Advertisement

Indian Journal of Plant Physiology

, Volume 23, Issue 2, pp 342–351 | Cite as

Antioxidant responses and isoenzyme activity of hydroponically grown safflower seedlings under copper stress

  • Sanskriti Gautam
  • Sameer S. Bhagyawant
  • Nidhi Srivastava
Original Article
  • 43 Downloads

Abstract

Safflower is an oilseed rabi crop. Continuous use of use of copper (Cu) containing fungicides and pesticides, due to high susceptibility of this crop of fungal attack has increased the accumulation of Cu in the soil. To investigate the effect of extra accumulated Cu on this particular crop, safflower seeds were germinated on different concentration of Cu (25, 50 and 100 µM) present in Hoagland’s solution (hydroponic) along with control (0.5 µM of Cu) and seedlings were harvested after 10 and 20 days of Cu treatment. The concentration of alpha-tocopherol was high in leaves as compared to roots in presence of Cu stress. The content of vitamin C was increased in both leaves and roots. In 10 and 20 day leaves, the CAT activity has been decreased in all the treatments. In 10 day roots, the CAT activity was increased but decreased abruptly in 20 day harvested roots. Isoenzymatic pattern of CAT showed three bands of CAT (CAT 1, CAT 2 and CAT 3) in control in 10 day leaves while CAT 1 was diminished in lane 2, 3 and 4. In 20 day leaves, we observed only one bands of CAT (CAT 2) in lane 1 (control) while CAT 1 was appeared at high concentration of Cu. We observed no more significant changes in the CAT isoforms in 10 and 20 day roots. The GPX activity was increased in both the day’s collected samples (leaves and roots). The analysis of GPX isoenzymes revealed no significant changes in isoenzymatic pattern but their band’s intensity was increased with an elevated metal treatment. In 20 day harvested leaves, we observed two bands of isoenzyme in lane 1 and four bands of GPX in lane 2, 3, 4 along with their increased intensities. In 10 day roots, we observed two band of GPX in all the treatment with elevated intensities as compared to control (lane 1). Similarly we observed two bands of GPX in all the treatments and control. But the intensity of bands was increased till lane 3 but in 20 day rootsat highest treatment (100 µM) the intensity of bands was decreased. The SOD activity was reduced in 10 and 20 day leaves. In 10 day roots, it was augmented but reduced at 100 µM as compared to control (100%). In 20 day harvested roots, the activity was decreased by 1.30, 11.11 and 15.68% with increased Cu concentration than control (100%). Three isoenzymes of SOD were appeared in 10 day leaves with more or less same intensity. While in 20 day leaves ex-plant has shown SOD 1, 2 and 3 activities. In 10 day treated roots SOD 1 with increasing intensity was appeared in lane 3 and 4 than control (lane 1) while SOD 2 was almost constantly present in all four lanes. Similarly in 20 day root ex-plant, SOD 1 has been observed with decreasing intensity in lane 2, 3 and 4 than control (lane 1). To cope up with the Cu stress, the antioxidant enzymes such as GPX and SOD were actively involved in safflower seedlings.

Keywords

Antioxidants enzymes Copper Isoenzymes Safflower 

Abbreviations

ROS

Reactive oxygen species

CAT

Catalase

POD

Peroxidase

GPX

Guaiacol peroxidase

SOD

Superoxide dismutase

NBT

Nitrobluetetrazolium

SDS

Sodium dodecyl sulfate

FW

Fresh weight

Notes

Acknowledgements

Authors would like to acknowledge Prof. Aditya Shastri, Vice Chancellor, Banasthali Vidyapith, for providing the facilities in the Department of Bioscience and Biotechnology for conducting the experiments. Also, we are grateful to Dr. K. Anjani, Principal Scientist, Directorate of Oilseed Research Institute, Hyderabad for providing the seeds to carry out the research study.

References

  1. Adrees, M., Ali, S., Rizwan, M., Ibrahim, M., Abbas, F., Farid, M., et al. (2015). The effect of excess copper on growth and physiology of important food crops: A review. Environment Science and Pollution Research, 22, 8148–8162.CrossRefGoogle Scholar
  2. Ahmad, P., Jaleel, C. A., Azooz, M. M., & Nabi, G. (2009). Generation of ROS and non-enzymatic antioxidants during abiotic stress in plants. Botany Research International, 2(1), 11–20.Google Scholar
  3. Al Chami, Z., Amer, N., Al Bitar, L., & Cavoski, I. (2015). Potential use of Sorghum bicolor and Carthamus tinctorius in phytoremediation of nickel, lead and zinc. International Journal of Environmental Science and Technology, 12(12), 3957–3970.CrossRefGoogle Scholar
  4. Alscher, R. G., Erturk, N., & Heath, L. S. (2002). Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. Journal of Experimental Botany, 53(372), 1331–1341.CrossRefPubMedGoogle Scholar
  5. Awasthy, J. M., Greeshma, G. M., Murukan, G., Krishan, V. G. M., & Murugan, K. (2015). Isozyme pattern of antioxidant enzymes and DNA damage in Octoblepharum albidum hedw. A bryophyte responses to cadmium and copper stress. Journal of Biological and Scientific Opinion, 3(1), 13–20.CrossRefGoogle Scholar
  6. Azeez, M. O., Adesanwo, O. O., & Adepetu, J. A. (2015). Effect of copper (Cu) application on soil available nutrients and uptake. African Journal of Agricultural Research, 10(5), 359–364.CrossRefGoogle Scholar
  7. Azooz, M. M., Abou-Elhamd, M. F., & Al-Fredan, M. A. (2012). Biphasic effect of copper on growth, proline, lipid peroxidation and antioxidant enzyme activities of wheat (Triticum aestivum cv. Hasaawi) at early growing stage. Australian Journal of Crop Science, 6(4), 688–694.Google Scholar
  8. Bano, S., Ashraf, M., & Akram, N. A. (2014). Salt stress regulates enzymatic and nonenzymatic antioxidative defense system in the edible part of carrot (Daucus carota L.). Journal of Plant Interactions, 9(1), 324–329.CrossRefGoogle Scholar
  9. Beauchamp, C., & Fridovich, I. (1971). Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry, 44(1), 276–287.CrossRefPubMedGoogle Scholar
  10. Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Analytical Biochemistry, 239(1), 70–76.CrossRefPubMedGoogle Scholar
  11. Bhaduri, A. M., & Fulekar, M. H. (2012). Antioxidant enzyme responses of plants to heavy metal stress. Reviews in Environmental Science & Biotechnology, 11, 55–69.CrossRefGoogle Scholar
  12. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate the antioxidant activity. LWT—Food Science and Technology, 28(1), 25–30.CrossRefGoogle Scholar
  13. Cakmak, I., & Horst, W. (1991). Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase and peroxidase activities in root tip of soybean (glysin max). Journal of Plant Physiology, 83, 463–468.CrossRefGoogle Scholar
  14. Cosgrove, D. J. (1997). Assembly and enlargement of the primary cell wall in plants. Annual Review of Cell Biology, 13, 171–201.CrossRefGoogle Scholar
  15. Dhindsa, R. S., Plumb-Dhindsa, P., & Thorpe, T. A. (1980). Leaf senescence correlated with increased levels of membrane permeability and lipid-peroxidation and decreased levels of superoxide dismutase and catalase. Journal of Experimental Botany, 32(1), 93–101.CrossRefGoogle Scholar
  16. Filek, M., Keskinen, R., Hartikainen, H., Szarejko, I., Janiak, A., Miszalski, Z., et al. (2008). The protective role of selenium in rape seedlings subjected to cadmium stress. Plant Physiology, 165(8), 833–844.CrossRefGoogle Scholar
  17. Fridovich, I. (1986). Biological effects of superoxide radical. Archives of Biochemistry and Biophysics, 247, 1–11.CrossRefPubMedGoogle Scholar
  18. Gao, S., Yan, R., Cao, M., Yang, W., Wang, S., & Chen, F. (2008). Effects of copper on growth, antioxidant enzymes and phenylalanine ammonia-lyase activities in Jatropha curcas L. seedling. Plant Soil and Environment, 54(3), 117–122.CrossRefGoogle Scholar
  19. Gautam, S., Anjani, K., & Srivastava, N. (2016). In vitro evaluation of excess copper affecting seedlings and their biochemical characteristics in Carthamus tinctorius L. (variety PBNS-12). Physiology and Molecular Biology of Plants.  https://doi.org/10.1007/s12298-016-0339-1.PubMedPubMedCentralCrossRefGoogle Scholar
  20. Gautam, S., Bhagyawant, S. S., & Srivastava, N. (2014). Detailed study on therapeutic properties, uses and pharmacological applications of safflower (Carthamus tinctorius L.). International Journal of Ayurveda and Pharma Research, 2(3), 1–12.Google Scholar
  21. Halliwell, B., & Gutteridge, J. M. C. (1999). Free radicals in biology and medicine (4th ed.). New York: Oxford University Press.Google Scholar
  22. Hanumantharaya, L., Balikai, R. A., Mallapur, C. P., Venkateshalu., & Kumar, C. J. (2008). Integrated pest management strategies against safflower aphid, Uroleucon compositae (Theobald). In 7th International safflower conference, Wagga Wagga, Australia.Google Scholar
  23. Jaishee, N., & Chakraborty, U. (2014). Evaluation of in-vitro antioxidant activities of Pteris biaurita L. International Journal of Pharmacy and Pharmaceutical Sciences, 6(2), 413–421.Google Scholar
  24. Jaleel, C. A. (2009). Non-enzymatic antioxidant changes in Withania somnifera with varying drought stress levels. American-European Journal of Scientific Research, 4(2), 64–67.Google Scholar
  25. Jayaraman, J. (1996). Laboratory manual in biochemistry fifth reprint. New Delhi: New Age International Ltd.Google Scholar
  26. Khatun, S., Ali, M. B., Hahn, E. J., & Paek, K. Y. (2008). Copper toxicity in Withania somnifera: Growth and antioxidant enzymes responses of in vitro grown plants. Environmental and Experimental Botany, 64(3), 279–285.CrossRefGoogle Scholar
  27. Kochhar, S., Kochhar, V. K., & Khanduja, S. D. (1979). Changes in the pattern of isoperoxidases during maturation of grape berries cv gulabi as affected by ethephon phosphonic acid. American Journal of Enology and Viticulture, 30(4), 275–277.Google Scholar
  28. Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of head bacteriophage T4. Nature, 227(5259), 680–685.CrossRefPubMedGoogle Scholar
  29. Lagrimini, L. M. (1991). Wound-induced deposition of polyphenols in transgenic plants overexpressing peroxidase. Plant Physiology, 96, 577–583.CrossRefPubMedPubMedCentralGoogle Scholar
  30. Li, Q., Li, Y., Li, C., & Yu, X. (2012). Enhanced ascorbic acid accumulation through overexpression of dehydroascorbate reductase confers tolerance to methyl viologen and salt stresses in tomato. Czech Journal of Genetics and Plant Breeding, 48, 74–86.CrossRefGoogle Scholar
  31. Li, S., Zhang, G., Gao, W., Zhao, X., Deng, C., & Lu, L. (2015). Plant growth, development and change in GSH level in safflower (Carthamus tinctorius L.) exposed to copper and lead. Archives of Biological Sciences, Belgrade, 67(2), 385–396.CrossRefGoogle Scholar
  32. Lin, C. C., & Kao, C. H. (2000). Effect of NaCl stress on H2O2 metabolism in rice leaves. Journal of Plant Growth Regultion, 30, 151–155.CrossRefGoogle Scholar
  33. Lombardi, L., & Sebastiani, L. (2005). Copper toxicity in Prunus cerasifera: Growth and antioxidant enzymes responses of in vitro grown plants. Plant Science, 168(3), 797–802.CrossRefGoogle Scholar
  34. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. The Journal of Biological Chemistry, 193, 265–275.PubMedGoogle Scholar
  35. Malar, S., Vikram, S. S., Favas, P. J., & Perumal, V. (2014). Lead heavy metal toxicity induced changes on growth and antioxidative enzymes level in water hyacinths [Eichhornia crassipes (Mart.)]. Botanical Studies, 55, 54.CrossRefPubMedPubMedCentralGoogle Scholar
  36. Mishra, S., Srivastava, S., Tripathi, R. D., Govindarajan, R., Kuriakose, S. V., & Prasad, M. N. V. (2006). Phytochelatin synthesis and response of antioxidants antioxidants during cadmium stress in Bacopa monnieri L. Plant Physiology and Biochemistry, 44(1), 24–37.CrossRefGoogle Scholar
  37. Munné-Bosch, S., & Alegre, L. (2002). The function of tocopherol and tocotrienols in plants. Critical Review in Plant Sciences, 21, 31–57.CrossRefGoogle Scholar
  38. Namdjoyan, S. H., Khavari-Nejad, R. A., Bernard, F., Nejadsattari, T., & Shaker, H. (2011). Antioxidant defense mechanisms in response to cadmium treatments in two safflower cultivars. Russian Journal of Plant Physiology, 58(3), 467–477.CrossRefGoogle Scholar
  39. Namdjoyan, S., Namdjoyan, S., & Kermanian, H. (2012). Induction of phytochelatin and responses of antioxidants under cadmium stress in safflower (Carthamus tinctorius) seedlings. Turkish Journal of Botany, 36, 495–502.Google Scholar
  40. Nosrati, S., Asli, E. D., & Pazoki, A. (2013). Studying the resistance, absorption and accumulation of cadmium in safflower (Carthamus tinctorius L.) plant. Annals of Biological Research, 4, 169–173.Google Scholar
  41. Polle, A., Otter, T., & Seifert, F. (1994). Apoplastic peroxidises and lignification in needles of Norway Spruce Picea abies L. Plant Physiology, 106(1), 53–60.CrossRefPubMedPubMedCentralGoogle Scholar
  42. Pulido, R., Bravo, L., & Saura-Calixto, F. (2000). Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. Journal of Agricultural and Food Chemistry, 48(8), 402–3396.CrossRefGoogle Scholar
  43. Quiroga, M., Guerrero, C., Botella, A., Barcelo, M., Amaya, I., Medina, M. I., et al. (2000). A tomato peroxidase involved in the synthesis of lignin and suberin. Plant Physiology, 122, 1119–1127.CrossRefPubMedPubMedCentralGoogle Scholar
  44. Rout, J. R., Ram, S. S., Das, R., Chakraborty, A., Sudarshan, M., & Sahoo, S. L. (2013). Copper-stress induced alternations in protein profile and antioxidant enzymes activities in the in vitro grown Withania somnifera L. Physiology and Molecular Biology of Plants, 19(3), 353–361.CrossRefPubMedPubMedCentralGoogle Scholar
  45. Schützendübel, A., Schwanz, P., Teichmann, T., Gross, K., Langenfeld-Heyser, R., Godbold, D. L., et al. (2001). Cadmium-induced changes in antioxidative systems, H2O2 content and differentiation in pine (pinus sylvestris) roots. Plant Physiology, 127, 887–892.CrossRefPubMedPubMedCentralGoogle Scholar
  46. Sharma, A., & Singh, G. (2013). Studies on the effect of Cu (II) ions on the antioxidant enzymes in chickpea (Cicer arietinum L) cultivars. Journal of Stress Physiology and Biochemistry, 9(1), 5–13.Google Scholar
  47. Singh, N., Ma, L. Q., Srivastava, M., & Rathinasabapathi, B. (2006). Metabolic adaptations to arsenic-induced oxidative stress in Pteris vittata L. and Pteris ensiformis L. Plant Science, 170(2), 274–282.CrossRefGoogle Scholar
  48. Velich, I., Lakatos, S., Vegvarl, A., Stefanovlts-Banyal, E., & Sardi, E. (2000). Study of peroxidase isozyme pattern on susceptible bean genotypes natural infected with Pseudomonas syringae pv. savastanoi. Annual Report-Bean Improvement Cooperative, 43, 188–189.Google Scholar
  49. Vitória, A. P., Lea, P. J., & Azevedo, R. A. (2001). Antioxidant enzymes responses to cadmium in radish tissues. Phytochemistry, 57, 701–710.CrossRefPubMedGoogle Scholar
  50. Wang, S. H., Yang, Z. M., Hong, Y., Lu, B., Li, S. Q., & Lu, Y. P. (2004). Copper-induced stress and antioxidative responses in roots of Brassica juncea L. Botanical Bulletin- Academia Sinica, 45, 203–212.Google Scholar
  51. Willekens, H., Chamnongpol, S., Davey, M., Schraudner, M., Langebartels, C., Van Montagu, M., et al. (1997). Catalase is a sink for H2O2 and is indispensable for stress defense in C3 plants. European Molecular Biology Organization, 16, 4806–4816.CrossRefGoogle Scholar
  52. Woodbury, W., Spencer, A. K., & Stahmann, M. A. (1971). An improved procedure using ferricyanide for detecting catalase isozymes. Analytical Biochemistry, 44(1), 301–305.CrossRefPubMedGoogle Scholar
  53. Zengin, F. K., & Munzuroglu, O. (2005). Effects of some heavy metals on content of chlorophyll, proline and some antioxidant chemicals in bean (Phaseolus vulgaris L.) seedlings. Acta Biologica Cracoviensia Series: Botanica, 47(2), 157–164.Google Scholar

Copyright information

© Indian Society for Plant Physiology 2018

Authors and Affiliations

  1. 1.Department of Bioscience and BiotechnologyBanasthali UniversityBanasthaliIndia
  2. 2.School of Studies in BiotechnologyJiwaji UniversityGwaliorIndia

Personalised recommendations