Abstract
Nicotine has practical applications relating to smoking cessation devices and alternative nicotine products. Genetic manipulation for increasing nicotine content in cultivated tobacco (Nicotiana tabacum L.) may be of value for industrial purposes, including the possibility of enhancing the efficiency of nicotine extraction. Biotechnological approaches have been evaluated in connection with this objective, but field-based results are few. Here, we report characterization of two genes encoding basic-helix-loop-helix (bHLH) transcription factors (TFs), NtMYC2a and NtMYC2b from tobacco. Overexpression of NtMYC2a increased leaf nicotine levels in T1 transgenic lines approximately 2.3-fold in greenhouse-grown plants of tobacco cultivar ‘NC 95′. Subsequent field testing of T2 and T3 generations of transgenic NtMYC2a overexpression lines showed nicotine concentrations were 76% and 58% higher than control lines, respectively. These results demonstrated that the increased nicotine trait was stably inherited to the T2 and T3 generations, indicating the important role that NtMYC2a plays in regulating nicotine accumulation in N. tabacum and the great potential of NtMYC2a overexpression in tobacco plants for industrial nicotine production. Collected data in this study also indicated a negative feedback inhibition of nicotine biosynthesis. Further enhancement of nicotine accumulation in tobacco leaf may require modification of the processes of nicotine transport and deposition.
Highlights
Methylputrescine oxidase (MPO) which catalyzes the deamination of methylputrescine
The yeast one-hybrid technique was used in this study to identify transcription factors that bind to the promoter sequence of NtQPT2
These findings prompted us to hypothesize that a major or master transcription factor related to the MeJA signaling pathway might exist to co-regulate the nicotine synthetic genes for nicotine formation
Summary
Methylputrescine oxidase (MPO) which catalyzes the deamination of methylputrescine. The pyridine ring is derived from nicotinic acid, an intermediate of the pyridine nucleotide cycle that is regulated by an entry enzyme of the cycle, quinolinic acid phosphoribosyltransferase (QPT)[11]. Several ERFs (ethylene response factors), another important class of transcription factors, were shown to positively regulate nicotine biosynthesis[18,19], but their effects on enhancement of nicotine content in cultivated tobacco plants have not been demonstrated. In another approach, overexpression of the allene oxide cyclase gene, a gene controlling a key step in jasmonate formation, was reported to cause a 4.8 fold increase in nicotine content in transgenic greenhouse-grown T0 N. tabacum cv. Strategies to further increase nicotine levels in tobacco are discussed
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