Nitric oxide production and antioxidants responses in maize under lead stress

Document Type : Research Paper

Authors

Department of Biology, Faculty of science, Urmia University, Urmia, Iran

Abstract

Lead pollution is an important issue in the world. In this research maize plants were treated with different concentrations of lead (0, 0. 25, 0.75, 1, 1.5, 2.5 and 5 mM) was applied as lead (II) nitrate for 10 days. The results showed that increase in lead concentration leads to reduction in growth, chlorophyll a and b content and increase in oxidative damages. Application of Pb significantly increased hydrogen peroxide content which followed by a significant level of lipid peroxidation. These changes were accompanied by increase in nitric oxide content in the low and its reduction in the high concentrations. Small increase in generation of hydrogen peroxide and nitric oxide are envisaged at low concentrations as messengers in signaling pathways that may act in triggering defense functions to detoxify lead. Therefore plant’s stress tolerance variables, including proline, ascorbic acid, flavonoid and anthocyanins content and activity of antioxidant enzymes enhanced under different lead applications.

Keywords


Article Title [فارسی]

تولید نیتریک اکسید و پاسخ های آنتی اکسیدان ها در گیاه ذرت تحت تنش سرب

Authors [فارسی]

  • رویا زنگنه
  • رشید جامعی
دانشگاه ارومیه- دانشکده علوم- گروه زیست شناسی
Abstract [فارسی]

آلودگی سرب یک چالش مهم در جهان محسوب می شود. در این پژوهش گیاه ذرت با غلظت های مختلف سرب (صفر، 0.25، 0.75، 1، 1.5، 2.5 و 5 میلی مولار) به مدت ده روز تیمار شدند. نتایج نشان داد که افزایش در غلظت سرب منجر به کاهش رشد، محتوای کلروفیل a و b و افزایش در خسارات اکسیداتیو گردید. کاربرد سرب به طور معنی داری میزان پراکسید هیدروژن را افزایش داد که سطح قابل توجهی از پراکسیداسیون لیپیدها را به دنبال داشت. این تغییرات با افزایش مقدار اکسید نیتریک در غلظت های پایین و کاهش آن در غلظت های بالا همراه بود. کمی افزایش در تولید پراکسید هیدروژن و نیتریک اکسید در غلظت های کم به عنوان پیامبر در مسیرهای سیگنالینگ انتظار می رود که در تحریک عملکردهای دفاعی برای سم زدایی سرب عمل کنند. بنابراین متغیرهای تحمل به تنش گیاهان، از جمله پرولین، اسید اسکوربیک، فلاونوئید و آنتوسیانین ها و فعالیت آنزیم های آنتی اکسیدانی تحت تیمار غلظت های مختلف سرب افزایش می یابد.

Keywords [فارسی]

  • آنتی اکسیدان ها
  • تنش سرب
  • نیتریک اکسید
  • تنش اکسیداتیو
  • گیاه ذرت


Introduction

Lead (Pb) is a dangerous heavy metal contaminant that leads to inhibition of metabolic processes, growth and productivity of plants (Sharma and Dubey 2005). One of the detrimental effects of Pb is the balanced cellular redox status disruption and oxidative stress induction (Verma and Dubey 2003). It is well characterized that H2O2 accumulation, as the most stable form of reactive oxygen species (ROS), has a harmful effect on plants, but can act as messengers involved in signal transduction pathways, especially at low amounts (Cuypers et al. 2016). Previous data have indicated the involvement of nitric oxide (NO) in plant acclimation and heavy metal tolerance (Yu et al. 2012). The activity of the signal transductor NO can cause changes in the antioxidant system and the activation of numerous defense mechanisms under stress conditions (Xiong et al. 2010). The interaction between NO and H2O2 could also be involved in response to abiotic stresses including Pb. At low Pb content, sometimes plants can show adaptive response. However, at high Pb concentration, plants cannot combat Pb-induced oxidative stress (Kaur et al. 2015).

Plants respond to heavy metal stress by activating multiple responses. Under metal stress conditions, metal ion chelation is one of the defense strategies in plants (Hassan and Aarts 2011). Proline is one of the chelating compounds, which contributes to metal chelation in the cytoplasm and is involved in the amelioration of detrimental effects owing to heavy metal excess (Kovacik et al. 2010). To alleviate oxidative stress, plants have enzymatic and non-enzymatic antioxidant defense mechanisms to quench ROS (Smirnoff 1993). So, this study aimed to evaluate lead stress effects on maize (Zea mays L.) plants by evaluating chlorophyll a and b, lipid peroxidation, endogenous signaling molecules content (NO and H2O2), non-enzymatic antioxidants and enzymatic antioxidants activity after exposure to different lead concentrations.

 

Materials and Methods

Experiment preparation

Seeds of maize (cv. 704) were provided from the Agriculture Research Center of Kerman, Iran. Uniform-sized seeds were surface sterilized in 10% (w/v) sodium hypochlorite, washed several times with sterilized distilled water and dipped into distilled water for 12 h. Thereafter, healthy seeds were germinated for three days. Then, seedlings were planted into plastic pots filled with sterilized sand and perlite (2:1 ratio). Growth chamber conditions for growth were set to day/night temperatures of 29/20 °C with a 16-h photoperiod, the relative humidity of 60/80% and light intensity of 150 µmol m-2.s. Six-days-old seedlings were used for exposure to different Pb concentrations (0, 0. 25, 0.75, 1, 1.5, 2.5 and 5 mM) for 10 days. Finally, all plants in each pot were harvested and the shoots and roots were separated and stored at ‐80 ˚C until use. 

 

Measurement of variables

Chlorophyll content was assayed using the methods of Lichtenthaler (1987). Anthocyanin content of leaves was determined according to Fulcki and Francis (1968). The determination of total flavonoid content was performed after reaction with the aluminum chloride according to the method of Chang et al. (2002) and catechin as the standard.

Hydrogen peroxide content was measured after reaction with potassium iodide (KI) according to the method of Alexieva et al. (2001). Malondialdehyde (MDA) content was measured by the method proposed by Heath and Packer (1968). Proline was extracted and estimated according to Bates et al. (1973). The determination of ascorbic acid (AsA) content was performed by the method of Omaye et al. (1979) using DTC (2,4-dinitrophenylhydrazine/thiourea/copper) reagent and AsA as the standard. Endogenous NO content was estimated by the method described by Ding et al. (1998) and Hu et al. (2003) with small changes using Greiss reagent and standard curve of NaNO2.

For antioxidant enzyme extraction, 500 mg of fresh samples were ground in 50 mM potassium phosphate buffer (pH 7.0) containing 1% soluble polyvinyl polypyrrolidone (PVP), 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (PMSF). The centrifugation of homogenate was performed at 10000 g for 20 min, and the supernatant was stored for different analyses of the enzyme activity.

The method of Dhindsa et al. (1981) was used for the assay of Catalase (CAT) activity by determining the decrease in absorbance due to H2O2 oxidation at 240 nm using the extinction coefficient of 40 mM-1cm-1 for H2O2. Ascorbate peroxidase (APX) activity was determined according to Nakano and Asada (1981) by observing the decrease in absorbance due to ascorbic acid oxidation for 1 min at 290 nm using the extinction coefficient of 2.8 mM-1cm-1.

 

Statistical analysis

All data were subjected to one-way analysis of variance and the means were compared using Duncan’s multiple range test. Statistical analyses were carried out with the SPSS 19 Program.

 

Results

The data in Table 1 display that lead stress decreased both root and shoot lengths significantly at all concentrations, except at 0.25 mM. Results showed that under lead stress, chlorophyll b content decreased significantly in maize plants at the three highest Pb concentrations, 1.5, 2.5 and 5 mM, and chlorophyll a content at the two highest Pb concentrations, 2.5 and 5 mM. While exposing maize plants to other concentrations of lead did not show any significant effect on the contents of chlorophyll (Table 1). Pb treatments significantly increased the level of anthocyanins in the maize leaves in comparison with non-stressed plants and the highest level was observed at 1.5 mM concentration (Table 3). A rise in the levels of total flavonoids was recorded in the Pb treatments as compared with the control in both roots and shoots of the maize plants. The increase descended at 5 mM concentration (Table 2). 

 

 

 

Table 1. Effects of different concentrations of Pb on root and shoot length, chlorophyll a and b and anthocyanin content ofmaize.

Treatment

Tissue

Length

(cm)

Chlorophyll a

(mg/gFW)

Chlorophyll b

(mg/gFW)

Anthocyanin

(µg/gFW)

Control

Root

Shoot

26.33a±0.88

36.33a±0.88

1.395ab±0.55

0.644a±0.55

38.222c±2.91

Pb0.25

Root

Shoot

25ab±0.57

37.33a±0.88

1.380ab±0.94

0.553ab±0.66

48.350b±0.50

Pb0.5

Root

Shoot

23bc±0.57

30b±0.57

1.452a±0.55

0.639ab±0.25

49.330b±1.19

Pb1

Root

Shoot

22cd±0.57

28.9b±0.60

1.321ab±0.78

0.511abc±0.29

54.130ab±0.35

Pb1.5

Root

Shoot

20.8cd±0.92

28.7b±0.69

1.296abc±0.12

0.451bc±0.32

59.550a±1.17

Pb2.5

Root

Shoot

19.7d±0.73

21.6c±1.45

1.210c±0.53

0.383c±0.53

54.440ab±1.51

Pb5

Root

Shoot

16.8e±1.58

18.5d±0.86

1.102c±0.58

0.361c±0.19

48.400b±1.80

Values followed by different letters within a column indicate a significant difference by Duncan's Multiple Range Test.

 

 

 

 

 

Proline concentration (Table 2) increased in roots and shoots of maize plants due to the Pb stress up to 2.5 mM, while the proline content at 5 mM concentration was not significantly different from the control. Generally, results showed that AsA content was increased with increasing Pb concentration. However, the changes in shoot AsA content at 0.25 and 5 mM concentrations and shoot and root AsA contents at 2.5 mM were not significant. Root AsA content decreased at the highest Pb concentration.

 

 

Table 2. Effects of different concentrations of Pb on proline, ASA, flavonoid and NO content ofmaize.

NO

(nmol/gDW)

Flavonoid (mg/gFW)

ASA (µg/gFW)

Proline (µg/gFW)

Tissue

Treatment

 

60.093b±1.61

52.870b±2.25

0.051c±0.004

0.123c±0.009

5.434b±0.06

7.141b±0.13

131.469d±10.70

73.118e±4.17

Root

Shoot

Control

 

78.981a±1.76

62.500a±3.20

0.075b±0.007

0.222b±0.01

6.817a±0.31

8.010ab±0.50

167.311c±9.93

157.276d±5.16

Root

Shoot

      Pb0.25

76.944a±5.67

68.796a±3.29

0.078b±0.007

0.225ab±0.008

7.121a±0.52

8.194a±0.23

218.207ab±8.72

190.967c±4.47

Root

Soot

Pb0.75

 

77.685a±1.61

68.611a±2.88

0.077b±0.007

0.228ab±0.01

7.296a±0.50

8.472a±0.15

223.942ab±13.62

214.623b±8.95

Root

Shoot

Pb1

 

75.463a±4.89

70.833a±1.60

0.082b±0.007

0.259a±0.004

7.713a±0.72

8.665a±0.27

250.465a±12.49

236.845a±6.12

Root

Shoot

Pb1.5

 

50.463b±3.33

42.685c±4.58

0.107a±0.002

0.244ab±0.01

4.123bc±0.26

7.799ab±0.26

210.322b±12.41

197.491bc±6.84

Root

Shoot

Pb2.5

 

39.166c±2.56

35.092c±1.61

0.075b±0.001

0.221b±0.002

3.510c±0.42

7.108b±0.26

96.344d±13.82

71.182e±5.21

Root

Shoot

Pb5

 

Values followed by different letters within a column indicate a significant difference by Duncan's Multiple Range Test.

 

 

Under Pb treatments, the root and shoot NO content increased at the concentrations of 0.25 to 1.5 mM and decreased at higher concentrations as compared with the control (Table 2). A linear increase inthe H2O2 level was observed with increasing Pb concentrations compared to the untreated plants in both roots and shoots of maize plants (Table 3). In the plants exposed to 0.25 mM of lead, no significant difference was observed between shoot MDA content and the control, whereas higher Pb concentrations significantly elevated MDA content in both roots and shoots. The highest MDA content was observed at the 5 mM Pb (Table 3).

Shoot APX activity was increased at 0.25 mM Pb concentration and decreased at 5 mM compared to control, while it did not exhibit significant variation in other treatments. In the case of roots, APX activity did not change significantly at 0.75, 1.5 and 5 Pb concentrations, but increased significantly at 0.25, 1 and 2.5 mM treatments. A significant increase of CAT activity under Pb treatments was observed in both roots and shoots of the maize plants as compared to the control (Table 3).

 

 

 

Table 3. Effects of different concentrations of Pb on H2O2 and MDA content and APX and CAT activity ofmaize.

CAT activity

(µmol min-1 g-1 FW)

APX activity

(µmol min-1 g-1 FW)

MDA (µg/gFW)

H2O2

(µg/gFW)

Tissue

Treatment

24.500d±0.90

33.500d±3.60

64.508c±3.46

76.116bc±4.86

0.951f±0.02

1.038e±0.03

0.163d±0.06

0.942e±0.05

Root

Shoot

Control

72.250a±4.75

79.000ab±6.33

100.223a±5.03

104.463a±4.35

1.086e±0.006

1.134ed±0.03

1.292c±0.07

1.010e±0.09

Root

Shoot

Pb0.25

46.625c±1.62

80.000ab±5.44

70.982c±2.78

86.830bc±1.94

1.122e±0.02

1.197d±0.01

1.610bc±0.08

1.588d±0.05

Root

Soot

Pb0.75

77.750a±1.95

83.750ab±6.15

83.035b±0.38

84.375b±2.78

1.379d±0.03

1.567c±0.01

2.153bc±0.23

2.041c±0.13

Root

Shoot

Pb1

71.750a±2.88

53.500c±2.53

71.428c±1.18

85.715b±2.04

1.590c±0.01

1.623c±0.01

2.225bc±0.44

2.368b±0.03

Root

Shoot

Pb1.5

78.500a±4.11

92.250a±2.63

89.062b±2.78

74.107cd±2.33

1.828b±0.06

1.746b±0.05

2.543b±0.33

2.624b±0.06

Root

Shoot

Pb2.5

57.750b±1.88

70.500bc±2.17

72.544c±2.51

64.955d±4.18

2.140a±0.07

2.113a±0.04

3.887a±0.51

2.717a±0.08

Root

Shoot

Pb5

Values followed by different letters within a column indicate a significant difference by Duncan's Multiple Range Test.

 

 


Discussion

The present study shows that Pb is more toxic to maize at high concentrations as shown by the toxicity symptoms such as decreases in the biomass and chlorophyll a and b content and elevation of hydrogen peroxide, which induced lipid peroxidation. One of the primary effects of lead stress on the plant is the aggressive reduction of plant growth. Such growth retardation is due to disturbance in various physiological and biochemical processes. John et al. (2012) also reported a notable decrease in early seedling growth and root and shoot length of Brassica juncea L. under Pb and Cd stress in a concentration-dependent manner.

In the present study, we observed Pb effects on the photosynthetic pigments and decreased levels of chlorophyll a and chlorophyll b at the highest Pb concentrations (Table 1). One of the biochemical alterations occurring when plants are treated to Pb stress is the production of reactive oxygen species (Verma and Dubey 2003). The ability of ROS to cause photooxidative damage in organic molecules could probably explain the structural damages in the chloroplasts and the reduction of leaf chlorophyll. Similarly, reductions in the level of photosynthetic pigments, including chlorophyll a and b, after exposure to heavy metals, including Pb, have been observed in many plant species (Sharma and Dubey 2005).

Generation and localization of anthocyanins cause plant resistance against abiotic stresses (Baek et al. 2012). Anthocyanin accumulation in stressed plants is believed to protect the plants from various types of stress (Baek et al. 2012). Other reports have also revealed that anthocyanins are accumulated in plants as a response to heavy metal stresses (Baek et al. 2012).

NO production has been well established to be an initial response of plants to multiple stresses. NO intercedes reactions to biotic and abiotic stresses (Yu et al. 2012). Low concentrations of NO act as a ROS inhibitor or scavenger and also improve plant resistance by inducing the expression of some genes (Xiong et al. 2010). Our results showed that long-term treatment with Pb significantly increased the endogenous level of NO (Table 2). A similar response to copper treatment (long-term) was previously noticed in two plants (Bartha et al. 2005). It is proposed that NO produced by plants challenged with low Pb concentrations could mediate signaling responses leading toward metal tolerance. But at higher concentrations, we observed a dose-dependent decrease in the production of NO. It has been reported that the effects of heavy metals on endogenous NO content depend on plant species, different metal concentrations and periods of metal treatment (Xiong et al. 2010). Similar results have been obtained by Lehotai et al. (2011) who showed that endogenous levels of NO decreased under high concentrations of Cu. They reported that the decrease in NO level could be attributed to the superoxide radicals by the reaction yielding peroxynitrite.

Our results demonstrated that accumulation of proline concentration (Table 2) increase in roots and shoots of the maize plants exposed to Pb concentrations up to 5 mM. Proline acts as an osmolyte, metal chelator, ROS scavenger, electron sink, protector of macromolecules and a component of the cell wall (Rejeb et al. 2014). Therefore, the increase in proline content might have a contribution to the improvement of plant defense. It has been reported that ROS and NO signaling induce proline accumulation in heavy metal stress (Zhang et al. 2008; Guzel and Terzi 2013). Therefore, H2O2 and NO signaling pathways and their interaction in a network cause accumulation of proline that increases plant’s tolerance against stress. In this investigation, the lowest proline content was observed at the 5-mM Pb treatment. Similar results of proline content enhancement by Cd2+ were also shown by John et al. (2012) in Brassica juncea L. They found that accumulation of proline reduced with exposure to higher concentrations of Cd. The decrease in proline content may be due to the increase in degradation. The proline degradation pathway takes place in mitochondria by the consecutive action of proline dehydrogenase (ProDH) and pyrroline-5-carboxylate dehydrogenase. The activity of ProDH coincides with the accumulation of ROS (Rejeb et al. 2014). Therefore, proline catabolism promotes ROS levels.

AsA is a soluble antioxidant in plants, and the most substantial reducing/substrate for detoxification of H2O2 (Singh et al. 2005). In the present investigation, lead treatment increased the concentration of AsA (Table 2). The icreasing concentration of ascorbate in wheat seedlings caused by Pb stress was noticed by Genisel et al. (2015). It has been reported that NO might serve as downstream of physiological events (Wang et al. 2017). Wang et al. (2017) reported that NO accumulation could be necessary for endogenous AsA production. The increase of AsA content in this investigation under low lead concentration could be attributed to NO accumulation.

Flavonoids function as non-enzymatic antioxidants (Michalak 2006). Zafari et al. (2016) showed that NO together with H2O2 trigger signal transduction pathways to enhance the content of phenolic compounds. Results showed that treatment of Pb significantly increased total flavonoid content (Table 2). Therefore, we assume that low stimulation of NO at low Pb concentration can be the reason for flavonoid and anthocyanin enhancement. The production of antioxidant compounds is normally operated with heavy metal stress, but it can be prevented at certain stress levels (Michalak 2006). The enhancement of flavonoids content probably is in charge of metal ions binding, and also for their sequestration within the vacuole (Michalak 2006).

Oxidative damage in plants is generally experienced when Pb and availability other metals to plants is high (Sharma and Dubey 2005). H2O2 acts as a signaling molecule and has a dual role in the mechanism of plant defense. It improves tolerance and stress adaptation at lower content, whereas, it acts as a ROS at high intracellular levels and induces cellular damage resulting in cell death (Stone and Yang 2006). The present study demonstrated that H2O2 and MDA levels increased further with the enhancement in the concentration of the Pb (Table 3), which probably can be critical in growth inhibition and lipid peroxidation. Several studies showed that the level of H2O2 and MDA increased in response to heavy metal stresses (Kaur et al. 2015). At low concentrations, H2O2 acts as a secondary messenger and responds to environmental stress (acclimation) through crosstalk with signaling molecules such as NO (Cuypers et al. 2016) and possibly stress signal transduction by NO and H2O2 interceded defense responses at low Pb concentrations in this study. It has been reported that NO can ameliorate the toxic effects of heavy metal-induced oxidative stress by regulation of general mechanisms for cellular redox homeostasis and H2O2 accumulation (Mazid et al. 2011). Thus, the decrease in NO levels at high Pb concentrations can be the reason for higher H2O2 accumulation.

In our study, the maize plants treated with Pb exhibited an increase in CAT activity at all concentrations, however, an increase in the APX activity was only observed at some concentrations (Table 3). Enhanced activity of these enzymes in stress conditions is responsible for H2O2 scavenging. In many studies, it has been confirmed that Pb could stimulate and increase the production of ROS leading to enhancement in the antioxidant enzyme activities as a defense system (Verma and Dubey 2003; Sharma and Dubey 2005). The same results have been obtained by Cho and Park (2000) in tomato plants grown under mercury stress. On the contrary, the decrease in shoot APX activity at higher Pb concentrations might be due to the down-regulation of gene expression or degradation and inactivation of these proteins (Lee et al. 2001).

 

Conclusions

The present results proved that the maize plants had a negative response to high Pb toxicity. The present study demonstrated that an increase in the concentration of lead caused a decrease in the NO content associated with more oxidative damage and membrane lipid peroxidation. Low concentrations of Pb resulted in a small stimulation of H2O2 and NO, which function as signal molecules implicated in the plant response to Pb stress.

 

Acknowledgements

The authors are thankful to Neda Farnad, MSc, for her assistance in accelerating this research.

 

Conflict of Interest

The author declare that they have no conflict of interest with any organization in relation to the subject of the manuscript.

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