Sugar Tech

pp 1–11 | Cite as

Hydrogen Peroxide-Induced Oxidative Stress in Sugarcane and Response Expression Pattern of Stress-Responsive Genes Through Quantitative RT-PCR

  • R. Manimekalai
  • Jini Narayanan
  • R. Ranjini
  • M. Gokul
  • A. Selvi
  • Pradheep Kumar
  • R. Gomathi
Research Article


Economic production of sugarcane is highly affected by oxidative stress due to biotic and abiotic stress factors. The main objective of this study was to determine which concentration of hydrogen peroxide can induce the oxidative stress conditions in sugarcane and determine whether several key gene expression responses reported in other species are triggered in sugarcane. Sixty-day-old sugarcane plants were sprayed with 30% H2O2 (300, 500 and 1000 ppm) for three consecutive days. Controls were maintained by spraying with water. Quantitative RT-PCR was performed to quantify the gene expression levels. The PCR products were cloned and sequenced to confirm the identity of the gene amplified. The representative sequences of the stress-responsive genes were deposited in GenBank (Acc. No KX828698 for HSP gene, KX828699 for APX gene, KX828700 for NAC transcription factor and KX828701 for ERF transcription factor). The expression pattern of stress-responsive key genes (HSP, NAC, ERF, GST, MYBAS and CAT) showed higher expression levels within 48 h of H2O2 treatment at 500 and 1000 ppm concentration. However, MYBAS transcription factor and catalase genes showed higher expression levels at a higher concentration of H2O2 (1000 ppm) of H2O2 at 48 h of treatment. Ascorbate peroxidase gene was up-regulated at 1000 ppm of H2O2 at 72 h of treatment. NAC transcription factor, heat shock proteins, ethylene-responsive factor and glutathione S-transferase showed a significant increase in expression at 500 ppm concentration of H2O2 at 48 h. Based on the expression pattern levels, 500 ppm of hydrogen peroxide at 48 h can induce the oxidative stress in sugarcane. The expression levels indicated that HSP, NAC, ERF, GST and MYBAS genes might play a role in signal transduction pathways, and APX and CAT are involved in scavenging reactive oxygen species.


Sugarcane Oxidative stress Reactive oxygen species Transcription factors Quantitative PCR 



Reactive oxygen species


Transcription factors


Quantitative Reverse-TranscriptionPolymerase Chain Reaction


Hydrogen peroxide


  1. Altschul, S.F., T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D.J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389–3402.CrossRefPubMedPubMedCentralGoogle Scholar
  2. Caverzan, A., P. Gisele, S.B. Rosa, C.W. Ribeiro, L. Fernanda, and M. Pinheiro. 2012. Plant responses to stresses: role of ascorbate peroxidase in the antioxidant protection. Genetics and Molecular Biology 35: 1010–1019.CrossRefGoogle Scholar
  3. Clemente, R., V. Vicente, Z. Saral, F. María, C. López, M. Valeria, and C. Gómez. 2012. Biotechnological approaches to study plant responses to stress. BioMed Research International 2012: 1–10.Google Scholar
  4. Deng, X.P., Y.J. Cheng, X.B. Wu, S.S. Kwak, W. Chen, and A.E. Eneji. 2012. Exogenous hydrogen peroxide positively influences root growth and metabolism in leaves of sweet potato seedlings. Australian Journal of Crop Science 6: 1572–1578.Google Scholar
  5. Desikan, R., A. Clarke, J.T. Hancock, and S.J. Neill. 1999. H2O2 activates a MAP kinase-like enzyme in Arabidopsis thaliana suspension cultures. Journal of Experimental Botany 50: 1863–1866.Google Scholar
  6. Ditt, R.F., A. Gentile, R.G. Tavares, S.R. Camargo, J.H. Fernandez, M.J. Silva, and M. Menossi. 2011. Analysis of the stress-inducible transcription factor SsNAC23 in sugarcane plants. Scientia Agricola 68: 454–461.CrossRefGoogle Scholar
  7. Dombrowski, J.E., and R.C. Martin. 2009. Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress. Plant Science 176: 390–396.CrossRefGoogle Scholar
  8. Foyer, C.H., and G. Noctor. 2005. Redox homeostasis and antioxidant signalling: a metabolic interface between stress perception and physiological responses. Plant Cell 17(7): 1866–1875.CrossRefPubMedPubMedCentralGoogle Scholar
  9. Gill, S.S., and N. Tudeja. 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry 48: 909–930.CrossRefPubMedGoogle Scholar
  10. Góemez, J.M., J.A. Hernández, A. Jiménez, L.A. del Río, and F. Sevilia. 1999. Differential response of antioxidative enzymes of chloroplasts and mitochondria to long-term NaCl stress of pea plants. Free Radical Research 31: S11–S18.CrossRefGoogle Scholar
  11. Gosset, D.R., S.W. Banks, E.P. Millhollon, and M.C. Lucas. 1996. Antioxidant response to NaCl stress in a control and NaCl-tolerant cotton cell line grown in the presence of paraquat, buthionine sulfoximine and exogenous glutathione. Plant Physiology 112: 803–809.CrossRefGoogle Scholar
  12. Guo, J., J. Wu, J. Qian, W. Chao, L. Lei, Y. Yi, W. Yonghua, and W. Jian. 2008. Genome-wide analysis of heat shock transcription factor families in rice and Arabidopsis. Journal of Genetics and Genomics 35: 105–118.CrossRefPubMedGoogle Scholar
  13. Harb, A., A. Dalal, and N. Samarah. 2016. Gene expression and activity of antioxidant enzymes in barley (Hordeum vulgare L.) under controlled severe drought. Journal of Plant Interactions 10: 109–116.CrossRefGoogle Scholar
  14. Hegedus, D., M. Yu, D. Baldwin, M. Gruber, A. Sharpe, I. Parkin, S. Whitwill, and D. Lydiate. 2003. Molecular characterization of Brassica napus NAC domain transcriptional activators induced in response to biotic and abiotic stress. Plant Molecular Biology 53: 383–397.CrossRefPubMedGoogle Scholar
  15. Hernandez, J.A., A. Jimenez, P. Mullineaux, and F. Sevilia. 2000. Tolerance of pea (Pisum sativumL.) to long-term salt stress is associated with induction of antioxidant defences. Plant Cell Environment 23: 853–862.CrossRefGoogle Scholar
  16. Hung, K.T., Y.T. Hsu, and C.H. Kao. 2006. Hydrogen peroxide is involved in methyl jasmonate- induced senescence of rice leaves. Physiologia Plantarum 127: 293–303.CrossRefGoogle Scholar
  17. James, G. 2004. Sugarcane, 2nd ed. London: Wiley Publishing. ISBN 978-0-632-05476-3.CrossRefGoogle Scholar
  18. Livak, K.J., and T.D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2∆∆C(T) method. Methods 25: 402–408.CrossRefPubMedGoogle Scholar
  19. Manimekalai, R., N. Jini, M. Gokul, Arun Selvi, A. Meena, R. Gomathi, and Bakshi Ram. 2017. Genome wide analysis of NAC gene family ‘sequences’ in sugarcane and its comparative phylogenetic relationship with rice, sorghum, maize and Arabidopsis for prediction of stress associated NAC genes. Agri Gene 3: 1–11.CrossRefGoogle Scholar
  20. Meng, X., B. Yin, H.L. Feng, X. Zhang, Q. Liang, and Q.C. Meng. 2014. Over-expression of R2R3-MYB gene leads to accumulation of anthocyanin and enhanced resistance to chilling and oxidative stress. Biologia Planetarium 58: 121–130.CrossRefGoogle Scholar
  21. Mhamdi, A., Q. Guillaume, S.V. Chaouch, S. Vanderauwera, F.V. Breusegem, and G. Noctor. 2010. Catalase function in plants: a focus on Arabidopsis mutants as stress-mimic models. Journal of Experimental Botany 10: 1–24.Google Scholar
  22. Mittler, R., S. Vanderauwera, M. Gollery, and B.F. Van. 2004. Reactive oxygen gene network of plants. Trends Plant Sciences 9(10): 490–498.CrossRefGoogle Scholar
  23. Mizoi, J., K. Shinozaki, and S.K. Yamaguchi. 2012. AP2/ERF family transcription factors in plant abiotic stress responses. Biochimica et Biophysica Acta 1819: 86–96.CrossRefPubMedGoogle Scholar
  24. Mukherjee, S.K. 1950. Search for wild relatives of sugarcane in India. International Sugar Journal 52: 261–262.Google Scholar
  25. Neill, S., R. Desikan, and J. Hancock. 2002. Hydrogen peroxide signalling. Current Opinion in Plant Biology 5: 388–395.CrossRefPubMedGoogle Scholar
  26. Noctor, G., and C. Foyer. 1998. Ascorbate and glutathione: keeping active oxygen under control. Annual Review Plant Physiology 49: 249–279.CrossRefGoogle Scholar
  27. Pegoraro, C., R.F.D.A. Daniel, L.M. Mertz, S.R.S. Railson, M.D.C. Luciano, V. Rombaldi, and O.C.D. Antonio. 2013. Ethylene response factors gene regulation and expression profiles under different stresses in rice. Theoretical and Experimental Plant Physiology 25: 261–274.CrossRefGoogle Scholar
  28. Prasad, T.K., M.D. Anderson, B.A. Martin, and C.R. Stewart. 1994. Evidence for chilling induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. Plant Cell 6: 65–74.CrossRefPubMedPubMedCentralGoogle Scholar
  29. Rocha, F., F.S. Terzi, Y. Nishiyama, Z.N. Vencio, M. Souza, et al. 2007. Signal transduction related responses to phytohormones and environmental challenges in sugarcane. BMC Genomics 8: 1471–2164.CrossRefGoogle Scholar
  30. Rouch, J.M., S.E. Bingham, and M.R. Sommerfeld. 2004. Protein expression during heat stress in thermo-intolerance and thermo-tolerance diatoms. Journal of Experimental Marine Biology and Ecology 306: 231–243.CrossRefGoogle Scholar
  31. Roy, S. 2015. Function of MYB domain transcription factors in abiotic stress and epigenetic control of stress response in plant genome. Plant Signalling and Behaviour 10: 1559–2324.Google Scholar
  32. Savouré, A., D. Thorin, M. Davey, X.J. Hua, S. Mauro, M. Van Montagu, D. Inzé, and N. Verbruggen. 1999. NaCl and CuZnSO4 treatments trigger distinct oxidative defence mechanism in Nicotiana plumbaginifolia L. Plant Cell Environment 22: 387–396.CrossRefGoogle Scholar
  33. Sharma, R., A. Sahoo, R. Devendran, and M. Jain. 2014. Over expression of rice tau class glutathione s-transferase gene improves tolerance to salinity and oxidative stress in Arabidopsis. PLoS ONE 9(3): e92900. Scholar
  34. Sofo, A., A. Scopa, M. Nuzzaci, and A. Vitti. 2015. Ascorbate peroxidase and catalase activities and their genetic regulation in plants subjected to drought and salinity stresses. International Journal of Molecular Sciences 16: 13561–13578. Scholar
  35. Upadhyaya, H., M.H. Khan, and S.K. Panda. 2007. Hydrogen peroxide induces oxidative stress in detached leaves of Oryza sativa L. Genetics and Plant Physiology 33: 83–95.Google Scholar
  36. Vanderauwera, S., P. Zimmermann, S. Rombauts, S. Vandenabeele, C. Langebartels, W. Gruissem, D. Inze, and F.V. Breusegem. 2005. Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis. Plant Physiology 139: 806–821.CrossRefPubMedPubMedCentralGoogle Scholar
  37. Vijayalakshmi, D., S. Srividhya, S. Muthulakshmi, and R. Satishraj. 2014. Induction of Oxidative stress by hydrogen peroxide treatment in rice genotypes to study the osmolyte accumulation pattern and antioxidant capacity. Journal of Stress Physiology and Biochemistry 10: 37–46.Google Scholar
  38. Wang, W., B. Vinocur, O. Shoseyov, and A. Altman. 2004. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends in Plant Science 9: 244–252.CrossRefPubMedGoogle Scholar
  39. Wang, X., H. Han, J. Yan, F. Chen, and W. Wei. 2015. A new AP2/ERF transcription factor from the oil plant Jatropha curcas confers salt and drought tolerance to transgenic tobacco. Applied Biochemistry and Biotechnology 176: 582–597. Scholar
  40. Xiang, J., L. Zhibin, Y. Yang, and L. Xufeng. 2012. Cloning and analysis of the ascorbate peroxidase gene promoter from Brassica napus. African Journal of Biotechnology 11: 6428–6433.CrossRefGoogle Scholar
  41. Yan, J., N. Tsuichihara, T. Etoh, and S. Iwai. 2007. Reactive oxygen species and nitric oxide are involved in ABA inhibition of stomatal opening. Plant, Cell and Environment 30: 1320–1325.CrossRefPubMedGoogle Scholar
  42. Zheng, X., B. Chen, G. Lu, and B. Han. 2008. Over expression of a NAC transcription factor enhances rice drought and salt tolerance. Biochemical and Biophysical Research communication 379: 985–989.CrossRefGoogle Scholar

Copyright information

© Society for Sugar Research & Promotion 2018

Authors and Affiliations

  • R. Manimekalai
    • 1
  • Jini Narayanan
    • 1
  • R. Ranjini
    • 1
  • M. Gokul
    • 1
  • A. Selvi
    • 1
  • Pradheep Kumar
    • 1
  • R. Gomathi
    • 2
  1. 1.Crop Improvement Division, ICAR- Sugarcane Breeding InstituteIndian Council of Agricultural Research (ICAR)CoimbatoreIndia
  2. 2.Crop Production Division, ICAR- Sugarcane Breeding InstituteIndian Council of Agricultural Research (ICAR)CoimbatoreIndia

Personalised recommendations