Increased Leaf Nicotine Content by Targeting Transcription Factor Gene Expression in Commercial Flue-Cured Tobacco (Nicotiana tabacum L.).

Hai Liu,Michael P Timko,Tatyana I Kotova

doi:10.3390/genes10110930

Abstract

Nicotine, the most abundant pyridine alkaloid in cultivated tobacco (Nicotiana tabacum L.), is a potent inhibitor of insect and animal herbivory and a neurostimulator of human brain function. Nicotine biosynthesis is controlled developmentally and can be induced by abiotic and biotic stressors via a jasmonic acid (JA)-mediated signal transduction mechanism involving members of the APETALA 2/ethylene-responsive factor (AP2/ERF) and basic helix-loop-helix (bHLH) transcription factor (TF) families. AP2/ERF and bHLH TFs work combinatorically to control nicotine biosynthesis and its subsequent accumulation in tobacco leaves. Here, we demonstrate that overexpression of the tobacco NtERF32, NtERF221/ORC1, and NtMYC2a TFs leads to significant increases in nicotine accumulation in T2 transgenic K326 tobacco plants before topping. Up to 9-fold higher nicotine production was achieved in transgenics overexpressing NtERF221/ORC1 under the control of a constitutive GmUBI3 gene promoter compared to wild-type plants. The constitutive 2XCaMV35S promoter and a novel JA-inducible 4XGAG promoter were less effective in driving high-level nicotine formation. Methyljasmonic acid (MeJA) treatment further elevated nicotine production in all transgenic lines. Our results show that targeted manipulation of NtERF221/ORC1 is an effective strategy for elevating leaf nicotine levels in commercial tobacco for use in the preparation of reduced risk tobacco products for smoking replacement therapeutics.

Highlights

Pyridine alkaloids are toxic compounds that play a key role in plant defense mechanisms against herbivore and insect attack [1,2,3,4]
In order highly regulated in the roots of wild and cultivated tobacco, and both APETALA 2/ethylene-responsive factor (AP2/ethylene response factor (ERF)) and basic helix-loop-helix (bHLH) transcription factor (TF) have determine if the overexpression of one or more specific TF is an effective means of increasing nicotine been shown to be involved in regulation of these genes (Supplementary Figure S2)
In order to levels in flue-cured tobacco (Nicotiana tabacum L.), we transformed K326 plants with a set of transgene constructs, in which the coding sequences of five TF genes previously nicotine levels in flue-cured tobacco (Nicotiana tabacum L.), we transformed K326 plants (a typical implicated in controlling nicotine gene expression (i.e., NtERF10, NtERF32, NtERF121, cultivated variety) with a set of biosynthetic transgene constructs, in which the coding sequences of five TF

Summary

Introduction

Pyridine alkaloids are toxic compounds that play a key role in plant defense mechanisms against herbivore and insect attack [1,2,3,4]. During the natural growth cycle, and in the absence of significant biotic or abiotic stress, cultivated tobacco plants produce only minimal basal levels of nicotine due to the high cost of metabolism. This level becomes elevated rapidly in response to insect or animal herbivory or in response to wounding, triggered by natural or human-mediated topping or decapitation [6,7,8]. The decapitation or wounding response is well known to result in the induced biosynthesis and transportation of jasmonic acid (JA) and its derivatives, such as methyljasmonic acid (MeJA), as part of a damage signal from shoot to root to promote the biosynthesis of nicotine and other alkaloids [9,10].

Methods

Results

Conclusion