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Genetic attenuation of alkaloids and nicotine content in tobacco (Nicotiana tabacum)

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Abstract

Main conclusion

The role of six alkaloid biosynthesis genes in the process of nicotine accumulation in tobacco was investigated. Downregulation of ornithine decarboxylase, arginine decarboxylase, and aspartate oxidase resulted in viable plants with a significantly lower nicotine content.

Abstract

Attenuation of nicotine accumulation in Nicotiana tabacum was addressed upon the application of RNAi technologies. The approach entailed a downregulation in the expression of six different alkaloid biosynthesis genes encoding upstream enzymes that are thought to function in the pathway of alkaloid and nicotine biosynthesis. Nine different RNAi constructs were designed to lower the expression level of the genes that encode the enzymes arginine decarboxylase, agmatine deiminase, aspartate oxidase, arginase, ornithine decarboxylase, and SAM synthase. Agrobacterium-based transformation of tobacco leaves was applied, and upon kanamycin selection, T0 and subsequently T1 generation seeds were produced. Mature T1 plants in the greenhouse were topped to prevent flowering and leaf nos. 3 and 4 below the topping point were tested for transcript levels and product accumulation. Down-regulation in arginine decarboxylase, aspartate oxidase, and ornithine decarboxylase consistently resulted in lower levels of nicotine in the leaves of the corresponding plants. Transformants with the aspartate oxidase RNAi construct showed the lowest nicotine level in the leaves, which varied from below the limit of quantification (20 μg per g dry leaf weight) to 1.3 mg per g dry leaf weight. The amount of putrescine, the main polyamine related to nicotine biosynthesis, showed a qualitative correlation with the nicotine content in the arginine decarboxylase and ornithine decarboxylase RNAi-expressing transformants. A putative early senescence phenotype and lower viability of the older leaves was observed in some of the transformant lines. The results are discussed in terms of the role of the above-mentioned genes in the alkaloid biosynthetic pathway and may serve to guide efforts to attenuate nicotine content in tobacco leaves.

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Abbreviations

ADC:

Arginine decarboxylase

AIC:

Agmatine deiminase

AO:

Aspartate oxidase

ARG:

Arginase

ODC:

Ornithine decarboxylase

SAM:

S'adenosyl-L:-methionine

WT:

Wild type

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Acknowledgements

We thank Christina Wistrom and her staff for the greenhouse support she provided during the cultivation and growth of both the T0 and T1 tobacco plants. We also wish to thank Jason W. Flora, Mingda Zhang, and Jesse Frederick for reading and commenting on the manuscript. Funding was provided by UCB grant (85992-13618-44-ME1AM).

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Authors and Affiliations

  1. Department of Plant and Microbial Biology, University of California, Berkeley, CA, 94720‑3102, USA

    Diego Hidalgo Martinez & Anastasios Melis

  2. Eurofins Lancaster Laboratories, Professional Scientific Service Division, 2425 New Holland Pike, Lancaster, PA, 17605, USA

    Raja S. Payyavula

  3. Biotechnology Division, Altria Client Services LLC, 601 East Jackson Street, Richmond, VA, 23219, USA

    Chengalrayan Kudithipudi, Yanxin Shen, Dongmei Xu, Ujwala Warek & James A. Strickland

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  1. Diego Hidalgo Martinez
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  2. Raja S. Payyavula
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  3. Chengalrayan Kudithipudi
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  4. Yanxin Shen
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  8. Anastasios Melis
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Correspondence to Anastasios Melis.

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Research did not involve human and/or animal subjects. Experimental protocols in this work were approved by the UC Berkeley Committee on Laboratory and Environmental Biosafety (CLEB).

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Hidalgo Martinez, D., Payyavula, R.S., Kudithipudi, C. et al. Genetic attenuation of alkaloids and nicotine content in tobacco (Nicotiana tabacum). Planta 251, 92 (2020). https://doi.org/10.1007/s00425-020-03387-1

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