Interactive effects of salicylic acid on enzymes of nitrogen metabolism in clusterbean (Cyamopsis tetragonoloba L.) under chromium(VI) toxicity

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Original Research Paper

Interactive effects of salicylic acid on enzymes of nitrogen metabolism in clusterbean (Cyamopsis tetragonoloba L.) under chromium(VI) toxicity Punesh Sangwan a,n, Vinod Kumar b, Deepika Gulati c, U.N. Joshi a a

Department of Biochemistry, C.C.S. Haryana Agricultural University, Hisar 125001, India Department of Biochemistry, G.B. Pant University of Agriculture and Technology, Pantnagar 163145, India c Department of Botany, C.C.S. Haryana Agricultural University, Hisar 125001, India b

art ic l e i nf o

a b s t r a c t

Article history: Received 27 March 2015 Accepted 1 June 2015

Chromium (Cr) toxicity is a major constraint to crop production. A pot experiment was conducted to examine the ameliorating effects of salicylic acid on Cr toxicity enzymes of nitrogen metabolism in clusterbean plant parts. For this study, salicylic acid (0.25 and 0.50 mM) was applied as foliar spray on control and Cr-stressed plants at 20, 35 and 55 days after sowing and its influence on Cr toxicity at vegetative, flowering and grain filling stages was examined. Cr treatment caused decrease in specific enzyme activity of nitrogenase, nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase and glutamate dehydrogenase in various plant organs at different growth stages with an increase in Cr(VI) levels from 0 to 2.0 mg Cr(VI) Kg
1 soil. However, exogenously added salicylic acid (0.25 and 0.50 mM) significantly alleviated Cr toxicity effects at all growth stages by increasing in enzymes specific activity. The treatments with 0.25 and 0.5 mM salicylic acid increased the specific activity of these enzymes in leaves, stem and root, when compared to those of Cr treatments alone. & 2015 Elsevier Ltd. All rights reserved.

Keywords: Salicylic acid Clusterbean Chromium phytotoxicity Nitrogen metabolism Amelioration

1. Introduction Heavy metal contamination in soil and water over prolonged time becomes hazardous to plants as their release from industrial units, metallurgical operations, mining activities and disposal of sewage leads to considerable reduction in crop yields (Chaffei et al., 2004). Among heavy metals, chromium (Cr) plays a major role in polluting environment as a consequence of above activities (Mehdi et al., 2003). It can exist in the environment in several oxidation states. Cr(VI) exists predominantly in the þIII and þVI oxidation states with Cr(þ III) as the most stable oxidation (Zayed and Terry, 2003). The Cr(VI) is extensively used in the industries such as electroplating, leather tanning, textile printing, textile preservation and metal finishing (Dixit et al., 2002). Cr element in very low amounts is useful for organisms but at higher concentrations, it is toxic and considered as a pollutant. Symptoms of Cr phytotoxicity include inhibition of seed germination or of early seedling development, reduction of root growth, leaf chlorosis and depressed biomass (Sharma et al., 1995). Cr affected physiological processes like seed germination, growth and nitrogen metabolism has also been reported in several studies n

Corresponding author. E-mail address: [email protected] (P. Sangwan).

(Seoccianti et al., 2006; Chidambaram et al., 2009; Akinci and Akinci, 2010; Sangwan et al., 2015). Nitrogen is considered to be a vital macronutrient for plants which determines growth, development and productivity of plants. Assimilation of nitrogen as NH+4 plays an important role in plant growth and development which determines yield and quality of grains (Balestrasse et al., 2003). Cr (VI) treatments adversely affected the growth of forage sorghum as a result of its interference with photosynthetic pigments and key enzymes of NH+4 assimilation (Kumar and Joshi, 2008). Cr(VI) treatments were also reported to affect the enzymes of nitrogen metabolism in clusterbean (Sangwan et al., 2014). Therefore, strategies are needed to alleviate the adverse effects of Cr toxicity, and also to decrease the Cr level in crops which may be helpful to minimize health risks and improvement of plant growth and development (Sangwan et al. 2013). Plant hormones have been reported as active members of signal transduction cascade involved in the plant stress responses responses (Mori and Schroeder, 2004). Exogenous application of plant hormones has emerged as a potential strategy to alleviate adverse effects of various abiotic stresses including heavy metal (Chakrabarti and Mukherji, 2003; Tuna et al., 2008; Gangwar et al., 2011; Sangwan et al., 2015). Salicylic acid (SA), an endogenous plant growth regulator, is involved in regulation of a wide range of metabolic and physiological responses in plants and thereby it affects growth and developmental processes. The importance and physiological

http://dx.doi.org/10.1016/j.bcab.2015.06.001 1878-8181/& 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: Sangwan, P., et al., Interactive effects of salicylic acid on enzymes of nitrogen metabolism in clusterbean (Cyamopsis tetragonoloba L.) under chromium(VI) toxicity. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.06.001i

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mechanism of SA in amelioration of abiotic stresses have been intensively studied (Zhou et al., 2009; Mohsenzadeh et al., 2011; Moradkhani et al., 2012). However, the possible role of salicylic acid application in alleviating Cr toxicity has not been explored yet. Therefore, it would be interesting to know more about the impact of exogenous application of SA in plants. Therefore, the present study was designed to explore and investigate the ameliorative responses of SA supplementation under Cr(VI) toxicity in clusterbean, a multipurpose fodder crop, which has recently assumed great industrial importance due to presence of gum, which is used extensively in paper, mining, explosive, food, pharmaceuticals, cosmetics, textiles and oil industries.

2. Material and methods 2.1 Chemicals, reagents and soil The chemicals and reagents used during the present investigation were of analytical grade. A nutrient deficient loamy sand soil from Regional Research Station, Gangwa block of Hisar district was used in the present study. The characteristics of soil were: pH (1:2) 8.50; organic carbon, 0.22; N, 4.0 mg kg
1 soil; P, 13.0 mg kg
1 soil; K, 163 mg kg
1 soil; Zn2 þ , 0.61 mg kg
1 soil; Fe2 þ , 0.9 mg kg
1 soil; Cu2 þ , 0.18 mg kg
1 soil; Mn2 þ , 3.6 mg kg
1 soil; EC, 1.5; CaCO3 3.5; Cr2 þ , 0.01 mg kg
1 soil; texture–sandy loam.

2.4 Protein estimation The soluble protein in the sample extract was precipitated by 20% TCA, centrifuged and resultant residue was dissolved in 0.1 N sodium hydroxide (NaOH) solution for its estimation following method of Lowry et al. (1951). 2.5 Chromium estimation One gram powdered sample was digested with 15 ml of di-acid mixture (4 HNO3:1 HClO4) in a conical flask by heating on hot plate in open space till clear white precipitates settled down at the bottom of the conical flask. The precipitates were then dissolved in 1 N HCl prepared in double glass distilled water, filtered and volume of the filtrate was made 50 ml with double glass distilled water. The content of Cr was estimated from the extract prepared by Atomic Absorption Spectrophotometer (Perkin-Elmer Model 2380). 2.6 Statistical analysis A two-factorial ANOVA in complete randomized block design was used to confirm the validity of the data using OPSTAT software available on CCSHAU website home Page (http://hau.ernet.in/op stat.html). The values used in graphs are mean of three replicates and shown as 7standard error.

2.2 Plant growth and environmental conditions

3. Results

Seeds of clusterbean (Cyamopsis tetragonoloba (L.) Taub.) cv HG 2-20 were procured from Forage Section, Department of Genetics and Plant Breeding, C.C.S. Haryana Agricultural University, Hisar and raised in pots filled with 5 kg of sandy loam soil in a naturally lit net house. Ten pots were used for each treatment. Two concentrations of SA i.e., 0.25 and 0.50 mM were prepared for foliar spray and 5 treatments of SA and Cr(VI) were made as follows; T0 ¼ 0 mg Cr(VI) Kg
1 soil; T1 ¼2.0 mg Cr(VI) Kg
1 soil; T2 ¼(0 mg soil þ0.25 mM SA); T3 ¼(2.0 mg Cr(VI) Kg
1 Cr(VI) Kg
1 soilþ 0.25 mM SA); T4 ¼ (0 mg Cr(VI) Kg
1 soil þ0.50 mM SA); T5 ¼ (2.0 mg Cr(VI) Kg
1 soil þ0.50 mM SA). The seeds were surface sterilized with mercuric chloride and after proper washing with distilled water, inoculated with Rhizobium culture. Equal amount of nutrient solution was supplied at weekly interval to each pot. The plants were irrigated with equal quantities of tap water as and when required. Foliar spray of SA was given at 20, 35 and 55 days after sowing. Plant samples from each treatment were collected at vegetative (30 DAS), flowering (50 DAS) and grain filling stages (65 DAS). The temperature and relative humidity during the experiment ranged from 11.0 to 35.6 °C and 34.5% to 95.2%, respectively. The light intensity ranged from 36,100 to 84,000 lx.

3.1 Effect of SA foliar spray on nitrogen metabolism enzymes Foliar spray of 0.25 mM SA (T3) on 2.0 mg Cr(VI) kg
1 soil stressed clusterbean plants leads to an increase in NR specific activity in leaves, stem and roots (Fig. 1). It was noticed that specific activity of NR increased from 0.016 to 0.018 units in leaves, 0.030 to 0.033 units in stem and 0.133 to 0.138 units in root at 50 DAS, respectively. In similar way, specific activity of NR also increased from 0.016 to 0.019 units in leaves, 0.030 to 0.037 units in stem and 0.133 to 0.146 units in root with spray of 0.50 mM SA (T5) over the 2.0 mg Cr(VI) kg
1 soil treated plants at 50 DAS, respectively (Fig. 1).

2.3 Enzyme activity measurement Specific activity of nitrogen metabolism enzymes i.e. Nitrogenase (E.C.1.7.99.2), nitrate reductase (NR; E.C.1.6.6.1), nitrite reductase (NiR; E.C. 1.7.7.1), glutamine synthetase (GS; E.C.6.3.1.2), glutamate dehydrogenase (GDH; E.C.1.4.1.4) and glutamate synthase (GOGAT; E.C.1.4.1.14) in leaves, shoot and root (Nitrogenase activity in nodules only) at different growth stages were measured by the standard methods as described in Sangwan et al. (2014). All observations were measured up to 2.0 mg kg
1 soil because plants treated with more than 2.0 mg Cr(VI) kg
1 soil concentration did not survive 20 days after sowing.

Fig. 1. Effect of SA foliar spray for amelioration of Cr(VI) toxicity on nitrate reductase activity in clusterbean plant parts at different stages of growth.

Please cite this article as: Sangwan, P., et al., Interactive effects of salicylic acid on enzymes of nitrogen metabolism in clusterbean (Cyamopsis tetragonoloba L.) under chromium(VI) toxicity. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.06.001i

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Fig. 4. Effect of SA foliar spray for amelioration of Cr(VI) toxicity on glutamate dehydrogenase activity in clusterbean plant parts at different stages of growth. Fig. 2. Effect of SA foliar spray for amelioration of Cr(VI) toxicity on nitrite reductase activity in clusterbean plant parts at different stages of growth.

The effects of Cr(VI) and SA on NiR specific activity at all the stages of growth are shown in Fig. 2. Foliar spray of 0.25 mM SA on Cr(VI) treated plants enhanced NiR specific activity of leaves, stem and roots of clusterbean plant. Its specific activity increased from 0.167 to 0.171 units in leaves, 0.329 to 0.336 units in stem and 0.375 to 0.397 units in root at 50 DAS, respectively, with spray of 0.25 mM SA on 2.0 mg Cr(VI) kg
1 soil treated plants (T3). Similar trend was also observed when spray of 0.50 mM SA was applied on 2.0 mg Cr(VI) kg
1 soil treated plants (T5), its specific activity increased from 0.167 to 0.184 units in leaves, 0.329 to 0.359 units in stem and 0.375 to 0.429 units in root at 50 DAS, respectively (Fig. 2). Specific activity of GS in leaves, stem and roots of clusterbean plant increased at all the stages of growth (i.e. 30, 50 and 65 DAS) when SA was sprayed on Cr(VI) treated plants (Fig. 3). The specific activity of this enzyme in leaves under study increased from 8.73 to 10.94, 12.34 to 14.85 and 11.00 to 14.32 units at 30, 50 and 65 DAS, respectively, with spray of 0.25 mM SA on 2.0 mg Cr(VI) kg
1 soil treated plants (T3). A similar trend for GS specific activity was observed in stem and root at all stages of growth i.e. GS specific activity increased from 12.70 to 13.30, 14.76 to 15.45 and 13.84 to

Fig. 3. Effect of SA foliar spray for amelioration of Cr(VI) toxicity on Glutamine synthetase activity in clusterbean plant parts at different stages of growth.

14.88 units in stem and in roots from 7.32 to 8.44, 10.10 to 15.19 and 8.97 to 13.10 units at 30, 50 and 65 DAS, respectively, with foliar spray of 0.25 mM SA (Fig. 3). Cr(VI) stress, significantly decreased GDH specific activity in leaves, stem and root of clusterbean plant at all stages of growth. However, GDH specific activity in leaves, stem and root were significantly increased, when 0.25 mM and 0.50 mM SA was sprayed on Cr(VI) treated plants (2.0 mg(VI) kg
1 soil) at 20, 35 and 55 DAS (Fig. 4). Foliar spray of 0.50 mM SA (T5) resulted into increase in GDH specific activity from 0.885 to 1.034, 1.179 to 1.420 and 0.683 to 0.929 units in leaves, in stem from 2.608 to 2.733, 3.522 to 3.644 and 2.916 to 3.253 units and in roots from 1.374 to 1.606, 2.383 to 2.687 and 1.270 to 1.536 units at 30, 50 and 65 DAS, respectively (Fig. 4). Cr(VI) addition significantly decreased the specific activity of GOGAT. But foliar spray of SA (0.25 mM and 0.50 mM) at 20, 35 and 55 DAS showed an increase in GOGAT specific activity in leaves, stem and roots at all stages of growth in clusterbean plants (Fig. 5). Spray of 0.25 mM SA in 2.0 mg Cr(VI) kg
1 soil treated plants (T3) resulted into increase in GOGAT specific activity in leaves from 0.272 to 0.296, 0.737 to 0.755 and 0.544 to 0.658 units at 30, 50 and 65 DAS, respectively. In stem, it increased from 1.266

Fig. 5. Effect of SA foliar spray for amelioration of Cr(VI) toxicity on Glutamate synthase activity in clusterbean plant parts at different stages of growth.

Please cite this article as: Sangwan, P., et al., Interactive effects of salicylic acid on enzymes of nitrogen metabolism in clusterbean (Cyamopsis tetragonoloba L.) under chromium(VI) toxicity. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.06.001i

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higher than in leaves and stem. SA significantly decreased bioaccumulation of Cr in leaves, stem and root. In roots, accumulation of Cr decreased with increasing SA concentration from 0.25 to 0.50 mM (Table 1). In leaves Cr concentration decreased from 1.16 to 1.13, 2.41 to 2.37 and 3.27 to 3.24 mg g
1 dry weight at 30, 50 and 65 DAS, respectively by foliar spray of 0.25 mM SA (T4). In stem, it decreased from 2.72 to 2.69, 3.26 to 3.20 and 3.82 to 3.79 mg g
1 dry weight at 30, 50 and 65 DAS, respectively by foliar spray of 0.25 mM SA on Cr (2.0 mg Cr(VI) kg
1 soil) treated plants and in roots it decreased from 7.76 to 7.70, 8.47 to 8.43 and 10.64 to 10.59 mg g
1 dry weight at 30, 50 and 65 DAS, respectively by foliar spray of SA (0.25 mM) on Cr(VI) treated plants. Foliar spray of 0.50 mM SA (T6) on Cr treated plants (2.0 mg Cr(VI) kg
1 soil) also decreased the accumulation of Cr content in leaves from 2.41 to 2.34 mg g
1 dry weight, in stem from 3.26 to 3.16 mg g
1 dry weight and in roots from 8.47 to 8.34 mg g
1 dry weight at 50 DAS. The decrease in Cr concentration was more with foliar spray of 0.50 mM SA as compared to 0.25 mM SA (Table 1). Fig. 6. Effect of SA foliar spray for amelioration of Cr(VI) toxicity on specific nodule nitrogenase activity in nodules of clusterbean plant at different stages of growth.

to 1.418, 1.930 to 2.123 and 1.143 to 1.328 units and in roots from 1.317 to 1.489, 1.860 to 2.080 and 1.423 to 1.519 units in roots at 30, 50 and 65 DAS, respectively by foliar spray of 0.25 mM SA. When, 0.50 mM SA was sprayed, it also resulted into increase in specific activity from 0.737 to 0.762 units in leaves, 1.930 to 2.476 units in stem and 1.860 to 2.198 units in roots (T5) at 50 DAS, respectively (Fig. 5). In clusterbean plants, 2.0 mg Cr(VI) kg
1 soil decreased specific nodule nitrogenase activity (SNA) as compared to that obtained at its normal levels in control plants (Fig. 6). When 0.25 mM and 0.50 mM SA was sprayed on 2.0 mg Cr(VI) kg
1 soil treated plants, the SNA increased, with maximum value in case of foliar spray of 0.50 mM SA. With spray of 0.25 mM SA (T3), the SNA increase by 29.93%, 10.13% and 11.3% as compared to their respective Cr(VI) treated plants at 30, 50 and 65 DAS, respectively. Similarly, with spray of 0.50 mM SA (T5), increase in SNA was 37.13%, 19.53% and 16.1% at 30, 50 and 65 DAS, respectively, as compared to 2.0 mg Cr (VI) kg
1 soil treated plants without spray of SA (Fig. 6). 3.2 Effect of foliar spray of salicylic acid on CR content Cr accumulation in leaves, stem and root of clusterbean plants is shown in Table 1. Cr accumulation in roots was substantially Table 1 Effect of treatments for amelioration of Cr(VI) toxicity on chromium content (mg g
1 dry weight) in clusterbean plant parts at different stages of growtha. Treatments

Days after sowing (DAS) Leaves

Stem

Root

30

50

65

30

50

65

30

50

65

T1 T2 T3 T4 T5 T6

0 1.16 0 1.13 0 1.08

0 2.41 0 2.37 0 2.34

0 3.27 0 3.24 0 2.19

0 2.72 0 2.69 0 2.64

0 3.26 0 3.20 0 3.16

0 3.82 0 3.79 0 3.75

0 7.76 0 7.70 0 7.64

0 8.47 0 8.43 0 6.39

0 10.64 0 10.59 0 10.52

SE (m) CD at 5%

A 0.01 0.03

B 0.03 0.08

AB 0.05 0.15

A 0.01 0.03

B 0.03 0.10

AB 0.06 0.17

A 0.03 0.09

B 0.08 0.23

AB 0.14 0.39

a Each value is the mean of five replicates; A ¼ Treatment, B ¼ Stages, A  B ¼ Interaction.

4. Discussion Importance of various enzymes of nitrogen metabolism and effect of Cr toxicity on these enzymes has been discussed earlier (Kumar and Joshi, 2008). Application of SA as seed pre-treatment and foliar spray was done to study its effect on enzymes of nitrogen metabolism under Cr toxicity. Its application at a concentration of 0.25 mM and 0.50 mM was inducing and led to increased NR specific activity in Cr stressed plants. It might be due to either NR activity was induced and/or degradation of enzyme was prevented. It might induce NR synthesis by mobilization of intracellular NO−3 , and provide protection to in vivo NR degradation in absence of NO−3 (Singh et al., 1997). Singh and Chaturvedi (2012) observed that SA at concentration of 10 mM was inducing NR activity. According to Fariduddin et al. (2003), low concentrations of SA increased NR activity while higher concentrations were inhibitory in Brassica juncea. The concentration of SA might play an active role in such a regulation, where the lower concentration favored an increase in the NR protein and higher quantity of SA decreased it by affecting the balance between its synthesis/activation and degradation/inactivation (Jain and Srivastava, 1981). The another appropriate reason that seems to explain the SA mediated elevation in the activity of NR is that it stabilizes the membrane structure and its fluidity, which could have facilitated the increased uptake of nutrients including nitrate thereby increasing its content in the roots which also acts as an inducer of NR (Campbell, 1999). The increase in the content of nitrates and thereby activity of NR due to exogenous SA treatment under normal growth conditions was also reported by Hayat et al. (2005, 2012). When SA was applied to the plants, NR activities also increased significantly in the leaves of salt-treated Artemisia annua plants (Aftab et al., 2011). The phytohormones are known to activate the establishment of nitrogen fixing mature nodules by the joint action of bacteria and host (Hopkins, 1995). Exogenous application of SA favors the legume-Rhizobium symbiosis thereby leading to the enhanced establishment and development of nodules which is expressed in terms of increased number and dry mass of nodules (Hayat et al., 2010). The healthy nodule development led to increased leghemoglobin content which eventually leads to increased activity of nitrogenase, an oxygen labile enzyme which is protected by leghemoglobin. All these processes together are thought to bring about an efficient nitrogen fixation and increased nitrogen content into nodules. Nitrogenase specific activity was also noticed to be increased with application of SA in present study on clusterbean plants

Please cite this article as: Sangwan, P., et al., Interactive effects of salicylic acid on enzymes of nitrogen metabolism in clusterbean (Cyamopsis tetragonoloba L.) under chromium(VI) toxicity. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.06.001i

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under Cr toxicity. According to Cooper and Scherer (2012), nitrogen fixing potential of each nodule is determined by three main factors: (a) photosynthates availability, which is maintained by enhanced photosynthesis, (b) low oxygen supply to the bacteroid, which at excessive level inhibits nitrogenase, is maintained by restricted O2 supply by the mediation of increase in leghemoglobin and (c) export of fixed nitrogen in the form of ammonia. Ammonia then diffuses across the peribacteroid membrane to the host cytosol by simple diffusion (Udvardi and Day, 1990). This ammonia is further metabolized in cytosol by two operative enzymatic systems (a) GDH and (b) GS/GOGAT. GDH causes direct reductive amination of α-ketoglutarate, giving glutamate, whereas, GS catalyses the addition of NH+4 to glutamate forming corresponding amide, glutamine. Transfer of amide group from glutamine to a molecule of α-ketoglutarate led to synthesis of glutamate again, Incorporation of NH+4 into glutamate is brought about by successive and highly-regulated actions of GS, GOGAT and GDH (Chaffei et al., 2004; Masclaux-Daubresse et al., 2006). SA, due to its action at transcriptional and/or translational level (Hayat et al., 2010), might have accelerated the synthesis and thereby activity of GDH, GS and GOGAT. Similarly, auxins are also known to enhance the activity of GS, GOGAT and GDH (Hayat et al., 2009, 2012). Hence, it might be possible that lower concentration of SA might enhance the release of auxins and increased the activity of GS, GOGAT and GDH enzymes in current study. Generally, Cr accumulation and toxic effect of Cr differ significantly in different plant species, cultivar, developmental stage and the experimental method. The heavy metals accumulation in cells is dependent on membrane integrity and cation transporter activity. The effect of SA on Cr accumulation is most probably indirect where possible causes of SA-induced decrease in Cr accumulation include: (a) deactivation of some divalent cation transporter capable to bind Cr (Hall and Williams, 2003), (b) enhanced Cr ion immobilization in leaf and especially root, intercellular spaces (Kevresan et al., 2003), or in the vacuole. Therefore, it could be credited to the enhancement of cation uptakes (Ca, Mg and Fe) in Cr-treated plants (Belkhadi et al., 2010).

5. Conclusion In conclusion, Cr(VI) alleviation potential of exogenous application of salicylic acid as improved activities of different enzymes of nitrogen metabolism at all growth stages has been observed in this study. It may have further implications in designing and conducting similar studies as well as understanding the detailed mechanism of this ameliorative process.

Conflict of interests The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgment The authors gratefully acknowledge Director Research, and Head, Department of Biochemistry for providing necessary infrastructural facility. Lead author is grateful to Indian Council of Agricultural Research (ICAR (F.N0.:7(1)/2011-Exam Cell)), for providing financial assistance in the form of ICAR-SRF. References Aftab, T., Khan, M.M.A., da Silva, J.A.T., Idrees, M., Naeem, M., 2011. Role of salicylic

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Please cite this article as: Sangwan, P., et al., Interactive effects of salicylic acid on enzymes of nitrogen metabolism in clusterbean (Cyamopsis tetragonoloba L.) under chromium(VI) toxicity. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.06.001i