional characterization
of putative PAOs from Brassica juncea (BJFAO) and
Arabidopsis (ATFAO1) are presented. The results show that
the ATFAO1 gene is temporally and spatially regulated and
is responsive to stress. Alteration of endogenous FAO
activity in Arabidopsis can change the levels of aliphatic
PAs and their relative ratios, resulting in different shoot
regeneration responses.
Materials and methods
Plant materials
Arabidopsis thaliana (ecotype Columbia) seeds were surface-
sterilized by sequential washes in 70% ethanol (v/v) for 1 min and
15% Clorox
(1% sodium hypochlorite) solution for 10 min, and
rinsed with sterile distilled water before being sown onto agar plates.
The MS agar medium contained Murashige and Skoog salts
(Murashige and Skoog, 1962), 3% sucrose, and 0.8% agar, pH 5.7.
The plates were incubated at 4 C for 2 d before being placed at room
temperature under white light for germination and growth. Two-
week-old seedlings were used for the experiments. For growth on
soil, seeds were sown on commercial potting compost and stratified at
4 C in a cold room for 4 d before being transferred to a growth
chamber under constant illumination at a light intensity of 50–100
mE m
2 s
1 and a temperature of 22 C. The humidity was maintained
at 60%. Three-week-old plants were used for the experiments. FAO
transgenic lines were grown on selection media consisting of MS agar
medium and kanamycin (50 mg l
1), while FAO transgenic lines
crossed with pER8-ESR1 lines were grown on selection media
consisting of MS agar medium, kanamycin (50 mg l
1), and
hygromycin (30 mg l
1) that select for S-BJFAO2 and AS-BJFAO8,
and pER8:ESR1 constructs, respectively.
Genomic DNA isolation and Southern analysis
Genomic DNA was isolated using the DNAzol ES kit according to
the manufacturer’s protocol (Molecular Research Centre, Inc.,
Cincinnati, OH, USA). For Southern analysis, 10 lg of genomic
DNA was digested with different restriction endonucleases and
separated on a 1% agarose gel by electrophoresis in 13 TRIS-borate-
EDTA buffer. The gel was treated with 0.25 N HCl for 10 min,
denatured twice in a solution of 0.5 N NaOH/1.5 M NaCl, each for
15 min with shaking, rinsed with sterile water, and transferred to the
neutralization solution [0.5 M TRIS–HCl (pH 7.5) and 3 M NaCl]
twice, each for 15 min with shaking. The DNA was transferred
overnight onto a positively charged membrane (Roche Diagnostics
GmbH, Mannheim, Germany) by capillary action in 103 SSC [3 M
NaCl and 0.3 M sodium citrate (pH 7.0)]. The membrane was cross-
linked, prehybridized at 42 C for 2 h in DIG Easy Hybridization

buffer (Roche Diagnostics GmbH), and hybridized overnight at 42 C
in the same buffer containing 25 ng ml
1 of a digoxigenein (DIG)-
labelled double-stranded DNA probe. The membrane was washed in
23 SSC/0.1% SDS twice, each for 5 min at room temperature,
followed by 0.53 SSC/0.1% SDS twice, each for 15 min at 68 C,
and 0.13 SSC/0.1% SDS for 15 min at 68 C. Detection of the
hybridized signal was carried out by capturing the chemiluminescent
signal on an X-ray film after CDP-Star (Roche Diagnostics GmbH)
was applied.
Stress and chemical treatments
Dehydration was induced by removing plants from the medium and
placing them on a dry filter paper, which was placed in a flow hood for
30 min. Osmotic stress was imposed by NaCl or polyethylene glycol
(PEG) (average molecular mass 6000 Da) treatment. Seedlings were
pulled out of agar and placed on filter papers soaked with different
concentrations of NaCl or PEG solutions. For abscisic acid (ABA), 1-
aminocyclopropane-1-carboxylate (ACC), silver nitrate (AgNO3),
2-aminoethoxyvinyl glycine (AVG), benzyladenine (BA), 2,4-
dichlorophenoxyacetic acid (2,4-D), gibberellic acid (GA3), H2O2,
methyl jasmonic acid (MeJA), Put, salicylic acid (SA), Spd, and Spm
treatment, different concentrations of the chemicals in appropriate
solvent solutions were sprayed on the seedlings. Seedlings were
immersed in water of different pHs for pH treatment. For temperature
treatment, 2-week-old seedlings in agar plates were incubated at 0 C
or 4 C in the dark, and 22, 28, 30, or 37 C under light, respectively.
For cold treatment, seedlings were thawed at room temperature for 2
h. Control treatments with seedlings soaked or sprayed with solvent
solutions or on agar plates under room temperature were also
conducted simultaneously. All treatments conducted on Petri dishes
were sealed and conducted under light at room temperature for 6 h
unless otherwise stated. For phenotypic analysis of stress treatments
of transgenic plants, 2-week-old wild-type and transgenic seedlings
on the same MS agar plates were subjected to optimum conditions or
different concentrations of stress and chemical treatments.
Shoot regeneration
Shoots were regenerated from Arabidopsis root culture as described by
Banno et al. (2001). Seven-day-old seedlings were transferred to B5
liquid medium and incubated for 15 d with shaking at 125 rpm. The
roots were cut into ~1 cm segments and six such segments were
incubated on callus-inducing medium (CIM) consisting of MS medium
(MS salts, Gamborg’s B5 vitamins, 1% sucrose, and 0.25% Phytagel as
gelling agent) supplemented with 2 lM 2,4-D. After 4 d on CIM, the
root segments were transferred to shoot-inducing medium (SIM)
comprising MS medium supplemented with 0.8 lM indole-3-acetic
acid (IAA) and 12.5 lM N6-D2-isopentyladenine. For shoot re-
generation of pER8-ESR1 and crossed lines, SIM was supplemented
with 10 lM 17-b-oestradiol for induction of ESR1 activity. For each
genotype, shoot regeneration experiments were repeated three times
with 48 root segments tested in each independent experiment.
RNA isolation and real-time PCR
Total RNA from plant tissues was isolated using the RNeasy Kit
(Qiagen, CA, USA) and reverse-transcribed by using the Thermo-
script real-time PCR System (Invitrogen Life Technologies,
Carlsbad, CA, USA). Real-time PCR assays were performed in
triplicate on a Bio-Rad iCycler iQ Real-Time Detection System (Bio-
Rad Laboratories Inc., Baltimore, MD, USA) using tubulin (TUB2) as
an internal standard. From the diluted cDNA, 1 ll (15 ng) was used as
template in a 20 ll PCR containing 13 SYBR Green PCR Master
Mix (Applied Biosystems, Foster City, CA, USA) and 0.25 lM
primer (each). The PCR thermal cycling parameters were 95 C for
3 min, 95 C for 20 s, followed by 55 cycles of 58 C for 20 s, 72 C for
Flavin-containing amine oxidases 4157
20 s, 95 C for 1 min, 55 C for 1 min, and 80 cycles of 55 C for 10 s.
The difference between the cycle threshold (Ct) of the target gene and
the Ct of tubulin, DCt¼Cttarget gene–Cttubulin, was used to obtain the
normalized expression of target genes, which corresponds to 2–DCt.
Each experiment was replicated at least three times using samples
collected separately. Primers designed for real-time PCR were as fol-
lows: SBJPRT1 (5#-TTCACCGTACCATACGCA-3#) and
SBJPRT2 (5#-GGCAGTGTAAAGCTTGTTCG-3#) for BJFAO,
ATP1RT1 (5#-CAACACCTTCGAGCTCTCAGT-3#) and
ATP1RT2 (5#-AACAGGGCTTCCACCGATTCC-3#) for ATFAO1,
and TUB2-P1 (5#-ATCCGTGAAGAGTACCCAGAT-3#) and
TUB2-P2 (5#-AAGAACCATGCACTCATCAGC-3#) for TUB2.
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Sequence analysis
The nucleotide sequence of cDNA was determined using the ABI
PRISM Big Dye and dRhodamine Terminator Cycle Sequencing
Ready Reaction Kit (Perkin-Elmer, Boston, MA, USA). Sequence
data were compared with the relevant sequences retrieved from the
National Center for Biotechnology Information (www.ncbi.nlm.nih.
gov) database. Phylogenetic analyses were carried out using the
program PAUP version 4.0b4 (Swofford, 2000) after alignment using
ClustalW (Thompson et al., 1994).
Plasmid construction
The FAO fragments that were amplified from mustard and the
Arabidopsis cDNA library were cloned into pGEM
-T Easy Vector
(Promega, Madison, WI, USA) prior to preparation of sense, anti-
sense, and double-stranded cDNA fragments. The sense BJFAO cDNA
of 1764 bp was amplified using Sense Fwd PAO (5#-GCTCTA-
GAGCTTCACCGTACCATACG-3#; XbaI site underlined) and SP6
(5#-TATTTAGGTGACACTATAG-3#) primers, respectively. After
digestion, the XbaI–SacI fragment was inserted in a sense orientation
between the cauliflower mosaic virus (CaMV) 35S promoter and the
nopaline synthase (NOS) terminator in the pBI121 vector pre-
digested with the same enzymes. The sense ATFAO cDNA of 1772
bp was amplified using ATSF (5#-CCCTTGGGAACACCGA-
CAAGGAGATTCC-3#; HindIII site underlined) and ATSR (5#-
TCCCCCGGGGGAGCTTCTCTAAACACATTACCC-3#; SmaI
site underlined). After digestion with HindIII and SmaI, the cDNA
was inserted in a sense orientation between two CaMV 35S pro-
moters and the CaMV terminator in pGreen 0229 IT vector pre-
digested with the same enzymes. For the antisense BJFAO plasmid,
the 967 bp cDNA was amplified by PCR using AS Fwd PAO (5#-
GCTCTAGAGCGGAGGACTAAACAAC-3#; XbaI site underlined)
and AS Rev PAO (5#-CGAGCTCGTTCACCGTACCATACG-3#;
SacI underlined). The resulting XbaI–SacI fragment was ligated into
the pBII121 vector predigested with the same enzymes. The double-
stranded construct of BJFAO was produced as described by Chuang
and Meyerowitz (2000). Generally, the construct consisted of one b-
glucuronidase (GUS) segment (nucleotides 787–1809) flanked by two
BJFAO fragments with the same sequence but in reverse directions.
The BJFAO fragment used for the double-stranded construct was 610
bp (
1 to 610 bp) in length.
The sense, antisense, and double-stranded constructs were desig-
nated as S-BJFAO, AS-BJFAO, and DS-BJFAO, respectively. All
constructs were introduced into Agrobacterium tumefaciens strain
(LBA4404) and used to transform wild-type (Col-0) Arabidopsis
thaliana by the floral dip method as described previously (Clough
and Bent, 1998).
Enzyme activity
The level of PAO activity in plant tissues was determined by
following the formation of a pink adduct (e515¼2.63104 m
1 cm
1),
resulting from the oxidation and condensation of 0.1 mM
4158 Lim et al.
4-aminoantipyrine (4-AAP) and of 1 mM 3,5-dichloro-2-hydroxy-
benzenzenesulphonic acid (DCHBS) catalysed by 0.08 mg ml
1 of
horseradish peroxidase in 0.2 M sodium phosphate buffer, pH 6.5, at
25 C (Smith and Barker, 1988). Plant materials were homogenized
in 0.2 M sodium phosphate buffer [tissue/buffer ratio 1:5 (w/v)]. The
homogenate was centrifuged at 12 000 g for 20 min. A 20 ll aliquot
of the supernatant was used for measurement of protein concentration
by Bradford assay (Bradford, 1976) and 40 ll was used for enzyme
assay. The reaction mixture comprised horseradish peroxidase (1 mg
ml
1), sodium phosphate buffer (pH 6.5, 0.2 M), 4-AAP (0.1 mM),
and DCHBS (1 mM) as well as 2 mM Spm as substrate. Enzyme
activity was expressed as U g
1FW.
Polyamine measurement
The levels of PAs, i.e. Put, Spd, and Spm, in cultured Arabidopsis
explants during shoot regeneration were determined based on the
method described previously (Flores and Galston, 1982). Tissue (200
mg) was extracted by homogenization with 1 ml of 5% (v/v) cold
perchloric acid. The extract was incubated on ice for 1 h and
centrifuged at 14 000 rpm. for 20 min at 4 C. Free PAs were
dansylated overnight by mixing 200 ll of supernatant with 400 ll of
dansyl chloride (5 mg ml
1 acetone) and 200 ll of saturated sodium
carbonate. After overnight incubation, 100 ll of proline (100
mg ml
1) was added and the mixture was allowed to stand for 30
min at room temperature to remove excess dansyl reagent. This was
followed by the addition of 0.5 ml of benzene to the mixture and
vortexing for 60 s, after which the organic phase was retained in
a glass vial and stored at
20 C until use. The dansylated PAs were
separated on silica gel thin-layer chromatography plates (Whatman
LK6D, Middlesex, UK) and developed for 70 min in chloroform:
triethylamine (25:2, v/v). The plates were dried for 5 min at 100 C,
and the dansyl-PA bands were identified under UV light, scraped, and
eluted with 2 ml of ethyl acetate. PAs in the eluant were quantified
using a fluorescent spectrophotometer (Shimadzu RF-1501, Shim-
adzu Incorporation, Tokyo, Japan) with excitation at 350 nm and
emission at 495 nm. The standard curve for each PA was plotted by
quantifying known concentrations of Put dihydrochloride, Spd
trihydrochloride, and Spm tetrahydrochloride (Sigma).
Ethylene measurement
The level of ethylene produced by cultured explants at different
intervals was measured as described previously (Chi et al., 1991). Six
root segments were cultured in a 50 ml Erlenmeyer flask with 20 ml
of CIM in the first 4 d and transferred to SIM on the fourth day. Prior
to ethylene measurement, the flasks were aerated for 1 h in the
laminar flow hood, sealed with airtight rubber stoppers, and allowed
to stand for an additional 2 h. A 1 ml gas sample was withdrawn from
each flask with a 1 ml hypodermic syringe. The ethylene content in
the gas sample was determined by gas chromatography (Chi et al.,
1991).
Results
Cloning and sequence analysis of BJFAO and ATFAO1
A partial 750 bp fragment homologous to Arabidopsis AO
was obtained from a specific cDNA pool that was created
from B. juncea leaf discs grown on shoot regeneration
medium for 5 d. Based on the above fragment, a 1747 bp
full-length cDNA was cloned using rapid amplification of
cDNA ends (accession no. AY188087). This cDNA con-
sisted of a 90 bp 5#-untranslated region, a 31 bp 3#-untranslated
region, and a 1626 bp open reading frame that encoded
a putative polypeptide of 542 amino acid residues with an
estimated mol. wt. of 59 390 Da and a theoretical pI of 5.32.
This mustard polypeptide (BJFAO) shared 81% sequence
identity with an Arabidopsis AO family protein designated
ATFAO1 (At4g29720). Conserved domain analysis re-
vealed that both FAOs possessed a flavin-containing amine
oxidoreductase, which is a fingerprint motif in the family
consisting of various AOs, including MPAO and other flavin-
containing monoamine oxidases (MAOs) (Fraaije et al.,
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2000). ATFAO1 and BJFAO shared 21% and 17% amino
acid identity with MPAO (Tavladoraki et al., 1998), and
20% and 18% with BPAO1 (Cervelli et al., 2001),
respectively (Fig. 1).
Phylogenetic analyses demonstrated that BJFAO and
ATFAO1 were clustered closely with AOs from other species
(Fig. 2). In addition, they revealed the presence of at least
eight other Arabidopsis AOs that were grouped together
with both ATFAO1 and BJFAO in the same cluster (Fig. 2).
These putative PAOs were clustered closely with MPAO
(ZM 064411; Tavladoraki et al., 1998), BPAO1 (HV1
AJ298131; Cervelli et al., 2001), and some animal MAOs,
which demonstrated AO activities in previous studies (Hsu
et al., 1988; Powell et al., 1989; Grimsby et al., 1991;
Hashizume et al., 2003; Chen et al., 2004; Okauchi et al.,
2004; Setini et al., 2005). Although MAO and PAO differed
in their specificity towards substrates, they could be grouped
in the same structural and functional class of flavin-
dependent oxidases (Tavladoraki et al., 1998).
DNA gel blot analyses of mustard and Arabidopsis
genomic DNA were performed to determine the relative
copy number of FAO genes (Fig. 3A). The full-length
BJFAO probe was hybridized to several distinct bands
(Fig. 3A), suggesting that BJFAO is encoded by a small gene
family, while the full-length ATFAO1 probe was hybridized
to single distinct bands, suggesting that ATFAO1 is en-
coded by a single gene (Fig. 3A). In particular, BJFAO
and ATFAO1 shared low homology (19–25% amino acid
identity) with eight Arabidopsis FAOs that were clustered
together in the same subgroup.
Spatial and temporal FAO expression in Arabidopsis
organs and in response to treatments
To investigate whether FAO transcripts were spatially
regulated, expression of ATFAO1 was examined by real-
time PCR from total RNA isolated from various organs
of wild-type Arabidopsis plants. Transcripts were detected
in all organs, among which the expression levels varied
greatly (Fig. 3B). While the gene expression was highest in
cauline leaves and stems, it was lower in flower buds, roots,
rosette leaves, and siliques, and least abundant in flowers.
In addition, ATFAO1 expression was detected in plants
from 1 week old up to 7 weeks old (Fig. 3C). The gene
 Flavin-containing amine oxidases 4159

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Fig. 1. Amino acid sequence comparison between MPAO, BPAO1, BJFAO, and ATFAO1. Identical residues are marked with asterisks. Conserved and
semi-conserved substitutions are denoted by ‘:’ and ‘.’, respectively. The fingerprint motif for flavoprotein is underlined. The absence of a cysteine
residue (filled triangle) indicates a non-covalent interaction of the flavin residue.

expression was significantly up-regulated in the second
week when the plants were at the floral transitional stage.
ATFAO1 expression was also investigated in response to
external stimuli by subjecting Arabidopsis seedlings to various
treatments such as exogenous phytohormones (Fig. 4A),
temperature (Fig. 4B), osmotic stress (Fig. 4C), PAs (Fig. 4D),
ethylene inhibitors (Fig. 4E), SA, H2O2, pH, and dehydration
(Fig. 4F). After responsive treatments, the ATFAO1 transcripts
were up-regulated in response to cold temperature (Fig. 4B,
0 C and 4 C), NaCl (Fig. 4C, 50–400 mM), PEG (Fig. 4C,
10–40%), and SA (Fig. 4F, 0.1–1 mM). In contrast, the
transcripts were down-regulated in response to ABA (Fig. 4A,
50–400 lM), ACC (Fig. 4A, 10–500 lM), 2,4-D (Fig. 4A,
0.01–2.5 mg l
1), GA3 (Fig. 4A, 0.01–2.5 mg l
1), MeJA
(Fig. 4A, 10–500 lM), high temperature (Fig. 4B, 28–42 C),
Put (Fig. 4D, 1–50 mM), Spd (Fig. 4D, 1–50 mM), Spm (Fig.
4D, 1–50 mM), and AVG (Fig. 4E, 5–50 lM). Dehydration
(Fig. 4F) treatment did not significantly affect ATFAO1 ex-
pression. In some cases, the expression fluctuated upon treat-
ment with different concentrations of chemicals, such as BA
(Fig. 4A), AgNO3 (Fig. 4E), H2O2 (Fig. 4F, 10–100 mM), and
pH (Fig. 4F, 5.5–8).
Molecular characterization of transgenic
Arabidopsis plants
To investigate the in vivo role of FAO in plants, FAO
expression was modulated by expressing sense, antisense,
and double-stranded BJFAO cDNAs under the control of
the CaMV 35S promoter in transgenic Arabidopsis plants
4160 Lim et al.

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Fig. 2. Phylogenetic analysis of AOs from different species. Arrows indicate the FAO sequences discussed in this study. Asterisks indicate maize PAO
(ZM 064411) and barley PAO1 (HV1 AJ298131), which are the two most studied members of plant PAOs. Numbers represent bootstrap values (>50 in
100 replicates). GenBank identifiers follow each entry. For non-plant species, the genus initial followed by the species name is given. For plant
sequences, hosts can be identified by the following abbreviations: AT, Arabidopsis; BJ, Brassica juncea; ZM, Zea mays; HA, Helianthus annuus; HV1,
Hordeum vulgare; OS, Oryza sativa; HV2, Hydrilla verticillata; NP, Narcissus pseudonarcissus; ST, Solanum tuberosum; NT, Nicotiana tabacum; PS,
Pisum sativum; LC, Lens culinaris; EC, Euphorbia characias; CA, Cicer arietinum.

Fig. 3. Southern and expression analyses of FAO. (A) Southern analyses
of the mustard and Arabidopsis genome using BJFAO and ATFAO1
probes, respectively. Genomic DNAs (10 lg) isolated from mustard leaf
tissue were digested with HaeIII (Ha), HindIII (H), and PvuII (Pv), and
hybridized with a 1696 bp BJFAO probe. Genomic DNAs (10 lg)
isolated from Arabidopsis whole tissue were digested with EcoRI (E),
EcoRV (Ev), SacI (S), and XbaI (X), and hybridized with a 1718 bp
ATFAO1 probe. (B) Expression of ATFAO1 in different Arabidopsis
organs. Cauline leaves (CL), flowers (FL), flower buds (FB), roots (R),
rosette leaves (RL), siliques (SI), and stems (ST). (C) Expression of
ATFAO1 in Arabidopsis seedlings from 1–7-week-old grown on soil.
Transcript levels were determined by real-time PCR and are shown
relative to expression of TUB2 in each sample. Values are the mean 6SE
from three replicates.
(Fig. 5A). The BJFAO cDNA used for antisense and
double-stranded constructs shared 85% and 84% identity,
respectively, with the corresponding sequences of Arabi-
dopsis ATFAO1. The copy number of transgenes was de-
termined in these transgenic plants by investigation of
genetic segregation in their progeny and Southern blot
analysis (see supplementary Fig. 1 at JXB online). The T3
homozygous lines were isolated for transformants with a
single insertion, while the T1 generation line was main-
tained by tissue culture for transformants with multiple
Flavin-containing amine oxidases 4161
insertions, because isolation of their homozyous progeny
was difficult due to complicated genetic segregation. In
addition, an Arabidopsis line containing a putative T-DNA
insert in At4g29720 (SALK_009671, designated atfao1)
was obtained from the SALK Institute’s SIGnAL collection
(Alonso et al., 2003; Fig. 5B), and was confirmed by PCR
analysis (data not shown).
Real-time PCR analysis revealed that the BJFAO tran-
scripts were highly accumulated in two sense transformants
(Fig. 5C; and supplementary Fig. S1 at JXB online),
while the endogenous ATFAO1 expression was not affected
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in these sense transformants (data not shown). The
ATFAO1 transcripts were reduced to various degrees in
all antisense (Fig. 5C, AS1–AS12) and double-stranded
transformants (Fig. 5C, DS2–DS4), and atfao1 mutants
(Fig. 5C, Salk). The mRNA expression of the other nine
Arabidopsis FAOs that were closely clustered with BJFAO
and ATFAO1 was also checked, and it was found that
their expression was not significantly affected in these
sense, antisense, and double-stranded transformants (data
not shown).
Enzyme activity assays of selected transgenic plants were
performed for every construct (Fig. 5D). It was observed
that most of the transgenic plants showed enzyme activity
relevant to the expression of the FAO mRNAs. However,
the enzyme activities of two antisense lines (AS4 and
AS12) and the atfao1 Salk line were higher than those in
wild-type plants (Fig. 5D). In view of the fact that the ex-
pression of the other nine Arabidopsis FAOs was not sig-
nificantly affected, it was deduced that ATFAO1 activity in
these lines was either post-transcriptionally regulated or
regulated via some unknown feedback mechanisms.
FAO activity does not affect plant development and
stress tolerance
Transgenic plants with different levels of FAO activity grew
normally and were phenotypically indistinguishable from
wild-type plants throughout plant growth and development.
Since plants usually respond to stress by increasing en-
dogenous PA content, an investigation was carried out
to determine whether the changes in PA content affected
the stress response of transgenic plants with modulated PA
levels (Bouchereau et al., 1999). These transgenic plants
were treated under different external stimuli, and transgenic
seedlings subjected to responsive stress treatments displayed
similar phenotypes to wild-type plants (data not shown).
Effect of FAO activity on shoot regeneration in
relation to ESR1, polyamines, and ethylene
To investigate whether shoot regeneration from cultured
explants was affected by the altered endogenous levels of
FAO activity, the regeneration capacity of root explants
excised from wild-type and transgenic plants was com-
pared. An obvious difference in shoot regeneration between
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4162 Lim et al.

wild-type and transgenic plants was observed after 20 d in
culture (Fig. 6A). Transgenic lines with down-regulated
FAO activity (AS-BJFAO6, AS-BJFAO8, DS-BJFAO2, DS-
BJFAO3, and DS-BJFAO4) were more regenerative (79–
89%) in culture compared with wild-type (65%) and pBI121
control explants (59%), while the transgenic lines with up-
regulated FAO activity (S-BJFAO2 and S-BJFAO5) were
poorly regenerative, with only 24% and 34% explants form-
ing shoots (Fig. 6A). In another independent experiment,
Arabidopsis transgenic lines overexpressing the sense
ATFAO1 cDNA were also generated (see supplementary
Fig. S2 at JXB online). Two transgenic lines, named S-
ATFAO1 and S-ATFAO4, respectively, were selected for fur-
ther shoot regeneration analyses. They exhibited comparable
low capacity of shoot regeneration with S-BJFAO2 and S-
BJFAO5 (see supplementary Fig. 3 at JXB online).
There is increasing evidence showing that shoot regenera-
tion is genetically regulated. One typical example is the in-
volvement of an ESR1 gene, which encodes a putative
transcription factor with an AP2/EREBP domain (Banno
et al., 2001). Expression of ESR1 could be up-regulated by
exogenous cytokinin. Overexpression of ESR1 in transgenic
plants conferred cytokinin-independent shoot formation from
root explants and increased shoot regeneration efficiency in
the presence of cytokinin (Banno et al., 2001). To elucidate
further the molecular basis of FAO function in shoot regen-
eration, two representative T3 homozygous transgenic lines,
S-BJFAO2 and AS-BJFAO8, that had a single insertion of a
transgene, were chosen for further crossing with an oestrogen
receptor-based inducible ESR1 overexpression line, desig-
nated pER8-ESR1 (Zuo et al., 2000; Banno et al., 2001). The
crossed plants were grown on selection media that selected
for S-BJFAO2, AS-BJFAO8, as well as the pER8:ESR1 con-
struct, and F3 homozygous hybrid plants were used for shoot
regeneration studies. The induction of ESR1 in the crossed
plants was also confirmed as previously published (Banno
et al., 2001). In accordance with the previous study (Banno
et al., 2001), induced pER8-ESR1 explants (80%) were
more regenerative than non-induced pER8-ESR1 explants
(63%) (Fig. 6B). Interestingly, when induced, pER8-ESR1
AS-BJFAO8 (83%) showed a similar regeneration rate to AS-
BJFAO8 (85%) and pER8-ESR1 (80%), whereas pER8-
ESR1 S-BJFAO2 (23%) exhibited s similar regeneration rate
to S-BJFAO2 (26%). Thus, the effect of FAO activity on
shoot regeneration was clearly downstream of ESR1 activity.
The altered FAO activity may be associated with dif-
ferent concentrations of PAs, which is a possible factor
causing the change of shoot regeneration responses in
generated transgenic plants, as shown in some previous
Fig. 4. ATFAO1 expression in Arabidopsis seedlings in response to various treatments. Seedlings were treated with various solutions for 6 h or left on
plates at room temperature (RT) without treatment. (A) Phytohormones, (B) temperature, (C) osmotic stress, (D) polyamines, (E) ethylene inhibitors, (F)
SA, H2O2, pH, and dehydration. The control for dehydration (Ctrl) was seedlings placed on filter papers in a covered Petri dish in the flow hood. To check
for the responsive treatments, different concentrations of the chemicals were used (except for pH and dehydration). Transcript levels were determined by
real-time PCR and are shown relative to expression of TUB2 in each sample. Values are the mean 6SE from three replicates.
Flavin-containing amine oxidases 4163
studies (Monteiro et al., 2002; Hunter and Burritt, 2005).
To verify the above possibility, the content of PAs in
cultured tissues was analysed during shoot organogenesis.
In general, all transgenic lines possessed higher levels of
Put than the wild type during culture (Fig. 7A). The Put
content in wild-type explants was shown to increase up to 5
d and decreased thereafter during the remaining culture
period (Fig. 7A). Unlike wild-type plants, the Put content in
S-BJFAO2 and AS-BJFAO8 decreased upon transfer to
SIM (day 5) and subsequently increased up to 8 d and 10 d,
respectively (Fig. 7A). With respect to Spd, there was a
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marked difference between wild-type and transgenic
explants after 4 d of culture in that Spd levels in the wild
type surged markedly (Fig. 7A). Wild-type explants were
shown to possess a significantly higher level of Spd than all
transgenic lines on day 5 (Fig. 7A), while all the transgenic
lines exhibited similar Spd levels throughout the culture
period (Fig. 7A). Like Spd, the Spm levels in the wild type
also increased greatly after 4 d of culture compared with
those in transgenic lines (Fig. 7A). Moreover, AS-BJFAO8
contained higher levels of Spm than S-BJFAO2 throughout
the culture period (Fig. 7A). In general, the individual con-
centrations of endogenous PAs were significantly different
between wild-type and FAO transgenic explants after they
were transferred from CIM (day 4) to SIM (day 5).
Since it has been suggested that high ratios of Put/Spd
and Put/Spd+Spm may cause low morphogenic capacity
(Bajaj and Rajam, 1996; Hunter and Burritt, 2005), the
ratios of different PAs in wild-type and transgenic explants
were compared further (Fig. 7B). It was found that S-
BJFAO2 possessed the highest Put/Spd and Put/Spd+Spm
throughout the entire culture period. The common trend of
all these plants after transfer to SIM (day 5) is the decreased
ratio of Put/Spd and Put/Spd+Spm (Fig. 7B), suggesting
that low ratios of Put/Spd and Put/Spd+Spm are markers of
shoot induction.
As evidence from previous studies showed that shoot
regeneration was associated with ethylene (Pua and Chi,
1993; Pua and Lee, 1995), the effect of BJFAO expression
on ethylene production in cultured explants of wild-type
and transgenic plants was also examined in this study.
Surprisingly, the patterns of ethylene production in both
wild-type and transgenic plants were similar during 20 d of
culture (Fig. 7C).
Discussion
Amine oxidation is important in a number of basic biological
processes ranging from lysyl oxidation in the cross-linking
4164 Lim et al.

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Fig. 5. Generation and molecular characterization of transgenic plants. (A) Schematic representation of chimeric genes used for plant transformation.
The numbers in the constructs indicate the position of nucleotides relative to the putative transcriptional start site (+1). (B) Relative position of a T-DNA
insertion of atfao1 in the Arabidopsis genome. (C) Expression of FAO mRNAs in transgenic plants at the T1 generation. Rosette leaves (RL) of wild-type
plants were used as a control. Transcript levels were determined by real-time PCR and are shown relative to expression of TUB2 in each sample. Values
are the mean 6SE from three replicates. (D) Enzyme activity in crude extracts of 2-week-old whole seedlings of selected transgenic plants at the T3
generation. Values are the mean 6SE from three measurements. WT, wild-type Arabidopsis plants.

of collagen to the catabolism of neurotransmitters and PAs.
The oxidation of biogenic amines is catalysed by either the
quinoprotein class of enzymes (usually primary amines)
(Hartmann and McIntire, 1997) or the flavin-containing AOs
(primary, secondary, or tertiary amines) (Massey, 2000).
AOs represent a class of enzymes heterogeneous in struc-
ture, catalytic mechanism, and mode of substrate oxidation.
They are found widespread in bacteria, fungi, animals, and
plants, and are classified into Cu-AO and FAD-AO. PAOs
belong to the latter subclass and they occur in high specific
activity in the Leguminosae and Graminae. To date, only
one study of dicotyledenous PAO has been reported in
alfalfa (Bagga et al., 1991). Although several putative
dicotyledenous PAO sequences can be retrieved from the
 Flavin-containing amine oxidases 4165

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Fig. 6. Effects of FAO activity on shoot regeneration. (A) Shoot regeneration of root segments from untransformed wild-type and transgenic plants
(pBI121, S-BJFAO2, S-BJFAO5, AS-BJFAO6, AS-BJFAO8, DS-BJFAO2, DS-BJFAO3, and DS-BJFAO4). The pBI121 control line contained only the
pBI121 vector without an insert. (B) Shoot regeneration of root segments from transgenic plants (pER8-ESR1, pER8-ESR1 S-BJFAO2, and pER8-ESR1
AS-BJFAO8) without (mock) and with an inducer of ESR1 transcription (10 lM 17b-oestradiol). Mock-treated plants were supplied with
dimethylsulphoxide, which is the solvent for 17b-oestradiol. In (A) and (B), representative plates taken after 20 d in culture are shown on the top, and
statistical analyses of shoot regeneration are shown below. Explants were evaluated in terms of percentage shoot regeneration, which was calculated
based on the number of explants forming shoots as a percentage of the total number of explants. Data were converted to Arcsin-transformed values before
being analysed statistically. Vertical bars represent the mean 6SE of three replicates.

Arabidopsis database, their concrete functions are un- MPAO is expressed constitutively in all organs tested,
known. In this study, ATFAO1 and Brassica BJFAO were with higher levels of expression in leaves and coleoptiles
characterized, both of which show close sequence similar- than in roots, mesocotyl outer tissue, and stele (Cervelli
ity to maize MPAO and barley BPAO1. et al., 2000). In barley, BPAO1 was expressed only in the
4166 Lim et al.

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Fig. 7. PA content and ethylene evolution in wild-type and transgenic plants. (A) Free PA content (Put, Spd, and Spm) in root explants of wild-type and
transgenic plants (S-BJFAO2 and AS-BJFAO8) during shoot regeneration. Vertical bars represent the mean 6SE of three measurements. (B) Ratios of
Put/Spd and Put/Spd+Spm. Ratios were calculated from the mean of Put, Spd, and Spm derived from (A). (C) Ethylene evolution in root explants of
Arabidopsis during 20 d of culture. All explants were prepared from 15-d-old root cultures and grown on CIM for 4 d, then transferred to SIM for the
specified period. Vertical bars represent the mean 6SE of three measurements.

ear while BPAO2 could be detected in all tissues examined
(Cervelli et al., 2001). In the present study, it was found that
ATFAO1 transcripts were detectable in all organs, with the
most abundant transcripts in cauline leaves. The variable
expression of ATFAO1 observed in whole seedlings from
1–7-weeks-old indicates that ATFAO1 expression may
be temporally regulated. In particular, the significantly in-
creased expression of ATFAO1 in the second week after
seed germination implies the involvement of the PA
metabolic pathway in floral transition of Arabidopsis. This
is consistent with the previous suggestion that optimum
levels of endogenous PA may be required for flowering
(Applewhite et al., 2000).
PAs have been suggested to play a protective role
against stress in plants because PA biosynthesis enzymes
and cellular PAs usually increase in response to stress
(Bouchereau et al., 1999). The present study showed that
the ATFAO1 gene was up-regulated in response to cold
temperature, NaCl, PEG, and SA, while it was down-
regulated in response to ABA, ACC, 2,4-D, GA3, MeJA,
high temperature, Put, Spd, Spm, and AVG. The down-
regulation of ATFAO1 by synthetic auxin (2,4-D) is in
agreement with the finding that MPAO1 expression in outer
tissues of maize mesocotyl was reduced by auxin treatment
(Cona et al., 2003). Some of the above changes of ATFAO1
expression may reflect the direct response of ATFAO1 in
defence against stress. However, some stress treatments
may indirectly affect ATFAO1 expression by regulating the
substrates or catabolic products of PAOs, because the
inhibitory effects of Put, Spd, Spm, and H2O2 on ATFAO1
could be clearly observed (Fig. 4). In addition, ATFAO1
expression was variable under the treatment with different
levels of ethylene (AgNO3) and BA (Fig. 4), indicating that
ATFAO1 expression may involve complex feedback
regulatory mechanisms.
For the last 15 years, there has been increasing evidence,
derived mainly from physiological studies, showing that
PAs are implicated in plant morphogenesis in vitro. In-
creased regeneration has been correlated with endogenous
PA accumulation (Pua et al., 1999) or exogenous application

of PAs (Chi et al., 1994). Furthermore, the inhibition of PA
synthesis has been shown to prevent regeneration (Pua et al.,
1996). The regulatory role of PAs in plant morphogenesis in
vitro has also been supported by transgenic studies, in which
carrot cells expressing mouse ODC cDNA showed increased
Put biosynthesis and promoted somatic embryogenesis
(Bastola and Minocha, 1995). The similar promoting effect
of increased PA titres on the plant regeneration potential
has been reported in rice callus expressing an ODC cDNA
(Kumria and Rajam, 2002). These findings have shown an
essential role for PAs in somatic embryogenesis and im-
plicated the importance of regulating the cellular PA levels.
The results of this study have provided direct evidence
showing that FAO is an important enzyme in the PA cata-
bolic pathway, and the activity of FAO can affect the
levels of individual PAs, thus regulating shoot regenera-
tion. Overexpression of BJFAO markedly decreased the
shoot regeneration capacity of the cultured tissue, while
down-regulation of ATFAO1 expression promoted regen-
eration. Moreover, transgenic plants overexpressing the
Arabidopsis homologue ATFAO1 under the control of
the 35S promoter showed similar shoot regeneration results
to S-BJFAO2 plants. This demonstrated that the results
obtained by the heterologous system are comparable with
those obtained by the homologous system.
In the transgenic plants, the changes of the individual
concentrations of endogenous PAs were significantly differ-
ent from those of wild-type plants upon transfer from CIM to
SIM, indicating that the levels of PAs are closely related to
shoot regeneration capacity. In addition, the demonstration of
higher Put/Spd and Put/Spd+Spm ratios in S-BJFAO2
throughout the culture period is in line with previous find-
ings that cultured tissues with a higher Put/Spd ratio dis-
played a low morphogenic capacity (Bajaj and Rajam, 1996;
Hunter and Burritt, 2005). It is noteworthy that FAO may
affect shoot regeneration downstream of the ESR1 gene,
which has been suggested to function in a branch of the
cytokinin signalling pathway that directs shoot regeneration
(Banno et al., 2001). This is supported by the genetic cross-
ing data showing that the effect of FAO activity on shoot
regeneration capacity was independent of overexpression of
ESR1 (Fig. 6). This result, together with the observation that
BA could up-regulate ATFAO1 expression (Fig. 4A), sug-
gests that the cytokinin signalling pathway may be involved
in the regulation of PA levels in plants.
Enhancement of shoot regeneration and somatic embryo-
genesis can also be achieved by inhibition of ethylene
synthesis or action using ethylene inhibitors (Pua and Chi,
1993), or down-regulation of genes of ethylene biosynthetic
enzymes (Pua and Lee, 1995). Although the mechanism of
ethylene action in plant morphogenesis is not clear, it has
been suggested that enhanced shoot regeneration by ethy-
lene inhibition may be attributed to increased PA synthesis,
in view of the scenario that PAs and ethylene may compete
for the same precursor S-adenosylmethionine for their
Flavin-containing amine oxidases 4167
synthesis (Pua et al., 1996). In the present study,
modulation of FAO activity regulated shoot regeneration,
but did not affect ethylene levels, indicating that altered PA
levels rather than decreased ethylene levels are directly
responsible for shoot regeneration.
Although the evidence thus far indicates the role of PAs
in shoot morphogenesis, the mechanism of PA action is not
clear. The results of several studies in recent years indicated
the possible implication of the PA oxidative product, H2O2,
in somatic embryogenesis. This assumption is in accor-
dance with the results of several recent studies showing that
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stress induces the occurrence of several morphogenic events
in vitro, including shoot formation in flax (Mundhara and
Rashid, 2001), somatic embryogenesis of Astragalus
adsurgens (Luo et al., 2001), cotton (Kumria et al., 2003),
and Arabidopsis (Ikeda-Iwai et al., 2003), and androgenesis
and subsequent plant regeneration in rye (Immonen and
Anttila, 1999) and triticale (Immonen and Robinson, 2000).
On the other hand, it was shown that overexpression of
glutathione-S-transferase (GST, EC 2.5.1.18), an enzyme
associated with limiting oxidative damage, enhanced the
capability of shoot regeneration from Arabidopsis explants
(Gong et al., 2005). Similarly, activation of the reactive
oxygen species-scavenging mechanism resulted in increased
regeneration of plant protoplasts (Papadakis and Roubelakis-
Angelakis, 2002). Therefore, it seems that H2O2 can play
a bivalent role in shoot regeneration, and its exact relevance
to the effect of PAs needs to be further investigated.
Supplementary data
Supplementary data can be found at JXB online.
Acknowledgements
This work was supported by the National University of Singapore
(research grants R-154-000-209-112 and R-154-000-232-101) and
intramural research funds from Temasek Life Sciences Laboratory.
TSL and PH were supported by postgraduate scholarships from the
National University of Singapore. The authors would like to thank the
SALK Institute Genomic Analysis Laboratory and the Arabidopsis
Biological Research Centre for providing the T-DNA insertion line,
Dr Nam Hai Chua from the Rockefeller University for the XVE-ESR1
stock/pER8-ESR1 line, Dr Banno Hiroharu of Chubu University for
useful advice on pER8-ESR1 line, and Drs Toshiro Ito and Wenwei
Hu for critical reading of the manuscript.
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