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Molecular Biotechnology

, Volume 56, Issue 2, pp 111–125 | Cite as

Overexpression of a Pea DNA Helicase (PDH45) in Peanut (Arachis hypogaea L.) Confers Improvement of Cellular Level Tolerance and Productivity Under Drought Stress

  • M. Manjulatha
  • Rohini Sreevathsa
  • A. Manoj Kumar
  • Chinta Sudhakar
  • T. G. Prasad
  • Narendra Tuteja
  • M. UdayakumarEmail author
Research

Abstract

Peanut, a major edible oil seed crop globally is predominantly grown under rainfed conditions and suffers yield losses due to drought. Development of drought-tolerant varieties through transgenic technology is a valid approach. Besides superior water relation traits like water mining, intrinsic cellular level tolerance mechanisms are important to sustain the growth under stress. To achieve this objective, the focus of this study was to pyramid drought adaptive traits by overexpressing a stress responsive helicase, PDH45 in the background of a genotype with superior water relations. PCR, Southern, and RT-PCR analyses confirmed stable integration and expression of the PDH45 gene in peanut transgenics. At the end of T3 generation, eight transgenic events were identified as promising based on stress tolerance and improvement in productivity. Several transgenic lines showed stay-green phenotype and increased chlorophyll stability under stress and reduced chlorophyll retardation under etherel-induced simulated stress conditions. Stress-induced root growth was also substantially higher in the case of transformants. This was reflected in increased WUE (low Δ13C) and improved growth rates and productivity. The transgenics showed 17.2 and 26.75 % increase in yield under non-stress and stress conditions over wild type ascertaining the feasibility of trait pyramiding strategy for the development of drought-tolerant peanut.

Keywords

Peanut In planta transformation PDH45 helicase Drought Yield Stress tolerance 

Notes

Acknowledgments

Authors acknowledge the financial support from Department of Biotechnology, Programme support (BT/01/COE/05/03), and the Niche Area of Excellence (ICAR) (F. No. 10-(6)/2005 EP&D).

References

  1. 1.
    Mahajan, S., & Tuteja, N. (2005). Cold, salinity and drought stresses: An overview. Archives of Biochemistry and Biophysics, 444, 139–158.CrossRefGoogle Scholar
  2. 2.
    Tuteja, N. (2007). Mechanisms of high salinity tolerance in plants. Methods in Enzymology, 428, 419–438.CrossRefGoogle Scholar
  3. 3.
    Bhatnagar-Mathur, P., Jyotsna Devi, M., Srinivas Reddy, D., Lavanya, M., Vadez, V., Serraj, R., et al. (2007). Stress-inducible expression of At DREB1A in transgenic peanut (Arachis hypogaeaL.) increases transpiration efficiency under water-limiting conditions. Plant Cell Reports, 26, 2071–2082.CrossRefGoogle Scholar
  4. 4.
    Akcay, U. C., Ercan, O., Kavas, M., Yildiz, L., Yilmaz, C., Oktem, H. A., et al. (2010). Drought-induced oxidative damage and antioxidant responses in peanut (Arachis hypogaea L.) seedlings. Plant Growth Regulation, 61, 21–28.CrossRefGoogle Scholar
  5. 5.
    Vadez, V., Rao, J. S., Bhatnagar-Mathur, P., & Sharma, K. K. (2013). DREB1A promotes root development in deep soil layers and increases water extraction under water stress in groundnut. Plant Biology, 15(1), 45–52.CrossRefGoogle Scholar
  6. 6.
    Qin, H., Gu, Q., Zhang, J., Sun, L., Kuppu, S., Zhang, Y., et al. (2011). Regulated expression of an isopentenyl transferase gene (IPT) in peanut significantly improves drought tolerance and increases yield under field conditions. Plant Cell Physiology, 52(11), 1904–1914.CrossRefGoogle Scholar
  7. 7.
    Asif, M. A., Zafar, Y., Iqbal, J., Iqbal, M. M., Rashid, U., Ali, G. M., et al. (2011). Enhanced expression of AtNHX1, in transgenic groundnut (Arachis hypogaea L.) improves salt and drought tolerance. Molecular Biotechnology, 49(3), 250–256.CrossRefGoogle Scholar
  8. 8.
    Tuteja, N. (2009). Cold, salt and drought stress. In H. Hirt (Ed.), Plant stress biology: From genomics towards system biology (pp. 137–159). Weinheim: WILEY-VCH Verlag GmbH & Co KGaA. ISBN 978-3-527-32290-9.CrossRefGoogle Scholar
  9. 9.
    Bartels, D., & Sunkar, R. (2005). Drought and salt tolerance in plants. Critical Reviews in Plant Sciences, 24, 23–58.CrossRefGoogle Scholar
  10. 10.
    Amuda, J., & Balasubramani, G. (2011). Recent molecular advances to combat abiotic stress tolerance in crop plants. Biotechnology and Molecular Biology Reviews, 6(2), 31–58.Google Scholar
  11. 11.
    Tuteja, N. (2010). A method to confer salinity stress tolerance to plants by helicase overexpression. Methods in Molecular Biology, 587, 377–387.CrossRefGoogle Scholar
  12. 12.
    Dang, H. Q., Tran, N. Q., Gill, S. S., Tuteja, R., & Tuteja, N. (2011). A single subunit MCM6 from pea promotes salinity stress tolerance without affecting yield. Plant Molecular Biology, 76, 19–34.CrossRefGoogle Scholar
  13. 13.
    Umate, P., Tuteja, R., & Tuteja, N. (2010). Genome-wide analysis of helicase gene family from rice and Arabidopsis: a comparison with yeast and human. Plant Molecular Biology, 73, 449–465.CrossRefGoogle Scholar
  14. 14.
    Amin, M., Elias, S. M., Hossain, A., Ferdousi, A., Rahman, M. S., Tuteja, N., et al. (2012). Overexpression of a DEAD box helicase, PDH45, confers both seedling and reproductive stage salinity tolerance to rice (Oryza sativa L.). Molecular Breeding, 30(1), 345–354.CrossRefGoogle Scholar
  15. 15.
    Tuteja, N. (2003). Plant DNA helcases: the long unwinding road. Journal of Experimental Botany, 54, 2201–2221.CrossRefGoogle Scholar
  16. 16.
    Sanan-Mishra, N., Pham, X. H., Sopory, S. K., & Tuteja, N. (2005). Pea DNA helicase 45 over expression in tobacco confers high salinity tolerance without affecting yield. Proceedings of the National Academy of Sciences of the United States of America, 102, 509–514.CrossRefGoogle Scholar
  17. 17.
    Tuteja, N., Vashisht, A., & Tuteja, R. (2008). Translation initiation factor 4A (eIF 4A): Prototype DEAD-Box RNA helicase. Physiology and Molecular Biology of Plants, 14, 101–107.CrossRefGoogle Scholar
  18. 18.
    Tuteja, N., Gill, S. S., & Tuteja, R. (2012). Helicases in improving abiotic stress tolerance in crop plants. In N. Tuteja, S. S. Gill, A. F. Tiburcio, & R. Tuteja (Eds.), Improving crop resistance to abiotic stress (pp. 433–445). Weinheim: Wiley. ISBN 978-3-527-32840-6.CrossRefGoogle Scholar
  19. 19.
    Gong, Z. Z., Dong, C. H., Lee, H., Zhu, J. H., Xiong, L. M., Gong, D. M., et al. (2005). A DEAD box RNA helicase is essential for mRNA export and important for development and stress responses in Arabidopsis. Plant Cell, 17, 256–267.CrossRefGoogle Scholar
  20. 20.
    Gong, Z. Z., Lee, H., Xiong, L. M., Jagendorf, A., Stevenson, B., & Zhu, J. K. (2002). RNA helicase-like protein as an early regulator of transcription factors for plant chilling and freezing tolerance. Proceedings of the National Academy of Sciences of the United States of America, 99, 11507–11512.CrossRefGoogle Scholar
  21. 21.
    Nakamura, T., Muramoto, Y., & Takabe, T. (2004). Structural and transcriptional characterization of a salt-responsive gene encoding putative ATP-dependent RNA helicase in barley. Plant Science, 167, 63–70.CrossRefGoogle Scholar
  22. 22.
    Vashisht, A. A., Pradhan, A., Tuteja, R., & Tuteja, N. (2005). Cold and salinity stress-induced pea bipolar pea DNA helicase 47 is involved in protein synthesis and stimulated by phosphorylation with protein kinase C. Plant Journal, 44, 76–87.CrossRefGoogle Scholar
  23. 23.
    Liu, H. H., Liu, J., Fan, S. L., Song, M. Z., Han, X. L., Liu, F., et al. (2008). Molecular cloning and characterization of a salinity stress-induced gene encoding DEAD-box helicase from the halophyte Apocynumvenetum. Journal of Experimental Botany, 59(3), 633–644.CrossRefGoogle Scholar
  24. 24.
    Luo, Y., Liu, Y. B., Dong, Y. X., Gao, X. Q., & Zhang, X. S. (2008). Expression of a putative alfalfa helicase increases tolerance to abiotic stress in Arabidopsis by enhancing the capacities for ROS scavenging and osmotic adjustment. Journal of Plant Physiology, 166(4), 385–394.CrossRefGoogle Scholar
  25. 25.
    Li, D., Liu, H., Zhang, H., Wang, X., & Song, Fengming. (2008). OsBIRH1, a DEAD box RNA helicase with functions in modulating defense responses against pathogen infection and oxidative stress. Journal of Experimental Botany, 59, 2133–2146.CrossRefGoogle Scholar
  26. 26.
    Rohini, V. K., & Rao, K. S. (2000). Transformation of peanut (Arachis hypogaea L.): A non-tissue culture based approach for generating transgenic plants. Plant Science, 15, 41–49.CrossRefGoogle Scholar
  27. 27.
    Jefferson, R. A., Kavanagh, T. A., & Bevan, M. W. (1987). GUS fusions: β-Glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal, 6, 3901–3907.Google Scholar
  28. 28.
    Dellaporta, S. L., Wood, J., & Hicks, J. B. (1983). A plant DNA mini preparation: version II. Plant Molecular Biology Reporter, 1, 19–21.CrossRefGoogle Scholar
  29. 29.
    Datta, K., Schmidt, A., & Marcus, A. (1989). Characterization of two soybean repetitive proline-rich proteins and a cognate cDNA from germinated axes. Plant Cell, 1, 945–952.Google Scholar
  30. 30.
    Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning, plain view. New York: Cold Spring Harbor Laboratory Press.Google Scholar
  31. 31.
    Hiscox, J. D., & Israelstam, G. F. (1979). Different methods of chlorophyll extraction. Journal of Botany, 57, 1332.CrossRefGoogle Scholar
  32. 32.
    Sullivan, C. Y., & Ross, W. M. (1979). Selecting for drought and heat resistance in grain sorghum. In H. Mussel & R. C. Staples (Eds.), Stress physiology in crop plants (pp. 263–281). NewYork: Wiley-Interscience.Google Scholar
  33. 33.
    Hoekstra, F. A., Golovina, E. A., & Buitink, J. (2001). Mechanisms of plant desiccation tolerance. Trends in Plant Science, 6, 431–438.CrossRefGoogle Scholar
  34. 34.
    Vadez, V., Krishnamurthy, K. J., Kholva, J., Devi, J. M., Sharma, K. K., Bhatnagar-Mathur, P., et al. (2007). Exploiting the functionality of root system for dry, saline, and nutrient deficient environments in a changing climate. SAT eJournal, 4(1), 1–61.Google Scholar
  35. 35.
    Sheshshayee, M. S., Bindumadhava, H., Shankar, A. G., Prasad, T. G., & Udayakumar, M. (2003). Breeding strategies to exploit water use efficiency for crop improvement. Journal of Plant Biology, 30(2), 253–268.Google Scholar
  36. 36.
    Reynolds, M., & Tuberosa, R. (2008). Translational research impacting on crop productivity in drought prone environments. Current Opinion in Plant Biology, 11, 171–179.CrossRefGoogle Scholar
  37. 37.
    Sheshshayee, M. S., Bindumadhava, H., Rachaputi, N. R., Prasad, T. G., Udayakumar, M., Wright, G. C., et al. (2006). Leaf chlorophyll concentration relates to transpiration efficiency in peanut. Annals of Applied Biology, 148(1), 7–15.CrossRefGoogle Scholar
  38. 38.
    Rasmussen, S., Barah, P., Suarez-Rodriguez, M. C., Bressendorff, S., Friis, P., Costantino, P., et al. (2013). Transcriptome responses to combinations of stresses in Arabidopsis. Plant Physiology, 161(4), 1783–1794.CrossRefGoogle Scholar
  39. 39.
    Sreenivasulu, N., Sopory, S. K., & Kavi Kishor, P. B. (2007). Deciphering the regulatory mechanisms of abiotic stress tolerance in plants by genomic approaches. Gene, 388(1–2), 1–13.Google Scholar
  40. 40.
    Manjulatha, M., Suma, T. C., Rahul, S. L., Madhura, J. N., Rohini, S., Prasad, T. G., et al. (2011). Development of drought tolerant peanut through pyramiding the desirable adaptive traits by transgenic approach. In K. Muralidharan & E. A. Siddiq (Eds.), Genomica and crop improvement: Relevance and reservations (pp. 338–352). Rajendranagar, Hyderabad, India: Institute of Biotechnology, Acharya NG Ranga Agricultural University.Google Scholar
  41. 41.
    Babitha, K. C., Ramu, S. V., Pruthvi, V., Mahesh, P., Nataraja, K. N., & Udayakumar, M. (2012). Co-expression of AtbHLH17 and AtWRKY28 confers resistance to abiotic stress in Arabidopsis. Transgenic Research. doi: 10.1007/s11248-012-9645-8.Google Scholar
  42. 42.
    Ramu, S. V., Rohini, S., Keshavareddy, G., Gowri, N. M., Shanmugam, N. B., Kumar, A. R. V., et al. (2012). Expression of a synthetic cry1AcF gene in transgenic Pigeon pea confers resistance to Helicoverpaarmigera. Journal of Applied Entomology. doi: 10.1111/j.1439-0418.2011.01703.x.Google Scholar
  43. 43.
    Keshamma, E., Sreevathsa, R., Kumar, M. A., Reddy, K. N., Manjulatha, M., Shanmugam, N. B., et al. (2012). Agrobacterium-mediated in planta transformation of field bean (Lablab purpureus L.) and recovery of stable transgenic plants expressing the cry1AcF gene. Plant Molecular Biology Reporter, 30(1), 67–78.CrossRefGoogle Scholar
  44. 44.
    Zhou, Q. Y., Tian, A. G., Zou, H. F., Xie, Z. M., Lei, G., Huang, J., et al. (2008). Soybean WRKY type transcription factor genes, GmWRKY13, GmWRKY21 and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnology Journal, 6, 486–503.CrossRefGoogle Scholar
  45. 45.
    Karaba, A., Dixit, S., Greco, R., Aharoni, A., Trijatmiko, K. R., Marsch- Martinez, N., et al. (2007). Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene. Proceedings of the National Academy of Sciences of the United States of America, 104(39), 15270–15275.CrossRefGoogle Scholar
  46. 46.
    Bao, A. K., Wang, S. M., Guo, Q. W., Xi, J. J., Zhang, J. L., & Wang, C. M. (2009). Overexpression of the Arabidopsis H+-PPase enhanced resistance to salt and drought stress in transgenic alfalfa (Medicago sativa). Plant Science, 176, 232–240.CrossRefGoogle Scholar
  47. 47.
    Yu, H., Chen, X., Hong, Y. Y., Wang, Y., Xu, P., Ke, S. D., et al. (2008). Activated expression of an Arabidopsis HD-START protein confers drought tolerance with improved root system and reduced stomatal density. Plant Cell, 20, 1134–1151.CrossRefGoogle Scholar
  48. 48.
    Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K., & Shinozaki, K. (1999). Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress inducible transcription factor. Nature Biotechnology, 17, 287–291.CrossRefGoogle Scholar
  49. 49.
    Nooden, L. D., Guiamet, J. J., & John, I. (1997). Senescence mechanisms. Physiologia Plantarum, 101(4), 746–753.CrossRefGoogle Scholar
  50. 50.
    Buchanan-Wollaston, V., & Ainsworth, C. (1997). Leaf senescence in Brassica napus: Cloning of senescence-related genes by subtractive hybridization. Plant Molecular Biology, 33, 21–834.CrossRefGoogle Scholar
  51. 51.
    Ma, Q. H. (2008). Genetic engineering of cytokinins and their application to agriculture. Critical Reviews in Biotechnology, 28, 213–232.CrossRefGoogle Scholar
  52. 52.
    Hajouj, T., Michelis, R., & Gepstein, S. (2000). Cloning and characterization of a receptor like kinase gene associated with senescence. Plant Physiology, 124, 1305–1314.CrossRefGoogle Scholar
  53. 53.
    Rivero, M. R., Shulev, V., & Blumwald, E. (2009). Cytokynin-dependent photorespiration and the protection of photosynthesis during water deficit. Plant Physiology, 150, 1530–1540.CrossRefGoogle Scholar
  54. 54.
    Udayakumar, M., Sheshshayee, M. S., Nataraja, K. N., BinduMadhava, H., Devendra, R., Aftab Hussain, I. S., et al. (1998). Why has breeding for water use efficiency not been successful? An analysis and alternate approach to exploit this trait for crop improvement. Current Science, 74, 994–999.Google Scholar
  55. 55.
    Farquhar, G. D. (1989). Use of stable isotopes in evaluating plant water use efficiency. Proceedings of an international symposium on the use of stable isotope in plant nutrition, soil fertility and environmental studies, International Atomic Energy Agency, FAO, Vienna.Google Scholar
  56. 56.
    Benbella, M., & Paulsen, G. M. (1998). Efficacy of treatments for delaying senescence of wheat leaves: Senescence and grain yield under field conditions. Agronomy Journal, 90, 11.Google Scholar
  57. 57.
    Borrell, A. K., Hammer, G. L., & Henzell, R. G. (2000). Does maintaining green leaf area in sorghum improve yield under drought? Dry matter production and yield. Crop Science, 40, 1037–1048.CrossRefGoogle Scholar
  58. 58.
    Haussmann, B. I. G., Mahalakshmi, V., Reddy, B. V. S., Seetharama, N., Hash, C. T., & Geiger, H. H. (2002). QTL mapping of stay-green in two sorghum recombinant inbred populations. Theoretical and Applied Genetics, 106, 133–142.Google Scholar
  59. 59.
    Verma, V., Foullces, M. J., Worland, A. J., Sylvester-Bradley, R., Caligari, P. D. S., & Snape, J. W. (2004). Mapping quantitative trait loci for flag leaf senescence as a yield determinant in winter wheat under optimal and drought-stressed environments. Euphytica, 135, 255–263.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • M. Manjulatha
    • 1
    • 5
  • Rohini Sreevathsa
    • 2
    • 6
  • A. Manoj Kumar
    • 3
  • Chinta Sudhakar
    • 1
  • T. G. Prasad
    • 2
  • Narendra Tuteja
    • 4
  • M. Udayakumar
    • 2
    Email author
  1. 1.Department of BotanySri Krishnadevaraya UniversityAnantapurIndia
  2. 2.Department of Crop PhysiologyUniversity of Agricultural SciencesBangaloreIndia
  3. 3.Department of Plant Molecular Biology, Institute of BiotechnologyUniversidad Nacional Autónoma de MéxicoCuernavacaMexico
  4. 4.International Centre for Genetic Engineering and BiotechnologyNew DelhiIndia
  5. 5.Highland Agricultural Research CenterNational Institute of Crop Science, RDAPyeongchangRepublic of Korea
  6. 6.National Research Centre for Plant BiotechnologyNew DelhiIndia

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