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Overexpression of Ran gene from Lepidium latifolium L. (LlaRan) renders transgenic tobacco plants hypersensitive to cold stress

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Abstract

Ran is a multifunctional small GTPase involved in important cellular activities like nucleocytoplasmic transport, mitotic spindle assembly, nuclear envelope formation, etc., but is also known to be differentially expressed in response to abiotic stress, particularly low temperature. We have over-expressed Lepidium latifolium (Fam. Brassicaceae) Ran gene in tobacco to study the response of the plants to cold stress (24 h; 4 °C). Transformation of the tobacco plants was verified using PCR targeting Ran gene and co-transformed selectable marker gene nptII. Segregation in Mendelian ratios was validated in five transgenic lines by germination of T1 and T2 seeds on moist filter papers containing 150 mg/l kanamycin. Higher levels of electrolyte leakage and lipid peroxidation pointed towards hypersensitivity of plants. Similarly, lesser proline accumulation compared to wild types also indicated susceptibility of plants to death under chilling conditions. Specific activity of antioxidant enzymes superoxide dismutase and glutathione reductase was also measured under stressed and control conditions. A variation was observed across the different lines, and four out of five lines showed lesser specific activity compared to wild type plants, thus indicating reduced capability of scavenging free radicals. In totality, a strong evidence on induced hypersensitivity to cold stress has been collected which may further be helpful in designing appropriate strategies for engineering crop plants for survival under cold stress conditions.

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Acknowledgments

Vimlendu Bhushan Sinha and Sadhana Singh thank Defence Research and Development Organization (DRDO), India for providing financial assistance.

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Author notes

  1. Vimlendu Bhushan Sinha

    Present address: University School of Biotechnology, Guru Gobind Singh Indraprastha University, Sector 16C, Dwarka, New Delhi, 110078, India

Authors and Affiliations

  1. Defence Institute of Bio-Energy Research, Goraparao, P.O. Arjunpur, Haldwani, 263139, India

    Vimlendu Bhushan Sinha, Atul Grover, Sadhana Singh & Zakwan Ahmed

  2. Department of Biotechnology, Kumaun University, Bhimtal Campus, Nainital, 263136, Uttarakhand, India

    Vimlendu Bhushan Sinha & Veena Pande

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  1. Vimlendu Bhushan Sinha
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  2. Atul Grover
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  3. Sadhana Singh
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  4. Veena Pande
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  5. Zakwan Ahmed
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Correspondence to Vimlendu Bhushan Sinha.

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Vimlendu Bhushan Sinha and Atul Grover have contributed equally to this work.

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Sinha, V.B., Grover, A., Singh, S. et al. Overexpression of Ran gene from Lepidium latifolium L. (LlaRan) renders transgenic tobacco plants hypersensitive to cold stress. Mol Biol Rep 41, 5989–5996 (2014). https://doi.org/10.1007/s11033-014-3476-z

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  • DOI: https://doi.org/10.1007/s11033-014-3476-z

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