overexpression of suadea salsa s-adenosylmethionine synthetase gene promotes salt tolerance in...
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ORIGINAL PAPER
Overexpression of suadea salsa S-adenosylmethionine synthetasegene promotes salt tolerance in transgenic tobacco
Yuan-Cheng Qi • Fei-Fei Wang • Hui Zhang •
Wei-Qun Liu
Received: 26 May 2009 / Revised: 2 September 2009 / Accepted: 15 September 2009 / Published online: 19 December 2009
� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2009
Abstract The suadea salsa full-length S-adenosylmethi-
onine synthetase (SsSAMS2) was introduced into tobacco
(Nicotiana tabacum L.) by Agrobacterium tumefaciens-
mediated transformation. The gene transformation and
expression in tobacco were confirmed by PCR, RT-PCR
and Northern blotting analysis. Several transgenic lines (ST
lines) overexpressing SsSAMS2 gene under the control of
cauliflower mosaic virus 35S promoter showed more seeds
number and weight, and accumulated higher free total
polyamines (PAs) than wild-type plants (WT lines) and
transformants with blank vector (BT lines). Salt stress-
induced damage was attenuated in these transgenic plants,
in the symptom of maintaining higher photosynthetic rate
and biomass. These results that the transgenic plants
overexpressing suadea salsa SAMS2 are more tolerant to
salt stress than wild-type plants suggest that PAs may play
an important role in contributing salt tolerance to plants.
Keywords Salt stress � Polyamines �S-adenosylmethionine synthetase � Transgenic tobacco
Abbreviations
PAs Polyamines
SAMS S-adenosylmethionine synthetase
WT Wild-type tobacco
ST Transgenic lines with with suadea salsa SAMS2
cDNA
BT Transgenic lines with blank vector
Introduction
The diamine putrescine (Put), triamine spermidine (Spd)
and tetramine spermine (Spm) are the main polyamines
(PAs) found in all living cells. Two alternative synthesis
pathways have been verified and the genes encoding
enzymes for the polyamine biosynthesis pathway have
been cloned and characterized from various plant species
(Liu et al. 2007; Bagni and Tassoni 2001; Bell and
Malmberg 1990; Michael et al. 1996). Putrescine is syn-
thesized through agmatine by three sequential reactions
catalyzed by arginine decarboxylase (ADC), agmatine
iminohydrolase (AIH) and N-carbamoylputrescine amido-
hydrolase (CPA), respectively. Starting from arginine, the
diamine putrescine can also be synthesized via ornithine by
arginase and ornithine decarboxylase (ODC). Putrescine is
converted to spermidine by the spermidine synthase
(SPDS); the required aminopropyl group is transferred
from the decarboxylated S-adenosylmethionine, which is
synthesized from methionine in two sequential reactions of
S-adenosylmethionine synthetase (SAMS) and S-adeno-
sylmethionine decarboxylase (SAMDC), respectively
(Kusano et al. 2008). The enzyme S-adenosylmethionine
synthetase (SAMS) catalyzes the nucleophilic substitution
Communicated by L.A. Kleczkowski.
Y.-C. Qi
College of Tobacco Science, Henan Agricultural University,
450002 Zhengzhou, China
e-mail: [email protected]
Y.-C. Qi � F.-F. Wang � W.-Q. Liu (&)
College of Life Science, Henan Agricultural University,
95 Wenhua Road, 450002 Zhengzhou, China
e-mail: [email protected]
H. Zhang
Key Laboratory of Plant Stress Research, College of Life
Science, Shandong Normal University, 250014 Jinan, China
123
Acta Physiol Plant (2010) 32:263–269
DOI 10.1007/s11738-009-0403-3
reaction between methionine and ATP into S-adenosyl-
methionine (SAM), first described by Cantoni in (1953).
Many genes encoding SAMS have been cloned from
monocots and dicots (Kawalleck et al. 1992; Van Breuse-
gem et al. 1994; Espartero et al. 1994; Izhaki et al. 1995;
Whittaker et al. 1995; Larsen and Woodson 1991).
Salt is one of the major abiotic stresses in agriculture.
A high salt concentration disrupts the integrity of cellular
membranes, the activity of various enzymes and the
function of the photosynthetic apparatus. Plants respond to
the salt-stressed environmental condition accumulating low
molecular weight osmolytes such as PAs (Flores 1991).
Salinity caused a significant increase in Spd and Spm in
almost all the plant species studied (Zapata et al. 2004;
Mutlu and Bozcuk 2005). Moreover, Mansour and
Al-Mutawa (1999) indicated that the cellular alterations
induced by NaCl in wheat roots were alleviated by low
concentrations of Spd or Spm and El-Shintinawy (2000)
also indicated that salinity greatly enhanced the accumu-
lation of Spm and Spd in wheat cultivars. At the same time,
some research indicated that endogenous Put also played
an important role in salt tolerance in plants (Urano et al.
2004; Verma and Mishra 2005; Tang and Newton 2005).
Therefore, these results would indicate a clear protective
role of polyamines for plants under salt-stressed condition.
Despite numerous reports trying to elucidate this essential
clue for many years, until now, it remains unclear which
component of salt stress is responsible for the accumulation
of PAs.
In plants, differential expression patterns for SAMS have
been found in the vascular tissues of Arabidopsis, prefer-
entially in the roots and stems (Peleman et al. 1989a, b) and
in leaves of rice (Dekeyser et al. 1990). But the relationship
between the expression of SAMS and the biosynthesis of
polyamines has been seldom reported till now. One SAMS
gene named SsSAMS2 was cloned (GenBank Accession
No. AF321001) from halophyte plant of Suaeda salsa L.
(Ma et al. 2003; Zhang et al. 2001). To understand the role
of SsSAMS2 during the biosynthesis of PAs and salt stress,
we obtained transgenic tobacco (Nicotiana tabacum L.
K326) plants overexpressing SsSAMS2.
Materials and methods
Plasmid construction
The SsSAMS2 cDNA fragment, 1,531 bp with an open
reading frame of 395 amino acids, was inserted into binary
plant vector pROK2 between the cauliflower mosaic virus
35S promoter and octopine synthase terminator. The
resulting plasmid was named pSAMS. The pSAMS and
blank vector pROK2 were mobilized to Agrobacterium
tumefaciens strain GV3101, respectively, and used for
Nicotiana tabacum L. (ecotype K326) transformation.
Transformation, regeneration of tobacco plants
and growth conditions
The leaf discs of Nicotiana tabacum L. (wild-type K326)
were infected with the A. tumefaciens strain GV3101 and
transformed tobacco plants were generated by the standard
method (Horsch et al. 1985). Homozygous T2 generation
plants were derived as described by Koo et al. (2002). The
seeds of these transgenic and wild-type plants were
allowed to germinate, and plants were then transplanted to
plastic pots filled with sand and watered daily with half-
strength Hoagland nutrient solution. T2 plants for assaying
the presence of transgene and the salt tolerance were grown
under 14-h light/10-h dark cycle with cool-white fluores-
cent light of 100 lmol m-2 s-1 at room conditions
(25 ± 5�C). Wild-type and transformation with blank
prok2 tobacco plants were used as control plants.
PCR analysis of SsSAMS2 in transgenic tobacco
Total genomic DNA was isolated from young leaves of T0
progeny and wild-type tobacco plants by cetyltrimethylam-
monium bromide (CTAB) method as described (Doyle and
Doyle 1990) and subjected to PCR analysis for the presence
of transgenes. PCR primers for the detection of SsSAMS2
were 35S-F (50-GTCTTGCGAAGGATAGTGG-30) and
SsSAMS2-R (50-ATACTCAACAGTGACTTGAG-30). PCR
was carried out using Taqr DNA polymerase (Takara, Shiga,
Japan) with 200 ng tobacco genomic DNA as template.
Plasmid DNA from the Agrobacterium carrying prok2 ?
SsSAMS2 was used as a positive control.
RT-PCR analysis of SsSAMS2 in transgenic tobacco
Total RNA was isolated from 80 to 100 mg of in vitro T2
progeny leaf tissue, using the TRIzol Reagent according to
the manufacturer’s specifications (Invitrogen; Carlsbad,
USA).
The accumulations of 18S rRNA, SsSAMS2 were ana-
lyzed by RT-PCR (One Step RT-PCR kit; Takara, Shiga,
Japan). The primers were 18S-F (50-ATG ATA ACT CGA
CGG ATC GC-30) and 18S-R (50-CTT GGA TGT GGT
AGC CGT TT-30) for 18S RNA; SsSAMS2-F (50-TCT
GAGTCTGTGAATGAAGG-30) and SsSAMS2-R (50-AT
GTAGGCACCACTTCTGTC-30) for SsSAMS2. After prior
tests to avoid interferences by saturation, 400 ng of total
RNA was used as template in each RT-PCR reaction and
20 cycles of amplification was chosen. A total of 50 lL of
reaction solution was bathed at 50�C for 30 min, 94�C for
2 min followed by 20 cycles of amplification (94�C for
264 Acta Physiol Plant (2010) 32:263–269
123
30 s, 58�C for 30 s and 72�C for 1 min). The PCR products
(15 lL) were separated on 1% agarose gels stained with
ethidium bromide. 18S rRNA was used in the RT-PCRs as
internal control.
Northern blotting analysis of SsSAMS2 in transgenic
tobacco
RNA (20 lg) was denatured, electrophoresed, transferred to
Hybond N? membrane (Amersham Pharmacia Biotech,
Piscataway, NJ, USA) in 209 SSPE (19 SSPE = 150 mM
NaCl, 10 mM NaH2PO4, and 1 mM EDTA, pH 7.4), and
fixed to the membrane by baking for 2 h at 80�C. A32P-labeled SsSAMS2 cDNA probe and a tobacco 18S rRNA
control probe were generated by the random priming tech-
nique. RNA blots were pre-hybridized in a solution con-
taining 50% (v/v) formamide, 0.25 M sodium phosphate (pH
7.2), 0.25 M NaCl, 7% (w/v) SDS and 1 mM EDTA at 50�C
for 4 h, and hybridized with 106 cpm ml-1 probes in the
same solution overnight. The membrane was washed at 55�C
four times, for 15 min each, in 0.29 SSPE and 0.1% (w/v)
SDS. The filter was exposed to X-ray film for 30–60 min.
The mRNA level of SsSAMS2 was quantified with a
Phosphorimager.
Northern blotting analysis of ADC in transgenic
tobacco
The detailed procedure was just as above. The ADC cDNA
prepared for A 32P-label was amplified with the primers
designed from ADC2 (GenBank Accession No. AF127241).
primers were ADC-F (50-TCAGGACCAAGCATTCAG
G-30) and ADC-R (50-AAGGGCATCTTCGTTGAGC-30).
Salt tolerance analysis
The positive transgenic plants were grown on medium
containing kanamycin. When the plants grew to a height of
about 6 cm, they were transplanted to plastic pots con-
taining a mixture of vermiculite, turf and humus (1:1:1; by
vol.) in a greenhouse, acclimated for 4 weeks and irrigated
with half-Hoagland solution. Thirty days after the trans-
plant, the plants were watered with half-Hoagland solution
containing 200 mM NaCl. The plants were irrigated at
5 days intervals up to 4 weeks. The half-Hoagland solution
without NaCl was used as control. At the end of the salt
treatment, the top fully expanded leaves were used for
measurement of photosynthetic rate and content of PAs.
The net photosynthesis rate was determined with an LI6400
portable apparatus (LI-COR, USA). After measuring the net
photosynthesis rate, the plants were harvested, dried and
then weighed. The greenhouse average photosynthetically
active radiation was above 1,000 lmol m-2 s-1 and
temperature was 30 ± 2�C day/28 ± 1�C night. Humidity
ranged from 40 to 85%. Six replicates were performed for
each treatment.
Determination of PA content
PAs (putrescine, spermidine and spermine) were analyzed
as described by Goren et al. (1982). Leaves (0.3 g) of
transgenic and control tobacco plants, which grew under
salt stress and normal condition, were homogenized in 2 ml
of 5% (v/v) perchloric acid and centrifuged at 12,0009g
for 30 min. Then, 0.2 ml of saturated sodium carbonate
and 0.4 ml of dimethylaminonaphthalene-1-sulfonyl chlo-
ride (1 mg ml-1 acetone) were added to 0.2 ml of super-
natant, and the mixture was incubated at 60�C for 1.5 h.
The dansylated product was extracted with benzene and
separated on thin layer chromatography in chloroform:
triethylamine (25:2, v/v). The separated PAs were scraped
off and quantified using a spectrophotofluorimeter (RF-
1501), by which the emission at 495 nm was recorded after
excitation at 350 nm.
Results
Fifteen kanamycin-resistant plants were obtained. Three
independent transgenic lines (ST8-9, ST14-2, ST3-5) and
two independent transgenic lines with blank vector (BT4-4,
BT5-7) were chosen for further analysis. For the confir-
mation of SsSAMS2 gene integration into the selected
transgenic lines, genomic DNA was isolated and subjected
to PCR analysis. The results of PCR analysis validated the
SsSAMS2 gene in transgenic tobacco, but did not show any
signal in WT and BT plants (Fig. 1). The expression of
SsSAMS2 gene was determined by RT-PCR and Northern
blotting analysis (Figs. 2, 3), and the SsSAMS2 transcript
could be detected in all transgenic tobacco plants, while no
signals were seen in both control plants.
1 2 3 4 5 6 7 8
740bp
Fig. 1 PCR analysis of SsSAMS2 in transgenic tobacco. Lanes from
1 to 8 are marker DL2000, ST8-9, ST14-2, ST3-5, WT, pSAMS,
BT4-4 and BT5-7. Primers used for detecting SsSAMS2 were
promoter 35S of prok2 and SsSAMS2. Total genomic DNA was
isolated from young leaves by CTAB (cetyltrimethylammonium
bromide) method. Plasmid pSAMS from the Agrobacterium was used
as a positive control
Acta Physiol Plant (2010) 32:263–269 265
123
The seeds produced from each flower of the T2 progeny
were counted. The transgenic control tobacco plants with
blank vector (prok2) produced almost the same number of
seeds with wild-type control tobacco plants, and three
transgenic lines (ST8-9, ST14-2, ST3-5) produced more
seeds than both controls (Fig. 4a). Seed mass also
increased in all transgenic lines compared with control
plants (Fig. 4b).
The decrease in productivity in many plants subjected to
excessive salinity is often associated with a diminished
photosynthetic capacity. As shown in Fig. 5, under normal
condition, both the control and transgenic plants exhibited
comparable photosynthetic rate; when the plants were
treated with 200 mM NaCl, transgenic lines showed sig-
nificantly higher photosynthetic rate than both control
plants. Figure 5 clearly showed that the photosynthetic rate
in the control plants was reduced by approximately 40%;
by contrast, the same treatment decreased the photosyn-
thetic rate in the gene transformed lines of ST8-9, ST14-2
and ST3-5 by 14, 24 and 30%, respectively. This indicates
that the transgenic plants maintain a greater photosynthetic
capacity than the wild-type plants under salt stress.
To test whether transformation of SsSAMS2 gene has an
impact on plant growth, the biomass of the transgenics and
wild-type was determined under both control and stress
conditions. As shown in Fig. 6, under normal condition,
both the control and transgenic plants maintained compa-
rable biomass; when the plants were treated with 200 mM
of NaCl, the whole-plant dry weight decreased in both the
transgenic and control plants. However, the reduction was
SsSAMS2
830bp
18S rRNA
Fig. 2 RT-PCR analysis for mRNA expressions of SsSAMS2 in
transgenic tobacco. Lanes from 1 to 6 are marker DL2000, WT, BT4-
4, ST8-9, ST14-2 and ST3-5. 18S rRNA PCR product was used as
native control
SsSAMS2
rRNA
1 2 3 4 5 6
Fig. 3 Northern blotting analysis of SsSAMS2 in transgenic tobacco.
Total RNA was isolated from leaves of non-transformed (WT),
vector-only (BT4-4, BT5-7) control and three transgenic lines (ST8-9,
ST14-2, ST3-5). RNA (20 lg) was gel-fractionated and blotted. The
filter was hybridized with 32P-labeled SsSAMS2 cDNA and 18S
rRNA PCR product control probes and exposed to X-ray film for 1 h.
Lanes from 1 to 6 are WT, BT4-4, BT5-7, ST8-9, ST14-2 and ST3-5
200
400
600
800A
B
Num
bers
of
seed
for
mat
ion
WT
BT4-4
BT5-7
ST8-9
ST14-2
ST3-5
d d d
a
b
c
4
6
8
10
Seed
wei
ght (
mg/
100
seed
s )
WT
BT4-4
BT5-7
ST8-9
ST14-2
ST3-5
dd d
a
b
c
Fig. 4 Numbers of seed formation and weights of seeds in individual
capsules of transgenic and control plants. a The seed number was
counted in each of five individual capsules of T2 transgenic and wild-
type plants; b The weight of 100 seeds was measured from each of
five individual capsules. Values with the same letter are not
significantly different at P B 0.05, by Duncan test. Data represent
the mean ± SE
0
2
4
6
8
10
12
(Pho
tosy
nthe
sis
Rat
e)
(µm
ol C
O2
m-2
s-1
)
WT BT4-4 BT5-7
ST8-9 ST14-2 ST3-5
WT+200mM NaCl BT4-4+200mM NaCl BT5-7+200mM NaCl
ST8-9+200mM NaCl ST14-2+200mM NaCl ST3-5+200mM NaCl
ab b b b bc
d ef f f
Fig. 5 Effects of salt stress on photosynthetic rate of the transgenic
and control tobacco plants. Tobacco plants were treated with 200 mM
NaCl for 4 weeks, and photosynthetic rate were determined. Values
with the same letter in each NaCl level are not significantly different
at P B 0.05, by Duncan test. Values shown are mean ± SE of five
replicates
266 Acta Physiol Plant (2010) 32:263–269
123
more remarkable in controls than in the transgenic plants:
under 200 mM of NaCl stress condition, that control plants
decreased by 38%, while transgenic lines of ST8-9, ST14-2
and ST3-5 decreased by 17, 27 and 34%, respectively. The
results demonstrate that transgenic plants displayed salt
tolerance to some degree.
Transgenic plants with high levels of suadea salsa
SsSAMS2 transcript showed higher levels of free putres-
cine, spermidine and spermine contents compared to con-
trol plants under 0 mM NaCl and 200 mM NaCl-stressed
conditions (Fig. 7a–c). To the same tobacco line, trans-
genic or control plants, the contents of each free poly-
amines determined under 200 mM of NaCl stress condition
was significantly increased compared to that under normal
condition (Fig. 7a–c). To the free total PAs, determined
under 200 mM NaCl stress condition compared to that
under normal condition, in control plants the increase was
10%, and in transgenic lines of ST8-9, ST14-2 and ST3-5,
respectively, the increase was by 17, 17 and 14%, respec-
tively (Fig. 7d).
The result of Northern blotting for SsSAMS2 showed
that ADC transcription was more abundant in transgenic
plants than in control plants (Fig. 8). It suggests that
SsSAMS2 overexpression in transgenic tobacco plants leads
to the up-regulated expression of ADC.
Discussion
Salt stress is one of the major environmental factors that
restrict plant growth and the worldwide productivity
(Epstein et al. 1980; Boyer 1982). Plants respond to
changes in this unfavorable environmental condition
accumulating low molecular weight osmolytes such as PAs
(Flores 1991). Until now, it remains unclear which com-
ponent of salt stress is responsible for the accumulation of
PAs. S-adenosylmethionine synthetase (SAMS) is an
enzyme that is required during the biosynthesis of sper-
midine and spermine, but little research in this field has
been reported.
To understand the role of SAMS in the biosynthesis of
polyamines, so as to overcome the effects of salt stress, we
used the suadea salsa full-length S-adenosylmethionine
synthetase (SsSAMS2) cDNA to get the transgenic tobacco
by Agrobacterium tumefaciens-mediated transformation.
The SsSAMS2 integration was confirmed by PCR analysis
(Fig. 1), and transgenic lines have many SsSAMS2 tran-
scripts, while both control lines do not (Figs. 2, 3). The
transgenic tobacco was very healthy and the seeds of
transgenic lines were more plentiful and heavier than that
of both control plants (Fig. 4).
S-adenosyl-L-methionine (SAM) is synthesized from
methionine and ATP by the enzyme S-adenosylmethionine
synthetase. Decarboxylated S-adenosylmethionine, synthe-
sized by S-adenosylmethionine decarboxylase, serves as an
aminopropyl donor in spermidine and spermine synthesis
(McKeon et al. 1995; Galston and Kaur-Sawhney 1995;
Chiang et al. 1996; Tiburcio et al. 1990). Accumulation of
PA is attributed, at least in part, to transcriptional activation
of genes encoding the PA biosynthetic enzymes, including
SAMDC in rice (Li and Chan 2000) and soybean (Tian et al.
2003) and ADC in mustard (Mo and Pua 2002). Transgenic
tobacco plants overexpressed SsSAMS gene accumulated
higher level of PAs than control plants (Fig. 7). This result
demonstrated that SsSAMS played an important role during
the biosynthesis of polyamines. To interpret why the trans-
genic tobacco plants overexpressed SsSAMS2 could accu-
mulate higher levels of putrescine content than control
plants, the ADC transcript levels in both transgenic and
control plants were determined by Northern blotting. The
result showed that ADC transcription was more abundant in
transgenic plants than in control plants (Fig. 8). Kumar et al.
(1996) reported an increase in putrescine content in sense
SAMDC potato transgenics and Thu-Hang et al. (2002)
reported an increase in putrescine levels in some of the
SAMDC rice transgenic, which could be directly correlated
to their increased ODC and ADC activities. So, the higher
putrescine level in transgenic tobacco plants was partly due
to the overexpression of SsSAMS2.
Transgenic plants overexpressing SsSAMS gene
enhanced salt tolerance, as indicated by the maintenance of
higher photosynthetic rate and accumulation of more bio-
mass (Figs. 5, 6). Therefore, increased accumulation of
PAs in transgenic plants correlated with an elevated
capacity for photosynthesis (Fig. 5) under both stress and
normal conditions, which were consistent with the sug-
gested role of PAs for increased tolerance to salt stress
WT BT4-4 BT5-7
ST8-9 ST14-2 ST3-5
WT+200mM NaCl BT4-4+200mM NaCl BT5-7+200mM NaCl
ST8-9+200mM NaCl ST14-2+200mM NaCl ST3-5+200mM NaCl
6
8
10
12
14
16
18
Bio
mas
s (g
/pla
nt D
W)
b b a b b
c
d ef f f
Fig. 6 Effects of NaCl stress on biomass of transgenic and control
tobacco plants. Tobacco plants were treated with 200 mM NaCl for
4 weeks, and the dry weight (DW) was determined. Values at the
same NaCl level with the same letter are not significantly different at
P B 0.05, by Duncan test. Values shown are means ± SE of five
replicates
Acta Physiol Plant (2010) 32:263–269 267
123
(Basu and Ghosh 1991; Krishnamurthy and Bhagwat
(1989). These findings demonstrated the feasibility of
engineering plants with increased salt tolerance and
enhanced productivity through overproduction of PAs.
In this report, we show that SsSAMS2 overexpression in
transgenic tobacco plants leads to increase in polyamines
content, and, as a result, promotes salt tolerance.
The salt tolerance mechanism for the plant is very
complicated, and besides some low molecular-weight
osmolytes, the role of some antioxidative enzymes should
not be neglected. The possible role analysis of antioxida-
tive enzymes on stress tolerance in SsSAMS2-overexpres-
sed transgenic tobacco may be a subject of continuing
study.
Acknowledgments This work was supported by the Hi-Tech
Research and Development (863) Program of China (2002AA629080)
and the State Key Basic Research and Development Plan of China
(G1995011700).
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to X-ray film for 1 h. Lanes from 1 to 6 are ST8-9 ? 200 mM NaCl,
ST8-9, WT ? 200 mM NaCl, WT, BT4-4 ? 200 mM NaCl and
BT4-4
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