Dataset Information

Development of a nornicotine-reduced flue-cured tobacco line via EMS mutagenesis of nicotine N-demethylase genes.

ABSTRACT: Substantial progress had been made in reducing nornicotine accumulation in burley tobacco, as nornicotine is a precursor of the carcinogen N-nitrosonornicotine (NNN). Three members of the CYP82E2 family encoding nicotine N-demethylase (NND) have been reported to be responsible for the majority of nicotine demethylation that forms nornicotine in burley tobacco. We had obtained a nonsense mutant of each NND member in flue-cured tobacco from an ethyl methanesulfonate (EMS)-mutagenized population. In this study, we developed dCAPS markers for each nonsense mutation. Using marker-assisted selection, NND mutants were crossed with each other to generate a triple mutant GP449. In line with previous reports, the triple knockout caused significantly decreased levels of nornicotine and NNN in flue-cured tobacco. With the decreased nornicotine, the nicotine level was expected to accumulate. However, the nicotine level in GP449 was significantly decreased to 72.80% of wild type. Realtime RT-PCR analysis showed that the nicotine reduction was correlated with inhibited expression of nicotine biosynthetic pathway genes. The triple mutant and dCAPS markers can be utilized to develop new flue-cured tobacco varieties with lower levels of nornicotine and NNN.

SUBMITTER: Song Z

PROVIDER: S-EPMC7053972 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Development of a nornicotine-reduced flue-cured tobacco line via EMS mutagenesis of nicotine N-demethylase genes.

Song Zhongbang Z Sui Xueyi X Li Meiyun M Gao Yulong Y Li Wenzheng W Zhao Lu L Li Feng F Yao Xuefeng X Liu Chunming C Wang Bingwu B

Plant signaling & behavior 20200103 2

Substantial progress had been made in reducing nornicotine accumulation in burley tobacco, as nornicotine is a precursor of the carcinogen N-nitrosonornicotine (NNN). Three members of the <i>CYP82E2</i> family encoding nicotine N-demethylase (NND) have been reported to be responsible for the majority of nicotine demethylation that forms nornicotine in burley tobacco. We had obtained a nonsense mutant of each NND member in flue-cured tobacco from an ethyl methanesulfonate (EMS)-mutagenized popula ...[more]

PMID: 31900036

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Project description:BackgroundContinuous cropping of the same crop leads to land degradation. This is also called the continuous-cropping obstacle. Currently, intercropping tobacco with other crops can serve as an effective strategy to alleviate continuous cropping obstacles.ResultsIn this study, tobacco K326 and insectary floral plants were used as materials, and seven treatments of tobacco monoculture (CK), tobacco intercropped with Tagetes erecta, Vicia villosa, Fagopyrum esculentum, Lobularia maritima, Trifolium repens, and Argyranthemum frutescens respectively, were set up to study their effects on rhizosphere soil chemical properties and composition and structure of rhizosphere soil microbial community of tobacco. The 16 S rRNA gene and ITS amplicons were sequenced using Illumina high-throughput sequencing. tobacco/insectary floral plants intercropping can influence rhizosphere soil chemical properties, which also change rhizosphere microbial communities. The CK and treatment groups tobacco rhizosphere soil microorganisms had significantly different genera, such as tobacco intercropping with T. repens and A. frutescens significantly increased the number of Fusarium and intercropping T. erecta, V. villosa, L. maritima, T. repens, and A. frutescens significantly increased the number of Sphingomonas and unknown Gemmatimonadaceae. Additionally, intercropping T. erecta, V. villosa and L. maritima changed the rhizosphere fungal and bacteria community and composition of tobacco and the positive correlation between tobacco rhizosphere the genera of fungi and bacterial were greater than CK. The pathway of the carbohydrate metabolism, amino acid metabolism, and energy metabolism in rhizosphere bacteria were significantly decreased after continuous cropping. Fungal symbiotic trophic and saprophytic trophic were significantly increased after intercropping V. villosa, L. maritima and plant pathogen and animal pathogen were increased after intercropping T. repens and A. frutescens. Additionally, bacterial and fungal communities significantly correlated with soil chemical properties, respectively.ConclusionThis study reveals that intercropping tobacco with insectary floral plants, particularly T. erecta, V. villosa, L. maritima and A. frutescens significantly affects soil chemical properties and alters rhizosphere microbial communities, increasing the abundance of certain microbial genera. Additionally, intercropping enhances pathways related to carbohydrate, amino acid, and energy metabolism in rhizosphere bacteria. These findings suggest that intercropping could provide a promising strategy to overcome challenges associated with continuous tobacco cropping by regulating the rhizosphere environment.

Project description:To study the relationship between the diversity of the surface microbial community and tobacco flavor, and to improve tobacco quality using microorganisms. The microbial community composition and diversity of 14 samples of flue-cured tobacco from tobacco-producing areas in Yunnan with varying grades were analyzed by high-throughput sequencing. PICRUSt was used for predicting microbial functions. A strain of Bacillus amyloliquefaciens W6-2 with the ability to degrade pectin was screened from the surface of flued-cured tobacco leaves from Yunnan reroasted tobacco leave. The enzyme preparation was prepared through fermentation and then applied for treating flue-cured tobacco. The improvement effect was evaluated by measuring the content of macromolecule and the changes in volatile components, combined with sensory evaluations. The bacterial communities on the surface of flue-cured tobacco exhibited functional diversity, consisting primarily of Variovorax, Pseudomonas, Sphingomonas, Burkholderia, and Bacillus. These bacterial strains played a role in the aging process of flue-cured tobacco leaves by participating in amino acid metabolism and carbohydrate metabolism. These metabolic activity converted complex macromolecules into smaller molecular compounds, ultimately influence the smoking quality and burning characteristics of flue-cured tobacco. The pectinase preparation produced through fermentation using W6-2 has been found to enhance the aroma and sweetness of flue-cured tobacco, leading to improved aroma, reduced impurities, and enhanced smoothness. Additionally, the levels of pectin, cellulose, and hemicellulose decreased, while the levels of water-soluble sugar and reducing sugar increased, and the contents of esters, ketones, and aldehydes increased, and the contents of benzoic acid decreased. The study revealed the correlation between surface microorganisms and volatile components of Yunnan tobacco leaves, and the enzyme produced by the pectin-degrading bacteria W6-2 effectively improved the quality of flue-cured tobacco.

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Dataset Information

Development of a nornicotine-reduced flue-cured tobacco line via EMS mutagenesis of nicotine N-demethylase genes.

Publications

Development of a nornicotine-reduced flue-cured tobacco line via EMS mutagenesis of nicotine N-demethylase genes.

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