Suppression of pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine in leaves of tobacco (N. tabacum L.).

BACKGROUND: Anatabine, although being one of four major tobacco alkaloids, is never accumulated in high quantity in any of the naturally occurring species from the Nicotiana genus. Previous studies therefore focused on transgenic approaches to synthetize anatabine, most notably by generating transgenic lines with suppressed putrescine methyltransferase (PMT) activity. This led to promising results, but the global gene expression of plants with such distinct metabolism has not been analyzed. In the current study, we describe how these plants respond to topping and the downstream effects on alkaloid biosynthesis. RESULTS: The surge in anatabine accumulation in PMT transgenic lines after topping treatment and its effects on gene expression changes were analyzed. The results revealed increases in expression of isoflavone reductase-like (A622) and berberine bridge-like enzymes (BBLs) oxidoreductase genes, previously shown to be crucial for the final steps of nicotine biosynthesis. We also observed significantly higher methylputrescine oxidase (MPO) expression in all plants subjected to topping treatment. In order to investigate if MPO suppression would have the same effects as that of PMT, we generated transgenic plants. These plants with suppressed MPO expression showed an almost complete drop in leaf nicotine content, whereas leaf anatabine was observed to increase by a factor of ~ 1.6X. CONCLUSION: Our results are the first concrete evidence that suppression of MPO leads to decreased nicotine in favor of anatabine in tobacco roots and that this anatabine is successfully transported to tobacco leaves. Alkaloid transport in plants remains to be investigated to higher detail due to high variation of its efficiency among Nicotiana species and varieties of tobacco. Our research adds important step to better understand pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine.

CLCNt2 Mediates Nitrate Content in Tobacco Leaf, Impacting the Production of Tobacco-Specific Nitrosamines in Cured Leaves.

Nitrate accumulation in tobacco (Nicotiana tabacum L.) leaf, particularly in the burley (BU) type, is a reservoir for the generation of nitrosating agents responsible for the formation of tobacco-specific nitrosamines (TSNAs). TSNAs are mainly produced via the nitrosation of alkaloids occurring during the curing of tobacco leaves. Additional formation of TSNAs may also occur during tobacco storage, leaf processing and in some circumstances via pyrosynthesis during combustion. Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are found in the tobacco products and have been documented to be animal carcinogens. A previous study showed that decreasing the accumulation of nitrate in tobacco leaf via the overexpression of a deregulated form of nitrate reductase is efficient to reduce the production of TSNAs. We pursue in finding another molecular genetic target to lower nitrate in BU tobacco. Suppressing expression or knocking-out CLCNt2 has a direct impact on leaf nitrate and TSNA reduction in cured leaves without altering biomass. This study provides now a straight path toward the development of new commercial tobacco varieties with reduced TSNA levels by breeding of variants deficient in active CLCNt2 copies.

Asparagine Synthesis During Tobacco Leaf Curing.

Senescence is a genetically controlled mechanism that modifies leaf chemistry. This involves significant changes in the accumulation of carbon- and nitrogen-containing compounds, including asparagine through the activity of asparagine synthetases. These enzymes are required for nitrogen re-assimilation and remobilization in plants; however, their mechanisms are not fully understood. Here, we report how leaf curing-a senescence-induced process that allows tobacco leaves to dry out-modifies the asparagine metabolism. We show that leaf curing strongly alters the concentration of the four main amino acids, asparagine, glutamine, aspartate, and glutamate. We demonstrate that detached tobacco leaf or stalk curing has a different impact on the expression of asparagine synthetase genes and accumulation of asparagine. Additionally, we characterize the main asparagine synthetases involved in the production of asparagine during curing. The expression of ASN1 and ASN5 genes is upregulated during curing. The ASN1-RNAi and ASN5-RNAi tobacco plant lines display significant alterations in the accumulation of asparagine, glutamine, and aspartate relative to wild-type plants. These results support the idea that ASN1 and ASN5 are key regulators of asparagine metabolism during leaf curing.

Impairing both HMA4 homeologs is required for cadmium reduction in tobacco.

In tobacco, the heavy metal P1B-ATPases HMA4.1 and HMA4.2 function in root-to-shoot zinc and cadmium transport. We present greenhouse and field data that dissect the possibilities to impact the two homeologous genes in order to define the best strategy for leaf cadmium reduction. In a first step, both genes were silenced using an RNAi approach leading to >90% reduction of leaf cadmium content. To modulate HMA4 function more precisely, mutant HMA4.1 and HMA4.2 alleles of a Targeting Induced Local Lesions IN Genomes (TILLING) population were combined. As observed with RNAi plants, knockout of both homeologs decreased cadmium root-to-shoot transfer by >90%. Analysis of plants with segregating null and wild-type alleles of both homeologs showed that one functional HMA4 allele is sufficient to maintain wild-type cadmium levels. Plant development was affected in HMA4 RNAi and double knockout plants that included retarded growth, necrotic lesions, altered leaf morphology and increased water content. The combination of complete functional loss (nonsense mutation) in one homeologous HMA4 gene and the functional reduction in the other HMA4 gene (missense mutation) is proposed as strategy to limit cadmium leaf accumulation without developmental effects.

Identification of CYP82E21 as a functional nicotine N-demethylase in tobacco flowers.

In the tobacco plant, nicotine N-demethylase enzymes (NND) belonging to the cytochrome P450 family catalyse the conversion of nicotine to nornicotine, the precursor of the carcinogenic tobacco-specific N-nitrosamine, N-nitrosonornicotine. To date three demethylase genes, namely CYP82E4, CYP82E5 and CYP82E10, have been shown to be involved in this process, while the related CYP82E2 and CYP82E3 genes are not functional. We have identified a further gene named CYP82E21 encoding a putative nicotine N-demethylase closely related to the CYP82E genes. The CYP82E21 gene was found in all Nicotiana tabacum cultivars analysed and originates from the tobacco ancestor Nicotiana tomentosiformis. We show that, in contrast to all other previously characterized NND genes, CYP82E21 is not expressed in green or senescent leaves, but in flowers, more specifically in ovaries. The nicotine N-demethylase activity of CYP82E21 was confirmed by ectopic expression of the coding sequence in a tobacco line lacking functional CYP82E4, CYP82E5 and CYP82E10 genes, resulting in an eightfold increase of nicotine demethylation compared to the control plants. Furthermore, nornicotine formation can be reduced in ovaries by introducing a CYP82E21-specific RNAi construct. Together, our results demonstrate that the CYP82E21 gene encodes a functional ovary-specific nicotine N-demethylase.