Diego Hidalgo Martinez1, Raja S Payyavula2, Chengalrayan Kudithipudi3, Yanxin Shen3, Dongmei Xu3, Ujwala Warek3, James A Strickland3, Anastasios Melis4. 1. Department of Plant and Microbial Biology, University of California, Berkeley, CA, 94720‑3102, USA. 2. Eurofins Lancaster Laboratories, Professional Scientific Service Division, 2425 New Holland Pike, Lancaster, PA, 17605, USA. 3. Biotechnology Division, Altria Client Services LLC, 601 East Jackson Street, Richmond, VA, 23219, USA. 4. Department of Plant and Microbial Biology, University of California, Berkeley, CA, 94720‑3102, USA. melis@berkeley.edu.
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. 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.
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. 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.
Authors: Jamie Waese; Jim Fan; Asher Pasha; Hans Yu; Geoffrey Fucile; Ruian Shi; Matthew Cumming; Lawrence A Kelley; Michael J Sternberg; Vivek Krishnakumar; Erik Ferlanti; Jason Miller; Chris Town; Wolfgang Stuerzlinger; Nicholas J Provart Journal: Plant Cell Date: 2017-08-14 Impact factor: 11.277
Authors: Raja S Payyavula; Duroy A Navarre; Joseph C Kuhl; Alberto Pantoja; Syamkumar S Pillai Journal: BMC Plant Biol Date: 2012-03-20 Impact factor: 4.215
Authors: Greta Nölke; Ivana Chudobova; Marcel Houdelet; Daniel Volke; Marcos Lusso; Jesse Frederick; Chengalrayan Kudithipudi; Yanxin Shen; Ujwala Warek; James A Strickland; Dongmei Xu; Helga Schinkel; Stefan Schillberg Journal: Plant Direct Date: 2021-05-27