Research
Nitrate reductase-mediated early nitric oxide burst alleviates oxidative damage induced by aluminum through enhancement of antioxidant defenses in roots of wheat (Triticum aestivum) Chengliang Sun1*, Lingli Lu1,2*, Lijuan Liu1, Wenjing Liu1, Yan Yu1, Xiaoxia Liu1, Yan Hu1, Chongwei Jin1,2 and Xianyong Lin1,2 1
MOE Key Laboratory of Environment Remediation and Ecological Health, College of Natural Resource & Environmental Sciences, Zhejiang University, Hangzhou 310058, China; 2Key
Laboratory of Subtropical Soil Science and Plant Nutrition of Zhejiang Province, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China
Summary Author for correspondence: Xianyong Lin Tel: +86 571 88982476 Email:
[email protected] Received: 6 October 2013 Accepted: 9 October 2013
New Phytologist (2014) 201: 1240–1250 doi: 10.1111/nph.12597
Key words: aluminum (Al), antioxidant, nitric oxide (NO), resistance, wheat (Triticum aestivum).
Nitric oxide (NO) is an important signaling molecule involved in the physiological processes
of plants. The role of NO release in the tolerance strategies of roots of wheat (Triticum aestivum) under aluminum (Al) stress was investigated using two genotypes with different Al resistances. An early NO burst at 3 h was observed in the root tips of the Al-tolerant genotype Jian-864, whereas the Al-sensitive genotype Yang-5 showed no NO accumulation at 3 h but an extremely high NO concentration after 12 h. Stimulating NO production at 3 h in the root tips of Yang-5 with the NO donor relieved Al-induced root inhibition and callose production, as well as oxidative damage and ROS accumulation, while elimination of the early NO burst by NO scavenger aggravated root inhibition in Jian-864. Synthesis of early NO in roots of Jian-864 was mediated through nitrate reductase (NR) but not through NO synthase. Elevated antioxidant enzyme activities were induced by Al stress in both wheat genotypes and significantly enhanced by NO donor, but suppressed by NO scavenger or NR inhibitor. These results suggest that an NR-mediated early NO burst plays an important role in Al resistance of wheat through modulating enhanced antioxidant defense to adapt to Al stress.
Introduction Aluminum (Al) toxicity is the primary constraint on agricultural production in acid soils (pH < 5.5), which constitute c. 50% of the world’s potential arable lands (Kochian et al., 2004; Ma, 2007). The most important and primary manifestation of Al phytotoxicity is inhibition of root apex growth, while mature root regions appear to be relatively unaffected by Al (Matsumoto, 2000). At the cellular level, a strong binding affinity of Al to cell components results in inhibition of cell wall synthesis, distortion of the cytoskeleton, destruction of plasma membrane integrity and disruption of calcium signaling cascades (Matsumoto, 2000; Rengel & Zhang, 2003; Ma, 2007). At the molecular level, Al stress causes major changes in the expression patterns of genes, some of which are important in oxidative stress response (Richards et al., 1998; Maron et al., 2008; Navascues et al., 2012). Aluminum increases peroxidation of membrane lipids and stimulates oxidative stress in plants if antioxidant defenses are overwhelmed (Yamamoto et al., 2001, 2002; Navascues et al., 2012). Plants have a number of strategies to respond to Al stress, for example, exclusion of Al in roots *These authors contributed equally to this work.
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and enhanced antioxidant capacity (Basu et al., 2001; Yamamoto et al., 2002). Although extensive efforts have been made to unravel the possible mechanisms of Al-induced primary toxicity during the past few years, the mechanisms of Al toxicity and tolerance are still not completely understood. Aluminum interferes with various signaling cascades in plants, such as 1,4,5-trisphosphate and cytosolic Ca2+ (Rengel & Zhang, 2003; Ma, 2007). Recently, several new signaling intermediates involved in Al tolerance in plants were identified (Tian et al., 2007; Ali et al., 2008), one of which is nitric oxide (NO), an important signaling molecule of increasing interest in plants. Studies have demonstrated that NO plays a myriad roles in modulating plant physiological and biochemical functions, including stimulation of seed germination, suppression of floral transition, mediation of stomatal movement, and modulation of plant development and senescence (Neill et al., 2003; Wilson et al., 2007). Responses and adaptations of plants to some abiotic stresses were also reported to be associated with NO. For example, NO triggers root ferric-chelate reductase activity in response to iron deficiency (Chen et al., 2010; Jin et al., 2011). It is also involved in cold acclimation and freezing tolerance through modulation of proline accumulation (Zhao et al., 2009). In addition, Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
New Phytologist it has been reported that NO protects plant cells against oxidative stress by reducing reactive oxygen species (ROS) accumulation under salt, drought, excessive zinc, and copper (Cu) stress (Zhang et al., 2009; Xu et al., 2010; Gonzalez et al., 2012). It has been shown that exogenous NO could protect plants against Al toxicity by preventing Al-induced oxidative stress (Wang & Yang, 2005; Wang et al., 2010) through altering cell wall polysaccharides or regulating hormonal equilibrium (He et al., 2012). However, reports on endogenous NO in response to Al stress are contradictory. It was found that the NO production peak disappeared completely after 60 min of Al treatment in the distal part of the transition zone of Arabidopsis (Illes et al., 2006). Reduced NO concentration in response to Al stress after 20 min was also reported in the roots of Hibiscus moscheutos (Tian et al., 2007). In other plant species, however, it has been reported that NO concentration significantly increased, for example, in the roots of Phaseolus vulgaris and Oryza sativa after Al treatment for 24 and 12 h, respectively (Wang et al., 2010; Yang et al., 2013). Different roles of NO involved in Al phytotoxicity were proposed because of its different responses to Al stress in plants (Tian et al., 2007; Wang et al., 2010; Zhou et al., 2012; Yang et al., 2013). It is possible that determination of NO content at a point in time during Al treatment does not completely reflect the specific role of NO in plant response to Al stress. Like other signaling molecules, such as cytosolic Ca2+, H2O2 and ethylene, responses of intracellular NO to stress conditions experience oscillations. NO oscillation has previously been reported in plants under both biotic and abiotic stresses. For example, when plants were infected by pathogens, only a strong NO burst and the following wave of secondary NO generation were associated with the stress resistance (Guo et al., 2004; Floryszak-Wieczorek et al., 2007). Recently, NO oscillation observed in plants under Cu stress was suggested to be a strategy in fighting against metal toxicity by increasing antioxidant protein gene transcripts (Gonzalez et al., 2012). However, the specific role of NO oscillation in plants with regard to resistance to Al toxicity has not been reported to date, although the change in NO production with time is probably involved in plants’ adaption to metal stress. In the present study, the endogenous NO production patterns were monitored in the root tips of two wheat genotypes with contrasting Al resistance, and the specific roles of endogenous NO produced at different time points during Al stress were investigated. Furthermore, the source of the time-dependent NO production under Al stress was examined.
Materials and Methods Plant materials and treatments Two winter wheat (Triticum aestivum L.) genotypes, Yang-5 (Al-sensitive) and Jian-864 (Al-resistant), were used. Seeds were surface-sterilized for 20 min in a 1% (v/v) sodium hypochlorite solution, rinsed thoroughly and soaked in deionized water overnight. The seeds were then geminated in the dark for 12 h at 25°C before being transplanted to plastic screens floating on a container filled with 2.5 l of 0.5 mM CaCl2 solution (pH = 4.3). CaCl2 solution is a frequently reported basal solution used to Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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investigate responses of plants under Al stress, because the simplicity of the basal medium allows a more precise computation of Al speciation than does a more complete nutrient medium (Kinraide & Parker, 1987). In order to keep the hydroponic system consistent, in this study the basal medium (CaCl2 solution) was used to precultivate plant seedlings, as previously reported (Osawa & Matsumoto, 2001). The solution was renewed daily. All experiments were conducted in a growth chamber with a 12 : 12 h, 25 : 22 °C day : night regime, a light intensity of 300 lmol m 2 s 1, and a relative humidity of 70%. After 3 d of pretreatment, uniform seedlings were selected for various treatments. To induce Al stress, AlCl3 (30 lΜ) was added to 0.5 mM CaCl2 (pH = 4.3). For NO treatment, the NO donor, sodium nitroprusside (SNP), was added to 0.5 mM CaCl2 (pH = 4.3) at a final concentration of 250 lΜ. To investigate the effects of various inhibitors and scavengers, wheat seedlings were pretreated with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, 100 lΜ), the nitrate reductase (NR) inhibitor tungstate (100 lΜ), or the nitric oxide synthase (NOS) inhibitor Nx-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 lΜ) for 2 h, and then exposed to 30 lΜ AlCl3 or 250 lΜ SNP treatment for 24 h under the same conditions as described earlier. Wheat seedlings were treated with 0.5 mM CaCl2 (pH = 4.3) under the same conditions for the duration of the experiment and served as controls. Each experiment was repeated at least three times. Measurement of root elongation Three-day-old seedlings were incubated in control solution (0.5 mM CaCl2, pH = 4.3) or treatment solutions for 24 h. Elongation of the primary root was measured before and after exposure of the roots to different chemicals. Relative root elongation was calculated as the percentage elongation of the root by various treatments as compared with the Al-free control. Root callose determination Callose content measurement was carried out following Jones et al. (2006). Briefly, root tips (50 mg) were cut and immediately fixed in 98% ethanol overnight. Ethanol was discarded, and the roots were homogenized in 400 ll of 1 M NaOH with a small pestle. After being heated at 80°C for 15 min, the samples were centrifuged at 1000 g for 10 min. Callose was determined in the following steps: 71 ll of supernatant, 142 ll of Aniline Blue (0.1% w/v), 75 ll of 1 M HCl and 210 ll of 1 M glycine-NaOH buffer (pH 9.5). After mixing, the samples were heated (50°C, 20 min) and left to cool before being measured on a fluorescence spectrophotometer (F 4600; Hitachi Ltd, Tokyo, Japan) at excitation 400 nm, emission 510 nm and slit width 10 nm. Determination of NO content Nitric oxide was visualized using the fluorescent probe diaminofluorescein-FM diacetate (DAF-FM DA), according to the method described by Luo et al. (2012). Briefly, root tips were loaded with New Phytologist (2014) 201: 1240–1250 www.newphytologist.com
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10 lM DAF-FM DA in 20 mM HEPES-NaOH buffer (pH 7.4) for 20 min, washed three times with fresh buffer, and observed under a Nikon Eclipse E600 epifluorescence microscope (Nikon, Tokyo, Japan) equipped with a Nikon B-2A filter block (450– 490 nm excitation filter, 505 nm dichroic mirror, 520 nm barrier filter). Treatments were repeated 10 times. Signal intensities of green fluorescence in the images were quantified according to the method of Guo & Crawford (2005) using Photoshop software (Adobe Systems, version 10.0, San Diego, CA, USA). Data were presented as the means of root fluorescence intensity relative to the root tips of control plants.
formed in the presence of H2O2 and are visible by transmission electron microscopy. Briefly, small segments 2–5 mm from the root tips were excised and incubated in freshly prepared 5 mM CeCl3 in 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.2 for 1 h. The root sections were then fixed in 2.5% (v/v) glutaraldehyde in 0.2 M sodium phosphate buffer (pH 7.2) overnight, and postfixed in 1% (w/v) OsO4 for 2 h. The specimens were dehydrated in a graded ethanol series (30–100%; v/v), followed by acetone and then infiltrated and embedded in Spurr’s resin. The samples were polymerized at 70°C for 16 h. Cross-sections (80 nm thick) were examined using a transmission electron microscope (H-7650, Hitachi).
Determination of ROS content H2O2 was measured by monitoring the A415 of the titanium–peroxide complex following the method described by Wang et al. (2010). Absorbance values were calibrated to a standard curve generated with known concentrations of H2O2. O2 content was measured as described by Liu et al. (2007) with some modifications. Root tips were homogenized in 2 ml of 65 mM phosphate buffer (pH 7.8) and then centrifuged at 5000 g for 10 min at 4°C. Then 1 ml supernatant was mixed with 0.9 ml of 65 mM phosphate buffer (pH 7.8) and 0.1 ml of 10 mM hydroxylamine hydrochloride. The mixture was placed at 25°C for 20 min. After that, 1 ml of the mixture, 1 ml of sulphanilamide (1% w/v in 1.5 M HCl) and 1 ml N-(1-naphthyl)-ethylenediaminedihy drochloride (0.02% w/v in 0.2 M HCl) were mixed and then incubated at 25°C for 30 min. The absorbance at 540 nm was measured with a spectrophotometer (Lambda 35; PerkinElmer, Waltham, MA, USA). The extent of OH determination is based on the degradation of 2-deoxyribose by OH , which yields a mixture of products among which malondialdehyde (MDA) is the most abundant (Liu et al., 2007). The extent of OH formation was calculated from the formation of the MDA at 532 nm. Histochemical analyses Callose accumulation was visualized by aniline blue staining as described in Jones et al. (2006). Root tips were transferred to 96-well plates and stained with 300 ll of 1 g l 1 aniline blue in 0.1 M H3PO4 (pH 9.0) for 30 min. The roots stained with aniline blue were observed under a fluorescence microscope (Nikon Eclipse E600 equipped with a Nikon G-2A filter block, 510–560 nm excitation filter, 575 nm dichroic mirror, 590 nm barrier filter). O2 was detected by incubating root tips with 10 lM dihydroethidium (DHE) in 10 mM Tris-HCl buffer (pH 7.4) for 30 min at 37°C in the dark, washed three times in fresh buffer, and observed under a Nikon Eclipse E600 epifluorescence microscope equipped with a Nikon U-2A filter block (380–420 nm excitation filter, 430 nm dichroic mirror, 450 nm barrier filter) as described by Rodrıguez-Serrano et al. (2009). Cytochemical detection of H2O2 H2O2 was visualized at the subcellular level using CeCl3 for localization (Zhang et al., 2007). Electron-dense CeCl3 deposits are New Phytologist (2014) 201: 1240–1250 www.newphytologist.com
Determination of oxidative damage The plasma membrane integrity was evaluated by Evans blue uptake according to Yamamoto et al. (2001). Membrane lipid peroxidation was estimated by measuring the concentration of MDA, according to the reaction with thiobarbituric acid as described by Yamamoto et al. (2001). Protein oxidative damage was determined by analysis of derivatization of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH) according to Levine et al. (1990). Determination of NR and NOS activity The activity of NR was determined as described by Tian et al. (2007). Root apexes (0–10 mm) were homogenized with a mortar and pestle on ice with 1.5 ml of extract buffer containing 50 mM HEPES-KOH (pH 7.5), 5% glycerol (v/v), 10 mM MgCl2, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 lM flavin adenine dinucleotide (FAD). Extract was centrifuged at 13 000 g 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 containing 50 mM HEPES-KOH (pH 7.5), 10 mM MgCl2, 1 mM DTT, 2 mM KNO3 and 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 0.5 M Zn-acetate. The nitrite produced was measured colorimetrically at 540 nm after adding 1 ml of 1% sulfanilamide in 3 M HCl plus 1 ml of 0.02% N-(1-naphthyl) ethylenediamine in 0.2 M HCl. To determine NOS activity, the total protein was extracted as described by Lin et al. (2012) 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. NOS activity was then measured after centrifugation at 13 000 g for 20 min at 4°C according to Gonzalez et al. (2012). Briefly, NOS activity was detected in 1 ml of reaction mixture containing 100 mM phosphate buffer (pH 7.0), 1 mM L-Arg, 2 mM MgCl2, 0.3 mM CaCl2, 4 lM BH4, 1 lM FAD, 1 lM flavin mononucleotide (FMN), 0.2 mM DTT, 0.2 mM NADPH, and 200 ll of protein extract. The decrease in absorbance as a result of 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). Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
New Phytologist Assay of antioxidant enzyme activities Fresh root (0.15 g) was frozen in liquid nitrogen and homogenized in 2 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1% PVP. The homogenate was centrifuged at 15 000 g for 20 min at 4°C and the supernatant was used for the following enzyme assays. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were determined as described by Jiang & Zhang (2001). Total SOD (EC 1.15.1.1) 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. GR (EC 1.6.4.2) activity was determined by monitoring the decrease in absorbance at 340 nm as a result of oxidized glutathione-dependent NADPH consumption (e = 6.22 mM 1 cm 1). Peroxidase (POD; EC 1.11.1.7) activity was monitored by oxidation of guaiacol using hydrogen peroxide (Upadhyaya et al., 1985). The enzyme activity was calculated using an extinction coefficient of 26.6 mM 1 cm 1 at 470 nm for tetraguaiacol. Lipoxygenase (LOX; EC 1.13.11.12) was assayed by monitoring the increase in absorbance resulting from 13(S)-hydroperoxylinolenic acid formation at 234 nm, using linolenic acid as the substrate at 25°C (Xu et al., 2005). The change of absorbance was monitored over 3 min. An increase of 0.1 absorbance unit min–1 is equivalent to one unit of LOX activity. Protein content determination Protein content in enzyme extracts was determined by Coomassie Brilliant Blue G-250 (Zhou et al., 2005). The protein content of enzyme extracts was calculated by comparison with a standard curve using BSA as a standard. Statistical analysis All data were statistically analyzed using the SPSS package (version 11.0; SPSS Inc., Chicago, IL, USA), ANOVA was performed on the data sets, and the mean and SD of each treatment as well as least significant difference (LSD; P < 0.05 and P < 0.01) for each set of corresponding data were calculated. The figures were drawn using the software Origin 8.0 (OriginLab Corporation, Northampton, MA, USA).
Results Endogenous NO production in root tips under Al stress Endogenous NO concentrations in root tips of two wheat genotypes with different Al tolerances were monitored by labeling NO using an NO-specific fluorescent probe, DAF-FM DA. Significant differences in endogenous NO production were observed in root tips of the two wheat genotypes with contrasting Al resistance after 30 lM Al treatment (Fig. 1). NO concentrations in root tips of the Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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Fig. 1 Responses of endogenous nitric oxide (NO) concentration in aluminium (Al)-tolerant and Al-sensitive genotypes of wheat (Triticum aestivum) to Al stress. (a) Detection of NO fluorescence using diaminofluorescein-FM diacetate (DAF-FM DA) staining and a fluorescence microscope. The arrow shows a significant NO increase at 3 h in the Al-tolerant wheat genotype. Bar, 250 lm. (b) NO production expressed as relative fluorescence. Roots of 3-d-old seedlings treated with 30 lM Al were harvested at 3, 6, 12, 24 h and loaded with 10 lM DAF-FM DA in 20 mM HEPES-NaOH buffer (pH 7.4) for 20 min. Representative images showing endogenous NO concentrations are given. The red dotted oval shows a significant NO increase at 3 h in the Al-tolerant wheat genotype. Data are means SD (n = 10). **, Significant difference between Al and control treatments at P < 0.01. CK, control.
two wheat genotypes with no Al treatment were relatively constant during a 24 h period. After treatment with 30 lM Al, significant NO was observed at 3 h in the root tips of the tolerant genotype Jian-864 h, whereas NO fluorescence was mostly observed after 6 h in the root tips of the sensitive genotype Yang-5 (Fig. 1a). When expressed as relative fluorescence of the controls (Fig. 1b), an early burst of NO at 3 h was 2.7-fold higher in the root tips of the tolerant genotype than in the sensitive genotype, whereas significantly higher NO was observed in the latter after 12 h of Al stress. A second experiment was performed to investigate the possibility of alarm signals caused by nutrient deficiency of CaCl2 precultivation. The results showed that there is no significant difference of either root elongation or Al-induced NO production between CaCl2 and 1/8 Hoagland’s solution precultivated seedlings for the two wheat genotypes (Supporting Information, Fig. S1). Effects of NO donor on root elongation and callose deposition To confirm the role of the early NO burst involved in Al tolerance, SNP, the most commonly used NO donor, was applied to New Phytologist (2014) 201: 1240–1250 www.newphytologist.com
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the roots of both wheat genotypes to mimic a similar early NO burst. In root tips of the Al-sensitive genotype treated with Al + SNP, a clear NO fluorescence was observed at 3 h, and decreased NO content was noted after 12 h when compared with those treated with Al alone (Fig. 2). Under Al stress, NO production rates were c. 3.8- and 3.3-fold higher at 3 h in the SNPtreated root tips of Jian-864 and Yang-5, respectively, than in the control (Fig. 2b). In addition to inducing an early NO burst, application of the NO donor SNP significantly reduced Al-induced root inhibition and callose deposition, which are two typical indicators of Al phytotoxicity. As shown in Fig. 3(a), measures of root elongation for Jian-864 and Yang-5 in the 250 lM
SNP+Al treatment were 72 and 58% of the control values, respectively, but only 55 and 37% of control values for the Al treatment alone. Similarly, SNP application significantly reduced callose deposit induced by Al stress in the root tips of the two wheat genotypes (Fig. 3b,c). To test whether the alleviating effect of SNP was a result of the triggered NO production, a special NO scavenger, cPTIO, was used. After pretreatment with 100 lM cPTIO for 2 h, the effect of SNP on the alleviation of Al-induced root inhibition was reversed (Fig. 3a). Depletion of endogenous NO by cPTIO strongly aggravated Al-induced root growth inhibition in the tolerant genotype Jian-864, but had no significant effect on that of the sensitive genotype Yang-5 (Fig. 3a).
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Effect of NO donor on ROS accumulation and oxidative damage
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Application of exogenous NO to the culture medium significantly reduced Al-induced ROS accumulation in the root tips of both wheat genotypes (Fig. 4). Al-induced accumulation of O2 , H2O2 and OH was detected in the root tips of both wheat genotypes after 24 h Al treatment, with the tolerance genotype Jian-864 accumulating much less ROS and experiencing less oxidative damage than the sensitive Yang-5 (Fig. 4). Adding the NO donor, SNP, significantly reduced ROS (O2 , H2O2 and OH ) contents in both genotypes (Fig. 4a–c). These results were further confirmed by histochemical analysis of O2 (Fig. 4d) and cytochemical analysis of H2O2 (Fig. 4e). Analysis of Evans blue uptake (Fig. 5a), MDA content (Fig. 5b), carbonyl concentration (Fig. 5c), and LOX activity (Fig. 5d) showed that Al caused severe oxidative damage to the plasma membrane, protein and lipid in root cells of the sensitive wheat genotype Yang-5. The damage was less pronounced in the Al-tolerant genotype Jian-864. Application of the NO donor SNP significantly reduced the amount of Al-induced oxidative damage in both wheat genotypes. Source of endogenous NO under Al stress
Fig. 2 Effect of sodium nitroprusside (SNP) on nitric oxide (NO) generation in wheat (Triticum aestivum) seedlings under aluminium (Al) stress. (a) Photographs of NO production after SNP application shown as green fluorescence in representative roots. Bar, 250 lm. (b) NO production expressed as relative fluorescence (Jian-864, white bars; Yang-5, gray bars). Three-day-old seedlings were treated either with or without SNP under Al stress. After 3 and 12 h of treatment, root tips of wheat were loaded with 10 lM diaminofluorescein-FM diacetate (DAF-FM DA) and NO fluorescence was imaged after 20 min. Data are means SD (n = 10). Different letters indicate significant differences (P < 0.05) among the treatments. CK, control. New Phytologist (2014) 201: 1240–1250 www.newphytologist.com
Nitrate reductase and NOS serve as two key enzymes responsible for NO biosynthesis in plants. To determine the possible source of an Al-induced early NO burst in the root tips of wheat, we first measured the effects of Al on NR and NOS activities. As shown in Fig. 6, the Al-tolerant genotype Jian-864 has a significantly higher NR activity than the sensitive genotype Yang-5 with or without Al stress. Different levels of NR and NOS activities in response to Al stress were noted in the root tips of the two wheat genotypes. Treatment of Al triggered significantly elevated NR activity after 3 h in the root tips of Jian-864, or 1.8-fold of the controls (Fig. 6a). By contrast, the activity of NOS, but not NR, increased c. twofold in the root tips of Yang-5 after treatment with Al for 12 h (Fig. 6b). Application of the NR inhibitor (tungstate) significantly eliminated endogenous NO production at 3 h and subsequently increased NO at 12 h in roots of the tolerant genotype Jian-864 under Al stress, but had no such effect on endogenous NO Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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Fig. 3 Effects of exogenous nitric oxide (NO) on the aluminium (Al)-induced root growth elongation and callose production of wheat (Triticum aestivum). (a) Effect of Al, NO donor (sodium nitroprusside, SNP) and NO scavenger (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, cPTIO) on the root growth of wheat seedlings. Three-day-old seedlings were treated with either 30 lM Al or 250 lM SNP, or both, for 24 h. For the NO scavenger treatment, the seedlings were pretreated with 100 lM cPTIO for 2 h, followed by NO or Al treatment. The elongations are expressed relative to root elongation in control solutions of 0.5 mM CaCl2, pH 4.3. The values shown are means SD (n = 20). (b) Effect of Al and NO donor (SNP) on callose production. Callose content was determined with aniline blue fluorescence by fluorescence spectrophotometer. Values represent means SD (n = 3). (c) Representative images of the surface view of aniline blue-stained roots taken under fluorescence microscope. Bar, 250 lm. Different letters indicate significant differences (P < 0.05) among the treatments. CK, control.
production for Yang-5 (Fig. 7a,b). Treatment with the NOS inhibitor (L-NAME) partially, but not completely, abolished NO production in roots of Yang-5, but had no significant effect on that in the roots of Jian-864 (Fig. 7a,b). Under Al stress, pretreatment with the NR inhibitor (tungstate) of Jian-864 aggravated its root growth inhibition, which could be subsequently reversed by addition of the NO donor SNP, while no such effect was observed in Yang-5 (Fig. 7c). No significant Al-induced root growth inhibition in Jian-864 or Yang-5 was found by pretreatment with L-NAME (Fig. 7d).
activity in Yang-5 treated with Al was 41% higher than that of the controls (Fig. 8d), which may result from a sufficient supply of H2O2, as POD could regulate cell-wall stiffening by catalyzing the oxidative cross-linking of cell wall polymers. Application of SNP further increased the activities of Al-induced antioxidant enzymes (Fig. 8b,c). Conversely, the activities of antioxidant enzymes tested were inhibited by the application of NO scavenger (cPTIO) and NR inhibitor (tungstate) in Jian-864 in the presence of Al treatment.
Discussion The effect of NO variation on the activities of antioxidative enzymes Analysis of antioxidant enzymes revealed that early NO production played an important role in protection against Al-induced oxidative stress by enhancing antioxidant enzyme activities (Fig. 8). Activities of SOD, CAT and GR in wheat roots increased after Al exposure for 24 h, by 54, 50 and 68% in Jian864, and 52, 20 and 7% in Yang-5, respectively (Fig. 8). POD Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
Exposure of Al resulted in inhibition of root elongation in both Al-sensitive (Yang-5) and Al-tolerant (Jian-864) wheat genotypes, with the effect being much less pronounced in the latter (Fig. 3a). It has been reported that Al tolerance in wheat was primarily associated with efflux of organic anions from root tips, with at least two independent strategies, Al-activated efflux of malate from root apices (Delhaize et al., 1993) and the constitutive efflux of citrate from root apices (Ryan et al., 2009). The results of the New Phytologist (2014) 201: 1240–1250 www.newphytologist.com
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Fig. 4 Reactive oxygen species (ROS) content in roots of wheat (Triticum aestivum) seedlings treated with aluminium (Al) and nitric oxide (NO). Threeday-old seedlings were treated either with or without sodium nitroprusside (SNP) under Al stress for 24 h. Contents of O2 (a), H2O2 (b) and OH– (c) in the root apexes of wheat seedlings after 24 h of different treatments were determined using spectrophotometry. Values represent means SD (n = 3). Different letters indicate significant differences (P < 0.05) among the treatments. (d) The endogenous O2 level was monitored by labeling O2 using dihydroethidium (DHE). Root tips were incubated in 10 lM DHE in 10 mM Tris-HCl buffer (pH 7.4) for 30 min at 37°C in darkness. Representative images of O2 -dependent red fluorescence were expressed. Bar, 1 mm. (e) Localization of H2O2 accumulation in wheat root tips by CeCl3-staining and transmission electron microscopy. The red rectangle shows amplification of the cell wall. CK, control; CW, cell wall. Bars, 1 lm.
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present study suggest that an early NO burst plays a crucial role in the initiation of a tolerance response of wheat to Al stress, and the improved Al tolerance by the NO donor was probably not New Phytologist (2014) 201: 1240–1250 www.newphytologist.com
Fig. 5 Oxidative damage parameters in roots of wheat (Triticum aestivum) seedlings treated with aluminium (Al) and nitric oxide (NO). Seedling root tips were collected 24 h after treatment, and then Evans blue uptake (a), malondialdehyde (MDA) content (b), carbonyl concentration (c) and lipoxygenase (LOX) activity (d) were determined. Values represent means SD (n = 3). Different letters indicate significant differences (P < 0.05) among the treatments. CK, control; Pr, protein.
associated with the efflux of organic anions, as suggested by a few previous studies (Wang & Yang, 2005; Tian et al., 2007). NO-regulated stress response is essential for plant performance Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
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Fig. 6 Effect of 30 lM aluminium (Al) on activities of nitrate reductase (NR) (a) and nitric oxide synthase (NOS) (b) in roots of wheat (Triticum aestivum) seedlings. Three-day-old seedlings were treated with 30 lM Al stress. Roots were collected for assaying activities of NR and NOS after 3 and 12 h exposure to the treatment solutions. Data are mean values SD (n = 3). Different letters indicate significant differences (P < 0.05) among the treatments. CK, control; Pr, protein.
under stress conditions. Production of stress-induced endogenous NO is generally time-dependent and plays differential roles in response to stress. The time-course of the endogenous NO concentrations in root tips indicates significant signal differences of endogenous NO production under Al stress in the two wheat genotypes (Fig. 1). Our results show a distinct early NO burst at 3 h in the root tips of the tolerant genotype, Jian-864, but no NO accumulation at 3 h and an extremely high NO concentration at 12 h in the sensitive genotype, Yang-5. The epicenter of NO was observed at the root meristem zone, which is the most sensitive region to Al toxicity. The difference in early NO burst between the two wheat genotypes was not simply induced by Al content in the root tips, as there was no significant difference of Al content in the root tips of the two genotypes before 6 h (Fig. S2). It is therefore possible that the NO signal induced by Al at the early time point (3 h) functions as an important factor associated with Al resistance in the tolerant genotype Jian-864. No genotypic differences in NO production were observed for roots of the two wheat genotypes when treated with 30 lΜ La3+, 30 lΜ Yb3+, or 20 lΜ Cd2+ (Fig. S3), suggesting that the early burst of NO in roots of Jian-864 is probablyl an Al-specific response. The role of the early NO burst involved in tolerance strategies to Al stress by wheat was further confirmed by using an NO donor or an NO scavenger. In the root tips of the sensitive
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Fig. 7 Effects of a nitrate reductase (NR) inhibitor (tungstate) and nitric oxide synthase (NOS) inhibitor (Nx-Nitro-L-arginine methyl ester hydrochloride, L-NAME) on NO content and root elongation of wheat (Triticum aestivum) seedlings. Three-day-old wheat seedlings were pretreated with an NR inhibitor (100 lM tungstate) or an NOS inhibitor (100 lM L-NAME) for 2 h, followed by Al treatment. (a) Representative images demonstrating the NOfluorescence. Bar, 250 lm. (b) NO production expressed as relative fluorescence. Data are means SD (n = 10). (c, d) Root elongation was measured after 24 h of Al treatment with tungstate (c) and L-NAME (d) pretreatment. Values shown are means SD (n = 20). Different letters indicate significant differences (P < 0.05) among the treatments. CK, control. Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
New Phytologist (2014) 201: 1240–1250 www.newphytologist.com
New Phytologist
1248 Research (a)
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Fig. 8 Effects of different treatments on antioxidant enzyme changes in wheat (Triticum aestivum) seedlings under aluminium (Al) stress. Three-day-old seedlings were used for the experiments. Seedlings were treated with 30 lM Al and with or without 250 lM sodium nitroprusside (SNP) for 24 h. For the nitric oxide (NO) scavenger and nitrate reductase (NR) inhibitor treatments, the seedlings were pretreated with 100 lM 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or 100 lM tungstate for 2 h, followed by 24 h of Al treatment. Roots were then collected to determine the activities of antioxidant enzymes: superoxide dismutase (SOD; a), glutathione reductase (GR; b), catalase (CAT; c) and peroxidase (POD; d). Values shown are means SD (n = 3). Different letters indicate significant differences (P < 0.05) among the treatments. CK, control; Pr, protein.
genotype Yang-5, application of SNP resulted in an increased early NO burst at 3 h, followed by a significantly reduced NO concentration at 12 h (Fig. 2). In addition, there was alleviation of Al-induced root growth inhibition and callose deposition (Fig. 3), which are well-known indicators of Al injury in plant roots. These results imply that reinforcing NO production at 3 h by a donor alleviates Al-induced toxicity in the root tips of the sensitive wheat genotype. Furthermore, NO scavenger (cPTIO) elimination of the early NO burst, either from an endogenous or an exogenous source (+SNP), significantly aggravated Al-induced root inhibition of the two wheat genotypes (Fig. 3a). These results provide additional evidence to suggest that an early NO burst plays a crucial role in the initiation of a tolerance response in wheat to Al stress. This is different from previous studies showing that Al-reduced NO concentration underpins Al-induced toxicity in the roots of Hibiscus moscheutos (Tian et al., 2007) and that exogenous NO exacerbates Al toxicity in rice bean (Zhou et al., 2012). It is possible that the discrepancy between the studies is related to the different time point for determining endogenous NO and the different plant species used. For example, Guo et al. (2004) suggested that only an early burst of NO enhanced the wheat immune defense response, whereas both an early burst of NO and a secondary NO generation were involved in the effective defense response of Pelargonium peltatum to Botrytis cinerea, a necrotrophic pathogen (Floryszak-Wieczorek et al., 2007). The early NO signal acts to maintain root function mainly through modulating enhanced antioxidant defense of roots for adaption to Al stress. Numerous studies have addressed the importance of antioxidant defense systems under Al stress, including elevated antioxidative enzyme activities in plants (Basu et al., 2001; Wang & Yang, 2005). The root tips of the sensitive wheat genotype Yang-5 experienced more serious oxidative New Phytologist (2014) 201: 1240–1250 www.newphytologist.com
damage as a result of the overwhelming production of ROS under Al stress than those of Jian-864, which showed higher activities of antioxidant enzymes (Figs 4,5,8). NO is reported to reduce Al toxicity by preventing oxidative stress in roots of Cassia tora (Wang & Yang, 2005) and Phaseolus vulgaris (Wang et al., 2010). Our results clearly show that increased NO production at 3 h by SNP application (Fig. 2) is followed by increased antioxidant enzyme activity (Fig. 8) and increased antioxidants such as ascorbate and glutathione (data not shown), thus reducing ROS concentrations and oxidative damage (Figs 4, 5). These findings suggest that the early NO burst plays an important role in enhancing the activities of antioxidant enzymes which subsequently act to prevent Al-induced oxidative stress. This is supported by the decreased GR and CAT activities after application of the NO scavenger (cPTIO) in the Al-tolerant genotype (Fig. 8). Recently, it was reported that Al resulted in the immediate production of ROS via activation of the plant plasma membrane NADPH oxidase (Tamas et al., 2004; Achary et al., 2008), and that NO suppressed NADPH oxidase-dependent ROS production by S-nitrosylation in human endothelial cells (Selemidis et al., 2007) and plants (Yun et al., 2011). Our results show that deposition of H2O2-derived CeCl3 precipitates were observed on the plasma membranes of the wheat root tips (Fig. 4e). Treatment with an inhibitor of NADPH oxidase significantly decreased ROS content in both wheat genotypes (data not shown). Therefore, it is possible that the early burst of NO controls the ROS contents through inactivation of NADPH oxidase. Our investigation also suggests that the early burst of NO in the root tips of the tolerant wheat genotype is mainly mediated by NR. NR and NOS are the two main enzymes responsible for NO synthesis in plants (Neill et al., 2003; Wilson et al., 2007). NR-dependent NO production is reported to be involved in Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
New Phytologist freezing tolerance (Zhao et al., 2009), whereas NOS-dependent NO participates in metal and salt tolerance (Zhao et al., 2004; Xu et al., 2010) and ABA-induced antioxidant defense (Zhang et al., 2007). It is further reported that NO produced from both NR and NOS activated root ferric-chelate reductase activity under iron deficiency (Chen et al., 2010). Wang et al. (2010) and Yang et al. (2013) suggested that NR-dependent NO production was associated with Al tolerance, while Tian et al. (2007) proved that decreased NOS-dependent NO was the main reason for Al toxicity. Several lines of evidence in this study suggest that synthesis of an early burst of NO (3 h) is mediated by NR in the root tips of the tolerant wheat genotype, whereas latent NO production at 12 h in the root tips of the sensitive genotype is probably associated with NOS and other sources. It was noted that in root tips of the Al-tolerant genotype, NR activity, but not NOS activity, was significantly elevated by Al stress at 3 h (Fig. 6). Application of an NR inhibitor (tungstate, Fig. 7a,b) completely eliminated NO production at 3 h, and thereby decreased root growth elongation of the Al-tolerant genotype (Fig. 7c), whereas no such effects of an NOS inhibitor (L-NAME) were observed (Fig. 7d). On the contrary, NOS rather than NR activity was significantly induced by Al stress in the sensitive genotype (Fig. 5), and the NOS inhibitor (L-NAME) reduced but did not completely eliminate NO production in the root tips at 12 h (Fig. 7), implying that NOS from other sources may contribute to the later NO production, such as mitochondrial electron transport and peroxisome (Corpas et al., 2001; Neill et al., 2003). In addition to being a signaling molecule, NO also belongs to reactive nitrogen species (RNS), which have toxic physiological consequences, namely nitrosative stress, when overproduced (Corpas et al., 2011). Our results show that the Al-sensitive genotype, Yang-5, accumulated extremely high NO after 12 h under Al stress, and this latent NO production can be partially alleviated by replenishing the early NO burst at 3 h using SNP (Fig. 2). Our results suggest different roles of the time-dependent NO production in roots of wheat under Al stress, with the early NO burst acting as a signal for enhanced antioxidant defense and late NO production possibly acting as nitrosative stress. The nitrosative stresses induced by NO have also been observed in plants under other forms of abiotic stress, such as salt, heat and mechanical wounding (Chaki et al., 2011; Corpas et al., 2011). In summary, our study is the first to reveal the important role of the NR-mediated early NO burst involved in the protection process against Al toxicity in roots of wheat. We found that the NR-mediated early burst of NO maintains root function under Al stress through differential expression of a number of tolerance mechanisms, including reduced ROS accumulation, probably through enhanced antioxidant enzyme activities and/or antioxidant pools. These mechanisms help to maintain membrane integrity and root elongation by reducing lipid peroxidation and callose deposition.
Acknowledgements This work was financially supported by the National Basic Research Program (973 Program) of China (no. 2013CB1274 Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
Research 1249
03), the National Natural Science Foundation of China (31272 237, 30771292), the Foundation for University PhD Granting Discipline of the Ministry of Education (20120101110130) and IPNI.
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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Effect of Al on wheat (Triticum aestivum) root elongation and NO production of CaCl2 and 1/8 Hoagland’s solution precultivated seedlings. Fig. S2 Accumulation of Al in root apexes in Al-resistant and Al-sensitive wheat (Triticum aestivum) genotypes at the indicated time after Al exposure. Fig. S3 Root elongation and NO generation in wheat (Triticum aestivum) seedlings under LaCl3, YbCl3 and CdCl2 treatments. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Ó 2013 The Authors New Phytologist Ó 2013 New Phytologist Trust
