Nitric oxide triggers a concentration-dependent differential modulation of superoxide dismutase (FeSOD and Cu/ZnSOD) activity in sunflower seedling roots and cotyledons as an early and long distance signaling response to NaCl stress

Abstract

Dark-grown sunflower (Helianthus annuus L.) seedlings exhibit modulation of total superoxide dismutase (SOD;_EC 1.15.1.1) activity in roots and cotyledons (10,000g supernatant) in response to salt stress (NaCl; 120 mM) through a differential, zymographically detectable, whole tissue activity of FeSOD and Cu/ZnSOD. Confocal laser scanning microscopic imaging (CLSM) has further shown that NaCl stress significantly influences differential spatial distribution of Cu/ZnSOD and MnSOD isoforms in an inverse manner. Dual action of nitric oxide (NO) is evident in its crosstalk with FeSOD and Cu/ZnSOD in seedling roots and cotyledons in control and NaCl⁻ stress conditions. Cu/ZnSOD activity in the roots of 2 d old NaCl⁻ stressed seedlings is enhanced in the presence of 125–1000 µM of NO donor (sodium nitroprusside; SNP) indicating salt sensitivity of the enzyme activity. Quenching of endogenous NO by cPTIO treatment (500, 1000 µM) lowers FeSOD activity in roots (-NaCl). Cotyledons from control seedlings show an upregulation of FeSOD activity with increasing availability of SNP (125–1000 µM) in the Hoagland irrigation medium. Quenching of NO by cPTIO provides evidence for an inverse correlation between NO availability and FeSOD activity in seedling cotyledons irrespective of NaCl stress. Variable response due to NO on SOD isoforms in sunflower seedlings reflects its concentration-dependent biphasic (pro- and antioxidant) nature of action. Differential induction of SOD isoforms by NO indicates separate intracellular signaling pathways (associated with their respective functional separation) operative in seedling roots as an early salt stress mechanism and in cotyledons as an early long-distance NaCl stress sensing mechanism.

Abbreviations

NO

Nitric oxide

ROS

Reactive oxygen species

SOD

Superoxide dismutase

O₂• −

Superoxide anion

Introduction

Variations in growth conditions (light intensity, temperature, etc.), severity and duration of biotic and abiotic stress conditions and ability of the tissue to acclimatize to energy imbalances are the major factors which regulate the balance between the production of reactive oxygen species (ROS) and their scavenging by enzymatic and non-enzymatic means in plants.¹ High salt concentrations in the growth medium result in an excessive generation of ROS in plant cells by the impairment of cellular electron transport within different subcellular compartments, such as chloroplasts and mitochondria, as well as induction of metabolic pathways, such as photorespiration. Salt stress can also lead to stomatal closure, and low chloroplastic CO₂/O₂ ratio resulting from it favors photorespiratory pathway, leading to increased production of ROS.^2,3 Salinity induces disruption of normal subcellular metabolism through lipid peroxidation and denaturation of proteins and nucleic acids in several plant species as a consequence of ROS production.^2,4,5 Antioxidant machinery comprising of ROS scavenging enzymes and non-enzymatic cell constituents, plays a crucial role in the detoxification of elevated ROS produced under salinity stress. Earlier investigations have demonstrated a correlation of salt tolerance in plants with the modulation of the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR).⁶

Superoxide dismutase (SOD;_EC 1.15.1.1) is a major scavenger of superoxide radical (O₂• −), and acts as the first line of defense against ROS by catalyzing the dismutation of 2 superoxide radicals (O₂• −) and water into H₂O₂ and O₂.^7,8 This metalloenzyme is ubiquitous and plays a crucial role in defense against oxidative stress in all aerobic organisms. Phospholipid membranes are impermeable to superoxide radicals (O₂• −), thereby leading to their accumulation in cells at toxic levels under stress conditions. Therefore, it is crucial that different SOD isoforms are active for the removal of O₂• − in the subcellular compartments where the radicals are being generated.⁹ According to the metal co-factor associated with the enzyme, three major SODs have been reported: iron SOD (FeSOD), manganese SOD (MnSOD) and copper-zinc SOD (Cu/ZnSOD). Cu/ZnSOD is primarily localized in the cytoplasm of the eukaryotic cells, MnSOD exists in the mitochondria of eukaryotic cells and the cytoplasm of prokaryotes, and FeSOD occurs mainly in the prokaryotes and the chloroplasts of eukaryotic plants.¹⁰

A possible signaling mechanism, which modulates sunflower seedling growth under salt stress involves a transiently enhanced NO signal in the cells of NaCl-treated root tips.¹¹ NO has also been observed to modulate lateral root development in sunflower seedlings depleted of endogenous NO by cPTIO (NO scavenger) treatment.¹² An upregulation of cPTIO-induced genes for lignin biosynthesis has also been observed accompanying its impact on lateral root formation. High reactivity of NO radical induces different post-translational modifications in a wide range of proteins, thereby affecting their structure and/or function.¹³ Protein modifications mediated by NO primarily include tyrosine nitration, S-nitrosylation and metal nitrosylation.¹⁴ Addition of an equivalent of NO₊ to an amine, thiol, or hydroxy aromatic group causes nitrosation. Removal of one or 2 electrons from the substrate as well as hydroxylation reactions come under oxidation whereas the addition of an equivalent of an NO₂₊ causes nitration.¹⁵ S-nitrosylation is a highly selective (restricted to specific Cys residues) and reversible post-translational modification which allows cells to flexibly and specifically respond and adapt to changes in environment signals.^16,17,18 Tyrosine nitration of proteins has been suggested as a biomarker of nitrosative stress as it can lead to either activation or inhibition of target proteins.¹⁹

NO has been shown to bring about a biphasic action in animal cells, it being protective at low concentrations (upto 500 µM) and harmful at high concentrations (>500 µM).²⁰ Simultaneous generation of NO and ROS during various stress conditions makes their interactions crucial, as they may result in protection or production of more toxic end products. When present in high concentration, NO reacts with superoxide anion to form highly reactive peroxynitrite (ONOO⁻), thereby acting as a pro-oxidant. At low concentrations, NO can inhibit oxidation and terminate chain reactions during lipid peroxidation, thereby acting as an anti-oxidant.²¹ Modulation of antioxidant activities and ROS detoxification system by nitric oxide (NO) has been reported to result in salinity tolerance.^22,23 Exogenous NO application results in an increase in the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CT), glutathione peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR), thereby improving plant growth under salinity stress.⁶

Present investigations focus on understanding the biochemical mechanisms of NaCl stress-induced seedling growth inhibition in sunflower. In this context, superoxide anion scavenging in the seedling cotyledons and roots has been examined by monitoring the activity of 2 isoforms of superoxide dismutase (FeSOD and Cu/ZnSOD). Influence of NaCl stress on spatial distribution of SOD isoforms (MnSOD and Cu/ZnSOD) has been examined in 2 d old seedling cotyledons by CLSM. Detailed analysis of crosstalk of SOD isoforms with NO has subsequently been undertaken. Biphasic, concentration-dependent differential action of NO on the 2 SOD isoforms demonstrates NO-SOD crosstalk during sensing of salt stress in sunflower seedlings by raising sunflower seedlings in various concentrations of NO scavenger (cPTIO), NO source (SNP), NOS inhibitor (aminoguanidine) and peroxynitrite, followed by zymographic analysis of FeSOD and Cu/ZnSOD activity. Results have also been discussed with reference to the dual nature of cPTIO in action as a pro- or antioxidant. Present work thus provides new information on the synchronous modulation of the activity of SOD isoforms by NaCl and its crosstalk with NO.

Results

Accompanying NaCl stress (120 mM) -induced visible reduction in the extent of seedling growth marked with reduced hypocotyl elongation and less proliferation of roots, seedling cotyledons and roots (10,000g supernatant) exhibit NaCl-induced inhibition of enzyme activity. This is evident from absolute values and more so from the relative change in per cent change with reference to enzyme activity in the cotyledons and roots (Fig. 1). NaCl stress significantly lowers total SOD activity at 2, 4 and 6 day stage of seedling roots. Thus, a noteworthy impact on NaCl stress of seedling growth inhibition accompanies reduced ability of seedlings to scavenge superoxide anions, although in absolute terms enzyme activity increases with advancing age of seedlings.

Figure 1.

Estimation of total SOD activity in the cotyledons and roots (10,000 g supernatant) of sunflower seedlings raised in the absence or presence of 120 mM NaCl. Inset depicts relative SOD activity at the 3 stages of seed germination in the respective tissues and in response to salt stress (calculated as percentage of SOD activity at 2d stage). Seedlings shown are at 6 d stage of growth in dark.

The three differentially spatially located SOD isoforms (Cu/ZnSOD, FeSOD and MnSOD) in the buffer soluble 10,000g supernatant of seedling cotyledons respond differently in their expression pattern both with reference to seedling age as well as their sensitivity to NaCl stress. Zymographically detectable activity of FeSOD is lowered by NaCl stress in seedling roots. Activity of FeSOD and Cu/ZnSOD is least in 2 d old roots and rises at later stages of development. In contrast with FeSOD activity, Cu/Zn SOD activity in roots is enhanced by NaCl stress, more so at later stage (6 d) of seedling development. No such NaCl sensitivity is evident in the modulation of these SOD isoforms in seedling cotyledons at the respective stages of development (Fig. 2). MnSOD isoform is zymographically not detectable (observed as faint bands) due to its relatively low activity in the tissue samples analyzed.

Figure 2.

Zymographic analysis of seedling development associated changes in the activity of Fe SOD (A) and Cu/Zn SOD (B) in sunflower seedling cotyledons and roots (10,000 g supernatant) raised in the absence or presence of 120 mM NaCl.

CLSM imaging of MnSOD and Cu/ZnSOD activities in the 7 µm thick cross sections of cotyledons from 2 d old seedlings highlights opposite impact of NaCl stress on the 2 isoforms. FeSOD activity could not be localized due to the specific growth condition (very low concentration of copper) required for the use of primary antibody against this isoform. NaCl stress enhances the overall distribution of MnSOD in the cells of cotyledons. High activity is evident in the specialized cells around the secretory canals. NaCl stress drastically lowers Cu/ZnSOD distribution in the cells of cotyledons (in contrast with its enhancing effect on MnSOD distribution). These novel observations on differential spatial distribution and opposite effect of NaCl on the distribution pattern of MnSOD and Cu/ZnSOD in 2 d old seedling cotyledons highlights their significant role in long distance sensing of salt stress (Fig. 3).

Figure 3.

Immunolocalization (CLSM imaging) of MnSOD (A) and Cu/ZnSOD (B) and its quantitation in the cells of the marginal region of cotyledons from 2d old sunflower seedlings raised in the absence or presence of 120mM NaCl. Seven µm thick sections were used for imaging by CLSM at pinhole 1. Magnification: 200X. Other details are as stated in ‘Materials and Methods.’

NO provided from outside as SNP (1000 µM) significantly stimulates FeSOD activity in NaCl stressed seedling roots but the impact remains marginal in control seedlings. The impact of NO on enhancing FeSOD activity is most likely linked with peroxynitrite since at its specific concentrations (250, 1000 µM) in 2d old control and salt stressed (125 µM) seedlings, FeSOD activity is enhanced by peroxynitrite treatment. Quenching of NO by raising seedlings in presence of cPTIO (125–1000 µM) lowers FeSOD activity in control seedlings but its impact is not evident in the roots of NaCl-stressed seedlings (Fig. 4). The impact of SNP is better evident and significant in enhancing the activity of Cu/ZnSOD. The fact that this enhancement of enzyme activity is evident only in the roots of NaCl stressed seedlings, indicates salt stress sensitivity of the response (Fig. 5). Thus, in case of both the isoforms (FeSOD and Cu/ZnSOD), the enzyme activity enhancement is sensitive to salt-stress, more so in case of Cu/ZnSOD. Seedling cotyledons exhibit a reverse trend on the influence of SNP on FeSOD and Cu/ZnSOD as compared to roots with respect to salt stress. FeSOD activity is higher in control seedling cotyledons in response to SNP and it slightly declines in salt-stressed seedling cotyledons. Enhanced activity due to aminoguanidine treatment (125–1000 µM ) in this situation (+NaCl) confirms negative regulation in control due to SNP. Thus, NaCl inversely modulates the impact of NO on FeSOD activity. The observation that cPTIO leads to quenching of endogenous NO and this coincides with the enhancement of enzyme activity, indicates an inverse correlation between NO and FeSOD activity (Fig. 6). Significant stimulation of Cu/ZnSOD activity in presence of 250–1000 µM of aminoguanidine (AG) in cotyledons indicates the critical role being played by endogenously produced NO from the NOS-like activity (Fig. 7).

Figure 8.

Summary of NO crosstalk with Cu/ZnSOD and FeSOD in sunflower seedling roots and cotyledons as affected by NaCl stress. Seedlings shown are at 2 d stage of growth in dark.

Figure 4.

Zymographic analysis and quantitation of the effect of variables concentrations of SNP, peroxynitrite, aminoguanidine and cPTIO on FeSOD activity in the tissue homogenates (10,000 g supernatant) of 2d old seedling roots. Quantitation (in relative terms) of the resolved SOD isoforms has been depicted as graphs following densitometric analysis of the gels.

Figure 5.

Zymographic analysis and quantitation of the effect of variables concentrations of SNP, peroxynitrite, aminoguanidine and cPTIO on Cu/ZnSOD activity in the tissue homogenates (10,000 g supernatant) of 2d old seedling roots. Quantitation (in relative terms) of the resolved SOD isoforms has been depicted as graphs following densitometric analysis of the gels.

Figure 6.

Zymographic analysis and quantitation of the effect of variables concentrations of SNP, peroxynitrite, aminoguanidine and cPTIO on FeSOD activity in the tissue homogenates (10,000 g supernatant) of 2d old seedling cotyledons. Quantitation (in relative terms) of the resolved SOD isoforms has been depicted as graphs following densitometric analysis of the gels.

Figure 7.

Zymographic analysis and quantitation of the effect of variables concentrations of SNP, peroxynitrite, aminoguanidine and cPTIO on Cu/ZnSOD activity in the tissue homogenates (10,000 g supernatant) of 2d old seedling cotyledons. Quantitation (in relative terms) of the resolved SOD isoforms has been depicted as graphs following densitometric analysis of the gels.

Discussion

Enhanced generation of reactive oxygen species (ROS) is induced by almost all abiotic stress factors, such as salinity, low and high temperatures, ultraviolet radiation, drought and exposure to ozone, leading to oxidative stress. ROS triggers various signaling pathways in addition to induction of several oxidatively destructive processes.^24,25 A noteworthy impact of NaCl stress on seedling growth inhibition in sunflower (present work) is the reduced total SOD activity although in absolute terms enzyme activity increases with advancing age of seedlings. The three differentially spatially located SOD isoforms in seedling cotyledons respond differently to NaCl stress with seedling development. NaCl stress enhances the overall distribution of MnSOD in the cells of cotyledons. In contrast, higher expression of Cu/ZnSOD is evident in control seedling cotyledons. Thus, differential sensitivity to NaCl stress is evident with reference to spatial distribution of SOD isoforms.

NO interacts with ROS in various ways and thus might have an antioxidant function under stress conditions in plant cells.²⁶ NO has earlier been reported to exhibit a protective role in heavy metals and salinity-stressed lupin (Lupinus sp.) roots by enhancing SOD activity.²³ NO stimulates the activitiy of Cu/Zn SOD in sweet potato (Ipomoea batatas) and reduces cell death triggered by H₂O₂.²⁷ It plays an important role in the mitigation of salinity stress in tomato (Solanum lycopersicum) plants by enhancing the activity of various antioxidant enzymes like SOD, APX, GR and POD, and some enzymes associated with nitrogen metabolism, like nitrate reductase and nitrite reductase.²⁸ Application of SNP to salt-stressed cucumber seedlings promotes SOD activity and hence assists in alleviation of oxidative stress.²⁹ In Brassica juncea, S-nitrosylation of FeSOD under salt stress leads to elevation of enzyme activity.³⁰ FeSOD is nitrated and thereby inactivated by peroxynitrite in a dose-dependent manner in Trypanosoma cruzi. Nitration occurs at the critical and universally conserved Tyr35 residue of the isozyme.³¹ Cu/ZnSOD quickly decomposes S-nitrosogluthathione (GSNO) in the presence of H₂O₂ to form oxidized glutathione (GSSG) and NO.³²

NO provided from outside as SNP (1000 µM) significantly stimulates FeSOD activity in salt stressed sunflower seedling roots but the impact is marginal in control seedlings (present work). Quenching of NO by raising seedlings in presence of cPTIO (125–1000 µM) lowers FeSOD activity in control seedling roots but NaCl is able to nullify its impact. Enhancement of Cu/ZnSOD activity by SNP is evident only in the roots of NaCl stressed seedlings, indicating salt stress sensitivity of the response. Seedling cotyledons exhibit an opposite influence of SNP on FeSOD as compared to roots. Thus, enzyme activity is higher in control seedling cotyledons in response to SNP and it slightly declines in salt-stressed seedling cotyledons. Quenching of endogenous NO by cPTIO coinciding with the enhancement of enzyme activity indicates an inverse correlation between NO availability and FeSOD activity. Differential induction of SOD isoforms thus indicates separate intracellular signaling pathways associated with their respective functional separation.

There are 2 possible mechanisms by which NO can counteract oxidative stress. First, it directly scavenges ROS, such as O₂⁻*, to form peroxynitrite (ONOO⁻), thus acting as an antioxidant itself.³³ Secondly, NO might also function as a signaling molecule thereby altering gene expression.^33-35 The overall mechanism by which varied SOD isoforms function has been called a “ping-pong” mechanism as it involves the sequential reduction and oxidation of the metal center with the concomitant oxidation and reduction of superoxide radicals at virtually diffusion controlled rates in a pH range of 5 to 9.5 where the rate is unchanging.³⁶ Reaction of NO and superoxide anions results in the formation of peroxynitrite (ONOO⁻), a potent oxidant and nitrating agent. The rate of peroxynitrite formation is determined by the concentrations of both superoxide anion and NO, and is at least 3–8 times faster as compared to the rate of dismutation of superoxide anions by superoxide dismutase.³⁷ Peroxynitrite-triggered NO-dependent signals modulate redox regulation in various signaling pathways but overproduction of ONOO⁻ can lead to oxidative and nitrosative stress in a cell.³⁸ The interaction between different SOD isoforms and NO seems to occur at multiple levels. SOD isoforms are differentially affected by NO donors and scavengers. A crosstalk between these 2 molcular entities is crucial to combat oxidative stress in plants facing biotic/abiotic stress. In turn, SOD isoforms can also regulate endogenous NO availability by competing for the common substrate, superoxide anion and by releasing NO from its donor compounds, S-nitrosoglutathione (GSNO). A competition has been observed between SOD and NO for superoxide anion and increase in SOD level has been shown to reduce peroxynitrite levels, thereby ameliorating oxidative stress:^39,40

$${\displaystyle {\text{ONOO}}^{-}\underset{6.7\times {10}^9{\text{M}}^{-1}{\text{S}}^{-1}}{\overset{\text{NO}}{\leftarrow }}{\text{O}}_{2^{-}}\underset{2\times {10}^9{\text{M}}^{-1}{\text{S}}^{-1}}{\overset{\text{SOD}}{\to }}+{\text{H}}_2{\text{O}}_2}$$

Lastly, it is important to note that variations in the impact of NO sources and quenchers can also be possible on the basis of various unique features of the pharmacological agents being commonly used for such investigations. Thus, cPTIO has been reported to act both as nitric oxide scavenger and nitric oxide source depending on its concentration.⁴¹ It can lead to the formation of additional N₂O₃ by the enhanced oxidation of NO. Oxidation of NO by cPTIO forms ^.NO₂ radical (NO + cPTIO → ^.NO₂ + cPTI). This radical can further react with NO to produce N₂O₃ (^.NO₂ + NO → N₂O₃). Aberrations in response to aminoguanidine treatment can be attributed to the fact that aminoguanidine specifically inhibits NOS-like activity. Keeping in view other enzymatic and non-enzymatic endogenous sources of NO, like nitrate reductase, apoplastic NO production at acidic pH, oxidative NO synthesis from L-arginine and nitrite-NO oxidoreductase (Ni-NOR), substantial level of NO is still expected to be available in the tissue subsequent to aminoguanidine treatment. Reaction of nitric oxide and superoxide anions results in the formation of peroxynitrite (ONOO⁻), a potent oxidant and nitrating agent.³⁷ Peroxynitrite-triggered NO-dependent signals modulate redox regulation in various signaling pathways but overproduction of ONOO⁻ can lead to oxidative and nitrosative stress in a cell.³⁸ Peroxynitrite mainly acts as an oxidizing agent at cysteine thiols and also causes tryosine nitration of numerous amino acids in proteins (O₂• − + NO → ONOO⁻ → Tyrosine nitration).⁴² MnSOD has been shown to be tyrosine-nitrated leading to its inactivation.^43,44 Possible tyrosine nitration of MnSOD by ONOO⁻ has also been demonstrated in sunflower hypocotyls.⁴⁵ Differential inhibition of mitochondrial MnSOD, peroxisomal Cu/ZnSOD and chloroplastic FeSOD has also been reported in Arabidposis thaliana due to peroxinitrite-mediated tyrosine nitration (Tyr63).⁴⁶

To sum up, total SOD activity in sunflower cotyledons or roots is modulated owing to differential response of the 2 isoforms (FeSOD and Cu/ZnSOD) to NaCl stress. The observed differential spatial distribution of the 2 isoforms (MnSOD and Cu/ZnSOD) in seedling cotyledons and their sensitivity to salt stress in 2d old seedlings indicates their crucial role in subcellular management of superoxide anion production as a rapid stress response. NO proves to be a critical regulatory molecule in the efficient and differential expression of FeSOD and Cu/ZnSOD activity for scavenging superoxide anions. NO acts both as a positive and negative modulator of the 2 isoforms depending on tissue (roots/cotyledons) or NaCl stress. Based on the present investigations using SNP, cPTIO, aminoguanidine and peroxynitrite over a wide concentration range, it is further evident that any conclusions drawn from using a specific concentration of these pharmacological agents need to be taken with a degree of caution. These findings provide significant information, necessary for further investigations on NO-SOD crosstalk at subcellular level.

Material and Methods

Seed germination

Seeds of sunflower (Helianthus annuus) variety KBSH 53 were treated with 0.005% HgCl₂ for sterilization and sown in plastic trays on germination paper. Seedlings were grown in dark at 25˚C and irrigated with half-strength Hoagland solution. For salt treatment, 120mM NaCl was added to the Hoagland solution.¹¹ Cotyledons and roots from seedlings with uniform growth pattern were harvested for further experiments. Various exogenous NO related treatments (sodium nitroprusside, SNP; aminoguanidine, peroxynitrite and cPTIO) were given at equimolar concentrations (100, 250, 500 and 1000 µM, prepared in half-strength Hoagland solution) after radical emergence from seeds.

Spectrophotometric estimation of total SOD activity

SOD activity was estimated in according to Nishikimi et al.,⁴⁷ with minor modifications. Cotyledons and roots obtained from etiolated seedlings grown in the absence or presence of NaCl (120mM) were homogenized in 50mM Tris-Cl buffer (pH 7.0) followed by centrifugation at 10,000g for 20 min. Total SOD activity was analyzed in the supernatant using non-enzymatic phenazine methosulfate (PMS)/ NADH-NBT reaction mixture as substrate. The decrease in absorbance at 560 nm in the presence of homogenate fraction was used to calculate the superoxide anion scavenging activity in the reaction mixture.

Zymographic detection of SOD isoforms

SOD isoforms were zymographically analyzed according to McCord.⁴⁸ Tissues (cotyledons/roots) were ground (500 mg of cotyledons and 1 g of roots) to powder in liquid nitrogen and homogenized in 3 ml Tris-Cl buffer (50 mM, pH 7.0). The homogenates were centrifuged at 10,000 g for 20 min at 4°C and protein estimation of the supernatant fraction (total soluble protein, TSP) was done using Bradford assay.⁴⁹ TSP from each homogenate (containing 50 µg and 30 µg of protein in cotyledons or root samples, respectively) was mixed with non-reducing Laemmli sample buffer and loaded on 12% flat polyacrylamide native gels. After vertical electrophoresis at 4°C, the gel was soaked in mixture of solution A (NBT prepared in phosphate buffer, pH 7.8 containing EDTA) and solution B (riboflavin) for 10 min in dark. The gel was then transferred to distilled water and exposed to light for 5–7 min. The gel was negatively stained in blue color and achromatic bands (clear area) depicting SOD activity were obtained. The band intensity was then quantified by performing densitometry using Quantity One software.

Immunolocalization of SOD isoforms in cotyledons

Wax sections of seedling cotyledons (7µm thick) were cut following fixation of the samples according to previously described method.⁵⁰ Blocking of sections was done using BloackAid solution (Invitrogen, USA) for 30 min. Subsequently the sections were incubated in anti-rabbit polyclonal primary antibody against MnSOD and Cu/ZnSOD (Abcam, Cambridge, UK) at a dilution of 1:200. Primary antibody detection was carried out using secondary antibody Cy3-labeled anti- rabbit IgG (GE Life Sciences, England) diluted to 1:1500 in PBS. Sections without primary antibody treatment were used as control. Sections were observed and CLSM analysis of the distribution of MnSOD and Cu/ZnSOD was undertaken using argon lasers (excitation: 535–550 nm and emission: 570 nm) at pinhole 1. Quantification of endogenous enzyme titer was done using standard imaging software (Leica QWin image analyzer, Leica, Germany) and expressed as relative optical density.

Funding

The authors are grateful to the Council of Scientific & Industrial Research (CSIR), University Grants Commission (UGC), Department of Science and Technology (DST) and Delhi University for financial support in the form of PURSE grant and Research and Development grant, respectively.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interests were disclosed.

Acknowledgments

DA and SCB were jointly responsible for the experimental design, data analysis and article writing. All experiments were performed by DA.