Interplay of Calcium and Nitric Oxide in improvement of Growth and Arsenic-induced Toxicity in Mustard Seedlings

In this study, Ca2+ mediated NO signalling was studied in response to metalloid (As) stress in Brassica seedlings. Arsenic toxicity strongly suppressed the growth (fresh weight, root and shoot length), photosynthetic pigments, Chl a fluorescence indices (Kinetic traits: Fv, Fm, Fv/Fo, Fm/Fo, ФPo or Fv/Fm, Ψo, ФEo, PIABS, Area and N and redox status (AsA/DHA and GSH/GSSG ratios) of the cell; whereas energy flux traits: ABS/RC, TRo/RC, ETo/RC and DIo/RC along with Fo, Fo/Fv, Fo/Fm, ФDo and Sm) were enhanced. Further, addition of EGTA (Ca2+ scavenger) and LaCl3 (plasma membrane Ca2+ channel blocker) to As + Ca; while c‒PTIO (NO scavenger) and l‒NAME (NO synthase inhibitor) to As + SNP treated seedlings, siezed recovery on above parameters caused due to Ca2+ and NO supplementation, respectively to As stressed seedlings thereby indicating their signalling behaviour. Further, to investigate the link between Ca2+ and NO, when c‒PTIO and l‒NAME individually as well as in combination were supplemented to As + Ca treated seedlings; a sharp inhibition in above mentioned traits was observed even in presence of Ca2+, thereby signifying that NO plays crucial role in Ca2+ mediated signalling. In addition, As accumulation, ROS and their indices, antioxidant system, NO accumulation and thiol compounds were also studied that showed varied results.

www.nature.com/scientificreports www.nature.com/scientificreports/ cascade. In proteins, NO induced post-translational modification is carried out by nitrosylation of their cysteine residue. Wu et al. 17 reported that NO application improves photosynthetic rate by (i) channelizing excess energy by increasing carotenoid or other antenna molecules and (ii) increasing quantum yield of PS II, under salinity stress in Solanum melongena seedlings.
From the available literature, it is clear that both Ca 2+ and NO plays multiple roles in regulating key physiological processes in stressed as well as non-stressed situations; however, their cumulative effect in orchestrating plant responses to different environmental cues have not been well established. Therefore, future studies are needed to understand the intensive interaction and interrelation of Ca 2+ and NO in various physiological, histochemical and metabolic approaches suffering from arsenic toxicity in plants. Towards this objective, the key components i.e. growth and growth regulating parameters: Chl a fluorescence, redox status of cell, enzymatic and non-enzymatic antioxidants and levels of thiol compounds were assessed in the present investigation.
Results and discussion ca 2+ and no recover As-induced damage in phenotypic appearance. To examine the Ca 2+ mediated NO signalling in alleviating As-induced toxicity, Brassica L. seedlings were treated with different donors, scavengers and inhibitors of Ca 2+ and NO. As expected, metalloid (As) stress caused deteriorating effect on growth and declined the fresh weight, root and shoot length by 29, 33 and 28%, respectively of test seedlings (Fig. 1), which is manifested by increased reactive oxygen species (ROS) production that caused lipid peroxidation, protein oxidation and loss to membrane integrity leading to electrolyte leakage (Figs. 2a,b). However, under As stress, both CaCl 2 and SNP treatment counteracted As-induced negative impact on FW, RL and SL of test seedlings (Figs. 1a-c), which agree with the greater accumulation of NO than As-stressed seedlings alone (Fig. 3). The Ca 2+ and NO induced positive response on growth have also been reported by Singh et al. 5 and Siddiqui et al. 15 in mustard and tomato seedlings, respectively. Further, a significant inhibition of growth after EGTA and LaCl 3 treatment indicated that both of them arrests Ca 2+ -induced positive impact on growth agreeing  3 : lanthanum chloride, a plasma membrane Ca channel blocker, c-PTIO: 2-4-carboxyphenyl-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide, a NO scavenger and L -NAME: N ω -nitro-L-arginine methyl ester hydrochloride, a nitric oxide synthase inhibitor). Data signifies the mean ± standard error of five replicates. Bars followed by different letters are significantly different at p < 0.05 level according to Tukey test. Where 'nd' is 'no detection' of As.
www.nature.com/scientificreports www.nature.com/scientificreports/ the fact of involvement of Ca 2+ as signalling molecule. Knight et al. 11 have also reported that lanthanum (La) and EGTA inhibit the salt-and mannitol-induced (Ca 2+ ) cyt elevations in Arabidopsis. In a previous study, Xu et al. 18 have reported that ABA protects tall fescue plant from oxidative injuries by promoting NO release (via activating NOS) thereby triggering the activities of antioxidant enzymes. Therefore, also in order to study the possible link between Ca 2+ and NO signalling, As + Ca treated Brassica seedlings were treated with NO scavenger: c-PTIO and synthase inhibitor: l-NAME. Interestingly, the growth of As + Ca treated seedlings was abolished in presence of c-PTIO and l-NAME (Figs. 1a-c), suggesting that NO is required for the maximal and sustained signalling of Ca 2+ , which corresponds to reduced NO accumulation in presence of c-PTIO and l-NAME (Fig. 3). Lanteri et al. 19 also reported that NO is required for the maximal activity of Ca 2+ -dependent protein kinase (CDPK) for adventitious root formation in Cucumis sativus. Results of As accumulation suggests that both CaCl 2 and SNP counteracted the As accumulation in test seedlings; however in presence of c-PTIO and/or l-NAME even Ca 2+ was unable in restricting As accumulation, therefore higher As content was observed in c-PTIO + l-NAME treated seedlings than As-stressed seedlings alone (Fig. 1d).  ) showing the effect of CaCl 2 and SNP on the leaves of As-challenged Brassica seedlings subjected to different modulators. [(I) Control, (II) As, (III) As + Ca, (IV) As + Ca + EGTA, (V) As + Ca + EGTA + LaCl 3 , (VI) As + Ca + c-PTIO, (VII) As + Ca + l-NAME, (VIII) As + Ca + c-PTIO + l-NAME, (IX) As + SNP, (X) As + SNP + c-PTIO and (XI) As + SNP + l-NAME].
www.nature.com/scientificreports www.nature.com/scientificreports/ ca 2+ and no rescue As-induced losses of chls and car and recovers photosynthetic rate and pS ii photochemistry. To further elucidate the role of NO in Ca 2+ -induced signalling on photosynthetic performance, photosynthetic pigments: Chls (a and b) and Car were examined. Arsenic induced reduction in the levels of Chl a, b and Car was in accordance with the reduced plant growth (Fig. 1), signifying that As impaired photosynthetic ability of plants (Table 1) by disrupting chloroplast structure and pigments' biosynthesis 6 . Calcium and NO, on the other hand, rescued the As-induced loss in photosynthetic pigments content that partly attributed to lowering of ROS, which might have prevented photo-oxidative damage of photosynthetic apparatus 14,15 . However, As-induced damage was severe when As + Ca treated seedlings were supplemented either with EGTA + LaCl 3 or c-PTIO + l-NAME, which corresponds to the decreased values of photosynthetic rate (Table 1).
Further, to reveal the Ca 2+ mediated NO role in structural and functional properties of PS II, the OJIP transient curves as well as biophysical traits deduced from OJIP were studied. A sharp drop in O-J, J-I and I-P transient curves, which denotes the sequential reduction of electron acceptor pool of PS II, indicates that PS II was the major target of As (Fig. 4a). The decline in O-J-I-P transient curve could be attributed to damage at the donor side of PS II restricting the flow of electron between OEC and PS II [20][21][22][23][24] . Moreover, the drop in OJIP transient curves was intense upon c-PTIO + l-NAME supplementation to As + Ca treated seedlings indicating that in absence of NO, the effect of As became more severe (even in presence of Ca 2+ ), which could be due to: (i) inhibition in electron transport rate on the donor side of PS II as reflected by decreased values for area over the fluorescence curve (Area), which consequently decreased the maximum quantum yield for primary photochemistry   spider plots for OJIP parameters inferred from chlorophyll a fluorescence OJIP transient in the leaves of Aschallenged Brassica seedlings subjected to different modulators. Data signifies the mean ± standard error of five replicates. Bars followed by different letters are significantly different at p < 0.05 level according to Tukey test. (c). The leaf model representing phenomenological energy fluxes per excited cross section (CS) in the leaves of As-challenged Brassica seedlings subjected to different modulators. [(I) Control, (II) As, (III) As + Ca, (IV) As + Ca + EGTA, (V) As + Ca + EGTA + LaCl 3 , (VI) As + Ca + c-PTIO, (VII) As + Ca + L -NAME, (VIII) As + Ca + c-PTIO + L -NAME, (IX) As + SNP, (X) As + SNP + c-PTIO and (XI) As + SNP + L -NAME]. The relative value for the measured parameters is the mean of quintuplicates (n = 5). The width of arrow corresponds to the intensity of flux parameters; ABS/CS: absorption flux per CS, TR 0 /CS: trapped energy flux per CS, ET 0 /CS, electron transport flux per CS and DI 0 /CS: dissipated energy flux per CS. Circles embedded in circle (RC/CS) are percentage of active/inactive RCs, where white circles are representing reduced Q A RCs (active) and red circles non-reducing Q A RCs (inactive). RCs: reaction centres as described by Sitko et al. 62 . www.nature.com/scientificreports www.nature.com/scientificreports/ (that designates trapped exciton moves an electron into the ETC beyond Q A − ) and the quantum yield of electron transport (ФE o ) was detected (Fig. 4b), which consequently declined the pool size of Q A − (acceptor side of the PS II 17,26,27 ); suggesting lethargic flow of electrons from PS II to PS I and restriction of Q A − reoxidation (Q A − -Q A ), which could be associated with poor diffusion of PQ across the thylakoid membranes 28 . Indeed, the higher F o / F m designates that Q A reduction rate was much higher than its reoxidation rate by Q B and PS I activity under As + Ca + c-PTIO + l-NAME treatment. Increasing values for the quantum yield of energy dissipation (ФD o ) and dissipated energy flux (DI o /RC) (Fig. 4b) under As + Ca + c-PTIO + l-NAME and As + Ca + EGTA + LaCl 3 treatment, advocate that excess excitation energy was converted to thermal dissipation in order to maintain the energy balance between absorption and consumption, and thus minimize the potential of photo-oxidative damage 8 . The decrease in F m /F o parameter reflects the damaging effect of As on the structural integrity of the PS II RCs 29 . Further, the increased S m (refers the pool of electron transporters between PS II and the acceptor side of PS I) value under As + Ca + c-PTIO + l-NAME and As + Ca + EGTA + LaCl 3 implies that heterogeneity of PQ increased the electron donation capacity and Q A reduction on acceptor side of PS II, suggesting that As along with c-PTIO + l-NAME and EGTA + LaCl 3 decreased the total electron accepting capacity 30 .
The progressive drop in overall performance of PS II (PI ABS ) upon NO scavenger and NOS inhibitors' treatment could have resulted from the inactivity of RCs (F v /F o ), and these RCs then changes into 'energy sinks/heat sink' , that absorb light but were unable to store the excitation energy and dissipate total energy as heat/fluorescence as deduced by ФP o (F v /F m ), which consequently changes the average antenna size linked to each active RC (ABS/RC 14,31 ). Therefore, ABS/RC was found to increase under above situation because the reduced number of active RCs favour for increasing the necessary numbers of RC turnovers for complete reduction of the PQ pool (N) (Fig. 4b). The TR o /RC, which refers only to active RCs (Q A -Q A − ), was increased suggesting that either (i) all the Q A might have been reduced (Q A − ) but were not able to oxidize back ( was inhibited under Ca 2+ and NO scavenger/synthase inhibitor treated As stressed seedlings, so that Q A was unable to transfer electrons efficiently to Q B 31 . The DIo/RC, which reflects the ratio of the dissipation of untrapped excitation energy from all the RCs with respect to the number of active RCs, was increased ( Fig. 4b) due to higher energy dissipation from the active RCs under As toxicity 14 . Furthermore, increased F o /F v refers to damaging effects on OEC, which could be due to the decline in uptake of mineral nutrients like Mn, an important component of OEC 6 , as it suggested by Samborska et al. 24 that mineral nutrient deficiency tends to affect the fluorescence parameters variably.
The leaf model for phenomenological energy fluxes showed that As toxicity caused an increase in absorption flux per CS (ABS/CS), trapped energy flux per CS (TR 0 /CS), electron transport flux per CS (ET 0 /CS) and dissipated energy flux per CS (DI 0 /CS) along with the number of inactive/closed RCs (RC/CS) (Fig. 4c).
Calcium and NO, on the other hand alleviated the negative impact of As by restoring the structural attributes of PS II as favoured by increased The positive role of Ca 2+ on ФP o may due to the mineral ion homeostasis as discussed by Ahmad et al. 13 in tomato seedlings. Upon CaCl 2 and SNP application, improvement in F v parameter was strongly supported by a reduction in F o , which favoured imitation of the PS II acceptor side 30 . Furthermore, an improvement in the electron transport rate of the photosynthetic ETC was noticed as deduced from the high F v /F o values (Fig. 4b). The restoration in F m value upon CaCl 2 and SNP application suggested that either they might have increased Mn ion and extrinsic proteins of OEC, which affected the electron donation from water to PS II 32 or might have caused the conformational changes in D1 protein, thereby altering the properties of PS II electron acceptors 33 , which augmented PS II activity 6 . The NO might have improved the electron transport rate from OEC to D1 protein and gene expression belongs to core reaction center (Psb) of PSII complex such as psbA, psbB and psbC as argued by Chen et al. 14 in heat-stressed tall fescue leaves. Upon CaCl 2 and SNP addition, a sharp drop in ABS/RC, TR o /RC, ET o /RC and DI o /RC (Fig. 4b) specify that PS II apparatus was able to tackle the balance of energy fluxes for absorption, trapping and transport of electrons through active PS II RCs under As toxicity 14,34 thereby improving overall performance of PS II, as agreed with higher PI ABS values, which can also be supported by improved Area value under similar conditions (Fig. 4b).
ca 2+ and no improve antioxidant defense system to counteract As-induced oxidative stress and injuries. In our study, the excess accumulation of As in leaf tissues of Brassica seedlings exhibited severe oxidative stress as evident by enhanced ROS: O 2˙− and H 2 O 2 levels (Fig. 2). The As-induced ROS production caused oxidative injuries by peroxidizing lipid membranes together with loss of membrane integrity which were correlated with significant increase in electrolyte leakage and MDA equivalents levels (Figs. 2a-b). Further, CaCl 2 and SNP addition counteracted the As-induced loss in cell structure and function by decreasing ROS and the indices of damage as evident by increased NO and decreased As content (Figs. 1d and 3). The increased NO might have formed a less toxic peroxynitrite (ONOO − ) 16,35 or induced various ROS-scavenging enzyme activities like superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) (Figs. 5a-c) as was discussed by Siddiqui et al. 15 and Lu et al. 36 . Interestingly, c-PTIO and l-NAME application arrested the effect of Ca 2+ and NO in As-stressed seedlings, which was further confirmed by in-vivo staining for SOR, H 2 O 2 , lipid peroxidation and injury of plasma membrane integrity in leaf tissues (Figs. 2a,b). To overcome the deleterious effect of As, the activities of enzymatic antioxidants: SOD and CAT were found to increase, which were further enhanced upon CaCl 2 and SNP addition and more importantly, still an increment in the enzyme activities was noticed after c-PTIO, l-NAME, EGTA and LaCl 3 treatment (Figs. 5a,b). The increased SOD and CAT activities, which established the frontline enzymatic network that dismutate O 2˙− into H 2 O 2 consecutively into H 2 O, speeded the reduction in ROS accumulation but it was not sufficient to overcome the massive As-induced c-PTIO, l-NAME, EGTA and LaCl 3 mediated ROS accumulation; therefore higher ROS accumulation were still noticed (Figs. 2a,b) under these conditions. www.nature.com/scientificreports www.nature.com/scientificreports/ ca 2+ and no recover As-induced losses of ascorbate and glutathione contents and maintain redox status. Ascorbate and glutathione are the important redox buffering agents, therefore were analyzed in the present study to know the redox status of the cell. The current study showed that, As seriously impaired the ROS detoxification process by reducing the contents of AsA and GSH along with their redox states: AsA/DHA and GSH/GSSG ratios (Table 2). Upon addition of NO chelator (c-PTIO) and inhibitor (l-NAME), further reduction in the contents of AsA and GSH and ratios of AsA/DHA and GSH/GSSG was reported even in presence of Ca 2+ (Table 2). Moreover, the activity of APX, which reduces H 2 O 2 to DHA on the expense of AsA, was increased under similar conditions (Fig. 5c) indicating that although APX activity was efficient for H 2 O 2 detoxification; however, this was not enough to counteract H 2 O 2 induced damage, which is obvious from the results of MDA content and electrolyte leakage (Fig. 2). In contrast to this, DHAR activity which recycled DHA into AsA in presence of GSH, was found to decrease suggesting the insufficient regeneration of AsA from DHA; therefore much lower AsA content was noticed under As + Ca + EGTA + LaCl 3 and As + Ca + c-PTIO + L -NAME treatment and obviously, low AsA/DHA ratio was found ( Table 2). The DHAR enzyme is susceptible to high H 2 O 2 concentration 37 ; therefore upon addition of NO chelator/synthesis inhibitor, a remarkable inhibition in DHAR activity was noticed that might have altered the rate of AsA-GSH cycle, which is apparent from decreased AsA/DHA ratio ( Table 2). www.nature.com/scientificreports www.nature.com/scientificreports/ Calcium and NO, on the other hand restored and up-regulated DHAR activity to maintain the higher level of AsA; therefore higher AsA/DHA ratio was noticed (Table 2), as was also argued by Ahmad et al. 13 . Further, during the conversion of DHA into AsA, two molecules of GSH by donating electron converted into GSSG, which in-turn re-reduced into GSH by glutathione reductase (GR) enzyme 38 . In the present investigation, a significant reduction in the content of GSH and subsequent increment in GSSG was found upon As treatment, and effect was more intense under NO scavenger and synthesis inhibitor, thereby causing a severe reduction in GSH/GSSG ratio ( Table 2). The decrease in GSH content might either be due to decrease in GSH recycling rate or increase in its degradation rate, as excessive GSH needed during DHA to AsA conversion 38 . Interestingly, GR activity was increased upon c-PTIO and l-NAME treatment (Fig. 5e), which suggests that it was not sufficient to manage the huge GSH consuming effect of As, such as GSH conjugation for GST and PCs synthesis (As-PCs complex); therefore low GSH/GSSG ratio was obtained (Table 2). Contrastingly, Ca 2+ and NO improved the GSH pool by speeding up the rate of GSH recycling from GSSG, which is evident by increased GR activity (Fig. 5e), thus greater GSH/ GSSG ratio was obtained to encounter As toxicity, as also suggested by Ahmad et al. 13 and Praveen and Gupta 4 . ca 2+ and no rescue As-induced damage by up-regulating synthesis of thiol compounds. The thiol compounds: Cys, NPTs and PCs act as first barrier against As toxicity thereby reducing the injurious effect to plants 2,4 . In the present investigation, the content of Cys, NPTs and PCs were found to increase under As stress and further enhancement was noticed when EGTA + LaCl 3 or c-PTIO + L -NAME were supplemented to As + Ca stressed seedlings (Table 2), which might be due to their demand for Fe-S cluster of photosynthetic apparatus 39 , protein synthesis, stabilizing tertiary structures of protein, synthesis of GSH, hydroxymethyl-PCs and other low molecular weight compounds 3,36 . Moreover, high PCs content demands more GSH to counteract the stressed situation and also higher As accumulation stimulates NPTs for scavenging, by using available GSH pool; thus lower GSH content was obtained upon EGTA + LaCl 3 or c-PTIO + L -NAME supplementation to As + Ca treated seedlings (Table 2). Additionally, CaCl 2 and SNP treatment to As-stressed test seedlings also showed an improvement in the contents of thiol compounds thereby justifying their role in reducing As toxicity by promoting peptides and proteins to chelate metalloid, which is corroborated with earlier findings of Lu et al. 36

and Praveen and Gupta 4 in
Amaranthus hypochondriacus and Oryza sativa seedlings, respectively. Table S2 shows the correlation between treatments and tested parameters in the B. juncea L. seedlings. All the treatments affected all the tested parameters significantly. The results clearly showed that arsenic negatively affected the growth and other growth regulating parameters, while Ca and SNP showed positive correlation with growth. Further, addition of EGTA and LaCl 3 to As + Ca; while c-PTIO and l-NAME to As + SNP treated seedlings significantly declined the growth (as depicted by negative correlation). Further when c-PTIO and l-NAME individually as well as in combination were supplemented to As + Ca treated seedlings, more negative values for pearson correlation were observed thereby signifying that NO plays crucial role in Ca 2+ mediated signalling.

Materials and methods
Experimental plant, growth conditions and treatments. Healthy seeds of Brassica juncea were surface sterilized with 5% (v/v) sodium hypochlorite (NaOCl) for 5 min, rinsed with distilled water (DW) and left overnight in dark for 48 h by wrapping them in a wet muslin cloth. The germinated seeds were sown in plastic cups having acid sterilized sand and kept in darkness for two days at 25 ± 1 °C. Seedlings were then transferred and allowed to grow in plant growth chamber (CDR model GRW-300 DGe, Athens, Greece) having photosynthetically active radiation (PAR) of 150 μmol photons m −2 s −1 with 16:8 h day-night regime and 65-70% relative humidity at 22 ± 1 °C. Seedlings were irrigated with 50% Hoagland and Arnon 40 solution on alternate days. After 25 days, seedlings were uprooted and acclimatized in 50% Hoagland solution for 24 h. After that, three healthy www.nature.com/scientificreports www.nature.com/scientificreports/ and uniform sized seedlings were transferred in each plastic cup having 50 ml of Hoagland solution with or without different combinations of donor, scavenger and inhibitors (doses of As, Ca and NO were selected on the basis of screening experiments; the experimental plants showing phenotypic variation as per the treatments have been shown in Supplementary Fig. S1). All the chemicals were prepared in 50% Hoagland solution. In hydroponic system, following combinations were made: (i) control (nutrient solution alone), (ii) As 50 µM, (iii) As + Ca, (iv) As + Ca + EGTA, (v) As + Ca + EGTA + LaCl 3 , (vi) As + Ca + c-PTIO, (vii) As + Ca + l-NAME, (viii) As + Ca + l-NAME + c-PTIO, (ix) As + SNP, (x) As + SNP + c-PTIO and (xi) As + SNP + l-NAME. Sodium arsenate (Na 2 HAsO 4 .7H 2 O: a source of As; 50 µM), calcium chloride (CaCl 2 : a Ca 2+ donor; 12 mM), sodium nitroprusside (SNP: a NO donor; 100 µM), ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA: a Ca 2+ scavenger; 0.10 mM), 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO: a NO scavenger; 0.10 mM), lanthanum chloride (LaCl 3 : a plasma membrane Ca 2+ channel blocker; 0.10 mM) and N ω -nitro-L-arginine methyl ester hydrochloride (l-NAME: a NO synthase inhibitor; 0.10 mM) were used as metal stress, donor, scavenger and inhibitors. Each treatment was performed in five sets and were transferred in growth chamber under similar condition as mentioned above. The seedlings were aerated regularly with air bubbler to avoid hypoxia condition and were harvested after four days of the treatments to examine the Ca 2+ and NO-induced mechanisms in modulating As-induced responses.
Growth analysis. After four days of the treatments, growth of Brassica seedlings was analyzed by measuring fresh weight (FW), root length (RL) and shoot length (SL). The FW of the seedling was recorded by single pan digital balance (Model CA 223, Contech, India), while RL and SL were recorded by meter scale.  45 and the amount was calculated with the help of standard curve of NaNO 2 and H 2 O 2 , respectively. The estimation of indices of damage: lipid peroxidation (measured in terms of MDA equivalents content) and loss of membrane integrity (measured in terms of electrolyte leakage) in leaf tissues were adopted from Heath and Packer 46 and Gong et al. 47 , respectively. www.nature.com/scientificreports www.nature.com/scientificreports/ Histochemical detection. To perform the histochemical detection for SOR, H 2 O 2 , lipid peroxidation and loss of membrane integrity, nitro blue tetrazolium (NBT; 0.1%), 3,3′-diaminobenzidine (DAB; 1%), Schiff 's reagent and Evan's blue tests were carried out as suggested by Frahry and Schopfer 48 , Thordal-Christensen et al. 49 , Pompella et al. 50 and Yamamoto et al. 51 , respectively. After staining, leaves were bleached with boiling ethanol and photographed.
One unit (U) of activity of SOD is the amount of SOD required to inhibit 50% NBT, CAT (U) is equivalent to 1 nmol H 2 O 2 dissociated min −1 , APX (U) is 1 nmol ascorbate oxidized min −1 , DHAR (U) is 1 nmol DHA reduced min −1 and GR (U) is defined as 1 nmol NADPH oxidized min −1 . estimation of non-enzymatic antioxidants: ascorbate, glutathione and their redox status.
The estimation of contents of ascorbate: reduced (AsA) and oxidised (dehydroascorbate: DHA) was carried out in acidic solution, which is mainly based on Fe 3+ reduction into Fe 2+ following the method of Gossett et al. 56 . The estimation of glutathione: reduced (GSH) and oxidized (GSSG) contents were based on the sequential oxidation of GSH by 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) into trinitrobenzoic acid (TNB) as suggested by Brehe and Burch 57 .
Estimation of thiol compounds: cysteine, non-protein thiols and phytochelatins. Estimation of cysteine (Cys) content was done in presence of glacial acetic acid (GAA), acid ninhydrin and toluene following the method of Gaitonde 58 . The content of non-protein thiols (NPTs) was measured according to Ellman 59 in presence of Ellman's reagent. The amount of total phytochelatins (PCs) was calculated using the formula: total PCs = NPTs-total GSH 60 . estimation of nitric oxide (no) content. The NO content was determined using the method described by Zhou et al. 61 . The 500 mg fresh leaf tissues were homogenized in 50 mM acetic acid buffer (pH 3.6) containing zinc diacetate (4%) and centrifuged for 15 min at 4 °C. The absorbance of reaction mixture containing charcoal and 1 ml of Greiss reagent was monitoring at 540 nm and NO content in the mixture was calculated with the help of standard curve of NaNO 2 .

Statistical analysis.
The experiments were performed in quintuplicates and the results displayed in figures and tables are the means ± standard error of the average values obtained from quintuplicates (n = 5) of individual experiment to check the reproducibility of result. The results were statistically analyzed by one-way analysis of variance (ANOVA) using software 'SPSS 16.0. Tukey alpha test was performed for the mean separation for significant differences among the treatments at p < 0.05 significance level. Pearson correlation coefficient (r) test was also applied to test the significance of treatments.

conclusion
From the present study, it can be concluded that As inhibits growth of Brassica seedlings; Ca 2+ and NO on the other hand recovered the growth and growth related parameters. However, with the addition of Ca 2+ chelator (EGTA), plasma membrane Ca 2+ channel blocker (LaCl 3 ) as well as NO chelator (c-PTIO) and synthetase inhibitor ( L-NAME), the improvement in growth caused by Ca 2+ and NO was further arrested, which suggests that both are involve in signalling network. When NO chelator and synthase inhibitor were added to As-stressed CaCl 2 supplemented seedlings, a steep decline in growth promoting processes: photosynthetic activity, Chl a fluorescence, redox status (AsA/DHA and GSH/GSSG) of the cell was noticed even in presence of Ca 2+ , thereby signifying that physiological and biochemical attributes of Brassica seedlings are mostly regulated by intensive Ca 2+ mediated NO signalling.