ORIGINAL PAPER

Physiological and biochemical responses induced by lead stressin Spirodela polyrhiza

Xuqiang Qiao • Guoxin Shi • Rong Jia •

Lin Chen • Xiuli Tian • Jun Xu

Received: 19 September 2011 / Accepted: 16 March 2012 / Published online: 10 April 2012

� Springer Science+Business Media B.V. 2012

Abstract The effects of increasing lead concentration on

the activities of superoxide dismutase (SOD), peroxides

(POD) and catalase (CAT), levels of ascorbate (AsA),

reduced glutathione (GSH), Pb accumulation and its influ-

ence on nutrient elements, polyamines (PAs) content, as well

as activities of polyamine oxidase (PAO) and ornithine

decarboxylase (ODC), were investigated in Spirodela

polyrhiza. POD and CAT activities increased progressively

followed by a decline, while SOD activity gradually fell. The

effect of Pb application on AsA content was similar to that

seen for POD and CAT activities. GSH content initially rose

but then declined. A significant enhancement in Pb accu-

mulation was observed, except in the 25 lM Pb treatments.

Nutrient elements were also affected. Moreover, Pb stress

induced a considerable decrease in total spermidine (Spd),

while the levels of total putrescine (Put) and spermine (Spm)

initially increased at 25 lM Pb but then declined. Free and

perchloric acid soluble conjugated (PS-conjugated) PAs

contents changed in a similar way to total PAs. In addition,

Pb stress induced a continuous accumulation of perchloric

acid insoluble bound (PIS-bound) Spm and an initial accu-

mulation of PIS-bound Put and Spd. The ratio of free

(Spd ? Spm)/Put significantly declined whereas the ratio of

total (Spd ? Spm)/Put rose at low Pb concentrations (25 and

50 lM). PAO activity rose gradually with an increase in Pb

concentration, reaching peak values at 100 lM, while ODC

activity first increased at 25 lM Pb and then declined. The

results indicated that the tolerance of S. polyrhiza to Pb

stress was enhanced by activating the antioxidant system,

preventing the entry of the Pb ion and altering the content of

polyamines.

Keywords Lemna minor � Lead � Polyamines �Antioxidant system � Nutrient elements

Introduction

Heavy metal contamination of water bodies is a serious

problem due to the application of pesticides in agriculture,

discharge of untreated industrial wastes and mining oper-

ations (Lou et al. 2004). Heavy metal toxicity may also

occur due to the fact that heavy metals induce secondary

oxidative stress by catalyzing the formation of harmful

reactive oxygen species (ROS) (Posmyk et al. 2009).

Excessive Pb induced phototoxic symptoms, such as

growth retardation, degradation of photosynthetic pig-

ments, lipid peroxidation etc., which are due to interference

with many metabolic processes (Fargasova 1994; Kumar

et al. 1993).

Plants have evolved various defense mechanisms to

cope with potential damage by Pb. To protect against

oxidative stress, plants have developed an antioxidative

system consisting of both antioxidative enzymes and non-

enzymatic antioxidants. Recently, considerable attention

has been focused on the involvement of plant polyamines

(PAs) in the acquisition of tolerance to various environ-

mental stresses. Polyamines (PAs) are small aliphatic

amines that are ubiquitous in plants, animals and micro-

organisms. Spermidine (Spd), spermine (Spm) and their

diamine obligate precursor, putrescine (Put), are major PAs

in plant cells. Polyamines (PAs) are low-molecular-weight

aliphatic amines that are involved in the regulation of plant

growth and development (Martin-Tanguy 2001). It has

X. Qiao � G. Shi (&) � R. Jia � L. Chen � X. Tian � J. Xu

Jiangsu Key Lab of Biodiversity and Biotechnology, College of

Life Science, Nanjing Normal University, No. 1 Wenyuan Road,

Nanjing 210046, Jiangsu, People’s Republic of China

e-mail: gxshi@njnu.edu.cn

123

Plant Growth Regul (2012) 67:217–225

DOI 10.1007/s10725-012-9680-8

been reported that PAs may undertake complex functions

in relation to plant adaptation to various abiotic and biotic

stress factors as a part of an integrated plant response

(Belle et al. 2004; Lefevre et al. 2001). As far as can be

ascertained, no work has so far been carried out to study

Pb-induced endogenous polyamine metabolic changes in

aquatic plants. Furthermore, it is not known whether

endogenous polyamines play a protective role against

heavy metal toxicity. Additionally, there are few available

reports on the interaction between Pb and essential metal

ions, which may cause severe nutrient deficiencies,

resulting in physiological disorders.

Spirodela polyrhiza, as a widespread aquatic floating

weed, is the first link in introducing metal elements found

in aquatic environments into the wider food chain (Singh

et al. 2006). They are small in size, easy to culture in a

laboratory and show rapid growth and high biomass pro-

duction. In particular, they are reported to accumulate toxic

metals and therefore are being used in experimental model

systems to investigate heavy metal induced responses (Jain

et al. 1998; Rahmani and Sternberg 1999; Severi 1997;

Sharma and Gaur 1995). Although existing studies have

been conducted on Pb accumulation in Spirodela polyrhiza

and the performance of Lemna minor in removing Pb from

aquatic systems (Leblebici and Aksoy 2011; Rahmani and

Sternberg 1999), the mechanism behind how aquatic plants

resist Pb toxicity is not known. In the present research, the

main objective was to study the effects of Pb contamination

on several biochemical and physiological parameters, and

explore possible strategies adopted by plants under Pb

stress. To address these issues, the antioxidant defense

system, and nutrient and polyamine metabolism under Pb

stress were investigated in S. polyrhiza.

Materials and methods

Plant material and lead treatments

Spirodela polyrhiza were collected from unpolluted bodies

of freshwater in Nanjing, China, washed with distilled

water, and acclimated in 1/10 Hoagland solution. They

were cultured in a totally enclosed incubator (Forma 3744,

UK) at a day/night temperature of 26/22 �C. The illumi-

nation procedure consisted of a 16/8 light/dark cycle and a

photon flux density of 50 l mol m-2 s-1 (Horvat 2007).

One week before the experiment, healthy colonies with 3–4

fronds were transferred to 2 l of 1/10 Hoagland solution

(without KH2PO4), treated with Pb(NO3)2, in concentra-

tions of 0, 25, 50, 100, and 400 lM, respectively, and then

placed in a growth chamber under the conditions described

above. All solutions were refreshed every 2 days and all

experiments were performed in triplicate. Physiological

and biochemical indexes were measured after the treat-

ments had been cultured for 7 days.

Measurement of protective enzyme activity

Spirodela polyrhiza (0.5 g fresh weight) was homogenized

on ice with a mortar and pestle in 50 mM sodium phos-

phate buffer (pH 7.8). The solid phase was separated

centrifugally at 10,000g for 20 min and the supernatant

was analyzed. The SOD activity was assayed by monitor-

ing the inhibition of the photochemical reduction of nitro

blue tetrazolium (NBT) according to the method used by

Giannopolitis and Ries (1977). The POD activity was

determined by using the guaiacol method (Maehly 1955).

CAT activity was measured at 405 nm by assaying the

concentration of hydrogen peroxide based on the formation

of its stable complex with ammonium molybdate (Goth

1991).

AsA and GSH determination

To determine the plant contents of AsA and GSH, the fresh

plants (0.5 g) were homogenized in ice-cold 5 % (w/v)

trichloroacetic acid and then centrifuged at 10,000g for

20 min at 4 �C. To measure total AsA, the supernatant was

initially treated with dithiothreitol (which reduces dehy-

droascorbate to ascorbate). Then 0.2 ml of treated super-

natant was added to 0.5 ml of 150 mM phosphate

buffer (pH 7.4) containing 5 mM EDTA and 0.1 ml of

0.5 mM N-ethylmaleimide. After adding 0.4 ml of 10 %

(w/v) tricholoroacetic acid, 0.4 ml of 44 % (v/v) ortho-

phosphoric acid, 0.4 ml of 4 % (w/v) dipyridyl in 70 %

(v/v) ethanol and 0.2 ml of 3 % (w/v) iron trichloride, the

mixture was incubated at 40 �C for 40 min. The color

developed was measured at 525 nm and the results were

expressed as AsA content in the tissue (l mol g-1 fresh

weight). GSH content was determined using a spectro-

photometer at 412 nm (Anderson 1985), after precipitation

with 0.1 M HCl, using GSH reductase, 5, 50-dithio-bis-(2-

nitrobenzoic acid) (DTNB) and NADPH. The content of

GSH was expressed as l mol g-1 fresh weight.

Measurement of Pb accumulation and nutrient element

contents

After 7 days incubation, both the control and the Pb-

exposed plants were washed thoroughly with 10 mM

EDTA to remove metals adsorbed to the surface. They

were oven-dried at 70 �C for 2 days and digested using 3:1

HNO3/HClO4 at 95 �C until the digest solution became

clear. The digested residue was dissolved in a minimal

volume of 7 % HCl and diluted with distilled water. The

solution samples were analyzed for nutrient concentration

218 Plant Growth Regul (2012) 67:217–225

123

by inductively coupled plasma atomic emission spectros-

copy (ICP-ES, Prodigy, Leemanlabsinc, Hudson city,

USA).

Polyamine determination

Plant material (1 g) was homogenized in 4 ml of 6 % (v/v)

cold perchloric acid (PCA), kept on ice for 1 h, and then

centrifuged at 21,000g for 30 min. The pellet was extracted

twice with 2 ml 5 % (v/v) PCA and centrifuged again. The

three supernatants were pooled and used to determine the

contents of free and PS-conjugated PAs, whereas the pellet

was used to determine the contents of PIS-bound PAs. The

pellet was re-suspended in 5 % (v/v) PCA and hydrolyzed

for 24 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 70 �C, and then re-suspended in 1 ml

of 5 % (v/v) PCA for analysis of PIS-bound PAs. For PS-

conjugated PAs, 2 ml of the supernatant were mixed with

2 ml of 12 N HCl and hydrolyzed for 24 h at 110 �C in

flame-sealed glass ampoules. The supernatant, hydrolyzed

supernatant and the pellet were then benzoylated (Aziz and

Larher 1995).

The benzoyl derivatives were separated and analyzed

using a HPLC system (Agilent 1100, USA) equipped with

an UV detector under the following conditions: 200 mm 9

4.6 mm C18 reverse-phase column (Kromasil, Sweden);

particle size, 5 lm; column temperature, 30 �C; mobile

phase, 64 % (v/v) methanol; a flow rate of 0.8 ml min-1, a

detected wavelength of 254 nm. The internal standard was

1, 6-hexanediamine.

Assay of PAO and ODC activities

PAO activities were determined according to the method

used by Gao et al. (2005), with some modifications. Fresh

samples were homogenized in 100 mM potassium phos-

phate buffer (pH 6.5). The homogenate was centrifuged at

10,000g for 20 min at 4 �C. The supernatant was used for

enzyme assay. The reaction mixture contained 2.5 ml of

potassium phosphate buffer (100 mM, pH 6.5), 0.2 ml

4-aminoantipyrine/N,N-dimethylaniline reaction solution,

0.1 ml of horseradish peroxidase (250 U ml-1) and 0.2 ml

of the enzyme extract. The reaction was initiated by the

addition of 0.1 ml of Spd (final concentration, 20 mM) for

PAO determination. One 0.001 absorbance unit change in

the optical density at 550 nm min-1 was considered as one

unit of enzyme activity.

ODC activity was determined according to Zhao

et al. (2003), with some modifications. The reaction mix-

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

100 mM Tris–HCl (pH 8.5), 5 mM EDTA, 40 lM pyri-

doxal phosphate and 5 mM DTT, 0.3 ml of the ODC

enzyme extract, and 0.2 ml of 25 mM Orn. The reaction

mixture was incubated at 37 �C for 60 min, and centri-

fuged at 3,000g for 10 min after which 0.5 ml of the

supernatant was mixed with 1 ml of 2 mM NaOH. Then

10 ll benzoyl chloride was added and the mixture was

stirred continuously for 20 s. After the reaction had pro-

ceeded 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. The mixture was then centrifuged at 1,500

g for 5 min, after which, 1 ml of the ether phase was

collected and evaporated at 50 �C. The remainder was

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

254 nm was measured using a HPLC system (Agilent

1100, USA). A standard curve for Put was used to calculate

the activity of ODC. The ODC activities was expressed as

l mol Put g-1 FW min-1 (U).

Statistical analysis

All assays were carried out in triplicate and results were

expressed as means ± SD. Statistical analysis was per-

formed using the ANOVA test in the Statistical Analysis

System (STATISTICA 6.0).To calculate the significance of

values, means were separated by Duncan’s Multiple Range

test at P \ 0.05.

Results

Effects of Pb on antioxidant enzyme activity

As the Pb concentration increased, the SOD activity

gradually declined after a transitory increase at 25 lM

(rSOD = -0.823, P \ 0.05) (Fig. 1a). When plants were

treated with 400 lM Pb, the reduction was 35.5 % com-

pared to the control. POD and CAT activities responded to

Pb stress differently compared to SOD (Fig. 1b, c). POD

and CAT activities increased initially, reaching peak values

at 50 lM Pb. However, as the concentration of Pb con-

tinued to rise, the activities of POD and CAT declined.

Effects of Pb on AsA and GSH contents

The effect of Pb application on AsA content was similar to

that seen for POD and CAT in S. polyrhiza (Fig. 2a). It

reached a peak value at 50 lM Pb and then declined.

Compared with the control, the GSH concentration

increased markedly with increasing Pb concentration

(Fig. 2b). However, GSH content declined rapidly when

the fronds were treated with 400 lM Pb, to only 72.9 % of

the control.

Plant Growth Regul (2012) 67:217–225 219

123

Effects of Pb on Pb accumulation and nutrient element

contents

All Pb treatments significantly increased the plant Pb

concentration compared with the control, except for the

25 lM Pb treatment. When grown in 400 lM Pb, the Pb

content rose to 19,223.8 lg g-1 FW (Table 1). In response

to the surrounding Pb concentration, the negative correla-

tion coefficients showed that the association between Mg,

Mo, Zn and Pb concentrations were found to be highly

significant (rMg = -0.9814, P \ 0.01; rMo = -0.9831,

P \ 0.05; rZn = -0.9521, P \ 0.01). Fe, Mn and Cu plant

contents dropped at low Pb concentrations, by 35.0, 24.7

and 26.1 % at 50 lM Pb respectively and were elevated at

higher Pb concentrations.

Fig. 1 Effects of Pb on activities of superoxide dismutase (SOD) (a),

peroxidase (POD) (b), and catalase (CAT) (c). Values represent

mean ± SD (n = 3). Value designated over the bars in different

letter are significant different at P \ 0.05

Fig. 2 Effects of Pb on the contents of AsA (a) and GSH (b).Values

represent mean ± SD (n = 3). Value designated over the bars in

different letter are significant different at P \ 0.05

220 Plant Growth Regul (2012) 67:217–225

123

Effects of Pb on polyamine contents

Compared with the control, Pb stress markedly decreased

the level of total Spd (Fig. 3a) (rSpd = -0.9844,

P \ 0.01). When plants were treated with 25 lM Pb, the

total Spm content reached a peak value, which was 3.4

times higher than the control, and then declined afterwards.

The effects of Pb application on total Put were similar to

those seen for total Spm (Fig. 3a) (rPut = -0.8906,

P \ 0.05). When treated with 25 lM Pb, total Put

increased slightly, and then decreased with further increa-

ses in the Pb concentration. Therefore, due to the combined

action of total Spm and total Put under induced Pb stress,

as well as the dramatic decrease in total Spd, the ratio of

total (Spd ? Spm)/Put first increased and then decreased

(Fig. 3b), reaching a peak value at 50 lM Pb.

The trends for free and PS-conjugated PAs were similar

to the trend seen for total PAs in S. polyrhiza (Fig. 3c and

e). The free Spd content declined significantly under Pb

treatment (rSpd = -0.9801, P \ 0.01). When the plants

were cultivated in 25 lM Pb, free Put and Spm contents

reached their peaks. Thus, a significant decrease of the ratio

of free (Spd ? Spm)/Put became apparent as the concen-

tration of Pb rose (Fig. 3d). The PS-conjugated Spd content

also showed a significant decrease (rspd = -0.9775,

P \ 0.01), dropping to 21.1 % of the control. However, the

changes in PS-conjugated Spd and Spm compared with the

control were similar to those seen for free Spd and Spm.

The maximum contents of PS-conjugated Put and Spm

increased to 118.5 and 390.0 % of the control, respectively.

In contrast, an initial accumulation in the Spd level and

a continuous accumulation in the Spm level were observed

on PIS-bound PAs in response to Pb stress. Compared with

the control, the maximum PS-bound Spd content increased

to 2.5 times that of the control when the plants were cul-

tivated in 50 lM Pb, while the PIS-bound Spm content

under 400 lM Pb stresses rose to 3.4 times that of the

control, indicating a marked difference (Fig. 3f). Pb

treatment increased the level of PIS-bound Put up to

100 lM but levels declined thereafter.

Effects of Pb on PAO and ODC activities

PAO activity increased gradually as Pb concentration

increased (Fig. 4a). The maximum increase was 3.9 times

that of the control in the plants which grew in a culture

medium containing 100 lM Pb. However, PAO activity

declined when the plants were treated with 400 lM Pb.

ODC activity reached a peak value at 25 lM Pb, increasing

by 74.5 % (Fig. 4b) compared to the control. However, as

the concentration of Pb rose above 50 lM Pb, ODC

activity declined.

Discussion

A common occurrence with both heavy metal and biotic

stress is the generation of reactive oxygen species (ROS)

(Mithufer et al. 2004). The protective mechanisms adapted

by plants to scavenge free radicals and peroxides involve

several antioxidant enzymes (SOD, POD and CAT) and

antioxidant compounds (AsA and GSH), which inactivate

excess ROS or decrease their generation (Malecka et al.

2001). SOD is the key antioxidant enzyme and is activated

by a number of stress factors (Scandalios 1993). However,

in the present study, the SOD activity was inhibited

(Fig. 1a), which was probably caused by either an inter-

action with specific groups of SOD (a metalloprotein) or

substituted for other functional divalent metal ions already

present in the SOD molecule, as suggested by a number of

reports (Cherian et al. 1999; Somashekaraiah et al. 1992).

In contrast to SOD, the increased POD and CAT activity

may catalyze the oxidation of excess H2O2 caused by Pb

and thus may play a detoxifying role (Fig. 1b, c). However,

POD and CAT activities began to decline as the Pb con-

centration rose to 100 lM, suggesting that the protective

Table 1 Effect of Pb supply on Pb accumulation and nutrient element (lg g-1 FW)

Element content

(lg g-1 FW)

Concentration (lM)

0 25 50 100 400

Pb 0a 1.1 ± 0.1a 958.9 ± 56.2b 1,759.3 ± 79.9b 19,223.8 ± 1,034.2c

Mg 478.4 ± 18.6a 422.9 ± 14.6b 289.4 ± 11.6c 274.0 ± 15.7c 170.4 ± 8.1d

Mo 5.47 ± 0.20a 4.84 ± 0.21b 3.77 ± 0.15c 3.57 ± 0.13c 2.89 ± 0.10d

Zn 33.7 ± 1.5a 28.6 ± 1.4b 21.28 ± 1.0c 20.7 ± 1.2c 18.4 ± 0.7d

Mn 561.5 ± 18.8a 473.1 ± 16.2b 422.8 ± 14.8c 441.1 ± 11.9c 617.2 ± 16.7d

Fe 223.3 ± 13.9a 175.0 ± 10.2b 145.3 ± 8.5c 244.6 ± 9.2d 378.2 ± 9.0e

Cu 2.3 ± 0.1a 2.1 ± 0.1a 1.5 ± 0.2b 1.9 ± 0.1c 3.3 ± 0.2d

Values represent mean ± SD (n = 3). Value designated over the bars in different letter are significant different at P \ 0.05

Plant Growth Regul (2012) 67:217–225 221

123

function of POD and CAT to harmonize and maintain the

stability of the membrane system had reached an upper

limit (Gu et al. 2002). The large increase in AsA and GSH

contents indicated that they might actively participate in

ROS detoxification. A decline seen at the higher Pb con-

centrations may be due to enzyme protein damage caused

by the high accumulation of Pb, which has been suggested

in a number of reports (Ding et al. 2007; Sgherri et al.

2007; Yang et al. 2011). These results suggest that at

higher concentrations of Pb, the protective systems in

S. polyrhiza lost their intrinsic balance and failed to resist

Pb toxicity effectively.

High levels of ROS can disturb metabolic events

through oxidative damage to lipids, proteins, and nucleic

acids (Fridovich 1986). The plasma membrane regulates

the passage of solutes between the cell and the external

environment by selectively absorbing nutrients into the cell

against a concentration gradient (Wang et al. 2007). In the

present study, there was no significant accumulation of

Pb at 25 lM Pb. However, it is clear that significant

Fig. 3 Effects of Pb on the

content of total PAs (a),

[(Spd ? Spm)/Put] total ratio

(b), Free PAs (c),

[(Spd ? Spm)/Put] free ratio

(d), PS-conjugated PAs (e) and

PIS-bound PAs (f). Values

represent mean ± SD (n = 3).

Value designated over the barsin different letter are significant

different at P \ 0.05

222 Plant Growth Regul (2012) 67:217–225

123

accumulation of Pb was induced in S. polyrhiza as Pb

concentrations continued to rise. Increase in toxic metals in

the culture medium resulted in a significant decline in the

uptake of various nutrients which was deleterious to the

plant (Gupta and Chandra 1998; Ouzounidou and Con-

stantinidou 1999). A similar result was observed in the

present study in relation to Mg, Mo and Zn content

(Table 1). This might result from the reduced availability

of energy (ATP), on which the membrane transport sys-

tems depend, and metal-induced disorder in cell metabo-

lism (Xu et al. 2010). Pb treatment also significantly

decreased the levels of Fe, Mn and Cu at low concentra-

tions. Recently, some metal transporters belonging to the

NRAMP (natural resistance associated macrophage pro-

tein) and ZIP (zinc regulated transporter/iron regulated

transporter related protein) families have been identified as

being able to transport Mn and Fe (Guerinot et al. 2000;

Pittman 2005). Competition or mutual interference with

such transporters may form the basis of the negative

interactions between Pb and mineral elements in plants. In

common with other heavy metals, Fe, Mn and Cu are toxic

when in excess and inhibit some metabolic processes, for

example photosynthesis (Adamski et al. 2011). They

increased in this study when Pb concentration rose to

100 lM, accompanied by decreases in the (Spd ? Spm)/

Put ratio and the activity of oxygen-scavenging systems. Pb

exposure stimulates ROS generation, which causes perox-

idative damage to membranes following the influx of Pb

into the cytoplasm.

Polyamines (PAs) are involved in the plant response to

environmental stresses (Roussos and Pontikis 2007; Sudha

and Ravishankar 2002). However, limited information is

currently available regarding how endogenous PA pools

are regulated and balanced under increasing concentrations

of Pb in S. polyrhiza. In the present experiment, a sharp

decline in Spd was observed while Put and Spm levels

increased at first and then declined as Pb concentration

rose. It has been suggested that Spd acts as a protectant for

the plasma membrane against stress damage by maintain-

ing membrane integrity, preventing the activation of

superoxide-generating NADPH oxidases (Roussos and

Pontikis 2007) and/or inhibiting protease and RNase

activity (Roy et al. 2005). In this study, Spd did not play a

positive role in the toxic effect induced by different con-

centrations of Pb. This could be a result of increases in

PAO activity (Fig. 4a), which accelerated the degradation

of Spd. A mass accumulation of Put is generally considered

toxic to plants and eventually leads to apoptotic cell death

if levels become too high (Panicot et al. 2002; Takao et al.

2006). However, as the Pb concentration continued to rise,

a decrease in Put content can be explained by a decrease in

the activity of ODC (Fig. 4b), which would improve

membrane stability and thus contribute to an increased

tolerance to lead poisoning. Spm interacts with many

negative charged molecules, thus modulating the surface

charge and consequently regulating membrane permeabil-

ity (Roy et al. 2005). In the present experiment, the Spm

level was elevated, indicating that Spm was involved in the

adaptive mechanisms of plants under heavy metal toxicity.

Based on these data, the elevation of the (Spd ? Spm)/Put

ratio may be critical in improving Pb tolerance in plants,

which has been suggested in several other reports (Bou-

chereau et al. 1999), indicating that increasing the ratio

might be beneficial to maintaining the structure and func-

tion of membranes. In this study, the ratio was elevated at

low concentrations but declined at higher Pb concentra-

tions (Fig. 3b).

The effects of Pb application on free and PS-conjugated

PAs were similar to those on total PAs in S. polyrhiza.

These results suggest that the effect of decreasing Spd and

Fig. 4 Effects of Pb on activities of PAO (a) and ODC (b). Values

represent mean ± SD (n = 3). Value designated over the bars in

different letter are significant different at P \ 0.05

Plant Growth Regul (2012) 67:217–225 223

123

Spm on free and PS-conjugated PAs made plants less

capable of resisting Pb toxicity. Based on these data, the

elevation of the free (Spd ? Spm)/Put ratio might be

critical in improving stress tolerance in plants. This con-

firms results found in other published papers (Bouchereau

et al. 1999; Wang et al. 2007). However, in this study, the

ratio of free (Spd ? Spm)/Put decreased (Fig. 3d), which

might be a disadvantage to maintaining the structure and

function of membranes, even though free Spm could ten-

tatively serve as a valuable protection countermeasure

against membrane deterioration (Roy et al. 2005). PIS-

bound PAs are thought to have an important role in pro-

tecting the plant against osmotic, salt, drought and oxida-

tion stresses (Roussos and Pontikis 2007; Zhao et al. 2008).

In the present study, PIS-bound Put and Spd gradually

increased followed by a decrease (Fig. 3f), while PIS-

bound Spm markedly increased, which could be an indi-

cation of reduced activity in the enzymes responsible for

their production, as well as of an increased activity of Spm

synthase, the enzyme which catalyzes the production of

Spm from Spd. PIS-bound Spd and Spm showed greater

stability compared to those on Free and PS-conjugated

PAs. Moreover, a continuous accumulation of Spm showed

a positive correlation with the Pb concentration, suggesting

that PIS-bound Spm is closely implicated in the protection

of S. polyrhiza from Pb stress. Spd and Spm might act as

signaling regulators in stress signaling pathways (Kasuk-

abe et al. 2004; Sanchez et al. 2005). The increase in

cellular Spd or Spm has been demonstrated to confer

tolerance to cadmium in sunflower leaf discs and in

leaves of Potamogeton crispus (Groppa et al. 2001; Yang

et al. 2010). Changes in PAs levels due to abiotic stress

may vary depending on the plant species and stress agents

involved.

In conclusion, Pb obviously altered physiological and

biochemical responses, indicating a strong phytotoxicity at

high concentrations in S. polyrhiza. Up-regulation of

scavenging enzyme activities (POD and CAT) and anti-

oxidative compounds (AsA and GSH) provided precise

information on the induction of oxidative stress. The

uptake of Pb was prevented when plants were treated with

12.5 lM Pb. Nevertheless, the intrinsic balance of nutrient

elements in cells was disturbed as the concentration of Pb

increased. Alterations in PAs contents indicated that they

play an important role in S polyrhiza adaptation to Pb-

induced stress. Pb caused a decrease in total Put accumu-

lation but the (Spd ? Spm)/Put ratio increased at low

concentrations. Finally, it seems apparent that PS-bound

Spm was very closely involved in the plant defense

mechanisms connected with Pb stress.

Acknowledgments This study was supported by Project 30870139

of the National Natural Science Foundation and Priority Academic

Program Development of Jiangsu Higher Education Institutions of

China.

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