Plant Cell Rep (2015) 34:367–379 DOI 10.1007/s00299-014-1715-3

ORIGINAL PAPER

Nitrate reductase-mediated nitric oxide production is involved in copper tolerance in shoots of hulless barley Yanfeng Hu • Jia You • Xiaolei Liang

Received: 11 September 2014 / Revised: 28 October 2014 / Accepted: 20 November 2014 / Published online: 2 December 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Key message An NR-mediated early NO production in the shoots of hulless barley plays an important role in protecting hulless barley from Cu toxicity through enhanced antioxidant enzyme activities and antioxidant pools. Abstract Nitric oxide (NO) has been identified as an important signaling molecule that is involved in multiple plant physiological responses, especially under some abiotic stress. Here, we investigated NO production and its effects on copper (Cu) excess in hulless barley shoots. An early NO burst at 24 h was observed in shoots of hulless barley, and the synthesis of early NO was mediated through nitrate reductase (NR), but not through nitric oxide synthase (NOS). Application of the NO donor sodium nitroprusside (SNP) efficiently alleviated Cu-induced shoot inhibition and decrease in chlorophyll content, as well as oxidative damage and reactive oxygen species (ROS) accumulation, while inhibiting NO accumulation by a specific NO scavenger or a NR inhibitor aggravated shoot

Communicated by Kang Chong. Y. Hu (&) Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150000, China e-mail: [email protected] J. You School of Life Science, Northwest Normal University, Lanzhou 730070, China X. Liang Key Laboratory of Cell Activities and Stress Adaptations, Ministry of Education, School of Life Sciences, Lanzhou University, Lanzhou 730000, Gansu, China

inhibition as well as the increase of hydrogen peroxide (H2O2) content, supporting the role of an NR-mediated early NO production in hulless barley responses to Cu toxicity. Furthermore, elevated antioxidant enzyme activities were induced by Cu stress in the shoots of hulless barley and further significantly enhanced by NO donor, whereas suppressed by NO scavenger or NR inhibitor. On the other hand, the application of NO scavenger significantly reduced Cu-induced accumulation of glutathione (GSH) and ascorbate (Asc) in the shoots of hulless barley. Taken together, our results indicate that NO may induce hulless barley seedling tolerance to Cu toxicity through modulating antioxidant enzyme activity and antioxidants accumulation. Keywords Antioxidant
Barley
Copper toxicity
Nitric oxide
Oxidative damage Abbreviations Asc Ascorbate APX Ascorbate peroxidase CAT Catalase cPTIO 2-(4-Carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide DTT Dithiothreitol FAD Flavin adenine dinucleotide GPX Glutathione peroxidase GR Glutathione reductase GSH Glutathione HbO2 Oxyhemoglobin H2O2 Hydrogen peroxide MetHb Methemoglobin L-NAME Nx-nitro-L-arginine methyl ester hydrochloride MDA Malondialdehyde

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NR NOS NO O 2POD PVP ROS SNP SOD TCA

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Nitrate reductase Nitric oxide synthase Nitric oxide Superoxide anion radical Peroxidase Polyvinylpyrrolidone Reactive oxygen species Sodium nitroprusside Superoxide dismutase Trichloroacetic acid

Introduction Heavy metal contamination is a serious problem for the environment because of its increasing level caused by anthropogenic activities such as mining, smelting, municipal waste disposal, electroplating, and phosphate fertilizer (Alloway 1995). Among the metallic elements, copper (Cu) is an essential micronutrient for normal plant growth and development (Maksymiec 1997). It is required in much important biological function since the element Cu has been selected as a cofactor in redox reactions of many enzymes and proteins like plastocyanin, cytochrome c, and Cu/Zn- superoxide dismutase (Cu/Zn¯SO). In addition, Cu plays important roles in many essential physiological processes such as photosynthesis, respiration, detoxification of free radicals, signaling of transcription, and protein trafficking machinery, cell wall remodeling and lignification (Burkhead et al. 2009). However, excess Cu is toxic to plants, producing a wide range of adverse effects, such as inhibition of photosynthetic and respiratory activity, DNA and membrane damage, and other metabolic disturbances (Demirevska-Kepova et al. 2004; Tanyolac¸ et al. 2007). Another important toxic feature of over-accumulation Cu is the induction of the generation of harmful reactive oxygen species (ROS) such as superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (HO) oxidative stress (Contreras et al. 2009; Madejo´n et al. 2009; Xu et al. 2011). Plants have evolved protective mechanisms including enzymatic and non-enzymatic antioxidant pathways to scavenge ROS and alleviate their deleterious effects induced by the heavy metals (Cobbett and Goldsbrough 2002). Several antioxidative enzymes, including catalase (CAT EC 1.11.1.6), peroxidase (POD EC 1.11.1.7), superoxide dismutase (SOD EC 1.15.1.1), ascorbate peroxidase (APX EC 1.11.1.11), glutathione reductase (GR), and guaiacol peroxidase (GPX), are reported to be involved in minimizing ROS damage or oxidative bursts during heavy metals stress responses (Xu et al. 2010; Iseri et al. 2011; Thounaojam et al. 2012; Sun et al. 2014). The non-

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enzymatic antioxidants scavengers such as ascorbate (Asc), glutathione (GSH), and hydrophobic molecules (tocopherols, carotenoids, xanthophylls, and polyamines) also play a central role in the antioxidant defense mechanism in plant cells (Foyer and Noctor 2005). Nitric oxide (NO) is a small, highly diffusible gas and has emerged as an important signaling molecule involved in many important physiological processes in plants, including regulation of seed germination, root growth and development, floral transition, stomatal movement, and senescence (Neill et al. 2003; Besson-Bard et al. 2008). In addition, NO was also reported to be involved in responses to abiotic stresses such as low temperature, salt, and heavy metals (Zhao et al. 2007, 2009; Xiong et al. 2010). It has been indicated that nitric oxide synthase (NOS) and nitrate reductase (NR), as two key enzymes for NO production, play an important role in responses to several stress (Neill et al. 2003; Besson-Bard et al. 2008; Xiong et al. 2010). NOS-like activity has been found widely in plants, and inhibitors of mammalian NOS were also suggested to inhibit NO generation and NO-mediated responses in plants under heavy metal stress (Xiong et al. 2010; Xu et al. 2010; Gonza´lez et al. 2012). Apart from NOS, NR is also capable of producing NO in plants. Several evidences were provided to indicate that NR-mediated NO production participated in heavy metal stress responses (Wang et al. 2013; Sun et al. 2014). Previous studies also suggested that NO protects plant cells against oxidative stress by reducing ROS accumulation (Xu et al. 2010; Wang et al. 2010; Sun et al. 2014), but alleviatory role of NO in Cu toxicity is still scanty and needs more attention. Recently, NO has been reported to counteract the toxicity of ROS generated by excessive Cu in tomato (Wang et al. 2010). However, very little is known about the specific role of different NO sources in plants with regard to tolerance to Cu toxicity. In present study, we used an ancient cereal crop, hulless barley which is widely distributed throughout QinghaiTibet in China (Sun et al. 1999; Yin et al. 2003), to investigate the relationships of NO, ROS, and antioxidants in Cu toxicity. Furthermore, the source of the timedependent NO production under Cu stress was examined. An effort was made to elucidate a possible physiological mechanism of NO in increasing hulless barley seedlings tolerance to Cu toxicity.

Materials and methods Plant materials and growth conditions Seeds of hulless barley (Hordeum vulgare L. var. nude) were purchased from Qinghai Academy of Agricultural Sciences, Qinghai Province, China. Seeds were surface

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sterilized by 1 % (v/v) sodium hypochlorite for 15 min, rinsed thoroughly five times with sterilized water and then germinated at 25 °C for 48 h in the dark. Uniformly germinated seeds were selected and were precultivated in plastic screens floating on a container filled with 1/4 Hoagland solution (pH 6.2). After 3 days of precultivation, uniform seedlings were selected for different treatments under a 13 h/24 °C day and a 11 h/18 °C night regime, a light intensity of 300 lmol m-2 s-1 photosynthetically active radiation and 70 % relative humidity in a growth chamber. The nutrient solution was renewed daily. Different concentrations of CuSO4 were added into the nutrient solution for Cu stress experiments. To test the effects of various inhibitors and scavengers, the seedlings of hulless barley were pretreated with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3-oxide (cPTIO, 150 lM) (Sigma), the NR inhibitor tungstate (150 lM) (Sigma), or the NOS inhibitor Nx-NitroL-arginine methyl ester hydrochloride (L-NAME, 150 lM) (Sigma) for 3 h before exposure to CuSO4 or 200 lM sodium nitroprusside (SNP, an NO donor) (Sigma) treatment for indicated time under the same conditions as described earlier. Each experiment was repeated at least three times. Growth and chlorophyll content analysis

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the digest solution became clear. The digest residue was dissolved in 2 M HCl and diluted with distilled water to 10 mL. The Cu content in the HCl solution was analyzed using an atomic absorption spectrophotometer (Perkin, Germany). Analysis of H2O2 content, the generation of O2and lipid peroxidation Determination of H2O2 was performed according to the method of Iseri et al. (2011). Lipid peroxidation was measured in terms of malondialdehyde (MDA) content following the method of Heath and Packer (1968). 0.2 g of the shoots was homogenized in 10 mL 10 % trichloroacetic acid (TCA) and centrifuged at 12,0009g for 15 min. A 2-mL aliquot of supernatant was mixed with 2 mL of 10 % TCA containing 0.6 % thiobarbituric acid. The mixture was boiled at 100 °C for 30 min then quickly cooled in an ice bath. After centrifugation at 15,0009g for 10 min, the absorbance of the supernatant was measured at 532 nm. The values were corrected for non-specific interference by subtracting absorbance reading at 600 nm. The amount of MDA was calculated using an extinction coefficient of 155 mM-1 cm-1. The generation rate of O2- was determined following the method of Sun et al. (2014).

Growth of the plant was determined by measuring the shoot length and fresh weight. Three-day-old seedlings were exposed to treatment solutions containing different concentrations of CuSO4 or various chemicals for indicated time. After treatment, the length of shoot was measured with a ruler, and a sample of 15–20 plants per treatment was used to record shoot length at indicated time. Fifty plants were harvested, and the fresh weight of the shoots was measured immediately. The chlorophyll was extracted and measured according to the method of Porra et al. (1989). After 4 days of various treatments, the fresh leaves (0.5 g) were ground to powder through adding a spot of quartz sand and then placed in 5 mL acetone (80 %) and incubated at 4 °C until the samples turned white. The samples were transferred into a 25-mL container, at the same time washing the mortar and the draff repeatedly. Finally, the volume of the samples was fixed using 80 % of 25 mL acetone and filtrated. Absorbance was measured under the wavelengths of 663, 645, 652 nm, respectively, using a UV–VIS spectrophotometer (UV-5300 PC, Shanghai Metash Instruments Co., Ltd.).

Determination of GSH and Asc content

Determination of Cu content

Determination of NO by hemoglobin

After treatment, the shoots were washed thoroughly with 10 mM EDTA three times. The samples were oven dried at 70 °C for 48 h and digested with 3:1 HNO3/HClO4 until

NO content was assayed by monitoring the conversion of oxyhemoglobin (HbO2) to methemoglobin (MetHb) as described by Murphy and Noack (1994). The method is

Total GSH content was measured according to a method from Griffith (1980). 0.5 g shoots was homogenized with 5 mL phosphoric acid and centrifuged at 15,0009g for 20 min at 4 °C. The supernatant was collected and used for assay preparation. The reaction mixture for GSH content contained plant extract, 0.1 M phosphate buffer (pH 6.8), 5, 5-dithiobis-2-nitrobenzoic acid and the absorbance was assayed spectrophotometrically at 412 nm. Asc content was determined according to the method described by Foyer et al. (1983). 0.5 g of shoots was ground in liquid nitrogen and 1 mL of 2.5 M perchloric acid was added. The crude extract was centrifuged at 4 °C for 15 min at 15,0009g, and the supernatant was neutralized with saturated Na2CO3 using methyl orange as the indicator. The reduced Asc was assayed spectrophotometrically at 265 nm in 1 M NaH2PO4 buffer, pH 5.6, with 1 unit ascorbate oxidase. The total Asc was assayed after incubation in the presence of 30 mM dithiothreitol (DTT) for 30 min.

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based on the direct reaction between NO and HbO2, which yields MetHb. Shoots of hulless barley (0.5 g) were incubated with 100 units of CAT and 100 units of SOD for 5 min to remove endogenous ROS before adding 10 mL of 5 mM HbO2. After incubation for 5 min, NO was calculated by measuring the conversion of HbO2 to MetHb spectrophotometrically at 401 and 421 nm, using an extinction coefficient of 77 mM-1 cm-1. Determination of NR and NOS activity The extraction and determination of NR activity were performed according to the method described by Mackintosh et al. (1995). 0.5 g shoots of hulless barley were homogenized with a mortar and pestle on ice with 1.5 mL of extract buffer [50 mM HEPES–KOH, pH 7.5, 5 % glycerol (v/v), 10 mM MgCl2, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine and 10 lM flavin adenine dinucleotide (FAD)]. Extract was centrifuged at 15,0009g for 20 min at 4 °C. The activity of NR was measured immediately by mixing 250 ll of supernatant with 250 ll prewarmed (25 °C) assay buffer (50 mM HEPES–KOH, pH 7.5, 10 mM MgCl2, 1 mM DTT, 2 mM KNO3, 200 lM NADH). The reaction was started by adding assay buffer, incubated at 30 °C for 30 min and then stopped by adding 50 ll of 0.5 M zinc acetate. The nitrite produced was measured colorimetrically at 540 nm after adding 150 ll of 1 % (w/v) sulfanilamide and 150 ll of 0.02 % (w/v) N-(1-naphthyl) ethylenediamine dihydrochloride. The NOS activity was measured according to Gonza´lez et al. (2012). The total protein was extracted using the buffer containing 100 mM HEPES–KOH (pH 7.5), 1 mM EDTA, 10 % glycerol (v/v), 5 mM DTT, 0.5 mM PMSF, 0.1 % Triton X-100 (v/v), 1 % polyvinylpyrrolidone (PVP) and 20 lM FAD. After centrifugation at 15,0009g for 20 min at 4 °C, NOS activity was detected in 1 mL of reaction mixture containing 100 mM phosphate buffer (pH 7.0), 0.5 mM L-Arg, 2 mM MgCl2, 0.3 mM CaCl2, 2 lM tetrahydrobiopterin, 1 lM FAD, 1 lM flavin mononucleotide, 0.2 mM DTT, 0.2 mM NADPH, and 200 ll of protein extract. The decrease in absorbance due to NADPH consumption was determined at 340 nm for 5 min. NOS activity was calculated using the extinction coefficient of NADPH (e = 6.22 mM-1 cm-1). Analysis of enzyme activities Fresh shoots (0.5 g) were frozen in liquid nitrogen and homogenized in 2 mL of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, 1 mM DTT and 2 % PVP. The homogenate was centrifuged at

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17,0009g for 20 min at 4 °C and the supernatant was used for the following enzyme assays. SOD (EC 1.15.1.1) activity was determined as described by Prochazkova et al. (2001). SOD activity was assayed by monitoring the inhibition of photochemical reduction of nitro blue tetrazolium (NBT). One unit of SOD activity was defined as the amount of enzyme required to cause 50 % inhibition of the reduction of NBT as monitored at 560 nm. CAT (EC 1.11.1.6) activity was determined by measuring the rate of decomposition of H2O2 at 240 nm for 3 min (Aebi 1984). APX (EC 1.11.1.11) activity was measured in the presence of 0.25 mM ascorbic acid and 0.5 mM H2O2 by monitoring the decrease of absorbance at 290 nm (Janda et al. 1999). POD (EC 1.11.1.7) activity was monitored by oxidation of guaiacol using H2O2 (Upadhyaya et al. 1985). The activities of GR and GPX determined as described by Thounaojam et al. (2012). The GR (EC 1.6.4.2) activity was determined by monitoring the decrease in absorbance at 412 nm as a result of oxidized GSH-dependent NADPH consumption. The reaction mixture for GPX (1.11.1.7) contained 2.1 mL of 50 mM phosphate buffer (pH 7.0), 0.3 mL of 1 % guaiacol, 0.3 mL of 1 % H2O2, 0.3 mL of enzyme extract and incubated for 5 min, and absorbance was monitored at 340 nm. Statistical analysis Each experiment was repeated at least three times. The data were analyzed by one-way analysis of variance (ANOVA) procedures and compared for level of significance by Duncan’s multiple range tests using SPSS version 17.0 software. In all figures, the error bars represent the standard deviation (SD) of means, and the confidence coefficient was set at p \ 0.05.

Results Growth inhibition of hulless barley shoots under Cu stress First, some symptoms of Cu toxicity were evaluated in the shoots of hulless barley seedlings with different CuSO4 treatment in comparison with controls. As shown in Table 1, a gradual decrease in the length and fresh weight of shoots was observed with the increase in Cu concentration after 4 days of treatment. A low Cu concentration (150 lM) did not significantly decrease the shoot length and weight. However, both the shoot length and weight reduced markedly in the presence of 300 lM or higher Cu concentrations (450 and 750 lM), the shoot length decreases by 15.56, 39.14 and 50.80 %, respectively. Exposure from 300 to 750 lM Cu treatment progressively

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Table 1 Effect of different concentrations of Cu on shoot length, fresh weight, contents of chlorophyll, GSH and Asc in shoots of hulless barley after 4 days of treatment Parameter

0 lM

150 lM

300 lM

Shoot length (cm)

18.19 ± 0.52

17.40 ± 0.75

15.36 ± 0.30a

11.07 ± 0.59a

8.95 ± 0.53a

2.36 ± 0.25

a

1.64 ± 0.07

a

1.28 ± 0.08a

4.82 ± 0.28

a

3.84 ± 0.25a

37.08 ± 1.45

a

44.18 ± 1.69a

0.86 ± 0.03

a

0.94 ± 0.05a

Fresh weight (g)

2.68 ± 0.13

Chlorophyll (mg g -1

GSH (lmol g Asc (mmol g

-1

-1

FW) FW)

FW)

8.10 ± 0.42 27.25 ± 2.06

7.14 ± 0.79 28.42 ± 1.69

0.78 ± 0.02

0.80 ± 0.06

450 lM

1.95 ± 0.12

a

5.61 ± 0.17

a

33.65 ± 0.61 0.83 ± 0.05

750 lM

The data presented are mean of three separate experiments ± SD a

Indicates significant differences from control at the 0.05 level according to LSD test

also reduced the chlorophyll content up to 52.59 % compared to untreated control plants (Table 1). Excess Cu induces ROS production and oxidative damage in shoots of hulless barley Because Cu toxicity inevitably caused the production of ROS in plants, we further investigated the accumulation of H2O2 and O2- in shoots exposed to 450 lM CuSO4 for different time periods. As indicated in Fig. 1, the production of H2O2 was detectable as early as 6 h of Cu treatment and reached a maximum at 48 h, and then exhibited a slight decline with extended time, but still remained significantly higher than that of the control for up to 96 h (Fig. 1a). The O2- accumulation showed a similar pattern as the production of H2O2 in shoots of hulless barley (Fig. 1b), but a significant increase in O2- content was observed at 12 h after exposure to Cu. When plants were subjected to environmental stress, oxidative damage resulted in membrane lipid peroxidation, which could be monitored by MDA content. The results showed that MDA content gradually increased with the increased treatment time under 450 lM Cu treatments in shoots, and it reached 249.25 % of the control at 96 h. These results suggested that excess Cu induced ROS production leading to oxidative damage in shoots of hulless barley. Elevations in GSH and Asc contents under Cu stress Under metal stress, the reduced GSH and Asc are important antioxidants and redox buffers in plant cells. The content of both antioxidant metabolites was determined in hulless barley shoots after 4 days exposure to different Cu concentrations. Treatment of the hulless barley plant with different concentrations of Cu increases total GSH content in shoots, maximum increase was observed at 750 lM Cu after 4 days of treatment by 62.13 % compared to the control (Table 1). No significant effect of lower concentrations Cu (150 and 300 lM) on Asc content was seen in shoots, but an significant increasing trend in total Asc

Fig. 1 Effects of Cu excess on ROS accumulation and MDA content in shoots of hulless barley. Three-day-old seedlings were treated with 450 lM CuSO4 for indicated time, and then contents of H2O2 (a), O2- (b) and MDA (c) in the treated shoots were measured. Values are the mean ± SD for three independent experiments. Error bars with different letters represent significant differences (p \ 0.05)

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production was observed under 450 and 750 lM Cu treatment (Table 1). Endogenous NO production in hulless barley shoots under Cu stress Previous studies demonstrated that NO was involved in responses to metal stress in plants (Wang et al. 2010). To elucidate the correlation between NO accumulation and Cu tolerance, endogenous NO production was determined in shoots exposed to Cu stress for different periods (Fig. 2). After treatment with 450 lM Cu, an early burst of NO was observed at 12 h in shoots of hulless barley and peaked at 24 h. The increase of NO production was sustained for 72 h, after which the production gradually declined. After 96 h of Cu treatment, NO levels were similar to levels in the control shoots (Fig. 2). Effects of NO on shoots growth and antioxidant contents under Cu stress To further confirm the role of the early NO burst during the hulless barley response to Cu stress, SNP, the most commonly used NO donor, was applied to the Cu-treated plants to mimic a similar early NO burst. Application of 200 lM SNP significantly reduced Cu-induced shoot inhibition and decrease in chlorophyll content, which are two typical toxic symptoms of Cu stress (Fig. 3). To investigate whether the alleviating effect of SNP was a result of the triggered NO production, a special NO scavenger, cPTIO, was used. Pretreatment with the NO scavenger 150 lM cPTIO

Fig. 2 Effects of Cu stress on NO production in shoots of hulless barley. Three-day-old seedlings were exposed to 450 lM CuSO4 for indicated time. Data are mean values ± SD of three independent experiments. Error bars with different letters indicate significant differences (p \ 0.05)

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for 3 h strongly reversed the beneficial effect of SNP on Cu-induced damage to hulless barley shoots, repressed the SNP-induced increase of shoot growth and chlorophyll content (Fig. 3a, b). SNP treatment did not affect Cuinduced total GSH and Asc contents in shoots, however, depletion of endogenous NO by cPTIO dramatically decreased the Cu-induced increase in GSH and Asc contents (Fig. 3c, d). These results showed that NO treatment enhances hulless barley tolerance to Cu stress. Application of NO donor reduces ROS accumulation and oxidative damage We further tested the production of H2O2 and O2- in shoots exposed to excess Cu and the role of NO in modulating the levels of H2O2 and O2-. Cu-induced accumulation of H2O2 and O2- was found in the shoots of hulless barley after 48-h Cu treatment. The application of SNP significantly reduced ROS (H2O2 and O2-) contents under Cu stress (Fig. 4a, b). However, supplementation with NO scavenger, cPTIO, further increased the production of H2O2 and O2- compared with the 450 lM Cu treatment alone in the shoots at 48 h of treatment. Similar to ROS change, SNP treatment also reduced Cu-induced increase in MAD content in the shoots (Fig. 4c). The data indicate that application of the NO donor SNP significantly relieved the amount of Cu-induced oxidative damage in hulless barley seedlings. Source of endogenous NO under Cu stress It has been known that NR and NOS serve as two key enzymes responsible for endogenous NO production in plants (Neill et al. 2003). To examine the possible source of early NO burst induced by Cu treatment, the NOS and NR activities were determined in the shoots of hulless barley seedlings exposed to 450 lM Cu for different times. As shown in Fig. 6, treatment of Cu triggered significant increase in NR activity after 12 h treatment. NR activity also displayed a similar pattern as NO did in shoots and they increased significantly during 48-h exposure to Cu, and peaked at 24 h (Figs. 4, 5a). By contrast, the activity of NOS only increased in the shoots after treatment with Cu for 12 h (Fig. 5b). To further elucidate the source of NO production induced by Cu, tungstate (an NR inhibitor) and L-NAME (an NOS inhibitor) were used in this study. As shown in Fig. 6a, application of tungstate significantly reduced the Cu-induced NO production in shoots at 24 h, but NOS inhibitor (L-NAME) did not, suggesting that NR pathway may be a major source for Cu-induced NO production in hulless barley shoots. Under Cu stress, pretreatment with tungstate aggravated its shoot growth inhibition, which

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Fig. 3 Effects of SNP and NO scavengers on Cu-induced shoot growth (a), chlorophyll (b), GSH (c) and Asc (d) contents in shoots of hulless barley. Threeday-old seedlings were treated with 450 lM CuSO4 or 200 lM SNP, or both, for 4 days. For the NO scavenger treatment, the seedlings were pretreated with 150 lM cPTIO for 3 h, followed by Cu treatment. Mean values and SD were calculated from three independent experiments. Within each set of experiments, bars with different letters indicate significant difference with p \ 0.05

could be subsequently reversed by application of SNP. While compared with Cu treatment alone, no significant Cu-induced shoot growth inhibition was observed by pretreatment with L-NAME (Fig. 6b). These results indicated that Cu-induced NO accumulation may be mediated by proteins similar to NR. NO affects the activities of antioxidative enzymes under Cu stress To evaluate the role of the antioxidative system in response to Cu stress, the antioxidative enzyme activities were analyzed. We found that the activities of SOD, CAT, POD, and APX in shoots were significantly increased by Cu treatment of 4 days (Fig. 7a–d). Application of SNP further increased the activities of SOD, CAT and APX under Cu stress. We also found that NO scavenger (cPTIO) and NR inhibitor (tungstate) pretreatment suppressed the activities of Cu-induced antioxidant enzymes (Fig. 7a–d). To further investigate how the intracellular Asc–GSH cycle is maintained in response to Cu stress in hulless barley shoots, the activities of GR and GPX were measured. Excess Cu increases the activities of GR and GPX, and exogenous NO supply further elevated GR activity, but not GPX in shoots of hulless barley. Conversely, the activities of GR and GPX tested were inhibited by the application of cPTIO or tungstate in the shoots in the presence of Cu treatment

(Fig. 7e, f). Our results revealed that an early NO production played an important role in protection against Cuinduced oxidative stress by enhancing antioxidant enzyme activities. NO did not affect Cu accumulation in the shoots exposed to excess Cu To test whether the alleviating effect of NO on Cu stress was due to the reduction of Cu content, we determined the accumulation of Cu in shoots of hulless barley. As shown in Fig. 8, Cu content in shoots was significantly increased by Cu stress compared to control, while addition of SNP, cPTIO or tungstate could not markedly alter Cu content. This observation suggested that Cu uptake and transport are not associated with altered NO levels in hulless barley seedling.

Discussion Production of NO is an early response to various biotic and abiotic stresses, such as pathogen attack, salt, low temperature, and heavy metal stress, suggesting that the NOmediated stress response is essential for stress tolerance and survival of plants (Neill et al. 2003; Besson-Bard et al. 2008). Many past studies on NO function in plants

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Fig. 5 Effect of Cu stress on activities of NR (a) and NOS (b) in shoots of hulless barley seedlings. Three-day-old seedlings were treated with 450 lM CuSO4 for different time. Shoots were collected for assaying activities of NR and NOS after indicated time exposure to the treatment solutions. Data are mean values ± SD of three independent experiments. Error bars with different letters indicate significant difference with p \ 0.05

Fig. 4 ROS content in shoots of hulless barley seedlings treated with Cu, SNP and cPTIO. Three-day-old plants were treated either with or without 200 lM SNP, 150 lM cPTIO under 450 lM CuSO4 stress for 48 h and then the contents of H2O2 (a), O2- (b), and MDA (c) in the shoots of hulless barley were determined using spectrophotometry. Values represent mean ± SD. Different letters indicate significant differences (p \ 0.05) among the treatments. CK, control

response to heavy metals were based on the use of root tissue (Xu et al. 2010; Wang et al. 2010; Gonza´lez et al. 2012; Sun et al. 2014). For example, Xu et al. (2010) suggested that only an early production of NO is associated with long-term Zinc tolerance in the roots of Solanum nigrum, whereas both an early burst of NO and a secondary

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NO generation in roots were involved in the tolerance of wheat to aluminum stress (Sun et al. 2014). Under Cu excess, an early strong NO burst and the following wave of secondary NO generation were also found in the Ulva compressa associated with the stress resistance (Gonza´lez et al. 2012). It is possible that the discrepancy of NO generation between the studies is related to the different time point for determining endogenous NO and the different plant species used. However, stress-induced endogenous NO production in overground tissues of plants might play specific roles in response to heavy metals. Here, we found that the rapid accumulation of NO induced by excess Cu was not only restricted to root tissue of hulless barley (unpublished data) but also detected in the shoots (Fig. 2), which indicated a specific role of NO in different plant tissue response to metal stress. Previous studies reported that ROS can induce NO accumulation in the plant response to heavy metals stress (Zhao et al. 2007). In this study, Cu triggered an early NO

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Fig. 6 Effects of NR inhibitor (Tungstate) and NOS inhibitor (LNAME) on NO production (a) and shoot length (b) of hulless barley seedlings under Cu stress. Three-day-old hulless barley seedlings were pretreated with 150 lM tungstate or 100 lM L-NAME for 3 h, followed by 450 lM CuSO4 treatments. After 24 h, seedlings were collected for determination of NO production. Shoot growth was measured after 4 days of Cu treatment with tungstate, L-NAME and SNP. Data are mean values ± SD of three independent experiments. Different letters indicate significant differences (p \ 0.05) among the treatments. CK, control

production in hulless barley shoots at 12 h, and it peaked at 24 h, after which the production significantly declined (Fig. 2). However, Cu-induced an early H2O2 production in shoots occurred within 6 h (Fig. 1). This time course supports the hypothesis that ROS plays a vital role in an early NO burst when plant exposes to stress (Zhao et al. 2007). In addition, these findings are consistent with the observation that NO synthesis in U. compressa exposed to Cu excess is activated by H2O2 (Gonza´lez et al. 2012). However, many

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groups have also found that NO acts upstream of ROS in plants under heavy metals stress and showed that NO stimulates ROS production in Arabidopsis suspension cells and Solanum nigrum (De Michele et al. 2009; Xu et al. 2010). This is conflicted with our results showing that supplementation with a NO scavenger, cPTIO, markedly increased ROS accumulation, but application of SNP reduced H2O2 production in hulless barley shoots exposed to Cu excess (Fig. 4). It is possible that the discrepancy between the studies is related to a complex crosstalk between NO and ROS as early intracellular signals in the plant stress response and the different plant species used. The importance of antioxidant enzymes (e.g., SOD, CAT, APX, and POD) implicated in H2O2 and O2- cleanup under Cu stress have been addressed in many plants, although there are conflicting results in these studies. For example, it was reported that antioxidative enzyme activity increased after Cu treatment in maize (Tanyolac¸ et al. 2007), Lycopersicum esculentum Mill. and Cucumis sativus L. plants (Is¸ eri et al. 2011), Arabidopsis seedlings (Cuypers et al. 2011) and rice roots (Thounaojam et al. 2012), but decreased in rice leaves (Chen and Kao 1999). In tomato seedlings, Cu stress markedly increased antioxidative enzyme activity in leaves (Wang et al. 2010), but did not affect these enzyme activities in roots and stems (Mazhoudi et al. 1997). The discrepancies may be attributed to differences in plant species and Cu concentrations used and in the time points detected in these studies. In the present experiment, activities of SOD, POD, CAT, and APX in shoots were significantly increased by Cu treatments (Fig. 7), implying that antioxidative enzymes induction might play an important role in hulless barley tolerance to Cu excess. Numerous studies have found that the function of NO alleviation of oxidative damage caused by heavy metal stresses was ascribed to induction of various ROSscavenging enzyme activities (Wang et al. 2010; Xiong et al. 2010; Xu et al. 2010; Sun et al. 2014). Recently, it was suggested that NO also induced the expression of antioxidant protein genes in U. compressa under Cu stress, which could be a strategy in fighting against Cu toxicity (Gonza´lez et al. 2012). In this work, increased NO production by application of SNP is followed by dramatically increased antioxidant enzymes activity (Fig. 7), thus reducing ROS (H2O2 and O2-) production and lipid peroxidation (Fig. 4). Similar results were reported in the experiment of NO enhancing the tolerance of tomato to Cu excess (Wang et al. 2010) and in the study of NO reducing Cu toxicity in rice leaves by preventing oxidative stress (Yu et al. 2005). These findings support an important role for NO in increasing the activities of antioxidant enzymes resulting decreased Cu-induced oxidative stress and alleviation of Cu toxicity. This is further supported by the

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Fig. 7 Changes in activities of SOD (a), CAT (b), POD (c), APX (d), GR (e) and GPX (f) in shoots of hulless barley seedlings under Cu stress. Three-day-old hulless barley seedlings were treated with 450 lM CuSO4 with or without 200 lM SNP, 150 lM cPTIO, 150 lM tungstate for 4 days, followed by 450 lM CuSO4 treatments.

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For the NO scavenger and NR inhibitor treatments, the seedlings were pretreated with cPTIO or Tungstate for 3 h, followed plus Cu treatment for 4 days. Each value is the mean ± SD of three independent experiments. Different letters indicate significant differences (p \ 0.05) between treatments. CK, control

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Fig. 8 Effects of exogenous NO supply on Cu accumulation in shoots of hulless barley seedlings. 450 lM CuSO4, 200 lM SNP, 150 lM cPTIO and 150 lM tungstate were used for various treatments. Three-day-old hulless barley seedlings were exposed to different treatments for 4 days and then shoots were collected for determination of Cu content. Data are mean values ± SD of three independent experiments. Error bars with different letters indicate significant difference (p \ 0.05). CK, control

decreased antioxidant enzymes activity after application of the NO scavenger (cPTIO) and NR inhibitor (tungstate) in the Cu-stressed hulless barley seedlings (Fig. 7). With respect to NO generation, plants have several potential sources for NO synthesis, including the NR enzyme and NOS-like enzyme pathways, and non-enzymatic pathways (Neill et al. 2003; Besson-Bard et al. 2008). A large number of reports have demonstrated that heavy metals such as zinc, cadmium, and aluminum induced NO synthesis in plants, which is dependent on an NOS-like activity (Xiong et al. 2010; Xu et al. 2010). It also has been shown that Cu induced NO production through an NOS-like enzyme in U. compressa (Gonza´lez et al. 2012). However, several lines of evidence in present study suggested that the early production of NO in the shoot of hulless barley is mainly mediated by NR-dependent route under Cu excess (Figs. 4, 5a, 6a). Very interesting, Wang et al. (2010) reported that the NR inhibitor and NOS-like inhibitor can efficiently suppress Cu-induced NO accumulation in leaves of tomato, suggesting that both NR and NOS-like may be required for NO accumulation. These observations differed from our results in hulless

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barley, implying that the contribution of NOS or NRmediated NO production in plants may be dependent upon environmental stimuli and species. Another ROS-scavenging system is small molecular mass non-enzymatic antioxidants, for instance, GSH and Asc, which are associated with maintaining an appropriate oxidative and reductive state in plants under various stresses (Noctor and Foyer 1998; Gill and Tuteja 2010; Li et al. 2010; Hasanuzzaman et al. 2011; Thounaojam et al. 2012). Because Asc and GSH function coordinately with other antioxidants scavengers and several enzymatic antioxidants to counteract O2-, which is generated by the Mehler reaction and photorespiration (Noctor and Foyer 1998), thereby the accumulation of GSH and Asc in the Cu-treated hulless barley shoots indicates its involvement in protecting against oxidative damages caused by the overproduction of ROS (Table 1). Similar finding has been reported in rice and Arabidopsis seedlings under metal stress (Cuypers et al. 2011; Thounaojam et al. 2012). Enzymatic antioxidants of the Asc–GSH cycle in shoots of hulless barley play very important roles in response to Cu excess. GR participated in the enzymatic detoxification of ROS and contributes to the maintenance of a higher GSH to GSSG ratio. GPX is the principle cellular enzyme capable of removing H2O2 and membrane lipid peroxidation repair and is generally considered to be the main line of enzymatic defense against oxidative damages (Islam et al. 2009; Hasanuzzaman et al. 2011). Multiple studies indicate a role for GR and GPX in the adaption process against heavy metal stress-induced oxidative stress (Li et al. 2006; Drazkiewicz et al. 2007; Cuypers et al. 2011; Thounaojam et al. 2012). The present study also showed that Cu stress induced the increase in the activities of GR and GPX (Fig. 7), which could be an acclimatization step against Cu toxicity. These results are in agreement with previous work (Drazkiewicz et al. 2007; Cuypers et al. 2011; Thounaojam et al. 2012), where Asc–GSH cycle plays an important role in reducing the oxidative damage of Cu toxicity. Here, we also provided evidences that Asc– GSH cycle involves in the NO-mediated tolerance to Cu stress in hulless barley seedlings. Application of SNP can significantly increase GR activity and the levels of GSH and Asc, but addition of an NO scavenger or NR inhibitor decreased the activities of GR and GPX, and reduced Cu induced the accumulation of Asc and GSH (Figs. 3, 7), indicating that NO may play an important role in activating Asc–GSH pathway during hulless barley response to Cu excess. Previous studies suggest that ROS detoxification could happen through the NO-dependent induction of expression of antioxidant genes such as GPX or GR under metal stress (Hu et al. 2007; Wang et al. 2010), which further supports the role for NO in reducing the toxic levels of ROS through Asc–GSH cycle. More recently, it was

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demonstrated that the Asc-deficient Arabidopsis mutant (vtc2-1, vtc2-3 and miox4), which altered Asc metabolism and subsequently changed ROS levels, exhibited the reduced NO contents under Cu stress (Pet} o et al. 2013), suggesting that under Cu stress the impact of a strictly regulated ROS level on NO production is associated with Asc metabolism. In conclusion, our study is the first to indicate the important role of the NR-mediated early NO burst involved in the protection process against Cu toxicity in shoots of hulless barley. We found that the NR-mediated early burst of NO exerted its protective effect under Cu stress through the activation of some antioxidative enzymes and/or antioxidant pools, not by preventing Cu uptake and transport (Fig. 8). These mechanisms help to maintain membrane integrity and shoot growth of hulless barley by reducing lipid peroxidation caused by ROS. Such an understanding is helpful for the breeding and cultivation of a Cu-resistant cereal crop. Author contribution statement Conceived and designed the experiments: YFH. Performed the experiments: YFH XLL and JY. Analyzed the data: YFH. Contributed to the writing of the manuscript: YFH. Acknowledgments This work was supported by Open Funds for Key Laboratory of Mollisols Agroecology of Chinese Academy of Sciences and the National Nature Science Foundation of China (No.31301252). Conflict of interest of interest.

The authors declare that they have no conflict

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