Regulation of redox homeostasis in cadmium stressed rice field cyanobacteria by exogenous hydrogen peroxide and nitric oxide

In the present study, defensive strategies of H2O2 mediated NO signaling were analyzed in Cd stressed Nostoc muscorum and Anabaena sp. Exogenously supplied SNP (10 µM) and H2O2 (1 µM) lessen the toxicity of Cd (6 µM) but without NO; H2O2 was unable to release the stress from cyanobacterial cells potentially. The reduced contents of exopolysaccharide, protein content, endogenous NO and enzymatic antioxidants (SOD, POD, CAT, and GST) due to Cd toxicity, were found increased significantly after exogenous application of H2O2 and SNP thereafter, cyanobacterial calls flourished much better after releasing toxic level of Cd. Moreover, increased level of ROS due to Cd stress also normalized under exogenous application of H2O2 and SNP. However, chelation of NO hindered the signaling mechanism of H2O2 that diminished its potential against Cd stress while signaling of NO has not been hindered by chelation of H2O2 and NO potentially released the Cd stress from cyanobacterial cells. In conclusion, current findings demonstrated the synergistic signaling between H2O2 and NO towards the improvement of cyanobacterial tolerance to Cd stress, thereby enhancing the growth and antioxidant defense system of test cyanobacteria that improved fertility and productivity of soil even under the situation of metal contamination.


Regulation of redox homeostasis in cadmium stressed rice field cyanobacteria by exogenous hydrogen peroxide and nitric oxide Nidhi Verma & Sheo Mohan Prasad *
In the present study, defensive strategies of H 2 O 2 mediated NO signaling were analyzed in Cd stressed Nostoc muscorum and Anabaena sp. Exogenously supplied SNP (10 µM) and H 2 O 2 (1 µM) lessen the toxicity of Cd (6 µM) but without NO; H 2 O 2 was unable to release the stress from cyanobacterial cells potentially. The reduced contents of exopolysaccharide, protein content, endogenous NO and enzymatic antioxidants (SOD, POD, CAT, and GST) due to Cd toxicity, were found increased significantly after exogenous application of H 2 O 2 and SNP thereafter, cyanobacterial calls flourished much better after releasing toxic level of Cd. Moreover, increased level of ROS due to Cd stress also normalized under exogenous application of H 2 O 2 and SNP. However, chelation of NO hindered the signaling mechanism of H 2 O 2 that diminished its potential against Cd stress while signaling of NO has not been hindered by chelation of H 2 O 2 and NO potentially released the Cd stress from cyanobacterial cells. In conclusion, current findings demonstrated the synergistic signaling between H 2 O 2 and NO towards the improvement of cyanobacterial tolerance to Cd stress, thereby enhancing the growth and antioxidant defense system of test cyanobacteria that improved fertility and productivity of soil even under the situation of metal contamination.
Cadmium (Cd) contamination in agricultural fields like rice crop fields has become a serious environmental issue. Potentially active Cd ions in the soil can accumulate in rice plants and ultimately reduce the uptake and translocation of vital minerals and nutrients for plants [1][2][3] . Cd ions in agricultural soil can also negatively affect the beneficial microbes present in the rice field 4 . Cyanobacteria are pioneer phototrophic prokaryotic microorganisms that are known to display their potential role in sustainable agricultural development especially for paddy fields. Nostoc muscorum and Anabaena sp. are categorized as heterocystous cyanobacteria and known for their nitrogen-fixing ability. They are mainly present on soil and water bodies and fix about 20-25 kg/ha atmospheric nitrogen. More efficiently, Anabaena sp. can fix about 60 kg/ha of nitrogen in a season 5 . Unfortunately, Cd ions present in rice soil are major threats to these nitrogen fixers. Previous studies demonstrated that some photosynthetic bacteria can tolerate up to 1 mg Cd kg −1 soil in the association with Hay bacillus (Bacillus subtilis) and some other lactic acid bacteria 6 . But the release of Cd is much higher (200 mg Cd kg −1 soil) in the soils near the leather and electro-plating factories 7 that creates a hazardous environment for paddy field cyanobacteria which ultimately results to the poor quality of rice crop. Cd interrupts the cell functioning and hangovers the regular metabolic processes of cyanobacteria. Thus, Cd ions can enter into the cell through P-type Cd 2+ ATPases (e.g. cadA1 and cadA2) and interrupt electron transport chain and produce excessive ROS (reactive oxygen species thereby disturbing the balance between antioxidants and ROS which tend to the cell death 8 . Study of Qiao et al. 9 shows that there are three possible ways through which Cd imposes ROS accumulation in cells: (1) excess level of Cd 2+ enhances the expression of miR398 that ultimately inhibits the functioning of Cu/Zn/SOD and finally, a drastic rise of ROS; (2) excess Cd 2+ in cells can inhibit the regulatory role of second messengers like Ca 2+ , or other signaling molecules like NO and H 2 O 2 ; (3) excess accumulation of Cd 2+ enhances the activity of NADPH oxidase, that is a major producer of ROS. So to enhance the tolerance against different environmental stresses the external applications of different signaling molecules like H 2 O 2 , NO, and H 2 S, are considered as one of the most beneficial methods 10 15 suggested that Cd toxicity has been removed expressively by pretreatment of H 2 O 2 in the seedlings of Brassica nupus. Pretreatment of H 2 O 2 possibly empowers the internal defense system of the organism, but its downstream signal transduction mechanisms and pathways are not effusively clear in cyanobacteria. The defense strategies in organisms are a much complex process in which different signaling molecules are linked together as reported in the study of Li et al. 16 ; it is proven that there is a multifaceted interaction among NO, H 2 S, and H 2 O 2 promoting thermotolerance in maize seedlings. Christou et al. 17 also noticed a faster and stronger response against salt stress by the priming of H 2 O 2 and SNP (donor of NO) in strawberry plants. Gonzalez et al. 18 have also revealed the cross linking mechanism of calcium, H 2 O 2 , and NO in copper treated Ulva compressa. However, in the case of cyanobacteria to the best of our knowledge the interacting role of signaling molecules H 2 O 2 and NO in Cd stress alleviation is lacking. Therefore, we have hypothesized that synergistically H 2 O 2 and NO could initiate certain protective mechanisms that may be associated with metal stress particularly Cd tolerance in cyanobacteria.
Hence, the prime focus of this study was to evaluate the potential role of H 2 O 2 and NO as defense signaling molecules to cadmium stress tolerance in cyanobacteria Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120. Also a key objective was to explore the probable mechanism and connective pathway between H 2 O 2 and NO.

Results
Effect of H 2 O 2 and NO on growth of Cd stressed cyanobacteria. In the present study growth of both the tested cyanobacteria was measured in terms of dry weight as shown in Fig. 1a,b which depicted that 6 µM Cd inhibited the growth by 28% in Nostoc muscorum ATCC 27893 and 30% in Anabaena sp. PCC 7120 (hereafter referred as Nostoc muscorum and Anabaena sp.) in comparison to control. However, on exogenous supplementation of H 2 O 2 , growth was recovered and inhibition was noticed only 9 and 11% respectively for Nostoc muscorum and Anabaena sp. Similar to this, exogenous SNP significantly reduced (P < 0.05) the inhibition exerted by Cd, and found only 11 and 7%, respectively as compared to control. Further, to know the regulatory role of endogenous ROS and NO on growth, Cd treated cells were subjected to NAC, DPI, PTIO and L NAME; therefore, a greater reduction in dry weight was noticed under PTIO and L NAME treatment in comparison to control (Fig. 1b). Furthermore, to clarify the cross-talk of H 2 O 2 and NO, cells were treated with scavengers (NAC and PTIO, respective scavengers of H 2 O 2 and NO) and inhibitors (DPI and L NAME, respective inhibitors of NADPH oxidase and nitric oxide synthase (NOS) enzymes) with Cd stress. In this case, our results showed a critical decline in growth under treatments of PTIO or L NAME i.e. 41 and 44% respectively in Nostoc muscorum and 44 and 46% respectively in Anabaena sp. even in the presence of H 2 O 2 . Contrastingly, growth was found improved moderately under SNP treatment even in the presence of NAC and DPI (Fig. 1a). Moreover, in a combined treatment of both the signaling molecules, H 2 O 2 and SNP along with both PTIO and L NAME under the same stress condition, inhibition in growth again found crucial i.e. 38 and 39% in Nostoc muscorum and Anabaena sp. respectively. Whereas, under similar stress, combined treatment of H 2 O 2 and SNP along with NAC and DPI inhibition in growth was reduced and found only 12 and 14% in Nostoc muscorum and Anabaena sp. respectively on comparison to control.
Effect of H 2 O 2 and NO on the secretion of exopolysaccharide (EPS) layer during Cd stress. Impact of exogenous supplementation of H 2 O 2 and SNP on secretion of defensive layer of EPS in test cyanobacteria has been portrayed in Fig. 1c. In both the test cyanobacteria Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120 secretion of the EPS layer was declined significantly (P < 0.05) by 23 and 25% respectively under Cd treatment in contrast to control values (Fig. 1c). Under the treatment of Cd + H 2 O 2 and Cd + SNP, the EPS secretion was enhanced significantly (P < 0.05) by 20 and 24% respectively in Nostoc muscorum and 17 and 19% respectively in Anabaena sp. comparative to control values. Contrastingly, under the same stress, the decline in EPS secretion was found more critical under the treatments of PTIO or L NAME i.e. 28 and 43% respectively in Nostoc muscorum and 33 and 51% respectively in Anabaena sp. even in the presence of H 2 O 2 . Whereas, treatments of NAC or DPI along with SNP do not hinder the signaling mechanism of NO and an increased EPS content was noticed i.e. 11 and 8% respectively in Nostoc muscorum and 10 and 7% respectively in Anabaena sp. Moreover, combined treatments of H 2 O 2 and SNP along with PTIO and L NAME under the same stress crucially declined the EPS content that showed the incapability of H 2

Effect of H 2 O 2 and NO on intracellular Cd accumulation under Cd stress. Histochemical analysis
of Cd inside the cells was observed in the form of red patches which were the insoluble red salt that appeared due to the complex of dithizone with Cd. Results pertaining to the in vivo visualization of Cd accumulation in both the tested organisms have been portrayed in Fig. 2. The appearance of intense red patches inside the vegetative cells of cyanobacteria showed the accumulation of Cd inside the cells. In Cd treated cells, red patches are found more intense than H 2 O 2 and SNP treated cells. Whereas in control, no red spots have appeared. Critically intense red patches appeared in the cells under treatments of PTIO or L NAME even in the presence of H 2 O 2 . Treatment of SNP along with NAC or DPI; reduced the appearance of red spots inside the cell. The patches were found more intense in Anabaena sp. than Nostoc muscorum which showed that Cd easily entered into the cells of Anabaena sp. than Nostoc muscorum.  (Figs. 3, 4). Blue patches were appeared by the staining with NBT for SOR, brown colored patches were appeared by DAB staining for H 2 O 2 while pink patches were the result of staining with Shiff 's reagent for MDA, and overall effects of oxidative stress were represented as electrolyte leakage (EL) shown by the sky blue spots (Figs. 3, 4). In the figure blue, brown, pink, and sky blue patches were appeared more intense under the exposure of Cd but spots were normalized under exogenous addition of H 2 O 2 or SNP along with Cd stress. But in the absence of NO inside the cell means under the exposure of PTIO and L NAME exogenously supplied H 2 O 2 cannot limit the ROS production and intense patches were noticed. While in lack of H 2 O 2 means under the exposure of NAC and DPI exogenously added SNP can able to limit the ROS production and faded patches have appeared inside the cells of both the organisms. This finding clearly showed the efficiency of NO towards controlling the ROS inside the cells and also clarified that   Under similar stress activities of these antioxidants were found extremely enhanced under the exposure of H 2 O 2 and SNP with a more pronounced effect of SNP. Contrary to this, activities of these antioxidants were got arrested under the exposure of PTIO or L NAME even in the presence of H 2 O 2 while enhanced activities of these enzymatic antioxidants were found on the exposure of SNP even in the presence of NAC or DPI in both the test organisms. The expressions of isoenzymes of SOD, POD, CAT and GST were more strongly supported the biochemical analysis of antioxidant enzymes clearly depicted in Fig. 6. where the intensity of bands of SOD, POD, CAT and GST were found nominally intense under Cd stress and band intensity was found extremely intense under the supplementation of H 2 O 2 and SNP with Cd. Unlikely, the bands were found negligibly appeared when NO is blocked while band slightly appeared where H 2 O 2 is blocked and SNP is provided. In Nostoc muscorum the activities of antioxidants were found more intense than Anabaena sp. showed the resistive behavior of Nostoc muscorum.

Effect of H 2 O 2 and NO on the activity of enzymatic antioxidants under Cd stress.
Results finally clarify the signaling role of H 2 O 2 and NO. They can detoxify the Cd stress and as it is evident form both the tested organisms by enhancing their defensive layer exopolysaccharides secretion as well as by boosting their antioxidant machinery and also by reducing the excessive level of ROS formed inside the cell. On the other hand, improvement was found more pronounced in the case of Nostoc muscorum showing its resistive behavior in comparison to Anabaena sp.  20 and Nahar et al. 21 . The possibility of reasons behind this improved growth can be summarized into the following points. (1) H 2 O 2 and NO can reduce the intracellular accumulation of Cd (Fig. 2) by enhancing the secretion of EPS (Fig. 1c); (2) protein content can also be enhanced by exogenous exposure of H 2 O 2 and NO (Fig. 1d); (3) H 2 O 2 and NO can enhance endogenous NO content (Fig. 1e) and (4) H 2 O 2 and NO can reduce the excessive ROS contents (Table 1) by enhancing enzymatic antioxidant activities (Fig. 5a-d). Similar to this, Christou et al. 17 reported that H 2 O 2 and NO reduced the salt stress in the strawberry plants. Several studies reported that a high dose of H 2 O 2 is used for eradication of algal bloom in ponds 22 . But against this, in the present study, a very low dose (1 µM) of H 2 O 2 (provided for a very short time; only for 3 h) is used and very satisfactory results have been obtained against the Cd toxicity and enhanced growth of test cyanobacteria have been found under H 2 O 2 treatment (Fig. 1a,b). Basal level of endogenous ROS and NO are very important to the cell functioning under stress condition which was proven by individual treatments of NAC, DPI, PTIO and L NAME along with Cd (Fig. 1b). Further experiments of the present study show the interlinked process of internal signaling of H 2 O 2 and NO. In this way, the application of inhibitor and scavenger of NO reversed the enhanced growth of cyanobacteria by worsening the Cd toxicity inside the cells that confirms the dependency of H 2 O 2 on NO. Our results are in consonance with the finding of www.nature.com/scientificreports/ H 2 O 2 promotes NO to enhance the EPS secretion which reduces the intracellular cd accumulation. The protective layer of EPS mainly comprises a group of biopolymers with high molecular weight which are secreted in response to any environmental stress that make a barrier against the stress 23 . Cadmium is a highly  www.nature.com/scientificreports/ toxic metal found on Earth that disrupts the protective layer of EPS in tested cyanobacteria (Fig. 1c) which can be related with the more Cd accumulation inside the cells (i.e. displayed by red patches) demonstrated in Fig. 2. Similar to this Patel et al. 24 also found the reduced EPS content along with increased As accumulation in Nostoc muscorum and Anabaena sp. Such disruptions in EPS content have been eliminated by the application of H 2 O 2 and SNP and red patches inside the cells are less appeared (Fig. 2)

H 2 O 2 up-regulates NO to enhance the protein content of Cd stressed cyanobacteria.
During adverse conditions, protein content of any organism is directly associated with the growth. In the present study, decreased protein content (Fig. 1d) subjected to Cd stress might be due to the direct impact of Cd on protein biosynthesis 25 . Contrary to this, exogenous H 2 O 2 and NO reversed the negative impact of Cd on protein content (Fig. 1d). H 2 O 2 and NO might be inducing the protein biosynthesis by controlling the ROS metabolism inside the cell 26 . Interestingly, ROS production was further induced ( Table 1) by supplementation of PTIO and L NAME that resulted to the damage in protein content (Fig. 1d) which collectively reduced the growth of Cd stressed cyanobacteria.
Exogenously supplied H 2 O 2 and SNP promote the endogenous production of NO for balancing of ROS and electrolyte leakage induced by Cd stress. As endogenous NO is known to play an important role in all the plant developmental activities and metal stress badly reduces the NO content by www.nature.com/scientificreports/ interfering with nitric oxide synthase enzyme 27 . Our present study revealed that endogenous NO content was reduced under Cd stress which was further improved by treatment of H 2 O 2 and SNP (Fig. 1e) and such increase was eliminated by adding PTIO and L NAME, which are in the same line with previous findings of Xuan et al. 28 and Li et al. 16 . Elimination of NO content by L NAME clearly shows that nitric oxide synthase enzyme involves in NO biosynthesis inside the tested organisms. Correspondingly, Bouchard and Yamasaki 29 have also clarified that microalgal NO is produced in a time-dependent manner under heat shock exposure which was further eliminated by adding cPTIO. Christou et al. 17 have also demonstrated that SNP and H 2 O 2 enhanced the NO content under NaCl stress in strawberry plants. Furthermore, exogenous SNP also dose a positive regulation of NO accumulation in the presence of NAC and DPI under Cd stress (Fig. 1e) 34 and Li et al. 35 . A previous study also shows that at very low concentration H 2 O 2 works as a signaling mediator and modulates deferent stress managing genes. H 2 O 2 up-regulate the NO facilitated ABA-induced mitogen-activated protein (MAP) kinase cascade pathway to empower the defensive mechanism of maize leaves. During stress situation H 2 O 2 , can regulate NO, and NO itself works as a ROS scavenger that is the point of interaction between these two incredible signaling molecules 15 .
Our results showed that H 2 O 2 and SNP alleviated electrolyte leakage ( Fig. 4; sky blue spots) which is supported by the previous finding of Song et al. 36 . Similarly, Li et al. 16 suggested that pretreatment of H 2 O 2 in heatstressed maize seedlings reduced the electrolyte leakage and endogenous MDA content to improve the overall photosynthesis and growth. Ali et al. 37 also showed that the application of SNP reduced the level of H 2 O 2 in the Triticum aestivum L. plant. Again the contents of these ROS species have been more pronouncedly increased under the treatment of PTIO and L NAME. However, in the current study, a reduced level of endogenous H 2 O 2 content have been found under the exposure of exogenous H 2 O 2 showed a reversible pathway in which H 2 O 2 www.nature.com/scientificreports/ signals may promote endogenous NO to enhance the activity of enzymatic antioxidants that ultimately expels out the overall ROS species from the cell as demonstrated by Verma et al. 12 .

H 2 O 2 and NO enhanced the activity of enzymatic antioxidants under Cd stress. The previous
study of Farooqui et al. 38 in Nostoc muscorum disclosed that SOD, CAT and POD antioxidants were found to decrease under high concentrations of Cd. In accordance with previous studies, the current results showed that Cd reduced the level of enzymatic antioxidants (SOD, POD, CAT and GST) and bands of their isoenzymes were also found less enhanced (Figs. 5, 6). The possible reason behind this disturbance in activities of enzymatic antioxidants might be due to the alterations in basal level of ROS and thereby increased oxidative stress 27 . Cd has a strong affinity to bind with -SH groups or proteins to disturb the activity and synthesis of various enzymes 39 . In present work increased activities of SOD, POD, CAT and GST (Fig. 5a-d) were recorded on exogenous exposure of H 2 O 2 and SNP which played a vital role in the reduction of ROS from the cells which is supported by the studies of Hasanuzzaman et al. 15 and Xu et al. 40 . Also, the study of Qian et al. 41 revealed the role of NO in positive regulation of several enzymatic antioxidants under herbicide stress in Chlorella vulgaris. SOD, POD, CAT and GST are known to directly catalyze the ROS scavenging reaction. A mild dose of H 2 O 2 supplied to cyanobacteria in the present study, enhanced the activities of these enzymatic antioxidants that may regulate the AsA-GSH cycle in Cd affected cyanobacteria which is directly related to ROS scavenging process and that is why the low amount of H 2 O 2 decreased the intracellular ROS levels [42][43][44] . The whole above mentioned results implied that the www.nature.com/scientificreports/ acquisition of Cd stress tolerance in cyanobacteria may be involved in cross-talk between H 2 O 2 and NO and a very complex feedback regulation is found between both signaling molecules for metabolic adaptation against abiotic stress.

Materials and methods
Experimental organisms and culture condition. Nostoc muscorum ATCC 27893 and Anabaena sp.
PCC 7120, heterocyst containing filamentous cyanobacteria were grown in BG-11 medium (pH 7.5). Their homogenous cultures were grown in controlled growth conditions; at a temp of 25   Measurement of growth. The growth of test cyanobacteria was measured in terms of dry weight. The 100 ml cultures of both the organisms were collected and centrifuged at 4000g for 10 min and then washed twice with distilled water. Further, pellets were dried at 80 ºC and weighed gravimetrically (Contech-CA 223, India).
Determination of exopolysaccharides content. Exopolysaccharides (EPS) content was determined according to Sharma et al. 45 . For EPS extraction, 100 ml samples were taken from each treatment, centrifuged at 3000g for 15 min supernatants were collected and then dried separately at 40 ºC. The obtained precipitates were washed thrice with isopropanol and dried again at 37 ºC. Further, hydrolysate was analyzed for glucose by Seifter et al. 46 and calculation was done by standard curve of glucose.
Estimation of protein content. For the extraction of protein, the method of Bradford 47 was adopted.
Cells were centrifuged and homogenized with 50 mM potassium phosphate buffer (PPB) (pH 7.8) containing 1 mM EDTA and 2% polyvinyl pyrrolidone at 4 °C. Further, 0.1 ml of obtained homogenates was mixed with 2.5 ml of Coomassie brilliant blue G-250 reagent, then kept for 2 min in dark at 25 °C and absorbance was read at 595 nm. Total soluble protein content was determined by using bovine serum albumin as standard protein solution.
Detection and quantification of NO. The NO content was determined by using Griess reagent (Sigma-Aldrich) by following the procedure of Zhou et al. 48 with some modifications. For NO estimation desired volume of cultures was centrifuged and pellets were crushed in 3 ml of 50 mM cool acetic acid buffer (pH 3.6, containing 4% zinc diacetate). Further, homogenates were centrifuged and supernatant was mixed with 50 mg of charcoal. After vortex and filtration, the filtrate was leached and collected. The 1 ml of filtrate and 1 ml of Greiss reagent was mixed and incubated at room temperature for 30 min. Absorbance was taken at 540 nm. NO content was calculated by the standard curve prepared by graded solution of NaNO 2 .
Histochemical detection of Cd. Histochemical analysis of Cd was conducted through the method of Seregin and Kozhevnikova 49 with some minor changes. To detect the intracellular Cd accumulation; cells were collected by centrifugation then gently washed twice or thrice with double distilled water (DDW) to remove excess Cd. Obtained cells were mixed with 1 ml of dithiozone stock solution containing 6 gm of dithiozone and DW (3:1). Further 1-2 drops of acetic acid glacial were added and after 4 h of incubation; cells were observed under microscope (Leica, model-DM 2500).  53 in which cells were suspended into nitrobluetetrazolium (NBT; Sigma) and 3, 3 diaminobenzidine (DAB) for in-vivo staining respectively. MDA equivalents contents and intensity of membrane damage were visualized by using Schiff 's reagent and Evan's blue by following the methods of Pompella et al. 54 and Yamamoto et al. 55 respectively. Images were taken in a high-quality microscope (Leica, model-DM 2500).

Estimation of oxidative biomarkers and indices of damage.
Enzymatic antioxidant essay. The activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), catalase (CAT; EC 1.11.3.6) and glutathione-S-transferase (GST, EC 2.5.1.18) were analyzed by following the methods of Giannopolitis and Ries 56 , Gahagan et al. 57 , Aebi 58 and Habig et al. 59 respectively. For SOD activity, the photoinhibition of NBT was recorded at 560 nm after 20 min illumination of light (100 µmol photons m −2 s −1 ). One unit of SOD activity is demarcated as the required amount of enzyme to cause 50% inhibition in the reduction of NBT. For POD, absorbance of the reaction mixture was recorded at 430 nm for 3 min. The activity of the enzyme was calculated by using an extinction coefficient of 25.5 mM −1 cm −1 and one unit of enzyme activity is defined as 1 nmol pyrogallol oxidized min −1 . Activity of CAT was determined by monitoring the decrease in absorbance at 240 nm in the reaction mixture 58 . The activity of enzyme was calculated by using an extinction coefficient of 39.4 mM −1 cm −1 . Here 1 nmol H 2 O 2 dissociated min −1 was equivalent to one unit of enzyme activity. For GST, absorbance of reaction mixture was recorded at 340 nm. Enzyme activity was calculated by an extinction coefficient of 9.6 mM −1 cm −1 and one unit of enzyme activity is equivalent to 1 nmol of CDNB conjugates formed min −1 .
Native polyacrylamide gel electrophoresis for isoenzyme profiling. Native-PAGE analysis was carried out on discontinuous polyacrylamide gels (PAGE) with 4.5% polyacrylamide in stacking and with its varying concentrations (10% for SOD and GST, 6% for CAT and 8% for POD). The separation of individual isoenzyme was performed in a vertical gel electrophoretic unit (GeNei, India) by considering the method of Laemmli 60 . A uniform amount (300 mg) of proteins mixed with sample buffer (0.5 M Tris-HCl, pH 6.8) was loaded in each well and proteins were electrophoretically separated at 80 V through stacking gel followed by 120 V in the separating gel at 4 ºC. For visualizing SOD isoenzymes, the gels were immersed in PPB (50 mM; pH 7.8) containing NBT (1.125 mM) in darkness for 20 min and followed by incubation in PPB containing TEMED (28 mM) and riboflavin (28 mM) in dark for 15 min. The gels were then placed in PPB containing mM EDTA (0.1 ml) and exposed to light for 20 min at 25 ºC 61 . For POD, gels were immersed in 1 mg ml −1 benzidine and 1 mM H 2 O 2 in 0.1 M Tris-acetate buffer (pH 5.0) at 25 ºC till the brown colored bands appeared 62 . For CAT isoenzymes gels were incubated with H 2 O 2 (0.01%) for 15 min, rinsed with water, and shaken in freshly prepared solutions each of 0.1% FeCl 3 and K 3 Fe(CN) 6 at 25 ºC until the achromatic bands appeared 63 . For GST isoenzyme staining a reaction mixture containing 0.1 M PPB (pH 6.5), GSH (5.0 mM), CDNB (1.0 mM) and NBT (1 mM) was used and bands were developed by illuminating the gel under 25 µmol photon m −2 s −1 of photon flux density 64 . Photographs of isoenzymes were captured with digital camera, photo-plate was prepared in CorelDRAW and image quality was enhanced in Photoshop 7.0. Statistical analysis. Statistical analysis of variance (ANOVA) was performed to test the significance at probability level P < 0.05. Duncan's multiple range test was applied to compare significant differences among the mean values. Graphical representations are the means of three independent experiments with three replicates in each experiment (n = 9). Lower case letter (a, b, c, d, e, f, g, h, i, j) shows statistical significance.

Conclusions
From the aforementioned results, it can be concluded that there is an interlinked pathway between NO and H 2 O 2 that exists. In Fig. 7, it is clear that Cd create oxidative stress inside the cell and produces more ROS and creates membrane damage by increasing MDA equivalents contents 15,21,65 . But exogenously supplied NO and H 2 O 2 promote the EPS secretion and check the entrance of Cd inside the cells after that they also enhance the antioxidant system and endogenous NO content that's why the level of ROS get minimized. Here, the low amount of exogenous H 2 O 2 works as a signal transmitter to enhance the beneficial amount of RNS species that indirectly respond to the balancing of antioxidants to cope up with cd stress. Afterward PTIO and L NAME with H 2 O 2 block the internal NO that creates a disturbance in the working of signaling mechanism of H 2 O 2 and NO here it is clear that exogenously supplied H 2 O 2 cannot work without NO. However, application of NAC and DPI cannot disturb the signaling of NO. Hence, from this it is clear that NO is a down-stream regulator of H 2 O 2 but the pathway of signaling between both the molecules is very complex because of their multidimensional roles in a stressful environment. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.