Abstract
Nicotiana glauca (Argentinean tree tobacco) is atypical within the genus Nicotiana, accumulating predominantly anabasine rather than nicotine and/or nornicotine as the main component of its leaf pyridine alkaloid fraction. The current study examines the role of the A622 gene from N. glauca (NgA622) in alkaloid production and utilises an RNAi approach to down-regulate gene expression and diminish levels of A622 protein in transgenic tissues. Results indicate that RNAi-mediated reduction in A622 transcript levels markedly reduces the capacity of N. glauca to produce anabasine resulting in plants with scarcely any pyridine alkaloids in leaf tissues, even after damage to apical tissues. In addition, analysis of hairy roots containing the NgA622-RNAi construct shows a substantial reduction in both anabasine and nicotine levels within these tissues, even if stimulated with methyl jasmonate, indicating a role for the A622 enzyme in the synthesis of both alkaloids in roots of N. glauca. Feeding of Nicotinic Acid (NA) to hairy roots of N. glauca containing the NgA622-RNAi construct did not restore capacity for synthesis of anabasine or nicotine. Moreover, treatment of these hairy root lines with NA did not lead to an increase in anatabine levels, unlike controls. Together, these results strongly suggest that A622 is an integral component of the final enzyme complex responsible for biosynthesis of all three pyridine alkaloids in Nicotiana.
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Acknowledgements
We thank Professor T. Hashimoto for a sample of N. tabacum A622 antibody and for general advice during this project. Drs A. Neale, S. Sinclair and K. Cane are acknowledged for their discussions and advice during the early stages of this project. We also thank R. Pickering and G. deGuzman for help with the western analysis and acknowledge the efforts of S. Ryan, N. deRycke and T. Crabb in assisting with aspects of this research.. This research was supported by an Australian Postgraduate scholarship to KdeB and research support grants from Monash University to JDH.
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DeBoer, K.D., Lye, J.C., Aitken, C.D. et al. The A622 gene in Nicotiana glauca (tree tobacco): evidence for a functional role in pyridine alkaloid synthesis. Plant Mol Biol 69, 299–312 (2009). https://doi.org/10.1007/s11103-008-9425-2
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DOI: https://doi.org/10.1007/s11103-008-9425-2