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Expression of the nos operon proteins from Pseudomonas stutzeri in transgenic plants to assemble nitrous oxide reductase

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Abstract

Nitrous oxide (N2O) is a stable greenhouse gas that plays a significant role in the destruction of the ozone layer. Soils are a significant source of atmospheric N2O. It is important to explore some innovative and effective biology-based strategies for N2O mitigation. The enzyme nitrous oxide reductase (N2OR), naturally found in soil bacteria, is responsible for catalysing the final step of the denitrification pathway, conversion of N2O to dintrogen gas (N2). To transfer this catalytic pathway from soil into plants and amplify the abundance of this essential mechanism (to reduce global warming), a mega-cassette of five coding sequences was assembled to produce transgenic plants heterologously expressing the bacterial nos operon in plant leaves. Both the single-gene transformants (nosZ) and the multi-gene transformants (nosFLZDY) produced active recombinant N2OR. Enzymatic activity was detected using the methyl viologen-linked enzyme assay, showing that extracts from both types of transgenic plants exhibited N2O-reducing activity. Remarkably, the single-gene strategy produced higher reductase capability than the whole-operon approach. The data indicate that bacterial N2OR expressed in plants could convert N2O into inert N2 without involvement of other Nos proteins. Silencing by homologous signal sequences, or cryptic intracellular targeting are possible explanations for the low activities obtained. Expressing N2OR from Pseudomonas stutzeri in single-gene transgenic plants indicated that such ag-biotech solutions to climate change have the potential to be easily incorporated into existing genetically modified organism seed germplasm.

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Acknowledgments

This work was supported by the Natural Sciences and Engineering Research Council (NSERC) and its industrial and government partners through the Green Crop Network. We also gratefully acknowledge Carlos Monreal (Agriculture and Agri-Food Canada) for guidance and Qing-yao Shu (Food and Agriculture Organisation of the United Nations/International Atomic Energy Agency) for catalysing our focus on nitrous oxide; Anastassia Voronova for purifying N2OR from P. stutzeri; W.G. Zumft (Karlsruhe Institute of Technology) for supplying anti-N2OR rabbit serum; and S. Sattar (University of Ottawa) for his excellent technical support and support with anaerobic chamber use.

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  1. Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, Centre for Research on Environmental Microbiology, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada

    Shen Wan, Yaseen Mottiar, Amanda M. Johnson, Kagami Goto & Illimar Altosaar

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  1. Shen Wan
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  2. Yaseen Mottiar
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  3. Amanda M. Johnson
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  4. Kagami Goto
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  5. Illimar Altosaar
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Correspondence to Illimar Altosaar.

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Wan, S., Mottiar, Y., Johnson, A.M. et al. Expression of the nos operon proteins from Pseudomonas stutzeri in transgenic plants to assemble nitrous oxide reductase. Transgenic Res 21, 593–603 (2012). https://doi.org/10.1007/s11248-011-9555-1

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  • DOI: https://doi.org/10.1007/s11248-011-9555-1

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