ORIGINAL RESEARCH

Regulation of exogenous spermidine on the reactive oxygen specieslevel and polyamine metabolism in Alternanthera philoxeroides(Mart.) Griseb under copper stress

Xiaoying Xu • Guoxin Shi • Chunxia Ding •

Ye Xu • Juan Zhao • Haiyan Yang •

Qiuhong Pan

Received: 26 April 2010 / Accepted: 31 August 2010 / Published online: 17 September 2010

� Springer Science+Business Media B.V. 2010

Abstract Effects of exogenous spermidine (Spd) on the

reactive oxygen species level and polyamine metabolism

against copper (Cu) stress in Alternanthera philoxeroides

(Mart.) Griseb leaves were investigated. Cu treatment

induced a marked accumulation of Cu and enhanced con-

tents of malondialdehyde (MDA), hydrogen peroxide

(H2O2) and the generation rate of O2�-. It also significantly

increased putrescine (Put) levels but lowered spermidine

(Spd) and spermine (Spm) levels. The activities of arginine

decarboxylase (ADC), ornithine decarboxylase (ODC) and

polyamine oxidase (PAO) were all elevated with the

increase of Cu concentration. However, application of

exogenous Spd effectively decreased H2O2 content and the

generation rate of O2�-, prevented Cu-induced lipid per-

oxidation and reduced Cu accumulation. Moreover, it

declined level of endogenous Put and increased levels of

Spd and Spm. Activities of ADC, ODC and PAO were all

inhibited by exogenous Spd. The results indicated that

application of exogenous Spd could enhance the tolerance

of A. philoxeroides to Cu stress by reducing the reactive

oxygen level and balancing polyamine metabolism.

Keywords Cu � Polyamine � Spermidine � Alternanthera

philoxeroides

Introduction

Human actions cause bioaccumulation of various heavy

metals such as copper (Cu) and zinc (Zn) in aquatic

ecosystems. Cu is an essential microelement for plant

metabolism, but excess Cu can interfere with numerous

physiological processes such as photosynthesis, pigment

synthesis, nitrogen and protein metabolism, membrane

integrity and mineral uptake (Shen et al. 1998; Nielsen

et al. 2003; Demirevska-Kepova et al. 2004). As a redox-

active metal, Cu can catalyze the generation of harmful

reactive oxygen species (ROS) such as superoxide anion

(O2�-), hydrogen peroxide (H2O2) and hydroxyl radical

(HO) (Schutzendubel and Polle 2002). These ROS react

with lipids, proteins and nucleic acids, causing lipid per-

oxidation, membrane damage and enzyme inactivation.

This toxic effect resulting from the oxidative state may be

allayed by several antioxidative systems to which poly-

amines (PAs) belong (Bouchereau et al. 1999; Chen et al.

2002; Kuthanova et al. 2004).

Polyamines (PAs) are ubiquitous low-molecular-weight

aliphatic amines that are involved in regulation of plant

growth and development (Martin-Tanguy 2001). Spermi-

dine (Spd), spermine (Spm) and their diamine precursor,

putrescine (Put), are major polyamines in plant cells

(Galston and Sawhney 1990). In plants, polyamines are

related to various kinds of environmental stresses including

acid stress (Shen et al. 1994), heavy metal stress (Groppa

et al. 2001, 2003), osmotic stress (Legocka and Kluk

2005), UV radiation (Lutz et al. 2005) and salt stress

(Jimenez-Bremont et al. 2007). It has been reported that

polyamine accumulation is due to ammonia detoxification,

which is released in plant cells after exposure to stress

(Slocum and Weinstein 1990). Polyamine metabolism

includes both synthesis and degradation. In plants, the

X. Xu � G. Shi (&) � C. Ding � Y. Xu � J. Zhao � H. Yang �Q. Pan

Jiangsu Key Lab of Biodiversity and Biotechnology,

College of Life Science, Nanjing Normal University,

1 Wenyuan Road, 210046 Nanjing, Jiangsu, People’s Republic

of China

e-mail: gxshi@njnu.edu.cn

123

Plant Growth Regul (2011) 63:251–258

DOI 10.1007/s10725-010-9522-5

initial step in polyamines biosynthesis is the decarboxyl-

ation of arginine or ornithine to produce putrescine (Put) by

arginine decarboxylase (ADC) or ornithine decarboxylase

(ODC). Spermidine (Spd) and spermine (Spm) are formed

by the subsequent addition of an aminopropyl moiety.

Polyamines degradation in plants is performed by poly-

amine oxidase (PAO) which oxidizes Spm and Spd to

diamine-propane, H2O2, pyrroline and 1, 5-diabicyclo-

nonane (Groppa et al. 2003). The levels of polyamines in

plants are altered in response to heavy metals (Sharma and

Dietz 2006). A close interrelationship between polyamines

and stress was documented by the finding that leaf necrosis

caused by ozone in tomato plants could be suppressed by

an exogenous supply of polyamines (Ormrod and Becker-

son 1986), and exogenous polyamines recovered browning

tissues into normal callus cultures of Virginia pine by

decreasing oxidative damage (Tang et al. 2004). Among

the three major polyamines, Spd in many cases has been

more closely associated with stress tolerance in plants

(Duan et al. 2008).

Most polyamine studies have focused on terrestrial

plants (Bezold et al. 2003; Liu et al. 2004; Swamy et al.

2004). Little is known about the role that polyamines play

in aquatic plants with little research on whether exoge-

nous polyamines play a protective role against heavy

metal toxicity. Alternanthera philoxeroides (Mart.) Griseb

is an aquatic and clonal weedy species native to South

America (Buckingham 1996) and was introduced into

China in the 1930s initially as a forage crop (Wang et al.

2005). Due to its strong reproductive abilities and wide-

spread distribution, it is a suitable plant for experiment

research. In this study, A. philoxeroides was used to

investigate whether exogenous Spd enhanced Cu toler-

ance with reference to: (1) change in uptake of copper;

(2) change in reactive oxygen species level; (3) change in

polyamine metabolism.

Materials and methods

Plant material

A. philoxeroides was collected from Tai lake in Suzhou,

China, washed with distilled water and acclimated for more

than 3 weeks under natural conditions (plants receiving

normal light/dark period, temperature, etc.), then main-

tained in aquaria containing 1/10 Hoagland solution. Plants

of similar height and weight were selected for experi-

mentation and grown in a controlled environmental growth

chamber (Forma 3744, England) with a photoperiod of

12 h light and 12 h dark and the day/night temperature of

24/18�C.

Treatments

Plant materials were treated as follows: (1) control: 1/10

Hoagland solution (containing 32 nmol Cu) and the leaves

were sprayed with either distilled water or 0.1 mmol L-1

Spd respectively; (2) Cu treatment: 1/10 Hoagland solution

containing 0.05, 0.1, 0.15, 0.2 mmol L-1 Cu and the leaves

were sprayed with distilled water; (3) Cu ? Spd treatment:

1/10 Hoagland solution containing 0.05, 0.1, 0.15,

0.2 mmol L-1 Cu and the leaves were sprayed with

0.1 mmol L-1 Spd. The spray of distilled water or Spd was

5 ml each time and took place at 9:00, 15:00 and 19:00

each day, respectively. The selected Cu and Spd concen-

trations were based on a preliminary experiment. All

solutions were refreshed every 2 days. After 5 days, the

fully expanded leaves were cut and sampled. All experi-

ments were performed in triplicate.

Determination of Cu content

Leaves were washed thoroughly with 10 mmol L-1 EDTA

to remove metals adsorbed to the surface. They were oven-

dried at 70�C for 2 days and digested with 3:1 HNO3/

HClO4 at 95�C until the digest solution became clear. The

digested residue was dissolved in 0.7 ml HCl and diluted

with distilled water to 10 ml. The solution samples were

analyzed for Cu by inductively coupled plasma spectros-

copy (Leeman, USA).

Determination of membrane lipid peroxidation,

H2O2 content and the generation rate of O2�-

The H2O2 content was measured according to Lin et al.

(1988). Membrane lipid peroxidation was measured by the

level of malondialdehyde (MDA), a product of lipid per-

oxidation, using a reaction with thiobarbituric acid (TCA)

as described by Hodges et al. (1999). Leaves (0.5 g FW)

were homogenized in 10 ml 10% TCA and centrifuged at

12,0009g for 20 min. After that, 2 ml 0.6% thiobarbituric

acid (TBA) in 10% TCA was added to an aliquot of 2 ml

from the supernatant. The mixture was heated in boiling

water for 30 min then quickly cooled in an ice bath. After

centrifugation at 1,8009g for 10 min, the absorbance of

the supernatant at 450, 532 and 600 nm was determined.

The generation rate of O2�- was determined following the

method of Wang and Luo (1990).

Analysis of polyamines

Plant material (1.5 g) was homogenized in 3 ml of 5%

(v/v) precooling perchloric acid (PCA), kept on ice for 1 h,

and then centrifuged at 12,0009g for 30 min. The pellet

was extracted three times with 2 ml 5% PCA and

252 Plant Growth Regul (2011) 63:251–258

123

re-centrifuged. The four supernatants were pooled and used

to determine levels of free and perchloric acid soluble con-

jugated polyamines (PS-conjugated PAs) whereas the pellet

was used to determine levels of perchloric acid insoluble

bound polyamines (PIS-bound PAs). The pellet was re-

suspended in 5% PCA and hydrolyzed for 18 h at 110�C

in flame-sealed glass ampoules after being mixed with 12 N

HCl (1:1, v/v). The hydrolyzates were filtered, dried at 80�C,

and then re-suspended in 0.5 ml of 5% PCA for analysis of

PIS-bound PAs. For PS-conjugated PAs, 2 ml of the super-

natant were mixed with 2 ml of 12 N HCl and hydrolyzed

under the conditions described above. The supernatant,

hydrolyzed supernatant and pellet were benzoylated in

accordance with the method of Aziz and Larher (1995).

The benzoy derivatives were separated and analyzed by

an HPLC system (Agilent 1100, USA) where 10 ll of

methanol solution of benzoyl PAs was injected into a 20 ll

loop, loaded onto a 200 9 4.6 mm, 5 lm particle size C18

reverse-phase column (Kromasil, Sweden). Column tem-

perature was maintained at 30�C. Samples were isocrati-

cally eluted from the column with 64% (v/v) methanol at a

flow rate of 0.8 ml min-1. Polyamine peaks were detected

with a UV detector at 254 nm. Three polyamine standards

(Sigma Chemical Co.) of Put, Spd and Spm were prepared

at different concentrations for the production of standard

curves. Final contents of Put, Spd and Spm were calculated

by the summation of free, PS-conjugated and PIS-bound

PAs, respectively.

Determination of ADC and ODC

Fresh samples (1.5 g) were homogenized in 50 mmol L-1

phosphate buffer (pH 6.3) containing 0.1 mmol L-1

phenylmethylsulfonyl fluoride (PMSF), 40 lmol L-1 pyr-

idoxal phosphate (PLP), 5 mmol L-1 dithiothreitol (DTT),

5 mmol L-1 ethylene diamine tetraacetic acid (EDTA),

20 mmol L-1 ascorbic acid (Vc) and 40 lmol L-1 poly-

vinylpyrrolidone (PVP). The homogenate was centrifuged

at 12,0009g for 40 min and the supernatant was used for

the enzyme assay.

The ADC and ODC activity was determined according to

Zhao et al. (2003) with some modifications. Reaction mix-

ture (1.5 ml) consisted of 1 ml of the assay buffer with

100 mmol L-1 Tris–HCl (pH 8.5), 5 mmol L-1 EDTA,

40 lmol L-1 pyridoxal phosphate and 5 mmol L-1 DTT,

0.3 ml of either the ADC or ODC enzyme extract and 0.2 ml

of 25 mmol L-1L-Arginine (Ornithine). The reaction mix-

ture was incubated at 37�C for 60 min, and centrifuged at

3,0009g for 10 min after which 0.5 ml of the supernatant

was mixed with 1 ml of 2 mmol L-1 NaOH, then 10 ll

benzoyl chloride was added to the mixture and stirred con-

tinuously for 20 s. After the reaction proceeded at 25�C for

60 min, 2 ml of saturated NaCl and 2 ml of ether were added

to the reaction mixture and stirred thoroughly, then centri-

fuged at 1,5009g for 5 min, 1 ml of ether phase was

collected and evaporated at 50�C. The remainder was dis-

solved in 0.5 ml of methanol, and its absorption value at

254 nm was measured by an HPLC system (Agilent 1100,

USA). A standard curve with Agm (Put) was used to calcu-

late the activity of ADC (ODC). ADC and ODC activities

were expressed as lmol Agm g-1 FW�h-1 (U) and lmol

Put g-1 FW�min-1 (U), respectively.

Determination of PAO

PAO activity was determined by the improved method of

Smith (1985). Leaves (0.5 g FW) were ground on ice, in

0.05 mmol L-1 Na2HPO4–NaH2PO4 buffer; then separated

centrifugally at 10,0009g for 20 min. The filtrate was used

to assay enzyme activity. For PAO measurement, 3 ml of

reaction mixture consisted of 2.5 ml phosphate buffer

(pH 6.5) containing 20 mmol L-1 Spd and 0.2 ml enzyme

extract. The reaction was conducted at 25�C for 30 min,

and stopped by adding 0.5 ml 10% TCA. After centrifu-

gation, anthranilic aldehyde at an equal volume was added

to the supernatant and measured spectropho-tometrically at

550 nm where 0.0014A435 g-1 FW min-1 was equal to

one enzyme activity unit (1 U).

Statistics

All experiments were repeated three times with three rep-

licates in each. The data reported in table and figures are

means of the values with standard deviation (SD). Results

were statistical analysis using analysis of variance

(ANOVA). Levels of significance were indicated by Dun-

can’s multiple range test at P \ 0.05. The coefficients of

correlation were expressed using r-values.

Results

Effects of exogenous Spd on copper accumulation

and membrane lipid peroxidation

After 5d of treatment, a marked accumulation of Cu was

observed in the leaves of A. philoxeroides and showed

concentration dependent characteristics (Table 1). The

application of exogenous Spd significantly suppressed Cu

accumulation compared to the corresponding Cu treat-

ment (Table 1). MDA content markedly increased in

plants exposed to low Cu concentrations (0.05 and

0.1 mmol L-1). When grown in higher Cu concentrations,

the MDA content decreased but was still higher than that of

the control (Fig. 1). Spd application efficiently suppressed

Cu-induced MDA accumulation in all treatments (Fig. 1).

Plant Growth Regul (2011) 63:251–258 253

123

Effects of exogenous Spd on H2O2 content

and the generation rate of O2�- under Cu stress

H2O2 content increased conspicuously in low Cu treat-

ments (0.05 and 0.1 mmol L-1), then slightly decreased

when Cu concentration was higher than 0.15 mmol L-1

(Fig. 2a). Application of exogenous Spd inhibited H2O2

accumulation to different extent. The generation rate

of O2�- increased gradually with increasing Cu concen-

tration and positively correlated with Cu concentration

(r = 0.9477, P \ 0.05) (Fig. 2b). When Cu treatment was

combined with exogenous Spd, the generation rate of

O2�-efficiently reduced compared with the corresponding

Cu treatment.

Effects of exogenous Spd on levels of endogenous

polyamines under Cu stress

In comparison with the control plants, a massive accumu-

lation of Put was induced by Cu treatments, which showed

a positive correlation with Cu concentration (r = 0.9408,

P \ 0.05). Exogenous Spd application decreased the level

of Put effectively. However, this decrease was not statis-

tically significant (Fig. 3a).

Cu treatment sharply declined levels of Spd and Spm,

which showed a negative correlation with Cu concentration

(rSpd = -0.9284, P \ 0.05; rSpm = -0.8497, P \ 0.05)

(Fig. 3b, c). When applied with exogenous Spd, the decline

was reversed. Exogenous Spd application generated a sta-

tistically significant difference in the level of endogenous

Spd at 0.15 mmol L-1 Cu treatment (Fig. 3b), but failed to

generate a statistically significant difference in the level of

Spm (Fig. 3c).

Effects of exogenous Spd on ADC and ODC activity

under Cu stress

ADC and ODC are important enzymes in the two pathways

of Put formation. ADC activity was elevated transiently at

0.05 mmol L-1 Cu then decreased gradually with further

Table 1 Effects of 0.1 mmol L-1 exogenous Spd on Cu accumulation in leaves of A. philoxeroides under Cu stress (lg g-1 DW) (r = 0.9261,

P \ 0.05)

Treatment Exogenous Cu concentrations (mmol L-1)

0 0.05 0.1 0.15 0.2

Cu 0.8 ± 0.17e 1.94 ± 0.12 e 60.80 ± 2.19 c 74.83 ± 0.95 b 187.67 ± 2.52 a

Cu ? Spd 0.62 ± 0.11 e 1.26 ± 0.14 e 25.40 ± 1.77 d 31.13 ± 3.50 d 59.33 ± 6.51 c

Each value is the mean ± SD of triplicates. Different letters indicate significant differences between treatments according to Duncan’s multiple

range test at P \ 0.05

Fig. 1 Effects of exogenous Spd on MDA content in leaves of

A. philoxeroides under Cu stress. Each value is the mean ± SD

of triplicates. Different letters indicate significant differences between

treatments according to Duncan’s multiple range test at P \ 0.05

Fig. 2 Effects of exogenous

Spd on H2O2 content (a) and the

generation rate of O2-(b) in

leaves of A. philoxeroides under

Cu stress. Each value is the

mean ± SD of triplicates.

Different letters indicate

significant differences between

treatments according to

Duncan’s multiple range test at

P \ 0.05

254 Plant Growth Regul (2011) 63:251–258

123

increase of Cu concentration. ODC activity was enhanced

significantly with increasing Cu concentration and showed

a positive correlation with Cu concentration (r = 0.9393,

P \ 0.05) (Fig. 4). When applied with exogenous Spd,

both ADC and ODC activities were inhibited. Exoge-

nous Spd application generated a statistically significant

difference in ADC activity in all Cu treatments (Fig. 4a),

but failed to generate a statistically significant difference in

ODC activity (Fig. 4b).

Effects of exogenous Spd on PAO activity under Cu

stress

PAO, oxidizing Spd or Spm, showed a steep increase

under Cu stress with significantly positive correlation with

Cu concentration (r = 0.9835, P \ 0.05). Application of

exogenous Spd lowered PAO activity, especially at 0.15

and 0.2 mmol L-1 Cu treatments which generated a sta-

tistically significant difference with the corresponding Cu

treatment (Fig. 5).

Discussion

As shown in this study, copper stress disrupted the selec-

tive uptake of the cells of A. philoxeroides leaves and

induced accumulation of copper. With increasing Cu con-

centration, the Cu content increased. Application of exog-

enous Spd significantly inhibited the accumulation of

Fig. 3 Effects of exogenous Spd on the levels of endogenous Put (a),

Spd (b) and Spm (c) in leaves of A. philoxeroides under Cu stress.

Each value is the mean ± SD of triplicates. Different letters indicate

significant differences between treatments according to Duncan’s

multiple range test at P \ 0.05

Fig. 4 Effects of exogenous

Spd on activities of ADC

(a) and ODC (b) in leaves of

A. philoxeroides under Cu

stress. Each value is the

mean ± SD of triplicates.

Different letters indicate

significant differences between

treatments according to

Duncan’s multiple range test at

P \ 0.05

Fig. 5 Effects of exogenous Spd on PAO activity in leaves of

A. philoxeroides under Cu stress. Each value is the mean ± SD

of triplicates. Different letters indicate significant differences between

treatments according to Duncan’s multiple range test at P \ 0.05

Plant Growth Regul (2011) 63:251–258 255

123

copper in cells (Table. 1). The accumulation of copper

might be related to membrane damage. The plasma mem-

brane regulates the passage of solutes between the cell and

the external environment by selectively absorbing nutrients

into the cell against a concentration gradient and prevent-

ing the entry of certain solutes present in the environment.

MDA is a cytotoxic decomposition product of polyunsat-

urated fatty acids (PUFA) of bio-membranes, which is

usually used as an indicator of membrane lipid peroxida-

tion caused by oxidative stress in heavy metal-treated plant

samples (Chaoui et al. 1997; Cuny et al. 2004). In the

present study, Cu treatments significantly enhanced the

MDA content. Although there was a little decrease at 0.15

and 0.2 mmol L-1 Cu treatments, the value of MDA was

still higher than that of control. The increasing MDA

content indicated the aggravation of membrane lipid per-

oxidation, resulting in disrupting the selective uptake of the

cells in A. philoxeroides leaves and inducing increased Cu

accumulation. Spd may act as a protectant for the plasma

membrane integrity (Roy et al. 2005). Polyamines are

highly protonated at physiological pHs, which should favor

electrostatic binding of polyamines to negatively charged

functional groups of membranes and proteins (Zhao and

Yang 2008). In this study, exogenous Spd evidently

decreased the MDA content compared with corresponding

Cu treatment. Thus polyamines can maintain membrane

stability and permeability through binding to the negatively

charged phospholipids head group. As a result, the accu-

mulation of copper was prevented by exogenous Spd

through alleviating membrane damage.

Membrane lipid peroxidation was induced by the pro-

duction of reactive oxygen species (ROS). It is well doc-

umented that most of biotic and abiotic stresses activate a

common mechanism involving the production of reactive

oxygen species (ROS) such as H2O2 and O2�- in plant cells.

Heavy-metal stress affects the normal translocation of

electrons, resulting in free-radical production that in turn

leads to lipid peroxidation (Atal et al. 1991). Data from the

present study indicated that Cu treatment induced an

accumulation of O2�- and H2O2 in A. philoxeroides

(Fig. 2). High levels of O2�- and H2O2 intra-cellular caused

membrane lipid peroxidation, thus the MDA content

increased under Cu2? treatment (Fig. 1). It is reported that

polyamines counteract oxidative damage in plants by act-

ing as direct free radical scavengers or binding to antiox-

idant enzyme molecules to scavenge free radical (Bors

et al. 1989). Polyamines can form a ternary complex with

Fe2? and the phospholipid polar heads that may change the

susceptibility of Fe2? to auto-oxidation and, thus, protect

the membrane from attack (Velikova et al. 2000). In this

study, application of Spd lessened the production of O2�-

and H2O2 (Fig. 2) and decreased MDA content (Fig. 1),

which indicated that application of Spd reduced the dam-

age of plasma membrane by lowering the level of ROS.

Copper accumulation induced by Cu stress also dis-

turbed the balance of PAs metabolism. As PAs are essential

for cellular growth and differentiation, deregulation of PAs

homeostasis may negatively affect cell proliferation and

eventually lead to cell death (Wallace et al. 2003; Takao

et al. 2006). Many types of environmental stresses caused

significant accumulation of Put in plant tissues, while the

levels of other PAs remained unchanged (Gorecka et al.

2007). In our previous study, increased mercury (Hg)

concentration in the culture medium resulted in a marked

increase of Put and a decrease of Spd and Spm in water

hyacinth leaves (Ding et al. 2010). A similar result was also

observed in the present work. Cu stress resulted in the

increase of Put and a decrease of Spd and Spm in leaves of

A. philoxeroides (Fig. 3). In plants, there are two alterna-

tive pathways leading to Put formation: decarboxylation of

either arginine or ornithine by ADC or ODC, respectively

(Slocum 1991). The accumulation of Put in Cu-treated

A. philoxeroides plants was attributed to high activities of

ODC and ADC (Fig. 4). The content of Put was mediated

by an alternate enhancement of ADC and ODC activity.

Both Spd and Spm contents decreased with the increasing

of Cu concentration (Fig. 3b, c) implicated the conversion

of Put to Spd and Spm was inhibited and PAs metabolism

was disordered. Another reason for the decrease of Spd and

Spm might be the large increase in PAO activity (Fig. 5)

which accelerated the degradation of Spd and Spm. A mass

accumulation of Put is generally considered toxic to plants

and eventually leads to apoptotic cell death if its level

becomes too high (Panicot et al. 2002; Takao et al. 2006).

Many reports indicated that exogenous application of Spd

could lower the level of Put (Ndayiragije and Lutts 2006;

Duan et al. 2008). In this study, we certified that exogenous

Spd inhibited the accumulation of Put under copper stress

(Fig. 3a). Firstly, exogenous Spd depressed activities of

ADC and ODC compared with corresponding Cu treatment

(Fig. 4). Secondly, application of Spd accelerated the

conversion of Put to Spd and Spm, as seen from the

enhancement of Spd and Spm contents in Spd-treated

plants (Fig. 3b, c). Otherwise, the enhancement of Spd and

Spm contents could be attributed not only to direct uptake

of Spd but also to a reduction of PAO activity in Spd-

treated plants (Fig. 5). Therefore, PAs metabolism balance

was restored by exogenous Spd via reducing accumulation

of Put and elevating levels of Spd and Spm in leaves of

A. philoxeroides. The similar effect of exogenous Spd on

PAs metabolism coincided with Ding et al. (2010). It was

indicated that these two aquatic plants responded in a

similar manner to heavy metal stress and exogenous Spd

effectively alleviated toxicity of heavy metal.

256 Plant Growth Regul (2011) 63:251–258

123

In conclusion, application of exogenous Spd lowered

reactive oxygen species level and balanced polyamine

metabolism to enhance copper tolerance in A. philoxero-

ides leaves. Our experiment indicated that Cu, which is an

essential microelement for plant growth, was strongly

phytotoxic at high concentration to A. philoxeroides leaves.

The application of Spd depressed the production of reactive

oxygen species and maintained the membranes stability.

Furthermore, it maintained the balance of PAs metabolism

by decreasing the level of endogenous Put and increasing

levels of endogenous Spd and Spm. Further studies con-

sidering expression of the corresponding resistance genes

are required to determine if the recorded change has any

relationship to heavy metal resistance.

Acknowledgments This research was supported by the National

Natural Science Foundation of China (No. 30670121 and No. 30870139).

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