Ecotoxicology and Environmental Safety 113 (2015) 95–102

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Interaction of nitric oxide and reactive oxygen species and associated regulation of root growth in wheat seedlings under zinc stress Xiaohui Duan 1, Xiaoning Li 1, Fan Ding, Jie Zhao, Aifeng Guo, Li Zhang, Jian Yao, Yingli Yang n School of Life Science, Northwest Normal University, Lanzhou 730070, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 2 July 2014 Received in revised form 23 November 2014 Accepted 25 November 2014

The inhibition of root growth was investigated in wheat seedlings exposed to 3 mM zinc (Zn). Zn treatment with or without 250 mM 2-phenyl-4,4,5,5,-tetrame-thylimidazoline-3-oxide-1-oxyl (PTIO) or 10 mM diphenylene iodonium (DPI) significantly inhibited growth, increased malondialdehyde content and lowered cell viability in roots. The most prominent changes of these three parameters at Zn þDPI treatment could be partly blocked by high PTIO concentration (1 mM). The production of nitric oxide (NO) and hydrogen peroxide (H2O2) influenced each other under different treatments, with the highest NO level and the highest H2O2 accumulation in Zn þDPI-treated roots. Compared with Zn-stressed roots, catalase, soluble peroxidase (POD), ascorbate peroxidase and superoxide dismutase decreased in Znþ DPI-treated roots, suggesting that ROS generation from plasma membrane (PM) NADPH oxidase was associated with the regulation of antioxidant enzyme activities. Additionally, Zn-treated roots exhibited significant decreases in cell wall-bound POD, diamine oxidase and polyamine oxidase activities. Our results suggested that Zn-induced effects on root growth resulted from NO interaction with H2O2 and that ZnþDPI-induced strongest inhibition could be explained by the highest increase in the endogenous NO content and the reduction of extracellular ROS production. & 2014 Elsevier Inc. All rights reserved.

Keywords: Hydrogen peroxide Nitric oxide Root growth Wheat Zinc

1. Introduction Nitric oxide (NO) is a stress factor or a signal molecule involved in a variety of plant processes including root growth and plant responses to abiotic stresses (Beligni et al., 2002; De Michele et al., 2009; Böhm et al., 2010). NO may also act as an antioxidant and quench reactive oxygen species (ROS) generated under oxidative stress (Beligni et al., 2002; Zheng et al., 2010). Moreover, in some cases, the effects of NO result from its interaction with ROS. For example, NO worked in oligosaccharide-induced hydrogen peroxide (H2O2) production and interacted with ROS to regulate oligosaccharide-induced artemisinin biosynthesis in Artemisia annua hairy roots (Zheng et al., 2010). Efficient activation of Abbreviations: ASA, ascorbate acid; Zn, zinc; DAF-FM DA, 3-Amino, 4-aminomethyl-2’,7’-difluorescein diacetate; DPI, diphenylene iodonium; DW, dry weight; EDTA, ethylenediaminetetraacetic acid; HNO3, nitric acid; H2O2, hydrogen peroxide; MDA, malondialdehyde; NBT, nitrobluetetrazolium; NO, nitric oxide; NOS, nitric oxide synthase;  OH, hydroxyl radical; O2   , superoxide anion; PBS, phosphate buffered solution; PM, plasma membrane; POD, peroxidase; PTIO, 2-phenyl4,4,5,5-tetrame-thylimidazoline-3-oxide-1-oxyl; PVP, polyvinylpyrrolidone; ROS, reactive oxygen species; TCA, trichloroacetic acid n Corresponding author. E-mail address: [email protected] (Y. Yang). 1 These authors contributed equally to the paper. http://dx.doi.org/10.1016/j.ecoenv.2014.11.030 0147-6513/& 2014 Elsevier Inc. All rights reserved.

hypersensitive cell death also required a balance between NO and ROS production in soybean suspension cultures (Delledonne et al., 2001). Recently, we observed that NO stimulated lead-treated seed germination and seedling shoot growth in wheat, indicating the protective role of exogenous NO against lead toxicity (Yang et al., 2010). The study of Xu et al. (2010) showed that NO was associated with the long-term Zn tolerance in Solanum nigrum. Although the formation of NO has been well documented in the experiments of different plant systems, the role of endogenous NO during plant responses to different heavy metals seems to be much more puzzling (Arasimowicz–Jelonek et al., 2011). The rapidly increasing generation of hydrogen peroxide (H2O2), hydroxyl radical (  OH) and superoxide anion (O2   ) is one of plant responses to heavy metal stresses. H2O2 acts as a secondary messenger that controls such different plant processes as growth and development, and stress responses (Gechev et al., 2006). Furthermore, ROS have the potential to cause oxidative damage to proteins, nucleic acids and other macromolecules, which can severely endanger cell health and viability (Halliwell and Gutteridge, 1999). A previous study demonstrated that ROS production was associated with phytotoxicity due to heavy metal stresses (Posmyk et al., 2009). As one of the micronutrients essential for normal growth and development of plants, zinc (Zn) becomes toxic to plants if it is

96

X. Duan et al. / Ecotoxicology and Environmental Safety 113 (2015) 95–102

excessive, which may retard plant growth and even lower agricultural products (Cherif et al., 2010; Todeschini et al., 2011). Wheat (Tritium aestivum L.) is one of the most important crops in China and many other countries in the world. More recently, we found that zinc treatment resulted in such phytotoxicity as growth inhibition in wheat seedlings and that the effects rose with the increasing concentration (Li et al., 2012a). DPI is a widely used selective NADPH oxidase inhibitor (Auh and Murphy, 1995) and PTIO is used as the scavenger of NO (Arasimowicz–Jelonek et al., 2012). In the present study, the relationship between the root growth and the H2O2 and NO generation was further investigated in wheat seedlings under 3 mM Zn treatment in the presence or absence of PTIO or DPI.

2. Material and methods 2.1. Seedling growth Wheat (Triticum aestivum, cv Xihan 3) seeds were purchased from Gansu Agricultrual Academy. The seeds were surface-sterilized with 0.1% (w/v) HgCl2 for 10 min and germinated in the dark at 2571.5 °C. Uniformly germinated seeds were transferred into Petri dishes and treated with 1/4 Hoagland solution containing 0 and 3 mM ZnSO4 in the presence or absence of 2-phenyl-4,4,5,5tetrame-thylimidazoline-3-oxide-1-oxyl (PTIO) or diphenylene iodonium (DPI) at 2572.5 °C under a light irradiance of 300 m M m  2 s  1 (12 h light: 12 h dark cycles). Root length was measured 6 days later.

O2   generation was determined according to Achary et al. (2012). To develop color resulting from reduction of NBT, wheat roots were immersed in the reaction mixture consisting of 50 mM Tris–HCl buffer (pH 6.4), 0.2 mM nitrobluetetrazolium (NBT), 0.2 mM NADH and 250 mM sucrose, and vacuum-infiltrated for 10–15 min and illuminated at 200 mM m  2 s  1 for 24 h. The absorbance of blue monoformazan formed in the reaction mixture was measured at 530 nm with an extinction coefficient of (ε ¼12.8 mM  1 cm  1), and the O2   content was expressed as m M g  1 fresh weight (FW). 2.5. Lipid peroxidation determination Lipid peroxidation was measured based on the method of Zhou (2001). Wheat roots (0.5 g) were homogenized in 0.25% (w/v) thiobarbituric acid, heated at 98 °C for 30 min, and then centrifuged at 10,000g for 10 min. The absorbance of the supernatant was measured at 450, 532 and 600 nm, respectively. The concentration of malondialdehyde (MDA) was calculated with an extinction coefficient (ε ¼ 155 mM  1 cm  1) and expressed as m M g  1 FW. 2.6. Cell viability analysis The loss of cell viability was assayed by Evans blue staining (Zanardo et al., 2009). Fresh roots (0.5 g) were incubated in 0.25% (w/v) Evans blue solution for 15 min, washed to remove the excessive and unbound dye. After root tips were soaked in 12 M N’Ndimethylformamide for 50 min at room temperature, the absorbance of released Evans blue was measured at 600 nm.

2.2. Zinc content analyses 2.7. Nitric oxide synthase activity measurement Roots and leaves were prepared for measuring Zn level according to Achary et al. (2008) with some modifications. Plant material was thoroughly washed with deionized water and dried to constant weight at 80 °C. The dry sample was dissolved in a solution containing 12 ml concentrated nitric acid (HNO3), 4 ml hydrofluoric acid and 4 ml H2O2 (30%), and was digested in a closed microwave digestion system (Multiwave 3000, Anton paar, Austria) for 2.5 h. The chilled sample was transferred into polytetrafluoroethylene beaker, and then evaporated to dryness. The ash residue was dissolved in 1% HNO3, and total Zn content was measured with flame Atomic Absorption Spectrophotometry (WFX210, China). 2.3. Fluorescence detection of nitric oxide Endogenous NO level was detected by use of diaminofluorescein diacetate (DAF-FM DA, Sigma),a specific NO fluorescent probe. Wheat roots were incubated with 10 mM DAF-FM DA at 37 °C for 20 min. The extra DAF-FM DA was washed and removed with NaH2PO4/Na2HPO4 buffer (PBS, pH 7. 4). A Leica MPS60 fluorescent microscope equipped with a red fluorescent protein filter (excitation 450–490 nm, emission 500–530 nm) was used to get fluorescent images. 2.4. Determination of Hydrogen peroxide and superoxide anion levels The measurement of H2O2 level was performed with the method of Sergiev et al. (1997). Plant roots were ground with 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged at 12,000g for 20 min. The supernatant was mixed with 10 mM PBS buffer (pH 7.0) and 1 M KI, and the absorbance of the solution was measured at 390 nm. H2O2 concentrations were calculated by use of a standard curve prepared with known H2O2 concentrations.

Nitric oxide synthase (NOS) activity was assayed according to the method described by Murphy and Noack (1994) with some modifications. Wheat roots (1 g) together with 1% (w/v) polyvinylpolypyrrolidone (PVP) were ground in liquid N2 and then resuspended in 5 ml extraction buffer (50 mM Tris–HCl (pH 7.4), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol, 1 μM leupeptin, 1 μM pepstatin, 320 mM sucrose and 1 mM phenylmethylsulfonyl fluoride). After centrifugation at 10,000g at 4 °C for 30 min, the supernatant was used to analyze NOS activity. 100 U catalase (CAT) and 100 U superoxide dismutase (SOD) were used to remove endogenous ROS for 5 min, and then 5 ml oxyhaemoglobin (5 mM) was added. NOS activity was analyzed by the hemoglobin assay at 401 nm and 421 nm with an extinction coefficient of 77 mM  1 cm  1. 2.8. Measurement of antioxidant enzyme activities Plant material (1 g) was ground with 1 ml of PBS buffer (50 mM, pH 7.8) containing 0.1 mM EDTA and 1% (w/v) PVP. After it was centrifuged at 15,000g for 30 min, the supernatant was collected to determine antioxidant enzyme activities. SOD activity was estimated based on the method of Dhindsa and Matowe (1981). The reaction mixture was made up of 50 mM PBS (pH 7.6), 13 mM methionine, 75 mM NBT, 0.1 mM EDTA-Na2 and an appropriate amount of enzyme extraction, and the reaction was started by the addition of 2 mM lactochrome. After the reaction was illuminated at 25 °C for 10 min with a non-illumination surface as a reference, the absorbance was recorded at 560 nm. The complete reaction medium without enzyme incubated in the dark was used as dark control. One unit (U) of enzyme activity was defined as the quantity of SOD required to produce a 50% inhibition of NBT reduction. A modification of the method of Aebi (1974) was used to assay CAT activity. The enzyme extraction was added to 50 mM PBS

X. Duan et al. / Ecotoxicology and Environmental Safety 113 (2015) 95–102

buffer (pH 7.0) and incubated at 25 °C for 5 min. The reaction was started by the addition of 6 mM H2O2 and the absorbance changes were recorded at 240 nm for 2 min. An absorbance change of 0.1 unit min  1 was defined as one unit of CAT activity. Peroxidase (POD) activity was analyzed according to Rao et al. (1996). The enzyme extraction was mixed with the reaction medium containing 50 mM PBS (pH 7.0) and 20 mM guaiacol. After the reaction mixture was pre-incubated at 25 °C for 5 min, 6 mM H2O2 was added to initiate the reaction. The absorbance changes at 470 nm within 2 min were recorded in order to calculate POD activity. One unit of soluble POD activity was defined as an absorbance change of 0.01 unit min  1. Wheat roots (1 g) were ground with 1 ml of chilled 50 mM PBS buffer (pH 7.0) containing 1 mM EDTA and 1 mM ascorbate (ASA). After it was centrifuged at 15,000g for 30 min, the supernatant was collected for the measurement of ascorbate peroxidase (APX) activity according to Nakano and Asada (1981). The assay was carried out in the reaction mixture consisting of 50 mM PBS (pH 7.0), 0.5 mM ASA, 3 mM H2O2 and an appropriate amount of the enzyme extraction. The absorbance changes at 290 nm were recorded at 25 °C for 1 min after the addition of H2O2. One unit of APX activity was defined as an absorbance change of 0.1 unit min  1. Fresh roots (0.5 g) were ground with 2 ml of 50 mM Tris–HCl buffer (pH 7.5) containing 0.1 mM EDTA and 0.1% (w/v) PVP. After centrifugation at 13,000g for 30 min, the supernatant was collected for the measurement of glutathione reductase (GR) activity, which was carried out by monitoring the oxidation of NADPH according to Schaedle and Bassham (1977). The reaction mixture was made up of 50 mM Tris–HCl (pH 7.5), 3 mM MgCl2, 0.5 mM oxidized glutathione and 0.15 mM NADPH. The absorbance changes at 340 nm within 3 min were recorded to calculate GR activity, and one unit of enzyme activity was defined as an absorbance change of 0.1 min  1.

97

1 mM NaCl. The reaction mixture was made up of 450 μl enzyme extraction, 50 U CAT and 250 μl 2-aminobenzaldehyde (0.1%, w/v), and then the reaction was started with 250 μl 10 mM spermidine. After the reaction mixture was incubated at 30 °C for 3 h, the reaction was stopped with 1 ml 10% (v/v) perchloric acid. After it was centrifuged at 6,500 g for 10 min, the absorbance was measured at 430 nm and the formation of Δ1-pyrroline product was determined. Control reactions were carried out without the enzyme extraction. 2.10. Protein content determination With bovine serum albumin as a standard, the amount of soluble proteins was estimated according to Bradford (1976). 2.11. Data analysis Each experiment was replicated at least three times. The data were expressed as the average7standard error (SE). Statistical comparisons were carried out by means of SPSS 13.0 software, and significant differences were indicated by different letters (p o0.05).

3. Results 3.1. Effects of PTIO and DPI on Zn level in Zn-treated seedlings Roots and leaves treated with 3 mM Zn exhibited about 32% and 23% increases in Zn content as compared with the control, respectively. The presence of PTIO significantly lowered Zn level in roots and leaves of Zn-treated seedlings in comparison with Zn treatment alone. Differently, DPI did not affect leaf Zn content, but significantly decreased root Zn level in Zn-treated seedlings (Table 1).

2.9. Determination of cell wall-bound POD, DAO and PAO activities 3.2. Effects of PTIO and DPI on root growth in Zn-treated seedlings Cell walls were prepared by homogenizing roots in 50 mM PBS (pH 5.8) as described by Lee and Lin (1995). The homogenate was centrifuged at 1,000g for 10 min and washed at least twice with 50 mM PBS (pH 5.8), and then the pellets were incubated in 1 M NaCl for 2 h. After it was centrifuged at 1,000g for 10 min, the supernatant containing cell wall-bound POD was collected. Enzyme activity was analyzed according to Dos Santos et al. (2008). The reaction mixture consisted of 25 mM PBS (pH 6.8), 2.58 mM guaiacol, 10 mM H2O2 and the enzyme extract. The absorbance changes at 470 nm within 2 min were recorded to calculate enzyme activity. One unit of cell wall-bound POD activity was defined as an absorbance change of 0.01 min  1. Diamineoxidase (DAO) activity in the fractions of cell walls was measured with the method of Naik et al. (1981). The reaction mixture consisting of 50 mM PBS buffer (pH 7.8), 10 mM putresine, 0.1 mM pyridoxal phosphate and enzyme extract was incubated at 30 °C for one hour, and then the reaction was terminated with 1 ml 20% (w/v) TAC. 30 min later, the incubation mixture was centrifuged at 5,000g for 15 min. One ml of ninhydrin mixture (250 mg ninhydrin in 6 ml acetic acid and 4 ml phosphoric acid) was added to the supernatant and the color was developed at 100 °C for 30 min. After 1 ml of acetic acid was added, the absorbance was measured at 510 nm. In the control, TAC was added prior to the enzyme solution. Polyamine oxidase (PAO) activity was assayed with spermidine as a substrate (Asthir et al., 2002). One gram of roots was homogenized in 2 ml 100 mM PBS (pH 7.0) containing 5 mM dithiothreitol, and centrifuged at 16,000g for 20 min. The residue pellet was sequentially extracted with 100 mM PBS (pH 7.0) containing

As shown in Table 2, 3 mM Zn remarkably reduced root length of wheat seedlings. The application of 250 mM PTIO led to further inhibition in root growth in Zn-treated seedlings in comparison with Zn-stressed ones. Additionally, wheat seedlings exhibited about 92% reduction in root length after treatment with 3 mM Zn in combination with 10 mM DPI, and only 56% decrease in root growth under 3 mM Zn stress alone, as compared with the control. Moreover, the presence of 1 mM PTIO partly blocked Zn þDPI-inhibitory effect on root growth. 3.3. Effects of PTIO and DPI on NO, H2O2 and O2   generation in Zntreated roots Wheat roots were incubated with fluorescent probe DAF-FM DA, and NO fluorescence was observed with a Leica MPS60 fluorescent microscope. As illustrated in Fig. 1A, a slight signal of Table 1 Zn content in roots and leaves of wheat seedlings (mg g  1 DW) exposed to different treatments (control, Zn, Znþ PTIO or Zn þDPI) for 6 days. Values are expressed as the average7 SE (n ¼5). Treatment

Root Zn content

Leaf Zn content

Control 3 mM Zn 3 mM Zn þ 250 mM PTIO 3 mM Zn þ 10 mM DPI

16107 19.8b 21207 30.3d 1850 7 16.4c 14107 21.3a

1660 7 41.4a 2050 7 16.6c 1750 7 27.9b 21407 23.0d

Different small letters in each column indicate significant difference at 0.05 levels.

98

X. Duan et al. / Ecotoxicology and Environmental Safety 113 (2015) 95–102

Table 2 Root length (cm), malondialdehyde (MDA) content (mmol g  1 FW) and cell viability (OD600) in wheat roots exposed to different treatments (control, Zn, Znþ PTIO, Zn þDPI or Znþ DPIþ PTIO) for 6 days. Values are expressed as the average 7 SE (n¼3). Treatment

Root length

MDA content Cell viability

Control 3 mM Zn 3 mM Znþ 250 mM PTIO 3 mM Znþ 10 mM DPI 3 mM Znþ 10 mM DPIþ 1 mM PTIO

8.86 7 0.20e 0.55 7 0.01a 3.90 7 0.06d 0.69 7 0.03b 3.38 7 0.07c 0.707 0.01b 0.69 7 0.04a 1.53 7 0.08d 1.577 0.07b 1.147 0.05c

0.058 7 0.002a 0.1177 0.002b 0.1397 0.001c 0.2727 0.003d 0.1547 0.005c

Different small letters in each column indicate significant difference at 0.05 levels.

DAF-FM DA fluorescence was detected in untreated roots, demonstrating that a basal level of NO production occurred. Compared with the control, 3 mM Zn-stressed roots exhibited the stronger NO fluorescence (Fig. 1B), while the application of PTIO lowered this signal in Zn-treated roots (Fig. 1C). Especially, the maximal fluorescence was observed in wheat roots exposed to 3 mM Zn together with 10 mM DPI (Fig. 1D). H2O2 content rose by about 132% in 3 mM Zn-stressed roots, while roots treated with Zn and PTIO in combination exhibited only 17% enhancement in H2O2 level, in comparison with the control (Fig. 2A). In addition, Zn treatment in combination with DPI resulted in the maximal increase in H2O2 content in wheat roots. Compared with the control, wheat roots showed about 28% reduction in O2   content after treatment with 3 mM Zn. The presences of PTIO and DPI resulted in about 35% and 23% decreases in this parameter in Zn-stressed roots in comparison with the control, respectively (Fig. 2B). Moreover, compared with the absence of DPI, the addition of DPI in the reaction solution led to about 46%, 44% and 43% reduction in O2   production in untreated, Zn-treated and Zn þPTIO-treated roots, respectively, but only about 25% decrease of O2   content in ZnþDPI-stressed ones (Fig. 2B). 3.4. Effects of PTIO and DPI on lipid peroxidation and cell viability in Zn-treated roots Compared with the control, root MDA content increased about 25% in seedlings stressed with 3 mM Zn (Table 2). The presence of 250 mM PTIO led to an insignificant increase in MDA level in Zntreated roots. In comparison with Zn stress alone, Znþ DPI treatment caused more prominent enhancement in the degree of lipid peroxidation. However, the presence of 1 mM PTIO partly reversed the enhancement of MDA content induced by the combined treatment with Zn and DPI. The loss of cell viability was evaluated by Evans blue staining in roots (Table 2). In comparison with untreated roots, the uptake of Evans blue in root cells increased in response to 3 mM Zn treatment. The addition of 250 mM PTIO further enhanced the uptake of Evans blue in Zn-treated roots. In addition, the most notable loss of Table 3 Antioxidant enzyme activities (U mg average7SE (n¼ 3).

1

cell viability was found in Zn-stressed roots in the presence of DPI. Differently, 1 mM PTIO could partly block the loss of cell viability in roots exposed to the combined treatment of Zn and DPI (Table 2). 3.5. Effects of PTIO and DPI on antioxidant enzyme activities in Zntreated roots CAT activity rose by about 111% in 3 mM Zn-stressed roots, while the addition of PTIO or DPI resulted in about 14% and 40% decrease in CAT activity in Zn-treated roots, respectively, compared with untreated roots (Table 3). Exposure to 3 mM Zn led to about 13% reduction in soluble POD activity in wheat roots. Zn treatment combined with PTIO did not affect this enzyme activity compared with Zn treatment alone. However, the addition of DPI resulted in about 56% decrease in soluble POD activity in Zn-stressed roots in comparison with the control. As showed in Table 3, APX activity increased in wheat roots after 3 mM Zn treatment. Compared with Zn treatment alone, the activity slightly decreased in Zn-treated roots in the presence of PTIO, whereas the activity of this enzyme significantly decreased in the roots treated with Zn and DPI in combination. The constitutive GR activity was 2.55 70.05 U mg  1 protein in the roots of wheat seedlings. In comparison with the control, a significant enhancement in GR activity was found when the seedlings were treated with 3 mM Zn. In addition, 250 mM PTIO further stimulated this enzyme, but compared with Zn treatment alone, DPI did not affect GR activity in Zn-stressed roots (Table 3). As shown in Tables 3, 3 mM Zn treatment stimulated SOD in wheat roots. A further enhancement in SOD activity was found in the roots exposed to Zn and PTIO treatment in combination, while the application of DPI resulted in the decrease of SOD activity in Zn-stressed roots, in comparison with Zn stress alone. 3.6. Effects of PTIO and DPI on NOS activity in Zn-treated roots As showed in Fig. 3, NOS activity significantly elevated in wheat roots after 3 mM Zn treatment. The addition of PTIO led to about 16% reduction in NOS activity, while the activity of this enzyme significantly increased in the roots treated with Zn and DPI in combination, compared with Zn treatment alone (Fig. 3). 3.7. Effects of PTIO and DPI on cell wall-bound POD, DAO and PAO activities in Zn-treated roots After treatment with 3 mM Zn, cell wall-bound POD activity remarkably decreased in wheat roots. PTIO did not affect Zn-inhibitory effect on cell wall-bound POD activity, whereas the application of DPI led to further reduction in this enzyme activity in Zn-treated roots (Table 4). The effects of Zn, Zn þPTIO or Zn þDPI on DAO activity were shown in Table 4. Wheat roots treated with 3 mM Zn exhibited 11% decrease in DAO activity, compared with the control. Similarly, when wheat seedlings were treated by 3 mM Zn in combination

protein) in wheat roots exposed to different treatments (control, Zn, Zn þ PTIO or Zn þ DPI) for 6 days. Values are expressed as the

Treatment

CAT

POD

APX

GR

SOD

Control 3 mM Zn 3 mM Znþ 250 mM PTIO 3 mM Znþ 10 mM DPI

5.36 7 0.30c 5.977 0.21d 4.60 7 0.08b 3.217 0.07a

1977 4.45c 1727 4.92b 1677 6.17b 86.8 7 0.27a

21.3 7 0.68a 28.4 7 0.83c 27.3 7 0.40c 24.97 0.98b

2.55 7 0.05a 3.52 7 0.10b 4.007 0.05c 3.45 7 0.04b

139 710.3a 174 72.06b 199 76.71c 131 70.67a

Different small letters in each column indicate significant difference at 0.05 levels.

X. Duan et al. / Ecotoxicology and Environmental Safety 113 (2015) 95–102

99

Fig. 1. Changes of NO fluorescence in wheat roots exposed to 3 mM Zn for 6 days in the absence or presence of PTIO or DPI: (A) control; (B) 3 mM Zn-treated root; (C) 3 mM Zn þ250 μM PTIO-treated root; and (D) 3 mM Znþ 10 μM DPI-treated root.

Fig. 3. Changes of NOS activity in wheat roots exposed to 3 mM Zn for 6 days in the absence or presence of 250 μM PTIO or 10 μM DPI. CK, control; Zn, 3 mM Zn; Znþ PTIO, 3 mM Zn þ250 μM PTIO; Zn þ DPI, 3 mM Zn þ10 μM DPI. Values are expressed as the average7 SE (n ¼3), and different letters on bars indicate significant difference (P o0.05). Table 4 Cell wall-bound POD, DAO and PAO activities (U mg  1 protein) in wheat roots exposed to different treatments (control, Zn, Zn þ PTIO or Zn þ DPI) for 6 days. Values are expressed as the average7SE (n¼ 3). Treatment

Cell wall-bound POD

DAO

PAO

Control 3 mM Zn 3 mM Zn þ 250 mM PTIO 3 mM Zn þ 10 mM DPI

44.0 7 2.05c 5.26 7 0.36b 5.687 0.54b 1.09 7 0.14a

4.157 0.19b 3.687 0.13a 3.65 7 0.10a 3.59 7 0.09a

5.88 7 0.03c 3.917 0.02a 4.26 7 0.11b 3.777 0.11a

Different small letters in each column indicate significant difference at 0.05 levels.

4. Discussion

Fig. 2. Changes of H2O2 (A) and O2   (B) content in wheat roots exposed to 3 mM Zn for 6 days in the absence or presence of 250 μM PTIO or 10 μM DPI. CK, control; Zn, 3 mM Zn; Zn þPTIO, 3 mM Zn þ250 μM PTIO; Znþ DPI, 3 mM Zn þ10 μM DPI. “with 7 DPI” means the addition of DPI in the reaction solution, “without DPI” means the absence of DPI in the reaction solution. Values are expressed as the average7SE (n¼ 3), and different letters on bars indicate significant difference (Po 0.05).

with PTIO or DPI, the activity of DAO significantly decreased in roots compared with the control. 3 mM Zn treatment caused a significant reduction in PAO activity in wheat roots. Addition of 250 mM PTIO partly alleviated Zn-induced inhibitory effect, but the application of DPI did not affect the decrease of PAO activity in response to Zn treatment (Table 4).

Roots may be the first part of plant to suffer from the toxicity of heavy metal and exhibit symptoms. More recently, Li et al. (2012a) observed the notable reduction of root length and the increase of Zn accumulation in wheat seedlings exposed to different Zn concentrations including 3 mM Zn. It has been demonstrated that the accumulation of heavy metal may be associated with the inhibition of plant growth (Phang et al., 2011). In the present study, 3 mM Znþ 250 mM PTIO treatment significantly lowered Zn levels in roots and leaves, and led to the further inhibition of root growth. Meanwhile, 3 mM Zn stress in combination with 10 mM DPI lowered the Zn level and the length in the roots, but did not affect the Zn content in leaves, in comparison with single Zn treatment. These results seemed to indicate that changes of NO and ROS might play an important role in root growth and Zn accumulation in wheat seedlings. They also suggested that a certain Zn accumulation was important to maintain root growth when wheat seedlings were

100

X. Duan et al. / Ecotoxicology and Environmental Safety 113 (2015) 95–102

exposed to Zn stress. In some cases, NO and H2O2 occur in response to the similar stress conditions, and these two molecules may interact in plant growth and stress responses (Liao et al., 2012). We observed that 3 mM Zn-stressed roots exhibited both the stronger NO fluorescence and the higher H2O2 production than untreated roots did. Similarly, a significant increase of NO content with H2O2 production accompanying was also observed in red kidney bean (Wang et al., 2010), in the roots of Matricaria chamomilla (Kováčik et al., 2010) and in Vicia faba L. (Zou et al., 2012) under heavy metal stresses. Moreover, the inhibition of NO syntheses partly resulted in the prevention of H2O2 increase in Arabidopsis suspension cultures (De Michele et al., 2009). The present data showed that the application of 250 mM NO scavenger PTIO efficiently lowered NO fluorescence and partly reduced H2O2 accumulation in Zn-treated roots. These results suggested that the generation of NO and H2O2 influenced each other in plants. The phytotoxicity of heavy metals arises partly from the excessive generation of ROS including H2O2. Moreover, several studies (Ruley et al., 2004; Li et al., 2012b) indicated that the increase of H2O2 level was correlated with the reduction of root length in plants exposed to different heavy metal stresses. However, the decrease of H2O2 accumulation due to the presence of PTIO did not block the Zn-induced inhibitory effect on root growth, suggesting that increased H2O2 production in response to Zn stress was not responsible for the reduction of root length in wheat seedlings. According to some reports, heavy metals including aluminum (Al), copper (Cu) and cadmium (Cd) triggered the inhibition of root growth and root formation, which was associated with the reduction of endogenous NO level in different plants (He et al., 2012; Zhang et al., 2012). This was supported by the study of Gouvêa et al. (1997), which demonstrated that the generation of NO promoted root growth in marigold. In the present study, the further reduction of root length due to the scavenging of NO with 250 mM PTIO in Zn-treated seedlings suggested that the increase of NO production to a certain extent might play an important role in alleviating the inhibition of root growth when wheat seedlings were exposed to 3 mM Zn stress. Moreover, the maximal NO fluorescence, the highest H2O2 level and the strongest inhibition of root growth were induced by 3 mM Zn and 10 mM DPI treatment in combination. Similarly, the study of Liszkay et al. (2004) showed that the elongation growth of maize roots depended on apoplastic O2   generation of NADPH oxidase and that DPI (the inhibitor of NADPH oxidase) suppressed elongation growth of maize roots. NO scavenging with the high PTIO concentration (1 mM) did partly prevent Znþ DPI-induced inhibitory effect on root growth. These results indicated that the excessive amount of endogenous NO in response to Znþ DPI was toxic and was responsible for the strongest inhibitory effect on root growth of wheat seedlings. In addition, 250 mM PTIO did not alleviate Zn þDPI-induced reduction of root length (data not shown) because NO scavenging with the low PTIO concentration (250 mM) might not be enough effective to block Znþ DPI-inhibitory effect. Wheat roots exhibited decreased O2   content after treatment with 3 mM Zn, Zn þPTIO or Zn þDPI (Fig. 2B). On the contrary, PTIO promoted Al-induced O2   production in the root of red kidney bean (Wang et al., 2010). PM NADPH oxidase is responsible for the formation of extracellular O2   , which is further dismutated to H2O2 and O2 either spontaneously or enzymatically modified by SOD (Matsumoto and Motoda, 2012). ROS accumulation, especially extracellular O2   or H2O2 formation, is mostly connected with the stimulation of NADPH oxidase in plants exposed to different heavy metal stresses (Remans et al., 2010). Further studies are needed to explore if PM NADPH oxidase is involved in regulating ROS production in Zn-stressed roots. We observed that the addition of DPI in the reaction solution led to

about 46%, 44% and 43% reduction in O2   level in untreated, and Zn-treated and Znþ PTIO-treated roots, respectively, but compared with the absence of DPI, it only led to about 25% decrease of O2   content in ZnþDPI-stressed roots (Fig. 2B). These findings suggested that PM NADPH oxidase activity in ZnþDPI-stressed roots was lower than that in untreated, Zn-stressed and Zn þPTIOstressed ones. Therefore, the reduction of O2   generation was partly due to the decrease of PM NADPH oxidase activity in Znþ DPI-treated roots. By scavenging ROS directly or by enhancing antioxidant enzyme activities, NO exerts a protective function against stress-induced oxidative damage (Hogg and Kalyanaraman, 1999; Liu et al., 2010). Stress-induced endogenous NO production was essential to lower membrane damage in some plants (Xu et al., 2012). However, 250 mM PTIO had no effect on Zn-induced oxidative damage. Interestingly, compared with Zn stress alone, Zn þDPI treatment caused more prominent lipid peroxidation in wheat roots, which could be partly reversed with the presence of 1 mM PTIO (Table 2). These results suggested that a certain increase of NO production was not responsible for lipid peroxidation in wheat roots exposed to 3 mM Zn stress, and that the excessive NO accumulation at Znþ DPI treatment caused oxidative damage to wheat roots. These conclusions could be supported by other studies, which indicated that NO might promote lipid peroxidation in case of its excessive concentration and the membrane environment (Stöhr, 2007). Besides, endogenous NO production was associated with the increase of cell death in plants or cells under abiotic stresses (Rodríguez– Serrano et al., 2012). An investigation of the uptake of Evans blue indicated an enhancement of the loss of cell viability in 3 mM Znstressed roots. Scavenging of endogenous NO with 250 mM PTIO further lowered cell viability in Zn-treated roots, suggesting increased NO production due to 3 mM Zn stress was required to maintain the cell viability of wheat roots. However, the greatest loss of cell viability with the strongest NO fluorescent was observed in Znþ DPI-treated roots, which could be partly blocked by 1 mM PTIO. These results indicated that the excessive NO generation in response to Zn þDPI was responsible for the remarkable decrease of cell viability in wheat roots. Moreover, in this study the observed changes of root growth were correlated with the loss of cell viability in wheat roots under different treatments. As one of potential enzymatic sources of NO, NOS was responsible for the endogenous NO generation in plants (Neill et al. 2008; Liao et al., 2013). The present data showed that NOS activity significantly elevated in Zn-treated wheat roots. Additionally, this enzyme activity reduced due to Znþ PTIO stress, but further increased in response to Zn þDPI treatment. This could be partly supported by the study of Liao et al. (2013), which indicated that NOS mediated NO generation in cut rose flowers. Therefore, NOS was associated with endogenous NO production in wheat seedling roots under different treatments. In the present study, increased CAT, APX, GR and SOD activities played important roles in scavenging ROS and protecting wheat roots against oxidative damage, and decreased POD activity along with increased SOD activity was responsible for increased H2O2 level in Zn-treated roots. In a recent study, Zn-induced NO production promoted the enhancement of H2O2 accumulation resulting from the stimulation of SOD as well as the inhibition of CAT and APX in Solanum nigrum roots (Xu et al., 2010). Similar results were obtained in Arabidopsis suspension cells, where Cd-induced NO production negatively affected the activities of CAT and APX (De Michele et al., 2009). The present data showed that the application of 250 mM PTIO resulted in the reduction of H2O2 and NO generation in Zn-treated wheat roots. Moreover, investigations of PTIO effects on antioxidant enzyme activities suggested that increased NO generation in Zn-stressed roots was necessary to stimulate CAT. This could be supported by the study of Xu (2010),

X. Duan et al. / Ecotoxicology and Environmental Safety 113 (2015) 95–102

which indicated that the abolition of endogenous NO accumulation significantly lowered the activity of CAT in cold-stored fruit. Differently, the application of PTIO further increased APX activity in Matricaria chamomilla roots (Kováčik et al., 2010). In this study, Zn þDPI treatment led to the significant inhibition of CAT, POD, APX and SOD compared with single Zn exposure, suggesting that extracellular ROS generation dependent on PM NADPH oxidase might be involved in the regulation of antioxidant enzyme activities in wheat roots. H2O2 accumulation was also linked with the production of extracellular ROS mediated by other enzymatic reactions. Cell wall-bound POD can produce H2O2 through the oxidation of NADH (Ranieri et al., 2001), and DAO and PAO play important roles in the generation of extracellular H2O2 via the catabolism of polyamines in plant tissues (Karuppanapandian et al., 2011; Cvikrová et al., 2012). Although some researchers believed that the production of NO and H2O2 was associated with the stimulation of DAO and PAO (Wimalasekera et al., 2011), the inhibition of cell wall-bound POD, DAO and PAO in this study suggested that these three enzymes were not responsible for increased NO and H2O2 levels in Zn-treated roots. In addition, NO scavenging with PTIO only partly blocked Zn-inhibitory effects on PAO activity, and the presence of DPI caused the further decrease in cell wallbound POD activity in Zn-treated roots (Table 4). It has been demonstrated that extracellular H2O2 and O2   are required for the emergence and growth of the radicals (Kranner et al., 2010) and the elongation of Arabidopsis roots (Dunand et al., 2007). Moreover, lowering the activities of DAO and PAO, which might be responsible for reducing extracellular H2O2 production, could inhibited the development of soybean lateral root (Su et al., 2006). Therefore, the reduction of extracellular ROS production resulting from the inhibition of cell wall-bound POD, DAO and PAO might be associated with the inhibition of root growth in wheat seedlings exposed to different treatments.

5. Conclusion This study suggested that the increase of NO production to a certain extent might play important roles in alleviating 3 mM Zninduced inhibitory effects on root growth in wheat seedlings, and that the excessive endogenous NO was toxic and caused the further reduction in root length when wheat seedlings were exposed to Zn þDPI treatment. The loss of cell viability was correlated with the reduction of root length in response to different treatments. Further studies showed that the elevation of NO content at 3 mM Zn stress was partly responsible for the increase of H2O2 level and the stimulation of CAT in wheat roots. The investigations of DPI effects on extracellular ROS level suggested that PM NADPH oxidase was associated with the regulation of NO and ROS production as well as NOS, CAT, POD, APX, SOD activities. In conclusion, the reduction of root growth was associated with the loss of cell viability in wheat seedlings under different treatments, but the inhibition was most sensitive to Zn þDPI treatment, which might be partly explained by the excessive endogenous NO correlated with the highest NOS activity and the decrease of extracellular ROS production as the result of the inhibition of PM NADPH oxidase, cell wall-bound POD, DAO and PAO.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Nos. 31470464, 31360094), Science and technology plan project of Gansu Province (144FKCA059) and Technology Development Plan Program of Lanzhou City (2011-1-142).

101

References Achary, V.M.M., Jena, S., Panda, K.K., Panda, B.B., 2008. Aluminium induced oxidative stress and DNA damage in root cells of Allium cepa L. Ecotoxicol. Environ. Saf. 70, 300–310. Achary, V.M.M., Patnaik, A.R., Panda, B.B., 2012. Oxidative biomarkers in leaf tissue of barley seedlings in response to aluminum stress. Ecotoxicol. Environ. Saf. 75, 16–26. Aebi, H., 1974. Catalase. In: Bergmeyer, H.U. (Ed.), In Methods of Enzymatic Analysis. Academic Press, New York, pp. 673–677. Arasimowicz-Jelonek, M., Floryszak-Wieczorek, J., Gwóźdź, E.A., 2011. The message of nitric oxide in cadmium challenged plants. Plant Sci. 181, 612–620. Arasimowicz-Jelonek, M., Floryszak-Wieczorek, J., Deckert, J., Rucinska-Sobkowiak, R., Gzyl, J., Pawlak-Sprada, S., Abramowski, D., Jelonek, T., Gwózd, E.A., 2012. Nitric oxide implication in cadmium-induced programmed cell death in roots and signaling response of yellow lupine plants. Plant Physiol. Biochem. 58, 124–134. Asthir, B., Duffus, C.M., Smith, R.C., Spoor, W., 2002. Diamine oxidase is involved in H2O2 production in the chalazal cells during barley grain filling. J. Exp. Bot. 53, 677–682. Auh, C.K., Murphy, T.M., 1995. Plasma membrane redox enzyme is involved in the synthesis of O2– and H2O2 by Phytophthora elicitor-stimulated rose cells. Plant Physiol. 107, 1241–1247. Beligni, M.V., Fath, A., Bethke, P.C., Lamattina, L., Jones, R.L., 2002. Nitric oxide acts as an antioxidant and delays programmed cell death in barley aleurone layers. Plant Physiol. 129, 1642–1650. Böhm, F.M.L.Z., Ferrarese, M.L.L., Zanardo, D.I.L., Magalhaes, J.R., Ferrarese-Filho, O., 2010. Nitric oxide affecting root growth, lignification and related enzymes in soybean seedlings. Acta Physiol. Plant 32, 1039–1046. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Cherif, J., Derbel, N., Nakkach, M., Bergmann, H.V., Jemal, F., Lakhdar, Z.B., 2010. . Analysis of in vivo chlorophyll fluorescence spectra to monitor physiological state of tomato plants growing under zinc stress. J. Photochem. Photobiol. B 101, 332–339. Cvikrová, M., Gemperlová, L., Dobrá, J., Martincová, O., Prášil, I., Gubiš, J., 2012. Effect of heat stress on polyamine metabolism in proline-over-producing tobacco plants. Plant Sci. 182, 49–58. De Michele, R., Vurro, E., Rigo, C., Costa, A., Elviri, L., Di Valentin, M., Careri, M., Zottini, M., Sanità di Toppi, L., Lo Schiavo, F., 2009. Nitric oxide is involved in cadmium-induced programmed cell death in Arabidopsis suspension cultures. Plant Physiol. 150, 217–228. Delledonne, M., Zeier, J., Marocco, A., Lamb, C., 2001. Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease-resistance response. Proc. Natl. Acad. Sci. 98, 13454–13459. Dhindsa, R.S., Matowe, W., 1981. Drought tolerance in two mosses: correlated with enzymatic defence against lipid peroxidation. J. Exp. Bot. 32, 79–91. Dos Santos, W.D., Ferrarese, M.L.L., Nakamura, C.V., Mourão, K.S.M., Mangolin, C.A., Ferrarese-Filho, O., 2008. Soybean (Glycine max) root lignification induced by ferulic acid. The possible mode of action. J. Chem. Ecol. 34, 1230–1241. Dunand, C., Crèvecoeur, M., Penel, C., 2007. Distribution of superoxide and hydrogen peroxide in Arabidopsis root and their influence on root development: possible interaction with peroxidases. New Phytol. 174, 332–341. Gechev, T.S., Van Breusegem, F., Stone, J.M., Denev, I., Laloi, C., 2006. Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. BioEssays 28, 1091–1101. Gouvêa, C.M.C.P., Souza, J.F., Magalhães, A.C.N., Martins, I.S., 1997. NO-releasing substances that induce growth elongation in maize root segments. Plant Growth Regul. 21, 183–187. Halliwell, B., Gutteridge, J.M.C., 1999. The chemistry of free radicals and related ‘reactive species'. Free Radic. Biol. Med. 3, 220. He, H.Y., Zhan, J., He, L.F., Gu, M.H., 2012. Nitric oxide signaling in aluminum stress in plants. Protoplasma 249, 483–492. Hogg, N., Kalyanaraman, B., 1999. Nitric oxide and lipid peroxidation. Biochim. Biophys. Acta 1411, 378–384. Karuppanapandian, T., Moon, J.C., Kim, C., Kumariah, M., Kim, W., 2011. Reactive oxygen species in plants: their generation, signal transduction, and scavenging mechanisms. Aust. J. Crop Sci. 5, 709–725. Kováčik, J., Grúz, J., Klejdus, B., Štork, F., Marchiosi, R., Ferrarese-Filhod, O., 2010. Lignification and related parameters in copper-exposed Matricaria chamomilla roots: role of H2O2 and NO in this process. Plant Sci. 179, 383–389. Kranner, I., Roach, T., Beckett, R.P., Whitaker, C., Minibayeva, F.V., 2010. Extracellular production of reactive oxygen species during seed germination and early seedling growth in Pisum sativum. Plant Physiol. 167, 805–811. Lee, T.M., Lin, Y.H., 1995. Changes in soluble and cell wall-bound peroxidase activities with growth in anoxia-treated rice (Oryza sativa L.) coleoptiles and roots. Plant Sci. 106, 1–7. Li, X.N., Yang, Y.L., Zhang, J., Jia, L.Y., Li, Q.X., Zhang, T.G., Qiao, K.X., Ma, S.C., 2012a. Zinc induced phytotoxicity mechanism involved in root growth of Triticum aestivum L. Ecotoxicol. Environ. Saf. 82, 198–203. Li, X.N., Ma, H.Z., Jia, P.X., Wang, J., Jia, L.Y., Zhang, T.G., Yang, Y.L., Chen, H.J., Wei, X., 2012b. Responses of seedling growth and antioxidant activity to excess iron and copper in Triticum aestivum L. Ecotoxicol. Environ. Saf. 86, 47–53. Liao, W.B., Huang, G.B., Yu, J.H., Zhang, M.L., 2012. Nitric oxide and hydrogen peroxide alleviate drought stress in marigold explants and promote its adventitious root development. Plant Physiol. Biochem. 58, 6–15.

102

X. Duan et al. / Ecotoxicology and Environmental Safety 113 (2015) 95–102

Liao, W.B., Zhang, M.L., Yu, J.H., 2013. Role of nitric oxide in delaying senescence of cut rose flowers and its interaction with ethylene. Sci. Hortic. 155, 30–38. Liu, Y.J., Jiang, H.F., Zhao, Z.G., An, L.J., 2010. Nitric oxide synthase like activity-dependent nitric oxide production protects against chilling-induced oxidative damage in Chorispora bungeana suspension cultured cells. Plant Physiol. Biochem. 48, 936–944. Liszkay, A., Van der Zalm, E., Schopfer, P., 2004. Production of reactive oxygen intermediates (O2  , H2O2, and OH) by maize roots and their role in wall loosening and their role in wall loosening and elongation growth. Plant Physiol. 136, 3114–3123. Matsumoto, H., Motoda, H., 2012. Aluminum toxicity recovery processes in root apices. Possible association with oxidative stress. Plant Sci. 185-186, 1–8. Murphy, M.E., Noack, E., 1994. Nitric oxide assay using hemoglobin method. Methods Enzymol. 233, 240–250. Naik, B.I., Goswami, R.G., Srivastawa, S.K., 1981. A rapid and sensitive colorimetric assay of amine oxidase. Anal. Biochem. 111, 146–148. Neill, S., Barros, R., Bright, J., Desikan, R., Hancock, J., Harrison, J., Morris, P., Ribeiro, D., Wilson, I., 2008. Nitric oxide, stomatal closure, and abiotic stress. J. Exp. Bot. 59, 165–176. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. Phang, I.C., Leung, D.W.M., Taylor, H.H., Burritt, D.J., 2011. Correlation of growth inhibition with accumulation of Pb in cell wall and changes in response to oxidative stress in Arabidopsis thaliana seedlings. Plant Growth Regul. 64, 17–25. Posmyk, M.M., Kontek, R., Janas, K.M., 2009. Antioxidant enzymes activity and phenolic compounds content in red cabbage seedlings exposed to copper stress. Ecotoxicol. Environ. Saf. 72, 596–602. Ranieri, A., Castagna, A., Baldan, B., Soldatini, G.F., 2001. Iron deficiency differently affects peroxidase isoforms in sunflower. J. Exp. Bot. 354, 25–35. Rao, M.V., Paliyath, G., Ormrod, D.P., 1996. Ultraviolet-band ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiol. 110, 125–136. Remans, T., Opdenakker, K., Smeets, K., Matgijsen, D., Vangronsveld, J., Cuypers, A., 2010. Metal-specific and NADPH oxidase dependent changes in lipoxygenase and NADPH oxidase gene expression in Arabidopsis thaliana exposed to cadmium or excess copper. Funct. Plant Biol. 37, 532–544. Rodríguez-Serrano, M., Bárány, I., Prem, D., Coronado, M.J., Risueño, M.C., Testillano, P.S., 2012. NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley. J. Exp. Bot. 63, 2007–2024. Ruley, A.T., Sharma, N.C., Sahi, S.V., 2004. Antioxidant defense in a lead accumulating plant, Sesbania drummondii. Plant Physiol. Biochem. 42, 899–906.

Schaedle, M., Bassham, J.A., 1977. Chloroplast glutathione reductase. Plant Physiol. 59, 1011–1012. Sergiev, I., Alexieva, V., Karanov, E., 1997. Effect of spermine, atrazine and combination between them on some endogenous protective systems and stress markers in plants. Compt. Rend. Acad. Bulg. Sci. 51, 121–124. Stöhr, C., 2007. Nitric oxide – a product of plant nitrogen metabolism. Plant Cell Monogr. 5, 15–34. Su, G.Q., Zhang, W.H., Liu, Y.L., 2006. Involvement of hydrogen peroxide generated by polyamine oxidative degradation in the development of lateral roots in Soybean. J. Integrat. Plant Biol. 48, 426–432. Todeschini, V., Lingua, G., Agostino, G.D., Carniato, F., Roccotiello, E., Bert, G., 2011. Effects of high zinc concentration on poplar leaves: a morphological and biochemical study. Environ. Exp. Bot. 71, 50–56. Wang, H.H., Huang, J.J., Bi, Y.R., 2010. Nitrate reductase-dependent nitric oxide production is involved in aluminum tolerance in red kidney bean roots. Plant Sci. 179, 281–288. Wimalasekera, R., Tebartz, F., Scherer, G.F.E., 2011. Polyamines, polyamine oxidase and nitric oxidase in development, abiotic and biotic stresses. Plant Sci. 181, 593–603. Xu, J., Yin, H.X., Li, Y.L., Liu, X.J., 2010. Nitric oxide is associated with long-term zinc tolerance in Solanum nigrum. Plant Physiol. 154, 1319–1334. Xu, M.J., Dong, J.F., Zhang, M., Xu, X.B., Sun, L.N., 2012. Cold-induced endogenous nitric oxide generation plays a role in chilling tolerance of loquat fruit during postharvest storage. Postharvest Biol. Technol. 65, 5–12. Yang, Y.L., Wei, X.L., Lu, J., You, J., Wang, W.R., Shi, R.X., 2010. Lead induced phytotoxicity mechanism involved in seed germination and seedling growth of wheat (Triticum aestivum L.). Ecotox. Environ. Saf. 73 (8), 1982–1987. Zanardo, D.I.L., Lima, R.B., Ferrarese, M.L.L., Bubna, G.A., Ferrarese-Filho, O., 2009. Soybean root growth inhibition and lignification induced by p-coumaric acid. Environ. Exp. Bot. 66, 25–30. Zhang, L., Chen, Z., Zhu, C., 2012. Endogenous nitric oxide mediates alleviation of cadmium toxicity induced by calcium in rice seedlings. J. Environ. Sci. 24, 940–948. Zheng, L.P., Zhang, B., Zou, T., Chen, Z.H., Wang, J.W., 2010. Nitric oxide interacts with reactive oxygen species to regulate oligosaccharide-induced artemisinin biosynthesis in Artemisia annua hairy roots. J. Med. Plants Res. 4, 758–766. Zhou, Q., 2001. The measurement of malondialdehyde in plants. In: Zhou, Q. (Ed.), Methods in Plant Physiology. China Agricultural Press, Beijing, pp. 173–174. Zou, T., Zheng, L.P., Yuan, H.Y., Yuan, Y.F., Wang, J.W., 2012. The nitric oxide production and NADPH-diaphorase activity in root tips of Vicia faba L. under copper toxicity. J. Plant Omics 5, 115–121.