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ORIGINAL PAPER Overexpression of suadea salsa S-adenosylmethionine synthetase gene 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 Ó Franciszek Go ´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako ´w 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

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Page 1: Overexpression of suadea salsa S-adenosylmethionine synthetase gene promotes salt tolerance in transgenic tobacco

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

Page 2: Overexpression of suadea salsa S-adenosylmethionine synthetase gene promotes salt tolerance in transgenic tobacco

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

Page 3: Overexpression of suadea salsa S-adenosylmethionine synthetase gene promotes salt tolerance in transgenic tobacco

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

Page 4: Overexpression of suadea salsa S-adenosylmethionine synthetase gene promotes salt tolerance in transgenic tobacco

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

Page 5: Overexpression of suadea salsa S-adenosylmethionine synthetase gene promotes salt tolerance in transgenic tobacco

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

Page 6: Overexpression of suadea salsa S-adenosylmethionine synthetase gene promotes salt tolerance in transgenic tobacco

(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).

References

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Bell E, Malmberg RL (1990) Analysis of a cDNA encoding arginine

decarboxylase from oat reveals similarity to the Escherichia coliarginine decarboxylase and evidence of protein processing. Mol

Gen Genet 224:431–436

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wei

ght)

WT BT4-4 BT5-7ST8-9 ST14-2 ST3-5WT+200mM NaCl BT4-4+200mM NaCl BT5-7+200mM NaClST8-9+200mM NaCl ST14-2+200mM NaCl ST3-5+200mM NaCl

a

bc

d e f g g g

h h h

2

4

6

8

10

Free

spe

rmin

e co

nten

t

(nm

ol g

-1 f

resh

wei

ght)

WT BT4-4 BT5-7ST8-9 ST14-2 ST3-5WT+200mM NaCl BT4-4+200mM NaCl BT5-7+200mM NaClST8-9+200mM NaCl ST14-2+200mM NaCl ST3-5+200mM NaCl

a

bcd

ef e e g

h h h

20

30

40

50

60

70

80

Tot

al f

ree

poly

amin

es c

onte

nt

(nm

ol g

-1 f

resh

wei

ght)

WT BT4-4 BT5-7ST8-9 ST14-2 ST3-5WT+200mM NaCl BT4-4+200mM NaCl BT5-7+200mM NaClST8-9+200mM NaCl ST14-2+200mM NaCl ST3-5+200mM NaCl

h h

a

bc

edf g g g

h

Fig. 7 Comparative analysis of free content of PAs in the leaves of

T2 transgenic progeny and control plants. The tenth leaves of each

plant were harvested and analyzed for free contents of PAs (a, b, c,

d). 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

mean ± SE of five replicates

1 2 3 4 5 6

ADC

rRNA

Fig. 8 Northern blotting analysis of ADC in tobacco plants. Total

RNA was isolated from leaves of non-transformed (WT), vector-only

(BT4-4) control and transgenic line (ST8-9) cultured under 200 mM

NaCl stressed and normal conditions. RNA (20 lg) was gel-

fractionated and blotted. The filter was hybridized with 32P-labeled

ADC cDNA and 18S rRNA PCR product control probes, and exposed

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

268 Acta Physiol Plant (2010) 32:263–269

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