Introduction

Abiotic stresses, such as drought, salinity, UV radiation, atmospheric pollutants, heavy metals, etc. cause molecular damage to plants, directly or indirectly through the overproduction of reactive oxygen species (ROS) (Ferreira et al. 2010). ROS cause oxidative modification in the biomolecules lipids, proteins, and nucleic acids leading to structural and functional changes.

Fig. 1
figure 1

Effect of Mn and NO treatment on a level of hydrogen peroxide (H2O2), b TBARS, c glutathione and d ascorbic acid in rice leaves in presence or absence of c-PTIO. The concentrations of MnCl2, SNP and c-PTIO were 15 mM, 100 μM and 100 μM, respectively. All measurements were done at 24 and 48 h after treatment in the light. Values are mean ± SD based on three independent determinations and bars indicate standard deviations. Asterisks represent significant differences among control and treatments at a particular stage at P ≤ 0.05, applying post hoc Tukey’s test

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Fig. 2
figure 2

Effect of Mn and NO treatment on specific activities of a SOD, b GPX and c CAT d APX, e GR and f DHAR in rice leaves in presence or absence of c-PTIO. The concentrations of MnCl2, SNP and c-PTIO were 15 mM, 100 μM and 100 μM, respectively. All measurements were done at 24 and 48 h after treatment in the light. Values are mean ± SD based on three independent determinations and bars indicate standard deviations. Asterisks represent significant differences among control and treatments at a particular stage P ≤ 0.05, applying post hoc Tukey’s test

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Manganese (Mn), an essential element for plant growth, becomes phytotoxic when present in excess in soil and consequently it represents an important constraint in crop productivity, especially in acid soils (Pittman 2005). Many reports in several crop plants have shown that excess Mn induces oxidative stress in the tissues characterized by enhanced lipid peroxidation, increased H2O2 level, ion leakage, DNA damage, and alteration in the levels of antioxidant enzymes (Demirevska-Kepova et al. 2004; Lei et al. 2007). Oxidative stress is an important phenomenon involved in the expression of Mn-toxicity in many plant species including rice (Srivastava and Dubey 2011). To avoid ROS-induced cellular injury, plants possess antioxidative defense system comprising of antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), peroxidases (PODs), glutathione reductase (GR) as well as low molecular weight antioxidants, such as ascorbate (AsA), glutathione (GSH), alpha-tocopherol, flavanoids, etc. (Xu et al. 2010).

Nitric oxide (NO) is a bioactive molecule involved in signaling processes in plants and plays a central role in a variety of physiological processes including responses to biotic and abiotic stresses (Wendehenne et al. 2004). It counteracts oxidative stress in tissues by acting as an antioxidant, directly scavenging ROS or as a signaling molecule in a cascade of events leading to gene expression changes (Laspina et al. 2005). It has been shown that exogenous application of NO provides a protection to the plants against the oxidative stresses caused by salinity (Shi et al. 2007), excess light (Xu et al. 2010), herbicides treatment (Ferreira et al. 2010) and heavy metals Cd, and Cu (Hsu and Kao 2004; Yu et al. 2005).

Rice is a staple food crop for the world population and its productivity is limited in acid and volcanic soils due to Mn-toxicity. Since NO acts as a signaling molecule and previous studies have demonstrated a protective role of NO in alleviating oxidative damage caused due to abiotic stresses in plants, to examine whether NO has a role in alleviating Mn-induced oxidative damage in rice, the present study was undertaken with the objectives to observe the effect of exogenously supplied NO on Mn-induced production of H2O2, lipid peroxides, and activity levels of key antioxidative enzymes in excised rice leaves.

Materials and methods

Plant materials and treatment conditions

Seeds of Indica rice (Oryza sativa L.) cv. Pant-12 were germinated in Petri dishes at 28°C for 5 days in a BOD incubator (York Scientific Instruments, New Delhi) (Srivastava and Dubey 2011). Uniformly germinated seeds were transferred to hydroponic cultures containing Yoshida rice nutrient solution (Yoshida et al. 1976) and maintained at pH 5.5 in 200 ml plastic pots. The nutrient solution contained 2 μM MnCl2. Pots were kept for 12 days in a green house at 28 ± 1°C under 80% relative humidity and 12 h light/dark cycle with 170–190 μmol m−2 s−1 PPFD. Seedlings were uprooted at 12th day, and 3 cm portions of the third leaf were used for all experiments. Excised rice leaves were cut into 3-mm segments; those weighing 200 mg were floated on 10 ml solution containing 15 mM MnCl2. Distilled water served as a control. Other treatment solutions comprised 100 μM SNP, a nitric oxide donor, 15 mM MnCl2 + 100 μM SNP and 15 mM MnCl2 + 100 μM SNP + 100 μM c-PTIO as NO scavenger. pH of all the treatment solutions was maintained at 4.5. Petri dishes were incubated at 28°C in the light (190–200 μmol m−2 s−1 PPFD). After 24 and 48 h of incubation, all biochemical determinations were carried out in leaf segments. Experiments were repeated thrice.

Determination of H2O2 and lipid peroxidation

H2O2 content was determined in the leaf segments spectrophotometrically using titanium sulphate following the method of Jana and Choudhuri (1981). The amount of H2O2 was calculated using an extinction coefficient 0.28 μmol−1 cm−1. The level of lipid peroxidation products was measured in terms of thiobarbituric acid reactive substances (TBARS) according to Heath and Packer (1968). The concentrations of lipid peroxides were expressed as nmol TBARS g−1 fresh weight of the leaf tissues using an extinction coefficient of 155 mM−1 cm−1.

Determination of ascorbate and glutathione content

The level of reduced ascorbate (AsA) was determined in rice leaves according to Law et al. (1983) and reduced glutathione (GSH) according to Griffith (1980).

Assays of antioxidative enzymes

SOD activity was determined using the method of Beauchamp and Fridovich (1971) based on the inhibition of p-nitro blue tetrazolium chloride (NBT) reduction by O .−2 under light. Excised leaves weighing 200 mg were homogenized using a chilled mortar and pestle in 5 mL of 100 mM K-phosphate buffer (pH 7.8) containing 0.1 mM EDTA, 0.1% (v/v) Triton X-100, and 2% (w/v) polyvinyl pyrrolidone (PVP). After centrifugation at 22,000g for 10 min at 4°C, supernatants were dialyzed in cellophane membrane tubings for 6 h against the extraction buffer in cold with 3–4 changes of the buffer. In the supernatant, total SOD activity was determined. One unit of SOD activity is expressed as the amount of enzyme required to cause 50% inhibition of the rate of NBT reduction measured at 560 nm. Catalase activity was assayed according to Beers and Sizer (1952) by measuring the decomposition of H2O2 at 240 nm (extinction coefficient of 0.036 mM−1 cm−1) and observing the decrease in absorbance using a UV-VIS spectrophotometer (Perkin Elmer, LAMBDA EZ 201, CA, USA). The activity of guaiacol peroxidase (GPX) was assayed in enzyme extracts according to Egley et al. (1983). Fresh tissue (±200 mg) was homogenized in 3 ml of chilled 50 mM Na-phosphate buffer (pH 7.0). After centrifugation and dialysis, enzyme was assayed by measuring increase in absorbance due to formation of tetraguaiacohinone at 420 nm (extinction coefficient of 26.6 mM−1 cm−1) at 30 s intervals up to 2 min using spectrophotometer (Bausch and Lomb, Spectronic 20, USA). Enzyme specific activity is expressed as μmol H2O2 reduced mg−1 protein min−1. Ascorbate peroxidase (APX) activity was assayed according to Nakano and Asada (1981). H2O2-dependent oxidation of AsA was followed by measuring the decrease in absorbance at 290 nm (extinction coefficient of 2.8 mM−1 cm−1). Enzyme specific activity is expressed as μmol AsA oxidized mg−1 protein min−1. The activity of dehydroascorbate reductase (DHAR) was assayed by measuring the reduction of DHA in the presence of GSH at 265 nm (Doulis et al. 1997) using a UV-VIS spectrophotometer after accounting for the non-enzymic reduction of DHA by GSH. Enzyme specific activity is expressed as nmol DHA reduced mg−1 protein min−1. Glutathione reductase (GR) activity was assayed using the method of Schaedle and Bassham (1977) following the oxidation of NADPH at 340 nm. Enzyme specific activity is expressed as nmol NADPH oxidized mg−1 protein min−1.

Protein content assay

In all enzyme preparations, protein was determined following the method of Bradford (1976) using bovine serum albumin (BSA; Sigma, St. Louis, USA) as standard.

Statistical analysis

All experiments were performed in triplicate. Values indicate mean ± SD based on three independent determinations. Differences among control and treatments were analyzed by one-way ANOVA applying post hoc Tukey’s test.

Results and discussion

Many abiotic stresses including excess levels of metals like Cd, Pb, Al, Ni, Mn in the soil cause molecular damage to plants through increased formation of ROS. Increasing evidences suggest that, at least partially, metal-toxicity is due to oxidative damage (Sandalio et al. 2009; Xiong et al. 2010). Our earlier studies have shown that oxidative stress is a major component in expression of Mn-toxicity in indica rice plants (Srivastava and Dubey 2011). In the present experiments, conducted to determine H2O2 level and lipid peroxidation in control and Mn treated detached rice leaves, it was revealed that MnCl2 treatment caused significant increase (P ≤ 0.05) in H2O2 level and lipid peroxidation in the leaves after 24 and 48 h of treatment as compared to controls (Fig. 1). Earlier reports have also indicated increased H2O2 production and increased peroxidation of lipids in response to different abiotic stresses in plants including toxicities due to metals Cd, Pb, Ni, Al, etc. (Shah et al. 2001; Verma and Dubey 2003; Laspina et al. 2005; Sharma and Dubey 2007; Maheshwari and Dubey 2009). In our experiments, a significant decline in the levels of nonenzymic antioxidants reduced glutathione and reduced ascorbate was observed in rice leaves with Mn treatment, which paralleled with the increase in H2O2 generation and lipid peroxidation.

Besides increase in H2O2 and lipid peroxidation, Mn treatment also resulted in significant increase (P ≤ 0.05) in activities of the antioxidative enzymes—SOD, GPX, CAT, APX, GR, and DHAR (Fig. 2). The enhanced activities of antioxidative enzymes have been observed in various plants like vigna, barley, and cucumber when exposed to Mn excess (Fecht-Christoffers et al. 2003; Demirevska-Kepova et al. 2004; Shi et al. 2007).

Evidences suggest that NO protects plant cells against oxidative stress and that exogenous NO prevents Cd-induced oxidative stress in rice leaves (Hsu and Kao 2004), Cu-induced toxicity and NH4 + accumulation in rice leaves (Yu et al. 2005) and reduces Al-toxicity by preventing oxidative stress in roots of Cassia tora (Wang and Yang 2005). In the present investigation, we observed that the application of exogenous NO (in the form of SNP) significantly ameliorated Mn-induced oxidative damage in rice leaves by reducing the level of H2O2 and lipid peroxides; reversing Mn-induced decrease in the levels of GSH, AsA and Mn-induced increase in the activities of antioxidative enzymes. Among the three antioxidative enzymes, NO treatment had better effect in restoring SOD activity level compared to GPX and CAT in Mn treated leaves. In the presence of c-PTIO, an NO scavenger, in the treatment medium along with SNP and MnCl2, induction of oxidative stress and altered levels of antioxidants and antioxidative enzymes were observed. This suggests that Mn induced oxidative stress in rice leaves and that amelioration of oxidative stress or the protective effect of SNP was only due to NO produced by SNP.

NO is a redox active, diffusible, small bioactive gaseous molecule that participates in several mechanisms of abiotic stress-tolerance in crop plants (Lamattina et al. 2003). As a signaling molecule, NO is shown to be involved in increase in the activities of antioxidative enzymes (Lamattina et al. 2003; Shi et al. 2007) and decrease in lipooxygenase activity (Zhao et al. 2008) to inhibit lipid peroxidation and membrane damage caused due to environmental stresses.

Exogenous NO has been shown to counteract the inhibitory effect of heavy metals on root growth of Lupinus luteus partly due to increased activity of SOD (Kopyra and Gwózdz 2003). Hsu and Kao (2004) observed that exogenous NO prevented the Cd-induced increase in contents of H2O2 and MDA; decrease in contents of GSH and AsA and increase in activities of antioxidative enzymes in rice leaves. Similarly, NO reduced Al-toxicity by preventing oxidative stress in of Cassia tora roots (Wang and Yang 2005). Exogenous NO also reduced Cu-induced NH4 + accumulation in rice leaves by scavenging ROS (Yu et al. 2005). In addition, Laspina et al. (2005) observed that in sunflower leaves, NO when applied before Cd exposure, significantly alleviated Cd-induced oxidative damage, mainly due to recovery of GSH level and CAT activity and enhancement of AsA level and APX activity. In hydroponically grown wheat roots, exogenous NO was shown to ameliorate Cd-toxicity by increasing ROS scavenging activity and reversing Cd-induced increase in the activities of antioxidative enzymes (Singh et al. 2008). It was shown by Singh et al. (2009) that in rice roots exogenous NO exhibited ROS scavenging activity and partially reversed As-induced increase in the activities of the antioxidative enzymes. These observations suggest that exogenous NO application ameliorates oxidative damage caused due to Cd, Al, Cu, As in different plant species by recovering the levels of antioxidants and modulating the levels of antioxidative enzymes to provide better ROS scavenging capacity.

Endogenous NO plays vital role in various plant processes, like induction of seed germination, regulation of plant metabolism and senescence, induction in cell death, regulation of mitochondrial functionality and multiple plant responses towards a variety of biotic and abiotic stresses and alleviating consequences provoked by oxidative stresses (Siddiqui et al. 2011). Our knowledge of the molecular and physiological mechanisms by which NO alleviates metal-toxicity and metal-induced oxidative stress is limited. However, the observations of various workers suggest that NO either directly or indirectly due to existence of an unpaired electron within the molecule, can react with and scavenge ROS, such as superoxide anion (O .−2 ), H2O2 and hydroxyl radical (.OH) (Martinez et al. 2000; Xiong et al. 2010) maintaining optimum content of antioxidants and activity levels of antioxidative enzymes in plants. According to another possible mechanism NO stimulates expression of GSH synthesis related genes and increases GSH content in plants under metal-toxicity (Xiong et al. 2010). GSH, a major low-molecular weight thiol present in plants, has crucial role in scavenging metal-induced ROS and in the regulation of redox homeostasis (Innocenti et al. 2007). In our studies as well, with exogenous NO, the level of GSH recovered in Mn-treated leaves.

In certain cases, activities of some antioxidative enzymes are shown to be inhibited by NO (Clark et al. 2000). However, our results show that SNP treatment alone did not affect the activities of antioxidative enzymes in rice leaves. Thus, the reduction of Mn-induced increase in activities of antioxidative enzymes by NO is unlikely due to a direct NO-mediated effect on enzymes. Evidences suggest that NO mediates its effects in plants through S-nitrosylation of Cys residues of target proteins, modulation of protein kinase activities, and mobilization of free Ca2+ and other second messengers (Courtois et al. 2008). It is a pertinent question, whether NO generation is advantageous at all to the plants undergoing metal stress. Our results suggest that in rice leaves, NO protects the tissues against Mn-induced oxidative stress. However, conflicting data exist concerning the impact of exogenous and endogenous NO in plants treated with metals. It has been documented that Cd enters root cells through transport proteins like ZIP transporters including IRT1, cation channels, calcium channels, etc. (Clemens 2006; Lux et al. 2011). In Arabidopsis thaliana Cd2+ application leads to NO synthesis and in turn NO promotes the up-regulation of iron acquisition-related genes expression (IRT1, FRO2 and FIT) and in consequence NO amplifies Cd uptake via IRT1 protein and contributes to Cd-toxicity by promoting its accumulation in roots (Besson-Bard et al. 2009). It is shown that IRT1 is a broad-range metal ion transporter in plants and when expressed in yeast, can also transport Mn (Korshunova et al. 1999). In rice, the iron transport proteins OsIRT1 and OsIRT2 have been shown to transport Cd under Fe2+ deficiency (Nakanishi et al. 2006). Exogenous NO in form of SNP enhances Cd-tolerance of rice by increasing pectin and hemicellulose content in the cell wall of roots, increasing Cd-accumulation in root cell wall and decreasing Cd-accumulation in soluble fraction of roots and leaves (Xiong et al. 2009), however, no such evidence is available in rice showing a possible relationship between Mn transport and endogenous NO level. To conclude with, the results of the present study suggest that Mn excess causes oxidative stress in excised rice leaves accompanied with increased production of H2O2, increased lipid peroxidation, decline in the level of antioxidants glutathione and ascorbate and increased activity of antioxidative enzymes. Further, exogenous NO shows potential ameliorating effect of Mn-induced oxidative stress in rice leaves by preventing Mn-induced increase in contents of H2O2, lipid peroxidation; recovering the contents of GSH and AsA and reducing Mn-induced increased activities of antioxidative enzymes.