Physiological responses of Egeria densa to high ammonium concentration and nitrogen deficiency

Chemosphere 86 (2012) 538–545

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Physiological responses of Egeria densa to high ammonium concentration and nitrogen deficiency Shengqi Su a,b,c, Yiming Zhou a, Jian G. Qin b,⇑, Wei Wang a, Weizhi Yao a,c, Liang Song b a

School of Animal Science and Technology, Southwest University, Chongqing 400715, China School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, 5001 SA, Australia c The Chongqing Municipal Key Laboratory in Fishery Science, Chongqing 400715, China b

a r t i c l e

i n f o

Article history: Received 16 March 2011 Received in revised form 14 October 2011 Accepted 20 October 2011 Available online 17 November 2011 Keywords: Ammonium Nitrogen deficiency Aquatic plants Egeria densa Physiological stress

a b s t r a c t High ammonia (i.e. the total of NH3 and NHþ 4 ) concentration or nitrogen deficiency in water can exert stress on growth and health of many aquatic plants. To investigate the physiological impacts of high ammonia-N (NH4Cl) concentration and nitrogen deficiency on plant physiology, apical shoots of submerged macrophyte Egeria densa were first treated with five levels of nitrogen: 0, 1, 10, 30, 60 mg L1 ammonia-N (NH4Cl) for 5 d. After having explored the stress range of ammonia-N, its effect on E. densa was further examined at three levels of ammonium (0, 1, 30 mg L1 ammonia-N) and at six exposure times (0, 1, 2, 3, 5 and 7 d). In testing the concentration-dependent stress, the increase of ammonia-N reduced the amounts of total chlorophyll (chl a and b), soluble proteins and soluble carbohydrates, but increased the activity levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase and peroxidase in E. densa. In the N-free medium, total chlorophyll, soluble proteins, soluble carbohydrates and the activities of SOD and peroxidase in E. densa decreased significantly compared with the control (1 mg L1 ammonia-N). When comparing the ammonia-N impacts over time, the plants showed a declining trend in total chlorophyll, soluble proteins and soluble carbohydrates, but an rising trend in MDA, SOD, peroxidase and catalase in 30 mg L1 ammonia-N over 7 d. Compared with the control, the N-free medium significantly decreased the amounts of total chlorophyll, soluble proteins, soluble carbohydrates, SOD and peroxidase in E. densa over time. Our study indicates that high ammonium (ammonia-N P 10 mg L1) affects the growth of E. densa through inducing oxidative stress and inhibiting photosynthesis, and nitrogen deficiency can also induce an abiotic stress condition for the E. densa growth by reducing photosynthetic pigments, soluble proteins, soluble carbohydrates, and the activity of antioxidant enzymes. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction In a changing environment, plants are inevitably exposed to various stress conditions. A stress situation does not only impose a prominent effect on plant growth, development and distribution (Litav and Agami, 1976; Ni, 2001; Britto and Kronzucker, 2002; Camargo and Alonso, 2006; Cao et al., 2007a; Nimptsch and Pflugmacher, 2007), but also induces reactive oxygen species (ROS) in cells and tissues to cause damage (Foyer et al., 1994; Huang et al., 2004). In plant cells, superabundant ROS including superoxide radical (O2 ), hydrogen peroxide (H2O2) and hydroxyl radical (OH) can damage tissues and seriously disrupt metabolism through oxidation to membrane lipids, proteins, pigments and nucleic acids (Misra

Abbreviations: MDA, malondialdehyde; SOD, superoxide dismutase; ROS, reactive oxygen species; O2 , superoxide radical; H2O2, hydrogen peroxide; OH, hydroxyl radical. ⇑ Corresponding author. Tel.: +61 882013045; fax: +61 882013015. E-mail address: jian.qin@flinders.edu.au (J.G. Qin). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.10.036

and Gupta, 2006). To eliminate or reduce ROS, plants have evolved various protective mechanisms. Hereinto, anti-oxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) compose a key protective anti-oxidant defense system in plant. The capacity of the anti-oxidant defense system often increases under a stress condition (Rout and Shaw, 2001; Misra and Gupta, 2006). In plants, therefore, fluctuations of many metabolites such as chlorophyll (Ferrat et al., 2003), soluble proteins (Amini and Ehsanpour, 2005), soluble carbohydrates (Costa and Spitz, 1997; Sativir et al., 2000), malondialdehyde (Bailly et al., 1996; Chen et al., 2003), and anti-oxidant enzymes are important indexes to measure the response of physiological oxidative stress. Ammonia (i.e. total NH3 and NHþ 4 ) is an important nitrogen source for plant growth and development. However, excessive ammonia often presents a stress condition for plant growth (Britto and Kronzucker, 2002). Total ammonia in aqueous solution consists of ammonium ion (NHþ 4 ) and un-ionized ammonia (NH3), with relative concentrations being regulated by pH and temperature.

S. Su et al. / Chemosphere 86 (2012) 538–545

Both forms are reported to become toxic at higher concentrations (Litav and Agami, 1976; Körner et al., 2001). In an aquatic environment, ammonia derived from agricultural run-off, atmospheric deposition, industrial discharge and urban sewage can reach 10– 200 mg L1 (Körner et al., 2001). High ammonia concentration often triggers eco-physiological stress on aquatic plants, resulting in a decline or loss of macrophytes (Litav and Agami, 1976; Ni, 2001; Camargo and Alonso, 2006; Cao et al., 2007a; Nimptsch and Pflugmacher, 2007). High ammonia can negatively impact survival, growth and reproductive capacity in plant (Best, 1980; Ni, 2001; Cao et al., 2007a; Li et al., 2007), and reduce the contents of chlorophylls (Wang et al., 2008; Wang et al., 2010), soluble proteins and soluble carbohydrates in plant tissues (Cao et al., 2004; Cao et al., 2007b; Yan et al., 2007). High ammonia often increases malondialdehyde (MDA) content (Cao et al., 2009; Jiao et al., 2009) in plant. Moreover, ROS induced by high ammonia stress can lead to the changes of antioxidant enzyme activities including SOD, CAT and POD for plant to cope with stress (Nimptsch and Pflugmacher, 2007; Wang et al., 2008; Cao et al., 2009; Wang et al., 2010). Previous studies have showed that the toxicity of 1.5–28 mg L1 ammonia-N to aquatic plants can be detected from 4 to 8 d of exposure, but toxic responses are species-specific (Nimptsch and Pflugmacher, 2007; Wang et al., 2008). However, the stress effect of high ammonia on aquatic plants that have potential use for environmental remediation in populated water is little known (Nimptsch and Pflugmacher, 2007). N deficiency is also a common stress for growth of many plants (Huang et al., 2004; Polesskaya et al., 2004). In terrestrial plants, N deficiency can suppress plant growth by reducing the photosynthetic capacity of leaves (Verhoeven et al., 1997; Lu and Zhang, 2000; Milroy and Bange, 2003; Huang et al., 2004), degrade photosynthetic pigments and proteins (Huang et al., 2004; Polesskaya et al., 2004), and reduce enzyme synthesis in plants (Verhoeven et al., 1997; Huang et al., 2004; Polesskaya et al., 2004). However, little is known on the role of N deficiency in regulating chlorophyll content, metabolites and antioxidant enzymes in aquatic plants. Egeria densa (Planch), commonly known as Brazilian waterweed, is a widely used plant in ornamental fish industry in the world via aquarium trades (Haramoto and Ikusima, 1988). It lives in an environment with moderately high light intensity, and conducts photosynthesis in stems and leaves. Because of its ease in propagation through double node, root crown or apical shoot (Getsinger and Dillon, 1984; Haramoto and Ikusima, 1988) and high capacity to absorb nutrients such as ammonium and phosphorus from water column (Feijoó et al., 2002), E. densa has been used as a model plant to assess water quality, heavy metal accumulation and toxicity, and metabolism of pesticides in plants (Malec et al., 2009). E. densa is able to live in 6 mg L1 ammonia-N, which sheds a hope as a candidate to remove ammonia from polluted water (Feijoó et al., 2002). However, the ammonia tolerance of this plant has not been further examined. In this study, we tested the hypothesis that either high ammonium or nitrogen deficiency stresses this model species of aquatic plants through regulating leaf chlorophyll content, metabolite levels, and plant growth. We first treated the E. densa apical shoots with a wider range of NH4Cl concentrations and described the plant response at the end of the experiment. Then, the plants were treated with a narrow range of NH4Cl concentrations, but with more frequent observations over time. In the whole experiment, NO 3 was excluded in the culture medium. The objectives of this study were (1) to investigate the effects of excessive ammonia in water on the content of total chlorophyll (i.e. chl a and b), soluble proteins, soluble carbohydrates, MDA and the activity of antioxidant enzymes (SOD, catalase and peroxidase) in E. densa; and (2) to evaluate the impact of nitrogen deficiency on the physiological stress response of E. densa.

539

2. Materials and methods 2.1. Plant material and experimental design The E. densa were obtained from an aquarium shop in Chongqing, China. Plants were cultured for over 2 weeks in two aquariums (50  40  40 cm) nourished with Tetra Plant Florapride (Tetra Werke Company, Germany). Prior to exposure, 8 cm of apical shoots were acclimatized to 1 mg L1 (N) NH4Cl for 5 d in 1-L glass beakers containing the primary culture medium. All beakers were placed in controlled incubators with a photoperiod of 12 h light and 12 h dark, temperature 25 ± 0.2 °C and 6 500 lx light intensity. The other ingredients of the culture medium included 1% Hoagland’s trace elements (Hoagland and Arnon, 1950) and the macro-nutrition elements adapted from the recipe of Smart and Barko (1985). The N-free macro-nutrition medium was composed of 0.008 mM KH2PO4, 0.4 mM MgSO47H2O, 0.5 mM CaCl22H2O and 0.5 mM CaCO3. The minimum requirement of ammonia for the normal growth of E. densa was above 1 mg L1 ammonia (Feijoó et al., 2002). Therefore, the range of 1–60 mg L1 (N) NH4Cl was chosen in this study to investigate the stress of high ammonia on E. densa and 1 mg L1 ammonia-N was set as a control representing the nutrient level at which normal growth was sustained. Meanwhile, the treatment of nitrogen free (N-free) was set to compare the stress of nitrogen deficiency on E. densa with that of normal ammonia supply. The whole study consisted of a concentration-dependent experiment and a duration-dependent experiment. The former included treatments of 1(control), 10, 30, 60 mg L1 (N) NH4Cl and N-free, and the experiment was terminated by d 5 after treatment. The latter narrowed the range of the ammonia concentrations to 1(control), 30 mg L1 (N) NH4Cl and N-free, but the plants were sequentially sampled on 1, 2, 3, 5, and 7 d after treatment.

2.2. Plant exposure and sampling The ammonia stock solution (1000 mg L1 N) was prepared by dissolving 3.9 g NH4Cl per liter in de-ionized water. After acclimation, 10 E. densa apical shoots of an equal size were planted in one beaker in triplicate. In the concentration-dependent experiment, after the plant biomass was weighed and recorded, the apical shoots were treated with 1, 10, 30, 60 mg L1 (N) NH4Cl and N-free in triplicate. Throughout the acclimation and exposure periods, the nutrient solution was renewed every 2 d and the pH of the solution was adjusted to 7.0 ± 0.1 with 0.5 M H2SO4 or 1 M NaOH twice a day. At the end of 5-d exposure period, plants in each beaker were harvested, rinsed with distilled water and blotted dry, and then the plant biomass was weighed to evaluate plant growth. Then, two apical shoots from each replicate were randomly collected and pooled into one sample (2.0–2.3 g) for biochemical measurements. All of the samples were stored at 20 °C before biochemical analyzes. In the time-dependent experiment, the plants were exposed to 1, 30 mg L1 (N) NH4Cl in triplicate and an N-free medium, and were sampled on 1, 2, 3, 5, and 7 d. On each sampling day, two apical shoots from each replicate were randomly collected, rinsed with distilled water, blotted dry, and pooled into one sample (2.0–2.3 g) for biochemical measurements. The renewal of nutrient solution and the management of pH were the same as above. According to the formula developed by Caicedo et al. (2000), the proportion of NH3-N was 0.05). Likewise, chlorophyll a/b ratio decreased quickly with increasing ammonia-N. Regarding the effect of N-free treatment, the total chlorophyll of the apical shoots decreased by 31.6% compared with the control (P < 0.05, Fig. 1A), but Chl a/b between the two treatments showed little change.

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Table 1 Growth characteristics of apical shoots in the concentration-dependent experiment. Unlike letters in the same row represent significant differences (P < 0.05). Values of biomass are mean ± SE (n = 10). Values of lateral bud number length are mean with the range in parentheses. Ammonia-N (mg L1)

N-free

1 (control)

10

30

60

Biomass gain (mg per apical shoot) No. of lateral buds per apical shoot Length (mm) of lateral buds per apical shoot

21.8 ± 2.2b 0 0

38.1 ± 3.2a 0.7 (0–2) 4.6 (4.0–5.5)

7.5 ± 2.9c 0.3 (0–1) 2.3 (1.7–3.2)

25.5 ± 2.7d 0.2 (0–1) 2.0 (1.5–2.5)

41.0 ± 3.5e 0.2 (0–1) 1.5 (1.5–1.5)

a

1.8

a b

2.4

1.4 1.2

SP (mg·g -1 FW)

1.5

1.3

0.8

1.2

0.4

a

a b

#

1.6

1.6

14 12

1.7

#

2.0

(A)

Chl a/b

Total Chl

a

Chl a/b

-1

Total Chl (mg L FW)

(A) 2.8

c

10 8 6 4

1.1

0.0

2

1.0 N-free

1 (control)

10

30

60

0

-1

Ammonia-N (mg L )

N-free

1 (control)

30

10

60

-1

Ammonia-N (mg L ) *

3

*

+

(B) 14

2.6

12

2.4 2.2

+ 2

+

+

2 1.8

1.6

1.6

1.2

Chl of N-free

Chl of 1 mg L ¹

Chl of 30 mg L ¹

Chl a/b of N-free

Chl a/b of 1 mg L ¹

Chl a/b of 30 mg L ¹

1.4 1.2

0.8

1 0

1

2

3

5

-1

2.4

2.8

SP (mg g FW)

-1

Total Chl mg L FW)

2.8

Chl a/b

(B) 3.2

*

10

+

+

*

*

+

+

8 6 4

N-free

1 mg L ¹ (control)

30 mg L ¹

2 0 -1

0

1

2

7

3

4

5

6

7

8

Time (d)

Time (d) Fig. 1. The contents of total chlorophyll (Chl) in E. densa apical shoots exposed to ammonia-N and N-free for 5 d (A); bars with different letters indicate significant differences among the concentrations of ammonia-N (P < 0.05); bars with a ‘‘#’’ sign show a significant difference between the N-free treatment and control (P < 0.05); changes of chlorophyll over time between treatments (B); bars with a asterisk () sign represent significant differences between the 30 mg L1 ammonia-N treatment and control, whereas bars with a ‘‘+’’ sign show a significant difference between the N-free treatment and control (P < 0.05). Values are mean ± SE (n = 3).

In the time-dependent experiment, total chlorophyll of apical shoots in 30 mg L1 ammonia-N was higher than that in the control on d 1 (P < 0.05, Fig. 1B), but became lower than that of the control by d 7 (P < 0.05). Meanwhile, total chlorophyll in the N-free treatment was significantly lower than the control from d 2 to 7 (P < 0.05). The chlorophyll a/b ratio of apical shoots in the control fluctuated little. In contrast, Chlorophyll a/b of apical shoots in other two ammonia treatments declined over time, especially in 30 mg L1 ammonia-N. 3.3. Soluble proteins (SP) In comparison to the control, the soluble proteins in the apical shoots treated with 30 or 60 mg L1 ammonia-N for 5 d was significantly declined (P < 0.05), but its change in 10 mg L1 ammonia-N was not significant (P > 0.05, Fig. 2A). The soluble proteins in the apical shoots in the N-free treatment were significantly lower than that in the control (P < 0.05). Considering the change over time (Fig. 2B), soluble proteins in the apical shoots treated with 30 mg L1 ammonia-N were significantly lower than that of the control from d 3 onward (P < 0.05).

Fig. 2. Soluble protein (SP) content in E. densa apical shoots exposed to ammonia-N and N-free at the end of 5 d (A) and its change over time in 7 d (B). Symbols are referred to Fig. 1.

However, soluble proteins of apical shoots in the N-free treatment were significantly lower than that in the control beyond d 2 (P < 0.05). 3.4. Soluble carbohydrates (SC) Comparing to the control, soluble carbohydrates in the apical shoots treated with 10, 30, and 60 mg L1 ammonia-N significantly decreased (P < 0.05, Fig. 3A). Similarly, soluble carbohydrates of the apical shoots in the N-free treatment were significantly lower than in the control (P < 0.05). Although the level of soluble carbohydrates in the 60 mg L1 ammonia-N was lower than that in the 10 mg L1 ammonia-N (P < 0.05), there was no significant difference in soluble carbohydrates between 30 and 60 mg L1 ammonia-N, or between 10 and 30 mg L1 ammonia-N (P > 0.05). In the time-dependent experiment (Fig. 3B), soluble carbohydrates of the apical shoots in the 30 mg L1 ammonia-N or in the N-free treatment were significantly lower than that in the control starting from d 2 (P < 0.05). 3.5. Malondialdehyde (MDA) Significant increases of MDA were observed in the treatments of 30 and 60 mg L1 ammonia-N when compared with the control

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(A) 7

(A)

a b

SC (%)

5

bc

c

#

4

4

3 2

a b

MDA (nmol g-1 FW)

6

3 c 2

c

1

1

0 0

N-free N-free

1 (control)

10

30

1 (control)

60

10

30

60

-1

Ammonia-N (mg L )

-1

Ammonia-N (mg L )

(B)

7 6

*

SC (%)

5 4

+

* *

*

+

+

+

3

4

*

3

1 N-free

1 mg L ¹ (control)

*

*

2

2

N-free

*

*

MDA (nmol g-1 FW)

(B)

30 mg L ¹

1 mg L ¹ (control)

30 mg L ¹

0

1

-1

0

1

2

3

4

5

6

7

8

Time (d)

0 2

3

4

5

6

7

8

Time (d) Fig. 3. Soluble carbohydrate (SC) content in E. densa apical shoots exposed to ammonia-N and N-free for 5 d (A) and their change over time for 7 d (B). Symbols are referred to Fig. 1.

(P < 0.05, Fig. 4A). MDA was not different between the control and the 10 mg L1 ammonia-N treatment (P > 0.05), but the level of MDA in the 30 mg L1 ammonia-N treatment was higher than that in the 60 mg L1 ammonia-N (P < 0.05). On the other hand, the MDA in N-free treatment was not different from that in the control (P > 0.05). In the time-dependent experiment (Fig. 4B), the MDA content in apical shoots in the treatment of 30 mg L1 ammonia-N was significantly higher than that in the control after treatment implementation for 1 d (P < 0.05), but the MDA in the control and in the N-free treatment was not different (P > 0.05). 3.6. Superoxide dismutase (SOD) Compared with the control, the SOD activity in the apical shoots treated with 30, and 60 mg L1 ammonia-N for 5 d was increased (P < 0.05), but the increase in the 10 mg L1 ammonia-N was not significant (P > 0.05, Fig. 5A). In contrast, there was also a significant SOD reduction in the N-free treatment compared with the control (P < 0.05). However, the SOD levels were not different between the control and 10 mg L1 ammonia-N or between 30 and 60 mg L1 ammonia-N for 5 d (P > 0.05). In the time-dependent experiment, the SOD activity in the apical shoots treated with 30 mg L1 ammonia-N was significantly higher than that in the control starting from d 1 and the ascending pattern lasted 7 d (P < 0.05, Fig. 5B). Yet, the SOD activity of the apical shoots in the N-free treatment was lower than that in the control from d 1 to 7, except on d 3 (P < 0.05). 3.7. Peroxidase (POD) Compared with the control, the peroxidase activity in the apical shoots treated with 30, and 60 mg L1 ammonia-N for 5 d increased (P < 0.05), but the increase in the 10 mg L1 NHþ 4 was not significant (P > 0.05, Fig. 6A). There was also a significant peroxidase reduction in the N-free treatment compared with the control (P < 0.05). However, the peroxidase levels were not

Fig. 4. The malondialdehyde (MDA) content in E. densa apical shoots exposed to ammonia-N and N-free for 5 d (A), and its change over time for 7 d (B). Symbols are referred to Fig. 1.

(A)

6 5

a a b

4

b

-1

1

SOD (nkat mg protein)

0

#

3 2 1 0

N-free

1 (control)

10

30

60

-1

Ammonia-N (mg L )

(B) SOD (nkat mg-1 protein)

-1

*

6 *

5 * 4 * *

3

+

+ 2 +

+

1 N-free

1 mg L ¹ (control)

30 mg L ¹

0 -1

0

1

2

3

4

5

6

7

8

Time (d) Fig. 5. The superoxide dismutase (SOD) activity in the E. densa apical shoots exposed to ammonia-N and N-free for 5 d (A) and its change over time in 7 d (B). Symbols are referred to Fig. 1.

different between the control and 10 mg L1 ammonia-N or between 30 and 60 mg L1 ammonia-N for 5 d (P > 0.05). In comparison to the control, the peroxidase activity in the apical shoots treated with 30 mg L1 ammonia-N increased on d 1 (P < 0.05), but this elevated peroxidase subsequently became insignificant until d 5 and 7 (P < 0.05, Fig. 6B). Compared with the control, the decrease of peroxidase activity in the apical shoots in the N-free treatment became significant after d 5 (P < 0.05).

S. Su et al. / Chemosphere 86 (2012) 538–545

-1

POD (nkat mg protein)

(A)

9

of 10 mg L1 ammonia-N or between 10 and 30 mg L1 ammonia-N was not significant (P > 0.05). Similarly, the catalase activity in the N-free treatment and the control was not different (P > 0.05). In the duration-dependent experiment (Fig. 7B), the treatment of 30 mg L1 ammonia-N led to a remarkable increase of the catalase activity from d 3 to 7 compared to the control (P < 0.05). The N-free treatment did not cause any significant decrease in the catalase activity compared with the control before d 5, but resulted in a significant reduction on d 7 (P < 0.05).

a a

8

b

b

7 6

543

#

5 4 3 2 1 0 N-free

1 (control)

10

30

60

4. Discussion

-1

Ammonia-N (mg L )

4.1. Response of E densa to ammonia-N stress

(B)

9

POD (nkat mg-1 protein)

8

*

*

7

*

6 5

+

4

+

3 2

N-free

1 mg L ¹ (control)

30 mg L ¹

1 0 -1

0

1

2

3

4

5

6

7

8

Time (d) Fig. 6. The peroxidase activity in E. densa apical shoots exposed to ammonia-N and N-free for 5 d (A) and its change over time for 7 d (B). Symbols are referred to Fig. 1.

3.8. Catalase (CAT)

(A)

17

CAT (nkat mg -1 protein)

After treated with ammonia-N for 5 d, catalase activities of in the apical shoots treated with 30 and 60 mg L1ammonia-N were higher than those in the control (P < 0.05, Fig. 7A), but the difference of catalase activities between the control and the treatment

16

a b

15

bc

14

c

13 12 11 10 N-free

1 (control)

10

30

60

-1

Ammonia-N (mg L )

(B) CAT (nkat mg-1 protein)

17 16

*

15

*

*

14 13

+

12

N-free

11

1 mg L ¹ (control)

30 mg L ¹

10 -1

0

1

2

3

4

5

6

7

8

Time (d) Fig. 7. The catalase activity in E. densa apical shoots exposed to ammonia-N and Nfree 5 d (A) and its change over time for 7 d (B). Symbols are referred to Fig. 1.

The existing literature in plant toxicology demonstrates that excessive ammonium is prone to trigger stress in aquatic plants, resulting a decrease of chlorophyll, a fluctuation of metabolites such as soluble carbohydrates and soluble proteins, MDA, SOD, peroxidase and catalase (Cao et al., 2003; Cao et al., 2007a, 2007b; Nimptsch and Pflugmacher, 2007; Wang et al., 2008). Our study has shown that high ammonia-N not only triggered the reduction of chlorophyll, soluble proteins and soluble carbohydrates, but also enhanced MDA and activities of SOD, peroxidase and catalase. Furthermore, plant lost biomass when ammonia-N was above 10 mg L1, indicating that the ammonia stress directly affects plant growth performance. These results suggest that excessive ammonia can produce negative impacts on the leaf processes, metabolite levels and the antioxidant system in aquatic plants. Previous studies showed that excessive ammonia can decrease total chlorophyll in aquatic plants such as Vallisneria natans (Wang et al., 2008) and Hydrilla verticillata (Yan et al., 2007). In this study, total chlorophyll in E. densa was reduced by high ammonia-N either in the concentration-dependent experiment or in the timedependent experiment. The high level of ammonia-N can inhibit the synthesis of pigments by suppressing plants to uptake and transport important cations such as magnesium and calcium necessary to pigment synthesis (Britto and Kronzucker, 2002). It can also lead to the chlorophyll degradation by scavenging of O2 induced by excessive ammonia-N (Polesskaya et al., 2004). Thus, our results support an early finding that ammonia-N stress can damage the photosynthetic system and inhibit photosynthesis in aquatic plants (Wang et al., 2008). The quantitative changes of soluble proteins in plants are responsible for adaptation in metabolic pathways under stress conditions (Amini and Ehsanpour, 2005). Protein synthesis and degradation respond differentially to ammonia-N depending on plant ammonia tolerance (Domínguez-Valdiviaa et al., 2008). Cao et al. (2004) reported that the contents of soluble proteins in Potamogeton crispus showed a unimodal response to elevated ammonia availability (0–20 mg L1 ammonia-N) in 48 h, with the highest protein content at 1 mg L1 ammonia-N. Yan et al. (2007) showed an increase of protein contents in H. verticillata at 0.5–4 mg L1 ammonia-N, but a remarkable reduction at 4–16 mg L1 ammonia-N. In the present study, we found the accumulation of proteins in E. densa apical shoots in the control and 10 mg L1 ammonia-N, but protein reduction at 30 and 60 mg L1 ammonia-N in the timedependent experiment. This supports the notion that E. densa, when exposed to a tolerant content of ammonia-N, may possibly strengthen detoxification of ammonia-N by increasing the assimilation of ammonia-N (Li et al., 2007; Cao et al., 2009), and that soluble proteins can be a form of nitrogen storage and can be reutilized as a nitrogen source when the ammonia stress is over (Amini and Ehsanpour, 2005). Given an excessive concentration of ammonia-N (>60 mg L1) or a prolonged-time exposure, the decrease in protein content in E. densa may be either caused by

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protein degradation under stress (Palma et al., 2002), or by protein fragmentation due to toxicity of reactive oxygen species (John et al., 2008). As important metabolites in plant metabolism (Harborne and Turner, 1984), soluble carbohydrates play a number of eco-physiologic roles in plant protection against wounds and infection, as well as in the detoxification of foreign substance (Sativir et al., 2000). Abiotic stress is liable to change the content of soluble carbohydrates in plants (Costa and Spitz, 1997). In the present study, E. densa exposed to 1–60 mg L1 ammonia-N for 5 d exhibited a continuous decline in soluble carbohydrates with elevated concentration of ammonia-N. In addition, the E. densa treated with 30 mg L1 ammonia-N in the duration-dependent experiment showed a remarkable decline in soluble carbohydrates over time. Our findings are also supported by previous studies that soluble carbohydrate reduction occurs in high ammonia-N in V. natans at 20 mg L1 (Cao et al., 2004) and in H. verticillata at 4 mg L1 ammonia-N (Yan et al., 2007). The dynamic of ammonia-N concentration and soluble carbohydrates in plant cells can be explained by the detoxification of ammonium in plant cells (Cao et al., 2009). Detoxification of ammonia-N in many plants involves active removal of NHþ 4 from the plant cells and synthesis of free amino acids and amines. The energy required to support these processes can lead to a reduction of soluble carbohydrates in plant cells (Harborne and Turner, 1984). MDA is the end product resulted from lipid peroxidation of extra free radicals, and the concentration of MDA is an important index of physiological stresses (Bailly et al., 1996; Chen et al., 2003). The change of MDA content indicates the degree of membrane damage of plant cells by stress (Huang et al., 2007). In this study, the MDA contents in E. densa significantly increased after exposing to ammonia-N P 30 mg L1 for 5 d. In the duration-dependent trial, the MDA contents in E. densa treated with 30 mg L1 ammonia-N continuously increased with exposure time. This suggests that high ammonia-N may strengthen the accumulation of extra free radicals leading to oxidative damage and production of MDA. Our results support the findings of increased MDA in Myriophyllum spicatum leaves (Jiao et al., 2009) and elevated MDA in H. verticillata leaves (Jin et al., 2008) under ammonia-N stress. In contrast, a reduction of MDA in plant leaves under ammonia-N stress was detected in M. mattogrossense (Nimptsch and Pflugmacher, 2007) and V. natans (Wang et al., 2008). It seems that the response of MDA in plants to ammonia-N is species specific and the underlying mechanism needs further investigation. As SOD in plant cells is a primary defense barrier against ROS and is an antioxidant response marker (Misra and Gupta, 2006; Domínguez-Valdiviaa et al., 2008), a high SOD activity reflects the level of stress (Apel and Hirt, 2004). Wang et al. (2008) found that the activity of SOD in V. natans was initially up-regulated under ammonia-N stress, but it decreased with a lengthened exposure or at an ammonia-N concentration >1.2 mM. A similar result was reported in P. crispus (Cao et al., 2004) and in H. verticillata (Wang et al., 2010). However, our study showed that the activity of SOD in E. densa apical shoots increased significantly with elevated ammonia-N and prolonged exposure. This implies that E. densa, under the oxidative stress caused by ammonia, is liable to enhance the ability of scavenging superoxide radical by increasing the activity of SOD to tolerate ammonia stress as suggested by Feijoó et al. (2002). In plant, the rapid removal of H2O2 is critical to cell function and otherwise H2O2 can diffuse across membranes to react with O2 resulting in the formation of destructive OH (Wang et al., 2008, 2010). Peroxidase and catalase can decompose H2O2 to H2O and O2 (Misra and Gupta, 2006). Peroxidase uses H2O2 as an electron donor to metabolize phenolic compounds (Quiroga et al., 2000) and catalase is able to cope with H2O2 from the photorespiratory cycle (Zhu et al., 2000). Nimptsch and Pflugmacher (2007) detected

that the activities of catalase and peroxidase in M. mattogrossense increased slowly below 10 mg L1 ammonia-N, but increased significantly above 30 mg L1 ammonia-N. In the present study, we discovered that the activities of peroxidase and catalase in E. densa apical shoots were enhanced when ammonia-N was over 30 mg L1 or by lengthened exposure to ammonia-N, which is consistent with the report by Nimptsch and Pflugmacher (2007). This result suggests that the E. densa stressed by high concentrations of ammonia-N can produce excess H2O2, resulting in more active decomposition of H2O2 by peroxidase and catalase, and also indicates the strong resistance of this plant to ammonia-N stress. Similar results were reported in other aquatic plants such as V. natans (Wang et al., 2008), H. verticillata (Wang et al., 2010). Therefore, we suggest that both elevated peroxidase activity and catalase activity be used as an ammonia-N stress marker enzyme with a high affinity to H2O2 (Domínguez-Valdiviaa et al., 2008). 4.2. Response of E. densa to nitrogen deficiency In terrestrial plants, N-deficient plants exhibit reduction in photosynthetic capacity relative to the N-replete control (Lu and Zhang, 2000; Milroy and Bange, 2003; Huang et al., 2004). For example, N deficiency in both rice Oryza sativa L. (Huang et al., 2004) and fingered citron Citrus medica var. sarcodactylis S. (Guo et al., 2009) causes significant reduction of chlorophyll and soluble proteins, and decline in the activities of SOD, peroxidase and catalase in the stressed leaves. In our study, the decreases of total chlorophyll, soluble proteins, and activity of SOD, peroxidase and catalase in the Nfree treatment over the experiment period exhibited a similar pattern as found in the above mentioned studies. This implies that N deficiency is also an abiotic stress for the E. densa growth. On the other hand, we detected no significant difference of the MDA content between the control and the N-free treatment over 7 d. This was similar to the result of Huang et al. (2004) who reported that the content of MDA in the rice treated with N deficiency changed little in the first 10 d, but exhibited a rapid increase afterwards. This suggests that a short-term (a week) N deficiency for E. densa may not lead to a serious lipid peroxidation. Still, some further studies need to be carried out to explore the prolonged time effects of N-free on the lipid peroxidation in E. densa. Verhoeven et al. (1997) pointed out that a key effect of N limitation was to lower the capacity for carbon assimilation due to the low synthesis of Calvin cycle enzymes. In the present study, we found that N deficiency remarkably decreased soluble carbohydrates in E. densa, and we regarded this as being caused by the lowered capacity for carbon assimilation under the condition of N deficiency. In conclusion, the results of this study indicate that E. densa can grow well at low concentrations of ammonia-N (1 mg L1) in the water column. At high ammonium (>10 mg L1), the excess ammonia-N can trigger oxidative stress in the E. densa tissues as indicated by the increase in antioxidant enzymes (SOD, peroxidase and catalase) and membrane lipid peroxidation (such as MDA), which may be partially responsible for the decline of growth, total chlorophyll, soluble proteins and carbohydrates. Likewise, nitrogen deficiency can also induce an abiotic stress situation for the E. densa growth by reducing the photosynthesis as evidenced by the degradation of photosynthetic pigments, soluble proteins and carbohydrates, and the decline in the activity of antioxidant enzymes. Acknowledgments This work was supported by the Fundamental Research Funds for the Central Universities of China (XDJK2009C170), the Doctor Initiation Fund of South West University, China (SWU20710902), and the State Scholarship Fund from the China Scholarship Council to support the senior author’s study at Flinders University.

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